Synthesis and Evaluation of the Biological Profile of Novel Analogues of Nucleosides and of Potential Mimetics of Sugar Phosphates and Nucleotides

Article abstract of DOI:10.1055/s-0035-1560591

The synthesis of purine/triazole 6'-isonucleosides and of glucuronic acid/glucuronamide-derived N-glycosylsulfonohydrazides through efficient and stereo- or regioselective methodologies is described. Their structures were envisaged to mimic nucleosides, s

Full text of DOI:10.1055/s-0035-1560591

SYNLETT
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
© Georg Thieme Verlag Stuttgart · New York
2015, 26, A–J
letter
A
N. M. Xavier et al.
Letter
Synlett
Synthesis and Evaluation of the Biological Profile of Novel
Analogues of Nucleosides and of Potential Mimetics of Sugar
Phosphates and Nucleotides
Nuno M. Xavier*a
Susana D. Lucasb
Radek Jordac
Stefan Schwarzd
Anne Loesched
René Csukd
M. Conceição Oliveirae
a Centro de Química e Bioquímica, Faculdade de Ciências,
Universidade de Lisboa, Ed. C8, 2º Piso, Campo Grande,
1749-016 Lisboa, Portugal
nmxavier@fc.ul.pt
b Instituto de Investigação do Medicamento, Faculdade de
Farmácia, Universidade de Lisboa, Av. Prof. Gama Pinto,
1649-003 Lisboa, Portugal
c Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Palacký University &
Institute of Experimental Botany AS CR, Šlechtitelů 27, 78371 Olomouc, Czech Republic
d Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, 06120 Halle (Saale), Germany
e Centro de Química Estrutural (CQE), Instituto Superior
Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisboa, Portugal
Received: 20.10.2015
Accepted after revision: 22.10.2015
Published online: 09.11.2015
DOI: 10.1055/s-0035-1560591; Art ID: st-2015-d0828-l
Abstract The synthesis of purine/triazole 6'-isonucleosides and of glu-
curonic
acid/glucuronamide-derived
N-glycosylsulfonohydrazides
through efficient and stereo- or regioselective methodologies is de-
scribed. Their structures were envisaged to mimic nucleosides, sugar
phosphates, or nucleotides, and were expected to provide potential in-
hibitors of therapeutically relevant enzymes, the active sites of which
could potentially bind their structural fragments or functional groups.
Such enzymes include cholinesterases, carbonic anhydrase II (CA-II) and
cyclin-dependent kinase 2 (CDK-2). A (triazolyl)methyl amide-linked di-
saccharide nucleoside, based on a new prospective structural frame-
work for analogues of nucleoside diphosphate sugars, was synthesized.
The synthetic strategies employed unprotected or partially protected
carbohydrate derivatives as precursors, including ribose, glucuronic ac-
id, glucuronolactone, and glycopyranosides and relied on stereoselec-
tive N-glycosylation, regioselective Mitsunobu coupling and ‘click
chemistry’ approaches. Some 6'-isonucleosides and triazole-containing
glycoderivatives displayed moderate selective acetylcholinesterase in-
hibitory activities. The best inhibitor was an aminomethyltriazole 6'-iso-
nucleoside with a Ki value of 11.9 μM. N-Glucuronylsulfonohydrazide
showed good inhibition of CA-II (Ki = 9.5 μM). Molecular docking of the
most active compounds into the effected enzymes showed interactions
with key amino acid residues for substrate recognition. In addition, the
tested compounds did not show toxicity to normal cells.
Nuno M. Xavier (born November 1982 in Vila Real, Portugal) graduat-
ed in chemistry from the University of Lisbon in 2005. He received a
dual Ph.D. degree in organic chemistry from the University of Lisbon
and from the National Institute of Applied Sciences of Lyon in 2011,
where he worked in the field of glycochemistry under the supervision of
Professor A. P. Rauter and Dr. Y. Queneau, respectively. During his
Ph.D. studies, he was also a visiting research student in the group of
Professor J. Thiem at the University of Hamburg for four months. He
joined Professor P. Kosma’s glycochemistry group at the University of
Natural Resources and Life Sciences of Vienna for one year as a postdoc-
toral member and worked on the synthesis of new heptose and sugar
phosphate derivatives toward novel antibacterial drug candidates. He
then returned to the University of Lisbon as postdoctoral fellow until
the end of 2013, whereupon he was awarded an Investigator Starting
Grant from the Portuguese Foundation for Science and Technology
(FCT). Since then he has been a researcher (FCT Investigator) at the Fac-
ulty of Sciences, University of Lisbon. His research interests and activi-
ties, reported in 23 publications and discussed in various
communications in several international scientific meetings and sym-
posia, are centred on the development of synthetic strategies for carbo-
hydrate-containing molecules of potential therapeutic interest. He was
awarded with the Scientific Award of the Groupe Lyonnais des Glycosci-
ences (France), a Young Scientist Award from the IUPAC/43rd IUPAC
World Chemistry Congress and the Young Investigator Award in Glyco-
sciences from the Alberta Ingenuity Centre for Carbohydrate Science
(Canada). He is a member of IUPAC.
Key words nucleosides, nucleotides, carbohydrates, mimetics, cholin-
esterases
Nucleotides and nucleosides are essential components
of cells and they play key roles in several fundamental bio-
logical processes including DNA and RNA synthesis, cell di-
vision, and metabolism.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

B
N. M. Xavier et al.
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The development of modified analogues as well as mi-
metics of nucleotides/nucleosides is an attractive approach
in drug discovery of compounds that aim to interfere with
such physiological pathways, which are uncontrolled in dis-
eases such as cancer or viral infections.1 These molecules
may act as antimetabolites, through incorporation into DNA
or RNA or by inhibition of enzymes involved in nucleotide
metabolism and nucleic acid synthesis such as DNA poly-
merase or ribonucleotide reductase. Antimetabolites con-
stitute a class of anticancer and antiviral agents1,2 that in-
clude structures such as pyrimidine3 and purine nucleo-
sides.4 Nucleobase analogues are also suggested to behave
as antimetabolites through diffusion into cells and subse-
quent conversion into analogues of nucleotides by enzymes
that are involved in the metabolism of natural purines and
pyrimidines.3c In addition to their potential to interfere in
nucleic acid synthesis, nucleoside and nucleotide analogues
can be exploited for targeting other nucleoside/nucleotide-
dependent enzymes such as glycosyltransferases, ATPases,
GTPases, phosphodiesterases or methyltransferases, which
are valuable therapeutic targets for a number of diseases.5
One of the most important targeted classes of nucleotide-
dependent enzymes, particularly in cancer therapy, are ki-
nases, which are ATP-dependent and responsible for pro-
tein phosphorylation.6 Among them, cyclin-dependent ki-
nases (CDKs) are directly involved in the regulation and in
driving the cell cycle, and due to their frequent overactiva-
tion or overexpression in tumor cells,7 CDK inhibition has
emerged as a potential anticancer therapeutic strategy.8
Some CDK inhibitors have demonstrated potent antitumor
efficacy8b,9 and mostly encompass small molecules that are
structurally based on aromatic or heteroaromatic motifs.9,10
Nucleobase analogues, including purine and pyrimidine de-
rivatives, such as (R)-roscovitine11 and the pyrazolopirymi-
dine dinaciclib,11,12 have attracted by far the highest atten-
tion as CDK inhibitors, among other heterocyclic derivatives
such as thiazoles, pyrazoles, indoles or flavones.
cules comprised of an N-heteroaromatic unit and a phos-
phate/diphosphate isostere as potential nucleotide mimet-
ics.
Biological evaluation of such compounds has focused on
enzymes of therapeutic relevance such as ChEs and carbon-
ic anhydrase II (CA-II). The latter is one of the CA isoforms
that catalyzes the reversible hydration of carbon dioxide
and it plays a crucial role in the regulation of the intraocu-
lar pressure. Its inhibition thus constitutes a therapeutic
approach towards glaucoma.16 Sulfonamide derivatives are
among established inhibitors such as the clinically used ac-
etazolamide.17 Glycosylsulfonamides were also reported to
inhibit various isoforms of CAs,18 which prompted us to test
the activity of sulfonohydrazide derivatives on CA-II.
As a nucleotide-dependent enzyme, CDK-2 was selected
for inclusion in the biological assays, because it has become
a privileged target for antitumor therapy among CDKs.19 Its
inhibition is suggested to induce selective cytotoxic effects
on tumor cells through an associated persistence of a tran-
scription factor (E2F) activity, the level of which is intolera-
ble in transformed cells but still acceptable in normal
cells.8a
Results on the bioactivity assessment of the new com-
pounds, i.e., their enzyme inhibitory effects as well as their
cytotoxicity on a panel of tumor and normal cells, are pre-
sented herein. For the active molecules, their possible in-
teractions with the enzymes were inspected by docking
simulations.
The general framework of one type of structure envis-
aged comprises a sugar moiety, a functional mimic of the
phosphate group, and an NH-containing moiety such as an
amide or an N-heteroaromatic unit (Figure 1). Molecules
combining two structural elements of the skeleton, having
a sugar unit and a phosphate isostere or possessing a sugar
moiety and an N-heteroaromatic system, were also planned
as mimetics of sugar phosphates and as nucleoside ana-
logues, respectively.
In addition to antitumor and antiviral properties, the
reported bioactivities exhibited by nucleoside and nucleo-
tide analogues also include antimicrobial13 and cholinester-
ase (ChE) inhibitory effects.14 ChEs hydrolyse acetylcholine
and are major drug targets for Alzheimer’s disease.15 Purine
nucleosides of D-glucuronic acid derivatives have been
shown to be effective and selective inhibitors of acetylcho-
linesterase,14a whereas mannosyl purine nucleosides have
been described as potent selective butyrylcholinesterase
inhibitors.14b
Figure 1 General skeleton of potential nucleoside mimetics (A–B),
sugar phosphate mimetics (B–C), and nucleotide mimetics (A–B–C)
This biological profile encourages the search for effi-
cient and straightforward approaches leading to new nucle-
oside/nucleotide-like derivatives. We report herein on the
synthesis of new nucleoside analogues, namely 6'-isonucle-
osides, sugar derivatives containing uncharged surrogates
for a phosphate moiety such as a sulfonohydrazide func-
tionality, as well as more elaborated sugar-containing mole-
In this study we concentrated on isonucleosides as ana-
logues of nucleosides. These regioisomers of nucleosides, in
which the nucleobase is linked to the sugar moiety at a
non-anomeric position, have been reported to display sig-
nificant biological properties including antiviral20 and anti-
cancer activities.21 In particular, based on in silico analysis,
adenine 3'-isonucleosides showed the potential to inhibit
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

C
N. M. Xavier et al.
Letter
Synlett
O
Cl
N
N
N
OH
HN
AcHN
N
N
N
O
a)
N
AcO
AcO
b)
O
HO
HO
AcHN
O
AcO
AcO
AcO
OMe
HO
AcO
OMe
OMe
2
3
1
O
N
R
OH
OH
O
OMe
O
OMe
O
HN
OAc
O
N
c), d)
OH
O
f), g)
N
6
O
O
AcHN
HO
HO
OMe
SPh
OMe
SPh
SPh
5 R = TIPSCl
6 R = H
4
7
e)
Scheme 1 Reagents and conditions: (a) PPh3, DEAD, 2-NHAc-6-Cl-purine, r.t., 16 h, 80%; (b) TFA (80% aq), 60 °C, 16 h, 57%; (c) TIPSCl, py, cat. DMAP,
r.t., 16 h; (d) Ac2O, py, r.t., 16 h, 95%, two steps; (e) TBAF, THF, r.t., 1 h, 56%; (f) PPh3, DEAD, 2-NHAc-6-Cl-purine, THF, reflux, 16 h; (g) TFA (80% aq),
60 °C, 16 h, 41%, two steps.
kinases,22 whereas 2'-isonucleoside triphosphates showed
the ability to be recognized by DNA polymerases.23 More-
over, these nucleoside analogues are likely to present better
stability towards enzymatic hydrolysis than their natural
counterparts. In the isonucleosides reported, the nucleo-
base is linked at either C-2 or C-3 to the sugar backbone, es-
pecially at furanose units.20c,21b,24 It was therefore appropri-
ate to consider other positions of the sugar moiety for the
coupling of a nucleobase towards new types of isonucleo-
sides. Hence, the synthesis of 6'-isonucleosides, namely gly-
copyranos-6-yl purines, was undertaken. Their construc-
tion was accomplished through Mitsunobu reaction of glu-
co- and manno-configured glycosides containing a free
hydroxyl group at C-6 with 2-acetamido-6-chloropurine
(Scheme 1).
Thus, methyl 2,3,4-O-acetyl-α-D-glucopyranoside (1),
available in three steps from methyl α-D-glucopyranoside,
was reacted with 2-acetamido-6-chloropurine in the pres-
ence of diethyl azodicarboxylate (DEAD) and triphenyl-
phosphine to give the N9-glucosylpurine 2 as the only
formed regioisomer in 80% yield.25 Evidence for the N9-C6'
linkage was provided by HMBC experiments, which showed
a key correlation between the sugar H-6' and C-4 of the pu-
rine moiety. Treatment of 2 with aqueous trifluoroacetic
acid (TFA) at 60 °C effected both deacetylation at the sugar
ring and hydrolysis at the chloro-containing 6-acetamido-
purine motif, leading to the N-acetylguanine moiety 3. The
guanine system of 3 was inferred by the significant differ-
ences in the chemical shifts in the signals of the nucleobase
moiety between 2 and 3 in their 13C NMR spectra. Whereas
C-6 of isonucleoside 3 (δ = 157.5 ppm) is deshielded relative
to that of 2 (δ = 151.5 ppm), the signal for C-5 appears at
higher field (δ = 120.4 ppm) than that of 2 (δ = 127.6 ppm).
HRMS analysis further supported the assignment of the
identity of 3, showing the corresponding peak for the [M +
Na]+ molecular ion and the loss of the Cl-associated isotopic
pattern.
An isonucleoside comprising a mannose-derived back-
bone was then synthesized. A suitable 6-monodeprotected
mannosyl precursor was prepared from the 3,4-O-butane
2',3'-diacetal derivative of phenyl 1-thiomannoside (4),
which could be accessed from mannose in four steps.26 Se-
lective protection of 4 at the primary hydroxyl group using
triisopropylsilyl chloride (TIPSCl) and a catalytic amount of
4-(dimethylamino)pyridine (DMAP) in pyridine and subse-
quent acetylation of the intermediate silyl ether, afforded
fully protected thiomannoside 5. Desilylation of 5 with tet-
ra-N-butylammonium fluoride (TBAF) gave 6, the coupling
of which with 2-acetamide-6-chloropurine using the
DEAD/triphenylphosphine system and further acid hydro-
lysis (aq TFA), employing similar conditions as with 2, fur-
nished the N9-linked mannosylguanine 7.
As potential mimetic structures of sugar phosphates, N-
glycosylsulfonohydrazides were chosen. The sulfonohydra-
zide moiety was predicted to be a surrogate for the phos-
phate group due to its polar nature and propensity for hy-
drogen bonding interactions with biological targets. The
anomeric sulfonohydrazide of ribose, the sugar unit present
in ATP (which is the natural nucleotide substrate of en-
zymes such as kinases) as well as that of glucuronic acid
were envisaged as target molecules.
The synthesis of N-ribosylsulfonohydrazide and of the
glucuronic acid counterpart was carried out by treatment
of ribose 8 and glucuronic acid 10 (Scheme 2), respectively,
with tosylhydrazide in the presence of acetic acid at 40 °C; a
methodology first reported for partially O-protected mono-
saccharides and more recently applied to fully unprotected
sugars.27,28 The reaction likely proceeds via the acyclic to-
sylhydrazone intermediate, which evolves to the more sta-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

D
N. M. Xavier et al.
Letter
Synlett
H
N
HN
OH
S
HO
O
O
a)
O
OH
O
OH
OH
OH
HO
9
8
O
O
S
HN
O
OH
OH
O
N
O
O
O
OAc
O
a)
b), c)
H
N
O
O
HO
HO
HO
HO
S
N
OH
H
OH
11
HO
OAc OAc
10
12
Scheme 2 Reagents and conditions: (a) TsNHNH2, cat. AcOH, DMF, 40 °C, 48 h, quantitative (9), 67% (11); (b) Ac2O, py, r.t., 5 min; (c) propargyl amine,
CH2Cl2, two steps.
ble cyclic glycosylhydrazine isomer by intramolecular at-
tack of the hydroxyl group at C-5 to the sp2 carbon of the
hydrazone. The β-N-glycosyl derivatives 9 and 11 were re-
gio- and stereoselectively obtained in quantitative and in
67% yields, respectively.
tons of the pyranose ring, thus confirming its 1C4 conforma-
tion and the HMBC correlation between the carbonyl car-
bon of the lactam functionality (δ = 167.0 ppm) with H-1,
which appeared at a rather high chemical shift value
(δ = 5.62 ppm), was in accordance with the presence of a
urono-1,6-lactam.
Based on the structure of 11, access to sulfonohydra-
zidyl glucuronamide derivatives bearing aromatic or N-het-
eroaromatic moieties, as molecules lying within the pro-
posed general skeleton for nucleotide mimetics (Figure 1),
was further targeted.
1
The C4 conformation adopted by 9 was revealed by its
1H NMR spectrum, particularly through the coupling con-
stants between H-4 and both of the protons at C-5 (JH-4,H-5a
and JH-4,H-5b of 4.2 and 2.0 Hz), indicating the absence of a H-
4–H-5a or H-5b trans-diaxial relationship as would be ex-
4
pected for a C1 conformation. The β-anomeric configura-
tion of 9 was established from the NOESY spectrum, in
which H-1 only presents correlations with H-5b and H-2.
Moreover, the small coupling constant between H-1 and H-
2 (J1,2 = 1.2 Hz) further confirmed the assigned conforma-
tion and anomeric configuration.
Glucofuranurono-6,3-lactone (13; Scheme 3) was the
precursor for the introduction of the aromatic moieties at
C-6 of the glycosyl unit through opening of the lactone moi-
ety with N-benzylamine or with N-propargylamine. The N-
benzyl glucofuranuronamide 1414a was subjected to acid-
mediated hydrolysis (aq TFA) to remove the 1,2-O- isopro-
pylidene functionality and cause subsequent intramolecu-
lar ring closure to the pyranose form, to give 15 in quantita-
tive yield. Glycosylation of tosylhydrazide with 15 led to the
N-benzyl sulfonohydrazidyl β-glucuronamide 16 in 46%
yield. The N-propargyl glucofuranuronamide 1730 was sub-
jected to a Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddi-
tion by treatment with benzyl azide in the presence of the
CuI/Amberlyst A-21 catalytic system,31 leading to the ex-
pected N-benzyl triazole derivative 18 in 87% yield. Cleav-
age of the acetonide functionality of 18 by aqueous TFA and
accompanying ring expansion afforded N-benzyltriazolyl-
methyl glucopyranuronamide (19), which was converted
into the corresponding anomeric tosylhydrazinyl derivative
20 by using similar N-glycosylation conditions to those
used for 15.32 Similar to that observed for the synthesis of 9
and 11, only β-anomers 16 and 20 were obtained, as was
confirmed by their J1,2 values (7.9 Hz) in their 1H NMR spec-
tra as well as by the NOE correlations between H-1, H-3,
and H-5. Of particular note is the use of simple, mild and
efficient coupling methods in the synthesis of 16 and 20
that fulfill the requirements of ‘click’ chemistry processes.33
The N-glucuronylsulfonohydrazide 11 adopted a 4C1
conformation, as judged by the coupling constants between
H-2–H3, H3–H4, and H4–H5, whereas the large J1,2 value
(8.8 Hz) was clearly indicative of a β-anomeric configura-
tion, supported by the NOESY correlations observed be-
tween H-1 and protons H-3 and H-5.
The sole formation of the β-anomers 9 and 11 may arise
from their stabilization through the exo-anomeric effect,
resulting from donation of electron density from the nitro-
gen lone pair to the endocyclic C1–O bond (nN1→σ*C1–O5); an
orbital interaction that occurs commonly in N-glycosyl
compounds.29 In the case of ribosyl sulfonamide 9 and de-
spite the fact that the sulfonamide function is in an axial
orientation, the anti-relationship between H-1 and NH
(since JNH,H-1 = 10.9 Hz) is consistent with an anti-periplanar
orientation between the nitrogen nonbonding orbital and
the C1–O bond, which allows the expression of this elec-
tronic effect.
Acetylation of 11 with acetic anhydride/pyridine fol-
lowed by treatment with N-propargylamine, furnished the
bicyclic glucuronolactam-based sulfonohydrazide 12. The
1H NMR spectrum of 12 presented broad singlets for all pro-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

E
N. M. Xavier et al.
Letter
Synlett
O
O
NHBn
NHBn
O
NHBn
O
O
O
O
O
OH
O
HO
c)
HO
a)
O
O
b)
H
HO
HO
HO
HO
S
N
N
O
OH
H
O
O
HO
HO
O
13
d)
15
16
14
Bn
N
Bn
O
O
N
N
Bn
N
H
N
H
N
NH
OH
O
NH
OH
O
N
N
O
HO
N
O
N
N
HO
HO
O
O
f)
g)
e)
H
O
O
HO
HO
S
N
HO
N
O
O
OH
H
HO
HO
20
O
O
19
18
17
Scheme 3 Reagents and conditions: (a) BnNH2, CH2Cl2, r.t., 16 h, quant.14a; (b) TFA (80% aq), r.t., 1.5 h, quant. (α/β = 1:0.60); (c) TsNHNH2, cat. AcOH,
DMF, 40 °C, 48 h, 46%; (d) propargylamine, CH2Cl2, r.t., 16 h; (e) BnN3, CuI/Amberlyst A-21 (cat.), CH2Cl2, r.t., 2 h, 87%; (f) TFA (80% aq), r.t., 1 h, quant.,
α/β ratio, 1:0.75; (g) TsNHNH2, cat. AcOH, DMF, 40 °C, 48 h, 45%.
In addition to the Cu(I)-catalyzed cycloaddition leading to
18, the formation of glucuronamide intermediates 14 and
17 by lactone ring opening with amines and the stereose-
lective coupling of lactols 15 and 19 with tosylhydrazide
also merit the ‘click’ reaction terminology.
The N-propargyl glucuronamide 17 was also a precursor
to the (triazolyl)methyl amide-linked disaccharide 24,
which is an analogue of 18 in which the benzyl group is re-
placed by a monosaccharide moiety (Scheme 4). Hence, the
triacetylated methyl 6-azido-glucopyranoside 23, which
was synthesized by tosylation of methyl 2,3,4-O-acetyl-α-
D-glucopyranoside (21) followed by nucleophilic replace-
ment with sodium azide, was subjected to ‘click’ cycloaddi-
tion with 17 using the CuI/Amberlyst A-21 catalytic system
to give 24 in 74% yield.34 Acetylation of 24 with acetic anhy-
dride and pyridine gave 25 in quantitative yield. Treatment
of 24 with aqueous TFA (80%) at 60 °C effected both
deacetylation and hydrolysis at the amide function, provid-
ing quantitatively the triazole isonucleoside 26, an amino-
methyl triazole analogue of the 6'-isonucleoside 3. Access to
26 constitutes a successful application of a coupling–decou-
pling pathway in which ‘click’ reactions are used to assem-
ble the different molecular units.35
The structural framework of 24 motivated attempts to
access to a nucleoside derivative with a structure resem-
bling that of nucleotide sugars; namely, the nucleoside di-
phosphate sugars (Figure 2,A), with the (triazolyl)methyl
amide fragment being envisaged as a potential metabolical-
ly and hydrolytically stable neutral surrogate of the diphos-
phate moiety (Figure 2,B).
N3
OR
O
O
b)
AcO
AcO
AcO
AcO
22
AcO
AcO
OMe
OMe
23
21 R = H
22 R = Ts
a)
O
O
OR
NH2
N
OR
O
NH
OH
O
H
N
N
N
N
HO
e)
c)
N
O
N
24
O
O
AcO
AcO
HO
HO
O
O
AcO
HO
OMe
O
OMe
24 R = H
25 R = Ac
17
26
d)
Scheme 4 Reagents and conditions: (a) TsCl, py, r.t., 16 h, 91%; (b) NaN3, DMF, 80 °C, 16 h, 83%; (c) 23, CuI/Amberlyst A21, CH2Cl2, r.t., 16 h, 74%;
(d) Ac2O, py, r.t., 1 h, quant.; (e) TFA (80% aq), 60 °C, 2 days.
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

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N. M. Xavier et al.
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NH
NH
NH
O
O
O
Cl
N
N
c)
a) b)
N
N
O
O
AcO
AcO
AcO
AcO
O
AcO
27
AcO
AcO
17
N
N
N
NHAc
AcO
AcO
OAc
N
NHAc
Cl
28
29
O
N
H
N
N
N
N
N
N
O
O
d)
AcO
AcO
N
NHAc
29
AcO
AcO
AcO
AcO
Cl
OMe
30
Scheme 5 Reagents and conditions: (a) TFA (70% aq), 40 °C, 30 min; (b) Ac2O, py, r.t., 30 min, 87%, two steps, α/β ratio: 1:0.5; (c) silylated 2-NHAc-6-
Cl-purine, TMSOTf, MeCN, 65 °C, MW, max. 150 W, 1.5 h, 30% (28) and 29% (29); (d) 23, CuI/Amberlyste A21, CH2Cl2, r.t., 16 h, 43%.
of trimethylsily trifluoromethanesulfonate (TMSOTf). The
O
O
O
P
regiochemistry of the nucleosidic linkage in 28–29 was un-
ambiguously assigned based on NMR data; particularly
through HMBC experiments that, in the case of the N9 nu-
cleoside 28, showed a correlation between the anomeric
proton (H-1') and C-4 of the purine. Diagnostic features of
the 1H spectra of 28 and 29 are the significant chemical
shift differences of H-1 and H-8 that are deshielded in the
N7 isomer. The N7-nucleoside was then coupled with the
previously synthesized 6-azido glucoside 23 under the
aforementioned Cu(I)-catalyzed cycloaddition conditions to
furnish 30 in moderate yield.
The 6′-isonucleosides 2, 3, 7, and 26, glycosyl sulfonohy-
drazides 9 and 11, sulfonohydrazidyl glucuronamide deriv-
atives 16 and 20, (triazolyl)methyl amide-linked disaccha-
rides 24 and 25, and related nucleoside derivative 30 were
subjected to enzyme inhibition and to cytotoxicity studies.
Cholinesterase assays were performed with acetylcho-
linesterase (AChE) from Electrophorus electricus and butyr-
ylcholinesterase (BChE) from equine serum. Galanthamine
hydrobromide, a clinically used ChE inhibitor, was used as
standard. The significant results are presented in Table 1.
Among the purine/guanine isonucleosides, only the
phenylthio mannopyranosid-6-yl guanine 7 showed signifi-
cant effects on ChEs, showing selective and moderate inhi-
bition of AChE (Ki = 18.8 μM). The aminomethyltriazole iso-
nucleoside 26 was the best ChE inhibitor of the series, with
a Ki value of 11.9 μM for AChE. With respect to the glycosyl-
sulfonohydrazides, the most active compounds were the N-
benzyltriazolylmethyl sulfonohydrazidyl glucuronamide
20, which showed moderate inhibition of AChE
(Ki = 31.0 μM) and glucuronolactam sulfonohydrazide 12,
which was the only compound to display significant inhibi-
tory effects on BChE. Selectivity to AChE was also exhibited
by the (triazolyl)methyl amide-linked disaccharide 24 with
moderate inhibition (Ki = 26.7 μM).
HO
N
nucleobase
P
O
O
O
O
-O
-O
HO OH
A
O
N
O
N
nucleobase
H
N
N
N
RO
B
O
RO
OR
Figure 2 (Triazolyl)methyl amide-linked disaccharide nucleosides (B)
as potential mimetics of nucleoside diphosphate sugars (A)
The ability of the triazole motif to act as a mimetic for
the phosphate group has already been shown in the mimic-
ry of glycosyl phosphate derivatives.36 Moreover, the nega-
tively charged diphosphate moiety is thus replaced by a less
polar and neutral system of similar length and space, ren-
dering the molecule more capable of cell penetration,
which is important for the targeting of an intracellular en-
zyme. As potential mimetics of nucleotide sugars, mole-
cules based on (triazolyl)methyl amide-linked disaccharide
nucleosides may act as potential inhibitors of enzymes such
as glycosyltransferases, which act on the biosynthesis of
glycans and glycoconjugates and are potential drug targets
against cancer, inflammation, and infection diseases.37
Access to structures based on the envisaged skeleton
was based on a convergent methodology (Scheme 5). A nu-
cleoside containing the N-propargyl moiety was synthe-
sized starting from 17, that underwent acid-mediated hy-
drolysis and further acetylation to afford the peracetylated
N-propargyl glucuronamide 27 as the glycosyl donor. The
latter was converted into N9- and N7-linked purinyl nucleo-
sides 28 and 29, which formed in a 1:1 ratio, by reaction
with silylated 2-acetamido-6-chloropurine in the presence
Molecular docking of the most active compound (26)
into the crystal structure of AChE was performed using
GOLD 5.2 software38 and AChE coordinates from the avail-
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

G
N. M. Xavier et al.
Letter
Synlett
able PDB structure 4BDT.39 Molecular interactions were in-
spected with the MOE 2012.13 package40 (Figure 3). Inter-
actions of 26 with amino acid residues located at the en-
zyme active site as well within the peripheral anionic site
were displayed. In the active site, the quaternary amonium
moiety is involved in cation-π interactions with His447 of
the esteratic subsite and also interacts with Glu202 of the
anionic subsite. The triazole ring has π-stacking contacts
with the indole system of Trp86, which is a critical amino
acid residue for substrate binding located at the anionic
subsite.41 The sugar moiety is positioned towards the pe-
ripheral anionic site of the enzyme, with interactions with
Asp74 through two hydroxyl groups, one of them also con-
tacting Tyr337 from the anionic subsite.
glucuronyl sulfonohydrazide (11) was an effective inhibitor,
with a Ki value of 9.5 μM (Table 2). The N-benzyl glucuro-
namide counterpart 16 showed only 11% inhibition (at
50 μM) whereas the remaining N-glycosylsulfonohydra-
zides exhibited virtually no inhibitory effect.
Table 2 Significant Results on the Carbonic Aanhydrase II Inhibition
Assays
Compounds
Ki (μM) (% Inhibition)a
[Ki′ (μM)]
(type of inhibition)
Acetazolamide
0.1 ± 0.0 (competitive)
11
9.5 ± 0.9
[49.6 ± 1.3]
(mixed-type)
16
> 50 (11%)
a % Inhibition at 50 μM.
These results suggest that the carboxyl function in 11 is
crucial for CA inhibition, since the presence of a sulfonohy-
drazide moiety in these molecules does not confer, by itself,
CA inhibitory ability. In the case of known primary sulfon-
amide CA inhibitors, their binding to CA is driven by the co-
ordination of the deprotonated sulfonamide function to the
enzyme-bound Zn2+ ion through the sulfonamide nitro-
gen.42 The glycosyl sulfonohydrazides are probably not as
prone to coordination to the Zn2+ cation through a sulfono-
hydrazide nitrogen atom via deprotonation, as glycosyl de-
rivatives comprising a primary sulfonamide function are,18a
and hence the presence of a more acidic functionality will
increase their binding affinities.
This assumption is supported by molecular docking
studies of the binding mode of N-glucuronylsulfonohydra-
zide (11) to the active site of CA-II, using available PDB
structure 2X7T43 (Figure 4). The carboxyl function in its
deprotonated form coordinates the Zn2+ and also interacts
through a hydrogen bond with Thr198, a key residue for the
catalytic activity of the enzyme, since it interacts with the
Zn2+-coordinated water, enhancing its nucleophilicity for
further attack to the substrate (CO2).16 Most of the known
sulfonamide CA-II inhibitors also interact with this residue
through a sulfonyl oxygen atom.
The compounds were also screened for their inhibitory
action towards recombinant CDK-2/cyclin E. However none
of the molecules showed significant inhibition of this com-
plex at concentrations below 100 μM.
All the compounds were tested for their cytotoxicity
against breast adenocarcinoma cell line, MCF-7, and a
chronic myeloid leukemia cell line, K562 and six of them,
namely 2, 3, 26, 11, 20, and 24, selected among each group
of molecules synthesized, were further tested on other can-
cer cell lines: G361 (melanoma), HeLa (cervix carcinoma),
HCT116 (colon carcinoma), CEM (T-cell leukemia), and
THP-1 (monocytic myeloid leukemia), and on nonmalig-
Figure 3 2D Schematic molecular interactions of the GOLD docking
pose for compound 26 in AChE. Picture prepared with MOE 2012.13
software.
Evaluation of the abilities of the compounds to inhibit
carbonic anhydrase II (CA-II, from bovine erythrocytes), us-
ing acetazolamide as standard inhibitor, revealed that only
Table 1 Significant Results on the Cholinesterase Inhibition Assays
Compound
AChE
BChE
Ki (μM) (% inhibition)a
[Ki′ (μM)]
(type of inhibition)
Galantamine HBr
0.5 ± 0.0 (competitive)
18.8 ± 2.0 (competitive)
11.9 ± 1.6 (competitive)
> 100 (31%)
9.4 ± 0.7 (competitive)
7
26
12
> 100 (7%)
> 100 (14%)
51.4 ± 5.7
[147.3 ± 8.9]
(mixed-type)
20
24
31.0 ± 9.3
[241.5 ± 42.5]
(mixed-type)
> 100 (18%)
26.7 ± 2.2
[109.4 ± 17.6]
(mixed-type)
> 100 (13%)
a % Inhibition at 50 μM
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

H
N. M. Xavier et al.
Letter
Synlett
The ability of the catalytic sites of such enzymes to bind
nucleoside analogues or molecules possessing functional
groups/motifs mimicking the structural fragments of nu-
cleotides was therefore shown. However, the compounds
did not exhibit significant CDK-2 inhibitory effects and cy-
totoxicity to tumor cells. Considering the structures of the
known CDK-2 inhibitors, mostly based on flattened and he-
teroaromatic systems, the high conformational freedom of
the synthesized molecules, arising particularly from the
carbohydrate unit, may be a reason for the lack of activity.
Hence, more constrained structures, obtained through
modifications at the sugar ring, may contribute to effective
binding as well as to target selectivity. Further investiga-
tions on this topic will address these aspects.
The lack of toxicity of the tested molecules to healthy
cells will prompt future structural optimization and syn-
thesis of analogues to attain improved bioactivities towards
the therapeutic targets on which efficacy was detected.
Figure 4 2D schematic of molecular interactions of the GOLD docking
pose for 11 in the CA-II active site. Picture prepared with MOE 2012.13
software.
Acknowledgment
‘Fundação para a Ciência e Tecnologia’ (FCT) is acknowledged for
funding through the FCT Investigator Program to N.M.X and to S.D.L.
(projects IF/01488/2013 and IF/00472/2014) and for financial support
of the strategic projects UID/MULTI/00612/2013 and Pest-
OE/SAL/UI4013/2014. The Node CQE-IST of RNEM is acknowledged
for facilities through the FCT projects RECI/QEQ-MED/0330/2012 and
REM 2013. R.J. is grateful to the project from the National Program for
Sustainability I (grant no. LO1204) provided by the Czech Ministry of
Education, Youth and Sports.
nant BJ fibroblasts and murine embryonic fibroblasts NIH
3T3. No cytotoxicity effect of the compounds was detected
at concentrations below 100 μM on MCF7 and K562 cell
lines, whereas additional tests on other cell lines still did
not reveal significant antiproliferative activity on tumor
cells or cytotoxicity to healthy cells, at concentrations be-
low 50 μM (compound 20) or at below 200 μM (compounds
2, 3, 26, 11, and 24).
In summary, in this contribution, novel molecules con-
structed on carbohydrate platforms; namely 6'-isonucleo-
sides, N-glycosylsulfonohydrazides derived from glucuronic
acid, and a (triazolyl)methyl amide-linked disaccharide nu-
cleoside, were synthesized as analogues and potential mi-
metics of nucleosides, glycosyl phosphates, and nucleo-
tides. Sulfonohydrazide and (triazolyl)methyl amide sys-
tems were proposed as novel bioisostere moieties for
phosphate or diphosphate functionalities contained in nu-
cleotides. A new structural skeleton for potential mimetics
of nucleoside diphosphate sugars was proposed and a syn-
thesis of a molecule based on such a framework was ac-
complished. These results are expected to motivate the syn-
thesis of structurally related compounds that could provide
potential new glycosyltransferase inhibitors.
Supporting Information
Supporting information for this article is available online at
http://dx.doi.org/10.1055/s-0035-1560591.
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References and Notes
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4.72 (m, J2',3' = 10.2 Hz, 2 H, H-2', H-4'), 4.45–4.29 (m, 2 H, H-6'a,
H-6'b), 4.11 (ddd, 1 H, H-5'), 3.17 (s, 3 H, OCH3), 2.51 (s, 3 H,
CH3, NHAc), 2.12, 2.06, 1.99 (3 × s, 9 H, CH3, OAc). 13C NMR (100
MHz, CDCl3): δ = 170.2, 170.1, 170.1 (CO, NHAc, CO, Ac), 152.8
(C-4), 152.2, 151.5 (C-2, C-6), 145.5 (C-8), 127.6 (C-5), 96.9 (C-
1'), 70.6 (C-2'), 69.7 (C-3'), 69.6 (C-4'), 67.5 (C-5'), 55.8 (OCH3),
43.9 (C-6'), 25.3 (CH3, NHAc), 20.9, 20.8, 20.7 (3 × CH3, OAc).
HRMS: m/z [M+Na]+ calcd for C20H24ClN5O9: 536.1155; found:
536.1152.
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Deoxy-1-(2-tosylhydrazin-1-yl)-β-D-glucopyranuronamide
(20) through N-Glycosylation: To a solution of N-(1-benzyl-
1H-1,2,3-triazol-4-yl)methyl-α,β-D-glucopyranuronamide (19;
250 mg. 0.69 mmol) in DMF (1.5 mL), p-toluenesulfonyl hydra-
zide (145 mg, 0.78 mmol, 1.1 equiv) and glacial acetic acid (4 μL,
0.07 mmol, 0.1 equiv) was added. The reaction mixture was
allowed to stand at 40 °C without stirring for 48 h, then the
solvent was evaporated under vacuum. Diethyl ether (20 mL)
was added to the residue and the mixture was vigorously
stirred for 24 h. The mixture was filtered and the white solid
was washed with diethyl ether, dichloromethane, and cold
methanol to give pure 20 (165 mg, 45%) as a white solid; mp
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20
154–155.7 °C; [α]D –2 (c = 0.4, MeOH). 1H NMR (400 MHz,
CD3OD): δ = 7.86 (s, 1 H, H-9), 7.77 (d, J = 8.1 Hz, 2 H, Ha, Ts),
7.43–7.27 (m, 7 H, Hb, Ts, Ph), 5.56 (s, 2 H, CH2, Bn), 4.50, 4.45
(2 × d, J = 15.7 Hz, AB system, CH2-7), 3.85 (d, J1,2 = 7.9 Hz, 1 H,
H-1), 3.65 (d, J4,5 = 8.5 Hz, 1 H, H-5), 3.49–3.36 (m, 2 H, H-2, H-
3, H-4), 2.43 (s, 3 H, Me, Ts). 13C NMR (100 MHz, CD3OD): δ =
172.3 (CO), 145.2 (Cq, Ts), 137.3 (2 × Cq, Ts, Ph), 130.7, 130.0,
129.6, 129.2, 129.0 (CH, Ts, Ph), 91.8 (C-1), 77.6 (C-3), 76.9 (C-5),
73.5, 71.0 (C-2, C-4), 55.0 (CH2, Bn), 35.2 (C-7), 21.5 (CH3, Ts).
HRMS: m/z [M+H]+ calcd for C23H28N6O7S: 533.1813; found:
533.1827.
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(25) Synthesis of 2-Acetamide-6-chloro-9-(methyl 2,3,4-O-acetyl-
6-deoxy-α-D-glucopyranosid-6-yl)purine (2) through Mitsu-
nobu Reaction: To a solution of methyl 2,3,4-O-acetyl-α-D-glu-
copyranoside (1; 100 mg, 0.31 mmol) in THF (5 mL) under
nitrogen, PPh3 (163 mg, 0.62 mmol), diethyl azodicarboxylate
(DEAD; 0.62 mmol, 0.1 mL) and 2-acetamido-6-chloropurine
(132 mg, 0.62 mmol) were sequentially added. The mixture was
stirred at r.r. under nitrogen for 16 h. The solvent was evapo-
rated and the residue was subjected to column chromatography
on silica gel (EtOAc–petroleum ether, 1:1 to 1:9) to afford 2 (128
(34) Synthesis of N-[1-(Methyl 2,3,4-O-acetyl-6-deoxy-α-D-gluco-
pyranosid-6-yl)-1H-1,2,3-triazol-4-yl]methyl-1,2-O-isopro-
pylidene-α-D-glucofuranuronamide
(34)
through
20
mg, 80%) as a white solid. [α]D +15 (c = 0.3, CHCl3). 1H NMR
CuI/Amberlyst A21-Catalyzed Cycloaddition: To a solution of
N-propargyl 1,2-O-isopropylidene-α-D-glucofuranuronamide
(17; 180 mg, mg, 0.66 mmol) in dichloromethane (7 mL),
(400 MHz, CDCl3): δ = 8.22 (s, 1 H, NH), 8.13 (s, 1 H, H-8), 5.46
(t, J = 9.4 Hz, 1 H, H-3'), 4.96 (d, J1',2' = 3.5 Hz, 1 H, H-1'), 4.84–
© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J

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N. M. Xavier et al.
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Synlett
methyl 2,3,4-O-acetyl-6-azido-6-deoxy-α-D-glucopyranoside
(23; 0.66 mmol, 229 mg) and CuI/Amberlyste A21 (126 mg)
were added. The suspension was stirred overnight at r.t., then
the catalyst was filtered off and the solvent was evaporated. The
residue was subjected to column chromatography on silica gel
(ethyl acetate to ethyl acetate–methanol, 9.5:0.5) to give tri-
azole-linked disaccharide 24 (304 mg, 74%) as a white solid.
(35) For reviews on ‘click’-coupling strategies and decoupling, see:
(a) Bielski, R.; Witczak, Z. J. Chem. Rev. 2013, 113, 2205.
(b) Bielski, R.; Witczak, Z. J. Paradigm and Advantage of Carbohy-
drate Click Chemistry Strategy for Future Decoupling, In Click
Chemistry in Glycoscience: New Developments and Strategies;
Witczak, Z. J.; Bielski, R., Eds.; John Wiley & Sons, Inc: Hoboken,
NJ, 2013, 3–30.
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20
Data for 24: mp 211–213 °C; [α]D = +25 (c = 0.3, CH3OH). 1H
NMR (400 MHz, MeOD): δ = 7.92 (s, 1 H, H-9), 5.93 (d,
J1,2 = 3.4 Hz, 1 H, H-1), 5.40 (t, J2',3' = J3',4' = 9.7 Hz, 1 H, H-3'),
5.01–4.80 (m, J1',2' = 3.7 Hz, J2',3' = 9.7 Hz, 3 H, H-1', H-2', H-4'),
4.62 (dd, part A of ABX system, J5',6'a = 2.0 Hz, J6'a 6'b = 14.2 Hz,
,
1 H, H-6'a), 4.57–4.46 (m, 4 H, H-2, H-6'b, H-7a, H-7b), 4.38 (d,
J4,5 = 6.3 Hz, 1 H, H-5), 4.25 (dd, J3,4 = 2.1 Hz, 1 H, H-4), 4.22–
4.12 (m, 1 H, H-3, H-5'), 3.14 (s, 3 H, Me), 2.07, 2.02, 1.98 (3 × s,
3 × 3 H, 3 × CH3, Ac), 1.46 (s, 3 H, CH3, i-Pr), 1.32 (s, 3 H, CH3, i-
Pr). 13C NMR (100 MHz, MeOD): δ = 171.7, 171.5, 171.4 (3 × CO,
Ac), 146.9 (C-8), 125.4 (C-9), 113.0 (Cq, i-Pr), 106.4 (C-1), 97.9
(C-1'), 86.5 (C-2), 82.4 (C-4), 75.8 (C-3), 71.9 (C-3'), 71.4, 71.3
(C-2', C-4'), 71.0 (C-5), 69.0 (C-5'), 55.9 (CH3, OMe), 51.7 (C-6'),
35.7 (C-7), 27.1, 26.4 (2 × CH3, i-Pr), 20.6, 20.6, 20.4 (3 × CH3,
Ac). HRMS: m/z [M+H]+ calcd for C25H36N4O14: 617.2301; found:
617.2304..
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Cambridge U. K., www.ccdc.cam.ac.uk/products/gold_suite.
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© Georg Thieme Verlag Stuttgart · New York — Synlett 2015, 26, A–J