8-Hydroxyquinoline glycoconjugates containing sulfur at the sugar anomeric position—synthesis and preliminary evaluation of their cytotoxicity

Article abstract of DOI:10.3390/molecules25184174

One of the main factors limiting the effectiveness of many drugs is the difficulty of their delivery to their target site in the cell and achieving the desired therapeutic dose. Moreover, the accumulation of the drug in healthy tissue can lead to serious side effects. The way to improve the selectivity of a drug to the cancer cells seems to be its conjugation with a sugar molecule, which should facilitate its selective transport through GLUT transporters (glucose transporters), whose overexpression is seen in some types of cancer. This was the idea behind the synthesis of 8-hydroxyquinoline (8-HQ) derivative glycoconjugates, for which 1-thiosugar derivatives were used as sugar moiety donors. It was expected that the introduction of a sulfur atom instead of an oxygen atom into the anomeric position of the sugar would increase the stability of the obtained glycoconjugates against untimely hydrolytic cleavage. The anticancer activity of new compounds was determined based on the results of the MTT cytotoxicity tests. Because of the assumption that the activity of this type of compounds was based on metal ion chelation, the effect of the addition of copper ions on cell proliferation was tested for some of them. It turned out that cancer cells treated with glycoconjugates in the presence of Cu²⁺ had a much slower growth rate compared to cells treated with free glycoconjugates in the absence of copper. The highest cytotoxic activity of the compounds was observed against the MCF-7 cell line.

Full text of DOI:10.3390/molecules25184174

molecules
Article
8-Hydroxyquinoline Glycoconjugates Containing
Sulfur at the Sugar Anomeric Position—Synthesis
and Preliminary Evaluation of Their Cytotoxicity
Monika Krawczyk 1,2,
Karol Erfurt 3
*
, Gabriela Pastuch-Gawołek 1,2 , Agnieszka Hadasik 1 and
1
Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology,
Silesian University of Technology, B. Krzywoustego 4, 44-100 Gliwice, Poland;
gabriela.pastuch@polsl.pl (G.P.-G.); agnieszkahadasik@gmail.com (A.H.)
Biotechnology Centre, Silesian University of Technology, B. Krzywoustego 8, 44-100 Gliwice, Poland
Department of Chemical Organic Technology and Petrochemistry, Silesian University of Technology,
B. Krzywoustego 4, 44-100 Gliwice, Poland; karol.erfurt@polsl.pl
2
3
*
Correspondence: monika.krawczyk@polsl.pl; Tel.: +48-32-237-1759
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editors: Robert Musioł and Giangiacomo Torri
Received: 7 August 2020; Accepted: 9 September 2020; Published: 11 September 2020
Abstract: One of the main factors limiting the effectiveness of many drugs is the difficulty of their delivery
to their target site in the cell and achieving the desired therapeutic dose. Moreover, the accumulation of
the drug in healthy tissue can lead to serious side effects. The way to improve the selectivity of a
drug to the cancer cells seems to be its conjugation with a sugar molecule, which should facilitate
its selective transport through GLUT transporters (glucose transporters), whose overexpression is
seen in some types of cancer. This was the idea behind the synthesis of 8-hydroxyquinoline (8-HQ)
derivative glycoconjugates, for which 1-thiosugar derivatives were used as sugar moiety donors.
It was expected that the introduction of a sulfur atom instead of an oxygen atom into the anomeric
position of the sugar would increase the stability of the obtained glycoconjugates against untimely
hydrolytic cleavage. The anticancer activity of new compounds was determined based on the results
of the MTT cytotoxicity tests. Because of the assumption that the activity of this type of compounds
was based on metal ion chelation, the effect of the addition of copper ions on cell proliferation was
tested for some of them. It turned out that cancer cells treated with glycoconjugates in the presence
of Cu2+ had a much slower growth rate compared to cells treated with free glycoconjugates in the
absence of copper. The highest cytotoxic activity of the compounds was observed against the MCF-7
cell line.
Keywords: quinoline; glycoconjugates; 1-thiosugars; click reaction; anticancer activity; targeted
drug delivery
1. Introduction
Despite significant progress in the treatment of various types of diseases, cancer is still one of the
most considerable clinical problems. Anticancer therapies introduced so far are not effective enough.
This is due to the low selectivity profile of approved chemotherapeutics, the use of which leads to
numerous undesirable side effects. Moreover, the resistance of cancer cells to available pharmaceuticals
results in their rapid proliferation and metastasis [1]. Therefore, it is necessary to develop new safe
drugs that target cancer cells directly without damaging healthy cells.
One of the main factors limiting the effectiveness of many drugs is the difficulty of their delivery
to their target site in the cell across the phospholipid cell membranes. A better understanding of
Molecules 2020, 25, 4174; doi:10.3390/molecules25184174
www.mdpi.com/journal/molecules

Molecules 2020, 25, 4174
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the processes ruling the delivery of substances to cancer cells can be helpful during planning new
treatment strategies. There are several ways to transport into the cell [ ]. The vast majority of available
2,3
chemotherapeutics penetrate cell membranes by passive diffusion. This is due to the presence of specific
gaps in the blood vessels of cancer cells resulting from chaotic neoangiogenesis. This is commonly
known as the effect of enhanced permeability and retention (EPR) [3–5]. On the other hand, a variety of
membrane proteins are anchored in the structure of lipid bilayer, which can act as carrier proteins or
channel proteins (facilitated diffusion). An attractive alternative to transporting chemotherapeutics
across a biological membrane may be their modification, which will increase affinity for binding to the
appropriate receptor [2–5].
One of the strategies developed for delivering anticancer drugs is the use of glucose transporting
proteins [6,7]. Compared to healthy cells, rapidly proliferating cancer cells have a different energy
metabolism, characterized by a high rate of glycolysis process, known as the Warburg effect [8,9
].
To ensure a sufficient amount of nutrients up to this process, cancer cells have an increased demand for
glucose. The phospholipid membrane surrounding the cells is impermeable to hydrophilic glucose
molecules by simple diffusion. Therefore, sugar to the cytosol is transferred by transmembrane
proteins-GLUTs or SGLTs (sodium-glucose linked transporters) [10]. In most tumor cells, these
transporters are overexpressed, especially GLUT1 [11]. Thanks to these transporters, it has been
proven that they even receive over 30 times more glucose than normal cells [12]. GLUT1 transport
substrates also include other sugars, such as galactose, mannose, and glucosamine [11]. Therefore,
an attractive system for targeted drug delivery directly to cancer cells is their conjugation with sugar
derivatives. The sugar fragment can be used as a drug carrier across the cell membrane by adapting
to the structure of the GLUT transporter [6]. The crucial question to be answered in the case of such
conjugates is whether the sugar-conjugated compound will be transported to the cancer cells by the
sugar transporters. The results of research on structural requirements for binding variously substituted
sugars to the sugar transporters indicate that β-configuration at the anomeric center of the sugar
derivatives is preferred for binding to the GLUT transporters [13–15].
Our research focuses on the use of small quinoline molecules as metal ion chelators and potential
anticancer agents [16,17]. The presence of copper ions is important in many biological processes and is
essential for cellular physiology [18]. Copper is a co-factor of many enzymes and takes part in a variety
of cellular processes in mammals [18]. Changes in the homeostasis of copper ions play an important
role in different types of diseases, such as inflammation, neurodegeneration, as well as in carcinogenesis
(copper is an essential cofactor for cancer growth and angiogenesis) [18
–21]. Therefore, the use of
metal chelating agents seems to be an ideal way to control the level of copper in the organism [21
–23].
The concept of using metal chelators is shown in Figure 1. The formation of metal complexes by
8-hydroxyquinoline (8-HQ) derivatives has been widely described in the literature and has become
a promising therapeutic strategy in clinical practice. Numerous 8-HQ derivatives have been shown
to have cytotoxic activity on many cancer cells [16,24,25]. However, a significant limitation of their
use is the lack of selectivity for healthy cells. We assumed that the therapeutic effectiveness of 8-HQ
derivatives could be improved by attaching a sugar moiety, which should increase the selectivity
of the obtained connections by targeting their transport to cancer cells through GLUT transporters.
8-Hydroxyquinoline glycoconjugates are of particular interest due to their simple synthesis procedures,
as well as facilitated intermembrane transport and improved solubility.
Our recent studies on the cytotoxic activity of 8-HQ derivatives connected via various linkers
with d-glucose or d-galactose derivatives containing an oxygen or nitrogen atom at the sugar anomeric
position indicated that some of them were able to inhibit the proliferation of tested cell lines [26,27].
Their biological activity depended on the structure of the linker connecting the sugar parts and the
quinoline moiety, as well as the type of protective groups in the attached sugar unit. The study of metal
complexing properties confirmed that the obtained glycoconjugates were capable of chelating copper
ions [27]. In the current research, 1-thiosugars derivatives were used for the synthesis of quinoline
glycoconjugates. It was expected that the introduction of a sulfur atom into the anomeric position

Molecules 2020, 25, 4174
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of the sugar would increase the stability of the obtained glycoconjugates against hydrolytic cleavage.
Compared to O-glycosyl derivatives, compounds with the S-glycosidic binding are less susceptible
to enzymatic degradation, especially under the action of glycosylhydrolases [28
,29]. 1-Thiosugars,
due to their enzymatic stability, exhibit great therapeutic potential [30 32]. The thiosugar moiety can
–
be found in many naturally occurring compounds, such as bacteriostatic antibiotics from the group of
lincosamides (lincomycin) or d-glucosinolates (sinigrin, gluconasturcin), which have chemopreventive
properties [30]. Synthetic thiosugars are effective inhibitors of numerous cellular enzyme pathways.
Besides, numerous studies have shown their significant antimicrobial, antiviral, antithrombotic,
and anticancer potential [33–36]. Therefore, increasingly sulfur derivatives of carbohydrates are used
for the synthesis of new pharmaceuticals. An example of a 1-thiosugar glycoconjugate that exhibits
antiproliferative effects in vivo is a gold-based compound 1-phenyl-bis(2-pyridyl)phosphole gold
chloride thio-β-d-glucose tetraacetate (GOPI-sugar). It inhibits the proliferation of glioblastoma cell
lines (NCH82, NCH89) to a much better extent than the drug used so far-carmustine, making it a
promising candidate in cancer therapy. Attachment of a sugar unit to the complex GOPI has improved
the solubility and bioavailability of this compound [37]. Previously reported quinolinecarboxylic
acid glycoconjugates containing in their structure a fragment derived from 1-thioglycosides have
shown a particularly interesting activity profile against colorectal cancer cells (HCT-116) more than
100 times exceeding the activity of the initial quinoline derivatives [38]. Considering the number of
reports devoted to the use of thiosugars in the treatment of a wide range of human diseases, it is worth
using this potential. In addition, compounds containing a sulfur atom have the ability to complex
metal ions, with sulfur preferring the so-called “soft” metals, such as gold, silver, or copper [39,40].
The introduction of sulfur to the designed glycoconjugates may improve their ability to complex
copper ions in relation to the previously obtained derivatives. In this article, we presented the results
of carried out cytotoxicity tests of new 8-HQ derivatives, allowing us to determine their potential
usefulness in anticancer therapy.
Figure 1. The concept of using metal chelators.
2. Results and Discussion
2.1. Synthesis
The structural modification of sugars used in this research concerned the introduction of a sulfur
atom into the anomeric position of the sugar, followed by its functionalization by chemical groups

Molecules 2020, 25, 4174
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involved in the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). The synthetic pathways
leading to the formation of sugar derivatives are presented in Schemes 1–3. All substrates were prepared
according to the previously published procedures thoroughly or making minor improvements.
Scheme 1. Synthesis of sugar derivatives 3–10. Reagents and Conditions: (i) CH3COONa, Ac2O, b.p.,
1 h; (ii) CH3COOH, 33% HBr/AcOH, 0 ◦C-r.t., 1 h; (iii) thiourea, acetone, b.p., 3 h; (iv) K2S2O5, CHCl3,
H2O, b.p., 1 h.
Scheme 2. Synthesis of 1-thiosugar derivatives 11–26. Reagents and Conditions: (i) DBU, CH2Cl2 or
(CH2)2Cl2, r.t., 1 h; (ii) NaN3, DMF, r.t., 24 h (15 and 16) or 60 ◦C, 72 h (17 and 18); (iii) 1. MeONa,
MeOH, r.t., 50 min; 2. Amberlyst-15; (iv) propargyl bromide, K2CO3, acetone, r.t., 24 h.
Scheme 3. Synthesisof1-thiosugarderivatives27
K2CO3, acetone, r.t., 2 h; (ii) Zn, AcOH, CH2Cl2, r.t., 1 h; (iii) chloroacetyl chloride, TEA, DCM, r.t., 1 h;
iv) NaN3, DMF, r.t., 24 h; ( ) 1. MeONa, MeOH, r.t., 1 h; 2. Amberlyst-15; (vi) propargyl chloroformate,
Hünig0s base, DCM, r.t., 2 h.
–38. ReagentsandConditions: (i)2-chloro-5-nitropyridine,
(
v

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2,3,4,6-Tetra-O-acetyl-1-thio-
sequence of reactions in which first glycosyl bromides
such prepared isothiouronium salts and were decomposed in the presence of potassium metabisulfite
in a two-phase water/chloroform system [41] (Scheme 1).
1-Thiosugars or 10 were substrates for the preparation of compounds used in the synthesis
β-d-glycopyranoses
9
and 10 were synthesized by carrying out a
5
or 6 reacted with thiourea in acetone, and then
7
8
9
of final glycoconjugates. Due to the susceptibility of these compounds to oxidation to symmetrical
disulfides, they were used immediately after purification by column chromatography for the next
stages of synthesis. For the glycoconjugates synthesis, the CuAAC reaction was used, in which the
substrates must possess an azide moiety or triple bond. Accordingly, it was necessary to receive
different 1-thiosugar derivatives substituted at the anomeric position with a substituent containing
either the azide moiety or triple bond.
In the beginning, chloromethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glycopyranosides 11 or 12 were
obtained in the SN2 reaction between corresponding glycosyl thiols
9
or 10 and dichloromethane,
in the presence of a strong base, such as DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) [42]. To lengthen
the alkyl chain in the structure of chloroalkyl 1-thioglycosides, an analogous reaction of 1-thiosugars
9
β
or 10 with 1,2-dichloroethane was carried out to obtain 2-chloroethyl 2,3,4,6-tetra-O-acetyl-1-thio-
-d-glycopyranosides 13 or 14. In these reactions, both dichloromethane and dichloroethane acted as a
reactant and solvent at the same time. As a result, products 11–14 were obtained in a short time and with
high yield. In the next step, the substitution of a chlorine atom with an azide moiety was carried out
using sodium azide and DMF (N,N-dimethylformamide) as a solvent, which resulted in azidomethyl
15
chlorine exchange to the azide moiety was the appearance in the 13C NMR spectra of the signal of
CH2N3 carbon with a shift of = 51.03–51.72 ppm for products 15 18, while the signal of CH2Cl carbon
was observed at δ = 43.31–45.45 ppm for substrates 11–14.
Propargyl 2,3,4,6-tetra-O-acetyl-1-thio- -d-glycopyranosides 23 and 24 were prepared by the
alkylation of 1-thiosugars or 10 with propargyl bromide. Minor modifications of the reaction
, 16 and 2-azidoethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glycopyranosides 17, 18. Confirmation of
δ
–
β
9
conditions, compared to the previously reported synthesis method [43], consisting of replacing
diisopropylethylamine (base) and dichloromethane (originally used as a solvent) with potassium
carbonate and acetone, allowed obtaining the appropriate propargyl 1-thioglycosides 23 and 24 with
high yields (97 and 94%, respectively, in comparison to 76 and 83% in cited position).
Removal of the acetyl protecting groups from compounds 15
a standard Zempl n procedure, involving the use of catalytic amounts of a methanolic solution of
sodium methoxide [44]. The reactions were carried out until the complete conversion of the substrate,
–18 and 23–24 was performed using
é
which was monitored by TLC (thin-layer chromatography). Crude products 19
–22 and 25–26 were
obtained with high yields (84–98%), sufficiently pure to be used in the next step.
Another direction of synthesis concerned the introduction of an additional aminopyridyl fragment
in the sugar part of the final structure. A simple and efficient synthesis of (5-nitro-2-pyridyl) 2,3,4,6-
tetra-O-acetyl-1-thio-β-d-glycopyranosides 27 and 28, followed by reduction to (5-amino-2-pyridyl)
2,3,4,6-tetra-O-acetyl-1-thio-β-d-glycopyranoside 29 and 30, was described a few years ago [45].
The presence of an amino group in the aglycone of 1-thioglycosides 29 and 30 allowed the addition of a
fragment containing an azide or propargyl moiety. This was possible as a result of the reaction leading
to the formation of an amide or carbamate bond. It is worth emphasizing that the introduction of an
amide bond additionally enhanced linker stability. Compounds 29 or 30 were reacted with chloroacetyl
chloride or propargyl chloroformate in anhydrous DCM (dichloromethane) (Scheme 3). The hydrogen
chloride produced during these reactions was neutralized by the addition to the reaction mixture of
a tertiary amine, such as triethylamine or N,N-diisopropylethylamine. Substitution of the chlorine
atom in compounds 31 and 32, leading to obtaining compounds 33 and 34, as well as the removal of
the acetyl protecting groups in the sugar part in order to obtain compounds 35 or 38, was carried out
under the same conditions as described above. Structures of sugar derivatives, obtained with high

Molecules 2020, 25, 4174
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yields, were confirmed by NMR spectra analysis. The spectroscopic data of obtained compounds are
presented in the experimental section.
For the synthesis of glycoconjugates, derivatives of 1-thioglycosides were used both with the
protected and deprotected hydroxyl groups in the sugar part. As the second structural element of
the glycoconjugates, corresponding derivatives of 8-hydroxyquinoline 39
8-OH position with propargyl or azide groups were used, obtained according to previously published
procedures [27 46]. Target glycoconjugates were prepared using the copper(I)-catalyzed 1,3-dipolar
azide-alkyne cycloaddition, described by Sharpless [47 48]. In these reactions, equimolar amounts of
sugar derivatives and 8-HQ derivatives were combined in the configuration shown in Scheme 4. The use
of CuSO4 5H2O as a catalyst allowed the carrying out of the reaction at room temperature and led to
–42 functionalized in the
,
,
·
obtaining only 1,4-disubstituted 1,2,3-triazoles. Sodium ascorbate (NaAsc), used as a reducing agent of
Cu(II) to Cu(I), avoided the formation of oxidation byproducts. All of the reagents were dissolved in
the THF:i-PrOH:H2O solvent system (tetrahydrofuran:isopropyl alcohol:water). The progress of the
reaction was controlled by TLC analysis so that the moment of the total conversion of the substrates
could be estimated. After purification of the crude products by column chromatography, in the result,
pure glycoconjugates 43–72 with high yields were obtained (Figure 2). The structures of all prepared
glycoconjugates were confirmed using NMR and HRMS spectroscopy methods. The formation of the
glycoconjugate was evidenced by the presence of a singlet at about
δ = 7.9 ppm from the H-C(5) proton
triazole ring in 1H NMR spectra and two characteristic carbon signals at about 123 ppm and 144 ppm for
C(4) and C(5) from triazole ring in the 13C NMR spectra, as well as the presence of signals characteristic
of the sugar and quinoline fragments. The obtained glycoconjugates contained the sugar unit with a
binding of the β-configuration at the anomeric center since such orientation is preferred for binding to
the GLUT transporters overexpressed in the cancer cells [13]. Such a structure was confirmed by the
large coupling constant from the H-1 proton, equal to about J = 9.0 Hz, observed in the 1H NMR spectra.
The NMR spectra of all the synthesized glycoconjugates are presented in the supporting information.
Their physicochemical properties, such as melting point and optical rotation, were also determined.
Scheme 4. Synthesis of glycoconjugates 43–58 (A), 59–66 (B), 67–69 (C), and 70–72 (D). Reagents and
Conditions: (i) CuSO4·5H2O, NaAsc, i-PrOH, THF, H2O, r.t., 24 h.

Molecules 2020, 25, 4174
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Figure 2. Structures and yields of obtained glycoconjugates 43–72.
2.2. Cytotoxicity Studies
In vitro cytotoxicity studies of the obtained building blocks and glycoconjugates were carried out
on cell lines characterized by the high expression of GLUT transporters and the strongly changed glucose
metabolism (strong Warburg effect) [10,49–51]. Based on this, colon cancer cell line (HCT-116) and
breast cancer cell line (MCF-7) were selected for the research. Compounds with a high ability to inhibit
the proliferation of tumor cells were also tested against the Normal Human Dermal Fibroblast-Neonatal
cells (NHDF-Neo) to assess the safety of their use. Cytotoxicity was evaluated using the MTT assay.
The results of the screening tests were used to determine the IC50 values (the half maximal inhibitory
concentration), which are summarized in Tables 1 and 2. These values represented 50% inhibition of cell
growth concerning the control system, which were the cells suspended in the medium supplemented
with DMSO in the amount used to dissolve the tested compounds.
Table 1. Summary of cytotoxicity of substrates used for glycoconjugation.
Activity IC50 [µM] a
Compound
MCF-7 c
HCT-116 b
NHDF-Neo b
15–26
33
34
35
36
>800
13.39 ± 0.10
541.37 ± 3.73
>800
124.79 ± 8.98
456.70 ± 3.85
>800
>800
>800
5.59 ± 0.14
>800
-
6.39 ± 0.07
382.69 ± 4.23
>800
38.33 ± 2.44
392.41 ± 5.29
>800
0.24 ± 0.01
43.18 ± 1.78
0.67 ± 0.01
11.20 ± 0.31
-
-
41.69 ± 3.40
37
38
-
-
>800
8HQ
2Me8HQ
Doxorubicin
346.77 ± 2.23
>20
a Cytotoxicity was evaluated using the MTT assay; b Incubation time 24 h; c Incubation time 72 h. Data are presented
as the mean ± standard deviation (n = 3).

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Table 2. Summary of cytotoxicity of tested glycoconjugates 43–72.
Activity IC50 [µM] a
Compound
MCF-7 c
HCT-116 b
NHDF-Neo b
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
106.71 ± 4.10
162.34 ± 1.35
268.54 ± 5.55
789.98 ± 2.33
791.73 ± 2.30
376.07 ± 7.16
>800
59.12 ± 1.46
85.65 ± 3.28
218.40 ± 6.24
235.24 ± 6.77
428.16 ± 8.84
204.88 ± 5.85
>800
54.62 ± 0.74
140.41 ± 7.25
-
-
-
-
-
-
>800
>800
127.05 ± 1.75
144.59 ± 4.84
338.97 ± 4.61
519.18 ± 0.90
394.77 ± 6.44
431.84 ± 5.10
>800
76.30 ± 1.33
93.97 ± 1.66
135.55 ± 1.58
204.79 ± 2.53
315.93 ± 1.66
172.46 ± 5.43
>800
332.85 ± 1.39
153.34 ± 0.25
212.15 ± 3.46
130.45 ± 0.20
243.65 ± 3.04
>800
105.32 ± 3.40
111.08 ± 1.24
-
-
-
-
-
-
>800
172.83 ± 3.48
304.80 ± 1.34
107.68 ± 1.62
324.00 ± 1.61
>800
229.12 ± 2.06
421.20 ± 0.14
260.43 ± 7.65
667.65 ± 6.43
-
>800
>800
>800
>800
>800
>800
-
-
-
146.16 ± 3.49
74.50 ± 0.16
130.31 ± 1.31
96.01 ± 0.59
210.39 ± 0.55
63.49 ± 2.37
73.87 ± 2.99
90.68 ± 0.52
82.99 ± 1.37
>800
69.72 ± 3.50
28.94 ± 1.52
96.32 ± 5.99
23.77 ± 0.48
144.42 ± 2.68
67.50 ± 1.58
25.90 ± 1.76
68.32 ± 3.68
20.51 ± 1.55
739.20 ± 3.79
71.81 ± 6.70
71.43 ± 1.35
110.60 ± 2.78
83.60 ± 3.03
163.81 ± 0.01
64.00 ± 5.34
68.43 ± 1.11
75.26 ± 0.38
69.31 ± 2.90
>800
67 + Cu2+ d
68
68 + Cu2+ d
69
70
70 + Cu2+ d
71
71 + Cu2+ d
72
Cytotoxicity was evaluated using the MTT assay; b Incubation time 24 h; c Incubation time 72 h; d Addition of
a
cooper acetate at 100 µM. Data are presented as the mean ± standard deviation (n = 3).
The screening tests of sugar substrates used for glycoconjugation indicated no toxicity of
compounds 15–26 and significant cytotoxicity of compounds 33 and 36 (Table 1). Probably due to
the small size, compounds 33 and 36 were able to penetrate through phospholipid membranes into
cells by passive diffusion. On the other hand, the presence of additional fragments in their structure
capable of coordinating divalent metal ions (an amidopyridyl part), compared to substrates 15–26,
affected their ability to inhibit cancer cell growth. As it turned out, these compounds were also highly
toxic to healthy cells. Interestingly, the d-galactose derivatives 34 and 37, as well as the compounds
35 and 38, without the acetyl protection of the hydroxyl groups were much less toxic and showed
no anti-proliferative activity. In the case of the deprotected derivatives 35 and 38, this result could
be explained by the fact that they are too little lipophilic to enter the cell by passive transport and
simultaneously, apparently, do not have sufficient affinity for GLUT transporters. However, the results
obtained for per-O-acetylated galactoconjugates 34 and 37 were surprising. The significant difference
in glycoconjugates cytotoxic activity observed with a simple replacement of the d-glucose residue with
d-galactose might indicate a significant influence of the type of sugar residue on the biological activity
of the whole compound. Cytotoxicity studies of 8-HQ and 2Me8HQ, the second building blocks of

Molecules 2020, 25, 4174
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glycoconjugates, showed that both compounds were non-toxic to HCT-116 cells and toxic to MCF-7
cells. 8-HQ appeared to be of particular interest as it exhibited high toxicity against the breast cancer
cell line, while not being toxic against the NHDF normal cell line. Thus, this compound is a good
starting point for structural modifications that should further improve its activity.
Taking into account the obtained results, it seemed necessary to develop a prodrug that would
improve the selectivity of a given compound. By starting to design prodrugs by functionalizing the
biologically active compound, we focused on differences in the properties of cancer and normal cells.
In this case, we paid attention to factors that increase the affinity of the prodrug for the interphase
space. It is known that there is a mildly acidic environment in cancer tissues that results from excessive
glycolysis in tumors (Warburg effect) [52,53]. In the case of healthy cells, this acidic environment does
not exist. Therefore, the presence of the 1,2,3-triazole ring having base properties should increase the
concentration of the prodrug at the interphase space of the cancer cells.
Dose-dependent cytotoxicity was observed for glycoconjugates derivatives of 8-HQ containing a
1,2,3-triazole fragment in the structure. The summary of cytotoxicity of tested glycoconjugates 43–72 is
shown in Table 2. In order to approximate the mechanism of glycoconjugate transport into the cell,
the influence of the presence of acetyl protecting groups in the sugar part of the glycoconjugate on
the activity was examined. High IC50 values of glycoconjugates with a deprotected sugar fragment
( –50, 55–58, 63–66) indicated that these compounds were not able to bind to glucose transporters
47
and be transported by them across biological membranes. Glycoconjugates with acetyl protecting
groups in the sugar part (43 46 51 54 59 62) turned out to be more active. This suggested that due to
their higher lipophilicity, they could cross the phospholipid bilayer by passive transport. Derivatives
43 44, and 51 52 revealed interesting antiproliferative properties against the tested tumor cell lines.
–
,
–
,
–
,
,
Unfortunately, at the same time, they also turned out to be toxic to healthy cells (NHDF-Neo). This was
probably because this type of transport is not preferred in the case of the designed prodrugs, as it does
not guarantee selectivity, and, as a result, the tested compounds damage both cancer cells as well as
healthy cells.
As part of the research, the effect of introducing a sulfur atom into the anomeric position of
sugar was assessed. Glycoconjugates 51–54 derivatives of azidoalkyl 1-thioglycosides showed higher
cytotoxic activity than the analogous glycoconjugates obtained earlier containing oxygen atom at
the sugar anomeric position [27]. This was particularly evident for the 8-HQ derivatives 51 and 52
for which the cytotoxic activity was about twice as high as that of their structural counterparts with
an anomeric oxygen atom (Figure 3). Unfortunately, an increase in cytotoxicity for glycoconjugates
containing anomeric sulfur was observed, both to neoplastic cells and to healthy cells. So, it can be
assumed that their stability in the presence of hydrolytic enzymes affects their activity and may suggest
that they were not prematurely degraded before entering the cell. In order to verify this assumption,
,
two galactoconjugates 56 and 64 (being the deprotected counterparts of galactoconjugates 52 and 60
and their two analogs with anomeric oxygen were chosen and subjected to hydrolysis reactions using
-galactosidase from Aspergillus oryzae [54]. The conducted experiments, for which the description
of the procedure is attached to Supplementary Materials, showed that glycoconjugates 56 and 64
)
β
,
containing the sulfur atom in the anomeric position, did not undergo hydrolysis in the presence of
the enzyme even after 24 h of the reaction, while their counterparts with the anomeric oxygen had
completely undergone hydrolysis after just 60 min of the enzymatic reaction.
In the case of glycoconjugates 59–62, their higher cytotoxicity, compared to the oxygen counterparts,
was not observed, despite the fact that also for a representative of these compounds, the positive influence
of the anomeric sulfur on the hydrolytic stability was confirmed experimentally. For glycoconjugates
59
anomeric propargyl moiety were used. In this reaction, an “inverted” 1,2,3-triazole system was formed
in relation to the one that was in the glycoconjugates 51 54. It can be assumed that for the cytotoxicity of
–62, to form a 1,2,3-triazole system in their structure, acetylated derivatives of 1-thiosugars with an
–
glycoconjugates, not only their hydrolytic stability is important but also the mutual spatial orientation
of the atom in the anomeric position of the sugar (sulfur or oxygen) and the 1,2,3-triazole ring, but
confirmation of this thesis requires further research.

Molecules 2020, 25, 4174
10 of 27
Figure 3. Comparison of IC50 activity glycoconjugates 51
atom at the sugar anomeric position.
(A) and 52 (B) containing sulfur or oxygen
The length of the aliphatic chain between the sugar and the 1,2,3-triazole ring did not significantly
affect the activity of glycoconjugates. It was found that d-glucose derivatives, in most cases, were more
active than d-galactose derivatives. Among them, greater cytotoxicity was demonstrated by derivatives
whose structure was based on the 8-HQ fragment, compared to derivatives with 2Me8-HQ units. Higher
antiproliferative activity of the tested compounds was found against the breast cancer cell line (MCF-7).
In this case, high toxicity to the cells of this line was observed for the quinoline aglycones themselves.
This indicated the huge sensitivity of the cells of this particular tumor line to 8-HQ.
Previously, we showed that 8-HQ derivative glycoconjugates containing 1,2,3-triazole fragment
in the linker structure were able to form complexes with copper ions and potentially inhibit the
multiplication of neoplastic cells by eliminating an important factor for their growth [27]. In the current
study, we decided to enhance this effect by creating new analogs, with an additional heteroaromatic
fragment in the structure 67
bond in the structure of glycoconjugate should improve their cytotoxic activity as their method of
action may be related to metal ion chelation. The protected glycoconjugates 67 68, and 70 71 showed
–72. The presence of sulfur, a pyridine ring, and an additional amide
,
,
good antiproliferative activity against HCT-116 and MCF-7 cancer cells. The unprotected glucose
derivative 69 showed moderate activity. Whereas, unprotected glucoconjugate 72 was not active in
the concentration range studied. Unfortunately, active compounds showed toxic activity also to the
NHDF-Neo cell line. Interestingly, the discussed compounds were created by connecting exhibiting
cytotoxic activity substrates 33 and 36 with quinoline derivatives 39 or 41. The cytotoxicity of the
resulting glycoconjugates was expected to increase relative to the substrates themselves, but this did
not happen. Perhaps the large size of the resulting glycoconjugates is one of the possible explanations
for the observed activity decrease.
Cultured cancer cells, in contrast to in vivo environment, are characterized by a low copper
level [55
mechanism of action is based on the chelation of metal ions. The antiproliferative activities of
glycoconjugates 67 68
70, and 71 were tested in the presence of Cu2+ ions. The effect of the interaction
,56]. Therefore, additional experiments were carried out for compounds whose expected
,
,
of glycoconjugate complexes with metal ions on cell proliferation was measured by adding a copper salt
solution to the growth medium and observing the rate of cell growth compared to copper-untreated cells.
First, measurements were made of the effect of different concentrations of copper on the
proliferation of cancer and non-cancer cells. Cell cultures under standard conditions were treated
for 24 h or 72 h with copper solutions of various concentrations, and their effect on cell proliferation
was verified by the MTT assay. Cells with the non-supplemented medium were used as controls. For
further experiments, a 100 µM copper acetate solution was used, as it did not affect the viability of any
of the tested cell lines in any way.
During the appropriate experiments, the cells (MCF-7, HCT-116, and NHDF-Neo) were treated
with solutions of glycoconjugate 67, 68, 70, or 71 in medium culture, supplemented with 100 µM
copper acetate, and then their proliferation was measured by the MTT assay. The control system

Molecules 2020, 25, 4174
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was cells suspended in medium supplemented with DMSO in the amount used to dissolve the
compounds and the addition of the same amount of copper. It turned out that cancer cells treated with
glycoconjugates in the presence of Cu2+ had a much slower growth rate compared to cells treated with
free glycoconjugates in the absence of copper. The highest cytotoxic activity of the compounds was
observed against the MCF-7 cell line. Their IC50 values ranged from 20.51
(Table 2). This confirmed the strong sensitivity of breast cancer cells to 8-HQ derivatives.
Figure 4 shows the cell viability treated with free glycoconjugate at 50 M and glycoconjugates
with the addition of Cu2+ at 100
M for MCF-7, HCT-116, and NHDF-Neo cells after 24 h (HCT-116 and
±
1.55
µM to 28.94 ± 1.52 µM
µ
µ
NHDF-Neo) or 72 h (MCF-7) of incubation. It can be seen that the greatest differences in cell survival
occurred in the case of MCF-7 cell lines. The detailed dependence of MCF-7 cells’ proliferation on the
concentration of a potential inhibitor is shown in Figure 5. A significant decrease in the proliferation
of MCF-7 cells was observed after treatment with glycoconjugates at a concentration of 50 µM in the
presence of Cu2+ (cell viability 10–28%), compared to experiments with glycoconjugates alone. In this
case, the cytotoxicity of glycoconjugates increased about three-fold in the presence of Cu(II) ions. Lower
concentrations of glycoconjugates also improved antiproliferative activity in the presence of Cu(II), while
concentrations above 50 µM led to complete inhibition of tumor cell growth. For HCT-116 and NHDF-Neo
cell lines, only slight differences in cell viability were observed after treatment with free compounds and
when Cu(II)(II) ions were added. This suggested that the cells’ viability of these lines was not affected by
the concentration of copper ions to the same extent as it was for the breast cancer cell line.
Figure 4. The cell viability treated with free glycoconjugate at 50
addition of Cu2+ at 100
M for MCF-7 ( ), HCT-116 ( ), and NHDF-Neo (
NHDF-Neo) or 72 h (MCF-7) incubation. Data are presented as the mean ± standard deviation (n = 3).
µM and glycoconjugates with the
µ
A
B
C) cells after 24 h (HCT-116 and
Figure 5. The dependence of MCF-7 cell proliferation on the concentration of the potential inhibitor 67
(A), 68 (B), 70 (C) or 71 (D) in the absence and presence of Cu2+
.

Molecules 2020, 25, 4174
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3. Materials and Methods
3.1. General Information
NMR spectra were recorded with an Agilent spectrometer at a frequency of 400 MHz using
TMS (tetramethylsilane) as the internal standards and CDCl3, CD3OD, or DMSO-d6 as the solvents.
NMR solvents were purchased from ACROS Organics (Geel, Belgium). Chemical shifts (δ) were
expressed in ppm and the coupling constants (J) in Hz. The following abbreviations were used to
explain the observed multiplicities: s: singlet, d: doublet, dd: doublet of doublets, ddd: doublet of
doublet of doublets, t: triplet, dd~t: doublet of doublets resembling a triplet (with similar values of
coupling constants), m: multiplet, p: pentet (quintet), b: broad. High-resolution mass spectra (HRMS)
were recorded with a WATERS LCT Premier XE system using the electrospray-ionization (ESI) technique.
Optical rotations were measured with a JASCO P-2000 polarimeter using a sodium lamp (589.3 nm)
at room temperature. Melting point measurements were performed on OptiMelt (MPA 100) Stanford
Research Systems. Reactions were monitored by thin-layer chromatography (TLC) on precoated plates
of silica gel 60 F254 (Merck Millipore, Burlington, MA, USA). The TLC plates were visualized under
UV light (λ = 254 nm) or by charring the plates after spraying with a 10% solution of sulfuric acid
in ethanol. Crude products were purified using column chromatography performed on Silica Gel 60
(70–230 mesh, Fluka, St. Louis, MI, USA), developed using toluene:EtOAc or CHCl3:MeOH as solvent
systems. All evaporations were performed on a rotary evaporator under diminished pressure at 40 ◦C.
The absorbance on MTT assay was measured spectrophotometrically at the 570 nm wavelength using a
plate reader (Epoch, BioTek, Winooski, VT, USA).
All used chemicals were purchased from Sigma-Aldrich (Saint Louis, MO, USA, ACROS Organics
(Geel, Belgium), and Avantor (Gliwice, Poland) and were used without purification. d-Glucose,
d-galactose, 8-hydroxyquinoline, and 8-hydroxyquinaldine are commercially available (Sigma-Aldrich).
2,3,4,6-Tetra-O-acetyl-1-thio-
2,3,4,6-tetra-O-acetyl-1-thio-
2,3,4,6-tetra-O-acetyl-1-thio-
-d-glycopyranosides 23, 24 [43], (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glycopyranosides
29 30 45], 8-(2-propyn-1-yloxy)quinoline 39 46], 2-methyl-8-(2-propyn-1-yloxy) quinoline 40 46],
8-(3-azidopropoxy)quinolone 41 27], and 2-methyl-8-(3-azidopropoxy)quinolone 42 27] were
β
-d-glycopyranoses
-d-glycopyranosides 15
-d-glycopyranosides 17, 18 [42], propargyl 2,3,4,6-tetra-O-acetyl-1-thio-
9
,
10 [41], chloromethyl- 11, 12 and azidomethyl
β
,
16 42], 2-chloroethyl- 13, 14 and 2-azidoethyl
[
β
β
,
[
[
[
[
[
prepared according to the respective published procedures.
3.2. Chemistry
3.2.1. General Procedure for the Synthesis of Sugar Derivatives 31 and 32
To a solution of (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
(5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio- -d-galactopyranoside 30 (685 mg, 1.5 mmol) in dry
CH2Cl2 (30 mL), triethylamine (625 L, 4.5 mmol) was added. The reaction mixture was cooled to
0 ◦C, and chloroacetyl chloride was added dropwise (175
L, 2.2 mmol), then stirring was continued at
room temperature. After 1 h, the resulting mixture was diluted with dichloromethane (50 mL) and
washed with brine (2 50 mL). The combined organic phases were dried over anhydrous MgSO4,
β-d-glucopyranoside 29 or
β
µ
µ
×
filtered, and concentrated in vacuo. The crude products were purified by column chromatography
(toluene:AcOEt; 2:1) to give products 31–32.
(5-chloroacetamide-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
β-d-glucopyranoside 31: Starting from (5-amino-2
-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio- -d-glucopyranoside 29, the product was obtained as an orange solid
β
25
(600 mg, 75%), m.p.: 145–146 ◦C; [ D = 0.4 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
α] δ 2.02, 2.02,
2,04, 2.05 (4s, 12H, CH3CO), 3.86 (ddd, 1H, J = 2.3 Hz, J = 4.6 Hz, J = 10.1 Hz, H-5Glu), 4.10 (dd, 1H,
J = 2.3 Hz, J = 12.4 Hz, H-6aGlu), 4.22 (s, 2H, CH2Cl), 4.27 (dd, 1H, J = 4.6 Hz, J = 12.4 Hz, H-6bGlu), 5.15
(dd~t, 1H, J = 9.4 Hz, J = 10.4 Hz, H-2Glu), 5.20 (dd~t, 1H, J = 9.3 Hz, J = 10.1 Hz, H-4Glu), 5.34 (dd~t,
1H, J = 9.3 Hz, J = 9.4 Hz, H-3Glu), 5.72 (d, 1H, J = 10.4 Hz, H-1Glu), 7.26 (d, 1H, J = 8.6 Hz, H-3Pyr), 7.97

Molecules 2020, 25, 4174
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(dd, 1H, J = 2.6 Hz, J = 8.6 Hz, H-4Pyr), 8.29 (bs, 1H, NH), 8.59 (d, 1H, J = 2.6 Hz, H-6Pyr); 13C NMR
(100 MHz, CDCl3): 20.60, 20.62, 20.69, 20.75 (CH3CO), 42.72 (CH2Cl), 61.94 (C-6Glu), 68.24, 69.47,
δ
74.09, 75.97 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 82.11 (C-1Glu), 123.65, 128.60, 131.53, 141.37, 151.09 (CPyr),
164.29 (C=O) 169.42, 169.50, 170.15, 170.66 (CH3CO); HRMS (ESI-TOF): calcd for C21H26N2O10SCl
([M + H]+): m/z 533.0997; found: m/z 533.0997.
(5-chloroacetamide-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-β-d-galactopyranoside 32: Starting from (5-amino-2-
pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
β
-d-galactopyranoside 30, the product was obtained as a beige solid (632 mg,
25
1
79%), m.p.: 49–51 ◦C; [
α]
= 13.6 (c = 1.0, CHCl3); H NMR (400 MHz, CDCl3):
δ
2.00, 2.01, 2,03,
D
2.17 (4s, 12H, CH3CO), 4.00–4.16 (m, 3H, H-5Gal, H-6aGal, H-6bGal), 4.22 (s, 2H, CH2Cl), 5.18 (dd, 1H,
J = 3.4 Hz, J = 9.9 Hz, H-3Gal), 5.40 (dd~t, 1H, J = 9.9 Hz, J = 10.4 Hz, H-2Gal), 5.49 (dd, 1H, J = 0.7 Hz
,
J = 3.4 Hz, H-4Gal), 5.69 (d, 1H, J = 10.4 Hz, H-1Gal), 7.28 (d, 1H, J = 8.6 Hz, H-3Pyr), 7.97 (dd, 1H,
J = 2.6 Hz, J = 8.6 Hz, H-4Pyr), 8.27 (s, 1H, NH), 8.57 (d, 1H, J = 2.6 Hz, H-6Pyr); 13C NMR (100 MHz,
CDCl3):
δ 20.60, 20.68, 20.70, 20.78 (CH3CO), 42.72 (CH2Cl), 61.29 (C-6Gal), 66.82, 67.30, 72.07, 74.58
(C-2Gal, C-3Gal, C-4Gal, C-5Gal), 82.67 (C-1Gal), 123.59, 128.54, 131.47, 141.47, 151.36 (CPyr), 164.30 (C=O)
169.70, 170.02, 170.26, 170.36 (CH3C
O); HRMS (ESI-TOF): calcd for C21H26N2O10SCl ([M + H]+): m/z
533.0997; found: m/z 533.1002.
3.2.2. General Procedure for the Synthesis of Sugar Derivatives 33 and 34
To a solution of compounds 31 or 32 (320 mg, 0.6 mmol) in dry DMF (10 mL), sodium azide (214 mg,
3.3 mmol) was added. The reaction mixture was stirred at room temperature for 24 h. After completion
of the reaction, the solvent was evaporated under reduced pressure, and the residue was dissolved
in ethyl acetate (50 mL) and extracted with water (2
organic phases were dried over anhydrous MgSO4, filtered, and concentrated in vacuo to afford the
corresponding azide 33 and 34, which were used for the next reaction without further purification.
×
30 mL) and brine (30 mL). The combined
(5-azidoacetamide-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-β-d-glucopyranoside 33: Starting from (5-chloroacetamide-
2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
β
-d-glucopyranoside 31, the product was obtained as an orange solid
25
(288 mg, 89%), m.p.: 75–78 ◦C; [
α
]
= 11.4 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
δ
2.02, 2.04,
D
2,04, 2.05 (4s, 12H, CH3CO), 3.86 (ddd, 1H, J = 2.3 Hz, J = 4.6 Hz, J = 10.1 Hz, H-5Glu), 4.10 (dd, 1H,
J = 2.3 Hz, J = 12.4 Hz, H-6aGlu), 4.19 (s, 2H, CH2N3), 4.27 (dd, 1H, J = 4.6 Hz, J = 12.4 Hz, H-6bGlu),
5.15 (dd~t, 1H, J = 9.3 Hz, J = 10.4 Hz, H-2Glu), 5.20 (dd~t, 1H, J = 9.3 Hz, J = 10.1 Hz, H-4Glu), 5.34
(dd~t, 1H, J = 9.3 Hz, J = 9.3 Hz, H-3Glu), 5.71 (d, 1H, J = 10.4 Hz, H-1Glu), 7.25 (d, 1H, J = 8.6 Hz
H-3Pyr), 7.97 (dd, 1H, J = 2.6 Hz, J = 8.6 Hz, H-4Pyr), 8.07 (bs, 1H, NH), 8.57 (d, 1H, J = 2.6 Hz, H-6Pyr);
13C NMR (100 MHz, CDCl3):
20.60, 20.63, 20.69, 20.75 ( H3CO), 52.85 (CH2N3), 61.95 (C-6Glu), 68.26,
69.49, 74.10, 75.96 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 82.15 (C-1Glu), 123.59, 128.41, 131.53, 141.33, 150.91
,
δ
C
(CPyr), 164.88 (C=O) 169.42, 169.50, 170.15, 170.66 (CH3
([M + H]+): m/z 540.1400; found: m/z 540.1396.
CO); HRMS (ESI-TOF): calcd for C21H26N5O10S
(5-azidoacetamide-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-β-d-galactopyranoside 34: Starting from (5-chloroacetamide
-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
β
-d-galactopyranoside 32, the product was obtained as a beige solid
25
(278 mg, 86%), m.p.: 57–59 ◦C; [
α
]
= 17.0 (c = 0.4, CHCl3); 1H NMR (400 MHz, CDCl3):
δ
2.00, 2.01,
D
2,03, 2.17 (4s, 12H, CH3CO), 4.04–4.16 (m, 3H, H-5Gal, H-6aGal, H-6bGal), 4.19 (s, 2H, CH2N3), 5.18
(dd, 1H, J = 3.4 Hz, J = 9.9 Hz, H-3Gal), 5.40 (dd~t, 1H, J = 9.9 Hz, J = 10.2 Hz, H-2Gal), 5.48 (dd, 1H,
J = 0.7 Hz, J = 3.4 Hz, H-4Gal), 5.68 (d, 1H, J = 10.4 Hz, H-1Gal), 7.27 (d, 1H, J = 8.6 Hz, H-3Pyr), 7.98
(dd, 1H, J = 2.7 Hz, J = 8.6 Hz, H-4Pyr), 8.08 (bs, 1H, NH), 8.57 (dd, 1H, J = 0.6 Hz, J = 2.7 Hz, H-6Pyr);
13C NMR (100 MHz, CDCl3):
67.30, 72.07, 74.57 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 82.71 (C-1Gal), 123.62, 128.43, 131.56, 141.35, 151.05
δ 20.60, 20.68, 20.70, 20.78 (CH3CO), 52.84 (CH2N3), 61.29 (C-6Gal), 66.83,
(CPyr), 164.94 (C=O) 169.69, 170.02, 170.25, 170.35 (CH3
([M + H]+): m/z 540.1400; found: m/z 540.1403.
CO); HRMS (ESI-TOF): calcd for C21H26N5O10S

Molecules 2020, 25, 4174
14 of 27
3.2.3. General Procedure for the Synthesis of Sugar Derivatives 36 and 37
To a solution of (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
(5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
CH2CL2 (20 mL), N,N-diisopropylethylamine (347
cooled to 0 ◦C, and propargyl chloroformate was added dropwise (117
β
-d-glucopyranoside 29 or
β
-d-galactopyranoside 30 (274 mg, 0.6 mmol) in dry
L, 2.1 mM) was added. The reaction mixture was
L, 1.2 mmol), then stirring was
µ
µ
continued at room temperature. After 2 h, the resulting mixture was diluted with dichloromethane
(60 mL) and washed with 1M HCl (40 mL), saturated NaHCO3 (40 mL), and brine (40 mL). The combined
organic phases were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude
products were purified by column chromatography (toluene:AcOEt; 5:1) to give products 36–37.
(5-(((prop-2-yn-1-yloxy)carbonyl)amino)-2-pyridyl)2,3,4,6-tetra-O-acetyl-1-thio-
β
-d-glucopyranoside36: Starting
-d-glucopyranoside 29, the product was obtained as an
D = 0.9 (c = 0.46, CHCl3); 1H NMR (400 MHz, CDCl3):
from (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
β
26
orange solid (265 mg, 82%), m.p.: 162–165 ◦C; [
α]
δ
2.02, 2.02, 2,03, 2.05 (4s, 12H, CH3CO), 2.54 (t, 1H, J = 2.5 Hz, CH), 3.85 (ddd, 1H, J = 2.3 Hz, J = 4.6 Hz
,
J = 10.1 Hz, H-5Glu), 4.10 (dd, 1H, J = 2.3 Hz, J = 12.4 Hz, H-6aGlu), 4.26 (dd, 1H, J = 4.6 Hz
,
J = 12.4 Hz,
H-6bGlu), 4.80 (d, 2H, J = 2.5 Hz, CH2), 5.15 (dd~t, 1H, J = 9.3 Hz, J = 10.4 Hz, H-2Glu), 5.19 (dd~t, 1H,
J = 9.3 Hz, J = 10.1 Hz, H-4Glu), 5.33 (dd~t, 1H, J = 9.3 Hz, J = 9.3 Hz, H-3Glu), 5.67 (d, 1H, J = 10.4 Hz
H-1Glu), 6.86 (bs, 1H, NH), 7.23 (d, 1H, J = 8.2 Hz, H-4Pyr), 7.84 (d, 1H, J = 8.2 Hz, H-3Pyr), 8.43 (d, 1H,
J = 2.5 Hz, H-6Pyr); 13C NMR (100 MHz, CDCl3):
20.61, 20.63, 20.70, 20.75 ( H3CO), 53.20 (CH2), 61.96
(C-6Glu), 68.27, 69.55, 74.11, 75.44 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 75.92, 77.40 (C CH), 82.37 (C-1Glu),
O); HRMS
,
δ
C
≡
123.95, 127.24, 132.48, 140.31, 149.35 (CPyr), 152.39 (C=O) 169.44, 169.52, 170.17, 170.68 (CH3
(ESI-TOF): calcd for C23H27N2O11S ([M + H]+): m/z 539.1336; found: m/z 539.1337.
C
(5-(((prop-2-yn-1-yloxy)carbonyl)amino)-2-pyridyl 2,3,4,6-tetra-O-acetyl-1-thio-
β
-d-galactopyranoside 37
:
Starting from (5-amino-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
β-d-galactopyranoside 30, the product was
26
obtained as a beige solid (258 mg, 80%), m.p.: 53–55 ◦C; [
α
]
D = 14.2 (c = 1.0, CHCl3); 1H NMR (400 MHz,
CDCl3):
δ 2.00, 2.00, 2,03, 2.17 (4s, 12H, CH3CO), 2.54 (t, 1H, J = 2.4 Hz, CH), 4.03–4.16 (m, 3H, H-5Gal,
H-6aGal, H-6bGal), 4.80 (d, 2H, J = 2.4 Hz, CH2), 5.17 (dd, 1H, J = 3.4 Hz, J = 9.9 Hz, H-3Gal), 5.39
(dd~t, 1H, J = 9.9 Hz, J = 10.4 Hz, H-2Gal), 5.48 (dd, 1H, J = 0.9 Hz, J = 3.4 Hz, H-4Gal), 5.64 (d, 1H,
J = 10.4 Hz, H-1Gal), 6.91 (bs, 1H, NH), 7.27 (d, 1H, J = 8.6 Hz, H-4Pyr), 7.85 (d, 1H, J = 8.6 Hz, H-3Pyr),
8.43 (d, 1H, J = 2.6 Hz, H-6Pyr); 13C NMR (100 MHz, CDCl3):
53.19 (CH2), 61.31 (C-6Gal), 66.90, 67.31, 72.08, 74.54 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 75.44, 77.41 (C
82.93 (C-1Gal), 123.98, 127.22, 132.49, 140.30, 149.52 (CPyr), 152.42 (C=O) 169.71, 170.04, 170.28, 170.37
(CH3CO); HRMS (ESI-TOF): calcd for C23H27N2O11S ([M + H]+): m/z 539.1336; found: m/z 539.1340.
δ
20.60, 20.67, 20.70, 20.79 (
C
H3CO),
CH),
≡
3.2.4. General Procedure for the Synthesis of Sugar Derivatives 35 and 38
(5-azidoacetamide-2-pyridyl) 2,3,4,6-tetra-O-acetyl-1-thio-
yn-1-yloxy)carbonyl)amino)-2-pyridyl 2,3,4,6-tetra-O-acetyl-1-thio-
was dissolved in MeOH (20 mL), and then 1 M solution of NaOMe in MeOH (200
β
-d-glucopyranoside 33 or (5-(((prop-2-
-d-glucopyranoside 36 (0.5 mmol)
L, 0.2 mmol) was
β
µ
added. The reaction mixture was stirred for 1 h at room temperature. After the reaction mixture was
neutralized with Amberlyst-15 ion exchange resin, the mixture was filtered off, and the filtrate was
concentrated in vacuo to give compounds 35 and 38, pure enough for further reactions.
(5-azidoacetamide-2-pyridyl) 1-thio-
β
-d-glucopyranoside 35: Starting from (5-azidoacetamide-2-pyridyl) 2,3,4,6-
tetra-O-acetyl-1-thio-
β-d-glucopyranoside 33, the product was obtained as an orange solid (150 mg, 81%), m.p.:
26
74–75 ◦C; [
α
]
D = -59.6 (c = 0.45, CH3OH); 1H NMR (400 MHz, CD3OD):
δ
3.32–3.42 (m, 3H, H-2Glu
,
,
H-4Glu, H-5Glu), 3.44 (dd~t, 1H, J = 8.6 Hz, J = 8.7 Hz, H-3Glu), 3.66 (dd, 1H, J = 5.6 Hz, J = 12.1 Hz
H-6aGlu), 3.85 (dd, 1H, J = 2.2 Hz, J = 12.1 Hz, H-6bGlu), 4.05 (s, 2H, CH2N3), 5.11 (d, 1H, J = 9.9 Hz,
H-1Glu), 7.48 (dd, 1H, J = 0.6 Hz, J = 8.7 Hz, H-3Pyr), 8.00 (dd, 1H, J = 2.6 Hz, J = 8.7 Hz, H-4Pyr), 8.63
(d, 1H, J = 2.6 Hz, H-6Pyr); 13C NMR (100 MHz, CD3OD):
δ 53.21 (CH2N3), 62.81 (C-6Glu), 71.35, 73.92,

Molecules 2020, 25, 4174
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79.77, 82.23 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 86.83 (C-1Glu), 125.20, 130.13, 134.33, 142.20, 153.64 (CPyr),
168.90 (C=O); HRMS (ESI-TOF): calcd for C13H18N5O6S ([M + H]+): m/z 372.0978; found: m/z 372.0979.
(5-(((prop-2-yn-1-yloxy)carbonyl)amino)-2-pyridyl 1-thio-β-d-glucopyranoside 38: Starting from (5-(((prop-2-
yn-1-yloxy)carbonyl)amino)-2-pyridyl 2,3,4,6-tetra-O-acetyl-1-thio-
β
-d-glucopyranoside 36, the product was
26
obtained as an orange solid (181 mg, 98%), m.p.: 69–71 ◦C; [
α
]
=
−
52.8 (c = 1.0, CH3OH); 1H NMR
D
(400 MHz, CD3OD):
δ 2.94 (t, 1H, J = 2.5 Hz, CH), 3.31–3.35 (m, 2H, H-2Glu, H-4Glu), 3.39 (ddd, 1H,
J = 2.3 Hz, J = 5.7 Hz, J = 9.7 Hz, H-5Glu), 3.43 (dd~t, 1H, J = 8.8 Hz, J = 8.8 Hz, H-3Glu), 3.66 (dd, 1H,
J = 5.7 Hz, J = 12.2 Hz, H-6aGlu), 3.85 (dd, 1H, J = 2.3 Hz, J = 12.2 Hz, H-6bGlu), 4.78 (d, 2H, J = 2.5 Hz,
CH2), 5.03 (d, 1H, J = 9.9 Hz, H-1Glu), 7.48 (d, 1H, J = 8.6 Hz, H-4Pyr), 7.89 (d, 1H, J = 8.6 Hz, H-3Pyr),
8.50 (d, 1H, J = 2.3 Hz, H-6Pyr); 13C NMR (100 MHz, CD3OD):
76.32, 79.03 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 79.74, 82.22 (C
δ
53.63 (CH2), 62.83 (C-6Glu), 71.36, 73.94,
≡
CH), 87.15 (C-1Glu), 125.86, 128.62, 135.43,
140.94, 151.80 (CPyr), 154.82 (C=O); HRMS (ESI-TOF): calcd for C15H19N2O7S ([M + H]+): m/z 371.0913;
found: m/z 371.0911.
3.2.5. General Procedure for the Synthesis of Glycoconjugates 43–72
The appropriate sugar derivatives 15
(1 eq.) were dissolved in a dry solvent system: THF (2 mL) and i-PrOH (2 mL). The catalyst systems
were prepared: sodium ascorbate (0.4 eq.) dissolved in H2O (1 mL) and CuSO4 5H2O (0.2 eq.) dissolved
–26, 33–38 (1 eq.) and 8-hydroxyquinoline derivatives 39–41
·
in H2O (1 mL), mixed, and immediately added to the reaction mixture. The reaction mixture was stirred
for 24 h at room temperature. Then, the solvents were evaporated in vacuo, and the crude products
were purified by column chromatography (dry loading; toluene:AcOEt, 2:1 and CHCl3:MeOH, 100:1
for fully protected glycoconjugates or CHCl3:MeOH, gradient: 50:1 to 2:1 for glycoconjugates with
unprotected sugar part) to give products 43–72.
Glycoconjugate 43: Starting from sugar derivative 15 and 8-HQ derivative 39, the product was obtained as a beige
23
solid (79% yield), m.p.: 191–196 ◦C; [
α
]
= δ 1.94,
70.4 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.99, 2.02, 2.05 (4s, 12H, CH3CO), 3.61 (ddd, 1H, J = 2.2 Hz, J = 4.8 Hz, J = 10.0 Hz, H-5Glu), 4.05 (dd, 1H,
J = 2.2 Hz, J = 12.5 Hz, H-6aGlu), 4.17 (dd, 1H, J = 4.8 Hz, J = 12.5 Hz, H-6bGlu), 4.55 (d, 1H, J = 10.1 Hz
,
H-1Glu), 5.02 (dd~t, 1H, J = 9.2 Hz, J = 10.0 Hz, H-4Glu), 5.06 (dd~t, 1H, J = 9.4 Hz, J = 10.1 Hz, H-2Glu),
5.15 (dd~t, 1H, J = 9.2 Hz, J = 9.4 Hz, H-3Glu), 5.29 i 5.71 (qAB, 2H, J = 14.5 Hz, CH2N3), 5.58 (s, 2H,
CH2O), 7.29 (dd, 1H, J = 1.9 Hz, J = 6.9 Hz, H-7Quin), 7.40–7.49 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.94
(s, 1H, H-5Triaz), 8.14 (dd, 1H, J = 1.6 Hz, J = 8.3 Hz, H-4Quin), 8.93 (dd, 1H, J = 1.6 Hz, 4.1 Hz, H-2Quin);
13C NMR (100 MHz, CDCl3):
δ 20.51, 20.55, 20.57, 20.74 (CH3CO), 48.05 (CH2N3), 61.58, 62.85 (C-6Glu,
CH2O), 67.91, 69.77, 73.56, 76.20 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 81.48 (C-1Glu), 109.93 (C-7Quin), 120.44
(C-5Quin), 121.71 (C-3Quin), 123.20 (C-5Triaz), 126.68 (C-6Quin), 129.52 (C-4aQuin), 136.00 (C-4Quin), 140.32
(C-8aQuin), 145.12 (C-4Triaz), 149.42 (C-2Quin), 155.63 (C-8Quin), 169.35, 169.40, 169.98, 170.54 (CH3CO);
HRMS (ESI-TOF): calcd for C27H31N4O10S ([M + H]+): m/z 603.1761; found: m/z 603.1766.
Glycoconjugate 44: Starting from sugar derivative 16 and 8-HQ derivative 39, the product was obtained as a beige
23
solid (93% yield), m.p.: 92–96 ◦C; [
α
]
= δ 1.95,
55.8 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.97, 2.03, 2.15 (4s, 12H, CH3CO), 3.67 (ddd, 1H, J = 0.8 Hz, J = 6.0 Hz, J = 6.9 Hz, H-5Gal), 3.93 (dd, 1H,
J = 6.0 Hz, J = 11.4 Hz, H-6aGal), 3.98 (dd, 1H, J = 6.9 Hz, J = 11.4 Hz, H-6bGal), 4.46 (d, 1H, J = 10.0 Hz
H-1Gal), 4.95 (dd, 1H, J = 3.4 Hz, J = 10.0 Hz, H-3Gal), 5.21 (dd~t, 1H, J = 10.0 Hz J = 10.0 Hz, H-2Gal),
,
,
5.31 and 5.71 (qAB, 2H, J = 14.6 Hz, CH2N3), 5.33 (dd, 1H, J = 0.8 Hz, J = 3.4 Hz, H-4Gal), 5.60 (s, 2H,
CH2O), 7.28 (dd, 1H, J = 2.7 Hz, J = 6.4 Hz, H-7Quin), 7.41–7.48 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.94
(s, 1H, H-5Triaz), 8.15 (dd, 1H, J = 1.6 Hz, J = 8.3 Hz, H-4Quin), 8.93 (dd, 1H, J = 1.6 Hz, 4.1 Hz, H-2Quin);
13C NMR (100 MHz, CDCl3): δ20.52, 20.60, 20.65, 20.69 (CH3CO), 47.93 (CH2N3), 61.36, 62.85 (C-6Gal
,
CH2O), 66.98, 67.13, 71.54, 74.92 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 81.81 (C-1Gal), 109.96 (C-7Quin), 120.40
(C-5Quin), 121.74 (C-3Quin), 123.25 (C-5Triaz), 126.69 (C-6Quin), 129.53 (C-4aQuin), 135.98 (C-4Quin), 140.34
(C-8aQuin), 144.99 (C-4Triaz), 149.45 (C-2Quin), 153.71 (C-8Quin), 169.60, 169.81, 170.12, 170.26 (CH3CO);
HRMS (ESI-TOF): calcd for C27H31N4O10S ([M + H]+): m/z 603.1761; found: m/z 603.1760.

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Glycoconjugate 45: Starting from sugar derivative 15 and 8-HQ derivative 40, the product was obtained as a
white solid (87% yield), m.p.: 159–163 ◦C; [α]23D = −84.8 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
δ
1.94, 1.99, 2.02, 2.07 (4s, 12H, CH3CO), 2.78 (s, 3H, CH3), 3.55 (ddd, 1H, J = 1.9 Hz, J = 4.8 Hz,
J = 10.1 Hz, H-5Glu), 4.01 (dd, 1H, J = 1.9 Hz, J = 12.5 Hz, H-6aGlu), 4.15 (dd, 1H, J = 4.8 Hz, J = 12.5 Hz,
H-6bGlu), 4.52 (d, 1H, J = 10.1 Hz, H-1Glu), 5.01 (dd~t, 1H, J = 9.3 Hz, J = 10.1 Hz, H-4Glu), 5.04 (dd~t,
1H, J = 9.3 Hz, J = 10.1 Hz, H-2Glu), 5.14 (dd~t, 1H, J = 9.3 Hz, J = 9.3 Hz, H-3Glu), 5.28 i 5.71 (qAB,
2H, J = 14.6 Hz, CH2N3), 5.59 i 5.62 (qAB, 2H, J = 13.4 Hz, CH2O), 7.22 (dd, 1H, J = 1.4 Hz, J = 7.2 Hz,
H-7Quin), 7.31 (d, 1H, J = 8.4 Hz, H-3Quin), 7.34–7.40 (m, 2H, H-5Quin, H-6Quin), 7.92 (s, 1H, H-5Triaz),
8.01 (d, 1H, J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, CDCl3):
25.74 (CH3), 47.97 (CH2N), 61.57, 63.21 (C-6Glu, CH2O), 67.90, 69.79, 73.55, 76.17 (C-2Glu, C-3Glu, C-4Glu
δ 20.51, 20.55, 20,57, 20.74 (CH3CO),
,
C-5Glu), 81.33 (C-1Glu), 110.48 (C-7Quin), 120.41 (C-5Quin), 122.62 (C-3Quin), 123.04 (C-5Triaz), 125.61
(C-6Quin), 127.76 (C-4aQuin), 136.17 (C-4Quin), 139.89 (C-8aQuin), 145.46 (C-4Triaz), 153.23 (C-2Quin), 158.29
(C-8Quin), 169.33, 169.40, 169.96, 170.51 (CH3CO); HRMS (ESI-TOF): calcd for C28H33N4O10S ([M +
H]+): m/z 617.1917; found: m/z 617.1915.
Glycoconjugate 46: Starting from sugar derivative 16 and 8-HQ derivative 40, the product was obtained as a beige
23
solid (92% yield), m.p.: 65–68 ◦C; [
α
]
= δ 1.95,
34.8 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.97, 2.06, 2.14 (4s, 12H, CH3CO), 2.78 (s, 3H, CH3), 3.49 (ddd, 1H, J = 0.8 Hz, J = 5.7 Hz, J = 7.1 Hz
,
H-5Gal), 3.83 (dd, 1H, J = 5.7 Hz, J = 11.5 Hz, H-6aGal), 3.93 (dd, 1H, J = 7.1 Hz, J = 11.5 Hz, H-6bGal), 4.38
(d, 1H, J = 10.0 Hz, H-1Gal), 4.93 (dd, 1H, J = 3.4 Hz, J = 10.0 Hz, H-3Gal), 5.19 (dd~t, 1H, J = 10.0 Hz
J = 10.0 Hz, H-2Gal), 5.28 (dd, 1H, J = 0.8 Hz, J = 3.4 Hz, H-4Gal), 5.30 and 5.70 (qAB, 2H, J = 14.5 Hz
,
,
CH2N3), 5.61 (dd, 2H, J = 2.5 Hz, J = 16.5 Hz, CH2O), 7.20 (dd, 1H, J = 1.4 Hz, J = 7.4 Hz, H-7Quin),
7.31 (d, 1H, J = 8.4 Hz, H-3Quin), 7.34–7.43 (m, 2H, H-5Quin, H-6Quin), 7.91 (s, 1H, H-5Triaz), 8.02 (d, 1H,
J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, CDCl3):
δ 20.52, 20.60, 20.63, 20.69 (CH3CO), 25.71 (CH3)
47.77 (CH2N3), 61.49, 63.13 (C-6Gal, CH2O), 67.03, 67.16, 71.51, 74.85 (C-2Gal, C-3Gal, C-4Gal, C-5Gal),
81.51 (C-1Gal), 110.47 (C-7Quin), 120.38 (C-5Quin), 122.70 (C-3Quin), 123.20 (C-5Triaz), 125.62 (C-6Quin),
127.77 (C-4aQuin), 136.15 (C-4Quin), 139.90 (C-8aQuin), 145.27 (C-4Triaz), 153.14 (C-2Quin), 158.37 (C-8Quin),
169.60, 169.80, 170.10, 170.24 (CH3CO); HRMS (ESI-TOF): calcd for C28H33N4O10S ([M + H]+): m/z
617.1917; found: m/z 617.1917.
Glycoconjugate 47: Starting from sugar derivative 19 and 8-HQ derivative 39, the product was obtained as
◦
23
1
a white solid (71% yield), m.p.: 123–126 C; [
α
]
= 127.0 (c = 1.0, DMSO); H NMR (400 MHz,
−
D
DMSO): 3.02–3.10 (m, 2H, H-2Glu, H-4Glu), 3.16 (m, 1H, H-5Glu), 3.26 (m, 1H, H-3Glu), 3.48 (m, 1H,
δ
H-6aGlu), 3.78 (m, 1H, H-6bGlu), 4.47 (d, 1H, J = 9.7 Hz, H-1Glu), 4.96 (t, 1H, J = 5.4 Hz, 6-OH), 5.02
(d, 1H, J = 5.5 Hz, OH), 5.10 (d, 1H, J = 4.9 Hz, OH), 5.29 (d, 1H, J = 6.2 Hz, OH), 5.36 (s, 2H, CH2O),
5.65 and 5.82 (qAB, 2H, J = 14.5 Hz, CH2N3), 7.42 (dd, 1H, J = 3.4 Hz, J = 5.4 Hz, H-7Quin), 7.51–7.58
(m, 3H, H-3Quin, H-5Quin, H-6Quin), 8.34 (dd, 1H, J = 1.4 Hz, J = 8.3 Hz, H-4Quin), 8.56 (s, 1H, H-5Triaz),
8.83 (d, 1H, J = 2.4 Hz, H-2Quin); 13C NMR (100 MHz, DMSO):
δ 47.81 (CH2N3), 61.39, 61.97 (C-6Glu,
CH2O), 70.16, 73.25, 78.05, 81.25 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 83.88 (C-1Glu), 110.01 (C-7Quin), 120.04
(C-5Quin), 121.88 (C-3Quin), 125.28 (C-5Triaz), 126.79 (C-6Quin), 129.08 (C-4aQuin), 135.97 (C-4Quin), 139.55
(C-8aQuin), 142.95 (C-4Triaz), 148.96 (C-2Quin), 153.74 (C-8Quin); HRMS (ESI-TOF): calcd for C19H23N4O6S
([M + H]+): m/z 435.1338; found: m/z 435.1339.
Glycoconjugate 48: Starting from sugar derivative 20 and 8-HQ derivative 39, the product was obtained
◦
23
1
as a beige solid (79% yield), m.p.: 142–145 C; [
α
]
= 58.7 (c = 1.0, DMSO); H NMR (400 MHz,
−
D
DMSO): 3.29 (m, 1H, H-3Gal), 3.39 (m, 1H, H-5Gal), 3.45 (m, 1H, H-2Gal), 3.53 (m, 1H, H-6aGal), 3.60
δ
(m, 1H, H-6bGal), 3.69 (m, 1H, H-4Gal), 4.40 (d, 1H, J = 9.6 Hz, H-1Gal), 4.52 (d, 1H, J = 4.5 Hz, OH),
4.84 (d, 1H, J = 5.2 Hz, OH), 4.91 (t, 1H, J = 5.2 Hz, 6-OH), 5.12 (d, 1H, J = 6.1 Hz, OH), 5.36 (s, 2H,
CH2O), 5.63 and 5.79 (qAB, 2H, J = 14.5 Hz, CH2N3), 7.41 (dd, 1H, J = 3.6, J = 5.4 Hz, H-7Quin), 7.50–7.58
(m, 3H, H-3Quin, H-5Quin, H-6Quin), 8.33 (dd, 1H, J = 1.7 Hz, J = 8.3 Hz, H-4Quin), 8.52 (s, 1H, H-5Triaz),
8.83 (dd, 1H, J = 1.7 Hz, J = 4.1 Hz, H-2Quin); 13C NMR (100 MHz, DMSO):
61.98 (C-6Gal, CH2O), 68.61, 70.01, 74.55, 79.68 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 84.39 (C-1Gal), 110.03
δ 47.88 (CH2N), 60.89,

Molecules 2020, 25, 4174
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(C-7Quin), 120.04 (C-5Quin), 121.88 (C-3Quin), 125.20 (C-5Triaz), 126.79 (C-6Quin), 129.08 (C-4aQuin), 135.95
(C-4Quin), 139.57 (C-8aQuin), 142.91 (C-4Triaz), 148.96 (C-2Quin), 153.72 (C-8Quin); HRMS (ESI-TOF): calcd
for C19H23N4O6S ([M + H]+): m/z 435.1338; found: m/z 435.1333.
Glycoconjugate 49: Starting from sugar derivative 19 and 8-HQ derivative 40, the product was obtained as a
23
white solid (83% yield), m.p.: 140–144 ◦C; [
δ
α
]
=
64.0 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO):
−
D
2.64 (s, 3H, CH3), 3.02–3.10 (m, 2H, H-2Glu, H-4Glu), 3.15 (m, 1H, H-5Glu), 3.24 (m, 1H, H-3Glu), 3.45
(m, 1H, H-6aGlu), 3.75 (m, 1H, H-6bGlu), 4.46 (d, 1H, J = 9.7 Hz, H-1Glu), 4.77 (t, 1H, J = 5.5 Hz, 6-OH),
5.01 (d, 1H, J = 5.4 Hz, OH), 5.10 (d, 1H, J = 4.8 Hz, OH), 5.28 (d, 1H, J = 6.2 Hz, OH), 5.35 (s, 2H, CH2O),
5.64 and 5.81 (qAB, 2H, J = 14.5 Hz, CH2N3), 7.37 (dd, 1H, J = 1.4 Hz, J = 7.6, H-7Quin), 7.42 (d, 1H,
J = 8.3 Hz, H-3Quin), 7.44 (dd~t, 1H, J = 7.3 Hz, J = 7.6 Hz, H-6Quin), 7.47 (dd, 1H, J = 1.2 Hz, J = 8.1 Hz,
H-5Quin), 8.19 (d, 1H, J = 8.4 Hz, H-4Quin), 8.49 (s, 1H, H-5Triaz); 13C NMR (100 MHz, DMSO):
δ 24.88
(CH3), 47.69 (CH2N3), 61.31, 61.65 (C-6Glu, CH2O), 70.10, 73.24, 78.04, 81.27 (C-2Glu, C-3Glu, C-4Glu
C-5Glu), 83.74 (C-1Glu), 110.22 (C-7Quin), 119.85 (C-5Quin), 122.49 (C-3Quin), 125.40, 125.66 (C-5Triaz
,
,
C-6Quin), 127.35 (C-4aQuin), 136.03 (C-4Quin), 139.14 (C-8aQuin), 142.91 (C-4Triaz), 153.29 (C-2Quin), 157.33
(C-8Quin); HRMS (ESI-TOF): calcd for C20H25N4O6S ([M + H]+): m/z 449.1495; found: m/z 449.1494.
Glycoconjugate 50: Starting from sugar derivative 20 and 8-HQ derivative 40, the product was obtained as a
beige solid (92% yield), m.p.: 134–138 ◦C; [α]23 = −90.0 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO):
D
δ 2.64 (s, 3H, CH3), 3.27 (m, 1H, H-3Gal), 3.34–3.44 (m, 2H, H-2Gal, H-5Gal), 3.49 (m, 1H, H-6aGal), 3.55
(m, 1H, H-6bGal), 3.67 (m, 1H, H-4Gal), 4.37 (d, 1H, J = 9.6 Hz, H-1Gal), 4.48 (d, 1H, J = 4.6 Hz, OH),
4.74 (t, 1H, J = 5.4 Hz, 6-OH), 4.84 (d, 1H, J = 4.9 Hz, OH), 5.11 (d, 1H, J = 6.1 Hz, OH), 5.36 (s, 2H,
CH2O), 5.62 and 5.78 (qAB, 2H, J = 14.5 Hz, CH2N3), 7.36 (dd, 1H, J = 1.7 Hz, J = 7.4 Hz, H-7Quin),
7.39–7.44 (m, 2H, H-3Quin, H-6Quin), 7.47 (dd, 1H, J = 1.6 Hz, J = 8.1 Hz, H-5Quin), 8.19 (d, 1H, J = 8.4 Hz,
H-4Quin), 8.44 (s, 1H, H-5Triaz); 13C NMR (100 MHz, DMSO):
δ 24.89 (CH3), 47.73 (CH2N3), 60.77,
61.65 (C-6Gal, CH2O), 68.53, 70.01, 74.54, 79.66 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 84.21 (C-1Gal), 110.26
(C-7Quin), 119.84 (C-5Quin), 122.49 (C-3Quin), 125.32, 125.66 (C-5Triaz, C-6Quin), 127.35 (C-4aQuin), 136.01
(C-4Quin), 139.17 (C-8aQuin), 142.87 (C-4Triaz), 153.25 (C-2Quin), 157.33 (C-8Quin); HRMS (ESI-TOF): calcd
for C20H25N4O6S ([M + H]+): m/z 449.1495; found: m/z 449.1496.
Glycoconjugate 51: Starting from sugar deri◦vative 17 and 8-HQ derivative 39, the product was obtained as
23
1
a brown solid (79% yield), m.p.: 150–152 C; [
α
]
=
−
31.2 (c = 1.0, CHCl3); H NMR (400 MHz,
D
CDCl3):
1H, J = 2.8 Hz, J = 4.6 Hz, J = 10.1 Hz, H-5Glu), 4.13 (dd, 1H, J = 2.8 Hz, J = 12.5 Hz, H-6aGlu), 4.19
(dd, 1H, J = 4.6 Hz J = 12.5 Hz, H-6bGlu), 4.47 (d, 1H, J = 10.0 Hz, H-1Glu), 4.50–4.67 (m, 2H, CH2N3),
5.02 (dd~t, 1H, J = 9.4 Hz, J = 10.1 Hz, H-4Glu), 5.04 (dd~t, 1H, J = 9.4 Hz, J = 10.0 Hz, H-2Glu), 5.21
(dd~t, 1H, J = 9.4 Hz J = 9.4 Hz, H-3Glu), 5.58 (s, 2H, CH2O), 7.32 (dd, 1H, J = 1.3 Hz, J = 7.2 Hz,
H-7Quin), 7.38–7.49 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.85 (s, 1H, H-5Triaz), 8.13 (dd, 1H, J = 1.4 Hz,
J = 8.3 Hz, H-4Quin), 8.93 (d, 1H, J = 2.7 Hz, H-2Quin); 13C NMR (100 MHz, CDCl3):
20.56, 20.57,
20.63, 20.66 ( H3CO), 30.28 (CH2S) 50.61 (CH2N3), 61.85, 62.97 (C-6Glu, CH2O), 68.11, 69.50, 73.56,
δ 2.00, 2.01, 2.03, 2.03 (4s, 12H, CH3CO), 3.05 (m, 1H, CHS), 3.25 (m, 1H, CHS), 3.70 (ddd,
,
,
δ
C
76.25 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 83.73 (C-1Glu), 110.07 (C-7Quin), 120.27 (C-5Quin), 121.65 (C-3Quin),
124.05 (C-5Triaz), 126.74 (C-6Quin), 129.51 (C-4aQuin), 135.98 (C-4Quin), 140.37 (C-8aQuin), 144.03 (C-4Triaz),
149.37 (C-2Quin), 153.87 (C-8Quin), 169.35, 169.38, 170.04, 170.49 (CH3CO); HRMS (ESI-TOF): calcd for
C28H33N4O10S ([M + H]+): m/z 617.1917; found: m/z 617.1918.
Glycoconjugate 52: Starting from sugar derivative 18 and 8-HQ derivative 39, the product was obtained as a
23
yellow solid (82% yield), m.p.: 90–95 ◦C; [
δ
α]
=
9.4 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.98, 1.99, 2.04, 2.15 (4s, 12H, CH3CO), 3.06 (m, 1H, CHS), 3.28 (m, 1H, CHS), 3.92 (ddd, 1H, J = 0.8 Hz
,
J = 6.0 Hz, J = 6.9 Hz, H-5Gal), 4.08 (dd, 1H, J = 6.0 Hz, J = 11.7 Hz, H-6aGal), 4.12 (dd, 1H, J = 6.9 Hz
J = 11.7 Hz, H-6bGal), 4.46 (d, 1H, J = 9.9 Hz, H-1Gal), 4.53–4.69 (m, 2H, CH2N3), 5.03 (dd, 1H, J = 3.3 Hz
J = 10.0 Hz, H-3Gal), 5.24 (dd~t, 1H, J = 9.9 Hz, J = 10.0 Hz, H-2Gal), 5.43 (dd, 1H, J = 0.8 Hz J = 3.3 Hz
,
,
,
,
H-4Gal), 5.58 (s, 2H, CH2O), 7.34 (d, 1H, J = 7.1 Hz, H-7Quin), 7.38–7.49 (m, 3H, H-3Quin, H-5Quin, H-6Quin),

Molecules 2020, 25, 4174
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7.85 (s, 1H, H-5Triaz), 8.13 (d, 1H, J = 8.0 Hz, H-4Quin), 8.94 (d, 1H, J = 1.9 Hz, H-2Quin); 13C NMR
(100 MHz, CDCl3): 20.56, 20.63, 20.68, 20.74 ( H3CO), 30.29 (CH2S) 50.66 (CH2N), 61.61, 62.93 (C-6Gal,
δ
C
CH2O), 66.76, 67.24, 71.64, 74.90 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 84.17 (C-1Gal), 109.99 (C-7Quin), 120.27
(C-5Quin), 121.66 (C-3Quin), 123.86 (C-5Triaz), 126.74 (C-6Quin), 129.51 (C-4aQuin), 135.96 (C-4Quin), 140.36
(C-8aQuin), 144.09 (C-4Triaz), 149.37 (C-2Quin), 153.89 (C-8Quin), 169.61, 169.94, 170.13, 170.33 (CH3CO);
HRMS (ESI-TOF): calcd for C28H33N4O10S ([M + H]+): m/z 617.1917; found: m/z 617.1920.
Glycoconjugate 53: Starting from sugar derivative 17 and 8-HQ derivative 40, the product was obtained as a
23
beige solid (77% yield), m.p.: 139–142 ◦C; [
δ
α
]
=
29.8 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
2.00, 2.01, 2.02, 2.03 (4s, 12H, CH3CO), 2.79 (s, 3H, CH3), 3.05 (m, 1H, CHS), 3.25 (m, 1H, CHS), 3.70
(ddd, 1H, J = 2.9 Hz, J = 4.5 Hz, J = 10.1 Hz, H-5Glu), 4.12 (dd, 1H, J = 2.9 Hz, J = 12.5 Hz, H-6aGlu),
4.18 (dd, 1H, J = 4.5 Hz, J = 12.5 Hz, H-6bGlu), 4.47 (d, 1H, J = 10.0 Hz, H-1Glu), 4.50–4.67 (m, 2H,
CH2N3), 5.01 (dd~t, 1H, J = 9.4 Hz, J = 10.1 Hz, H-4Glu), 5.04 (dd~t, 1H, J = 9.4 Hz, J = 10.0 Hz,
H-2Glu), 5.20 (dd~t, 1H, J = 9.4 Hz, J = 9.4 Hz, H-3Glu), 5.59 (s, 2H, CH2O), 7.26 (dd, 1H, J = 3.3 Hz
J = 5.6 Hz, H-7Quin), 7.31 (d, 1H, J = 8.4 Hz, H-3Quin), 7.33–7.39 (m, 2H, H5Quin, H-6Quin), 7.83 (s, 1H,
H-5Triaz), 8.01 (d, 1H, J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, CDCl3):
20.56, 20.57, 20.63, 20.66
H3CO), 25.73 (CH3), 30.33 (CH2S) 50.60 (CH2N3), 61.86, 63.33 (C-6Glu, CH2O), 68.12, 69.50, 73.57,
,
δ
(C
76.24 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 83.74 (C-1Glu), 110.65 (C-7Quin), 120.26 (C-5Quin), 122.59 (C-3Quin),
123.91 (C-5Triaz), 125.70 (C-6Quin), 127.76 (C-4aQuin), 136.17 (C-4Quin), 139.93 (C-8aQuin), 144.37 (C-4Triaz),
153.37 (C-2Quin), 158.19 (C-8Quin), 169.36, 169.40, 170.04, 170.50 (CH3CO); HRMS (ESI-TOF): calcd for
C29H35N4O10S ([M + H]+): m/z 631.2074; found: m/z 631.2075.
Glycoconjugate 54: Starting from sugar derivative 18 and 8-HQ derivative 40, the product was obtained as
23
a white solid (70% yield), m.p.: 56–61 ◦C; [
δ
α
]
=
9.3 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.98, 1.99, 2.04, 2.15 (4s, 12H, CH3CO), 2.79 (s, 3H, CH3), 3.06 (m, 1H, CHS), 3.28 (m, 1H, CHS), 3.90
(ddd, 1H, J = 0.7 Hz, J = 6.0 Hz, J = 7.0 Hz, H-5Gal), 4.07 (dd, 1H, J = 6.0 Hz, J = 11.9 Hz, H-6aGal), 4.11
(dd, 1H, J = 7.0 Hz, J = 11.9 Hz, H-6bGal), 4.45 (d, 1H, J = 9.9 Hz, H-1Gal), 4.53–4.69 (m, 2H, CH2N3),
5.02 (dd, 1H, J = 3.3 Hz, J = 10.0 Hz, H-3Gal), 5.24 (dd~t, 1H, J = 9.9 Hz, J = 10.0 Hz, H-2Gal), 5.42
(dd, 1H, J = 0.7 Hz, J = 3.3 Hz, H-4Gal), 5.60 (s, 2H, CH2O), 7.28 (m, 1H, H-7Quin), 7.31 (d, 1H, J = 8.4 Hz,
H-3Quin), 7.33–7.39 (m, 2H, H-5Quin, H-6Quin), 7.83 (s, 1H, H-5Triaz), 8.01 (d, 1H, J = 8.4 Hz, H-4Quin); 13
NMR (100 MHz, CDCl3): 20.56, 20.63, 20.67, 20.74 ( H3CO), 25.79 (CH3), 30.30 (CH2S) 50.65 (CH2N),
C
δ
C
61.62, 63.33 (C-6Gal, CH2O), 66.75, 67.24, 71.64, 74.89 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 84.15 (C-1Gal),
110.59 (C-7Quin), 120.25 (C-5Quin), 122.58 (C-3Quin), 123.69 (C-5Triaz), 125.70 (C-6Quin), 127.75 (C-4aQuin),
136.17 (C-4Quin), 139.90 (C-8aQuin), 144.44 (C-4Triaz), 153.36 (C-2Quin), 158.20 (C-8Quin), 169.61, 169.93,
170.12, 170.33 (CH3C
O); HRMS (ESI-TOF): calcd for C29H35N4O10S ([M + H]+): m/z 631.2074; found:
m/z 631.2070.
Glycoconjugate 55: Starting from sugar derivative 21 and 8-HQ derivative 39, the product was obtained as a
23
brown solid (99% yield), m.p.: 65–69 ◦C; [
δ
α
]
=
4.0 (c = 1.0, CH3OH); 1H NMR (400 MHz, DMSO):
−
D
2.97–3.24 (m, 6H, H-2Glu, H-3Glu, H-4Glu H-5Glu, CH2S), 3.45 (m, 1H, 6aGlu), 3.72 (m, 1H, 6bGlu), 4.37
(d, 1H, J = 9.6 Hz, H-1Glu), 4.57–4.73 (m, 3H, CH2N3, 6-OH), 4.95 (d, 1H, J = 5.3 Hz, OH), 5.03 (d, 1H,
J = 4.8 Hz, OH), 5.21 (d, 1H, J = 5.8 Hz, OH), 5.35 (s, 2H, CH2O), 7.41 (dd, 1H, J = 3.8 Hz, J = 5.3 Hz,
H-7Quin), 7.50–7.57 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 8.32 (dd, 1H, J = 1.8 Hz, J = 8.3 Hz, H-4Quin),
8.34 (s, 1H, H-5Triaz), 8.83 (dd, 1H, J = 1.8 Hz, J = 4.1 Hz, H-2Quin); 13C NMR (100 MHz, DMSO):
δ
29.78
(CH2S), 49.91 (CH2N3), 61.28, 61.91 (C-6Glu, CH2O), 70.06, 72.95, 78.10, 81.00 (C-2Glu, C-3Glu, C-4Glu
,
C-5Glu), 85.36 (C-1Glu), 110.06 (C-7Quin), 120.01 (C-5Quin), 121.84 (C-3Quin), 125.39 (C-5Triaz), 126.75
(C-6Quin), 129.06 (C-4aQuin), 135.81 (C-4Quin), 139.70 (C-8aQuin), 142.21 (C-4Triaz), 148.95 (C-2Quin), 153.86
(C-8Quin); HRMS (ESI-TOF): calcd for C20H25N4O6S ([M + H]+): m/z 449.1495; found: m/z 449.1492.
Glycoconjugate 56: Starting from sugar derivative 22 and 8-HQ derivative 39, the product was obtained
◦
23
1
as a beige solid (77% yield), m.p.: 125–128 C; [
α
]
= 6.2 (c = 1.0, CH3OH); H NMR (400 MHz,
D
DMSO): 3.06 (m, 1H, CHS), 3.19 (m, 1H, CHS), 3.28 (m, 1H, H-3Gal), 3.36–3.46 (m, 2H, 2Gal, 5Gal),
δ

Molecules 2020, 25, 4174
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3.47–3.58 (m, 2H, 6aGal, 6bGal), 3.69 (m, 1H, H-4Gal), 4.30 (d, 1H, J = 9.4 Hz Hz, H-1Gal), 4.45 (d, 1H,
J = 4.5 Hz, OH), 4.58–4.73 (m, 3H, CH2N3, 6-OH), 4.82 (d, 1H, J = 5.4 Hz, OH), 5.06 (d, 1H, J = 5.7 Hz,
OH), 5.35 (s, 2H, CH2O), 7.42 (dd, 1H, J = 4.2 Hz, J = 4.9 Hz, H-7Quin), 7.50–7.57 (m, 3H, H-3Quin
H-5Quin, H-6Quin), 8.32 (dd, 1H, J = 1.8 Hz, J = 8.3 Hz, H-4Quin), 8.34 (s, 1H, H-5Triaz), 8.83 (dd, 1H,
J = 1.8 Hz, J = 4.1 Hz, H-2Quin); 13C NMR (100 MHz, DMSO):
29.66 (CH2S), 50.04 (CH2N3), 60.82,
,
δ
61.91 (C-6Gal, CH2O), 68.57, 69.65, 74.64, 79.36 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 85.80 (C-1Gal), 110.07
(C-7Quin), 120.06 (C-5Quin), 121.92 (C-3Quin), 125.37 (C-5Triaz), 126.83 (C-6Quin), 129.11 (C-4aQuin), 135.89
(C-4Quin), 139.71 (C-8aQuin), 142.31 (C-4Triaz), 149.03 (C-2Quin), 153.88 (C-8Quin); HRMS (ESI-TOF): calcd
for C20H25N4O6S ([M + H]+): m/z 449.1495; found: m/z 449.1494.
Glycoconjugate 57: Starting from sugar derivative 21 and 8-HQ derivative 40, the product was obtained as a
23
brown solid (87% yield), m.p.: 91–95 ◦C; [
α
]
= 5.0 (c = 1.0, CH3OH); 1H NMR (400 MHz, DMSO):
D
δ
2.63 (s, 3H, CH3), 2.98–3.23 (m, 6H, H-2Glu, H-3Glu, H-4Glu H-5Glu, CH2S), 3.44 (m, 1H, 6aGlu), 3.72
(m, 1H, 6bGlu), 4.38 (d, 1H, J = 9.6 Hz, H-1Glu), 4.55–4.73 (m, 3H, CH2N3, 6-OH), 4.95 (d, 1H, J = 5.3 Hz
,
OH), 5.03 (d, 1H, J = 4.8 Hz, OH), 5.20 (d, 1H, J = 5.8 Hz, OH), 5.35 (s, 2H, CH2O), 7.37 (dd, 1H,
J = 1.7 Hz, J = 8.1 Hz, H-7Quin), 7.39–7.44 (m, 2H, H-3Quin, H-6Quin), 7.47 (dd, 1H, J = 1.7 Hz, J = 7.4 Hz,
H-5Quin), 8.19 (d, 1H, J = 8.4 Hz, H-4Quin), 8.33 (s, 1H, H-5Triaz); 13C NMR (100 MHz, DMSO):
δ
24.83
(CH3), 29.70 (CH2S), 49.82 (CH2N), 61.19, 61.68 (C-6Glu, CH2O), 69.96, 72.85, 78.01, 80.91 (C-2Glu, C-3Glu
,
C-4Glu, C-5Glu), 85.30 (C-1Glu), 110.21 (C-7Quin), 119.74 (C-5Quin), 122.36 (C-3Quin), 125.34 (C-5Triaz),
125.57 (C-6Quin), 127.24 (C-4aQuin), 135.88 (C-4Quin), 139.10 (C-8aQuin), 142.21 (C-4Triaz), 153.28 (C-2Quin),
157.16 (C-8Quin); HRMS (ESI-TOF): calcd for C21H27N4O6S ([M + H]+): m/z 463.1651; found: m/z
463.1651.
Glycoconjugate 58: Starting from sugar derivative 22 and 8-HQ derivative 40, the product was obtained as a
23
brown solid (88% yield), m.p.: 83–87 ◦C; [
α
]
D = 10.2 (c = 1.0, CH3OH); 1H NMR (400 MHz, DMSO):
δ
2.63 (s, 3H, CH3), 3.05 (m, 1H, CHS), 3.18 (m, 1H, CHS), 3.28 (m, 1H, H-3Gal), 3.36–3.46 (m, 2H,
H-2Gal, H-5Gal), 3.48–3.58 (m, 2H, 6aGal, 6bGal), 3.68 (m, 1H, H-4Gal), 4.30 (d, 1H, J = 9.5 Hz, H-1Gal),
4.47 (d, 1H, J = 4.6 Hz, OH), 4.57–4.72 (m, 3H, CH2N3, 6-OH), 4.86 (d, 1H, J = 5.6 Hz, OH), 5.09 (d, 1H,
J = 5.8 Hz, OH), 5.34 (s, 2H, CH2O), 7.37 (dd, 1H, J = 1.8 Hz, J = 7.4 Hz, H-7Quin), 7.40–7.46 (m, 2H,
H-3Quin, H-6Quin), 7.47 (dd, 1H, J = 1.8 Hz, J = 8.1 Hz, H-5Quin), 8.19 (d, 1H, J = 8.4 Hz, H-4Quin),
8.33 (s, 1H, H-5Triaz); 13C NMR (100 MHz, DMSO):
δ 24.97 (CH3), 29.67 (CH2S), 50.01 (CH2N3), 60.77,
61.71 (C-6Gal, CH2O), 68.52, 69.60, 74.63, 79.36 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 85.84 (C-1Gal), 110.23
(C-7Quin), 119.85 (C-5Quin), 122.52 (C-3Quin), 125.43, 125.72 (C-5Triaz, C-6Quin), 127.36 (C-4aQuin), 136.03
(C-4Quin), 139.19 (C-8aQuin), 142.34 (C-4Triaz), 153.39 (C-2Quin), 157.32 (C-8Quin); HRMS (ESI-TOF): calcd
for C21H27N4O6S ([M + H]+): m/z 463.1651; found: m/z 463.1654.
Glycoconjugate 59: Starting from sugar derivative 23 and 8-HQ derivative 41, the product was obtained as
23
a brown oil (94% yield), [
α
]
= 36.8 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
δ 1.98, 2.00, 2.02,
D
2.06 (4s, 12H, CH3CO), 2.63 (p, 2H, J = 6.3 Hz, CH2), 3.67 (ddd, 1H, J = 2.2 Hz, J = 4.6 Hz, J = 10.0 Hz,
H-5Glu), 3.85 and 4.05 (qAB, 2H, J = 14.3 Hz, CH2S), 4.07 (dd, 1H, J = 2.2 Hz, J = 12.4 Hz, H-6aGlu),
4.20 (dd, 1H, J = 4.6 Hz, J = 12.4 Hz, H-6bGlu), 4.24 (t, 2H, J = 5.9 Hz, CH2O), 4.60 (d, 1H, J = 10.1 Hz,
H-1Glu), 4.73 (t, 2H, J = 6.7 Hz, CH2N), 5.02 (dd~t, 1H, J = 9.3 Hz, J = 10.1 Hz, H-2Glu), 5.08 (dd~t, 1H,
J = 9.4 Hz, J = 10.0 Hz, H-4Glu), 5.19 (dd~t, 1H, J = 9.3 Hz, J = 9.4 Hz, H-3Glu), 7.05 (dd, 1H, J = 2.1 Hz,
J = 6.7 Hz, H-7Quin), 7.39–7.52 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.67 (s, 1H, H-5Triaz), 8.17 (dd, 1H,
J = 1.6 Hz, J = 8.3 Hz, H-4Quin), 8.96 (dd, 1H, J = 1.6 Hz, J = 4.0 Hz, H-2Quin); 13C NMR (100 MHz,
CDCl3):
δ 20.60, 20.62, 20.66, 20.77 (CH3CO), 24.39 (CH2), 29.61 (CH2S) 47.28 (CH2N), 61.85 (C-6Glu),
65.19 (CH2O), 68.15, 69.89, 73.82, 75.76 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 82.75 (C-1Glu), 109.40 (C-7Quin),
120.34 (C-5Quin), 121.74 (C-3Quin), 123.03 (C-5Triaz), 126.71 (C-6Quin), 129.56 (C-4aQuin), 136.08 (C-4Quin),
140.30 (C-8aQuin), 144.53 (C-4Triaz), 149.41 (C-2Quin), 154.20 (C-8Quin), 169.40, 169.81, 170.14, 170.65
(CH3CO); HRMS (ESI-TOF): calcd for C29H35N4O10S ([M + H]+): m/z 631.2074; found: m/z 631.2070.

Molecules 2020, 25, 4174
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Glycoconjugate 60: Starting from sugar derivative 24 and 8-HQ derivative 41, the product was obtained as a
25
beige solid (79% yield), m.p.: 56–58 ◦C; [
δ
α
]
=
27.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.97, 1.99, 2.02, 2.14 (4s, 12H, CH3CO), 2.63 (p, 2H, J = 6.3 Hz, CH2), 3.80 (m, 1H, H-5Gal), 3.88 and 4.07
(qAB, 2H, J = 14.2 Hz, CH2S), 3.96–4.07 (m, 2H, CH2O), 4.16–4.31 (m, 2H, H-6aGal, H-6bGal), 4.55 (d, 1H,
J = 10.0 Hz, H-1Gal), 4.73 (t, 2H, J = 6.7 Hz, CH2N), 5.00 (dd~t, 1H, J = 3.4 Hz, J = 10.0 Hz, H-3Gal), 5.22
(dd~t, 1H, J = 10.0 Hz, J = 10.0 Hz, H-2Gal), 5.38 (d, 1H, J = 2.9 Hz, H-4Gal), 7.06 (dd, 1H, J = 1.6 Hz
,
J = 7.0 Hz, H-7Quin), 7.40–7.51 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.66 (s, 1H, H-5Triaz), 8.16 (dd, 1H,
J = 1.5 Hz, J = 8.3 Hz, H-4Quin), 8.96 (dd, 1H, J = 1.4 Hz, J = 4.0 Hz, H-2Quin); 13C NMR (100 MHz,
CDCl3):
δ 20.58, 20.67, 20.69, 20.74 (CH3CO), 24.24 (CH2), 29.65 (CH2S) 47.25 (CH2N), 61.42 (C-6Gal),
65.19 (CH2O), 67.30, 71.78, 74.45, 77.23 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 83.04 (C-1Gal), 109.44 (C-7Quin),
120.38 (C-5Quin), 121.77 (C-3Quin), 123.04 (C-5Triaz), 126.74 (C-6Quin), 129.59 (C-4aQuin), 136.06 (C-4Quin),
140.35 (C-8aQuin), 144.38 (C-4Triaz), 149.43 (C-2Quin), 154.26 (C-8Quin), 169.58, 169.97, 170.21, 170.31
(CH3CO); HRMS (ESI-TOF): calcd for C29H35N4O10S ([M + H]+): m/z 631.2074; found: m/z 631.2076.
Glycoconjugate 61: Starting from sugar derivative 23 and 8-HQ derivative 42, the product was obtained as a
23
brown solid (77% yield), m.p.: 48–49 ◦C; [
α
]
= 36.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
D
δ
1.97, 2.00, 2.02, 2.06 (4s, 12H, CH3CO), 2.56 (p, 2H, J = 6.3 Hz, CH2), 2.79 (s, 3H, CH3), 3.65 (ddd, 1H,
J = 2.1 Hz, J = 4.5 Hz, J = 10.0 Hz, H-5Glu), 3.85 and 4.05 (qAB, 2H, J = 14.3 Hz, CH2S), 4.06 (dd, 1H,
J = 2.1 Hz, J = 12.4 Hz, H-6aGlu), 4.19 (dd, 1H, J = 4.5 Hz, J = 12.4 Hz, H-6bGlu), 4.24 (t, 2H, J = 5.4 Hz
CH2O), 4.60 (d, 1H, J = 10.1 Hz, H-1Glu), 4.74 (t, 2H, J = 6.7 Hz, CH2N), 5.01 (dd~t, 1H, J = 9.3 Hz, J =
10.1 Hz, H-2Glu), 5.08 (dd~t, 1H, J = 9.4 Hz, J = 10.0 Hz, H-4Glu), 5.18 (dd~t, 1H, J = 9.3 Hz J = 9.4 Hz
H-3Glu), 7.04 (dd, 1H, J = 2.8 Hz, J = 6.1 Hz, H-7Quin), 7.30–7.43 (m, 3H, H-3Quin, H-5Quin, H-6Quin),
7.74 (s, 1H, H-5Triaz), 8.04 (d, 1H, J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, CDCl3):
20.60, 20.61,
20.65, 20.77 ( H3CO), 24.42 (CH2), 25.75 (CH3), 29.63 (CH2S) 47.20 (CH2N), 61.84 (C-6Glu), 65.34
,
,
,
δ
C
(CH2O), 68.14, 69.91, 73.83, 75.72 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 82.74 (C-1Glu), 110.06 (C-7Quin), 120.32
(C-5Quin), 122.63 (C-3Quin), 123.08 (C-5Triaz), 125.70 (C-6Quin), 127.76 (C-4aQuin), 136.19 (C-4Quin), 139.91
(C-8aQuin), 144.47 (C-4Triaz), 153.64 (C-2Quin), 158.23 (C-8Quin), 169.40, 169.77, 170.14, 170.65 (CH3CO);
HRMS (ESI-TOF): calcd for C30H37N4O10S ([M + H]+): m/z 645.2230; found: m/z 645.2233.
Glycoconjugate 62: Starting from sugar derivative 24 and 8-HQ derivative 42, the product was obtained as a
24
beige solid (80% yield), m.p.: 56–58 ◦C; [
δ
α
]
=
26.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.97, 1.98, 2.02, 2.14 (4s, 12H, CH3CO), 2.60 (p, 2H, J = 6.3 Hz, CH2), 2.79 (s, 3H, CH3), 3.78 (m, 1H,
H-5Gal), 3.88 and 4.07 (qAB, 2H, J = 14.1 Hz, CH2S), 3.96–4.10 (m, 2H, CH2O), 4.15–4.31 (m, 2H, H-6aGal
,
H-6bGal), 4.54 (d, 1H, J = 10.0 Hz, H-1Gal), 4.75 (t, 2H, J = 6.7 Hz, CH2N), 5.00 (dd~t, 1H, J = 3.2 Hz,
J = 10.0 Hz, H-3Gal), 5.22 (dd~t, 1H, J = 10.0 Hz, J = 10.0 Hz, H-2Gal), 5.37 (d, 1H, J = 3.2 Hz, H-4Gal),
7.04 (m, 1H, H-7Quin), 7.33 (d, 1H, J = 8.4 Hz, H-3Quin), 7.36–7.43 (m, 2H, H-5Quin, H-6Quin), 7.72 (s, 1H,
H-5Triaz), 8.04 (d, 1H, J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, CDCl3):
δ
20.58, 20.62, 20.68, 20.74
(CH3CO), 24.26 (CH2), 25.70 (CH3), 29.67 (CH2S) 47.18 (CH2N), 61.41 (C-6Gal), 65.32 (CH2O), 67.30,
67.32, 71.78, 74.42 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 83.02 (C-1Gal), 110.07 (C-7Quin), 120.34 (C-5Quin),
122.65 (C-3Quin), 123.07 (C-5Triaz), 125.73 (C-6Quin), 127.80 (C-4aQuin), 136.20 (C-4Quin), 139.93 (C-8aQuin),
144.31 (C-4Triaz), 153.70 (C-2Quin), 158.25 (C-8Quin), 169.58, 169.97, 170.22, 170.30 (CH3CO); HRMS
(ESI-TOF): calcd for C30H37N4O10S ([M + H]+): m/z 645.2230; found: m/z 645.2233.
Glycoconjugate 63: Starting from sugar derivative 25 and 8-HQ derivative 41, the product was obtained as a
25
beige solid (81% yield), m.p.: 59–62 ◦C; [
δ
α
]
=
51.0 (c = 1.0, CH3OH); 1H NMR (400 MHz, DMSO):
−
D
2.41 (p, 2H, J = 6.5 Hz, CH2), 3.02–3.10 (m, 2H, H-3Glu, H-5Glu), 3.11–3.19 (m, 2H, H-2Glu, H-6aGlu),
3.45 (m, 1H, H-6bGlu), 3.73 (m, 1H, H-4Glu), 3.85 and 3.98 (qAB, 2H, J = 14.2, CH2S), 4.19 (t, 2H,
J = 6.1 Hz CH2O), 4.27 (d, 1H, J = 9.6 Hz, H-1Glu), 4.61 (t, 2H, J = 7.0 Hz, CH2N), 4.75 (t, 1H, J = 5.7
Hz, 6-OH), 4.97 (d, 1H, J = 5.3 Hz, OH), 5.00 (d, 1H, J = 4.9 Hz, OH), 5.17 (d, 1H, J = 5.9 Hz, OH),
7.20 (dd, 1H, J = 1.8 Hz, J = 7.1 Hz, H-7Quin), 7.48–7.54 (m, 2H, H-5Quin, H-6Quin), 7.59 (dd, 1H, J =
4.1 Hz, J = 8.3 Hz, H-3Quin), 8.20 (s, 1H, H-5Triaz), 8.33 (dd, 1H, J = 1.7 Hz, J = 8.3 Hz, H-4Quin), 8.90
(dd, 1H, J = 1.7 Hz, J = 4.1 Hz, H-2Quin); 13C NMR (100 MHz, DMSO):
δ 23.16 (CH2), 29.61 (CH2S),

Molecules 2020, 25, 4174
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46.59 (CH2N), 61.35 (C-6Glu), 65.38 (CH2O), 70.14, 73.08, 78.16, 81.06 (C-2Glu, C-3Glu, C-4Glu, C-5Glu),
84.26 (C-1Glu), 109.80 (C-7Quin), 119.89 (C-5Quin), 121.86 (C-3Quin), 123.52 (C-5Triaz), 126.82 (C-6Quin),
129.05 (C-4aQuin), 135.85 (C-4Quin), 139.72 (C-8aQuin), 144.39 (C-4Triaz), 149.07 (C-2Quin), 154.15 (C-8Quin);
HRMS (ESI-TOF): calcd for C21H27N4O6S ([M + H]+): m/z 463.1651; found: m/z 463.1653.
Glycoconjugate 64: Starting from sugar derivative 26 and 8-HQ derivative 41, the product was obtained as a
26
white solid (89% yield), m.p.: 150–153 ◦C; [
δ
α
]
=
30.0 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO):
−
D
2.42 (p, 2H, J = 6.5 Hz, CH2), 3.25 (m, 1H, H-3Gal), 3.34–3.46 (m, 2H, H-2Gal, H-5Gal), 3.45–3.58 (m,
2H, H-6aGal, H-6bGal), 3.65 (m, 1H, H-4Gal), 3.84 and 3.96 (qAB, 2H, J = 14.1 Hz, CH2S), 4.13–4.24
(m, 2H, CH2O), 4.20 (d, 1H, J = 9.5 Hz, H-1Gal), 4.42 (d, 1H, J = 3.8 Hz, OH), 4.61 (t, 2H, J = 7.0 Hz,
CH2N), 4.72 (bs, 1H, 6-OH), 4.78 (d, 1H, J = 4.4 Hz, OH), 5.00 (d, 1H, J = 5.5 Hz, OH), 7.19 (dd, 1H,
J = 2.2 Hz, J = 6.8 Hz, H-7Quin), 7.47–7.54 (m, 2H, H-5Quin, H-6Quin), 7.59 (dd, 1H, J = 4.2 Hz, J = 8.3 Hz,
H-3Quin), 8.18 (s, 1H, H-5Triaz), 8.32 (dd, 1H, J = 1.7 Hz, J = 8.3 Hz, H-4Quin), 8.90 (dd, 1H, J = 1.7 Hz,
J = 4.2 Hz, H-2Quin); 13C NMR (100 MHz, DMSO):
δ 23.08 (CH2), 29.59 (CH2S), 46.58 (CH2N), 60.86
(C-6Gal), 65.34 (CH2O), 68.60, 69.84, 74.64, 79.38 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 84.70 (C-1Gal), 109.78
(C-7Quin), 119.90 (C-5Quin), 121.87 (C-3Quin), 123.53 (C-5Triaz), 126.82 (C-6Quin), 129.05 (C-4aQuin), 135.85
(C-4Quin), 139.73 (C-8aQuin), 144.41 (C-4Triaz), 149.09 (C-2Quin), 154.16 (C-8Quin); HRMS (ESI-TOF): calcd
for C21H27N4O6S ([M + H]+): m/z 463.1651; found: m/z 463.1651.
Glycoconjugate 65: Starting from sugar derivative 25 and 8-HQ derivative 42, the product was obtained as a
26
brown solid (91% yield), m.p.: 55–58 ◦C; [
δ
α
]
=
40.0 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO):
−
D
2.40 (p, 2H, J = 6.6 Hz, CH2), 2.67 (s, 3H, CH3), 2.99–3.19 (m, 4H, H-2Glu, H-3Glu, H-4Glu, H-5Glu),
3.43 (m, 1H, H-6aGlu), 3.71 (m, 1H, H-6bGlu), 3.84 and 3.98 (qAB, 2H, J = 14.1 Hz, CH2S), 4.19 (t, 2H,
J = 6.2 Hz, CH2O), 4.26 (d, 1H, J = 9.6 Hz, H-1Glu), 4.61 (t, 2H, J = 7.0 Hz, CH2N), 4.69 (t, 1H, J = 5.8 Hz,
6-OH), 4.94 (d, 1H, J = 5.1 Hz, OH), 5.01 (d, 1H, J = 4.7 Hz, OH), 5.15 (d, 1H, J = 5.8 Hz, OH), 7.15 (dd, 1H,
J = 1.3 Hz, J = 7.5 Hz, H-7Quin), 7.38–7.49 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 8.17 (s, 1H, H-5Triaz), 8.19
(d, 1H, J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, DMSO):
δ 23.10 (CH2), 25.07 (CH3), 29.54 (CH2S),
46.58 (CH2N), 61.31 (C-6Glu), 65.54 (CH2O), 70.13, 73.09, 78.16, 81.06 (C-2Glu, C-3Glu, C-4Glu, C-5Glu),
84.21 (C-1Glu), 110.33 (C-7Quin), 119.83 (C-5Quin), 122.47 (C-3Quin), 123.45 (C-5Triaz), 125.75 (C-6Quin),
127.33 (C-4aQuin), 136.00 (C-4Quin), 139.31 (C-8aQuin), 144.36 (C-4Triaz), 153.67 (C-2Quin), 157.32 (C-8Quin);
HRMS (ESI-TOF): calcd for C22H29N4O6S ([M + H]+): m/z 477.1808; found: m/z 477.1806.
Glycoconjugate 66: Starting from sugar derivative 26 and 8-HQ derivative 42, the product was obtained as
23
a beige solid (90% yield), m.p.: 83–87 ◦C; [
α
]
= 10.2 (c = 1.0, DMSO); 1H NMR (400 MHz, DMSO):
D
δ
2.41 (p, 2H, J = 6.3 Hz, CH2), 3.43 (s, 3H, CH3), 3.23 (m, 1H, H-3Gal), 3.34–3.42 (m, 2H, H-2Gal, H-5Gal),
3.43–3.58 (m, 2H, H-6aGal, H-6bGal), 3.65 (m, 1H, H-4Gal), 3.83 and 3.96 (qAB, 2H, J = 14.1 Hz, CH2S),
4.12–4.24 (m, 2H, CH2O), 4.19 (d, 1H, J = 9.4 Hz, H-1Gal), 4.41 (d, 1H, J = 4.0 Hz, OH), 4.61 (t, 2H,
J = 7.0 Hz, CH2N), 4.68 (bs, 1H, 6-OH), 4.77 (d, 1H, J = 4.7 Hz, OH), 4.99 (d, 1H, J = 5.6 Hz, OH), 7.16
(dd, 1H, J = 1.3 Hz, J = 7.6 Hz, H-7Quin), 7.38–7.45 (m, 2H, H-3Quin, H-6Quin), 7.47 (dd, 1H, J = 1.4 Hz,
J = 8.2 Hz, H-5Quin), 8.16 (s, 1H, H-5Triaz), 8.19 (d, 1H, J = 8.4 Hz, H-4Quin); 13C NMR (100 MHz, DMSO):
δ
23.02 (CH2), 25.08 (CH3), 29.52 (CH2S), 46.56 (CH2N), 60.81 (C-6Gal), 65.50 (CH2O), 68.57, 69.83, 74.64,
79.36 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 84.65 (C-1Gal), 110.31 (C-7Quin), 119.83 (C-5Quin), 122.47 (C-3Quin),
123.43 (C-5Triaz), 125.73 (C-6Quin), 127.33 (C-4aQuin), 135.99 (C-4Quin), 139.31 (C-8aQuin), 144.37 (C-4Triaz),
153.67 (C-2Quin), 157.33 (C-8Quin); HRMS (ESI-TOF): calcd for C22H29N4O6S ([M + H]+): m/z 477.1808;
found: m/z 477.1807.
Glycoconjugate 67: Starting from sugar derivative 33 and 8-HQ derivative 39, the product was obtained as a
20
brown solid (84% yield), m.p.: 114–115 ◦C; [
δ
α
]
=
2.2 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
−
D
1.97, 2.00, 2.00, 2.02 (4s, 12H, CH3CO), 3.80 (ddd, 1H, J = 2.2 Hz, J = 4.4 Hz, J = 10.1 Hz, H-5Glu), 4.07
(dd, 1H, J = 2.2 Hz, J = 12.4 Hz, H-6aGlu), 4.22 (dd, 1H, J = 4.4 Hz, J = 12.4 Hz, H-6bGlu), 5.05–5.20
(m, 4H, H-2Glu, H-4Glu, CH2O), 5.29 (dd~t, 1H, J = 9.3 Hz, J = 9.3 Hz, H-3Glu), 5.40 (s, 2H, CH2O), 5.59
(d, 1H, J = 10.4 Hz, H-1Glu), 6.89 (d, 1H, J = 8.4 Hz, H-3Pyr), 7.34 (d, 1H, J = 6.6 Hz, H-7Quin), 7.44–7.59

Molecules 2020, 25, 4174
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(m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.76 (d, 1H, J = 8.4 Hz, H-4Pyr), 7.97 (s, 1H, H-5Triaz), 8.25 (d, 1H,
J = 7.7 Hz, H-4Quin), 8.41 (s, 1H, H-6Pyr), 8.84 (bs, 1H, H-2Quin), 10.84 (bs, 1H, NH); 13C NMR (100 MHz,
CDCl3):
δ 20.60, 20.62, 20.71, 20.71 (CH3CO), 52.80 (CH2N), 61. 87, 62.04 (CH2O, C-6Glu), 68.20,
69.47, 74.11, 75.83 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 82.22 (C-1Glu), 109.56 (C-7Quin), 120.45 (C-5Quin),
122.10 (C-3Quin), 123.27 (C-5Triaz), 125.92 (CPyr), 127.29 (CPyr), 128.09 (C-6Quin), 129.73 (C-4aQuin), 132.80
(C-7Quin), 136.99 (C-4Quin), 139.54 (C-7Quin), 141.30 (C-8aQuin), 142.92 (C-4Triaz), 148.63 (C-7Quin), 149.61
(C-2Quin), 153.69 (C-8Quin), 163.96 (C=O), 169.44, 169.53, 170.11, 170.65 (CH3
calcd for C33H35N6O11S ([M + H]+): m/z 732.2085; found: m/z 732.2084.
CO); HRMS (ESI-TOF):
Glycoconjugate 68: Starting from sugar derivative 34 and 8-HQ derivative 39, the product was obtained as a
21
beige solid (83% yield), m.p.: 128–129 ◦C; [
α
]
= 9.6 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3):
D
δ
1.94, 1.98, 2,01, 2.15 (4s, 12H, CH3CO), 4.01 (m, 1H, H-5Gal), 4.05–4.10 (m, 2H, H-6aGal, H-6bGal), 5.11
(s, 2H, CH2O), 5.14 (dd, 1H, J = 3.4 Hz, J = 10.1 Hz, H-3Gal), 5.36 (dd~t, 1H, J = 10.1 Hz, J = 10.2 Hz,
H-2Gal), 5.40 (s, 2H, CH2N), 5.45 (dd, 1H, J = 0.7 Hz, J = 3.4 Hz, H-4Gal), 5.58 (d, 1H, J = 10.2 Hz, H-1Gal),
7.00 (d, 1H, J = 8.7 Hz, H-3Pyr), 7.33 (d, 1H, J = 7.4 Hz, H-7Quin), 7.44–7.58 (m, 3H, H-3Quin, H-5Quin
,
H-6Quin), 7.81 (dd, 1H, J = 2.3 Hz, J = 8.7 Hz, H-4Pyr), 7.97 (s, 1H, H-5Triaz), 8.24 (d, 1H, J = 8.3 Hz,
H-4Quin), 8.43 (d, 1H, J = 1.9 Hz, H-6Pyr), 8.32 (d, 1H, J = 3.0 Hz, H-2Quin), 10.77 (bs, 1H, NH); 13C NMR
(100 MHz, CDCl3):
δ 20.60, 20.64, 20.68, 20.80 (CH3CO), 52.83 (CH2N), 61. 23, 62.07 (CH2O, C-6Gal),
66.84, 67.29, 72.04, 74.46 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 82.75 (C-1Gal), 109.57 (C-7Quin), 120.49 (C-5Quin),
122.08 (C-3Quin), 123.29 (C-5Triaz), 125.85 (CPyr), 127.24 (CPyr), 128.21 (C-6Quin), 129.73 (C-4aQuin), 132.76
(C-7Quin), 136.96 (C-4Quin), 139.55 (C-7Quin), 141.36 (C-8aQuin), 143.02 (C-4Triaz), 148.65 (C-7Quin), 149.93
(C-2Quin), 153.67 (C-8Quin), 163.91 (C=O), 169.72, 169.98, 170.24, 170.33 (CH3CO); HRMS (ESI-TOF):
calcd for C33H35N6O11S ([M + H]+): m/z 723.2085; found: m/z 723.2086.
Glycoconjugate 69: Starting from sugar derivative 35 and 8-HQ derivative 39, the product was obtained as a
22
brown solid (77% yield), m.p.: 143–144 ◦C; [
δ
α
]
=
24.0 (c = 0.5, H2O); 1H NMR (400 MHz, DMSO):
−
D
3.13 (m, 1H, H-2Glu), 3.21–3.29 (m, 2H, H-3Glu, H-4Glu), 3.37–3.50 (m, 2H, H-6aGlu, H-6bGlu), 3.66
(m, 1H, H-5Glu), 4.51 (t, 1H, J = 5.7 Hz, 6-OH), 5.00 (d, 1H, J = 5.3 Hz, OH), 5.07 (d, 1H, J = 9.9 Hz,
H-1Glu),5.13 (d, 1H, J = 4.9 Hz, OH), 5.36 (d, 1H, J = 6.1 Hz, OH), 5.39 (t, 2H, J = 6.0 Hz, CH2O),
5.42 (t, 2H, J = 6.9 Hz, CH2N), 7.40 (d, 1H, J = 8.6 Hz, H-3Pyr), 7.44 (dd, 1H, J = 4.4 Hz, J = 8.9 Hz,
H-7Quin), 7.50–7.58 (m, 3H, H-3Quin, H-5Quin, H-6Quin), 7.92 (dd, 1H, J = 2.6 Hz, J = 8.6 Hz, H-4Pyr),
8.32 (dd, 1H, J = 1.8 Hz, J = 8.3 Hz, H-4Quin), 8.35 (s, 1H, H-5Triaz), 8.63 (d, 1H, J = 2.6 Hz, H-6Pyr),
8.84 (dd, 1H, J = 1.7 Hz, J = 4.1 Hz, H-2Quin), 10.73 (s, 1H, NH); 13C NMR (100 MHz, DMSO):
δ 52.10
(CH2N), 60. 83, 61.77 (CH2O, C-6Glu), 69.71, 72.39, 78.28, 81.05 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 84.60
(C-1Glu), 109.94 (C-7Quin), 119.98 (C-5Quin), 121.85 (C-3Quin), 122.17 (C-5Triaz), 126.51 (CPyr), 126.74
(CPyr), 127.76 (C-6Quin), 129.06 (C-4aQuin), 132.35 (C-7Quin), 135.77 (C-4Quin), 139.73 (C-7Quin), 140.29
(C-8aQuin), 142.49 (C-4Triaz), 148.98 (C-7Quin), 152.52 (C-2Quin), 153.87 (C-8Quin), 164.69 (C=O); HRMS
(ESI-TOF): calcd for C25H27N6O7S ([M + H]+): m/z 555.1662; found: m/z 555.1667.
Glycoconjugate 70: Starting from sugar derivative 36 and 8-HQ derivative 41, the product was obtained as a
21
white solid (72% yield), m.p.: 93–95 ◦C; [
α
]
= δ 1.96,
3.0 (c = 1, CHCl3); 1H NMR (400 MHz, DMSO):
−
D
1.97, 1.98, 2.00 (4s, 12H, CH3CO), 2.43 (bs, 2H, CH2), 4.02 (m, 1H, H-5Glu), 4.10–4.17 (m, 2H, H-6aGlu
,
H-6bGlu), 4.21 (bs, 2H, CH2O), 4.67 (t, 2H, J = 6.0 Hz, CH2N), 4.93–5.02 (m, 2H, H-2Glu, H-4Glu), 5.22
(s, 2H, CH2OCO), 5.41 (dd~t, 1H, J = 9.4 Hz, J = 9.4 Hz, H-3Glu), 5.69 (d, 1H, J = 10.3 Hz, H-1Glu), 7.21
(d, 1H, J = 8.0 Hz, H-7Quin), 7.37 (d, 1H, J = 8.7 Hz, H-3Pyr), 7.50 (dd~t, 1H, J = 7.5 Hz, J = 8.0 Hz,
H-3Quin), 7.47–7.64 (m, 2H, H-5Quin, H-6Quin), 7.83 (dd, 1H, J = 2.4 Hz, J = 8.7 Hz, H-4Pyr), 8.32–8.40
(m, 2H, H-4Quin, H-6Pyr), 8.54 (s, 1H, H-5Triaz), 8.91 (bs, 1H, H-2Quin), 10.00 (s, 1H, NH); 13C NMR
(100 MHz, DMSO):
δ 20.19, 20.24, 20.28, 20.24 (CH3CO), 29.51 (CH2), 46.63 (CH2N), 57.63 (CH2OCO),
61.73 (C-6Glu), 65.36 (CH2O), 67.94, 69.33, 72.91, 74.41 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 81.26 (C-1Glu),
109.82 (C-7Quin), 119.92 (C-5Quin), 121.81 (C-3Quin), 123.05 (C-5Triaz), 125.00 (CPyr), 126.60 (CPyr), 126.78
(C-6Quin), 128.98 (C-4aQuin), 133.84 (CPyr), 135.92 (C-4Quin), 139.43 (CPyr), 139.68 (C-8aQuin), 141.91

Molecules 2020, 25, 4174
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(C-4Triaz), 148.01 (CPyr), 148.84 (C-2Quin), 153.10 (C-8Quin), 153.99 (C=O), 168.99, 169.18, 169.38, 169.80
(CH3CO); HRMS (ESI-TOF): calcd for C35H39N6O12S ([M + H]+): m/z 767.2347; found: m/z 767.2344.
Glycoconjugate 71: Starting from sugar derivative 37 and 8-HQ derivative 41, the product was obtained as a
21
white solid (73% yield), m.p.: 97–99 ◦C; [
α
]
D = 9.8 (c = 1.0, CHCl3); 1H NMR (400 MHz, DMSO):
δ
1.94,
1.95, 2.00, 2.14 (4s, 12H, CH3CO), 2.43 (bs, 2H, CH2), 3.97–4.07 (m, 2H, H-6aGal, H-6bGal), 4.21 (bs, 2H,
CH2O), 4.35 (m, 1H, H-5Gal), 4.67 (t, 2H, J = 6.5 Hz, CH2N), 5.13 (dd~t, 1H, J = 9.9 Hz, J = 10.2 Hz
,
H-2Gal), 5.22 (s, 2H, CH2OCO), 5.30–5.38 (m, 2H, H-3Gal, H-4Gal), 5.65 (d, 1H, J = 10.2 Hz, H-1Gal), 7.21
(d, 1H, J = 8.4 Hz, H-7Quin), 7.38 (d, 1H, J = 8.7 Hz, H-3Pyr), 7.50 (dd~t, 1H, J = 7.5 Hz, J = 8.1 Hz,
H-3Quin), 7.50–7.65 (m, 2H, H-5Quin, H-6Quin), 7.84 (dd, 1H, J = 2.4 Hz, J = 8.7 Hz, H-4Pyr), 8.32–8.40
(m, 2H, H-4Quin, H-6Pyr), 8.54 (s, 1H, H-5Triaz), 8.90 (bs, 1H, H-2Quin), 10.00 (s, 1H, NH); 13C NMR
(100 MHz, DMSO):
δ 20.33, 20.39, 20.41, 20.43 (CH3CO), 29.60 (CH2), 46.72 (CH2N), 57.72 (CH2OCO),
61.51 (C-6Gal), 65.44 (CH2O), 66.80, 67.55, 71.01, 73.66 (C-2Gal, C-3Gal, C-4Gal, C-5Gal), 81.77 (C-1Gal),
109.89 (C-7Quin), 120.00 (C-5Quin), 121.91 (C-3Quin), 123.04 (C-5Triaz), 125.08 (CPyr), 126.67 (CPyr), 126.85
(C-6Quin), 129.08 (C-4aQuin), 133.87 (CPyr), 135.95 (C-4Quin), 139.36 (CPyr), 139.78 (C-8aQuin), 142.00
(C-4Triaz), 148.29 (CPyr), 148.90 (C-2Quin), 153.19 (C-8Quin), 154.18 (C=O), 169.31, 169.39, 169.75, 169.94
(CH3CO); HRMS (ESI-TOF): calcd for C35H39N6O12S ([M + H]+): m/z 767.2347; found: m/z 767.2345.
Glycoconjugate 72: Starting from sugar derivative 38 and 8-HQ derivative 41, the product was obtained as a
22
white solid (78% yield), m.p.: 152–154 ◦C; [
δ
α
]
=
36.0 (c = 1.0, CH3OH); 1H NMR (400 MHz, DMSO):
−
D
2.43 (p, 2H, J = 6.3 Hz, CH2),3.09–3.19 (m, 2H, H-2Glu, H-4Glu), 3.21–3.29 (m, 2H, H-3Glu, H-5Glu), 3.44
(m, 1H, H-6aGlu), 3.66 (m, 1H, H-6bGlu), 4.19 (t, 2H, J = 6.0 Hz, CH2O), 4.51 (t, 1H, J = 5.7 Hz, 6-OH),
4.67 (t, 2H, J = 6.9 Hz, CH2N), 4.99 (d, 1H, J = 5.3 Hz, OH), 5.03 (d, 1H, J = 9.9 Hz, H-1Glu), 5.11 (d, 1H,
J = 4.8 Hz, OH), 5.21 (s, 2H, CH2OCO), 5.34 (d, 1H, J = 6.1 Hz, OH), 7.19 (dd, 1H, J = 1.7 Hz
H-7Quin), 7.36 (d, 1H, J = 8.7 Hz, H-3Pyr), 7.45–7.60 (m, 2H, H-5Quin, H-6Quin), 7.56 (dd, 1H, J = 4.0 Hz
J = 8.3 Hz, H-3Quin), 7.76 (dd, 1H, J = 2.1 Hz, J = 8.7 Hz, H-4Pyr), 8.32 (dd, 1H, J = 1.3 Hz, J = 7.0 Hz
,
J = 7.1 Hz
,
,
,
H-4Quin), 8.37 (s, 1H, H-5Triaz), 8.49 (d, 1H, J = 2.1 Hz, H-6Pyr), 8.89 (bs, 1H, H-2Quin), 9.92 (bs, 1H,
NH); 13C NMR (100 MHz, DMSO):
δ
29.51 (CH2), 46.62 (CH2N), 57.56 ( H2OCO), 60.76 (C-6Glu),
C
65.32 (CH2O), 69.65, 72.30, 78.20, 80.97 (C-2Glu, C-3Glu, C-4Glu, C-5Glu), 84.71 (C-1Glu), 109.75 (C-7Quin),
119.86 (C-5Quin), 121.77 (C-3Quin), 122.19 (C-5Triaz), 124.99 (CPyr), 126.58 (CPyr), 126.68 (C-6Quin), 128.96
(C-4aQuin), 132.91 (C-7Quin), 135.71 (C-4Quin), 139.38 (C-7Quin), 139.68 (C-8aQuin), 141.96 (C-4Triaz), 148.95
(C-7Quin), 150.91 (C-2Quin), 153.12 (C-8Quin), 154.09 (C=O); HRMS (ESI-TOF): calcd for C27H31N6O8S
([M + H]+): m/z 599.1924; found: m/z 599.1919.
3.3. Biological Assays
3.3.1. Cell Lines
The human colon adenocarcinoma cell line HCT-116 was obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA). The human cell line MCF-7 was obtained from collections at
the Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, branch in Gliwice,
Poland. The Normal Human Dermal Fibroblasts-Neonatal (NHDF-Neo) was purchased from LONZA
(Cat. No. CC-2509, NHDF-Neo, Dermal Fibroblasts, Neonatal, Lonza, Poland). The culture media
consisted of RPMI 1640 or DMEM+F12 medium, supplemented with 10% fetal bovine serum and 1% of
standard antibiotics (penicillin and streptomycin). The culture media were purchased from EuroClone
and HyClone. Fetal bovine serum (FBS) was delivered by EURx, Poland, and Antibiotic Antimycotic
Solution (100 ) by Sigma-Aldrich, Germany. The cells were cultured under standard conditions at
×
37 ◦C in a humidified atmosphere at 5% CO2.
3.3.2. MTT Assay
The viability of the cells was determined using an MTT (3-[4,5–dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide) test (Sigma-Aldrich). Stock solutions of tested compounds were prepared

Molecules 2020, 25, 4174
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in DMSO and diluted with the appropriate volumes of the growth medium directly before the experiment.
The cells were seeded into 96-well plates at concentration 1
×
104 (HCT 116, NHDF-Neo) or 5
×
103
(MCF-7) per well. The cell cultures were incubated for 24 h at 37 ◦C in a humidified atmosphere of 5%
CO2. Then, the culture medium was removed, replaced with the solution of the tested compounds in
medium with varying concentrations, and incubated for further 24 h or 72 h. After that, the medium
was removed, and the MTT solution (50 µL, 0.5 mg/mL in PBS) was added. After 3 h of incubation,
the MTT solution was removed, and the precipitated formazan was dissolved in DMSO. Finally, the
absorbance at the 570 nm wavelength was measured spectrophotometrically with the plate reader.
The experiment was conducted in at least three independent iterations with four technical repetitions.
The IC50 values were calculated using CalcuSyn software (version 2.0, Biosoft, Cambridge, UK).
3.3.3. Influence of Metal Ions on Cellular Proliferation
The cells were seeded into 96-well plates at concentration 1
×
104 (HCT 116, NHDF-Neo) or
5 × 103 (MCF-7) per well. The cell cultures were incubated for 24 h at 37 ◦C in a humidified atmosphere
of 5% CO2. Then, the culture medium was removed, replaced with the solution of the tested compounds
in medium with varying concentrations. Additionally, 100 µM solution of Cu(CH3COO)2 was added
into wells with tested compounds. After 24 h or 72 h incubation, the MTT assay was performed.
4. Conclusions
Glycoconjugation of quinoline derivatives with appropriately functionalized 1-sugar derivatives
was supposed to increase the selectivity of such obtained prodrugs in relation to the cancer cells,
thanks to the facilitation of its transport through GLUT transporters, whose overexpression is seen in
some types of cancer. The cytotoxic activity in vitro of the new quinoline glycoconjugates was tested
against the MCF-7, HCT-116, and NHDF-Neo cell lines. In order to approximate the mechanism of
glycoconjugates transport into the cell, both types of glycoconjugates: Protected in the sugar part and
their unprotected counterparts were investigated. Based on the obtained results, it can be stated that
only compounds with acetyl protection of hydroxyl groups in the sugar part have the ability to inhibit
the proliferation of tumor cells. Thus, lipophilicity has a significant influence on the biological activity
of the tested compounds, which can be explained by the facilitation of passive transport through
biological membranes into the cells. Derivatives with an unprotected sugar fragment showed low
activity, probably due to their high hydrophilicity and low affinity for the cell membrane, as well as the
inability to bind to GLUT transporters. It is likely that the functionalization of the anomeric position
in sugar and the use of the compounds thus obtained to binding with 8-HQ derivatives reduces the
affinity of the sugar residue for GLUT transporters. In this situation, the improvement of the hydrolytic
stability of the obtained glycoconjugates by introducing a sulfur atom into the anomeric position
of the sugar did not affect their selectivity, but only in some cases (compounds 51–54), increased
cytotoxicity compared to their oxygen counterparts. This can be explained by the fact that they were not
prematurely degraded before entering the cell, and, as a consequence of facilitated passive transport,
their concentration in the cells was higher.
Among the tested compounds, the glycoconjugates 67–71 containing an additional heteroaromatic
(5-amine-2-pyridyl) moiety in the linker structure turned out to be the most active. For these compounds,
the additional experiments of antiproliferative activity in the presence of Cu2+ ions were carried out. It was
observed that the activity of glycoconjugates increased significantly in the presence of copper compared to
cells treated with alone glycoconjugates in the absence of Cu2+. The highest cytotoxicity of the compounds
was observed against the MCF-7 cell line. This confirmed the strong sensitivity of breast cancer cells to the
presence of copper ions as well as their sensitivity to compounds capable of complexing these ions, such
as 8-HQ or sugar derivatives containing 2-thio-5-amino-pyridine moiety. Unfortunately, in the case of the
latter, cytotoxicity to non-cancer cells was also observed. In such a case, the research on the dependence of
glycoconjugates’ cytotoxic activity on their structure should be extended in the direction that allows better
matching of glycoconjugates to GLUT transporters, which should improve their selectivity. According to

Molecules 2020, 25, 4174
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some literature reports, the 6-OH group is the position in the sugar that is least involved in binding to
the GLUT transporter. Perhaps the use of this position for binding with quinoline derivatives will prove
effective and allow to increase the selectivity of the obtained glycoconjugates.
Supplementary Materials: Determination of glycoconjugates stability under the action of
Aspergillus oryzae, and the 1H NMR and 13C NMR spectra of all obtained compounds.
β-galactosidase from
Author Contributions: Conceptualization and methodology, M.K. and G.P.-G.; synthesis and characterization of
chemical compounds, M.K. and A.H.; cytotoxicity tests, M.K.; mass spectra, K.E.; supervision, G.P.-G.; analysis
and interpretation of the results, M.K. and G.P.-G.; writing—original draft preparation, M.K.; writing—review and
editing, G.P.-G. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Grants BKM No. 04/020/BKM19/0095 (BKM-526/RCH2/2019) and
04/020/BKM20/0138 (BKM-611/RCH2/2020) as part of a targeted subsidy for conducting scientific research or
development works and related tasks for the development of young scientists and participants of doctoral studies
granted by the Ministry of Science and Higher Education, Poland.
Conflicts of Interest: The authors declare no conflict of interest.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).