Synthesis of N-(β-d-glucopyranosyl) monoamides of dicarboxylic acids as potential inhibitors of glycogen phosphorylase

Article abstract of DOI:10.1016/j.carres.2006.03.002

O-Peracetylated N-(β-d-glucopyranosyl)imino trimethylphosphorane obtained in situ from 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl azide and PMe₃ was reacted with saturated and unsaturated aliphatic and aromatic dicarboxylic acids, or their anhyd

Full text of DOI:10.1016/j.carres.2006.03.002

Carbohydrate Research 341 (2006) 947–956
Synthesis of N-(b-D-glucopyranosyl) monoamides
of dicarboxylic acids as potential inhibitors
of glycogen phosphorylase
a
a
b
c
d
´
´
Katalin Czifrak, Zsuzsa Hadady, Tibor Docsa, Pal Gergely, Jurgen Schmidt,
¨
d
a,
*
´
´
´
Ludger Wessjohann and Laszlo Somsak
aDepartment of Organic Chemistry, Faculty of Science, University of Debrecen, PO Box 20, H-4010 Debrecen, Hungary
bSignalling and Apoptosis Research Group of the Hungarian, Academy of Sciences, Research Center for Molecular Medicine,
University of Debrecen, H-4012 Debrecen, Nagyerdei Rrt. 98, Hungary
cDepartment of Medical Chemistry, Medical and Health Science Centre, University of Debrecen, POB 7, H-4012 Debrecen, Hungary
dDepartment of Bioorganic Chemistry, Leibniz Institute of Plant Biochemistry, Weinberg 3, D-06120 Halle (Saale), Germany
Received 6 January 2006; received in revised form 27 February 2006; accepted 2 March 2006
Available online 29 March 2006
z
´
´
Dedicated to the memory of Professor Dr. Zoltan Gyo¨rgydeak
Abstract—O-Peracetylated N-(b-D-glucopyranosyl)imino trimethylphosphorane obtained in situ from 2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl azide and PMe3 was reacted with saturated and unsaturated aliphatic and aromatic dicarboxylic acids, or their
anhydrides, or monoesters to give the corresponding N-(b-D-glucopyranosyl) monoamides of dicarboxylic acids or derivatives.
´
The acetyl protecting groups were removed according to the Zemplen protocol to give a series of compounds which showed mod-
erate inhibitory effects against rabbit muscle glycogen phosphorylase b. The best inhibitor was 3-(N-b-D-glucopyranosyl-carb-
amoyl)propanoic acid (7) with Ki = 20 lM.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: N-Glycosylamides; Glucose analogues; Inhibitors; Glycogen phosphorylase
1. Introduction
inhibition of GP2 as a new concept of a possible basis
of a novel treatment3 for type 2 diabetes4,5 has emerged
and intensively been studied both in academia and
industry.6 One of the most populated class of inhibitors
of GP is represented by derivatives of D-glucose
Many efforts devoted to develop carbohydrate based
therapeutics aim at finding inhibitors of glycoprocessing
enzymes and discovering their structure–activity rela-
tionships (SAR). Special types of glycoenzymes are the
glycogen phosphorylases1 (GP), which transfer a glucose
unit from the nonreducing end of the storage polysac-
charide glycogen to an inorganic phosphate. This is a
most important event in glycogen metabolism, and the
liver GP, as part of a complex regulatory system is
directly responsible for the regulation of hepatic glucose
output and thereby of blood sugar levels. Therefore,
,7–11
(Chart 1).
N-(b-D-glucopyranosyl)amides A and B are among
the first efficient glucose analogue inhibitors.12 An
important structural feature in binding the amides to
the catalytic site of GP is a strong H-bond between
the amide NH and the main chain carbonyl of
His377.2,13 A recent study on a series of various
N-glucopyranosyl amides14 has revealed that a properly
*
Corresponding author. Tel.: +36 52 512 900/22348; fax: +36 52 453
836; e-mail: somsak@tigris.unideb.hu
For other inhibitors of GP the reader is kindly referred to recent
review articles2,7,8 and the literature quoted there.
z Passed away on October 20th, 2005 at the age of 63 years.
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.002

948
K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
the preparation of N-glycosyl monoamides of some
OH
dicarboxylic acids: anhydrides of phthalic, succinic18
and maleic acid19,20 were used for the acylation of vari-
ously protected b-D-glucopyranosylamines; b-D-galacto-
pyranosylamine was acylated by a mixed anhydride
obtained from monomethyl succinate.21 O-Peracetylated
b-D-galactopyranosyl isothiocyanates were also trans-
formed to N-galactosylated monoamides of C3–C6 and
C9 a,x-dicarboxylic acids.22
O
HO
HO
NH
R
HO
O
C R =
A R = CH3 Ki = 32 μM
B R = C6H5 Ki = 81 μM
Ki = 3.5 μM
OH
O
HO
NH
HO
NH
R
A more advantageous method to produce N-glycosyl
amides uses glycosyl azides23 following the Staudinger
protocol,24 that is, reaction of an N-glycosyl iminophos-
phorane with activated carboxylic acid derivatives. An
elegant modification of this method has recently been
elaborated introducing the use of PMe3 that results in
enhanced nucleophilicity of the intermediate phospho-
rane16 allowing carboxylic acids to be applied as the
acylating agents. Easier work-up of the reaction mix-
tures due to the volatile nature of the by-product
P(@O)Me3 represents another advantage. Following
the successful synthesis of several new N-glycosyl amides
of monocarboxylic acids as well as a-amino acid deriva-
tives14 by this procedure we have now extended the
method to dicarboxylic acids and their derivatives. It
is to be noted that a similar reaction with phthalic anhy-
dride resulted in several N-substituted phthalimides
from diverse azido sugars excluding glycosyl azides.25,26
Formation of the intermediate N-glucosyl iminophos-
phorane was effected in each case at room temperature
by reacting 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl
azide27 (3) dissolved in CH2Cl2 with a 1 M toluene or
THF solution of PMe3. Treatment of the iminophos-
phorane with glutaric or adipic acid (Scheme 1) gave
the corresponding N-glucopyranosyl monoamides 1
and 2, and subsequent removal of the protecting groups
HO
O
O
F R =
D R = CH3 Ki = 370 μM
E R = C6H5 Ki = 4.6 μM
Ki = 0.4 μM
OH
O
HO
NH
COOH(Me)
G
linker
HO
HO
O
Chart 1. Inhibitors of glycogen phosphorylase.
positioned and large enough hydrophobic group
attached to the amide moiety as in C makes the inhibi-
tion one order of magnitude stronger than that of A.
On the other hand, this compound is still much less
efficient than the best known glucose analogue inhibitor
of GP N-(b-D-glucopyranosyl)-N0-(2-naphthoyl) urea
F.7,8 Other compounds of the urea type15 (D, E) show
weaker activity than that of F. Binding of the aglycon
of these compounds takes place in the so called b-
channel of the enzyme. This channel of mixed character
is surrounded by both polar and apolar amino acid side
chains.2 Therefore, carefully designed inhibitors need
proper arrangements of groups capable of interacting
with the residues in the b-channel.
´
using the Zemplen method afforded 9 and 10, respec-
In this paper, we wish to map the b-channel with a
series of N-(b-D-glucopyranosyl) monoamides of dicarb-
oxylic acids (G). Such compounds offer the possibility
to place a strongly polar group (COOH) at different dis-
tances from the sugar moiety while the ability to form
the important H-bond from the amide can be main-
tained. The carbon skeleton (linker) of the diacid may
also contribute to the binding and, furthermore, simple
esterification (to COOMe) can modify the character of
the polar group.
tively. Reaction of the iminophosphorane with succinic
or glutaric anhydride gave N-glucopyranosyl succini-
mide§ 4 or -glutarimide 5, respectively. This contrasts
with the reported formation of N-glycosyl monoamides
of dicarboxylic acids in the reaction of glycosylamines
with succinic anhydride18 and in similar reactions men-
tioned in the first paragraph. During removal of the ace-
tyl protecting groups by NaOMe/MeOH from 4 and 5
the cyclic imide moiety was opened to give methylester
amides 6 and 8, respectively. Free acid 7 was obtained
from 6 by NaOH in MeOH followed by acidification
with Amberlyst 15 (H+).
2. Results and discussion
2.1. Synthesis
§ Compound 4 was prepared earlier by reacting succinimide (as the Ag
salt) with 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide.28 The
benzoylated analogue of 4 was reported to form in a reaction of the
corresponding ethyl 1-thioglucopyranoside with NIS–TfOH.29
Diverse other N-glucopyranosyl succinimide derivatives were
obtained by acylation of D-glucopyranosylamines with succinic
anhydride followed by cyclization.30
N-Glycosyl amidesà are frequently prepared by acyla-
tion of glycosylamines.17 This method was applied for
à For a critical survey of recent literature see Ref. 16.

K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
949
OAc
O
1. Me3P
AcO
AcO
NaOMe
MeOH
(CH2)n CO2H
NH
2. HO2C-(CH2)n-CO2H
OAc
O
1 n = 3 (92 %)
2 n = 4 (73 %)
O
OAc
O
OAc
O
(CH2)n
OH
O
AcO
1. Me3P
AcO
AcO
N
HO
HO
N3
NaOMe
MeOH
(CH2)n CO2R
NH
AcO
OH
OAc
O
OAc
O
O
4 n = 2 (41 %)
5 n = 3 (43 %)
3
6 n = 2, R = CH3 (80 % from 4)
7 n = 2, R = H (61 %)
O
(CH2)n
2.
NaOH, MeOH
8 n = 3, R = CH3 (75 % from 5)
9 n = 3, R = H (88 % from 1)
10 n = 4, R = H (99 % from 2)
O
1. Me3P
2. HO2C [CC] CO2R
OAc
O
OH
O
AcO
AcO
HO
HO
[CC] CO2R
[CC] CO2R
NH
NaOMe
MeOH
NH
OAc
OH
O
O
[E]
[E]
11 [CC] = HC=CH, R = C2H5 (79 %)
14 [CC] = HC=CH, R = CH3 (86 %)
[E]
[E]
15 [CC] = HC=CH, R = H (27 %)
12 [CC] = HC=CH, R = H (54 %)
13 [CC] = C=C, R = H, (35%)
1. Me3P
O
O
2.
OAc
O
OH
O
NH
NH
AcO
AcO
HO
HO
O
NaOMe
MeOH
OAc
OH
O
CO2H
O
CO2H
17 (96 %)
16 (51 %)
1. Me3P
2.
CO2H
OAc
O
OH
O
m, p
CO2CH3
NH
NH
AcO
AcO
HO
HO
NaOMe
MeOH
m, p
CO2R
m, p
CO2CH3
OAc
OH
O
O
18 m (92 %)
19 p (88 %)
20 m R = CH3 (77 %)
21 m R = H (99 %)
22 p R = CH3 (91 %)
23 p R = H (84 %)
NaOH, MeOH
NaOH, MeOH
Scheme 1.
To get similar derivatives of unsaturated aliphatic
diacids, reactions of the iminophosphorane with fumaric
acid and butyndioic acid were investigated first. In both
cases, the transformation was rather sluggish and gave
complex mixtures from which the expected acids 12
and 13, respectively, were isolated in moderate yields.
On the other hand, in a reaction with monoethyl fuma-
rate, ethylester amide 11 was formed in good yield.
which no discrete product could be isolated. These
observations with the unsaturated acids and derivatives
might be due to the sensitivity of the electron poor dou-
ble bonds toward nucleophilic attacks, and are in accord
with the reported unstability of O-peracetylated N-
glucopyranosyl maleimide.–,33
Possibilities to obtain derivatives of aromatic dicarb-
oxylic acids (phthalic-, isophthalic-, or terephthalic
´
Deprotection according to the Zemplen method gave
methylester amide 14 from 11, free acid 15 from 12,
while in the case of 13 decomposition took place. With
maleic anhydride, complex mixtures were formed from
– For the preparation of other N-glucopyranosyl maleimide deriva-
tives, see as leading references: 19,20,31,32.

950
K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
Table 1. Inhibitory effects of N-(b-D-glucopyranosyl) monoamides of dicarboxylic acid derivatives tested with rabbit muscle GPb
OH
O
HO
NH
linker COOR
Ki (lM)
IC50 (mM)
HO
HO
O
Compound
–Linker–
R
6
7
–CH2CH2–
–CH2CH2–
CH3
H
170
20
8
–CH2CH2CH2–
–CH2CH2CH2–
–CH2CH2CH2CH2–
CH3
H
H
83
9
10
No effect in 625 lM
7.9
14
15
CH3
H
1
No effect in 625 lM
No effect in 625 lM
17
20
H
CH3
580
329
21
H
4
22
23
CH3
H
No effect in 625 lM
acid) were investigated next. Reaction of either isomer
of the diacids with the iminophosphorane resulted in
decomposition. Phthalic anhydride furnished acid 16
in acceptable yield what is contrary to the formation
of N-glycosyl imides 4 and 5 with the corresponding ali-
phatic anhydrides.k This, we speculate, might be due to
stereoelectronic reasons: attack of the iminophospho-
rane nucleophile may open the anhydro ring in each
case; this can be followed by a ring closure to give the
cyclic imide by an intramolecular nucleophilic attack
of the nitrogen atom on the other carbonyl group; while
with the flexible aliphatic derivatives this can occur
were converted to acids 21 and 23, respectively, by
NaOH in MeOH followed by treatment with Amberlyst
15 (H+) resin.
Stuctural elucidation of the new compounds was
straightforward by NMR spectroscopy and needs no
special comment. For assignments of the proton and
carbon spectra, see Section 4.
2.2. Enzyme tests and evaluation of the inhibitors
Enzyme tests were carried out as described earlier37 (see
also Section 4). Compound 7 has proven the best inhib-
itor of the investigated series (Table 1) suggesting that a
polar group with an ability to act as both hydrogen
bond donor and acceptor (compare 6 and 7) at three
bond distance (compare 7, 9 and 10) from the amide
moiety is beneficial for the strong binding of a glucose
analogue. The flexibility of the linker seems also impor-
tant since a less mobile carbon chain as in 15 results in
loss of the activity. Changing the linker to aromatic resi-
dues again makes less efficient inhibitors (17, 20–23).
The role of the flexibility of the carbon chain cannot
be evaluated on the basis of the present data. One can
assess two possibilities: (a) the formation of intramolec-
ular hydrogen bonds either with the amide NH or C@O
to form ring shaped structures attached to the sugar
along the favorable Burgi–Dunitz trajectory,36 in the
¨
conjugated and therefore planar aromatic derivative this
stereoelectronic condition could only be met with signi-
ficant loss of conjugative stabilization which cannot be
provided energetically under the mild reaction condi-
tions. Acylation of the iminophosphorane by mono-
methylesters of iso- and terephthalic acid gave good
yields of methylester amides 18 and 19, respectively.
´
Zemplen deacetylations of 18 and 19 gave the deprotec-
ted methylester amides 20 and 22, respectively, which
k For the preparation of N-glycosyl phthalimide derivatives, see Refs.
28,31,34,35.

K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
951
ring; (b) ‘open chain’ bound structures with more exten-
sive interactions in the b-channel of the enzyme. Such
binding properties can be revealed by protein crystallo-
graphic investigation of the enzyme–inhibitors com-
plexes, and these studies are in progress.
in one portion. The mixture was stirred at rt until nitro-
gen evolution had ceased and TLC (1:1 EtOAc–hexane)
had indicated complete transformation of 3 (approx.
15 min). This soln was then reacted with a dicarboxylic
acid, or its monoester, or anhydride (1.1 equiv) as indi-
cated with the particular compounds till the disappear-
ance of the iminophosphorane (ꢁ2 d, TLC 1:1
EtOAc–hexane). The volatiles were removed under
diminished pressure and the residue purified by column
chromatography.
3. Conclusion
Extension of the modified Staudinger methodology to
the synthesis of N-(b-D-glucopyranosyl) monoamides
of various dicarboxylic acids proved efficient and
resulted in a new series of compounds which were tested
as inhibitors of rabbit muscle glycogen phosphorylase b.
The best inhibitor was compound 7 with a flexible linker
between the amide and the carboxylic acid group.
´
4.3. General procedure II for the Zemplen-deacetylation
To a soln of an acetyl protected compound in dry
MeOH, 1–2 drops of a ꢁ1 M methanolic NaOMe soln
were added, and the reaction mixture was kept at rt until
completion of the transformation (TLC 1:1 CHCl3–
MeOH). Amberlyst 15 (H+ form) was then added to
remove sodium ions, the resin was filtered off, and the
solvent removed under diminished pressure. If the
residue was chromatographically not uniform it was
purified by column chromatography or crystallization.
4. Experimental
4.1. General methods
Melting points were measured in open capillary tubes or
on a Kofler hot-stage and are uncorrected. Optical rota-
tions were determined with a Perkin–Elmer 241 polari-
meter at room temperature. NMR spectra were recorded
4.4. General procedure III for the hydrolysis of methyl-
ester groups
1
with a Bruker 360 (360/90 MHz for H/13C) spectro-
A methylester was suspended in distilled MeOH (2 mL/
mmol), and NaOH (1.5 equiv of a 1 M methanolic soln)
was added. The reaction mixture was stirred at rt or
heated at 60 ꢁC until disappearance of the starting mate-
rial (TLC 1:1 CHCl3–MeOH). Amberlyst 15 (H+ form)
was then added to remove sodium ions, the resin was fil-
tered off, and the solvent removed under diminished
pressure. The crude product was purified by crystalliza-
tion or column chromatography.
meter. Chemical shifts are referenced to Me4Si (1H), or
to the residual solvent signals (13C). TLC was performed
on DC-Alurolle Kieselgel 60 F254 (E. Merck), and the
plates were visualized under UV light and by gentle
heating. For column chromatography, Kieselgel 60 (E.
Merck, particle size 0.063–0.200 mm) was used. Organic
solutions were dried over anhyd MgSO4 and concen-
trated under diminished pressure at 40–50 ꢁC (water
bath). PMe3 (1 M soln in toluene or THF) as well as
dicarboxylic acids and their derivatives were purchased
from Aldrich. The high resolution electrospray (ESI)
mass spectra were obtained from a Bruker Apex III
70e Fourier transform ion cyclotron resonance mass
spectrometer (Bruker Daltonics, Billerica, USA)
equipped with an InfinityTM cell, a 7.0 Tesla supercon-
ducting magnet (Bruker, Karlsruhe, Germany), an RF-
only hexapole ion guide and an external APOLLO elec-
trospray ion source (Agilent, off axis spray). Nitrogen
was used as drying gas at 150 ꢁC. The sample solns were
introduced continuously via a syringe pump with a flow
rate of 120 lL hꢀ1. All data were aquired with 256k data
points and zero filled to 1024k by averaging 32 scans.
4.5. 4-(N-20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl-
carbamoyl)butyric acid (1)
Prepared from 3 (1.5 g, 4.02 mmol) and glutaric acid
(0.58 g, 4.42 mmol) according to General procedure I.
Eluent for the column chromatography: 95:5 CHCl3–
MeOH. Yield: 1.7 g (92%) white crystalline product,
mp: 119–122 ꢁC; [a]D +1 (c 0.58, CHCl3); 1H NMR
(CDCl3): d (ppm) 9.31 (1H, s, COOH), 6.74 (1H, d,
J = 9.3 Hz, NH), 5.33, 5.28, 5.06, 4.95 (4H, 4 pseudo
t, J ꢁ 9.3 Hz in each, H-10, H-20, H-30, H-40), 4.29
(1H, dd, J = 12.4, 3.9 Hz, H-60a), 4.09 (1H, dd,
J = 12.4, 1.5 Hz, H-60b), 3.83 (1H, ddd, J = 9.3, 3.9,
1.5 Hz, H-50), 2.39–2.27 (4H, m, 2 · CH2), 2.08, 2.06,
2.03, 2.02 (12H, 4s, 4 · CH3), 1.96–1.92 (2H, m, CH2);
13C NMR (CDCl3): d (ppm) 177.1 (COOH), 171.0,
(NHCO), 170.6, 169.9, 169.8, 169.6 (CO), 169.6
(NHCO), 77.9 (C-10), 73.4, 72.9, 70.6, 68.1 (C-20 to
C-50), 61.7 (C-60), 34.9, 32.5 (CH2), 20.6, 20.5, 20.4,
20.3 (CH3), 19.9 (CH2); HRMS: m/z 484.14163
4.2. General procedure I for the reaction of 2,3,4,6-tetra-
O-acetyl-b-D-glucopyranosyl azide (3) with trimethyl
phosphane and dicarboxylic acid derivatives
To a soln of 3 in dry CH2Cl2 (5–7 mL/mmol), PMe3
(1.1 equiv of a 1 M toluene or THF soln) was added

952
K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
([M+Na]+, calcd for C19H27NO12Na+ 484.14255). Anal.
Calcd for C19H27NO12 (461.43): C, 49.46; H, 5.90; N,
3.04. Found: C, 49.34; H, 5.86; N, 3.12.
4.15 (1H, dd, J = 12.5, 2.3 Hz, H-60b), 3.77 (1H, ddd,
J = 9.4, 4.7, 2.3 Hz, H-50), 2.66–2.62 (6H, m, 3 · CH2),
2.08, 2.05, 2.04, 1.99 (12H, 4s, 4 · CH3); 13C NMR
(CDCl3): d (ppm) 172.1, 170.5, 170.4, 169.8, 169.6,
169.2 (CO), 78.7 (C-10), 74.5, 73.4, 68.8, 67.9 (C-20 to
C-50), 61.8 (C-60), 33.9, 32.9 (CH2) 20.8, 20.7,
20.6, 20.5 (CH3), 16.8 (CH2); ESIMS: m/z 466
([M+Na]+). Anal. Calcd for C19H25NO11 (443.41): C,
51.47; H, 5.68; N, 3.16. Found: C, 51.22; H, 5.26; N,
3.10.
4.6. 5-(N-20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl-
carbamoyl)pentanoic acid (2)
Prepared from 3 (0.5 g, 1.34 mmol) and adipic acid
(0.42 g, 1.46 mmol) according to General procedure I.
Eluent for the column chromatography: 9:1 CHCl3–
MeOH. Yield: 0.46 g (73%) colourless oil, Rf = 0.52
1
(EtOAc); [a]D +23 (c 0.40, CHCl3); H NMR (CDCl3):
4.9. Methyl 3-(N-b-D-glucopyranosyl-carbamoyl)-
propanoate (6)
d (ppm) 6.51 (1H, d, J = 9.5 Hz, NH), 5.28, 5.24, 5.03,
4.88 (4H, 4 pseudo t, J ꢁ 9.5 Hz in each, H-10, H-20,
H-30, H-40), 4.27 (1H, dd, J = 12.6, 4.4 Hz, H-60a),
4.05 (1H, dd, J = 12.6, 2.0 Hz, H-60b), 3.81 (1H, ddd,
J = 9.5, 4.4, 2.0 Hz, H-50), 2.33–2.20 (4H, m, 2 · CH2),
2.05, 2.02, 2.01, 1.99 (12H, 4s, 4 · CH3), 1.62–1.60
(4H, m, 2 · CH2); 13C NMR (CDCl3): d (ppm) 177.8,
172.3 (COOH, NHCO), 170.8, 170.5, 169.7, 169.4
(CO), 77.9 (C-10), 73.4, 72.7, 70.6, 68.1 (C-20 to C-50),
61.7 (C-60), 36.0, 33.5, 24.4, 24.0 (CH2), 20.8, 20.7,
20.6 (2) (CH3); ESIMS: m/z 498 ([M+Na]+).
Prepared from 4 (0.13 g, 0.30 mmol) according to Gen-
eral procedure II. Eluent for the column chromatogra-
phy: 7:3 CHCl3–MeOH. Yield: 0.07 g (80%) white
crystalline product, mp: 160–163 ꢁC; [a]D ꢀ19 (c 0.41,
H2O); 1H NMR (D2O):
d (ppm) 4.95 (1H, d,
J = 9.3 Hz, H-10), 3.87 (1H, dd, J = 12.4, 2.2 Hz, H-
60a), 3.71 (1H, dd, J = 12.4, 5.3 Hz, H-60b), 3.70 (3H,
s, OCH3), 3.54 (1H, pseudo t, J = 9.3, 9.1 Hz, H-20),
3.50 (1H, ddd, J = 9.3, 5.3, 2.2 Hz, H-50), 3.44–3.38
(2H, m, H-30, H-40), 2.71–2.62 (4H, m, 2 · CH2); 13C
NMR (D2O): d (ppm) 176.8, 176.4 (COOCH3, NHCO),
80.2 (C-10), 78.5, 77.5, 72.7, 70.2 (C-20 to C-50), 61.5
(C-60), 53.2 (OCH3) 31.2, 29.9 (CH2); ESIMS: m/z 316
([M+Na]+). Anal. Calcd for C11H19NO8 (293.28): C,
45.05; H, 6.53; N, 4.78. Found: C, 45.14; H, 6.66; N,
4.52.
4.7. N-(20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl)-
succinimide (4)
Prepared from 3 (0.8 g, 2.14 mmol) and succinic anhy-
dride (0.23 g, 2.30 mmol) according to General proce-
dure I. Eluent for the column chromatography: 3:1
EtOAc–hexane. Yield: 0.28 g (41%) yellow crystalline
product, mp: 203–204 ꢁC; [a]D +10 (c 0.43, CHCl3);
1H NMR (CDCl3): d (ppm) 5.89, 5.28, 5.20 (3H, 3
pseudo t, J ꢁ 10.0 Hz in each, H-20, H-30, H-40), 5.26
(1H, d, J = 10.0 Hz, H-10), 4.19–4.16 (2H, m, H-60a,
H-60b), 3.79 (1H, ddd, J = 10.0, 7.0, 3.5 Hz, H-50),
2.70–2.68 (4H, m, 2 · CH2), 2.13, 2.08, 2.05, 1.97
(12H, 4s, 4 · CH3); 13C NMR (CDCl3): d (ppm)
175.1 (2), 170.4, 169.8, 169.3, 169.1 (CO), 78.2
(C-10), 74.6, 73.2, 67.9, 67.8 (C-20 to C-50), 61.6 (C-60),
27.9 (2) (CH2), 20.8, 20.7, 20.6, 20.5 (CH3); ESIMS:
m/z 452 ([M+Na]+). Anal. Calcd for C18H23NO11
(429.38): C, 50.35; H, 5.40; N, 3.26. Found: C, 49.98;
H, 5.26; N, 3.32.
4.10. 3-(N-b-D-Glucopyranosyl-carbamoyl)propanoic acid
(7)
Prepared from 6 (0.05 g, 0.17 mmol) according to
General procedure III. Eluent for the column chromato-
graphy: 1:1 CHCl3–MeOH. Yield: 0.03 g (61%) colour-
less oil, Rf = 0.21 (1:1 CHCl3–MeOH); [a]D +2 (c 0.38,
MeOH); 1H NMR (D2O): d (ppm) 4.96 (1H, d,
J = 9.1 Hz, H-10), 3.87 (1H, dd, J = 12.4, 2.2 Hz, H-
60a), 3.72 (1H, dd, J = 12.4, 5.1 Hz, H-60b), 3.55 (1H,
pseudo t, J = 9.1, 8.9 Hz, H-20), 3.52 (1H, ddd,
J = 9.1, 5.1, 2.2 Hz, H-50), 3.45–3.36 (2H, m, H-30, H-
40), 2.60–2.56 (4H, m, 2 · CH2); 13C NMR (D2O): d
(ppm) 179.6 (COOH), 177.4 (NHCO), 80.2 (C-10),
78.5, 77.4, 72.8, 70.2 (C-20 to C-50), 61.5 (C-60), 31.5,
32.1 (CH2); ESIMS: m/z 302 ([M+Na]+).
4.8. N-(20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl)-
glutarimide (5)
Prepared from 3 (0.8 g, 2.14 mmol) and glutaric anhy-
dride (0.27 g, 2.37 mmol) according to General proce-
dure I. Eluent for the column chromatography: 3:1
EtOAc–hexane. Yield: 0.41 g (43%) white crystalline
4.11. Methyl 4-(N-b-D-glucopyranosyl-carbamoyl)-
butyrate (8)
Prepared from 5 (0.10 g, 0.22 mmol) according to
General procedure II. Eluent for the column chromato-
graphy 7:3 CHCl3–MeOH. Yield: 0.05 g (75%) colour-
less oil, Rf = 0.67 (7:3 CHCl3–MeOH); [a]D ꢀ8 (c 0.33,
H2O); 1H NMR (D2O): d (ppm) 4.96 (1H, d, J =
1
product, mp: 129–132 ꢁC; [a]D +14 (c 0.41, CHCl3); H
NMR (CDCl3): d (ppm) 5.83 (1H, d, J = 9.4 Hz, H-
10), 5.90, 5.28, 5.20 (3H, 3 pseudo t, J ꢁ 9.4 Hz in each,
H-20, H-30, H-40), 4.21 (1H, dd, J = 12.5, 4.7 Hz, H-60a),

K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
953
8.9 Hz, H-10), 3.89 (1H, dd, J = 12.4, 1.8 Hz, H-60a),
3.72 (1H, dd, J = 12.4, 4.9 Hz, H-60b), 3.71 (3H, s,
OCH3), 3.56 (1H, pseudo t, J = 9.1, 8.9 Hz, H-20),
3.52 (1H, ddd, J = 9.1, 4.9, 1.8 Hz, H-50), 3.42–3.38
(2H, m, H-30, H-40), 2.46–1.93 (6H, m, 3 · CH2); 13C
NMR (D2O): d (ppm) 178.0, 177.1 (COOCH3, NHCO),
80.2 (C-10), 78.5, 77.5, 72.7, 70.2 (C-20 to C-50), 61.5 (C-
60), 53.2 (OCH3) 35.7, 33.8, 21.3 (CH2); HRMS: m/z
330.11593 ([M+Na]+, calcd for C12H21NO8Na+
330.11594).
(1H, d, J = 15.8 Hz, CH), 5.39, 5.32, 5.06, 4.98 (4H, 4
pseudo t, J ꢁ 9.2 Hz in each, H-10, H-20, H-30, H-40),
4.28 (1H, dd, J = 11.9, 5.3 Hz, H-60a), 4.24 (2H, q,
J = 6.6 Hz, CH2), 4.09 (1H, dd, J = 11.9, 1.5 Hz, H-
60b), 3.88 (1H, ddd, J = 9.5, 5.3, 1.5 Hz, H-50), 2.08,
2.04 (2), 2.03, (12H, 3s, 4 · CH3), 1.45 (3H, t,
J = 6.6 Hz, CH3); 13C NMR (CDCl3): d (ppm) 170.6,
170.5, 169.8, 169.5, 165.0, 163.9 (CO, NHCO,
COOCH2CH3), 135.0, 131.9 (CH), 78.0 (C-10), 73.5,
72.7, 70.5, 68.0 (C-20 to C-50), 61.6 (C-60), 61.3
(CH2CH3), 20.5, 20.4 (3) (CH3), 13.9 (CH2CH3);
HRMS: m/z 496.14226 ([M+Na]+, calcd for C20H27-
NO12Na+ 496.14255). Anal. Calcd for C20H27NO12
(473.44): C, 50.74; H, 5.75; N, 2.96. Found: C, 50.43;
H, 5.86; N, 3.00.
4.12. 4-(N-b-D-Glucopyranosyl-carbamoyl)butyric acid (9)
Prepared from 1 (0.40 g, 0.86 mmol) according to
General procedure II. Eluent for the column chromato-
graphy: 1:1 CHCl3–MeOH. Yield: 0.26 g (88%) yellow-
ish oil, Rf = 0.11 (1:1 CHCl3–MeOH); [a]D +1 (c 0.69,
MeOH); 1H NMR (D2O): d (ppm) 4.95 (1H, d,
J = 8.9 Hz, H-10), 3.87 (1H, dd, J = 11.4, 2.2 Hz, H-
60a), 3.70 (1H, dd, J = 11.4, 3.8 Hz, H-60b), 3.56 (1H,
pseudo t, J = 9.4, 8.9 Hz, H-20), 3.51 (1H, ddd,
J = 9.4, 3.8, 2.2 Hz, H-50), 3.42–3.38 (2H, m, H-30, H-
40), 2.37–2.28 (4H, m, 2 · CH2), 1.89–1.85 (2H, m,
2 · CH2); 13C NMR (D2O): d (ppm) 181.1 (COOH),
178.1 (NHCO), 79.8 (C-10), 78.1, 77.0, 72.3, 69.8 (C-20
to C-50), 61.1 (C-60), 35.0 (2), 21.8 (CH2); HRMS:
m/z 316.10037 ([M+Na]+, calcd for C11H19NO8Na+
316.10029).
4.15. 3-(N-20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl-
carbamoyl)-(E)-propenoic acid (12)
Prepared from 3 (0.8 g, 2.14 mmol) and fumaric acid
(0.27 g, 2.35 mmol) according to General procedure I.
Eluent for the column chromatography: 4:1 EtOAc–
hexane. Yield: 0.52 g (54%) yellowish oil, Rf = 0.12
1
(EtOAc); [a]D ꢀ21 (c 0.22, CHCl3); H NMR (CDCl3):
d (ppm) 7.42 (1H, d, J = 9.2 Hz, NH) 7.08 (1H, d,
J = 15.8 Hz, CH), 6.91 (1H, d, J = 15.8 Hz, CH), 5.39,
5.31, 5.10, 5.03 (4H, 4 pseudo t, J ꢁ 9.2 Hz in each,
H-10, H-20, H-30, H-40), 4.30 (1H, dd, J = 13.2, 5.3 Hz,
H-60a), 4.11 (1H, dd, J = 13.2, 2.6 Hz, H-60b), 3.88
(1H, ddd, J = 9.2, 5.3, 2.6 Hz, H-50), 2.08, 2.05, 2.03
(2) (12H, 3s, 4 · CH3); 13C NMR (CDCl3): d (ppm)
171.4, 170.7, 169.6, 167.6, 167.4, 164.0 (COOH, NHCO,
CO), 136.8, 131.3 (CH), 78.0 (C-10), 73.7, 73.0, 70.6, 68.0
(C-20 to C-50), 61.7 (C-60), 20.5 (4) (CH3); HRMS: m/z
468.11154 ([M+Na]+, calcd for C18H23NO12Na+
468.11125).
4.13. 5-(N-b-D-Glucopyranosyl-carbamoyl)pentanoic acid
(10)
Prepared from 2 (0.25 g, 0.52 mmol) according to
General procedure II. Eluent for the column chromato-
graphy: 1:1 CHCl3–MeOH. Yield: 0.16 g (99%) colour-
less oil, Rf = 0.37 (1:1 CHCl3–MeOH); [a]D ꢀ16 (c 0.39,
H2O); 1H NMR (D2O):
d (ppm) 4.94 (1H, d,
J = 9.1 Hz, H-10), 3.86 (1H, dd, J = 12.4, 2.0 Hz, H-
60a), 3.71 (1H, dd, J = 12.4, 5.3 Hz, H-60b), 3.53 (1H,
pseudo t, J = 9.1, 8.9 Hz, H-20), 3.52 (1H, ddd,
J = 9.1, 5.3, 2.0 Hz, H-50), 3.43–3.37 (2H, m, H-30, H-
40), 2.34–1.61 (8H, m, 4 · CH2); 13C NMR (D2O): d
(ppm) 182.3 (COOH), 178.9 (NHCO), 80.2 (C-10),
78.5, 77.5, 72.7, 70.2 (C-20 to C-50), 61.6 (C-60), 36.7,
36.5, 25.8, 25.7 (CH2); ESIMS: m/z 330 ([M+Na]+).
4.16. 3-(N-20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl-
carbamoyl)propynoic acid (13)
Prepared from 3 (0.5 g, 1.34 mmol) and acetylenedicarb-
oxylic acid (0.17 g, 1.47 mmol) according to General
procedure I. Eluent for the column chromatography:
3:1 EtOAc–hexane. Yield: 0.20 g (35%) colourless oil,
Rf = 0.40 (3:1 EtOAc–hexane); [a]D ꢀ33 (c 0.27,
Me2SO); 1H NMR (CDCl3): d (ppm) 7.30 (1H, d,
J = 9.1 Hz, NH), 5.40, 5.30, 5.07, 4.99 (4H, 4 pseudo
t, J ꢁ 9.5 Hz in each, H-10, H-20, H-30, H-40), 4.31
(1H, dd, J = 12.4, 3.8 Hz, H-60a), 4.11 (1H, dd,
J = 12.4, 2.2 Hz, H-60b), 3.85 (1H, ddd, J = 9.5, 3.8,
2.2 Hz, H-50), 2.09, 2.08, 2.04, 2.03 (12H, 4s, 4 · CH3);
13C NMR (CDCl3): d (ppm) 170.4, 169.7 (2), 169.4 (2)
(COOH, CO), 152.0 (NHCO), 77.5 (C-10, „C–), 75.5
(„C–), 73.5, 72.6, 70.1, 67.9 (C-20 to C-50), 61.5 (C-
60), 20.4 (2), 20.2 (2) (CH3); HRMS: m/z 422.10615
([M+NaꢀCO2]+, calcd for C17H21NO10Na+ 422.10577).
4.14. Ethyl 3-(N-20,30,40,60-tetra-O-acetyl-b-D-gluco-
pyranosyl-carbamoyl)-(E)-propenoate (11)
Prepared from 3 (1.0 g, 2.68 mmol) and fumaric acid
monoethyl ester (0.42 g, 2.91 mmol) according to Gen-
eral procedure I. Eluent for the column chromatogra-
phy: 1:1 EtOAc–hexane. Yield: 1.0 g (79%) white
crystalline product, mp: 123–126 ꢁC; [a]D ꢀ8 (c 0.26,
CHCl3); 1H NMR (CDCl3): d (ppm) 7.27 (1H, d,
J = 9.2 Hz, NH) 6.92 (1H, d, J = 15.8 Hz, CH), 6.83

954
K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
4.17. Methyl 3-(N-b-D-glucopyranosyl-carbamoyl)-(E)-
propenoate (14)
73.3, 72.8, 70.6, 68.2 (C-20 to C-50), 61.8 (C-60), 20.6,
20.5 (3) (CH3); HRMS: m/z 518.12714 ([M+Na]+, calcd
for C22H25NO12Na+ 518.12690). Anal. Calcd for
C22H25NO12 (495.44): C, 53.34; H, 5.09; N, 2.83. Found:
C, 53.14; H, 5.16; N, 2.92.
Prepared from 11 (0.2 g, 0.42 mmol) according to Gen-
eral procedure II. Crystallized from MeOH to give
0.11 g (86%) white solid, mp: 176–179 ꢁC; [a]D ꢀ54 (c
0.22, H2O); 1H NMR (D2O): d (ppm) 7.04 (1H, d,
J = 15.8 Hz, CH), 6.84 (1H, d, J = 15.8 Hz, CH), 5.07
(1H, d, J = 9.2 Hz, H-10), 3.87 (1H, dd, J = 11.9,
2.6 Hz, H-60a), 3.83 (3H, s, OCH3), 3.73 (1H, dd,
J = 11.9, 5.3 Hz, H-60b), 3.57 (1H, t, J = 9.2, 9.2 Hz,
H-20), 3.55 (1H, ddd, J = 9.2, 5.3, 2.6 Hz, H-50), 3.47–
3.42 (2H, m, H-30, H-40); 13C NMR (D2O): d (ppm)
168.1, 167.6 (COOCH3, NHCO), 136.0, 131.7 (CH),
80.0 (C-10), 78.3, 77.0, 72.4, 69.8 (C-20 to C-50), 61.1
(C-60), 53.40 (OCH3); HRMS: m/z 314.08493
([M+Na]+, calcd for C11H17NO8Na+ 314.08464). Anal.
Calcd for C11H17NO8 (291.26): C, 45.36; H, 5.88; N,
4.81. Found: C, 45.20; H, 5.56; N, 4.62.
4.20. 2-(N-b-D-Glucopyranosyl-carbamoyl)benzoic acid
(17)
Prepared from 16 (0.2 g, 0.40 mmol) according to Gen-
eral procedure II. Eluent for the column chromatogra-
phy: 1:1 CHCl3–MeOH. Yield: 0.13 g (96%) colourless
oil, Rf = 0.2 (1:1 CHCl3–MeOH); [a]D +4 (c 0.21,
1
MeOH); H NMR (D2O): d (ppm) 7.76–7.52 (4H, m,
aromatics), 5.10 (1H, d, J = 9.5 Hz, H-10), 3.89 (1H,
dd, J = 11.6, 1.3, Hz, H-60a), 3.74 (1H, dd, J = 11.6,
4.8 Hz, H-60b), 3.70–3.43 (4H, m, H-20, H-30, H-40, H-
50); 13C NMR (D2O): d (ppm) 174.2, 173.3 (COOH,
NHCO), 135.4 (C-1), 133.7 (C-2), 131.8, 130.3, 129.9,
128.0 (C-3 to C-5), 80.3 (C-10), 78.3, 77.0, 72.6, 69.9
(C-20 to C-50), 61.2 (C-60); HRMS: m/z 350.08501
([M+Na]+, calcd for C14H17NO8Na+ 350.08464).
4.18. 3-(N-b-D-Glucopyranosyl-carbamoyl)-(E)-propenoic
acid (15)
4.21. Methyl 3-(N-20,30,40,60-tetra-O-acetyl-b-D-gluco-
pyranosyl-carbamoyl)benzoate (18)
Prepared from 12 (0.1 g, 0.22 mmol) according to Gen-
eral procedure II. Eluent for the column chromatogra-
phy: 2:1 CHCl3–MeOH. Yield: 0.03 g (27%) colourless
oil, Rf = 0.47 (2:1 CHCl3–MeOH); [a]D ꢀ22 (c 0.37,
Prepared from 3 (0.5 g, 1.34 mmol) and isophthalic acid
monomethyl ester (0.17 g, 1.47 mmol) according to Gen-
eral procedure I. Eluent for the column chromatogra-
phy: 1:1 EtOAc–hexane. Yield: 0.63 g (92%) white
crystalline product, mp: 148–150 ꢁC; [a]D ꢀ10 (c 0.67,
H2O); 1H NMR (D2O):
d (ppm) 6.77 (1H, d,
J = 15.8 Hz, CH), 6.61 (1H, d, J = 15.8 Hz, CH), 5.00
(1H, d, J = 9.2 Hz, H-10), 3.81 (1H, dd, J = 11.9,
2.6 Hz, H-60a), 3.67 (1H, dd, J = 11.9, 5.3 Hz, H-60b),
3.53 (1H, t, J = 9.1, 9.1 Hz, H-20), 3.52 (1H, ddd,
J = 9.1, 5.3, 2.6 Hz, H-50), 3.41–3.36 (2H, m, H-30, H-
40); 13C NMR (D2O): d (ppm) 173.5, 169.2 (COOH,
NHCO), 138.6, 131.0 (CH), 80.1 (C-10), 78.2, 77.0,
72.4, 69.7 (C-20 to C-50), 61.1 (C-60); HRMS: m/z
1
CHCl3); H NMR (CDCl3): d (ppm) 8.44–7.52 (4H, m,
aromatics), 7.27 (1H, d, J = 9.0 Hz, NH), 5.48, 5.41,
5.11, 5.05 (4H, 4 pseudo t, J ꢁ 9.0 Hz in each, H-10,
H-20, H-30, H-40), 4.34 (1H, dd, J = 12.6, 4.1 Hz, H-
6a0), 4.12 (1H, dd, J = 12.6, 1.5 Hz, H-6b0), 3.95 (3H,
s, OCH3), 3.94 (1H, ddd, J = 9.0, 4.1, 1.5 Hz, H-50),
2.07, 2.06 (2), 2.03 (12H, 3s, 4 · CH3); 13C NMR
(CDCl3): d (ppm) 171.3, 170.6, 169.8, 169.6, 166.3,
166.0 (NHCO, COOCH3, CO), 133.2 (C-1), 131.5 (C-
3), 133.1, 130.8, 128.9, 128.2 (C-2, C-4 to C-6), 78.9
(C-10), 73.7, 72.6, 70.8, 68.2 (C-20 to C-50), 61.7 (C-60),
52.4 (COOCH3) 20.6 (2), 20.5 (2) (CH3); HRMS: m/z
532.14262 ([M+Na]+, calcd for C23H27NO12Na+
532.14255). Anal. Calcd for C23H27NO12 (509.47): C,
54.22; H, 5.34; N, 2.75. Found: C, 54.34; H, 5.11; N,
2.66.
276.07305 ([MꢀH]ꢀ, calcd for C10H14NO 276.07249).
ꢀ
8
4.19. 2-(N-20,30,40,60-Tetra-O-acetyl-b-D-glucopyranosyl-
carbamoyl)benzoic acid (16)
Prepared from 3 (0.8 g, 2.14 mmol) and phthalic anhy-
dride (0.35 g, 2.36 mmol) according to General proce-
dure I. Eluent for the column chromatography: 95:5
CHCl3–MeOH. Yield: 0.54 g (51%) white crystalline
1
product, mp: 158–160 ꢁC; [a]D +5 (c 0.83, CHCl3); H
NMR (CDCl3): d (ppm) 7.94–7.51 (4H, m, aromatics),
7.46 (1H, d, J = 10.4 Hz, NH), 5.46, 5.38, 5.05, 4.97
(4H, 4 pseudo t, J ꢁ 10 Hz in each, H-10, H-20, H-30,
H-40), 4.30 (1H, dd, J = 12.5, 3.6 Hz, H-60a), 4.11 (1H,
ddd, J=10.4, 3.6, 2.1 Hz, H-50), 3.95 (1H, dd, J = 12.5,
2.1 Hz, H-60b), 2.17 (2), 2.10, 2.05 (12H, 3s, 4 · CH3);
13C NMR (CDCl3): d (ppm) 171.4, 171.0, 169.9 (2),
169.6 (2) (COOH, NHCO, CO), 136.8 (C-1) 132.1 (C-
2) 132.0, 130.7, 130.2, 127.4 (C-3 to C-6), 78.2 (C-10),
4.22. Methyl 4-(N-2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranosyl-carbamoyl)benzoate (19)
Prepared from 3 (0.5 g, 1.34 mmol) and terephthalic acid
monomethyl ester (0.17 g, 1.47 mmol) according to Gen-
eral procedure I. Eluent for the column chromato-
graphy: 1:1 EtOAc–hexane. Yield: 0.60 g (88%) white

K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
955
crystalline product, mp: 187–190 ꢁC; [a]D ꢀ16 (c 0.55,
4.25. Methyl 4-(N-b-D-glucopyranosyl-carbamoyl)-
benzoate (22)
CHCl3); 1H NMR (CDCl3): d (ppm) 8.11 (2H, d,
J = 7.8 Hz, H-2, H-6), 7.83 (2H, d, J = 7.8 Hz, H-3,
H-5), 7.21 (1H, d, J = 9.0 Hz, NH), 5.44, 5.40, 5.11,
5.06 (4H, 4 pseudo t, J ꢁ 9.3 Hz in each, H-10, H-20,
H-30, H-40), 4.34 (1H, dd, J = 12.3, 3.9 Hz, H-60a),
4.11 (1H, dd, J = 12.3, 2.5 Hz, H-60b), 3.95 (3H, s,
OCH3), 3.94 (1H, ddd, J = 9.0, 3.9, 2.5 Hz, H-50),
2.17, 2.08, 2.05, 2.05 (12H, 4s, 4 · CH3); 13C NMR
(CDCl3): d (ppm) 171.5, 170.5, 169.8, 169.6, 166.2,
166.0 (COOCH3, NHCO, CO), 136.5 (C-1), 133.5 (C-
4) 129.9 (C-2, C-6), 127.3 (C-3, C-5), 78.9 (C-10), 73.6,
72.5, 70.8, 68.2 (C-20 to C-50), 61.5 (C-60), 52.4
(COOCH3) 20.6 (2), 20.5 (2) (CH3); HRMS: m/z
532.14257 ([M+Na]+, calcd for C23H27NO12Na+
532.14255). Anal. Calcd for C23H27NO12 (509.47): C,
54.22; H, 5.34; N, 2.75. Found: C, 54.14; H, 5.24; N,
2.80.
Prepared from 19 (0.2 g, 0.39 mmol) according to Gen-
eral procedure II. Crystallized from MeOH to give
0.115 g (91%) white crystalline product, mp: 245–
1
248 ꢁC; [a]D +12 (c 0.38, Me2SO); H NMR (Me2SO-
d6): d (ppm) 8.04 (4H, br s, aromatics), 4.98 (1H, d,
J = 8.9 Hz, H-10), 3.88 (3H, s, OCH3), 3.67 (1H, dd,
J = 12.1, 3.7 Hz, H-60a), 3.40 (1H, dd, J = 12.1,
5.3 Hz, H-60b), 3.36–3.10 (4H, m, H-20, H-30, H-40, H-
50); 13C NMR (Me2SO-d6): d (ppm) 165.7 (2) (COOCH3,
NHCO), 138.1 (C-1), 131.9 (C-4), 128.9 (C-2, C-6),
127.9 (C-3, C-5), 80.2 (C-10), 78.7, 77.4, 71.9, 69.9 (C-
20 to C-50), 60.9 (C-60), 52.3 (OCH3); HRMS: m/z
364.10027 ([M+Na]+, calcd for C15H19NO8Na+
364.10029). Anal. Calcd for C15H19NO8 (341.32): C,
52.79; H, 5.61; N, 4.16. Found: C, 52.60; H, 5.55; N,
4.22.
4.23. Methyl 3-(N-b-D-glucopyranosyl-carbamoyl)-
benzoate (20)
4.26. 4-(N-b-D-Glucopyranosyl-carbamoyl)benzoic acid
(23)
Prepared from 18 (0.2 g, 0.39 mmol) according to Gen-
eral procedure II. Crystallized from Et2O to give
0.10 g (77%) white crystalline product, mp: 222–
Prepared from 22 (0.17 g, 0.49 mmol) according to Gen-
eral procedure III. Eluent for the column chromatogra-
phy: 1:2 CHCl3–MeOH. Yield: 0.14 g (84%) colourless
oil, Rf = 0.17 (1:1 CHCl3–MeOH); [a]D ꢀ5 (c 0.67,
MeOH); 1H NMR (D2O): d (ppm) 7.92 (2H, d,
J = 7.9 Hz, H-2, H-6), 8.40 (2H, d, J = 7.9 Hz, H-3,
H-5), 5.20 (1H, d, J = 7.7 Hz, H-10), 3.89 (1H, dd,
J = 12.5, 1.1, Hz, H-60a), 3.69 (1H, dd, J = 12.5,
4.2 Hz, H-60b), 3.66–3.45 (4H, m, H-20, H-30, H-40, H-
50); 13C NMR (D2O): d (ppm) 180.9 (COOH), 172.1
(NHCO), 140.4 (C-1), 135.5 (C-4), 129.5 (C-2, C-6),
128.0 (C-3, C-5), 80.6 (C-10), 78.3, 77.1, 72.4, 69.9 (C-
20 to C-50), 61.2 (C-60); HRMS: m/z 350.08403
([M+Na]+, calcd for C14H17NO8Na+ 350.08464).
1
224 ꢁC; [a]D +10 (c 0.44, MeOH); H NMR (D2O): d
(ppm) 8.33–7.56 (4H, m, aromatics), 5.23 (1H, d,
J = 8.0 Hz, H-10), 3.95 (3H, s, OCH3), 3.93 (1H, dd,
J = 11.1, 2.5 Hz, H-60a), 3.83 (1H, dd, J = 11.1,
3.7 Hz, H-60b), 3.68–3.59 (4H, m, H-20, H-30, H-40, H-
50); 13C NMR (D2O): d (ppm) 170.4, 168.1 (COOCH3,
NHCO), 133.2 (C-1), 129.9 (C-3), 133.1, 132.2, 129.2,
128.4 (C-2, C-4 to C-6), 79.9 (C-10), 77.7, 76.5, 71.8,
69.2 (C-20 to C-50), 60.5 (C-60), 52.8 (OCH3); HRMS:
m/z 364.09963 ([M+Na]+, calcd for C15H19NO8Na+
364.10029). Anal. Calcd for C15H19NO8 (341.32): C,
52.79; H, 5.61; N, 4.16. Found: C, 52.54; H, 5.41; N,
4.06.
4.27. Enzyme assays
4.24. 3-(N-b-D-Glucopyranosyl-carbamoyl)benzoic acid
(21)
Glycogen phosphorylase b was prepared from rabbit
skeletal muscle according to the method of Fischer
and Krebs,38 using 2-mercaptoethanol instead of L-cys-
teine, and recrystallized at least three times before use.
The kinetic studies with glycogen phosphorylase were
performed as described previously.37 Kinetic data for
the inhibition of rabbit skeletal muscle glycogen phos-
phorylase by monosaccharide compounds were col-
lected using different concentrations of a-D-glucose-1-
phosphate (4, 6, 8, 10, 12 and 14 mM) and constant con-
centrations of glycogen (1% w/v) and AMP (1 mM). The
enzymatic activities were presented in the form of dou-
ble-reciprocal plots (Lineweaver–Burk) applying a non-
linear data-analysis programme. The inhibitor constants
(Ki) were determined by Dixon plots, by replotting the
Prepared from 20 (0.17 g, 0.49 mmol) according to Gen-
eral procedure III. Eluent for the column chromatogra-
phy: 1:2 CHCl3–MeOH. Yield: 0.16 g (99%) colourless
oil, Rf = 0.17 (1:1 CHCl3–MeOH); [a]D +8 (c 0.29,
1
MeOH); H NMR (D2O): d (ppm) 8.22–7.45 (4H, m,
aromatics), 5.20 (1H, d, J = 8.2 Hz, H-10), 3.91 (1H,
dd, J = 11.3, 1.5 Hz, H-60a), 3.79 (1H, dd, J = 11.3,
3.8 Hz, H-60b), 3.65–3.49 (4H, m, H-20, H-30, H-40, H-
50); 13C NMR (D2O): d (ppm) 174.9, 171.9 (COOH,
NHCO), 136.6 (C-1), 132.9 (C-3), 132.8, 130.0, 128.8,
127.7 (C-2, C-4 to C-6), 80.5 (C-10), 78.3, 76.2, 72.3,
69.8 (C-20 to C-50), 61.2 (C-60);ꢀHRMS: m/z 326.08746
([MꢀH]ꢀ, calcd for C14H16NO 326.08814).
8

956
K. Czifra´k et al. / Carbohydrate Research 341 (2006) 947–956
´
14. Gyo¨rgydeak, Z.; Hadady, Z.; Felfo¨ldi, N.; Krakomperger,
slopes from the Lineweaver–Burk plots against the
inhibitor concentrations. The means of standard errors
for all calculated kinetic parameters averaged to less
than 10%.39,40 IC50 values were determined in the pres-
ence of 4 mM glucose 1-phosphate, 1 mM AMP, 1%
glycogen, and varying concentrations of an inhibitor.
´
´
A.; Nagy, V.; Toth, M.; Brunyanszky, A.; Docsa, T.;
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Leonidas, D. D.; Somsak, L.; Nagy, V.; Praly, J.-P.;
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This work was supported by the Hungarian Scientific
Research Fund (OTKA T43550). Z.H. thanks CMS
Chemicals Ltd., Oxford, UK, for financing her PhD
studies with L.S. K.C. and L.W. thank the German Aca-
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