Concise syntheses of selective inhibitors against α-1,3- galactosyltransferase

Article abstract of DOI:10.1039/c0ob00042f

Several iminosugar-based uridine diphosphate galactose (UDP-Gal) mimetics 1-4 including d- and l-epimers were designed and synthesized by concise routes, and these synthetic compounds were evaluated for the inhibition of α-1,3- and β-1,4-galactosyltransfe

Full text of DOI:10.1039/c0ob00042f

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Concise syntheses of selective inhibitors against a-1,3-galactosyltransferase†
Guo-Liang Zhang, Li-He Zhang and Xin-Shan Ye*
Received 25th April 2010, Accepted 8th July 2010
DOI: 10.1039/c0ob00042f
Several iminosugar-based uridine diphosphate galactose (UDP-Gal) mimetics 1–4 including D- and
L-epimers were designed and synthesized by concise routes, and these synthetic compounds were
evaluated for the inhibition of a-1,3- and b-1,4-galactosyltransferases in vitro. The experimental data
demonstrated that L-epimer 2 displayed the strongest inhibitory activity with moderate selectivity
against a-1,3-galactosyltransferase.
Introduction
Oligosaccharides which are attached to proteins and lipids on
cell surfaces play pivotal roles in many important molecular
recognition processes such as bacterial and viral infections,
immune response, cell growth, cell–cell adhesion in inflammation
and metastasis, and many other intercellular communication and
signal transductions.1 Glycosyltransferases as the oligosaccharide-
synthesizing enzymes have been actively studied.2 Selective in-
hibitors against glycosyltransferases may lead to the discovery of
new therapeutic agents. The synthetic inhibitors of glycosyltrans-
ferases known to date involve acceptor analogues,3 donor sugar
nucleotide analogues,4,5 or transition-state analogues6,7 including
Fig. 1 The structures of designed target molecules 1–4.
bisubstrate8 and trisubstrate analogues.9
Compound 4 was designed because the vicinal diol moiety16a was
proved to benefit inhibition against a1,3GalT.
We have chosen a-1,3-galactosyltransferase (a1,3GalT) as
the target enzyme because of its biochemical importance in
xenotransplantation.10 a1,3GalT catalyzes the transfer of galac-
tose from UDP-galactose to an acceptor with the retention of the
anomeric configuration of the galactose moiety transferred.11,12
a1,3GalT is an enzyme expressed on the cell surface of most
mammals including primates and New World monkeys, but not
humans, apes, and Old World monkeys.13 This enzyme leads to
the Gala1,3Galb1,4GlcNAc epitope, which can react with about
1% of circulating endogenous antibodies in species lacking this
enzyme.14 It will cause the hyperacute rejection response when
pig organs are transplanted into human.15 Although selective
inhibitors against a1,3GalT are very important to xenotransplan-
tation, only limited success has been achieved so far.16 To search
for better inhibitors, herein we report the syntheses of several
iminosugar-based UDP-galactose mimetics 1–4 (Fig. 1).
Results and discussion
The preparation of UDP-galactose mimetic 1 is shown in
Scheme 1. The key materials iminosugar derivatives 5 and
6 were obtained from D-galactose according to the proce-
dure described previously.19 Treatment of commercially available
Iminosugars were chosen as the galactose mimetics since they
could mimic the transition state of the enzymatic reaction.17 On the
other hand, the O-sulfamoylamide group18 was used as the surro-
gate of pyrophosphate in UDP-galactose because this structure is
more stable under physiological conditions. To check the effect of
configurations of iminosugars on inhibitory activities, compounds
1 (D-epimer) and 2 (L-epimer) were designed. Compound 3 was
designed to examine whether an increase of the carbon atom
number of the acyl group can improve the inhibitory ability.
State Key Laboratory of Natural and Biomimetic Drugs, Peking University,
and School of Pharmaceutical Sciences, Peking University, Xue Yuan Road
#38, Beijing 100191, China. E-mail: xinshan@bjmu.edu.cn; Fax: +86 10
62014949; Tel: +86 10 82801570
† Electronic supplementary information (ESI) available: Experimental
procedures and 1H and 13C NMR spectra. See DOI: 10.1039/c0ob00042f
Scheme 1 Synthesis of UDP-galactose mimetic 1.
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2¢,3¢-isopropylidene-uridine with sulfamoyl chloride (H2NSO2Cl)
in dry pyridine gave the desired O-sulfamoylamide 7 in 58%
yield.20 The alcohol 5 was oxidized by 2,2,6,6-tetramethyl-1-
piperidinyloxy (TEMPO) free radical/(diacetoxyiodo)benzene
(BAIB) or pyridinium chlorochromate (PCC)21 to provide alde-
hyde 8 in high yield, which was readily converted to the acid
9 in 78% isolated yield by treatment with NaClO2/NaH2PO4
in the presence of 30% hydrogen peroxide.19 Next, the key
coupling reaction of
9 and 7 was carried out. Initially,
when the coupling reagents (1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate(HBTU)/diisopropyl
ethylamine (DIPEA) or N-hydroxysuccinimide (HOSu)/1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) were employed, no desired
product was obtained. There was still no reaction when compound
9 was fully deprotected which was followed by the coupling with 7
under the above-mentioned conditions. Eventually, the acid 9 was
reacted with (COCl)2 to afford the carbonyl chloride intermediate,
which was successfully converted to the coupling product 10 with
the benzyloxycarbonyl group deprotected in satisfactory yield
(84%) by the treatment with 7 in the presence of DIPEA. Full
deprotection of compound 10 was one of the critical steps of the
synthetic route. When the NaBrO3/NaS2O4 system22 was used
to cleave the benzyl groups, no desired product was produced.
Catalytic hydrogenolysis was also used to remove the benzyl
groups, but the double bond in compound 10 was always reduced
in spite of many efforts. Finally, this problem was successfully
solved by using BCl3 as the deprotection reagent.23 All benzyl
groups and the isopropylidene group were deprotected in one step
and the target compound 1 was obtained in 86% isolated yield in
the form of hydrochloride salt.
The preparation of mimetic 2 is displayed in Scheme 2. The
alcohol 6 was oxidized by TEMPO/BAIB. It was found that
different oxidation products were obtained by the use of different
solvents. The alcohol 6 was able to be directly converted to the
acid 11 in 78% isolated yield in acetonitrile–water mixed solvent,
whereas only the aldehyde product was collected in 90% yield
when dichloromethane was used as the solvent. Subsequently,
similar to the preparation of 10, the coupling reaction of 11 and
7 afforded compound 12 in 77% yield. Full deprotection of 12
by BCl3 solution achieved the target molecule 2 in satisfactory
yield. It should be noted that the control of reaction time was very
important, a long reaction time makes the product decompose
Scheme 2 Synthesis of UDP-galactose mimetic 2.
and a short reaction time leads to at least one benzyl group
remaining.
The preparation of UDP-galactose mimetic 3 is shown
in Scheme 3. Compound 8 underwent Wittig reaction with
(EtO)2P(O)CH2CO2Et24 in the presence of NaH to provide the
product, but the yield was not satisfactory (less than 70%). The
decomposition of aldehyde 8 caused by NaH might be the major
reason. Therefore, an alternative reagent Ph3P CHCO2Me25 was
used for the reaction, in which the use of NaH was avoided,
and compound 13 was afforded in excellent yield (92%) as a
chromatographically inseparable mixture of Z and E isomers. The
double bond in 13 was selectively reduced by 4% NiCl2·6H2O in the
presence of NaBH4, yielding the saturated ester 14. By treatment
of 14 with 1.0 M NaOH aqueous solution at 60 ◦C, the methyl
ester 14 was saponified and the crude product was directly reacted
with (COCl)2 followed by the coupling with O-sulfamoylamide 7
in the presence of DIPEA, successfully providing compound 15 in
good yield (73% for three steps). Global deprotection of 15 under
the above-mentioned conditions afforded the target molecule 3.
The synthesis of UDP-galactose mimetic 4 is exhibited
in Scheme 4. The aldehyde 8 was readily converted to the
oxime 16 in quantitative yield by treatment with hydroxylamine
hydrochloride.21 Reduction of 16 using NiCl2·6H2O/NaBH4 pro-
duced the amine 17 which was used for the next reaction. On the
other hand, starting from commercially available L-tartaric acid
diethyl ester, the uridine derivative 18 was synthesized via six steps
according to the reported procedure.16a Using the Dess–Martin
Scheme 3 Synthesis of UDP-galactose mimetic3.
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Scheme 4 Synthesis of UDP-galactose mimetic 4.
reagent compound 18 was oxidized to the aldehyde, which was
then coupled with the amine 17 by way of a reductive amination
approach, providing 19 in moderate yield (48% for two steps).
Deprotection of 19 using BCl3 afforded the target compound 4
in 34% yield and the by-product 20 with one benzyl group intact
in 43% yield. The structure of compound 20 was identified based
on the analyses of the 1D- (1H, 13C) and 2D- (COSY, HSQC,
HMBC) NMR spectra. Both increasing the amount of BCl3 and
prolonging the reaction time failed to improve the yield of the
target compound 4. Besides, when compound 20 was further
treated with BCl3 in the hope of getting 4, there was no reaction
and the material was fully recovered.
The synthetic compounds 1–4 and by-product 20 were eval-
uated in vitro for the inhibition of a-1,3-galactosyltransferase
(a1,3GalT)26,27 and b-1,4-galactosyltransferase (b1,4GalT)28,29 by
using radiolabeled UDP-Gal as the donor and N-acetyllactos-
amine (LacNAc) or N-acetyl-D-glucosamine(GlcNAc) as the
acceptors (see Experimental section for details). The results are
shown in Table 1. It was found that all the compounds showed
some inhibitory activities and compound 2 displayed the strongest
inhibitory ability (IC50 = 320 mM) (Fig. 2) among the five synthetic
compounds. It seemed that compounds 1, 2, 4 and 20 showed
moderate selectivity for inhibition. The experimental data showed
that the addition of the uridine moiety improved the inhibition
by comparing the inhibition of compound 2 (78% inhibition) with
that of the fully-deprotected product of 11 (5.5% inhibition at
1.0 mM against a1,3GalT). Comparing the inhibition of D-epimer
1 (63.5% inhibition) with that of L-epimer 2 (78% inhibition), it
was suggested that the configuration of the hydroxymethyl group
has effects on the inhibition against a1,3GalT. Prolonging the
carbon chain of the acyl group decreased the inhibitory activity
based on the fact that compound 1 (63.5% inhibition at 1.0 mM
against a1,3GalT) exhibited better inhibition than compound 3
(23.5% inhibition at 1.0 mM against a1,3GalT). Comparing the
inhibition of compound 4 with that of 20, it was clear that the
existence of the benzyl group can improve the inhibitory activity
against b1,4GalT, suggesting that the hydrophobic character may
be important to the inhibition.
Table 1 Inhibition of the synthetic compounds against a-1,3- and b-1,4-
galactosyltransferases
a1,3GalT
b1,4GalT
compound
concentration
inhibition concentration inhibition
1
1.0 mM
50 mM
IC50 = 320 mM
63.5%
NI
1.0 mM
50 mM
1.0 mM
50 mM
1.0 mM
50 mM
1.0 mM
50 mM
1.0 mM
50 mM
36%
NI
38%
18%
16%
12%
6%
NI
28%
22%
2
3
1.0 mM
50 mM
1.0 mM
50 mM
1.0 mM
50 mM
23.5%
NI
21%
NI
25%
NI
4
20
Fig. 2 Inhibition of a1,3GalT by compound 2: I = inhibition, C =
concentration of inhibitor.
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reaction mixture was stirred for 36 h at 40 ◦C. The solvent
was removed under reduced pressure, the residue was purified
by column chromatography on silica gel (petroleum ether/EtOAc
Conclusion
In summary, we designed and synthesized four stable iminosugar-
based UDP-Gal mimetics in high yields using concise synthetic
routes. These compounds were evaluated in vitro for the inhibition
of a-1,3- and b-1,4-galactosyltransferases. It was clear that the L-
epimer 2 displayed the strongest inhibitory ability (IC50 = 320 mM)
with moderate selectivity against a-1,3-galactosyltransferase. In
addition, prolonging the carbon chain of the acyl group decreased
the inhibitory activity. The experimental data also suggested that
the hydrophobic character may be important to the inhibition
activity against b-1,4-galactosyltransferase. The disclosed infor-
mation may benefit the discovery of more potent and selective
galactosyltransferase inhibitors.
1
18 : 1 to 15 : 1) to provide 8 (278 mg, 91%) as a yellow oil. H
NMR (500 MHz, CDCl3) d 3.67 (br.s, 1H), 3.81–3.92 (m, 3H),
3.99 (t, J = 10.0 Hz, 1H), 4.09 (d, J = 3.0 Hz, 1H), 4.38–4.61 (m,
8H), 4.75–4.85 (m, 1H), 4.99 (d, J = 12.0 Hz, 1H), 5.17 (d, J =
12.0 Hz, 1H), 7.16–7.36 (m, 25H), 9.76 (s, 1H). The data were in
good agreement with those reported.19
(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glycero-D-galacto-heptitolyl)formic acid (9). To a solu-
tion of aldehyde 8 (68.0 mg, 0.10 mmol) in acetonitrile (1.5 mL),
sodium dihydrogen phosphate in water (1.5 mL, 100 mg mL-1),
and 30% H2O2 (0.38 mL) was added dropwise a solution of sodium
chlorite (3.0 mL, 130 mg mL-1) in water. After stirring for 10 h
at room temperature, the aqueous solution was extracted with
ethyl acetate. The combined organic layers were dried (Na2SO4)
and evaporated in vacuo. The residue was purified by column
chromatography on silica gel (petroleum ether/EtOAc 3 : 1 to 1 : 1)
Experimental
General
All chemicals were purchased and used without further pu-
rification. Tetrahydrofuran (THF) was distilled over sodium/
benzophenone, methylene chloride (CH2Cl2) over calcium hydride.
All reactions were carried out under an argon atmosphere with
dry, freshly distilled solvents under anhydrous conditions, unless
otherwise noted. Reactions were monitored with analytical TLC
on silica gel 60-F254 precoated aluminium plates and visualized un-
der UV (254 nm) and/or by staining with acidic ceric ammonium
molybdate. Column chromatography was performed on silica gel
(35–75 mm). NMR spectra were recorded on a Varian VXR-
300M or Varian INOVA-500M spectrometer. Mass spectra were
recorded using a PE SCLEX QSTAR spectrometer. Elemental
analysis data were recorded on PE-2400C elemental analyzer. a-
1,3-Galactosyltransferase recombinant from E. coli (0.15 U mL-1)
and b-1,4-galactosyltransferase I human (14.6 U g-1), recombinant
from Saccharomyces cerevisiae, were purchased from Sigma. Uri-
dine 5¢diphosphate galactose-[6-3H] was purchased from Amer-
ican Radiolabeled Chemicals (specific activity: 20 Ci mmol-1,
concentration: 1 mCi mL-1). OPTi-Fluor scintillation cocktail
was purchased from Perkin Elmer. The scintillation counter used
was the Packard Tri-Carb 2100 TR. Other reagents were from
commercial sources.
1
to yield 9 (54.2 mg, 78%) as a yellow oil. H NMR (500 MHz,
CDCl3) d 3.55 (dd, J = 8.0, 3.0 Hz, 1H), 3.80–4.06 (m, 4H), 4.12
(dd, J = 7.0, 4.5 Hz, 1H), 4.28–4.44 (m, 3H), 4.51 (d, J = 11.5 Hz,
1H), 4.57 (d, J = 11.5 Hz, 1H), 4.61 (d, J = 12.0 Hz, 1H), 4.63–
4.70 (m, 2H), 4.78 (br., 1H), 4.84 (d, J = 12.0 Hz, 1H), 5.02 (d,
J = 12.5 Hz, 1H), 7.11–7.24 (m, 25H). The 1H NMR data were in
good agreement with those reported.19
2¢,3¢-O-Isopropylidene-5¢-O-(2,3,4,6-tetra-O-benzyl-1,5-did-
eoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)formamidosulfa-
moyluridine (10). To a solution of 9 (93 mg, 0.13 mmol) in
dry toluene (6.0 mL) was added (COCl)2 (95 mL, 1.30 mmol).
The reaction mixture was stirred for 10 h at 40 ◦C. The solvent
was removed under reduced pressure, the residue was dissolved
in CH2Cl2 (6.0 mL). To this solution, compound 7 (70 mg,
0.20 mmol) and DIPEA (46 mL, 0.26 mmol) were added. After
stirring overnight at room temperature, the solvent was removed
and the residue was purified by column chromatography on silica
gel (petroleum ether/EtOAc/methanol 2 : 1 : 0.08 to 2 : 1 : 0.1) to
provide 10 (102 mg, 84%) as a colorless oil. 1H NMR (500 MHz,
DMSO): d 1.26 (s, 3H), 1.46 (s, 3H), 3.69–3.71 (m, 4H), 3.95–3.98
(m, 2H), 4.09 (dd, J = 5.0, 10.5 Hz, 1H), 4.14–4.17 (m, 2H), 4.21
(dd, J = 4.5, 9.0 Hz, 1H), 4.43 (d, J = 12.5 Hz, 1H), 4.50 (d, J =
12.5 Hz, 1H), 4.51 (d, J = 12.0 Hz, 1H), 4.52 (d, J = 11.5 Hz,
1H), 4.54 (d, J = 12.0 Hz, 1H), 4.58 (d, J = 12.0 Hz, 1H), 4.62
(d, J = 11.5 Hz, 1H), 4.63 (d, J = 12.0 Hz, 1H), 4.76 (dd, J =
3.5, 6.0 Hz, 1H), 4.94 (dd, J = 2.5, 6.5 Hz, 1H), 5.61 (d, J =
8.0 Hz, 1H), 5.83 (d, J = 2.5 Hz, 1H), 7.24–7.37 (m, 20H), 7.77
(d, J = 8.0 Hz, 1H), 8.86 (br.s, 2H), 11.40 (br.s, 1H). 13C NMR
(125 MHz, DMSO): d 25.14, 26.97, 54.14, 67.38, 70.74, 72.04,
72.30, 72.62, 80.73, 83.46, 84.16, 91.74, 101.93, 113.18, 127.27,
127.45, 127.65, 128.09, 128.17, 128.24, 128.30, 138.15, 142.42,
150.31, 163.19. HRMS (ESI, positive) Calcd for C47H53N4O13S
[(M + H)+] 913.3324, found: 913.3328.
2¢,3¢-O-Isopropylidene-5¢-O-sulfamoyluridine
(7). 2¢,3¢-O-
Isopropylidene-uridine (570 mg, 2.0 mmol) was suspended in
CH2Cl2 (20 mL) and dry pyridine (0.57 mL). Sulfamoyl chloride
(463 mg, 4.0 mmol) was added to the stirred reaction mixture.
After stirring for one day at 40 ◦C, the solvent was removed and
the residue was purified by column chromatography on silica gel
(CH2Cl2–methanol 80 : 1 to 60 : 1) to provide 7 (420 mg, 58%) as a
white solid. 1H NMR (500 MHz, d6-DMSO): d 1.20 (s, 3H), 1.49
(s, 3H), 4.15 (dd, J = 2.5, 11.5 Hz, 1H), 4.22–4.25 (m, 2H), 4.80
(dd, J = 3.5, 6.5 Hz, 1H), 5.06 (dd, J = 1.5, 6.5 Hz, 1H), 5.64 (dd,
J = 2.0, 8.0 Hz, 1H), 5.82 (d, J = 2.0 Hz, 1H), 7.60 (s, 2H), 7.71
(d, J = 4.0 Hz, 1H), 11.44 (br.s, 1H). The 1H NMR data were in
good agreement with those reported.20
5¢-O-(1,5-Dideoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)for-
mamidosulfamoyluridine (1). To a solution of 10 (33 mg,
0.04 mmol) in dry CH2Cl2 (3.0 mL) was added BCl3 in hexane
(1 M solution, 0.48 mL, 0.48 mmol). The reaction mixture
was stirred for 3 h at 0 ◦C, and then the solids formed were
(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glycero-D-galacto-heptitolyl)aldehyde (8). To a solution
of5 (307 mg, 0.45 mmol) in CH2Cl2 (20 mL) was added BAIB
(260 mg, 0.81 mmol) and TEMPO (63 mg, 0.41 mmol). The
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dissolved by the addition of MeOH (1.0 mL) and water (1.0 mL).
The solvent was removed and the residue was purified by flash
column chromatography on C18-reverse phase silica gel (H2O as
eluent), providing 1 (17 mg, 86%) as a white solid in the form of
70.21, 70.71, 70.94, 74.18, 82.48, 89.64, 103.23, 142.37, 152.48,
166.97. HRMS (ESI, positive) Calcd for C16H25N4O13S [(M + H)+]
513.1133, found: 513.1133.
(2,3,4,6-Tetra-O-benzyl-N -benzyloxycarbonyl-1,5-dideoxy-
1,5-imino-D-glycero-D-galacto-heptitolyl)methyl acrylate (13). To
a solution of 8 (170 mg, 0.25 mmol) in dry THF (5.0 mL) was
added Ph3P CHCOOMe (165 mg, 0.50 mmol). The reaction
mixture was stirred overnight at room temperature. The reaction
was quenched by several drops of NH4Cl aqueous solution, and
the mixture was extracted with EtOAc (3 ¥ 50 mL) and washed
with aqueous NaHCO3 (20 mL) and NaCl (20 mL). The organic
phases were combined, dried (Na2SO4), filtered and concentrated.
The residue was purified by column chromatography on silica gel
(petroleum ether/EtOAc 15 : 1 to 13 : 1) to provide 13 (170 mg,
92%) as a colorless oil.1H NMR (500 MHz, CDCl3): d 3.47 (dd,
J = 3.0, 9.0 Hz, 1H), 3.73 (s, 3H), 3.80–3.88 (m, 1H), 4.00–4.07 (m,
3H), 4.19–4.57 (m, 4H), 4.59–4.65 (m, 1H), 4.68 (d, J = 11.5 Hz,
2H), 4.72 (d, J = 12.0 Hz, 2H), 4.78 (d, J = 11.5 Hz, 1H), 4.89 (br.s,
1H), 5.08 (d, J = 7.0 Hz, 1H), 5.20 (d, J = 2.0 Hz, 1H), 5.98–6.03
(m, 1H), 7.18–7.34 (m, 25H). MS-ESI: 742 [M + H+]. Anal. Calcd
for C46H47NO8: C, 74.47; H, 6.39; N, 1.89. Found: C, 74.52; H,
6.35; N, 1.88%.
1
hydrochloride salt. H NMR (500 MHz, D2O): d 3.81–3.84 (m,
1H), 3.94–4.02 (m, 3H), 4.25–4.28 (m, 2H), 4.31–4.35 (m, 4H),
4.40 (dd, J = 1.5, 11.5 Hz, 1H), 4.48 (d, J = 11.5 Hz, 1H), 5.93–
5.95 (m, 2H), 7.81 (dd, J = 1.5, 8.5 Hz, 1H). 13C NMR (125 MHz,
D2O): d 56.32, 58.00, 58.28, 65.40, 68.37, 69.31, 70.11, 70.50, 74.15,
82.24, 89.85, 103.20, 142.36, 152.37, 166.89, 172.70. HRMS (ESI,
positive) Calcd for C16H25N4O13S [(M + H)+] 513.1133, found:
513.1138.
(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glycero-L-galacto-heptitolyl)formic acid (11). To a solu-
tion of 6 (82 mg, 0.12 mmol) in CH3CN and H2O (v/v 1 : 1,
20 mL) was added BAIB (98 mg, 0.30 mmol) and TEMPO (19 mg,
0.12 mmol). The reaction mixture was stirred for 24 h at room
temperature. The solvent was removed under reduced pressure,
and the residue was extracted with EtOAc (3 ¥ 50 mL) and washed
with brine (20 mL). The organic layers were combined, dried
(Na2SO4), filtered, and concentrated. The residue was purified
by column chromatography on silica gel (petroleum ether/EtOAc
6 : 1 to 3 : 1) to provide 11 (65 mg, 78%) as a yellow oil. 1H NMR
(500 Hz, CDCl3) d 3.54–3.55 (m, 1H), 3.86–3.87 (m, 1H), 3.99 (br.s,
1H), 4.36–4.52 (m, 10H), 4.72–4.80 (m, 2H), 5.10–5.21 (m, 2H),
(2, 3, 4, 6 - Tetra - O - benzyl - N - benzyloxycarbonyl - 1, 5 - dide -
oxy-1,5-imino-D-glycero-D-galacto-heptitolyl)methyl propionate
(14). To a solution of 13 (170 mg, 0.23 mmol) in dry CH2Cl2
(1.3 mL) and MeOH (8.0 mL) was added 4% solution of
NiCl2·6H2O in MeOH (2.3 mL, 0.39 mmol) at 0 ◦C. After
stirring for 20 min, a portion of NaBH4 (35 mg, 0.92 mmol)
was added. The reaction mixture was stirred for another 5 h at
room temperature. The reaction was quenched by several drops
of HOAc. The solvent was removed and the residue was purified
by column chromatography on silica gel (petroleum ether/EtOAc
18 : 1 to 16 : 1) to provide 14 (145 mg, 85%) as a yellow oil.
1H NMR (400 MHz, d6-DMSO, 70 ◦C): d 1.97–2.00 (m, 2H),
2.24–2.27 (m, 2H), 3.52 (s, 3H), 3.63 (br.s, 1H), 3.73–3.82 (m, 2H),
3.91 (br.s, 2H), 4.01 (br.s, 1H), 4.36 (d, J = 12.0 Hz, 2H), 4.42 (d,
J = 12.4 Hz, 1H), 4.47 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 12.4 Hz,
1H), 4.61 (d, J = 12.0 Hz, 1H), 4.65 (d, J = 12.0 Hz, 2H), 4.69 (d,
J = 8.3 Hz, 1H), 4.86 (d, J = 12.8 Hz, 1H), 5.01 (d, J = 12.4 Hz,
1H), 7.20–7.35 (m, 25H). 13C NMR (125 MHz, d6-DMSO): d
18.99, 30.19, 51.17, 53.85, 54.44, 66.48, 67.70, 71.67, 71.88, 71.95,
73.42, 74.29, 76.20, 79.68, 127.28, 127.36, 127.43, 127.69, 128.02,
128.17, 128.24, 129.12, 129.45, 134.54, 136.68, 138.26, 138.48,
138.87, 154.96, 157.00, 173.03. MS-ESI: 766 [M + Na+]. Anal.
Calcd for C46H49NO8: C, 74.27; H, 6.64; N, 1.88. Found: C, 74.53;
H, 6.87; N, 1.79%.
1
7.26–7.32 (m, 25H). The H NMR data were in good agreement
with those reported previously.19
2¢,3¢-O-Isopropylidene-5¢-O-(2,3,4,6-tetra-O-benzyl-1,5-did-
eoxy-1,5-imino-D-glycero-L-galacto-heptitolyl)formamidosulfa-
moyluridine (12). Compound 12 was prepared from 11 (20 mg,
0.03 mmol) as described in the preparation of 10, providing 12
(20 mg, 77%) as a colorless oil.1H NMR (500 MHz, d6-DMSO): d
1.26 (s, 3H), 1.46 (s, 3H), 3.70 (d, J = 4.5 Hz, 2H), 3.77 (br.s, 2H),
4.04 (br.s, 1H), 4.08 (dd, J = 5.5, 11.0 Hz, 1H), 4.15 (dd, J = 4.0,
10.5 Hz, 1H), 4.21 (dd, J = 4.0, 8.5 Hz, 1H), 4.31 (d, J = 11.5 Hz,
2H), 4.44 (d, J = 12.0 Hz, 1H), 4.48 (d, J = 12.5 Hz, 2H), 4.55 (d,
J = 12.0 Hz, 2H), 4.61 (d, J = 11.5 Hz, 1H), 4.63 (d, J = 11.5 Hz,
1H), 4.67 (d, J = 11.5 Hz, 1H), 4.76 (dd, J = 3.5, 6.0 Hz, 1H), 4.95
(dd, J = 2.5, 6.0 Hz, 1H), 5.61 (dd, J = 1.5, 8.0 Hz, 1H), 5.83 (d,
J = 2.5 Hz, 1H), 7.14–7.36 (m, 20H), 7.76 (d, J = 8.5 Hz, 1H),
8.67 (br.s, 2H), 11.40 (d, J = 1.5 Hz, 1H). 13C NMR (125 MHz,
d6-DMSO): d 25.12, 26.95, 52.17, 58.08, 67.40, 70.33, 72.13, 72.29,
72.58, 80.75, 83.43, 84.15, 91.77, 101.89, 113.15, 127.52, 127.62,
127.79, 127.94, 128.02, 128.14, 128.19, 128.27, 137.86, 142.42,
150.29, 163.15. HRMS (ESI, positive) Calcd for C47H53N4O13S
[(M + H)+] 913.3324, found: 913.3337.
5¢O-(1,5-Dideoxy-1,5-imino-D-glycero-L-galacto-heptitolyl)for-
mamidosulfamoyluridine (2). Compound 2 was prepared from 12
(32 mg, 0.04 mmol) as described in the preparation of 1, providing
light yellow solids. The crude product was purified by HPLC (95%
water and 5% methanol as eluent), giving 2 (15 mg, 80%) as a
white solid in the form of hydrochloride salt after lyophilization.
1H NMR (500 MHz, D2O): d 3.18 (br.s, 1H), 3.86 (dd, J = 5.5,
12.0 Hz, 1H), 3.92 (br.s, 2H), 3.95 (dd, J = 2.5, 10.5 Hz, 1H), 4.06
(t, J = 3.5 Hz, 1H), 4.31–4.36 (m, 5H), 4.43 (d, J = 11.0 Hz, 1H),
5.94 (s, 1H), 5.96 (d, J = 4.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H).
13C NMR (125 MHz, D2O): d 55.38, 59.37, 60.16, 64.72, 68.82,
2¢,3¢-O-Isopropylidene-5¢-O-(2,3,4,6-tetra-O-benzyl-N-benzylo-
xycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)-
propionamidosulfamoyluridine (15). To a solution of 14 (55 mg,
0.07 mmol) in MeOH (2.0 mL) was added 1 M NaOH aqueous
solution (0.28 mL, 0.28 mmol). After stirring for 5 h at 60 ◦C,
a portion of cation exchange resin was added until the pH
value was between 5 and 6. After the resin was filtered off, the
solvent was removed and the residue was purified by short column
chromatography on silica gel (petroleum ether/EtOAc 3 : 1 to
1 : 1) to provide the crude product (51 mg). The product was
treated under the conditions as described in the preparation of 10,
5066 | Org. Biomol. Chem., 2010, 8, 5062–5068
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yielding 15 (58 mg, 73% for three steps) as a colorless oil.1H NMR
(500 MHz, d6-DMSO): d 1.26 (s, 3H), 1.46 (s, 3H), 1.98 (br.s,
2H), 2.27–2.31 (m, 2H), 3.75–3.79 (m, 4H), 3.99–4.05 (m, 2H),
4.22–4.25 (m, 2H), 4.37 (dd, J = 7.0, 10.5 Hz, 3H), 4.47 (dd, J =
4.0, 10.5 Hz, 2H), 4.57 (br.s, 2H), 4.70 (br.s, 4H), 4.79 (dd, J = 4.0,
6.5 Hz, 1H), 5.06 (dd, J = 2.0, 6.5 Hz, 2H), 5.61 (dd, J = 2.5, 8.0 Hz,
1H), 5.80 (d, J = 2.0 Hz, 1H), 7.23–7.35 (m, 20H), 7.70 (d, J =
8.0 Hz, 1H), 11.44 (d, J = 2.0 Hz, 1H), 12.21 (br.s, 1H). 13C NMR
(125 MHz, d6-DMSO): d 25.01, 26.81, 32.07, 54.42, 71.59, 71.88,
72.02, 74.14, 80.72, 83.53, 84.22, 93.31, 101.81, 113.30, 127.30,
127.47, 127.79, 128.00, 128.13, 128.17, 128.21, 128.27, 138.86,
143.23, 150.29, 163.23, 171.02. HRMS (ESI, positive) Calcd for
C57H66N5O15S [(M + NH4)+] 1092.4271, found: 1092.4254.
heptitolyl)ethylamine-4¢,5¢-O-isopropylidene-vicinal diolyl)uridine
(19). To a solution of 18 (20 mg, 0.05 mmol) in CH2Cl2 (2.0 mL)
was added Dess–Martin periodinane (30 mg, 0.08 mmol) at
0
◦C. After stirring for 3 h, the mixture was extracted with
EtOAc (3 ¥ 40 mL) and washed with NaHCO3 aqueous solution
(10 mL) and brine (10 mL). The organic phases were combined,
dried (Na2SO4), filtered and concentrated. The crude product
was dissolved in MeOH (2.0 mL), and a solution of compound
17 (32 mg, 0.05 mmol) in MeOH (2.0 mL) was added. Then a
portion of HOAc was added dropwise until the pH value was
between 5 and 6. After stirring for 1 h at room temperature,
a portion of NaBH3CN (10.0 mg, 0.15 mmol) was added. The
reaction mixture was stirred overnight at room temperature, and
quenched with 1 N HCl aqueous solution (0.2 mL). The mixture
was extracted with EtOAc (3 ¥ 30 mL), washed with NaHCO3
aqueous solution (10 mL) and brine (10 mL). The organic phases
were combined, dried (Na2SO4), filtered and concentrated. The
residue was purified by column chromatography on silica gel
(petroleum ether/EtOAc 1 : 1 to 1 : 1.5) to provide 19 (24 mg, 48%
for two steps) as a colorless oil. 1H NMR (500 MHz, d6-DMSO):
d 1.24 (s, 3H), 1.27 (s, 3H), 1.28 (s, 3H), 1.46 (s, 3H), 2.66–2.70 (m,
3H), 2.78–3.20 (m, 5H), 3.76–4.13 (m, 8H), 4.40–4.64 (m, 9H), 4.71
(dd, J = 4.0, 6.5 Hz, 1H), 4.91 (br.s, 1H), 5.02 (dd, J = 2.0, 6.5 Hz,
2H), 5.62 (d, J = 8.0 Hz, 1H), 5.75 (s, 1H), 5.78 (d, J = 2.0 Hz, 1H),
7.27–7.32 (m, 25H), 7.69 (d, J = 8.0 Hz, 1H). 13C NMR (125 MHz,
DMSO): d 25.10, 26.90, 27.03, 27.07, 34.21, 34.42, 50.77, 54.88,
66.31, 71.86, 73.95, 78.73, 79.54, 82.83, 83.46, 85.74, 92.03, 101.89,
108.09, 113.26, 127.24, 127.30, 127.37, 127.66, 128.08, 128.14,
128.17, 128.24, 136.61, 138.21, 138.47, 138.60, 138.73, 142.80,
150.21, 163.16. HRMS (ESI, positive) Calcd for C62H73N4O15S
[(M + H)+] 1113.4889, found: 1113.4902.
5¢-O-(1,5-Dideoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)-
propionamidosulfamoyluridine (3). Compound 3 was prepared
from 15 (39 mg, 0.04 mmol) as described in the preparation of
1, providing light yellow solids. The crude product was purified
by HPLC (70% water and 30% methanol as eluent), giving 3
(15 mg, 72%) as a white solid in the form of hydrochloride salt
1
after lyophilization. H NMR (500 MHz, D2O): d 1.91–1.99 (m,
1H), 2.15–2.22 (m, 1H), 2.53–2.67 (m, 2H), 3.60–3.63 (m, 1H), 3.70
(dd, J = 6.5, 11.5 Hz, 1H), 3.87 (dd, J = 3.5, 8.5 Hz, 1H), 3.91–3.97
(m, 2H), 4.11 (dd, J = 4.5, 8.5 Hz, 1H), 4.20 (t, J = 3.0 Hz, 1H),
4.29–4.34 (m, 2H), 4.36 (t, J = 4.5 Hz, 1H), 4.46 (dd, J = 3.5,
11.5 Hz, 1H), 4.53 (dd, J = 2.0, 11.5 Hz, 1H), 5.91 (d, J = 4.0 Hz,
1H), 5.93 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz, 1H). 13C NMR
(125 MHz, D2O): d 20.74, 34.16, 54.23, 56.61, 58.58, 66.28, 67.45,
69.62, 69.86, 71.09, 73.91, 81.62, 90.42, 103.14, 142.52, 152.31,
166.90, 175.97. HRMS (ESI, positive) Calcd for C18H29N4O13S
[(M + H)+] 541.1446, found: 541.1452.
(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glycero-D-galacto-heptitolyl)oxime (16). To a solution
of 8 (275 mg, 0.40 mmol) in MeOH (8.0 mL) was added
hydroxylamine hydrochloride (42 mg, 0.60 mmol) and KHCO3
(60 mg, 0.60 mmol). The reaction mixture was stirred for 5 h at
room temperature. The mixture was extracted with EtOAc (3 ¥
80 mL) and washed with NaHCO3 aqueous solution (20 mL) and
brine (20 mL). The organic phases were combined, dried (Na2SO4),
filtered and concentrated. The residue was purified by column
chromatography on silica gel (petroleum ether/EtOAc 6 : 1 to 5 : 1)
to provide 16 (281 mg, 100%) as a colorless oil. The 1H NMR data
were in good agreement with those reported previously.21
5¢-S-((1,5-Dideoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)-
ethylamine-vicinal diolyl)uridine (4) and 5¢-S-((2-O-benzyl-1,5-
dideoxy-1,5-imino-D-glycero-D-galacto-heptitolyl)ethylamine-vici-
nal diolyl)uridine (20). Compounds 4 and 20 were prepared from
19 (22 mg, 0.02 mmol) as described in the preparation of 1,
providing light yellow solids 4 (3.9 mg, 34%) and white solids 20
(5.7 mg, 43%) in the form of hydrochloride salt after lyophilization.
For compound 4:1H NMR (500 MHz, D2O): d 2.79 (dd, J = 8.0,
13.5 Hz, 1H), 2.86 (dd, J = 5.5, 13.5 Hz, 1H), 2.93–2.97 (m, 1H),
3.04–3.08 (m, 1H), 3.31–3.37 (m, 2H), 3.49–3.52 (m, 2H), 3.78–
3.93 (m, 5H), 4.09–4.15 (m, 2H), 4.19–4.22 (m, 3H), 4.28 (dd, J =
5.0, 9.0 Hz, 1H), 4.41–4.43 (m, 1H), 5.86 (d, J = 4.5 Hz, 1H), 5.91
(d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H). 13C NMR (125 MHz,
D2O): d 34.59, 35.69, 44.90, 51.74, 57.10, 59.32, 65.60, 66.92, 67.02,
68.04, 69.88, 71.70, 72.66, 73.75, 83.37, 90.95, 103.17, 142.96,
152.28, 166.90. HRMS (ESI, positive) Calcd for C20H35N4O11S
[(M + H)+] 539.2018, found: 539.2022. For compound 20:1H NMR
(500 MHz, D2O): d 2.76 (dd, J = 8.0, 14.0 Hz, 1H), 2.83 (dd, J =
5.5, 14.0 Hz, 1H), 2.93–2.97 (m, 1H), 3.04–3.07 (m, 1H), 3.13–
3.16 (m, 3H), 3.35 (dd, J = 9.0, 13.5 Hz, 1H), 3.41 (dd, J = 5.0,
13.0 Hz, 1H), 3.70–3.79 (m, 4H), 3.84 (dd, J = 3.0, 10.0 Hz, 1H),
3.97 (dt, J = 2.5, 7.0 Hz, 1H), 4.02 (dd, J = 5.5, 9.5 Hz, 1H),
4.06–4.07 (m, 1H), 4.18–4.21 (m, 2H), 4.40 (t, J = 4.5 Hz, 1H),
4.68 (d, J = 11.5 Hz, 1H), 4.80 (d, J = 11.5 Hz, 1H), 5.85 (d, J =
4.5 Hz, 1H), 5.88 (d, J = 8.5 Hz, 1H), 7.42–7.49 (m, 5H), 7.72 (d,
J = 8.0 Hz, 1H). 13C NMR (125 MHz, D2O): d 34.61, 35.68, 44.75,
50.56, 54.87, 61.02, 68.21, 68.98, 69.96, 71.71, 72.66, 73.77, 74.43,
(2,3,4,6-Tetra-O-benzyl-N-benzyloxycarbonyl-1,5-dideoxy-1,5-
imino-D-glycero-D-galacto-heptitolyl)ethylamine (17). To a solu-
tion of 16 (280 mg, 0.40 mmol) in MeOH (8.0 mL) was added
NiCl2·6H2O (485 mg, 2.0 mmol) and NaBH4 (15 mg, 4.0 mmol).
The reaction mixture was stirred for 5 h at room temperature. The
reaction was quenched by several drops of HOAc. The mixture
was extracted with EtOAc (3 ¥ 80 mL) and washed with NaHCO3
aqueous solution (20 mL) and brine (20 mL). The organic phases
were combined, dried (Na2SO4), filtered and concentrated. The
residue was purified by flash column chromatography on silica gel
(petroleum ether/EtOAc 1.5 : 1 to 1 : 1.5 containing 1% NH3·H2O)
to provide 17 (220 mg) as a yellow oil, which was unstable and
directly used for the next reaction.
2¢¢,3¢¢-O -Isopropylidene-5¢¢-S -((2,3,4,6-tetra-O -benzyl-N -
benzyloxycarbonyl-1,5-dideoxy-1,5-imino-D-glycero-D-galacto-
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Org. Biomol. Chem., 2010, 8, 5062–5068 | 5067
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View Article Online
76.57, 83.45, 90.96, 103.15, 129.43, 129.55, 129.68, 137.89, 142.91,
152.20, 166.83. HRMS (ESI, positive) Calcd for C27H41N4O11S
[(M + H)+] 629.2487, found: 629.2483.
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Med. Chem., 2006, 14, 7293.
Activity measurements of inhibitors against a-1,3-
galactosyltransferase
The assays contained MnCl2 (10 mM), a-1,3-galactosyltransferase
(0.0004 U mL-1), UDP-[6-3H]-galactose (0.05 mM), UDP-
galactose (49.5 mM), LacNAc (0.5 mM), inhibitor (0.025–
2.0 mM), bovine serum albumin (1.0 mg mL-1), MES buffer
(50 mM, pH 6.0) in a total assay volume of 0.05 mL. Assays
were performed at 37 ◦C for 30 min. Reactions were quenched by
the addition of cold water (0.3 mL). The reaction mixtures were
transferred onto a Dowex 1 ¥ 8 column (1.0 mL). The column
was washed with an additional 0.6 mL of water (2 ¥ 0.3 mL). The
flow-through and column washes were collected and added OPTi-
Fluor scintillation cocktail (4.0 mL). A control experiment without
enzyme and inhibitor was used to establish the background count.
The IC50 was determined by plotting the ratio of inhibition versus
concentration of inhibitor.
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Activity measurements of inhibitors against b-1,4-
galactosyltransferase
The assays contained MnCl2 (10 mM), b-1,4-galactosyltransferase
(0.004 U mL-1), UDP-[6-3H]-galactose (0.05 mM), UDP-galactose
(49.5 mM), GlcNAc (0.5 mM), inhibitor (0.05–1.0 mM), bovine
serum albumin (1.0 mg mL-1), HEPES buffer (50 mM, pH 7.4)
in a total assay volume of 0.05 mL. Reaction temperature, work
up, etc. are the same as the above-described inhibitory assay for
a-1,3-galactosyltransferase.
Acknowledgements
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 90713010) and the grant
(2009ZX09501-011) from the Ministry of Science and Technology
of China.
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5068 | Org. Biomol. Chem., 2010, 8, 5062–5068
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The Royal Society of Chemistry 2010
©