Synthesis of some novel D-glucuronic acid acetylated derivatives as potential anti-tumor agents

Article abstract of DOI:10.1002/ardp.201000367

A structurally diverse series of Δ^4,5-uronamide derivatives have been chemically synthesized starting from D-glucuronic acid itself by means of acetylation, activation, amide bond formation and base-catalyzed elimination protocols. Structure el

Full text of DOI:10.1002/ardp.201000367

648
Arch. Pharm. Chem. Life Sci. 2011, 344, 648–657
Full Paper
Synthesis of Some Novel D-Glucuronic Acid Acetylated
Derivatives as Potential Anti-Tumor Agents
Ahmed O. H. El-Nezhawy1,2, Frady G. Adly1, Ahmed F. Eweas1,2, Atef G. Hanna3,
Yehya M. El-Kholy4, Shahenaz H. El-Sayed4, and Tarek B. A. El-Naggar5
1 Chemistry of Natural and Microbial Products Department, National Research Center, Dokki, Cairo, Egypt
2 Pharmaceutical Chemistry Department, College of Pharmacy, Taif University, KSA
3 Chemistry of Natural Compounds Department, National Research Center, Dokki, Cairo, Egypt
4 Chemistry Department, Faculty of Science, Helwan University, Egypt
5 Pharmacology Department, Faculty of Pharmacy, Complutense University, Spain
A structurally diverse series of D4,5-uronamide derivatives have been chemically synthesized starting
from D-glucuronic acid itself by means of acetylation, activation, amide bond formation and base-
catalyzed elimination protocols. Structure elucidation for all products along with optimization of the
synthetic steps is described. The synthesized compounds were evaluated for their in-vitro anti-tumor
activity against MCF-7, TK-10 and UACC-62 cell lines. The compounds 5, 11, 13, 15 and 16 were the most
active against TK-10 cell line. On the other hand, the most active compounds against the MCF-7 cell
line were 11 and 15. However, compounds 5, 7, 11, 13, 15 and 16 were the most active against the
UACC-62 cell line.
Keywords: Amide linkage / Anti-tumor / D-Glucuronamide / D-Glucuronic acid / MCF-7 / TK-10 / UACC-62
Received: November 26, 2010; Revised: December 24, 2010; Accepted: January 19, 2011
DOI 10.1002/ardp.201000367
Introduction
firmed by the discovery of a wide range of proteins that
recognize them. The importance of sialic acids in these pro-
cesses, especially with respect to human disease states, has
led to interest in the synthesis of natural and modified sialic
acids [13, 14] both as probes of sialic acid-recognizing
proteins, and as potential glycopharmaceuticals. The 2,3-
dehydro-2-deoxy-sialic acids Neu5Ac2en, Neu5Gc2en, and
their acetates are widely distributed in nature [15] and dis-
play interesting biological activities as transition state inhibi-
tors of the enzyme, sialidase [16]. A 2,3-dehydro-sialic acid,
such as Neu2en5Ac, Neu2en5Gc and Kdn2en mimetics, is
essentially derived from 4,5-unsaturated-D-glucopyranosi-
duronic acid (Fig. 2). Combination of non-carbohydrate
moieties with sialic acid derivative would be anticipated to
give rise to selective interaction towards target biological
molecules and target tumor cells [17–20].
Cancer has been a major lethal threat worldwide till date.
This malady is not a single ailment, but rather a collection of
hundreds of different diseases as a result of uncontrolled cell
proliferation. Chemotherapy is a major cancer treatment
besides surgery and radiation therapy. The majority of the
chemotherapeutic drugs that are currently in use are organic
drugs or natural products. Some examples of natural prod-
ucts as potential anti-tumor agents are illustrated in (Fig. 1).
The synthesis of new compounds and testing their biological
and pharmacological activities are the major goals of drug
development projects [1]. Sialic acids are involved in a range
of biological processes including cell-cell, cell-microbe inter-
actions [2–4], masking effects for cell surface antigen [5, 6],
differentiation of cells [7–9], and neoplastic transformation
[10–12]. These biological importances were further con-
Research within our group has been recently directed
towards the preparation of a series of indole-pyrimidine
(Meridianin D analogues) and phthalazinone-amino acid con-
jugates. These conjugates were tested as anti-tumor agents
and showed moderate activities against caucasian breast
adenocarcinoma (MCF-7) cell line [21–24]. As a part of our
ongoing research on drug discovery, we have been interested
Correspondence: Dr. Ahmed O. H. El-Nezhawy, Pharmaceutical
Chemistry Department, College of Pharmacy, Taif University, KSA and
Chemistry of Natural and Microbial Products Department, National
Research Center, Dokki, Cairo, Egypt
E-mail: nzhawy7@yahoo.com
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Arch. Pharm. Chem. Life Sci. 2011, 344, 648–657
Novel D-Glucuronic Acid Acetylated Derivatives
649
Ph
O
OH
O
OH
O
OH
OH
Ph
H
O
(+)-Altholactone
O
O
O
HO
H
HO
NH
O
O
R
O
HO
H
N
HO
HO
R = H
R = OH
Lycoricidine
Narciclasine
(+)-Gonipypyrone
OH
OH
5-Aza-D-glucuronic acid
Figure 1. Examples of natural products containing sugar ring as potential antitumor agents.
Figure 2. Structures of Neu5Ac2en, Neu5Gc2en and Kdn2en.
in developing a family of compounds containing an acety-
lated sialic acid nucleus through a facile and rapid synthetic
pathway to new drug-like small organic molecules.
Results and discussion
Scheme 1. Acetylation of D-glucuronic acid.
Synthesis of the saccharide conjugates
Studies were initiated by the acetylation of the commercially
available D-glucuronic acid using acetic anhydride and a
catalytic amount of iodine as an acetyl transfer reagent
[25] to give the 1,2,3,4-tetra-O-acetyl mixed anhydride 1 in
98% yield (Scheme 1). The formation of 1 was confirmed by
1H-NMR spectroscopy. In particular, the acetoxy methyl sig-
nal that appeared at relatively low field (2.28 ppm) indicates
the existence of the COOCOCH3 group, in addition to the
higher field signals observed at 2.13 (3H), 2.05 (6H) and
2.04 ppm (3H) which corresponds to the four O-acetyl groups.
Compound 1 was subjected to reaction with water to afford
the corresponding 1,2,3,4-tetra-O-acetyl-b-D-glucuronic acid 2
in 99% yield (Scheme 1). The formation of 2 was proven using
1H-NMR which revealed the absence of the acetoxy methyl
signal at 2.28 ppm and the appearance of a broad signal at
3.44 ppm corresponding to the carboxyl group proton [25].
Glucuronates derivatives were reported to readily undergo b-
cis-elimination to give the corresponding 4,5-unsaturated
species by treatment with a base [26–32]. The electron-with-
drawing effect of the carbonyl group makes the a-proton (H-5)
sufficiently acidic to be removed by a base. It has been
proposed that there are three steps involved in the elimi-
nation. Firstly, removal of H-5 forming a carbanion, followed
by conformational inversion, and finally, elimination to give
the 4,5-unsaturated derivative [26] (Fig. 3).
On this basis, a straightforward one pot procedure was
employed starting from acid 2 in which, in addition to the
amide bond formation, a base-promoted elimination of acetic
acid was achieved. The coupling of 2 with different amines in
the presence of ethyl chloroformate and TEA at ꢀ208C pro-
duced, after extractive work-up, the desired 4,5-unsaturated
amides 3, 5, 7, 9, 11 and 13 along with the non-elimination
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Figure 3. b-Elimination in D-glucuronic acid derivatives.
Figure 4. Spin-spin coupling interactions of the methylene protons.
onamide (5) were obtained, respectively. The structural
assignment for 3 and 5 were confirmed on the basis of their
NMR spectral data. The 1H-NMR spectrum of compound 3
showed the anomeric proton at 6.21 ppm. The other H-3 and
H-2 ring protons appeared at 5.17 and 5.04 ppm, respectively,
while the olefinic proton appeared at 6.18 ppm. The remain-
ing three acetoxy groups appeared as three singlets at 1.99,
1.98 and 1.97 ppm, while the methylene protons resonated at
4.39 ppm.
The splitting pattern of the methylene group was ddd (double
of doublets for each methylene proton) which corresponds to
an ABX system and it was reduced to a double of doublets after
proton exchange experiment. This was accounted by the pres-
ence of asymmetric centers in the vicinity of the methylene
group which makes a ‘non-equivalence’ between the two
methylene protons and in turn a diastereotopic effect will be
reflected in the 1H-NMR spectrum and the CH2 group will
appear as a double of doublets (doublet for each methylene
proton). Further coupling with the neighboring amide NH
proton will give the observed ddd pattern (Fig. 4).
Additionally, 13C-NMR interpretation assisted with DEPT
confirmed the structure of 3 by the observation of a signal at
88.7 ppm corresponding to C-1 atom. The other two signals at
67.5 and 64.1 ppm corresponds to C-2 and C-3, respectively,
while the two signals at 144.8 and 103.8 ppm corresponds to
C-5 and C-4 olefinic carbons, respectively. The four signals
appearing in the 170.0–160.6 ppm region are due to the four
carbonyl carbon atoms, while the signals at 21.2 and 21.1 are
due to the acetate methyl carbons. The benzyl group was also
observed in the 13C-NMR by the appearance of four signals at
137.9, 129.2, 128.4 and 128.1 ppm corresponding to the
phenyl ring carbons, and a signal at 43.9 ppm corresponding
to the methylene carbon atom. On the other hand, the
1H NMR spectrum for compound 5 showed the anomeric
proton at 6.30 ppm with as spin-spin coupling constant of
3.1 Hz which is typical axial–quasi-axial coupling between H-
1 and H-2 protons [33], while the other H-2 and H-3 ring
protons appeared at 5.14 and 5.26 ppm, respectively.
Additionally, 13C-NMR interpretation assisted with DEPT
confirmed the structure of 5 by the observation of a signal
Scheme 2. Reaction of 2 with benzyl, allyl, substituted aromatic
and heterocyclic aromatic amines.
products 4, 6, 8, 10, 12 and 14 which were also identified
(Scheme 2). Fortunately, these products could be separated by
a slow silica gel column chromatography.
By using benzyl or allyl amine, the corresponding N-
(benzyl)- (3) or N-(allyl)-1,2,3-tri-O-acetyl-D4,5-b-D-glucopyranur-
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Novel D-Glucuronic Acid Acetylated Derivatives
651
at 88.2 ppm corresponding to C-1 atom. The other two signals
at 67.0 and 63.5 ppm corresponds to C-2 and C-3, respectively,
while the two signals at 144.2 and 103.1 ppm corresponds to
C-5 and C-4 olefinic carbons, respectively. The four signals
appearing in the 169.6–160.0 ppm region are due to the four
carbonyl carbon atoms, while the signals at 20.7 and 20.6 are
due to the acetate methyl carbons. The allyl carbons were also
observed by the appearance of two signals at 133.2 and
116.9 ppm corresponding to the two olefinic carbons and
a signal at 41.7 ppm for the methylene carbon.
By using 4-bromoaniline or 2-chloro-4-nitroaniline as aro-
matic amines, the corresponding N-(4-bromophenyl)- (7) or
N-(2-chloro-4-nitrophenyl)-1,2,3-tri-O-acetyl-D4,5-b-D-glucopyra-
nuron-amide (9) were obtained, respectively. Again, the struc-
tural assignment for 7 and 9 were confirmed on the basis of
their NMR spectral data. In particular, the 1H-NMR for
compound 7 showed the anomeric proton at 6.37 ppm over-
lapped with the olefinic proton H-4, while the other H-2 and
H-3 ring protons appeared at 5.16 and 5.28 ppm, respectively.
Additionally, 13C-NMR interpretation confirmed the struc-
ture of 7 by the observation of a signal at 88.3 ppm corre-
sponding to C-1 atom, while the two signals at 143.9 and
104.3 ppm corresponds to C-5 and C-4 olefinic carbons,
respectively. The 1H-NMR for compound 9 showed the anome-
ric proton at 6.44 ppm with as spin-spin coupling constant of
4.6 Hz which is typical axial-quasi-axial coupling between H-1
and H-2 protons [33]. The olefinic proton appeared at
6.41 ppm, while the other H-2 and H-3 ring protons appeared
at 5.20 and 5.32 ppm, respectively.
Scheme 3. Reaction of 2 with 2-aminopyrazine.
nuronamide (15), but instead it gave ethyl 1,2,3-tri-O-acetyl-
4,5-b-D-glucopyranuronate (16) (Scheme 3). The formation of
D
16 was an unexpected result, and the mechanism of its
formation requires further investigation.
The structure of compound 16 was assigned on the basis of
its NMR spectral data. In particular the 1H-NMR spectrum
revealed nine signals; a quartet at 4.31 ppm and a triplet at
1.34 ppm corresponding to the ethyl group, three signals at
6.41, 5.23 and 5.15 ppm corresponding respectively to H-1, H-
3, H-2 ring protons and a signal at 6.27 ppm corresponding to
the olefinic ring proton. Additionally, the 13C-NMR spectra
confirmed the suggested structure of 16. The two signals at
62.0 and 14.13 ppm were assigned for the ethyl carbon
atoms, while the signal at 161.2 ppm was assigned for the
ethyl ester carbonyl carbon.
By using 2-aminothiazole or 2-aminobenzimidazole as het-
erocyclic aromatic amines, the corresponding N-(thiazol-2-yl)-
(11) or N-(1H-benzimidazol-2-yl)-1,2,3-tri-O-acetyl-D4,5-b-D-glu-
copyranuronamide (13) were obtained, respectively, and
the structural assignments were confirmed on the basis of
The focus was shifted to find an alternative route for the
preparation of N-(pyrazin-2-yl)-1,2,3-tri-O-acetyl-D4,5-b-D-gluco-
pyranuronamide (15) and a two step procedure was suggested
(Scheme 4). N-(Pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-b-D-glucopyr-
anuronamide (17) was obtained from the reaction of 2-amino-
pyrazine and 1 in anhydrous DCM at room temperature.
After product purification, a solution of 17 in anhydrous
DCM maintained at 08C was then treated with DBU. After
30 min, TLC analysis of the reaction mixture indicated a
complete consumption of the starting material and a clean
conversion to a product with a higher Rf. To work-up the
reaction, the solvent was removed in vacuo and the residue
was subjected to column chromatography to give the desired
product 15. The NMR spectroscopic analysis of the product
1
their NMR spectral data. In particular, the H-NMR for com-
pound 11 showed the anomeric proton overlapped with the
olefinic proton in the 6.34–6.32 ppm region. The other H-2
and H-3 ring protons appeared at 5.12 and 5.22 ppm, respec-
tively. Additionally, 13C-NMR interpretation confirmed the
structure of 11 by the observation of a signal at 88.8 ppm
corresponding to C-1 atom, while the two signals at 143.6 and
106.1 ppm corresponds to C-5 and C-4 olefinic carbons, respec-
tively. The three thiazole ring carbons were also observed at
158.1, 138.4 and 115.0 ppm. On the other hand, the 1H-NMR for
compound 13 showed the anomeric proton at 6.35 ppm
with as spin-spin coupling constant of 4.6 Hz which is typical
axial-quasi-axial coupling between H-1 and H-2 protons [33].
Interestingly, the attempt to use 2-aminopyrazine as the
amine in this reaction gave a single reaction product as
indicated by TLC, but after purification and spectral data
inspection, it was found that the reaction did not give the
desired N-(pyrazin-2-yl)-1,2,3-tri-O-acetyl-D4,5-b-D-glucopyra-
1
confirmed the elimination as the H-NMR spectrum showed
an olefinic H-4 resonates at ꢁ6.3 ppm and the absence of
resonances for H-5. Additionally, the 13C-NMR showed down-
field signals for olefinic carbons at 105.4 ppm and 143.6 ppm
which are consistent with the C-4–C-5 double bond.
1
Additionally, both the H-NMR and 13C-NMR showed signals
which corresponds to only three acetyl groups. The DBU
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A. O. H. El-Nezhawy et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 648–657
Figure 5. The concluded conformation for the prepared 4,5-
unsaturated glucuronamide derivatives.
ance of two downfield signals is consistent with C-4–C-5
double bond carbons. The ring conformation of each product
was determined by comparing their coupling constants with
those of known glycosides of hex-4-enopyranuronate [29, 34].
The coupling between H-2 and H-3 was small (J2,3 < 3.0 Hz),
and within the range expected for 4,5-unsaturated glucuro-
nides having 1H2 conformation. Further, long range (W)
coupling is also observed between H-2 and H-4, as well as,
between H-1 and H-3 in some products, which is also con-
Scheme 4. Preparation of compound 15.
promoted elimination procedure was also tried for the prep-
aration of the other 4,5-unsaturated compounds (3, 5, 7, 9, 11
and 13) from the corresponding 1,2,3,4-tetra-O-acetyl-b-D-glu-
copyranuronamides which successfully produced the same
compounds as a single product in moderate yields except
for N-(1H-benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl-b-D-glucuron-
amide (14) in which the reaction suffered from series of
complications (Scheme 5).
1
sistent with the H2 conformation [29] (Fig. 5).
Anti-tumor evaluation and discussion
The newly synthesized compounds were examined for in-vitro
activity against several human cancer cell lines, including
renal adenocarcinoma (TK-10), human breast adenocarcinoma
(MCF-7) and human melanoma (UACC-62). Three response
parameters (GI50, TGI, and LC50) were calculated for each cell
line. The GI50 value (growth inhibitory activity) corresponds to
the concentration of the compounds causing 50% decrease in
net cell growth, the TGI value (cytostatic activity) is the
concentration of the compounds resulting in total growth
inhibition and the LC50 value (cytotoxic activity) is the con-
centration of the compounds causing net 50% loss of initial
cells at the end of the incubation period. Table 1 lists the GI50,
TGI and LC50 values obtained for all tested compounds.
Regarding the activities of the 4,5-unsaturated derivatives
(3, 5, 7, 9, 11, 13, 15 and 16) in Table 1, compounds 7, 9, 11, 15
and 16 showed a growth inhibitory activity on the three
mentioned tumoral cell lines at the tested doses. However,
b-Elimination was accompanied by a conformational
change of the ring from 4C1 chair to half-chair to accommo-
date the newly formed double bond. This was reflected in the
1H-NMR spectrums of all products through the reduction in
the magnitude of coupling between ring protons that are no
longer in an axial–axial relationship with their neighbors.
Further indication for b-elimination was provided by the
observed downfield shift of H-4 signal and the disappearance
of the H-5 signal. The conformational change was also
reflected in 13C-NMR spectrum by the upfield shifts of signals
corresponding to C-1, C-2 and C-3. Furthermore, the appear-
compounds 3,
5 and 13 showed only an inhibitory
effect against the growth of the TK-10 and UACC-62 cells,
but did not posses any activity on the MCF-7 cell lines. The
compounds 5, 11, 15 and 16 were the most cytotoxic on TK-10
(GI50 ¼ 0.23, 1.51, 2.38 and 0.14 mM, respectively). On the
other hand, the most compounds which were affected on the
MCF-7 cell line were 11 and 15 (GI50 ¼ 0.32 and 3.04 mM,
respectively). However, all tested 4,5-unsaturated derivatives,
with exception of compounds 3 and 9, showed a high growth
inhibitory activity against the UACC-62 cell line. The growth
Scheme 5. Reaction of N-(1H-benzimidazol-2-yl)-1,2,3,4-tetra-
O-acetyl-b-D-glucuronamide with DBU.
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Table 1. Compound concentrations (mM) required to cause different inhibitions.
Compound
number
Inhibition
parameter
Cell Line
TK-10
MCF-7
UACC-62
3
GI50
TGI
LC50
84.96 ꢃ 13.48
>100
>100
>100
>100
>100
24.38 ꢃ 2.01
>100
>100
5
GI50
TGI
LC50
0.23 ꢃ 0.01
>100
>100
>100
>100
>100
0.036 ꢃ 0.01
0.71 ꢃ 0.03
15.06 ꢃ 1.97
7
GI50
TGI
LC50
14.43 ꢃ 3.31
34.63 ꢃ 8.22
83.05 ꢃ 6.19
99.73 ꢃ 21.63
>100
>100
2.14 ꢃ 0.12
8.55 ꢃ 0.50
34.12 ꢃ 6.18
9
GI50
TGI
LC50
32.83 ꢃ 6.81
72.13 ꢃ 12.07
>100
25.71 ꢃ 2.77
>100
>100
16.09 ꢃ 3.17
36.31 ꢃ 7.68
81.91 ꢃ 16.00
11
13
15
16
GI50
TGI
LC50
1.51 ꢃ 0.80
3.44 ꢃ 0.10
7.85 ꢃ 1.18
0.32 ꢃ 0.31
1.06 ꢃ 0.10
3.55 ꢃ 0.18
0.803 ꢃ 0.21
2.57 ꢃ 0.18
8.22 ꢃ 1.92
GI50
TGI
LC50
6.00 ꢃ 0.09
>100
>100
>100
>100
>100
1.53 ꢃ 0.03
11.46 ꢃ 2.81
85.67 ꢃ 11.50
GI50
TGI
LC50
2.38 ꢃ 0.04
7.71 ꢃ 1.50
25.00 ꢃ 5.16
3.04 ꢃ 0.09
80.87 ꢃ 19.11
>100
0.62 ꢃ 0.02
4.67 ꢃ 0.99
24.75 ꢃ 4.75
GI50
TGI
LC50
0.14 ꢃ 0.05
>100
>100
9.03 ꢃ 1.11
>100
>100
0.18 ꢃ 0.02
>100
>100
The range of doses assayed was 10ꢀ4, 10ꢀ5, 10ꢀ6, 10ꢀ7 and 10ꢀ8 M. Results are mean ꢃ S.E.M. (n ¼ 3).
of TK-10 cells was totally inhibited by 7, 9, 11 and 15
(TGI ¼ 34.63, 72.13, 3.44, and 7.71 mM, respectively). At the
same time, all the tested 4,5-unsaturated derivatives, with
exception of compounds 3 and 16, showed a cytostatic and a
cytotoxic activities against the UACC-62 cell line. Compounds
11 and 15 demonstrated a cytostatic activity against the
MCF-7 cell line with TGI values of 1.06 and 80.87 mM, respectively.
Furthermore, only 11 produced a cytotoxic activity (LC50)
against MCF-7 cell line at the dose of 3.55 mM.
However, compounds 5, 7, 11, 13, 15 and 16 were the most
active against the UACC-62 cell line.
Experimental
Chemistry
General methods
All starting materials and reagents were purchased from Sigma-
Aldrich, BDH and Fluka and used without further purification.
All solvents were either of analytical grades or dried and distilled
immediately prior to use: DCM from calcium hydride and THF
from sodium/benzophenone. All of the reactions were performed
using oven-dried glassware. Melting points were measured on a
Stuart-SMP10 melting point apparatus and are uncorrected.
TLC was performed using Merck precoated Silica gel 60 F254
aluminum sheets (20 ꢂ 20 cm, layer thickness 0.2 mm) and
spots were visualized by UV (254 nm), KMNO4 solution and/or
charring with H2SO4/EtOH (5% v/v). Column chromatography was
carried out on Silica Gel 60 (particle size 0.063–0.200 mm, 70–
230 mesh ASTM, Merck) using the specified eluents. All reaction
products were stored refrigerated under 48C. NMR spectra were
recorded with JEOL ECA-500, JEOL EX-270, and Varian Mercury-
200BB spectrometers at room temperature in solvents given.
Summary
A highly efficient and practical method was described for the
preparation of D4,5-uronamide derivatives according to stan-
dard protocols. The newly synthesized compounds were
examined for in-vitro activity against several human cancer
cell lines, including TK-10, MCF-7 and UACC-62. The com-
pounds 5, 11, 13, 15 and 16 were the most active against
TK-10 cell line. On the other hand, the most active com-
pounds against the MCF-7 cell line were 11 and 15.
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Chemical shifts were expressed in parts per million (ppm)
and reported either relative to an internal tetramethylsilane
standard (TMS d ¼ 0.0) or relative to solvent peaks (CDCl3
d ¼ 7.2, DMSO-d6 d ¼ 2.5) for 1H and (CDCl3 d ¼ 77.0) for 13C.
Multiplicities are denoted as follows: s ¼ singlet, d ¼ doublet,
t ¼ triplet, q ¼ quartet, dd ¼ double doublet, ddd ¼ double
double doublet, m ¼ multiplet, br ¼ broad, apt ¼ apparently.
13C signals were assigned with the aid of DEPT. Coupling con-
stants (J) were reported in Hertz (Hz).
129.2 (2 ꢂ aromatic CH), 128.4 (2 ꢂ aromatic CH), 128.1 (aro-
matic CH), 103.8 (C-4), 88.7 (C-1), 67.5 (C-2), 64.1 (C-3), 43.9
(CH2), 21.2, 21.1 (3 COCH3). Anal. calcd. for C19H21NO8 (391.3):
C, 58.31; H, 5.41; N, 3.58. Found: C, 58.51; H, 5.49; N, 3.68.
N-Benzyl-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (4)
White solid; (15%); mp 115–1178C; Rf ¼ 0.50 (EtOAc/petroleum
ether, 1:1); 1H-NMR (500 MHz, DMSO-d6): d 8.67 (t, 1H, NH), 7.26–
7.17 (m, 5H, aromatic H), 5.94 (d, 1H, J1-2 ¼ 8.4 Hz, H-1), 5.40 (apt
t, 1H, H-3), 5.12 (apt t, 1H, H-4), 4.95 (apt t, 1H, H-2), 4.33 (d, 1H,
J5-4 ¼ 10.0 Hz, H-5), 4.20 (ddd, 2H, CH2) 2.03, 1.97, 1.91, 1.83
(4s, 12H, 4 COCH3); 13C-NMR (50 MHz, CDCl3): d 169.8, 169.6,
169.2, 168.7 (4 COCH3), 165.8 (CONH), 137.4 (aromatic C), 128.7
(2 ꢂ aromatic CH), 127.8 (2 ꢂ aromatic CH), 127.6 (aromatic CH),
91.2 (C-1), 72.9, 71.9, 70.1, 68.9 (C-2–C-5), 42.9 (CH2), 20.6, 20.5
(4 COCH3). Anal. calcd. for C21H25NO10 (451.4): C, 55.87; H, 5.58;
N, 3.10. Found: C, 55.67; H, 5.45; N, 3.39.
1,2,3,4-Tetra-O-acetyl-b-D-glucopyranuronic acetic
anhydride (1) [25]
D-Glucuronic acid (2 g, 10.30 mmol) was suspended in acetic
anhydride (30 mL) and stirred at 0 C. Iodine (140 mg, 0.55 mmol)
was added and the red solution was left to stir for 30 min at 08C
and a further 2 h at room temperature. Acetic anhydride was
mostly removed in vacuo and the formed solid was taken up
in methylene chloride (50 mL), washed with 1 M Na2S2O3
(2 ꢂ 30 mL), dried over anhydrous Na2SO4, filtered and evapo-
rated to afford the title compound as a white solid (4.08 g, 98%).
1H-NMR (270 MHz, CDCl3): d 5.80 (d, 1H, J1-2 ¼ 6.9 Hz, H-1), 5.32
(m, 2H, H-3 and H-4 overlapping), 5.12 (apt t, 1H, H-2), 4.31 (d, 1H,
J5-4 ¼ 8.7 Hz, H-5), 2.28 (s, 3H, COCOOCH3), 2.13 (s, 3H, COCH3),
2.05 (s, 6H, 2 COCH3), 2.04 (s, 3H, COCH3).
N-Allyl-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (5)
Colorless oil; (45%); Rf ¼ 0.44 (EtOAc/petroleum ether, 1:1);
1H-NMR (500 MHz, CDCl3): d 6.67 (t, 1H, NH), 6.30 (d, 1H,
–
J1-2 ¼ 3.1 Hz, H-1), 6.25 (d, 1H, H-4), 5.85 (m, 1H, CH CH ),
–
5.26 (m, 1H, H-3), 5.22–5.15 (m, 2H, CH CH ), 5.14 (br s, 1H,
2
–
–
H-2), 3.96 (m, 2H, OCH –CH CH ), 2.13, 2.12, 2.09 (3s, 9H,
2
–
–
1,2,3,4-Tetra-O-acetyl-b-D-glucopyranuronic acid (2) [25]
1,2,3,4-Tetra-O-acetyl-b-D-glucuronic acetic anhydride (1, 4.08 g,
10.09 mmol) was dissolved in water and THF (90 mL, 1:2) and
stirred overnight. The solution was concentrated and the pro-
duct was extracted into methylene chloride (3 ꢂ 30 mL), the
combined organic layers were dried over anhydrous Na2SO4,
filtered and evaporated in vacuo to yield the title compound as
white foam (3.61 g, 99%). 1H-NMR (270 MHz, DMSO-d6): d 6.00
(d, 1H, J1-2 ¼ 8.1 Hz, H-1), 5.48 (apt t, 1H, H-3), 5.05 (apt t, 1H, H-4),
4.95 (apt t, 1H, H-2), 4.52 (d, 1H, J5-4 ¼ 8.2 Hz), 3.44 (br s, 1H,
COOH), 2.08 (s, 3H, COCH3), 2.01 (s, 3H, COCH3), 1.97 (s, 6H,
2 COCH3).
2
2
3 COCH3); 13C-NMR (50 MHz, CDCl3): d 169.6, 169.1, 168.7
–
(3 COCH ), 160.0 (CONH), 144.2 (C-5), 133.2 (CH CH ), 116.9
–
–
(CH CH ), 103.1 (C-4), 88.2 (C-1), 67.0 (C-2), 63.5 (C-3), 41.7
3
2
–
(OCH –CH CH ), 20.7, 20.6 (3 COCH3). Anal. calcd. for
2
–
–
C15H19NO8 (341.3): C, 52.78; H, 5.61; N, 4.10. Found: C, 52.46;
2
2
H, 5.43; N, 3.99.
N-Allyl-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (6)
White solid; (18%); mp 182–1848C; Rf ¼ 0.46 (EtOAc/petroleum
ether, 1:1); 1H-NMR (270 MHz, CDCl3): d 6.40 (t, 1H, NH), 5.84–5.81
–
(m, 1H, CH CH ), 5.75 (d, 1H, J ¼ 7.1 Hz, H-1), 5.35–5.09 (m, 5H,
–
2
1-2
–
General procedure for preparation of N-substituted-1,2,3-
tri-O-acetyl-D4,5-b-D-glucopyranuronamide
CH CH , H-2, H-3 and H-4), 4.10 (d, 1H, J ¼ 8.1 Hz, H-5), 3.86
–
(m, 2H, OCH –CH CH ), 2.15, 2.08, 2.05, 2.03 (4s, 12H, 4 COCH ).
2
5-4
–
–
2
2
3
Anal. calcd. for C17H23NO10 (401.3): C, 50.87; H, 5.78; N, 3.49.
Found: C, 50.58; H, 5.49; N, 3.28.
Triethylamine (1 equiv.) was added to a stirred solution of 1,2,3,4-
O-tetra-b-D-glucuronic acid (2) and ethyl chloroformate (1 equiv.)
in methylene chloride (5 mL/0.1 g) cooled to ꢀ208C. After
15 min, the amine (1 equiv.) was added, and the mixture was
allowed to warm to ꢀ58C over a period of 1 h. The reaction was
then diluted with methylene chloride, transferred to a sepa-
rating funnel and washed with 1 M HCl, deionized water, satu-
rated NaHCO3, deionized water again, dried over anhydrous
Na2SO4 and filtered. The solvent was removed and the residue
was purified by slow column chromatography (EtOAc/petroleum
ether) to afford the title compounds.
N-(4-Bromophenyl)-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (7)
Yellow oil; (41%); Rf ¼ 0.74 (EtOAc/petroleum ether, 1:1); 1H-NMR
(500 MHz, CDCl3): d 8.2 (s, 1H, NH), 7.51 (d, 2H, aromatic H), 7.46
(d, 2H, aromatic H), 6.37 (m, 2H, H-1 and H-4 overlapping), 5.28
(m, 1H, H-3), 5.16 (br s, 1H, H-2), 2.14, 2.12, 2.10 (3s, 9H, 3 COCH3);
13C-NMR (50 MHz, CDCl3): d 169.6, 169.2, 168.8 (3 COCH3), 157.9
(CONH), 143.9 (C-5), 135.8 (aromatic C-Br), 132.0 (2 ꢂ aromatic
CH), 121.6 (2 ꢂ aromatic CH), 117.6 (aromatic C–NH), 104.3 (C-4),
88.3 (C-1), 67.0 (C-2), 63.4 (C-3), 20.7, 20.6 (3 COCH3). Anal. calcd.
for C18H18BrNO8 (456.2): C, 47.39; H, 3.98; Br, 17.51; N, 3.07.
Found: C, 47.71; H, 3.78; Br, 17.46; N, 3.24.
N-Benzyl-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (3)
White solid; (46%); mp 97–1008C; Rf ¼ 0.5 (EtOAc/petroleum
ether, 1:1); 1H-NMR (500 MHz, CDCl3): d 7.24–7.17 (m, 5H, aro-
matic H), 7.05 (t, 1H, NH), 6.21 (s, 1H, H-1), 6.18 (d, 1H, H-4), 5.17
(m, 1H, H-3), 5.04 (br s, 1H, H-2), 4.39 (ddd, 2H, CH2), 1.99, 1.98,
1.97 (3s, 9H, 3 COCH3); 13C NMR (50 MHz, CDCl3): d 170.0, 169.6,
169.1 (3 COCH3), 160.6 (CONH), 144.8 (C-5), 137.9 (aromatic C),
N-(4-Bromophenyl)-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (8)
White solid; (18%); mp 1738C (dec.); Rf ¼ 0.52 (EtOAc/petroleum
ether, 1:1); 1H-NMR (270 MHz, DMSO-d6): d 10.30 (s, 1H, NH), 7.52
ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2011, 344, 648–657
Novel D-Glucuronic Acid Acetylated Derivatives
655
(br s, 4H, aromatic H), 6.06 (d, 1H, J1-2 ¼ 8.4 Hz, H-1), 5.53 (apt t,
1H, H-4), 5.26 (apt t, 1H, H-3), 5.08 (apt t, 1H, H-2), 4.46 (d, 1H,
J5-4 ¼ 9.7 Hz, H-5), 2.09, 2.03, 1.97, 1.92 (4s, 12H, 4 COCH3). Anal.
calcd. for C20H22BrNO10 (516.3): C, 46.53; H, 4.29; Br, 15.48; N,
2.71. Found: C, 46.41; H, 4.51; Br, 15.62; N, 2.79.
N-(1H-Benzimidazol-2-yl)-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (14)
White solid; (10%); mp 142–1448C; Rf ¼ 0.29 (EtOAc/petroleum
1
ether, 1:1); H-NMR (270 MHz, DMSO-d6): d 7.50 (s, 2H, aromatic
H), 7.23 (s, 2H, aromatic H), 6.09 (d, 1H, J1-2 ¼ 9.7 Hz, H-1), 5.56
(apt t, 1H, H-4), 5.35 (apt t, 1H, H-3), 5.10 (apt t, 1H, H-2),
4.65 (d, 1H, J5-4 ¼ 11.5 Hz, H-5), 2.10, 2.04, 1.98, 1.96 (4s, 12H,
4 COCH3). Anal. calcd. for C21H23N3O10 (477.4): C, 52.83; H, 4.86;
N, 8.80. Found: C, 52.66; H, 5.02; N, 8.65.
N-(2-Chloro-4-nitrophenyl)-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (9)
Yellow oil; (43%); Rf ¼ 0.85 (EtOAc/petroleum ether, 1:1); 1H-NMR
(500 MHz, CDCl3): d 9.15 (s, 1H, NH), 8.74 (d, 1H, aromatic H),
8.33 (d, 1H, aromatic H), 8.20 (dd, 1H, aromatic H), 6.44 (d, 1H,
J1-2 ¼ 4.6 Hz, H-1), 6.41 (dd, 1H, H-4), 5.32 (m, 1H, H-3), 5.20 (br s,
1H, H-2), 2.15, 2.14, 2.12 (3s, 9H, 3 COCH3). Anal. calcd. for
C18H17ClN2O10 (456.8): C, 47.33; H, 3.75; Cl, 7.76 N, 6.13.
Found: C, 47.50; H, 3.52; Cl, 7.49; N, 6.22.
Ethyl 1,2,3-tri-O-acetyl-D4,5-b-D-glucpyranuronate (16)
Colorless oil; (49%); Rf ¼ 0.29 (EtOAc/petroleum ether, 1:3);
1H-NMR (500 MHz, CDCl3): d 6.41 (d, 1H, J1-2 ¼ 2.3 Hz, H-1),
6.27 (dd, 1H, H-4), 5.23 (m, 1H, H-3), 5.15 (br s, 1H, H-2), 4.31
(q, 2H, CH2), 2.12, 2.11, 2.10 (3s, 9H, 3 COCH3), 1.34 (t, 3H, CH3);
13C-NMR (125 MHz, CDCl3): d 169.7, 169.1, 168.4 (3 COCH3), 161.2
(COOEt), 143.2 (C-5), 106.6 (C-4), 88.5 (C-1), 66.7 (C-2), 63.6 (C-3),
62.0 (OCH2CH3), 20.8, 20.7 (3 COCH3), 14.13 (OCH2CH3). Anal.
calcd. for C14H18O9 (330.3): C, 50.91; H, 5.49. Found: C, 50.76;
H, 5.14.
N-(2-Chloro-4-nitrophenyl)-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (10)
White solid; (11%); mp 169–1708C; Rf ¼ 0.25 (EtOAc/petroleum
ether, 1:3); 1H-NMR (500 MHz, CDCl3): d 8.90 (s, 1H, NH), 8.52
(d, 1H, aromatic H), 8.29 (s, 1H, aromatic H), 8.15 (m, 1H, aromatic
H), 5.84 (d, 1H, J1-2 ¼ 7.5 Hz, H-1), 5.35 (m, 2H, H-3 and H-4
overlapping), 5.18 (apt t, 1H, H-2), 4.28 (d, 1H, J5-4 ¼ 9.8 Hz,
H-5), 2.15, 2.10, 2.06, 2.02 (4s, 12H, 4 COCH3). Anal. calcd. for
C20H21ClN2O12 (516.8): C, 46.48; H, 4.10; Cl, 6.86 N, 5.42. Found:
C, 46.59; H, 4.01; Cl, 6.58; N, 5.61.
N-(Pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (17)
To a solution of 1,2,3,4-tetra-O-acetyl-b-D-glucuronic acetic anhy-
dride (1, 0.5 g, 1.23 mmol) in dry methylene chloride (30 mL)
and under argon, 2-aminopyrazine (0.118 g, 1.23 mmol) was
added and the reaction mixture was stirred overnight at room
temperature. The mixture was then diluted with methylene
chloride, transferred to a separating funnel and washed with
1 M HCl (2 ꢂ 30 mL), saturated NaHCO3 (2 ꢂ 30 mL), deionized
water (2 ꢂ 30 mL), dried over anhydrous Na2SO4 and filtered.
The solvent was then removed and the residue was purified by
column chromatography (EtOAc/petroleum ether, 1:3) to afford
the title compound as a white solid (0.23 mg, 42%); mp 108–
1108C; Rf ¼ 0.37 (EtOAc/petroleum ether, 1:1); 1H-NMR
(270 MHz, CDCl3): d 9.40 (s, 1H, aromatic H), 8.61 (s, 1H, NH),
8.38 (d, 1H, aromatic H), 8.27 (d, 1H, aromatic H), 5.82 (d, 1H,
J1-2 ¼ 8.1 Hz, H-1), 5.32 (m, 2H, H-3 and H-4 overlapping), 5.15
(apt t, 1H, H-2), 4.24 (d, 1H, J5-4 ¼ 8.8 Hz, H-5), 2.15, 2.10, 2.05,
2.02 (4s, 12H, 4 COCH3). Anal. calcd. for C18H21N3O10 (439.3):
C, 49.20; H, 4.82; N, 9.56. Found: C, 49.41; H, 4.77; N, 9.46.
N-(Thiazol-2-yl)-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (11)
White solid; (43%); mp 103–1048C; Rf ¼ 0.5 (EtOAc/petroleum
ether, 1:1); 1H-NMR (500 MHz, CDCl3): d 7.44 (d, 1H, aromatic
H), 6.98 (d, 1H, aromatic H), 6.34–6.32 (m, 2H, H-1 and H-4
overlapping), 5.22 (m, 1H, H-3), 5.12 (br s, 1H, H-2), 2.05, 2.04,
2.01 (3s, 9H, 3 COCH3); 13C-NMR (50 MHz, CDCl3): d 170.0, 169.5,
168.8 (3 COCH3), 158.1 (aromatic C), 157.5 (CONH), 143.6 (C-5),
138.4 (aromatic C), 115.0 (aromatic C), 106.1 (C-4), 88.8 (C-1), 67.3
(C-2), 63.8 (C-3), 21.2, 21.1 (3 COCH3). Anal. calcd. for C15H16N2O8S
(384.3): C, 46.87; H, 4.20; N, 7.29; S, 8.34. Found: C, 46.68; H, 4.12;
N, 7.14; S, 8.56.
N-(Thiazol-2-yl)-1,2,3,4-tetra-O-acetyl-b-D-
glucopyranuronamide (12)
N-(Pyrazin-2-yl)-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (15)
White solid; (10%); mp 178–1808C; Rf ¼ 0.37 (EtOAc/petroleum
ether, 1:1); 1H-NMR (270 MHz, DMSO-d6): d 12.49 (s, 1H, NH), 7.50
(d, 1H, aromatic H), 7.31 (d, 1H, aromatic H), 6.08 (d, 1H,
J1-2 ¼ 8.1 Hz, H-1), 5.55 (apt t, 1H, H-4), 5.34 (apt t, 1H, H-3),
5.12 (apt t, 1H, H-2), 4.61 (d, 1H, J5-4 ¼ 8.9 Hz, H-5), 2.08, 2.03,
DBU (0.05 mL, 0.33 mmol) was added to a stirred solution of
N-(pyrazin-2-yl)-1,2,3,4-tetra-O-acetyl-b-D-glucopyranuronamide (9,
0.1 g, 0.23 mmol) in dry methylene chloride (5 mL) cooled to
08C. After stirring for 30 min at 08C, the solvent was removed
in vacuo and the dark brown residue was purified by column
chromatography (EtOAc/petroleum ether, 1:3) to afford the title
compound as a colorless oil (73 mg, 81%); Rf ¼ 0.38 (EtOAc/
petroleum ether, 1:1); 1H-NMR (500 MHz, CDCl3): d 9.51 (s, 1H,
aromatic H), 8.77 (s,1H, NH), 8.33 (d, 1H, aromatic H), 8.25 (s, 1H,
aromatic H), 6.36 (m, 2H, H-1 and H-4 overlapping), 5.26 (m, 1H,
H-3), 5.14 (br s, 1H, H-2), 2.09, 2.07, 2.05 (3s, 9H, 3 COCH3); 13C-NMR
(50 MHz, CDCl3): d 169.5, 169.1, 168.5 (3 COCH3), 158.2 (CONH),
147.2 (aromatic C), 143.6 (C-5), 142.3 (aromatic CH), 140.8 (aromatic
CH), 137.1 (aromatic CH), 105.2 (C-4), 88.3 (C-1), 66.8 (C-2), 63.3 (C-3),
20.7, 20.6 (3 COCH3). Anal. calcd. for C16H17N3O8 (379.3): C, 50.66;
H, 4.52; N, 11.08. Found: C, 50.39; H, 4.75; N, 10.96.
1.97, 1.93 (4s, 12H, 4 COCH3). Anal. calcd. for C17H20N2O10
S
(444.4): C, 45.94; H, 4.54; N, 6.30; S, 7.22. Found: C, 45.70; H,
4.81; N, 6.58; S, 6.98.
N-(1H-Benzimidazol-2-yl)-1,2,3-tri-O-acetyl-D4,5-b-D-
glucopyranuronamide (13)
Yellow oil; (39%); Rf ¼ 0.35 (EtOAc/petroleum ether, 1:1); 1H-NMR
(500 MHz, CDCl3): d 7.42 (m, 2H, aromatic H), 7.15 (m, 2H,
aromatic H), 6.35 (d, 1H, J1-2 ¼ 4.6 Hz, H-1), 6.27 (d, 1H, H-4),
5.23 (m, 1H, H-3), 5.12 (br s, 1H, H-2), 2.06, 2.02, 2.00 (3s, 9H,
3 COCH3). Anal. calcd. for C19H19N3O8 (417.3): C, 54.68; H, 4.49;
N, 10.07. Found: C, 54.78; H, 4.41; N, 9.96.
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656
A. O. H. El-Nezhawy et al.
Arch. Pharm. Chem. Life Sci. 2011, 344, 648–657
compound. The data are given as the mean of two or three
different assays ꢃS.E.M.
Biological evaluation
Assay for cytotoxic activity: human cell lines
The following three human cancer cell lines were used in these
experiments: the human renal adenocarcinoma (TK-10), the
human breast adenocarcinoma (MCF-7) and the human mela-
noma (UACC-62) cell lines. The human tumor cytotoxicities were
determined following protocols established by NCI [35]. TK-10,
MCF-7, UACC-62 cell lines were cultured in RPMI 1640 medium
(BioWhittaker1) containing 20% fetal calf serum (FCS), 2 mmol/L
L-glutamine, 100 U/mL penicillin and 100 mg/mL streptomycin.
All cell lines were maintained at 378C in a 5% CO2 atmosphere
with 95% humidity. Maintenance cultures were passaged weekly,
and the culture medium was changed twice a week. According to
their growth profiles, the optimal plating densities of each cell
line was determined (15 ꢂ 103, 5 ꢂ 103 and 100 ꢂ 103 cells/well
for TK-10, MCF-7 and UACC-62, respectively) to ensure exponen-
tial growth throughout the experimental period and to ensure a
linear relationship between absorbance at 492 nm and cell num-
ber when analyzed by the sulphorhodamine B (SRB) assay.
The Authors gratefully acknowledge the financial support of the National
Research Centre (NRC), Dokki, Giza, Egypt. Also, they would like to express
special thanks to Prof. Emilia Carretera, Pharmacology Department,
Faculty of Pharmacy, Complutense University, for her help and support
concerning the biological activity tests. Further, they are grateful in every
possible way to the members of the NMR spectroscopy unit for their
continuous help regarding the necessary NMR measurements especially
Prof. Sahar Awad-Allah Mahmoud.
The authors have declared no conflict of interest.
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