Study on glycosylated prodrugs of toxoflavins for antibody-directed enzyme tumor therapy

Article abstract of DOI:10.1016/j.carres.2007.03.006

Eight novel toxoflavin glycosides, which are potential prodrugs in antibody directed enzyme prodrug therapy (ADEPT), were synthesized. The structures of all toxoflavin glycosides were characterized by ¹³C NMR spectroscopy, elemental analysis, and MS. Their enzymatic hydrolysis activities were tested against β-glucosidase (EC.3.2.1.21).

Full text of DOI:10.1016/j.carres.2007.03.006

Carbohydrate Research 342 (2007) 1254–1260
Note
Study on glycosylated prodrugs of toxoflavins for
antibody-directed enzyme tumor therapy
Shusheng Wang,a,b Dan Liu,a,b Xu Zhang,a Shengyu Li,a Yongxu Sun,a Jia Li,a
Yifa Zhoua,* and Liping Zhanga,*
aSchool of Life Sciences, Northeast Normal University, Changchun 130024, PR China
bSchool of Life Sciences, Anhui University, Hefei 230039, PR China
Received 21 January 2007; received in revised form 24 February 2007; accepted 5 March 2007
Available online 12 March 2007
Abstract—Eight novel toxoflavin glycosides, which are potential prodrugs in antibody directed enzyme prodrug therapy (ADEPT),
were synthesized. The structures of all toxoflavin glycosides were characterized by 13C NMR spectroscopy, elemental analysis, and
MS. Their enzymatic hydrolysis activities were tested against b-glucosidase (EC.3.2.1.21).
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Toxoflavins; Toxoflavin glycosides; Prodrugs; b-Glucosidase; Anticancer
Toxoflavin, a yellow crystalline solid isolated in 1933
from Pwsendomoras cacovenenan, was determined by
total synthesis in 1961.1 In previous studies, toxoflavin
has stimulated considerable interest because of its broad-
spectrum antibacterial activity.2,3 Recently, toxoflavin
(1) and its analogs (2) (Scheme 1) were identified as po-
tential novel anticancer agents by their action against
Polo-like kinase (PLK1). However, there are still several
limitations for their clinical use because of their high
toxicity and poor solubility.4 As glycosylation usually
increases the solubility and changes the activities of
compounds, it is possible to overcome these drawbacks
if we convert toxoflavin into glycosides and use them in
ADEPT.5 Thus, in this paper we report a trial for the
synthesis of toxoflavin glycosides and their hydrolysis
catalyzed by b-glucosidase.
We have synthesized eight toxoflavin glycosides (10a–
d), (17a–d) containing glucose and lactose as the carbo-
hydrate residue. For the toxoflavin glycosides (10a–d),
glucose and lactose were linked to the toxoflavin by aro-
matic space-armed and the synthetic route is described
in Scheme 2. Here, the corresponding 2,3,4,6-tetra-O-
acetyl-D-glucopyranosyl bromide (3a) and 2,3,4,6,20,
30,60-hepta-O-acetyllactosyl bromide (3b) were firstly
prepared by the method described in the literature and
used as glycosyl donor.6 The commercially available
compound 4a (4-hydroxybenzaldehyde) and 4b (4-
hydroxy-3-methoxy-benzaldehyde, Vanillin) as glycosyl
accepters were separately coupled to the glycosyl donors
3a and 3b by using TBAB (tetrabutylammonium bro-
mide) as phase-transfer catalyst in satd aq K2CO3 and
dichloromethane.7,8 The reactions were completed as
indicated by TLC monitoring and subjected to flash
chromatographyic purification to afford the four glyco-
sides (5a–d) as the white solid in yields of 26–62%. The
toxoflavin glycosides (10a–d) were synthesized according
to the known procedure.9 Stirring of glycosides (5a–d)
O
N
O
N
Me
O
N
N
Me
O
N
N
R
N
N
N
N
Me
Me
Benzene or
Methoxy-benzene
R=
1
2
Scheme 1. Toxoflavin and toxoflavin analogs.
*
Corresponding authors. Tel./fax: +86 431 85098212; e-mail:
zhouyf383@nenu.edu.cn
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.03.006

S. Wang et al. / Carbohydrate Research 342 (2007) 1254–1260
1255
O
Me
N
R1
HO
NH2
R1
O
H
O
N
H
N
OAc
OAc
O
O
H
Me
R2O
AcO
O
R2O
AcO
O
6
4
Br
OAc
OAc
b
a
3
5
O
Me
O
AcO
O
OAc
OR2
N
N C
O
R1
N
H
N
H
AcO
Me
7
c
AcO
O
OAc
AcO
O
OR2
OAc
O
O
N
OR2
+
O
N
O
Me
O
R1
AcO
Me
O
N
N
N
R1
AcO
N
AcO
OAc
O
OAc
N
N
N
O
N
5d,7d,8d,9d: R1=OMe, R2= AcO
Me
9
Me
8
10a: R1=H, R2=OH
d
OH
HO
HO
3a: R2=Ac
AcO
O
e
10b: R1=H, R2=
OH
OH
OR2
OAc
O
HO
O
10c: R1=OMe, R2=OH
3b: R2=
AcO
4a: R1=H
4b: R1=OMe
5a,7a,8a,9a: R1=H, R2=Ac
AcO
O
OH
HO
10d: R1=OMe, R2=
HO
O
OAc
HO
O
OH
Me
N
N
R1
N
O
N
N
10
Me
OAc
O
OAc
5b,7b,8b,9b: R1=H, R2=
AcO
5c,7c,8c,9c: R1=OMe, R2=Ac
Scheme 2. Reagents and conditions: (a) TBAB (tetrabutylammonium bromide), CH2Cl2: satd aq K2CO3 = 1:1, 60 ꢁC, 3 h; (b) anhydrous acetic acid,
5 ꢁC, 90 min; (c) NaNO2, rt, 90 min; (d) Na2S2SO4, CH2Cl2:water = 1:1, rt, overnight; (e) MeONa in MeOH, rt, overnight.
and compound
6
(3-methyl-6-(1-methylhydrazino)-
white solid in yields 89–94% without purification. The
synthesis of toxoflavin glycosides (17a–d) were carried
out according to the synthetic method of (10a–d). Stir-
ring of glycosides (13a–d) and compound 6 in anhydrous
acetic acid at 5 ꢁC for 90 min, the Schiff bases (14a–d)
were formed then, by nitrative cyclization, afforded the
compounds (15a–d) and its 4-oxide analogs (16a–d).
Followed via reduction and deprotection, we obtained
the water soluble toxoflavin glycosides (17a–d) as yellow
solid in yields 60–80%.
In the ADEPT approach, a key step is that the anti-
cancer drug must be released from inactive prodrug by
enzyme. The b-glucosidase (EC.3.2.1.21) was used to
hydrolyze the eight toxoflavin glycosides (10a–d),
(17a–d) in this study. The kinetics were measured via
analysis of generating of reduced sugar by 3,5-dinitro-
salicyclic acid assay (Scheme 4).14 the result showed only
10a and 10c can be hydrolyzed at pH 7.4 (0.002 M phos-
phate buffer) and 37 ꢁC in vitro, the other six toxoflavin
glycosides 10b, 10d and (17a–d) would not be hydro-
lyzed in the same condition. Furthermore, for the same
glycosides concentration (0.01 mmol/mL) and enzyme
concentration (0.69 lm/mL), the speed of enzymatic
hydrolysis indicated that glycoside 10a was more
quicker than 10c (Fig. 1). From the result, it clearly
uracil) in anhydrous acetic acid at 5 ꢁC for 90 min gave
the Schiff bases (7a–d). In the acid condition, after
sodium nitrite was added and stirring for 90 min at
room temperature, obtained compounds (8a–d) and its
4-oxide analog compounds (9a–d). Then compounds
(9a–d) were reduced to compounds (8a–d) by the mild
reducer hyposulphite in dichloromethane and water.10
Finally compounds (8a–d) were deprotected in
NaOMe/MeOH.11 The water soluble toxoflavin glyco-
sides (10a–d) were synthesized as the yellow solid in
yields of 56–70%.
For the other toxoflavin glycosides (17a–d), glucose
and lactose were linked to the toxoflavin by an aliphatic
spacer-arm. The synthetic route is described in Scheme
3. The glycosyl donors 3a, 3b were reacted, respectively,
with glycol 11a and diethylene glycol 11b in the presence
of HgBr2 as catalyst in dry dichloromethane (under
Koenigs–Knorr conditions), by flash chromatographic
purification to give the four glycosides (12a–d) as the
white solids in yields 22–30%.12 Then, the free hydroxyl
group of glycosides (12a–d) was converted into aldehyde
by using a highly efficient oxidant Dess–Martin period-
inane in anhydrous dichloromethane at room tempera-
ture,13 the glycosides (13a–d) were obtained as the

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S. Wang et al. / Carbohydrate Research 342 (2007) 1254–1260
OAc
O
OAc
OAc
HO
OR3
R2O
AcO
OR3
O
R2O
AcO
R2O
AcO
O
R4
O
O
11
Br
OAc
OAc
13
OAc
12
3
a
b
O
Me
N
NH2
O
O
N
H
N
AcO
Me
O
OAc
OR2
OAc
O
AcO
AcO
R5
Me
N
12d: R2 =
O
3
O
CH CH OH
R =
6
N C
H
2
2
N
N
Me
AcO
OAc
c
13a: R2 =Ac,R4 =CHO
14
OAc
AcO
AcO
O
13b: R2=
R4 =CHO
d
OAc
CH OCH CHO
2
4
13c: R =Ac, R =
2
2
OAc
OR2
AcO
O
O
N
O
N
AcO
OAc
O
N
R5
OR2
OAc
R5
Me
AcO
13d: R2 =
AcO
Me
O
N
N
O
N
O
O
4
N
O
CH OCH CHO
R =
+
AcO
2
2
N
AcO
N
O
N
OAc
R5 =CH2
Me
14a,15a,16a: R2 =Ac,
Me
15
16
OAc
AcO
AcO
e
14b,15b,16b: R2 =
O
R5 =CH2
3a: R2=Ac
AcO
AcO
11a: R3 =H
OAc
14c,15c,16c: R2 =Ac,
5
OAc
f
CH OCH CH
2 2 2
R =
3b: R2 =
O
OAc
AcO
AcO
OAc
14d,15d,16d: R2 =
R =
5
O
CH OCH CH
2
2
2
HO
OH
OR2
OAc
O
R5
11b: R3 =
CH2CH2OH
17a: R2 =H, R5 =CH2
Me
O
N
O
O
N
HO
HO
HO
12a: R2 =Ac,R3 =H
OAc
OH
N
17b: R2=
N
N
O
R5 =CH
2
AcO
12b: R2=
17
Me
OH
O
OAc
R3=H
2
6
AcO
CH OCH CH
2
17c: R =H, R =
2
2
12c: R2=Ac,R3= CH2CH2OH
OH
HO
17d: R2 =
HO
R5 = CH2OCH2CH2
O
OH
˚
Scheme 3. Reagents and conditions: (a) HgBr2, CH2Cl2, 4 A molecular sieves, rt, overnight; (b) Dess–Martin Periodinane, CH2Cl2, rt, 90 min; (c)
anhydrous acetic acid, 5 ꢁC, 90 min; (d) NaNO2, rt, 90 min; (e) Na2S2SO4, CH2Cl2:water = 1:1, rt, overnight; (f) MeONa in MeOH, rt, overnight.
OH
OH
HO
O
OH
R1
O
N
O
N
O
OH
OH
β-glucosidase
Me
O
N
N
Me
O
N
N
N
N
O
HO
HO
R1
N
N
+
OH
OH
Me
Me
10a: R1=H
10c: R1=OMe
Scheme 4. Enzymatic hydrolysis of toxoflavin glucosides 10a and 10c.
appears that toxoflavin glucosides 10a and 10c are the
suitable substrates for b-glucosidase (EC.3.2.1.21) and
they can be used as the potential prodrugs for ADEPT
in vitro studies and consequently in vivo studies.
expressed in parts per million (ppm) based on internal
TMS (0.00 ppm) in CDCl3. 13C chemical shifts are
expressed in parts per million (ppm) based on solvent
signal of D2O or DMSO. Mass spectra were obtained
at 70 eV with a Finnigan MAT spectrometer. IR spectra
were registered on a Specord IR 75 spectrometer in KBr
pellets. Optical rotations were measured on a Perkin El-
mer Model 341LC polarimeter in a 1-dm cell on solvent
H2O at 20 ꢁC. Elemental analyses were performed on a
VarioEL analyzer. All reactions were monitored by thin
layer chromatography (TLC) on Silica Gel60 F254 (E.
Merck), with detection by UV light or by visualizing
1. Experimental
1.1. General methods
NMR spectra were recorded with the Varian INOVA
1
500 MHz and Bruke 600 MHz. H chemical shifts are

S. Wang et al. / Carbohydrate Research 342 (2007) 1254–1260
1257
washed with H2O (100 mL · 2), dried (anhydrous
Na2SO4) and concentrated. Purification by chromato-
graphy (petroleum:ethyl acetate = 1:1) gave compound
0.14
0.12
0.1
1
5b (1.4 g, 1.9 mmol, 27%) as a white solid. H NMR
10a
10c
(500 MHz, CDCl3) d: 1.98–2.17 (m, 24H, OAc), 3.90
(d, 1H, H-5Gal), 3.91 (d, 1H, H-5Glc), 4.07–4.25 (m,
4H, H-6aGal, H-6bGal, H-6aGlc, H-6bGlc), 4.51–4.65
(m, 2H, H-4Gal, H-3Gal), 4.99–5.01 (m, 2H, H-3Glc,
H-2Gal), 5.20–5.22 (m, 2H, H-2Glc, H-4Glc), 5.30 (d,
1H, H-1Gal), 5.36 (d, 1H, H-1Glc), 7.08 (d, 2H, H-
arom6, H-arom2 J2,3 = J5,6 = 5), 7.84 (d, 2H, H-arom3,
H-arom5 J2,3 = J5,6 = 5), 9.92 (s, 1H, CHO). Anal.
Calcd for C33H40O19: C, 53.51; H, 5.44. Found: C,
53.58; H, 5.36.
0.08
0.06
0.04
0.02
0
0
12
24
36
48
60
72
84
96
Time (min)
Figure 1. Kinetic result of enzymatic hydrolysis toxoflavin glucosides
10a and 10c.
1.4. 3-Methoxy-4-(2,3,4,6-tetra-O-acetyl-D-glucopyranos-
yloxy)benzaldehyde (5c)
with 10% H2SO4 in ethanol and heating. b-glucosidase
(EC.3.2.1.21) purchased from Sigma–Aldrich Co. All
solvents were dried and freshly distilled prior to use.
Compound 3a (3 g, 7.69 mmol) was reacted with com-
pound 4b (1.6 g, 10.8 mmol) as described for compound
5a to give 5c (2.3 g, 4.77 mmol) as a white solid in yield
1
1.2. 4-(2,3,4,6-Tetra-O-acetyl-D-glucopyranosyl-
oxy)benzaldehyde (5a)
of 62%. H NMR (500 MHz, CDCl3) d: 2.04 (s, 3H,
CH3CO), 2.05 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO),
2.08 (s, 3H, CH3CO), 3.86–3.88 (m, 3H, OCH3), 3.83–
3.87 (m, 1H, H-5Glc), 4.17–4.20 (dd, 1H, H-6aGlc,
J5,6a = J6a,6b = 5), 4.25–4.29 (dd, 1H, H-6bGlc,
J5,6b = J6a,6b = 5), 5.09–5.10 (d, 1H, H-4Glc), 5.16–
5.20 (m, 1H, H-3Glc), 5.29–5.35 (m, 2H, H-2Glc, H-
1Glc), 7.04 (d, 1H, H-arom6, J5,6 = 5), 7.20 (d, 1H, H-
arom5, J5,6 = 5), 7.42 (s, 1H, H-arom2), 9.89 (s, 1H,
CHO). Anal. Calcd for C22H26O12: C, 54.77; H, 5.43.
Found: C, 54.82; H, 5.24.
2,3,4,6-Tetra-O-acetyl-D-glucopyranosyl bromide 3a
(3 g, 7.69 mmol) was dissolved in CH2Cl2 (50 mL) in
which TBAB (2.49 g, 7.69 mmol), 4-hydroxybenzalde-
hyde 4a (1.2 g, 10.6 mmol) and satd aq K2CO3
(50 mL) were added. The emulsion was stirred at
60 ꢁC for 3 h after addition of H2O (50 mL), the aque-
ous layer was separated and extracted with CH2Cl2
(50 mL · 2). Combined organic layers were washed with
H2O (100 mL · 2), then dried (anhydrous Na2SO4) and
concentrated. Purification by chromatography (petro-
leum ether:ethyl acetate = 2:1), afforded compound 5a
1.5. 3-Methoxy-4-(2,3,4,6,20,30,60-hepta-O-acetyl-b-
lactosyloxy)benzaldehyde (5d)
1
(1.6 g, 3.59 mmol) as a white solid in yield of 46%. H
NMR (500 MHz, CDCl3) d: 2.04 (s, 3H, CH3CO),
2.06 (s, 3H, CH3CO), 2.07 (s, 3H, CH3CO), 2.09 (s,
3H, CH3CO), 3.91–3.94 (m, 1H, H-5Glc), 4.15–4.18
(d, 1H, H-6aGlc), 4.27–4.30 (dd, 1H, H-6bGlc), 5.16–
5.22 (m, 2H, H-3Glc, H-4Glc), 5.29–5.34 (m, 2H, H-
1Glc, H-2Glc), 7.09 (d, 2H, H-arom2, H-arom6,
J2,3 = J5,6 = 8), 7.84 (d, 2H, H-arom3, H-arom5,
J2,3 = J5,6 = 8), 9.92 (s, 1H, CHO). Anal. Calcd for
C21H24O11: C, 55.75; H, 5.35. Found: C, 55.82; H, 5.31.
Compound 3b (5 g, 7 mmol) was reacted with com-
pound 4b (1.6 g, 10.5 mmol) as described for compound
5b to give 5d (1.9 g, 2.48 mmol) as a white solid in yield
1
of 35%. H NMR (500 MHz, CDCl3) d: 1.97–2.16 (m,
24H, OAc), 3.76–3.88 (m, 1H, H-5Gal), 3.88 (s, 3H,
OCH3), 3.91–3.97 (m, 1H, H-5Glc), 4.06–4.17 (m, 4H,
H-6aGal, H-6bGal, H-6aGlc, H-6bGlc), 4.50–4.54 (m,
2H, H-4Gal, H-3Gal), 4.95–4.98 (m, 1H, H-3Glc), 5.07
(d, 1H, H-2Gal), 5.11–5.21 (m, 2H, H-2Glc, H-4Glc),
5.29 (d, 1H, H-1Gal), 5.35 (s, 1H, H-1Glc), 7.16 (d,
1H, H-arom6, J5,6 = 5), 7.40 (s, 1H, H-arom5), 7.42 (s,
1H, H-arom2), 9.88 (s, 1H, CHO). Anal. Calcd
for C34H42O20: C, 52.99; H, 5.49. Found: C, 52.92; H,
5.43.
1.3. 4-(2,3,4,6,20,30,60-Hepta-O-acetyl-b-lactosyl-
oxy)benzaldehyde (5b)
2,3,4,6,20,30,60-Hepta-O-acetyl-b-lactosyl bromide 3b
(5 g, 7 mmol) was dissolved in CH2Cl2 (50 mL) in which
TBAB (2.2 g, 7 mmol), 4-hydroxybenzaldehyde 4a
(1.2 g, 10.5 mmol) and satd aq K2CO3 (50 mL) were
added. Stirring at 60 ꢁC for 3 h after addition of H2O
(50 mL), separated the aqueous layer and extracted with
CH2Cl2 (50 mL · 2). The combined organic phase was
1.6. 3-(40-(b-D-Glucopyranosyloxy)phenyl)-1,6-dimethyl-
pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (10a)
Compound 6 (3-methyl-6-(10-methylhydrazino)-uracil)
(85 mg, 0.5 mmol) and compound 5a (485 mg,

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S. Wang et al. / Carbohydrate Research 342 (2007) 1254–1260
0.75 mmol) were stirred in 6 mL acetic acid under cool-
ing at 5 ꢁC for 90 min. The Schiff base 7a was obtained
and was stirred for 90 min after the sodium nitrite
(138 mg, 2 mmol) was added. The mixture of the com-
pound 8a and compound 9a were obtained. Then the
solvent were neutralized with aq satd K2CO3 and
extracted with CH2Cl2 (30 mL · 2). The dried (anhydrous
Na2SO4) extracts were evaporated in vacuum and puri-
fication by chromatography. The mixture of 8a and 9a
was a yellow solid. They were dissolved in 10 mL
CH2Cl2 which 10 mL Na2S2O4 (200 mg, 1.2 mmol) sol-
vent was added. After stirring continuously overnight
at room temperature, compound 9a was all reduced to
compound 8a. The aqueous layer was separated and
extracted with CH2Cl2 (10 mL · 2). Combined organic
layers were washed with H2O (10 mL · 2), dried and
concentrated, afforded 8a (240 mg, 0.39 mmol) as yellow
solid. To dissolve 8a in 10 mL NaOMe/MeOH (4 mg/
mL) and was stirred overnight at room temperature.
The solution was neutralized by acetic acid and concen-
trated. After purified by chromatography, a yellow solid
10a (152 mg, 0.35 mmol) was give in yield of 70%. [a]D
ꢀ160; 13C NMR (600 MHz, DMSO) d: 20.2, 28.1,
61.5, 67.8, 70.5, 70.9, 71.8, 96.3, 116.4, 131.3, 137.6,
148.3, 150.4, 160.9, 159.57–169.93 (multi peaks, C ring
C-3, C-4a, C-5, C-7, C-8a). IR (KBr): 3413, 1728,
1381, 1280, 1073 cmꢀ1. ESIMS (m/z): 448.3 (M+H+).
Anal. Calcd for C19H21N5O8: C, 51.01; H, 4.73; N,
15.65. Found: C, 52.17; H, 4.41; N, 15.62.
Calcd for C20H23N5O9: C, 50.31; H, 4.86; N, 14.67.
Found: C, 50.27; H, 4.78; N, 14.53.
1.9. 3-(30-Methoxy-40-(b-D-lactosyloxy)phenyl)-1,6-di-
methylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione
(10d)
Compound 10d (296 mg, 0.32 mmol) as a yellow solid in
yield of 56%. [a]D ꢀ24; 13C NMR (600 MHz, DMSO) d:
20.3, 28.1, 55.9, 60.8, 61.9, 67.0, 68.8, 69.6, 70.2, 70.7,
72.0, 72.1, 76.0. 97.1, 99.9, 111.3, 116.0, 124.6, 131.7,
137.6, 149.7–170.1 (multi peaks C ring C-3, C-4a, C-5,
C-7, C-8a). IR (KBr): 3422, 1554, 1139, 1077 cmꢀ1
.
ESIMS (m/z): 640.2 (M+H+). Anal. Calcd for
C26H33N5O14: C, 48.83; H, 5.20; N, 10.95. Found: C,
48.86; H, 5.03; N, 10.89.
1.10. 2-Hydroxy-ethyl 2,3,4,6-tetra-O-acetyl-b-D-gluco-
pyranoside (12a)
A mixture of 3a (4.5 g, 10 mmol) and glycol 11a (10 mL)
in dry CH2Cl2 (50 mL), containing actived powdered
˚
4 A molecular sieves (5 g), was stirred under N2 for
15 min. Then HgBr2 (3.6 g, 10 mmol) was added and
stirring was continue overnight at room temperature.
The mixture was diluted with CH2Cl2 (50 mL), filtered
through Celite and the filtrate was washed with aq 5%
KI (30 mL · 3) and water (30 mL · 3), dried (anhydrous
Na2SO4) and concentrated, purification by chromato-
graphy to yield 12a (1.02 g, 2.60 mmol, 26%) as a white
solid. 1H NMR (500 MHz, CDCl3) d: 2.01 (s, 3H,
CH3CO), 2.03 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO),
2.10 (s, 3H, CH3CO), 3.72–3.76 (m, 3H, H-5Glc, H-
6aGlc, H-6bGlc), 3.85 (d, 2H, CH2), 4.19 (d, 2H,
CH2), 4.55 (d, 1H, OH), 5.00–5.08 (m, 3H, H-2Glc, H-
3Glc), 5.22 (t, 1H, H-1Glc1, J1,2 = 10). Anal. Calcd
for C16H24O11: C, 48.98; H, 6.17. Found: C, 49.04; H,
6.09.
The compounds (10b–d) were synthesized as described
for compound 10a.
1.7. 3-(40-(b-D-Lactosyloxy)phenyl)-1,6-dimethylpyr-
imido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (10b)
Compound 10b (180 mg, 0.3 mmol) as a yellow solid in
yield of 60%. [a]D ꢀ120; 13C NMR (600 MHz, D2O) d:
19.7, 22.6, 59.2, 60.6, 68.1, 70.5, 70.9, 73.0, 73.6, 74.4,
74.9, 77.4, 98.9, 102.8, 116.1, 126.1, 128.2, 130.5,
149.7–173.3 (multi peaks C ring C-3, C-4a, C-5, C-7,
C-8a). IR (KBr): 3415, 1609, 1559, 1240 cmꢀ1. ESIMS
(m/z): 609.9 (M+H+). Anal. Calcd for C25H31N5O13:
C, 49.26; H, 5.13; N, 11.49. Found: C, 49.44; H, 5.03;
N, 11.45.
1.11. 2-Hydroxy-ethyl 2,3,4,6,20,30,60-hepta-O-acetyl-b-
lactoside (12b)
A mixture of 3b (5 g, 7.37 mmol), 11a (10 mL) in dry
˚
CH2Cl2 (50 mL), containing actived powdered 4 A
molecular sieves (5 g), was stirred under N2 for
15 min. After adding HgBr2 (1.7 g, 7 mmol) and stirring
overnight at room temperature, the mixture was diluted
with CH2Cl2 (50 mL), filtered through Celite and the fil-
trate was washed with aq 5% KI (30 mL · 3) and water
(30 mL · 3), dried (anhydrous Na2SO4) and concen-
trated, purification by chromatography, afforded 12b
(1.17 g, 1.72 mmol, 23%) as a white solid. 1H NMR
(500 MHz, CDCl3) d: 1.97–2.16 (m, 21H, OAc), 3.66–
3.74 (m, 4H, CH2CH2), 3.76–3.89 (m, 2H, H-5Gal, H-
5Glc), 4.04–4.14 (m, 4H, H-6aGal, H-6bGal, H-6aGlc,
H-6bGlc), 4.48–4.55 (m, 3H, H-4Gal, H-3Gal, H-
1.8. 3-(30-Methoxy-40-(b-D-glucopyranosyloxy)phenyl)-
1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-
dione (10c)
Compound 10c (180 mg, 0.38 mmol) as a yellow solid in
yield of 76%. [a]D ꢀ48; 13C NMR (600 MHz, DMSO) d:
22.7, 28.3, 55.5, 60.6, 69.6, 73.2, 76.8, 77.2, 99.7, 109.9,
115.1, 119.2, 129.5, 130.6, 141.5–174.0 (multi peaks, C
ring, C-3, C-4a, C-5, C-7, C-8a). IR (KBr): 3425, 1605,
1557, 1075 cmꢀ1. ESIMS (m/z): 477.4 (M+H+). Anal.

S. Wang et al. / Carbohydrate Research 342 (2007) 1254–1260
1259
Glc3), 4.92–4.97 (m, 2H, H-2Gal, H-2Glc), 5.10 (d, 1H,
1.15. 2-(2,3,4,6,20,30,60-Hepta-O-acetyl-b-D-lactosyl-
oxy)acetaldehyde (13b)
H-4Glc), 5.19–5.23 (m, 1H, H-1Gal), 5.35 (d, 1H, H-
1Glc). Anal. Calcd for C28H40O19: C, 49.41; H, 5.92.
Found: C, 49.45; H, 5.89.
Compound 13b (422 mg, 0.62 mmol) as a white solid.
1
Yield: 89%. H NMR (500 MHz, CDCl3) d: 1.97–2.15
1.12. 5-Hydroxy-3-oxa-pentyl 2,3,4,6-tetra-O-acetyl-b-D-
glucopyranoside (12c)
(m, 21H, OAc), 3.70–3.82 (m, 2H, CH2), 3.87–4.25 (m,
6H, H-5Gal, H-5Glc, H-6aGal, H-6bGal, H-6aGlc, H-
6bGlc), 4.45–4.55 (m, 3H, H-4Gal, H-3Gal, H-Glc3),
4.94–5.00 (m, 2H, H-2Gal, H-2Glc), 5.09–5.13 (m, 1H,
H-4Glc), 5.20–5.23 (m, 21H, H-1Gal, H-1Glc), 9.65 (s,
1H, CHO). Anal. Calcd for C28H38O19: C, 49.56; H,
5.64. Found: C, 49.68; H, 5.60.
Compound 3a (4.5 g, 10 mmol) was reacted with glycol
11b (10 mL) as described for compound 12a to give
12c (1.29 g, 2.96 mmol) as a white solid. Yield: 30%.
1H NMR (500 MHz, CDCl3) d: 2.01 (s, 3H, OAc),
2.03 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.09 (s, 3H,
OAc), 3.56–3.75 (m, 8H, CH2CH2OCH2CH2), 3.95–
3.98 (m, 1H, H-5Glc), 4.13–4.28 (m, 2H, H-6aGlc, H-
6bGlc), 4.60 (d, 1H, OH), 4.99–5.11 (m, 2H, H-4Glc,
H-3Glc), 5.12–5.23 (m, 1H, H-2Glc, H-1Glc). Anal.
Calcd for C18H28O12: C, 49.54; H, 6.47. Found: C,
49.62; H, 6.46.
1.16. 2-((2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
oxy)ethoxy)acetaldehyde (13c)
Compound 13c (290 mg, 0.66 mmol) as a white solid.
1
Yield: 94%. H NMR (500 MHz, CDCl3) d: 2.00–2.08
(m, 12H, OAc), 3.69–3.80 (m, 6H, CH2CH2OCH2),
3.98–4.01 (m, 1H, H-5Glc), 4.12–4.27 (m, 2H, H-6aGlc,
H-6bGlc), 4.58 (d, 1H, H-4Glc), 5.00 (t, 1H, H-3Glc,
J2,3 = J3,4 = 5), 5.08 (t, 1H, H-2Glc, J1,2 = J2,3 = 10),
5.19 (d, 1H H-1Glc), 9.69 (s, 1H, CHO). Anal. Calcd for
C18H26O12: C, 49.77; H, 6.03. Found: C, 49.80; H, 6.01.
1.13. 5-Hydroxy-3-oxa-pentyl 2,3,4,6,20,30,60-hepta-O-
acetyl-b-lactoside (12d)
Compound 3b (5 g, 7.37 mmol) was reacted with glycol
11b (10 mL) as described for compound 12b to give
12d (1.48 g, 1.59 mmol) as a white solid. Yield: 22%.
1H NMR (500 MHz, CDCl3) d: 1.95–2.47 (m, 21H,
OAc), 3.56–3.69 (m, 8H, CH2CH2OCH2CH2), 3.71–
3.91 (m, 2H, H-5Gal, H-5Glc), 4.04–4.13 (m, 4H, H-
6aGal, H-6bGal, H-6aGlc, H-6bGlc), 4.49–4.56 (m,
3H, H-4Gal, H-3Gal, H-Glc3), 4.87–4.95 (m, 2H, H-
2Gal, H-2Glc), 5.08 (d, 1H, H-4Glc), 5.16–5.20 (m,
1H, H-1Gal), 5.32 (d, 1H, H-1Glc). Anal. Calcd for
C30H44O20: C, 49.72; H, 6.12. Found: C, 49.87; H, 6.08.
1.17. 2-((2,3,4,6,20,30,60-Hepta-O-acetyl-b-lactosyl-
oxy)ethoxy)acetaldehyde (13d)
Compound 13d (464 mg, 0.64 mmol) as a white solid.
1
Yield: 91%. H NMR (500 MHz, CDCl3) d: 1.96–2.18
(m, 21H, OAc), 3.72–3.85 (m, 6H, CH2CH2OCH2),
3.87–4.16 (m, 6H, H-5Gal, H-5Glc, H-6aGal, H-6bGal,
H-6aGlc, H-6bGlc), 4.47–4.55 (m, H-4Gal, H-3Gal, H-
Glc3), 4.88–4.96 (m, 2H, H-2Gal, H-2Glc), 5.09–5.12
(1H, H-4Glc), 5.18–5.30 (m, 2H, H-1Gal, H-1Glc),
9.69 (s, 1H, CHO). Anal. Calcd for C30H42O20: C,
49.86; H, 5.86. Found: C, 49.93; H, 5.86.
1.14. 2-(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl-
oxy)acetaldehyde (13a)
The compounds (17a–d) were synthesized as described
for compound 10a.
To a stirred solution of 12a (300 mg, 0.77 mmol) in dry
CH2Cl2 (10 mL) under N2 at temperature, a solution of
Dess–Martin periodinane (326 mg, 0.77 mmol) in dry
CH2Cl2 (5 mL) was added. After 90 min the solution
was added to a saturated aqueous solution of NaHCO3
(10 mL) which contained 10% Na2S2O3. The mixture
was stirred vigorously for 5 min and then extracted with
CH2Cl2 (10 mL · 2), the dried (anhydrous Na2SO4)
extracts were evaporated in vacuum and gave 13a
(280 mg, 0.7 mmol) as a white solid in yield of 91%.
1H NMR (500 MHz, CDCl3) d: 2.02–2.10 (m, 12H,
OAc), 3.72–4.10 (m, 2H, CH2), 4.12–4.33 (m, 3H, H-
5Glc, H-6aGlc, H-6bGlc), 4.59 (d, 1H, H-4Glc), 5.00–
5.09 (m, 2H, H-3Glc, H-2Glc), 5.21 (d, 1H, H-1Glc),
9.67 (s, 1H, CHO). Anal. Calcd for C16H22O11: C,
49.23; H, 5.68. Found: C, 49.32; H, 5.64.
1.18. 3-((b-D-Glucopyranosyloxy)methyl)-1,6-dimethyl-
pyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (17a)
Compound 17a (141 mg, 0.37 mmol) as a yellow solid.
Yield: 74%. [a]D ꢀ12; 13C NMR (600 MHz, D2O) d:
19.6, 22.6, 56.9, 60.1, 69.8, 70.1, 72.7, 75.1, 101.9, 133.0–
172.6 (multi peaks, C ring, C-3, C-4a, C-5, C-7, C-8a).
IR (KBr): 3411, 1642, 1564, 1076 cmꢀ1. ESIMS (m/z):
386.1 (M+H+). Anal. Calcd for C14H19N5O8: C, 43.64;
H, 4.97; N, 18.17. Found: C, 43.75; H, 4.94; N, 18.08.
1.19. 3-((b-D-Lactosyloxy)methyl)-1,6-dimethylpyr-
imido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione (17b)
The compounds (13b–d) were synthesized as described
for compound 13a.
Compound 17b (162 mg, 0.3 mmol) as a yellow solid.
Yield: 60%. [a]D ꢀ6; 13C NMR (600 MHz, D2O) d:

1260
S. Wang et al. / Carbohydrate Research 342 (2007) 1254–1260
20.1, 22.8, 59.0, 60.5, 68.1, 69.9, 70.4, 72.0, 72.2, 73.6,
73.7, 74.8, 77.7, 99.4, 102.4, 133.1–173.1 (multi peaks
C ring C-3, C-4a, C-5, C-7, C-8a). IR (KBr): 3411,
1561, 1146, 1115 cmꢀ1. ESIMS (m/z): 546.2 (M+H+).
Anal. Calcd for C20H29N5O13: C, 43.88; H, 5.34; N,
12.79. Found: C, 43.74; H, 5.31; N, 12.62.
by incubating at 37 ꢁC. Analysis of reduced sugar was
performed by 3,5-dinitrosalicyclic acid assay. It is about
12 min that a test tube was heated at 100 ꢁC for 10 min,
after rapid cooling to room temperature. The first test
tube as contrast, determine the absorbance at 570 nm
and the result gave 8 point.
1.20. 3-(((b-D-Glucopyranosyloxy)ethoxy)methyl)-1,6-
dimethylpyrimido[5,4-e][1,2,4]triazine-5,7(1H,6H)-dione
(17c)
Acknowledgements
This work was financed by the Natural Science Founda-
tion of Jilin Province (Grant No. 20040546).
Compound 17c (160 mg, 0.4 mmol) as a yellow solid.
Yield: 80%. [a]D ꢀ56; 13C NMR (600 MHz, D2O) d:
20.1, 22.8, 59.8, 60.2, 68.2, 69.1, 70.9, 72.6, 75.1, 75.4,
101.8, 133.2–170.6 (multi peaks, C ring, C-3, C-4a, C-
References
5, C-7, C-8a). IR (KBr): 3402, 1563, 1110, 1076 cmꢀ1
.
ESIMS (m/z): 430.0 (M+H+). Anal. Calcd for
C16H23N5O9: C, 44.76; H, 5.40; N, 16.31. Found: C,
44.72; H, 5.39; N, 16.22.
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