The first general synthesis of 2-C-(β-d-glycopyranosyl)pyrimidines and their evaluation as inhibitors of some glycoenzymes

Article abstract of DOI:10.1039/c8nj04035d

A systematic study was performed on the preparation of unknown 2-C-(β-d-glucopyranosyl)pyrimidines. Pinner type cyclisation of O-perbenzylated C-(β-d-glucopyranosyl)formamidine with β-ketoesters, dimethyl malonate, and β-diketone derived α,β-unsaturated β-chloroketones followed by catalytic hydrogenation resulted in various substituted 2-C-(β-d-glucopyranosyl)-pyrimidin-4(3H)-ones, and 2-C-(β-d-glucopyranosyl)-4,6-disubstituted-pyrimidines, respectively, in moderate to good yields. The above pyrimidine derivatives were also obtained by ring closure of the unprotected C-(β-d-glucopyranosyl)formamidine with the same 1,3-dielectrophiles. In addition, a continuous one-pot three-step procedure starting from O-peracylated d-glycopyranosyl cyanides was also elaborated to give representatives of the aforementioned pyrimidines with various sugar configurations in acceptable to excellent overall yields (25-94%). Due to the versatility of the applied 1,3-dielectrophiles, these synthetic routes represent the first expansible method to obtain the target compounds. The new C-glycopyranosyl pyrimidines showed moderate inhibition against α-glucosidase and β-galactosidase enzymes, had, however, no activity against glycogen phosphorylase. The obtained molecule library is ready for further biological testing.

Full text of DOI:10.1039/c8nj04035d

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Jason B. Benedict et al.
The role of atropisomers on the photo-reactivity and fatigue of
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First general synthesis of 2-C-(β-D-glycopyranosyl)pyrimidinDeOsI: 1a0.n10d39/C8NJ04035D
their evaluation as inhibitors of some glycoenzymes
Eszter Szennyes,a Éva Bokor,a* Peter Langer,b Gyöngyi Gyémánt,c Tibor Docsa,d Ádám
Sipos,d László Somsáka*
a Department of Organic Chemistry, University of Debrecen, H-4002 POB 400, Debrecen,
Hungary
b Department of Chemistry, University of Rostock, Albert-Einstein-Str. 3a, 18059 Rostock,
Germany
c Department of Inorganic and Analytical Chemistry, University of Debrecen, H-4002 POB
400, Debrecen, Hungary
d Department of Medical Chemistry, Faculty of Medicine, University of Debrecen, Egyetem
tér 1, H-4032 Debrecen, Hungary
Abstract
A systematic study was performed on the preparation of unknown 2-C-(β-D-
glucopyranosyl)pyrimidines. Pinner type cyclisation of O-perbenzylated C-(β-D-
glucopyranosyl)formamidine with β-ketoesters, dimethyl malonate, and β-diketone derived
α,β-unsaturated β-chloroketones followed by catalytic hydrogenation resulted in variously
substituted 2-C-(β-D-glucopyranosyl)-pyrimidin-4(3H)-ones, and 2-C-(β-D-glucopyranosyl)-
* Corresponding authors: Bokor, É. tel - +3652512900 ext 22474; fax - +3652512744; e-mail –
bokor.eva@science.unideb.hu; Somsák, L. tel - +3652512900 ext 22348; fax - +3652512744; e-mail –
somsak.laszlo@science.unideb.hu
1

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DOI: 10.1039/C8NJ04035D
4,6-disubstituted-pyrimidines, respectively, in moderate to good yields. The above pyrimidine
derivatives were also achieved by ring closure of the unprotected C-(β-D-
glucopyranosyl)formamidine with the same 1,3-dielectrophiles. In addition, a continuous one-
pot three-step procedure starting from O-peracylated D-glycopyranosyl cyanides was also
elaborated to give representatives of the aforementioned pyrimidines with various sugar
configurations in acceptable to excellent overall yields (25-94 %). Due to the versatility of the
applied 1,3-dielectrophiles these synthetic routes represent the first expansible method to get
the target compounds. The new C-glycopyranosyl pyrimidines showed moderate inhibition
against α-glucosidase and β-galactosidase enzymes had, however, no activity against
glycogen phosphorylase. The obtained molecule library is ready for further biological testing.
Keywords
Pyrimidine, Amidine, 1,3-Dicarbonyl compounds, Pinner synthesis, C-Glucopyranosyl
derivative
2

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Introduction
Today’s chemical biology and drug discovery is in an urgent need to identify new small
molecules and molecular architectures to extend chemical space in order to modulate the
functions of target proteins. The traditional scenario of active ingredients comprising
heterocyclic assemblies is continuously being broadened towards unconventional conjugates,
among which especially sugar derivatives may open up ways to new targets, new ways of
action and unprecedented selectivities.1
C-Glycosyl compounds2 in general and C-glycopyranosyl arenes3 and hetarenes4 in particular
continuously attract intense interest of synthetic and medicinal chemists due to many types of
biological activities as well as to the use of such compounds as glycomimetics and also as
active ingredients of marketed drugs. While C-glycosyl derivatives of five-membered
heterocycles are widely represented, much less attention has been devoted to six-membered
heterocyclic C-glycosyl compounds.4 In the latter class C-glycofuranosyl heterocycles5, 6
make the overwhelming majority of the known derivatives. This general trend is also valid for
pyrimidines and especially for their 2-C-glycosylated derivatives. Thus, among 2-C-glycosyl
pyrimidines, 2-(3',5'-di-O-benzoyl-2'-deoxy-α-D-ribofuranosyl)pyrimidine (together with its
4-substituted counterpart and the β-D-anomer of the latter in 56 % yield for the three
products)7 as well as 2-(1'-hydroxy-1',2';3',5'-di-O-isopropylidene-β-L-arabinofuranosyl)-4-
methyl-pyrimidine (together with the 6-glycosyl derivative in 65 % combined yield with a
ratio of ~7 : 3 for the 2-and 6-glycosyl isomers)8 were obtained by Minisci type radical
glycosylations of protonated pyrimidines. C-Glycofuranosyl formamidines (2,5-anhydro-
aldonimidamides) were condensed with 1,3-dielectrophiles (e. g. Me2NCH=CHCOCOOEt,9
RCOCH2COR10 to the expected 2-C-glycosyl pyrimidine derivatives. A 2-C-ribofuranosyl-4-
hydroxy-6-methyl-pyrimidine was obtained by a ring transformation of a related 1,3-oxazine
3

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11
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derivative. C-Glycofuranosylation in other positions of the pyrimidine ring is surveyed in
excellent reviews.5, 6
On the other hand, to the best of our knowledge, C-glycopyranosyl derivatives of pyrimidine
are even more scarce in the literature. Thus, only one report described a 2-(3',4'-di-O-benzoyl-
2'-deoxy-β-D-ribopyranosyl)pyrimidine as a minor product beside the 4-substituted
counterpart (combined yield 56 %, ratio 3:7).7 Some 5-C-glycopyranosyl pyrimidines were
achieved by cyclocondensations of C-glycopyranosylated enaminoketones with guanidine or
acetamidine.12, 13 A set of C-glycopyranosylated dihydropyrimidines (having the sugar
moieties at C-4 or C-6, or in both positions) was synthetized by Biginelli-type
cyclocondensations of C-glycopyranosyl formaldehydes and β-ketoesters, respectively.4
In the light of the practical non-existence of 2-C-glycopyranosylated pyrimidine type
compounds we envisaged a series of investigations to obtain this class of compounds by
reactions of C-(β-D-glycopyranosyl)formamidines which became available by our recently
published procedures.14-16 In addition, we also wished to study these compounds as inhibitors
of different enzymes acting on carbohydrate substrates, such as glycogen phosphorylase (GP)
since N-(β-D-glucopyranosyl)pyrimidines were shown to have low micromolar inhibition of
rabbit muscle GPb.17-19 In this work the reactions of C-(β-D-glycopyranosyl)formamidines
with β-dicarbonyl derivatives and analogous 1,3-dielectrophiles to give the expected C-
glucosyl pyrimidines and the study of the latter as inhibitors of GP and some glycosidases are
described.
Results and Discussion
The construction of the pyrimidine ring from various precursors is a very active field of
research and within this the archetypal Pinner method (reaction of an amidine with 1,3-
4

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dicarbonyl compounds) is being intensively investigated even today.20-22 The Pinner synthesis
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DOI: 10.1039/C8NJ04035D
of pyrimidines generally requires strongly basic conditions,10, 23, 24 therefore, the recently
prepared 2,6-anhydro-3,4,5,7-tetra-O-benzyl-D-glycero-D-gulo-heptonimidamide15, 16 (O-
perbenzylated C-(β-D-glucopyranosyl)formamidine, 1) was chosen as the starting material.
The reactions of 1 with 3-ketoesters in the presence of 3 equiv of NaOMe in MeOH gave the
corresponding ring-closed products 3 in acceptable to good yields (Scheme 1). Removal of
the O-benzyl protecting groups was effected by catalytic hydrogenation over Pd(OH)2,
however, the use of elevated temparature was necessary to obtain the expected products 4a
and 4d in good yields. To avoid possible reductive hydrodehalogenation25 of 3b and 3c during
deprotection, hydrogenolytic O-debenzylation of 1 was performed in an acidified mixture of
EtOAc and EtOH at room temperature to give the unprotected amidine 2 as a hydrochloride
salt in quantitative yield. The ring closure of 2 with 3-ketoesters was achieved under the same
conditions as with the protected 1 and pyrimidines 4 were obtained in good to excellent
yields. Besides facilitating to avoid hydrodehalogenation, this latter route resulted in
significantly higher overall yields of the target products (1→3a→4a: 49 % vs 1→2→4a: 87
%; 1→3d→4d: 33 % vs 1→2→4d: 64 %).
5

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Scheme 1. a) H2, Pd(OH)2/C, dry EtOAc-EtOH (1 : 2), 1 drop of ccHCl, rt; b) 3-ketoester (2
equiv), 1M NaOMe in dry MeOH (3 equiv), dry MeOH, rt; c) H2, Pd(OH)2/C, dry EtOAc-
EtOH (1 : 2), reflux.
The reaction of 1 with an excess of dimethyl malonate (Scheme 2) proceeded similarly to give
pyrimidine 5 which was O-deprotected by catalytic hydrogenolysis to 6. The unprotected
amidine 2 and dimethyl malonate gave 6 to show again that in terms of overall yields
debenzylation followed by ring closure is more efficient than the opposite reaction sequence
(1→2→6: 70 % vs 1→5→6: 39 %).
When malononitrile or a cyanoacetic acid ester were used as the dielectrophile to react with 1
or 2 no ring closure could be achieved. With 2 complex mixtures were formed while with 1
these reagents replaced one of the nitrogens in the amidine moiety to give enamino
6

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ester/nitrile type derivatives 7. A similar transformation was observed with simple aliphatic or
DOI: 10.1039/C8NJ04035D
aromatic substrates.26 In 7b the (Z)-configuration of the double bond unequivocally followed
from the different chemical shifts of the NH2 protons (8.89 and 6.37 ppm) due to formation of
a H-bond with the COOEt group.27
Scheme 2. Reagents and conditions: a) dimethyl malonate (10 equiv), 1 M solution of
NaOMe in dry MeOH (10 equiv), dry MeOH, rt; b) malononitrile (2 equiv) or ethyl-2-
cyanoacetate (10 equiv), 1M NaOEt in dry EtOH (2 or 10 equiv), dry EtOH, rt; c) H2,
Pd(OH)2/C, dry EtOAc-EtOH (1 : 2), reflux.
The experiences of reactions of amidines 1 and 2 with 1,3-diketones are summarized in Table
1. Since the applied 1,3-diketones gave no reaction with the above substrates activation of the
reagents was necessary as described previously.10 The diketones were treated with a
chlorination agent (PCl5, (COCl)2 or SOCl2) and the so obtained crude α,β-unsaturated β-
chloroketones were reacted with 1 or 2 to give the expected 8 or 10, respectively. In both
series the yields were in the 62-75 % range. O-Debenzylation of compounds 8 was effected
7

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by catalytic hydrogenation. While at room temperature and under neutral conditioDnOsI:n1o0.1039/C8NJ04035D
reaction took place, boiling of the reaction mixtures gave very good yields of the desired 10a
and 10b. On the other hand, these conditions resulted in a mixture of compounds which
contained partially saturated pyrimidines 9c,d and 10c,d (vide infra Fig 1 for stereochemical
assignments). When these hydrogenolyses were carried out under acidic conditions only the
formation of inseparable mixtures of 9c,d could be observed in keeping with the known
general behaviour of pyrimidines to take up two moles of hydrogen under catalytic
hydrogenation conditions in acidic media.28
8

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Table 1. Reactions of amidines 1 and 2 with 1,3-diketones
a) PCl5 (1 equiv), dry Et2O, rt; b) (COCl)2 (1.2 equiv), DMF (1.3 equiv), dry CH2Cl2, 0 °C to
rt; c) SOCl2 (3 equiv), dry CHCl3, cat. DMF, reflux; d) K2CO3 (4 equiv), α,β-unsaturated
chloroketone (1.2 equiv), 4 Å molecular sieves, dry DMF, 0 °C to rt; e) H2, Pd(OH)2/C, dry
EtOAc-EtOH, (1 : 2), reflux; f) H2, Pd(OH)2/C, 1 drop of ccHCl, dry EtOAc-EtOH (1 : 2), rt.
Conditions and yields (%)
10
a
R1
R2
9b
8
from 2
from 8
CH3
CH3
a
b
c
d
d
d
65
d
d
d
62
a
b
c
e
e
--
--
79
CH3
Ph
CF3
68
74
63
69
92
CH3
e
f
27
82
36
--
Ph
CF3
c
d
72
d
75
d
e
f
43
79
19
--
a Conditions for the activation of the 1,3-diketone.
b Mixture of two diastereomers (see Fig 1).
9

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The formation of tetrahydropyrimidines 9 was clearly indicated by the mass spectra of the
compounds showing an increase of the molecular masses by ~4 Da. The 1H NMR spectra
exhibited two series of resonances in a ratio of ~5 : 1 for 9c and 1 : 1 for 9d (see supporting
material). These were supposed to be originated from the presence of diastereomers rather
than tautomers that were not to be detected in CD3OD applied as the solvent. The coupling
constants identified in the proton spectra (e. g. for 9d: 4.62-4.57 ppm, 2 dd, J4,5ax = 11.3 Hz,
J4,5eq = 3.8 Hz in each, 2 × Py-H-4; 2.31-2.25 ppm, 2 dt, J5ax,5eq = 13.0 Hz, J4,5eq = J5eq,6 = 3.8
Hz in each, 2 × Py-H-5eq; 1.81-1.67 ppm, 2 dt, J5ax,5eq = 13.0 Hz, J4,5ax = J5ax,6 = 11.3 Hz in
each, 2 × Py-H-5ax) indicated that the tetrahydropyrimidine rings were 4,6-cis-disubstituted
diastereomers, and this was in accord with the expected suprafacial addition of the hydrogens
under the heterogenous catalytic conditions. In addition, these coupling constants were also
consistent with the existence of the tetrahydropyrimidine moieties in E5 and 5E conformations.
The envelope-like conformations of 1,4,5,6-tetrahydropyrimidine derivatives were firmly
determined by NMR29 and X-ray29-32 studies and it was also pointed out that the
conformations were the same in solution and in the solid phase.29 The established structures
for compounds 9 are shown in Fig. 1.
10

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Figure 1. Stereochemical assignment of tetrahydropyrimidines 9.
Since in the above studies the best results were obtained with the unprotected amidine 2,
experiments were also directed to getting this compound from the easily available O-
perbenzoylated β-D-glucopyranosyl cyanide 13.33 To this end the Pinner-type conversion of
13 to the imidate 11 was investigated first (Scheme 3). Contrary to the experiences with the
analogous O-benzoyl protected ribofuranosyl cyanide, where the crystalline imidate could be
isolated from a reaction with catalytic NaOMe in MeOH,34 under similar conditions 13 gave a
crude mixture from which the isolation of 11 by crystallization failed. A trial to treat the crude
mixture by NH3/NH4Cl transformed the imidate to the expected amidine 2, however, a minor
amount of the unsaturated 12 inseparable from 2 was also detected by the presence of a
resonance35 at 6.07 ppm of H-2 (d, J = 2.6 Hz) in the 1H NMR spectrum of the mixture. The
elimination most probably took place from molecules still having the 2-OBz group since it is
well known that base-induced eliminations can be brought about from O-peracylated glycosyl
cyanides36, 37 and related compounds.38 Following these experiences, we attempted the
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transformation of 13 to pyrimidines 4 and 10 in continuous operations by additionDoOfI: 1N0.a10O39M/C8eNJ04035D
in MeOH, followed by NH3/NH4Cl and subsequent reaction with the corresponding 1,3-
dielectrophile. This procedure gave 4a and 4b in 43 % and 25 % isolated yield, respectively,
for the three steps. From the final reaction mixture of 13 with benzoylacetone besides the
expected 10c (30 % isolated overall yield) the unsaturated by-product 14 could also be
obtained (4 %) by column chromatography.
Scheme 3. Reagents and conditions: a) 1M NaOMe in dry MeOH (20 mol %), dry, MeOH,
dry CHCl3, rt; b) NH4Cl (1 equiv), sat. NH3 in dry MeOH; c) K2CO3 (4 equiv), α,β-
unsaturated chloroketone (1.2 equiv) freshly prepared from 1-phenylbutane-1,3-dione (see in
Table 1.), 4 Å molecular sieves, dry DMF, 0 °C to rt; d) RCOCH2COOEt (2 equiv), 1M
NaOMe in dry MeOH (3 equiv), dry MeOH, rt.
12

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This method was also extended to the preparation of further 2-C-glycopyranosyl-pyrimidin-
4(3H)-ones (Table 2). Starting from various O-peracylated glycosyl cyanides 15,39 16,40 17,41
and 1836, 42-44 the one-pot three-step procedures were smoothly accomplished to get the
corresponding C-glycopyranosyl heterocycles 19-22 in moderate to excellent yields.
Table 2. Synthesis of 6-methyl-2-C-glycopyranosyl-
pyrimidin-4(3H)-ones
a) 1M NaOMe in dry MeOH (20 mol %), dry MeOH, dry
CHCl3, rt; b) NH4Cl (1.2 equiv), dry MeOH; c)
CH3COCH2COOEt (2 equiv), 1M NaOMe in dry MeOH
(3 equiv), dry MeOH, rt.
Gly
Yield (%)
Starting material
Product
70
15
19
27
43
94
16
17
18
20
21
22
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The unprotected pyrimidines were assayed as inhibitors of some glycoenzymes. CDoOmI: 1p0o.1u03n9d/Cs8NJ04035D
4, 6, and 10 had no inhibitory effect against rabbit muscle glycogen phosphorylase b up to
625 μM concentration. Pyrimidines 4, 10, 19 and 20 did not inhibit almond β-glucosidase in a
low mM concentration range, while weak inhibitions of yeast α-glucosidase and bovine liver
β-galactosidase by the same molecules were observed (Table 3). Among the tested molecules,
4d and 10d proved to be the most potent ones, displaying submillimolar inhibitory activities
(IC50 = 0.7 and 0.34 mM, respectively) against the indicated glycosidase enzymes.
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Table 3. Inhibitory effects of 2-C-(β-D-glycopyranosyl)-pyrimidines against glycosidase
enzymes
Code Chemical structure
Inhibition
α-glucosidase
β-galactosidase
(yeast)
(bovine liver)
27 % at 2.1 mM
IC50 = 0.7 mM
33 % at 3.1 mM
30 % at 1.6 mM
90 % at 5.7 mM
54 % at 6.8 mM
10 % at 1.3 mM
no inh. at 2.1 mM
56 % at 3.2 mM
45 % at 3.1 mM
20 % at 1.6 mM
56 % at 5.7 mM
IC50 = 0.34 mM
no inh. at 1.3 mM
no inh. at 0.78 mM
4a
4d
10a
10b
10c
10d
19
14 % at 0.78 mM
20
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Although the inhibitory potency of the newly synthesized componds was not outstanding
against the studied enzymes, the observed activities demonstrate the interaction of 2-C-(β-D-
glucopyranosyl)pyrimidines with proteins, notably with glycosidase enzymes, whose
analogues play important roles in human diseases such as lysosomal disorders, diabetes, and
several neurological disorders.45
Conclusion
This work represents the first systematic study on the synthesis of 2-C-(β-D-
glucopyranosyl)pyrimidines. The reactions of O-perbenzylated C-(β-D-
glucopyranosyl)formamidines with β-ketoesters, dimethyl malonate, and β-diketone derived
α,β-unsaturated β-chloroketones gave the expected pyrimidines in good yields. Removal of
the O-benzyl protecting groups under catalytic hydrogenation in neutral media gave the
unprotected 2-C-glucosyl-4-substituted-6-hydroxy-pyrimidines and 2-C-glucosyl-4,6-
dihydroxy-pyrimidine in their respective keto tautomeric forms as well as some 2-C-glucosyl-
4,6-disubstituted-pyrimidines in good to excellent yields. Under similar hydrogenolytic
conditions 2-C-glucosyl-4-phenyl-6-substituted-pyrimidines gave mixtures of two 4,6-cis-
disubstituted diastereomers of the corresponding 1,4,5,6-tetrahydropyrimidines. Unprotected
C-(β-D-glucopyranosyl)formamidine proved also a suitable substrate to give good to high
yields of the aforementioned pyrimidines. Preparation of other 2-C-(β-D-glycopyranosyl)-
pyrimidines was also achieved from O-peracylated D-glycopyranosyl cyanides in a one-pot
three-step continuous operation to give the expected products in 25-94 % overall yields. The
compounds showed no activity against glycogen phosphorylase, however, weak inhibition of
some glycosidase enzymes was observed. By this work a new compound class has been made
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available for biological investigations. Further studies for the synthesis of other tyDpOeIs: 1o0.f10C39-/C8NJ04035D
glycopyranosyl pyrimidines as well as their biological assays are in progress in our
laboratory.
Experimental
Syntheses
General Methods
Melting points were measured on a Kofler hot-stage and are uncorrected. Optical rotations were
determined with a Jasco P-2000 polarimeter at rt. NMR spectra were recorded with Bruker 360
1
1
(360/90 MHz for H/13C) or Bruker 400 (400/100 MHz for H/13C) spectrometers. Chemical
shifts are referenced to the internal TMS (1H), or to the residual solvent signals (13C). Proton-
signal assigments for compounds 3, 5, 7 are based on COSY correlations. Microanalyses were
performed on an Elementar Vario Micro cube instrument. Mass spectra were obtained by
Thermo Scientific LTQ XL or 284 MicroTOF-Q type Qq-TOF MS (Bruker Daltonik, Bremen,
285 Germany) instruments. TLC was performed on DC-Alurolle Kieselgel 60 F254 (Merck),
and the plates were visualised under UV light and by gentle heating (generally no spray reagent
was used but, if more intense charring was necessary, the plate was sprayed with the following
solution: abs. EtOH (95 mL), ccH2SO4 (5 mL) anisaldehyde (1 mL)). For column
chromatography Kieselgel 60 (Merck, particle size 0.063-0.200 mm) was used. EtOAc, CHCl3
and CH2Cl2 were distilled from P4O10 and stored over 4 Å molecular sieves. MeOH was purified
by distillation after refluxing for a couple of hours with magnesium turnings and iodine. EtOH
and DMF were purchased from Sigma-Aldrich. 1M solution of NaOMe in anhydrous MeOH
and NaOEt in anhydrous EtOH was freshly prepared before using. Organic solutions were dried
over anhydrous MgSO4 and concentrated under diminished pressure at 40-60 ºC (water bath).
C-(2,3,4,6-Tetra-O-benzyl-β-D-glucopyranosyl)formamidine hydrochloride15, 16 (1) and O-
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33
39
40
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peracylated glycosyl cyanides (11, 15, 16, 17, 18 ) were synthesized basedDoOnI: 1l0it.1e0r3a9/tCu8rNeJ04035D
procedures. The α,β-unsaturated β-chloroketones were obtained by chlorination of the
corresponding
1,3-diketones
(pentane-2,4-dione/PCl5,46
1,1,1-trifluoropentane-2,4-
dione/(COCl)2,10 1-phenylbutane-1,3-dione/(COCl)2,47 4,4,4-trifluoro-1-phenylbutane-1,3-
10
dione/SOCl2 ) according to the cited protocols.
General procedure I for the synthesis of 2-(β-D-glucopyranosyl)-pyrimidin-4(3H)-ones (3,
4) by cyclocondensation of C-(β-D-glucopyranosyl)formamidines (1, 2)
The corresponding C-(β-D-glucopyranosyl)formamidine hydrochloride (115, 16 or 2) was
dissolved in anhydrous MeOH (2 mL/100 mg amidine), and a 1M solution of NaOMe in MeOH
(3 equiv) was added. After 10 min, the appropriate 3-ketoester (2 equiv) was added to the
reaction mixture and the stirring was continued at rt until the TLC showed complete conversion
of the starting material (9 : 1 CHCl3-MeOH and 1 : 1 hexane-EtOAc for compounds 1, 3, and
7 : 3 CHCl3-MeOH for compounds 2, 4, respectively). The reaction mixture was then
neutralized with glacial AcOH, and concentrated under diminished pressure. The crude product
was purified by column chromatography.
General procedure II for the removal of O-benzyl protecting groups by catalytic
hydrogenation under neutral conditions to get compounds 4, 6, 10
To a solution of the corresponding O-perbenzylated C-(β-D-glucopyranosyl)pyrimidine (3, 5 or
8) in a mixture of anhydrous EtOAc (2 mL/100 mg substrate) and EtOH (4 mL/100 mg
substrate) 20 % Pd(OH)2/C (50 weight % of substrate) was added. The reaction mixture was
then vigorously stirred at reflux temperature under H2 atmosphere. After completion of the
reaction monitored by TLC (1 : 1 hexane-EtOAc and 3 : 1 EtOAc-MeOH), the hot mixture was
filtered through a pad of celite, and washed thoroughly with MeOH. The solvent was then
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evaporated under reduced pressure and the crude product was purified by column
DOI: 10.1039/C8NJ04035D
chromatography.
General procedure III for the reaction of C-(2,3,4,6-tetra-O-benzyl-β-D-
glucopyranosyl)formamidine (1) with malononitrile and ethyl cyanoacetate to get
compounds 7a,b
To a solution of C-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)formamidine hydrochloride15,
16 (1, 200 mg, 0.33 mmol) in anhydrous EtOH (5 mL) a 1M solution of NaOEt in EtOH (2 or
10 equiv, depending on the quantity of the reagent, see below) was added. After stirring the
reaction mixture for 15 min at rt malononitrile (2 equiv) or ethyl cyanoacetate (10 equiv) was
added. The stirring was continued at rt until the TLC (9 : 1 CHCl3-MeOH, 1 : 1 hexane-EtOAc)
indicated complete conversion of the starting material. The reaction mixture was then
neutralized with glacial AcOH, and evaporated under diminished pressure. The resulting crude
product was purified by column chromatography.
General procedure IV for the synthesis of 4,6-disubstituted-2-(β-D-glucopyranosyl)-
pyrimidines (8, 10) by cyclocondensation of C-(β-D-glucopyranosyl)formamidines (1, 2)
The corresponding C-(β-D-glucopyranosyl)formamidine hydrochloride (115, 16 or 2) and K2CO3
(4 equiv) in the presence of activated molecular sieves (4 Å) were suspended in anhydrous DMF
(3 mL/100 mg substrate). This mixture was then cooled to 0 °C and the freshly prepared α,β-
unsaturated β-chloroketone (1.2 equiv) obtained from the corresponding 1,3-diketone was
added. The reaction mixture was allowed to warm up to rt and the stirring was continued. After
completion of the reaction (~ 2 d), monitored by TLC (9 : 1 CHCl3-MeOH and 1 :1 hexane-
EtOAc for compounds 1, 8, and 7 : 3 CHCl3-MeOH for compounds 2, 10, respectively), the
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inorganic precipitates were filtered off, washed with MeOH, and the solvent was evaporated
DOI: 10.1039/C8NJ04035D
under reduced pressure. The crude product was purified by column chromatography.
General procedure V for the removal of O-benzyl protecting groups by catalytic
hydrogenation under acidic conditions to get compounds 9c,d
A degassed, vigorously stirred suspension of 20 % Pd(OH)2/C (50 weight % of substrate) in a
mixture of anhydrous EtOAc (2 mL/100 mg substrate) and EtOH (5 mL/100 mg substrate) was
saturated with H2. To this mixture a solution of the corresponding O-perbenzylated C-β-D-
glucopyranosyl derivative in anhydrous EtOAc (3 mL/100 mg substrate), and a drop of ccHCl
were added. The reaction mixture was then stirred under H2 atmosphere at rt overnight. The
completion of the reaction was judged by TLC (1 :1 hexane-EtOAc and 7 : 3 CHCl3-MeOH)
and the mixture was neutralized by the addition of NaHCO3. The catalyst and the inorganic
salts were filtered off through a pad of celite, and washed with MeOH. The solvent was removed
under diminished pressure, and the resulting crude product was purified by column
chromatography.
General procedure VI for the synthesis of 2-glycosyl-6-methylpyrimidin-4(3H)-ones (19-
22) from glycosyl cyanides (15-18) by a one-pot three-step procedure
To a stirred solution of the corresponding cyanide (15-18) in a mixture of anhydrous CHCl3 (2
mL / 1 g substrate) and MeOH (15 mL / 1 g substrate) a 1M solution of NaOMe in MeOH (20
mol %) was added at rt. After conversion of the starting material into the unprotected methyl
C-glycopyranosyl formimidate (1 day) NH4Cl (1.2 equiv) was added to the reaction mixture
and the stirring was continued at rt. When TLC (7 : 3 CHCl3-MeOH) indicated complete
conversion of formimidate into the appropriate formamidine derivative (1 day) ethyl
acetoacetate (2 equiv) and a 1M solution of NaOMe in MeOH (3 equiv) were added to the
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stirred reaction mixture. After 8 h the solution was neutralized with glacial aceticDaOcI:id10.a10n3d9/Ct8hNeJ04035D
solvents were removed under reduced pressure. The residual crude product was purified either
by column chromatography or by crystallization.
Enzyme assays
Glycogen phoshorylase b was obtained from rabbit skeletal muscle by some modifications
(application of 2-mercaptoethanol instead of L-cysteine, and recrystallization at least three
times before use) of the purification protocol developed by Fischer and Krebs.48 The kinetic
measurements were carried out in the direction of glycogen synthesis as described earlier49 with
maximal inhibitor concentrations of 625 µM.
The glycosidase enzymes used were purchased from Sigma-Aldrich.
In the glycosidase assays typically a 10 µL aliquot of each inhibitor’s stock solution was mixed
with 370 µL of the buffer and 20 µL enzyme stock solution in a plastic UV cuvette. After
equilibration at 37 °C for 5 min, a 100 µL aliquot of the substrate stock solution was added.
The resulting solutions were thoroughly mixed, and the change in absorbance was followed at
400 nm over 24s in 2s intervals using the Parallel Kinetics Analysis program of a JASCO V550
(JASCO Tokyo, Japan) spectrophotometer. Progress curves were plotted and fitted to a straight
line. ΔA/min values, proportional to initial rate, were considered to be enzyme activities. In a
control experiment, the aliquot of the inhibitor solution was replaced by the same amount of
buffer. The initial rate data for the enzymatic substrate hydrolysis in presence and absence of
inhibitor were transferred into percentages of overall inhibition, and plotted against the inhibitor
concentration in logarithmic scale for IC50 determination.
Further details of the experimental procedures and compound characterization can be found in
the supporting material.
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DOI: 10.1039/C8NJ04035D
Conflicts of interest
There are no conflicts of interest to declare.
Acknowledgement
This work was supported by the National Research, Development and Innovation Office of
Hungary (Grant OTKA FK 125067), by the EU co-financed by the European Regional
Development Fund under the projects GINOP-2.3.2-15-2016-00008 and GINOP-2.3.3-15-
2016-00004, as well as by the Alexander von Humboldt Foundation (Germany).
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DOI: 10.1039/C8NJ04035D
NH . HCl
NH2
BnO
BnO
59-92 %
for two steps
R1
N
O
OBn
R2
R3
The first systematic study on the synthesis
N
of 2-C-( -D-glycopyranosyl)pyrimidines
either from amidine A or glycosyl cyanides
B and 1,3-dicarbonyl compounds.
OBn
A
O
(HO)n
CN
O
25-94 %
for three steps in
(AcylO)n
one continuous operation
B