Synthesis, characterization, and biological evaluation of new N-glycosides derived from O-pivaloylated β-D-glucopyranosylamine

Article abstract of DOI:10.1007/s11164-011-0423-7

The novel synthesis of N-(2,3,4,6-tetra-O-pivaloyl-β-D-glucopyranosyl) benzo[d] oxazol-2-amine 4 was described in this study. The new compounds N-arylthioureas 3a-c and 4, along with a series of glucose-modified imines 5a-g, were evaluated for their antitumor activity against human myeloid leukemia cell lines (HL-60 cells), gastric carcinoma (BGC-823 cells), liver carcinoma (Bel-7402 cells), and oral carcinomas (KB cells). The antibacterial potency of these compounds was also determined using an inhibition zone diameter test. Although none of the compounds were active against human cancer cells, compound 4 was found to be the most active compound against Escherichia coli. Springer Science+Business Media B.V. 2011.

Full text of DOI:10.1007/s11164-011-0423-7

Res Chem Intermed (2012) 38:863–870
DOI 10.1007/s11164-011-0423-7
Synthesis, characterization, and biological evaluation
of new N-glycosides derived from O-pivaloylated
b-D-glucopyranosylamine
•
•
•
Chao Shen Guobin Zhou Xinzhi Chen
Pengfei Zhang
Received: 22 September 2011 / Accepted: 10 October 2011 / Published online: 30 October 2011
Ó Springer Science+Business Media B.V. 2011
Abstract The novel synthesis of N-(2,3,4,6-tetra-O-pivaloyl-b-D-glucopyranosyl)
benzo[d] oxazol-2-amine 4 was described in this study. The new compounds
N-arylthioureas 3a-c and 4, along with a series of glucose-modified imines 5a-g,
were evaluated for their antitumor activity against human myeloid leukemia cell
lines (HL-60 cells), gastric carcinoma (BGC-823 cells), liver carcinoma (Bel-7402
cells), and oral carcinomas (KB cells). The antibacterial potency of these com-
pounds was also determined using an inhibition zone diameter test. Although none
of the compounds were active against human cancer cells, compound 4 was found to
be the most active compound against Escherichia coli.
Keywords Glucosyl thioureas ꢀ Glucose-modified benzoxazole ꢀ
Glucose-modified imines ꢀ Antitumor activity ꢀ Antimicrobial activity
Introduction
Carbohydrates and their derivatives exerted important effects on many complex
biological events [1], such as cellular recognition in the processes of immune
response [2], cell migration [3], inflammation [4], carbohydrate recognition [5],
tumor metastasis, and viral infections [6]. Meanwhile, glycoconjugates play a key
role as receptors for proteins and enzymes on the cell surface [7], and they have
C. Shen ꢀ G. Zhou ꢀ X. Chen (&)
Department of Chemical and Biological Engineering, Zhejiang University,
Hangzhou 310027, China
e-mail: shenchaozju@hotmail.com
C. Shen ꢀ P. Zhang (&)
College of Material, Chemistry and Chemical Engineering,
Hangzhou Normal University, Hangzhou 310036, China
e-mail: chxyzpf@hotmail.com
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C. Shen et al.
been shown to interact with either RNA or the backbone phosphate of DNA [8].
These derivatives, bearing nitrogen scaffolds in different ring positions on the
sugar skeleton, are called N-glycosides, and many N-glycosides derivatives are of
increasing interest because of their diverse biological properties such as antimicro-
bial [9], anti-inflammatory [10], anti-influenza [11], antiviral [12], and antitumor
activities [13]. All this explains that certain series of N-glycosides are regarded as
major target molecules for many years, owing to their biological and medicinal
importance [14, 15]. Therefore, studies of the synthesis and pharmacology of N-
glycosides derivatives have attracted more interest in recent years [3, 16, 17].
Following our interesting on synthesis of glycoconjugates [18–20], we were
eager to extend our approach to the synthesis of other new classes of sugar-based
heterocycles, which are of considerable importance in medicinal chemistry [21]. We
anticipated that some glucosyl heterocycles could be synthesized in the presence of
Pd catalyst through new cyclization model.
Results and discussion
Firstly, the reaction of 2,3,4,6-tetra-O-pivaloyl-b-D-glucopyranosylamine 1 with
2-hydroxyphenylisothiocyanate affords exclusively the glucosyl thiourea 3a in 81%
yield in isopropanol at room temperature (Table 1, entry 1). Then, the effects of
various solvents were studied for the preparation of 3a and comparative results are
presented in Table 1. The reaction of glucopyranosylamine 1 proceeded smoothly in
acetonitrile within 3 h at room temperature to afford the desired products in very
high yield (Table 1, entry 4). In other cases, the yields varied from 81 to 92%. These
results encouraged further studies with various other substituted phenylisothiocy-
anates and these phenylisothiocyanates readily reacted with glucopyranosylamine
Table 1 Synthesis of glucose-modified thioureas 3a-c
OPiv
OPiv
NCS
H
N
H
r.t. 3 h
O
O
+
PivO
PivO
PivO
PivO
NH2
N
R
solvent
S
OPiv
OPiv
R
1
2a-c
3a-c
Entry
R
Product
Solvent
Yield(%)a
1
2
3
4
5
6
o-OH
o-OH
o-OH
o-OH
o-CH3
p-OH
3a
3a
3a
3a
3b
3c
Isopropanol
Toluene
CH2Cl2
81
90
92
95
91
93
CH3CN
CH3CN
CH3CN
All reactions were carried out using 1 (1 mmol), 2 (1.1 mmol), in 5 mL solvent
a
Isolated yield after chromatography
123

Evaluation of new N-glycosides
865
under identical conditions to produce glucosyl thioureas 3b and 3c in high yields
(Table 1, entries 5 and 6).
The glucose-modified benzoxazoles 4 was obtained in 86% yield in the presence
of 15% Pd(OAc)2 in dimethyl sulfoxide from 3a. Noteworthy, the other glucosyl
thioureas cannot give the corresponding product in the selected condition. The
structures of the products 4 were established and confirmed on the basis of their
elemental analysis and spectral data (MS, 1H NMR and 13C NMR). 1H NMR
1
spectroscopy was used to confirm the product and the H NMR spectrum of 4
showed the anomeric proton as a doublet at d = 5.34 ppm with a J = 9.2 Hz
indicating the b-configuration. The other six protons of the glucopyranosyl ring
resonate in the d = 3.95–5.52 ppm region. In addition, the right NH groups of the
glucosyl thioureas 3a with a singlet at 8.41 ppm disappeared in the product and the
left NH groups of glucose-modified benzoxazoles 4 show distinctive chemical shifts
at 6.05 ppm. Complementarily, a singlet at 182.39 ppm on the 13C NMR spectrum
is assigned to the C=S bond also disappeared and the new C=N bond of benzoxazole
ring can also be detected as a carbon resonance at 160.19 ppm in the 13C NMR
spectrum, which should be attributed to the formation of compound 4 (Fig. 1)
(Scheme 1).
Next, we also prepared another series of glucose-modified imines using aromatic
aldehydes instead of substituted phenylisothiocyanates. The reaction of glucopyr-
anosylamine 1 with aromatic aldehydes 5a-g proceeded smoothly in isopropanol to
afford the desired glucose-modified imines 6a-g in high yields (Scheme 2) [19].
Finally, the newly synthesized glucose-modified benzoxazoles 4, glucosyl
thiourea 3b-c and glucose-modified imines 6a-g were screened for their cytostatic
activities against human myeloid leukemia cell lines (HL-60 cells) and other three
different cell lines derived from various human solid tumors including gastric
carcinoma (BGC-823 cells), liver carcinoma (Bel-7402 cells), and oral carcinomas
(KB cells). However, the IC50 values of selected compounds against four types of
Fig. 1 1H NMR spectra of compound 3a and 4 in CDCl3
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C. Shen et al.
OH
OPiv
OPiv
H
N
15% Pd(OAc)2
DMSO, 80 °C
H
N
H
N
O
O
PivO
PivO
N
O
PivO
PivO
S
OPiv
4
OPiv
3a
Scheme 1 Synthesis of glucose-modified benzoxazoles 4
OPiv
CHO
OPiv
O
r.t., 12 h
PivO
PivO
O
+
PivO
PivO
N
R
NH2
R
Isopropanol
OPiv
OPiv
e:
R = p-Cl
6a: R = H
1
5a-g
f: R = m-CH3
g:
b
: R = p-CH3
R = o-OH
c: R = p-OCH3
d
: R = p-NO2
Scheme 2 Synthesis of glucose-modified imines 6a-g
Table 2 Antimicrobial activity of N-glycosides against Escherichia coli
Microorganism EC
Compounds
3a
3b
3c
4
6a
6c
6e
6f
Zone diameter
9
8
6
20
8
11
ND
5
Test microorganism: EC Escherichia coli; ND not determined
tumor cells in culture were larger than 30, which indicated none of the compounds
were active against human myeloid leukemia cell.
Determination of the antimicrobial activities of the new compounds was carried
out in vitro by the agar well diffusion method [22]. The antimicrobial activity of
these compounds was evaluated by measuring the inhibition zone diameter observed
and were found to be significant at 5 mM concentration. It is obvious that our
synthesized compounds showed significant activity against the tested microorgan-
isms with inhibition zones ranging from 5 to 20 mm. Among the N-glycosides
derivatives studied, the most active compound was 4 that showed good antimicro-
bial activity (Table 2).
Conclusions
In summary, the novel N-glycosides containing benzoxazole ring were synthesized
in good yield. A series of glucosyl thioureas and imines were also obtained from
the same starting materials. All of the target compounds were screened for their
123

Evaluation of new N-glycosides
867
antitumor activities against HL-60, BGC-823, Bel-7402, and KB cells. Among the
N-glycosides derivatives studied, glucose-modified benzoxazoles 4 possess better
antibacterial activity than other compounds. This study forms part of our research
program to evolve new methodologies for the construction of novel glycoconjugates
and to discover new molecules with antitumor activities. Further synthesis and
biological investigation of new glycoconjugates for antitumor activity is continuing
within our group.
Experimental section
2,3,4,6-Tetra-O-pivaloyl-b-D-glucopyranosylamine 1 and glucose-modified imines
6a-g were prepared according our previous reports [18–20]. Other chemicals and
solvents were either purchased or purified by standard techniques. Analytical TLC
was performed on a Merck precoated TLC (silica gel 60 F254) plate. Melting points
were recorded on an X4-Data microscopic melting point apparatus and are
uncorrected. IR spectra were recorded on a Nicolet 380 FT-IR spectrophotometer
using KBr discs. ESI–MS were acquired on a Bruker Esquire 3000 plus
spectrometer. 1H and 13C NMR were recorded on a Bruker Avance 400
spectrometer in CDCl3 using TMS as internal standard. Elemental analysis were
performed on Carlo-Erba 1106.
General procedure for the synthesis of glucosyl thiourea 3a–b
To a glass vial charged with acetonitrile (5 mL) was added glucosyl glucopyrano-
sylamine (1.00 mmol) and substituted isothiocyanate (1.10 mmol). The resulting
mixture was stirred at room temperature and monitored by TLC. Upon completion
of the reaction (*3 h), the mixture was poured into water (10 mL), and extracted
three times with dichloromethane (10 9 3 mL). The combined organic layers were
washed with water, dried over MgSO4, and filtered. The solvents were removed via
rotary evaporation and the product was purified by silica gel column chromatog-
raphy using hexane–AcOEt (5:1) as an eluent to yield the desired products.
N-(2,3,4,6-Tetra-O-pivaloyl-b-D-glucopyranosyl)-N0-o-hydroxyphenyl
thiourea 3a
1
White solid; yield: 95%; mp 181–183 °C. H NMR (400 MHz, CDCl3):d 8.41
(s, 1H, NH), 7.25 (t, J = 7.6 Hz, 1H, ArH), 7.11 (d, J = 7.6 Hz, 1H, ArH), 7.06
(d, J = 8.4 Hz, 1H, ArH), 6.97 (t, J = 7.6 Hz, 1H, ArH), 6.77 (s, 1H, NH), 5.82
(t, J = 8.8 Hz, 1H, G1H), 5.44 (t, J = 9.2 Hz, 1H, G2H), 5.04 (t, J = 9.6 Hz, 1H,
G4H), 4.91 (t, J = 9.6 Hz, 1H, G3H), 4.18-4.11 (m, 2H, G6H), 3.85 (m, 1H, G5H),
1.21–1.09 (m, 36H); 13C NMR (100 MHz, CDCl3): d 182.39, 178.72, 178.52,
176.81, 176.52, 150.95, 129.60, 126.94, 121.30, 118.33, 83.07, 74.10, 72.01, 70.50,
67.68, 61.60, 38.94, 38.89, 38.73, 38.65, 27.11, 27.05, 26.98, 26.90; m/z (EI):
667.23 [M ? H]?; Anal. Calcd. For : C33H50N2O10S: C, 59.42; H, 7.59; N, 4.11;
Found: C, 59.40; H, 7.61; N, 4.16.
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C. Shen et al.
N-(2,3,4,6-Tetra-O-pivaloyl-b-D-glucopyranosyl)-N0-omethoxyphenyl
thiourea 3b
1
White solid; yield 91%; mp 166–167 °C; H NMR (400 MHz, CDCl3):d 7.83 (s,
1H, NH), 7.27–7.24 (m, 2H, ArH), 7.02–6.94 (m, 2H, ArH), 6.68 (s, 1H, NH), 5.88
(t, J = 9.6 Hz, 1H, G1H), 5.44 (t, J = 10.0 Hz, 1H, G2H), 5.09 (t, J = 10.0 Hz, 1H,
G4H), 4.96 (t, J = 9.6 Hz, 1H, G3H), 4.14–4.13 (m, 2H, G6H), 3.89 (m, 1H, G5H),
3.82 (s, 3H, OCH3), 1.20–1.07 (m, 36H); 13C NMR (100 MHz, CDCl3): d 182.05,
181.79, 178.84, 178.07, 176.64, 175.88, 152.51, 128.37, 124.98, 120.99, 111.87,
83.32, 74.04, 72.07, 70.45, 67.66, 61.61, 55.55, 38.88, 38.74, 38.63, 27.12, 27.07,
26.95, 26.85; m/z (EI): 681.33 [M ? H]?; Anal. Calcd. For C34H52N2O10S: C,
59.98; H, 7.71; N, 4.11. Found: C, 59.99; H, 7.72; N, 4.12.
N-(2,3,4,6-Tetra-O-pivaloyl-b-D-glucopyranosyl)-N0-p-
hydroxyphenylthiourea 3c
1
White solid; yield: 93%; mp 193–195 °C. H NMR (400 MHz, CDCl3):d 8.71 (s,
1H, NH), 7.69(d, J = 9.2 Hz, 2H, ArH), 7.44 (d, J = 8.4 Hz, 2H, ArH), 6.90 (s, 1H,
NH), 5.82 (t, J = 8.8 Hz, 1H, G1H), 5.44 (t, J = 9.2 Hz, 1H, G2H), 5.04 (t,
J = 9.6 Hz, 1H, G4H), 4.91 (t, J = 9.6 Hz, 1H, G3H), 4.18–4.11 (m, 2H, G6H),
3.85 (m, 1H, G5H), 1.21–1.09 (m, 36H); 13C NMR (100 MHz, CDCl3): d 181.60,
178.98, 178.18, 17679, 176.48, 140.66, 133.60, 124.16, 118.21, 82.99, 74.47, 70.73,
67.64, 61.77, 39.03, 38.86, 38.77, 38.71, 27.13, 27.09, 26.97; m/z (EI): 667.50
[M ? H]?; Anal. Calcd. For : C33H50N2O10S: C, 59.39; H, 7.51; N, 4.22; Found: C,
59.51; H, 7.69; N, 4.12.
General procedure for the synthesis of glucose-modified benzoxazoles 4
A mixture of 3a (0.5 mmol), and Pd(OAc)2 (16.73 mg, 0.075 mmol) in DMSO
(5 mL) was stirred at 80 °C under air and the reaction progress was monitored
by TLC. Upon completion of the reaction (ca. 12 h), the mixture was cooled to
room temperature and poured into 10 mL of water. The mixture was extracted
with AcOEt (5 mL 9 3) and the combined organic layer was washed with brine
(10 mL), then dried over MgSO4. The solvent was removed under reduced pressure
and the residue was purified by silica gel column chromatography using hexane–
AcOEt (10:1) as an eluent to yield the desired product glucose-modified
benzothiazoles 4.
N-(2,3,4,6-Tetra-O-pivaloyl-b-D-glucopyranosyl) benzo[d] oxazol-2-amine 4
1
White solid; yield: 86%; mp 191–193 °C. H NMR (400 MHz, CDCl3):d 7.39 (d,
J = 7.6 Hz, 1H, ArH), 7.26 (d, J = 8.0 Hz, 1H, ArH), 7.19 (d, J = 7.6 Hz, 1H,
ArH), 7.06 (t, J = 7.6 Hz, 1H, ArH), 6.05 (s, 1H, NH), 5.49 (t, J = 9.2 Hz, 1H,
G1H), 5.34 (d, J = 9.2 Hz, 1H, G2H), 5.18 (t, J = 9.6 Hz, 1H, G4H), 5.12 (t,
J = 9.2 Hz, 1H, G3H), 4.18–4.15 (m, 2H, G6H), 3.98–3.95 (m, 1H, G5H), 1.18–1.09
(m, 36H); 13C NMR (100 MHz, CDCl3): d 178.56, 178.24, 177.15, 176.70, 160.19,
123

Evaluation of new N-glycosides
869
148.80, 142.22, 124.47, 122.11, 117.49, 109.44, 83.04, 74.26, 72.38, 70.83, 68.01,
61.93, 39.14, 39.06, 39.02, 38.97, 27.37, 27.27, 27.13; m/z (EI): 633.12 [M ? H]?;
Anal. Calcd. For C33H48N2O10: C, 62.60; H, 7.52; N, 4.41; 4. Found: C, 62.55; H,
7.42; N, 4.50.
Biological activity assays
Cell culture
Four different human carcinoma cell lines: HL-60, Bel-7402, BGC-823 and KB
were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum,
100 units/mL of penicillin and 100 lg/mL of streptomycin. Cells were maintained
at 37 °C in a humidified atmosphere of 5% CO2 in air.
Solutions
The selected complexes were then dissolved in DMSO at a concentration of 5 mM
as stock solution, and diluted in culture medium at concentrations of 1.0, 10, 100,
and 500 lM as working-solution. To avoid DMSO toxicity, the concentration of
DMSO was less than 0.1% (v/v) in all experiments.
Cytotoxicity analysis
The cells harvested from exponential phase were seeded equivalently into a 96-
well plate, and then the complexes were added to the wells to achieve final
concentrations. Control wells were prepared by the addition of culture medium.
Wells containing culture medium without cells were used as blanks. All
experiments were performed in quintuplicate. The MTT assay was performed as
described by Mosmann for HL-60 [23]. Upon completion of the incubation for 44 h,
stock MTT dye solution (20 mL, 5 mg/mL) was added to each well. After 4 h
incubation, 2-propanol (100 mL) was added to solubilize the MTT formazan. The
OD of each well was measured on a microplate spectrophotometer at a wavelength
of 570 nm. The SRB assay was performed as previously described for Bel-7402,
BGC-823, and KB. Upon completion of the incubation for 44 h, the cells were fixed
in 10% trichloroacetic acid (100 mL) for 30 min at 4 °C, washed five times and
stained with 0.1% SRB in 1% acetic acid (100 mL) for 15 min. The cells were
washed four times in 1% acetic acid and air-dried. The stain was solubilized in
10 mM unbuffered Tris base (100 mL) and OD was measured at 540 nm as above.
The IC50 value was determined from plot of % viability against dose of compounds
added.
Acknowledgments For financial support, we thank the Science and Technology Plan of Zhejiang
Province (2011C24004), Key Sci-tech Innovation Team of Zhejiang Province (2010R50017) and the
National Natural Science Foundation of China (No. 21076052). We also thank Prof. Zhang J. C. and Prof.
Zhu Q. for providing Cytotoxicity Analysis for research.
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