Synthesis and cytotoxicities of mannose conjugated S-Nitroso-N-acetylpenicillamine (SNAP)

Article abstract of DOI:10.1016/S0968-0896(02)00064-0

A series of mannose conjugated S-nitroso-N-acetylpenicillamines (SNAPs) has been synthesized, and their cytotoxicities were assessed for DU 145 human prostate cancer cells and Hela R cancer cells.

Full text of DOI:10.1016/S0968-0896(02)00064-0

Bioorganic & Medicinal Chemistry 10 (2002) 2303–2307
Synthesis and Cytotoxicities of Mannose Conjugated
S-Nitroso-N-acetylpenicillamine (SNAP)
Xuejun Wu,a Xiaoping Tang,a Ming Xian,a
Paul G. Brauschweigerb and Peng George Wanga,*
aDepartment of Chemistry, Wayne State University, Detroit, MI 48202, USA
bDepartment of Radiation Oncology, University of Miami, Miami, FL 33136, USA
Received 4 December 2001; accepted 10 January 2002
Abstract—A series of mannose conjugated S-nitroso-N-acetylpenicillamines (SNAPs) has been synthesized, and their cytotoxicities
were assessed for DU 145 human prostate cancer cells and Hela R cancer cells. # 2002 Elsevier Science Ltd. All rights reserved.
In the last few years, S-nitrosothiols (RSNOs) have
been paid special attention because they play important
roles in nitric oxide (NO) storage and transport in a
wide range of physiological and pathophysiological
processes.1ꢀ3 Recently, we have reported that fructose
and glucose conjugates of SNAP are more stable than
SNAP and they exhibit much stronger effects on killing
cancer cell lines.4ꢀ7 For example, Glu-2-SNAP has been
shown to be 5000-fold more potent than SNAP in kill-
ing A2780S human ovarian cancer cells.6 The enhanced
cytotoxicity can be explained by the expression of
GLUT-1 transporter in the cancer cells. The biological
activities of glyco-SNAPs are also being investigated in
detail by other researchers. Babich et al. have evaluated
the cytotoxicities of glucose-1-SNAP, glucose-2-SNAP
and fructose-1-SNAP towards the human gingival epi-
thelioid S-G cell line and three human carcinoma cell
lines derived fromtissues of the oral cavity. They found
that glucose-SNAPs are more cytotoxic than SNAP.
They also reached a conclusion that the death of S-G
cells exposed to glucose-2-SNAP apparently occurred
by apoptosis.8 Incubation of aldose reductase (AR) with
glyco-SNAP results in an increase in enzyme activity
which is accompanied by the nitrosation of the active-
site residue, Cys-298, and the formation of a mixed dis-
ulfide with glyco-AP.9 Treatment of brain-derived cells
with glyco-1-SNAP can inhibit the DNA binding activity
of transcription factor AP-1 through a NO-dependent
mechanism.10
Herein, we will report the syntheses and cytotoxicities of
three mannose SNAPs: Man-1-SNAP, Man-2-SNAP
and Man-6-SNAP (Fig. 1).
Synthesis and Characterization
The synthetic routes toward mannose SNAPs are illus-
trated in Scheme 1. The 3-acetamido-4, 4-dimethylthie-
tan-2-one (cyclic AP) was synthesized according to our
previous method.4 The b-1-mannosylamine was pre-
pared following known precedure.11ꢀ13 Coupling of b-1-
mannosylamine with cyclic AP in pyridine provided
Man-1-AP (2). Subsequent nitrosation of 2 with
NaNO2/HCl gave the Man-1-SNAP in quantitative
yield. The Man-2-SNAP was synthesized by nitrosation
of Man-2-AP (4), which was prepared by coupling the
d-mannosamine to cyclic-AP in DMF. The compound 4
is a mixture of a- and b-anomers with a mole ratio of
13:5 as indicated by 1H and 13C NMR. Finally, the
Man-6-AP was synthesized by a relatively long pro-
cedure. The 6-OH group of methyl-a-d-mannopyrano-
side (5) was tosylated with tosyl chloride in pyridine.
Nucleophilic substitution of the tosyl group with NaN3
*Corresponding author. Tel.: +1-313-577-6759; fax: +1-313-577-
2554; e-mail: pwang@chem.wayne.edu
Figure 1. Structures of Man-SNAPs.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00064-0

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X. Wu et al. / Bioorg. Med. Chem. 10 (2002) 2303–2307
Scheme 1. Synthetic methodology toward Man-1-SNAP, Man-2-SNAP, and Man-6-SNAP.
Figure 2. NO generation by a 0.2 mM solution of mannose SNAPs in
a phosphate buffer (0.1 M, pH 7.4) and 1.0 mM EDTA.
Figure 3. NO generation by a 0.2 mM solution of mannose SNAPs in
a phosphate buffer (0.1 M, pH 7.4) and 50 mM Cu2+
.
in DMF at 60 C, followed by Pd/C catalyzed hydro-
genation, provided 6-amino-a-d-methyl mannoside
(8). Coupling of 8 with cyclic-AP gave Man-6-AP (9),
which was further nitrosated to give Man-6-SNAP.
The configuration of the anomeric carbon was estab-
lished by 2-D NOSEY analysis. Man-1-SNAP was
assigned as b-anomer, based on the fact that there was
strong NOE between H-1 and H-3, as well as between
H-1 and H-5.
a product of World Precision Instruments, Inc. (Sara-
sota, FL, USA). The sample was dissolved in small
amount of methanol, one portion of the solution was
then injected into the sealed O2-free aqueous buffer
solutions. Figure 2 shows the release of NO by a 0.2
mM solution of mannose SNAP in a phosphate buffer
solution (0.1 M, pH 7.4) and 1.0 mM EDTA.
Figure 3 illustrates the NO release profiles of mannose
SNAPs in a phosphate buffer solution containing Cu2+
but without EDTA. The results indicate that the NO
release rate could be accelerated about 20- to 30-fold
when the Cu2+ was presented in the buffer solution.
Among the three tested compounds, Man-1-SNAP
released NO most efficiently.
Nitric Oxide Measurement
NO measurement was carried out with an Electro-
chemical ISO-NO Mark II Isolated Nitric Oxide Meter,

X. Wu et al. / Bioorg. Med. Chem. 10 (2002) 2303–2307
2305
cancer cells, Man-1-SNAP appears to be the most
potent agent. It is believed that the potency of the com-
pound is correlated to both NO releasing efficacy and
steric configuration, which may affect its interaction
with sugar transporters.
Experimental
General
All reagents were purchased from commercial suppliers
and were used without further purification. 1H and
13C NMR were recorded on a Varian Gemini-300, a
Mercury-400, or a Varian Unity-500 spectrometer.
Mass spectra were recorded on a Kratos MS 80RFT
spectrometer. Silica gel F254 plates (Merck) and Silica
Gel 60 (70–230 mesh, Merck) were used in analytical
thin-layer chromatography (TLC) and flash column
chromatography, respectively.
Figure 4. The dose-dependent cytotoxicity of mannose SNAPs against
DU-145 human prostate cancer cells.
Man-1-AP (2). A mixture of 1-b-d-mannosylamine
(3.27 g) and cyclic-AP (1.25 g, 7.22 mmol) was dissolved
in 25 mL of pyridine. The reaction mixture was stirred
vigorously at roomtemperature for 16 h, it was then
evaporated to give an oily residue, which was purified
by column chromatography eluted with CH2Cl2/
CH3OH (10:1–7:1). The product 2 was obtained as a
white foam (710 mg, 2.02 mmol, yield 28.0%). 1H NMR
(400 MHz, CD3OD) dppm 5.17 (s, 1H, H-1), 4.62 (s, 1H,
H-20), 3.83 (dd, 1H, J=11.2, 2.4 Hz, H-6), 3.76 (d, 1H,
J=1.6 Hz, H-2), 3.67 (dd, 1H, 11.2, 5.6 Hz, H-6), 3.56
(t, 1H, J=9.6 Hz, H-4), 3.52 (dd, 1H, J=9.6, 2.4 Hz, H-
3), 3.29–3.25 (m, 1H, H-5), 2.03 (s, CH3 of Ac), 1.45 (s,
CH3-40), 1.39 (s, CH3-50); 13C NMR (100 MHz,
CD3OD) dppm 171.99 (C¼O), 170.27 (C¼O), 78.90 (C-5),
78.02 (C-1), 74.46 (C-3), 71.14 (C-2), 66.89 (C-4), 61.61
(C-6 and C-20), 45.24 (C-3), 29.61 (CH3-40), 28.15 (CH3-
5), 21.26 (CH3 of Ac). MS (ESI) m/z found 353
(M+H+), 375 (M+Na+), 391 (M+K+).
Figure 5. The dose-dependent cytotoxicity of mannose SNAPs against
Hela R cancer cells.
Man-2-AP (4). d-Mannosamine hydrochloride (3.24 g,
15.0 mmol) was stirred with sodium bicarbonate (1.26 g,
15.0 mmol) in 30 mL of dry DMF for 1.5 h, to which
was added cyclic-AP (2.60 g, 15.0 mmol). After TLC
indicated the disappearance of d-mannosamine the sol-
vent was removed under vacuum and the crude was
purified by silica gel chromatography eluted with
Cytotoxicities
The cytotoxicities of mannose SNAPs were assessed by
in vitro clonogenic cell survival assays as described pre-
viously.5 Figures 4 and 5 plot the surviving fraction
versus dose for DU-145 human prostate cancer cells and
Hela R cancer cells.
CH2Cl2/CH3OH (9:1–7:1). The compound
4 was
obtained as a white powder (1.74 g, 4.94 mmol, yield
32.9%). NMR indicated that the product was a mixture
of a-, b-anomers with a molar ratio of 13:5. a-Anomer:
1H NMR (CD3OD, 400 MHz) dppm 5.03 (d, 1H, J=1.6
Hz, H-1), 4.64 (s, 1H, H-20), 4.30 (dd, 1H, J=4.8 Hz,
1.6 Hz, H-2), 4.02 (dd, 1H, J=9.4, 4.6 Hz, H-3), 3.84
(dd, 1H, J=14.4, 4.4 Hz, H-6), 3.78–3.73 (m, 2H, H-5
and H-6), 3.60 (t, 1H, J=9.6 Hz, H-4), 2.03 (s, 3H, CH3
of Ac), 1.46 (s, 3H, CH3-40), 1.41 (s, 3H, CH3-50); 13C
NMR (CD3OD, 100 MHz) dppm 171.89 (C¼O), 170.82
(C¼O), 93.53 (C-1), 72.25 (C-5), 69.61 (C-3), 67.26 (C-
4), 61.77 (C-2), 60.95 (C-6), 53.91 (C-2), 45.58 (C-30),
29.20 (CH3 of Ac), 28.56 (CH3-40), 21.42 (CH3-5). MS
(ESI) m/z found 375 (M+Na+), 391 (M+K+).
In the DU-145 human prostate cancer cells, the MEDs
(median effect doses) are approximately 13, 42 and 22
mM for Man-1-SNAP, Man-2-SNAP and Man-6-SNAP,
respectively. Compared with our previous data of other
sugar SNAPs, the mannose conjugated SNAPs are
approximately as potent as Glu-2-SNAP or Fru-
2-SNAP.
In the Hela R cancer cells, the MEDs are approxi-
mately 4, 6 and 15 mM for Man-1-SNAP, Man-
2-SNAP and Man-6-SNAP, respectively. For both

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X. Wu et al. / Bioorg. Med. Chem. 10 (2002) 2303–2307
6-O-Toluenesulfonyl-methyl-ꢀ-D-mannopyranoside (6).
Methyl-a-d-mannopyranoside (20 g, 103.0 mmol) was
dissolved in 200 mL of pyridine and cooled to 0 ꢁC, to
which was added toluenesulfonyl chloride (24 g, 125.9
mmol) in portions. The reaction was monitored with
TLC. After the removal of solvent, the crude product
was purified by silica gel column chromatography eluted
with CH2Cl2/CH3OH (16:1). The product 6 was
obtained as a colorless syrup (33.38 g, 95.92 mmol,
CH3OH (14:1–12:1). The compound 9 was obtained as
a white powder (346.2 mg, 0.95 mmol, yield: 86.8%). 1H
NMR (D2O, 400 MHz) dppm 4.67 (s, 1H, H-1), 4.40 (s,
1H, H-20), 3.87 (d, 1H, J=1.6 Hz, H-2), 3.68 (dd, 1H,
J=9.6, 3.2 Hz, H-3), 3.63 (d, 1H, J=14.4, H-6), 3.56
(dd, 1H, J=8.8, 7.6 Hz, H-5), 3.51 (dd, 1H, J=9.6, 8.8
Hz, H-4), 3.42 (dd, 1H, J=14.4, 7.2 Hz, H-6), 3.31 (s,
3H, OCH3), 2.03 (s, 3H, Ac) 1.43 (s, 3H, CH3-40), 1.36
(s, 3H, CH3-50); 13C NMR (D2O, 100 MHz) dppm 173.93
(C¼O), 171.31 (C¼O), 100.94 (C-1), 71.02 (C-5), 70.34
(C-3), 69.91 (C-2), 68.10 (C-4), 62.38 (C-20), 54.91
(OCH3), 45.16 (C-30), 40.01 (C-6), 29.69 (CH3-40), 29.02
(CH3-50), 21.86 (CH3 of Ac). MS (ESI) m/z found 389
(M+Na+), 755 (2M+Na+).
1
yield 93.1%). H NMR (CDCl3, 400 MHz) dppm 7.79
(d, 2H, J=8.8 Hz), 7.31 (d, 2H, J=8.8 Hz), 4.76 (s,
1H), 4.65 (s, 1H), 4.82 (s, 1H), 4.31 (s, 3H), 3.91 (s,
1H), 3.75 (s, 1H), 3.71 (s, 1H), 3.26 (s, 3H, OCH3), 2.40
(s, 3H); 13C NMR (CDCl3, 400 MHz) dppm 145.22,
132.83, 130.15, 128.25, 101.17 (C-1), 71.85 (CH), 70.70
(CH), 70.32 (CH), 70.05 (CH2), 55.21 (OCH3), 21.86
(CH3). MS (ESI) m/z found 371 (M+Na+), 719
(2M+Na+).
Nitrosation of glyco-N-acetyl-penicilliamines. The man-
nose AP was dissolved in methanol and cooled to
ꢀ20 ꢁC using a dry ice/acetone/water bath, to which was
added 1.0 M HCl. Then a aqueous solution of sodium
nitrite was added dropwise. After stirring for several
hours under dark, the solvent was removed under
vacuum. Cold methanol was added to precipitate the
salt. After filtration and solvent removal the mannose
SNAP was obtained as a green powder in quantitative
yield.
6-Azido-6-dexoxy-methyl-ꢀ-D-mannopyranoside (7). The
compound 6 (5.15 g, 14.8 mmol) was dissolved in 45 mL
of dry DMF, and the solution was heated to 60 C. Then
sodium azide (10.64 g, 163.7 mmol) was added in por-
tions. When TLC indicated that all of the starting
material disappeared, the white precipitate was removed
by vacuumfiltration and the solvent was evaporated
under vacuum. Column chromatography purification of
the crude provided 7 (3.04 g, 13.9 mmol, yield 93.9%) as
1
Man-1-SNAP. H NMR (CD3OD, 500 MHz) dppm 5.37
(s, 1H, H-2), 5.16 (d, 1H, J=1.0 Hz, H-1), 3.83 (dd, 1H,
J=11.6, 2.5 Hz, H-6), 3.73 (dd, 1H, J=3.0, 1.0 Hz, H-2),
3.69 (d, 1H, J=11.6, 5.6 Hz, H-6), 3.58 (t, 1H, J=9.6
Hz, H-4), 3.52 (dd, 1H, J=9.6, 3.0 Hz, H-3), 3.32–3.28
(m, 1H, H-5), 2.06 (s, 3H, CH3-40), 2.00 (s, 3H, CH3-50),
1.96 (s, 3H, CH3 of Ac); 13C NMR (CD3OD, 125 MHz)
dppm 172.03 (C¼O), 169.49 (C¼O), 78.84 (C-5), 78.09
(C-1), 74.39 (C-3), 71.06 (C-2), 66.81 (C-4), 61.40 (C-6),
60.09 (C-20) 58.25 (C-30), 26.01 (CH3-40), 24.46 (CH3-50),
21.19 (CH3 of Ac). UV lmax: 229 nm, 350 nm. MS (ESI)
m/z found 404 (M+Na+), 785 (2M+Na+).
1
a colorless syrup. H NMR (CD3OD, 500 MHz) dppm
4.64 (d, 1H, J=1.5 Hz, H-1), 3.80 (dd, 1H, J=3.0, 1.5
Hz, H-2), 3.65–3.60 (m, 2H, H-3 and H-5), 3.58 (t, 1H,
J=9.1 Hz, H-4), 3.48 (dd, 1H, J=13.2, 2.5 Hz, H-6),
3.43 (dd, 1H, J=13.2, 7.1 Hz, H-6); 13C NMR
(CD3OD, 125 MHz) dppm 101.64 (C-1), 72.50 (C-5),
71.18 (C-3), 70.75 (C-2), 68.26 (C-4), 54.19 (OCH3),
51.71 (C-6). MS (ESI) m/z found 242 (M+Na+).
6-Amino-6-dexoxy-methyl-ꢀ-D-mannopyranoside (8). The
compound 7 (0.56 g, 2.56 mmol) was dissolved in 50 mL
of methanol, to which was added 50 mg of Pd/C as
catalyst. The reaction vessel was shaken at roomtem-
perature for 17 h under 52 psi of hydrogen atmosphere.
The mixture was filtered through Celite, and the filtrate
was concentrated to a very small volume. Flash column
chromatography of the residue with CH2Cl2/CH3OH
(2:1–1:1) as eluting solvent provided 8 (356 mg, 1.84
mmol) as a colorless solid. 1H NMR (CD3OD,
300 MHz) dppm 4.64 (d, 1H, J=1.2 Hz, H-1), 3.79 (dd,
2H, J=3.0, 1.8 Hz, H-2), 3.65 (dd, 1H, J=9.0 Hz, 3.0
Hz, H-3), 3.52 (t, 1H, J=9.0 Hz, H-4), 3.47–3.38 (m,
1H, H-5), 3.37 (s, 3H, OCH3), 2.98 (dd, 1H, J=13.2, 2.4
Hz, H-6), 2.79 (dd, 1H, J=13.2, 6.6 Hz, H-6); 13C
NMR (CD3OD, 75 MHz) dppm 101.63 (C-1), 72.98 (C-
5), 71.30 (C-3), 70.87 (C-2), 68.63 (C-4), 54.17 (OCH3),
42.43 (C-6). MS (ESI) m/z found 194 (M+H+), 216
(M+Na+).
1
Man-2-SNAP. H NMR (CD3OD, 500 MHz) dppm 5.38
(s, 1H, H-20), 5.03 (s, 1H, H-1), 4.32 (d, 1H, J=4.1 Hz,
H-2), 4.04 (dd, 1H, J=9.4, 4.8 Hz, H-3); 3.86 (dd, 1H,
J=10.9, 3.8 Hz, H-6), 3.81–3.73 (m, 2H, H-5 and H-6),
3.61 (t, 1H, J=9.6 Hz, H-4), 2.16 (s, 3H, CH3-40), 2.02
(s, 3H, CH3-50), 1.92 (s, 3H, CH3 of Ac); 13C NMR
(CD3OD, 125 MHz) dppm 171.74 (C¼O), 169.95 (C¼O),
93.54 (C-1), 72.21 (C-5), 69.52 (C-3), 67.25 (C-4), 60.88
(C-6), 60.20 (C-20), 58.99 (C-30), 54.04 (C-2), 26.95
(CH3-40), 23.96 (CH3-50), 21.16 (CH3 of Ac). UV lmax
229 nm, 350 nm. MS (ESI) m/z found 404 (M+Na+),
785 (2M+Na+).
1
Man-6-SNAP. H NMR (CD3OD, 400 MHz) dppm 5.32
(s, 1H, H-20), 4.61 (s, 1H, H-1), 3.78 (d, 1H, J=1.6 Hz,
H-2), 3.69–3.56 (m, 2H, H-3 and H-6), 3.56–3.44 (m,
3H, H-5, H-4 and H-6), 3.34 (s, 3H, OCH3), 2.07 (s, 3H,
CH3 of Ac), 2.00 (s, 3H, CH3-40), 1.96 (s, 3H, CH3-50);
13C NMR (CD3OD, 100 MHz) dppm 174.35 (C¼O),
171.90 (C¼O), 101.63 (C-1), 71.32 (C-5), 70.94 (C-3),
70.82 (C-2), 68.32 (C-4), 60.30 (C-20), 58.33 (C-30), 54.35
(OCH3), 40.23 (C-6), 26.28 (C-40), 22.38 (C-50), 21.24
(CH3 of Ac). UV lmax 228 nm, 346 nm. MS (ESI) m/z
found 418 (M+Na+), 753 (2M+Na+).
Man-6-AP (9). The compound 8 (210 mg, 1.09 mmol)
was dissolved in 15 mL of dry DMF, to which was
added cyclic AP (226 mg, 1.31 mmol). The reaction
mixture was stirred for one day, then the solvent was
removed under vacuum. The crude was purified by silica
gel column chromatography eluted with CH2Cl2/

X. Wu et al. / Bioorg. Med. Chem. 10 (2002) 2303–2307
2307
Acknowledgement
6. Cantuaria, G.; Magalhaes, A.; Angioli, R.; Mendez, L.;
Mirhashemi, R.; Wang, J. Q.; Wang, P. G.; Penalver, M.;
Averette, H.; Braunschweiger, P. G. Cancer 2000, 88, 381.
7. Hou, Y. C.; Wu, X. J.; Xie, W. H.; Braunschweiger, P. G.;
Wang, P. G. Tetrahedron Lett. 2001, 42, 825.
This work was supported by grants fromthe National
Institutes of Health (GM 54074).
8. Babich, H.; Zuckerbraun, H. L. Toxicol. Vitro 2001, 15,
181.
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