Simple and efficient synthesis of novel glycosyl thiourea derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2008.01.011

The practical synthesis of pseudonucleosides incorporating thiourea derivative by coupling of monosaccharides (d-galactose, d-glucose and d-xylose) per-O-acetylated glycosyl isothiocyanates and different heterocyclic hydrazide derivatives is reported. The method involves the preparation of per-O-acetylated glycosyl isothiocyanates from per-O-acetylated sugars (two-step synthesis), which couple with heterocyclic hydrazides from amines to give thiourea-linked pseudonucleosides. All newly synthesized pseudonucleosides were assayed against human lung cancer-cell lines (PG) and human liver cancer-cell lines (BEL-7402) in vitro. The 2-(4-methoxybenzamide)-benzoimidazole-1-yl-acetyl pseudonucleosides showed moderate inhibition against these two cancer-cell lines with EC₅₀ from 22.8 to 76.4 μM and from 54.9 to 82.4 μM, respectively. And the other compounds did not demonstrate any significant cytotoxicity even at concentrations up to 200 μM.

Full text of DOI:10.1016/j.ejmech.2008.01.011

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2778e2783
http://www.elsevier.com/locate/ejmech
Original article
Simple and efficient synthesis of novel glycosyl thiourea derivatives as
potential antitumor agents
*
Zhang Shusheng, Zhan Tianrong, Cheng Kun, Xia Youfeng, Yang Bo
Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and
Technology, Zhengzhou Road No. 53, Qingdao 266042, Shandong, PR China
Received 26 September 2007; received in revised form 18 December 2007; accepted 10 January 2008
Available online 25 January 2008
Abstract
The practical synthesis of pseudonucleosides incorporating thiourea derivative by coupling of monosaccharides (D-galactose, D-glucose and
D-xylose) per-O-acetylated glycosyl isothiocyanates and different heterocyclic hydrazide derivatives is reported. The method involves the prep-
aration of per-O-acetylated glycosyl isothiocyanates from per-O-acetylated sugars (two-step synthesis), which couple with heterocyclic hydra-
zides from amines to give thiourea-linked pseudonucleosides. All newly synthesized pseudonucleosides were assayed against human lung
cancer-cell lines (PG) and human liver cancer-cell lines (BEL-7402) in vitro. The 2-(4-methoxybenzamide)-benzoimidazole-1-yl-acetyl pseu-
donucleosides showed moderate inhibition against these two cancer-cell lines with EC50 from 22.8 to 76.4 mM and from 54.9 to 82.4 mM, re-
spectively. And the other compounds did not demonstrate any significant cytotoxicity even at concentrations up to 200 mM.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Glycosyl bromides; Glycosyl isothiocyanates; Heterocyclic hydrazides; Thiourea-linked pseudonucleosides; Cytotoxicity
1. Introduction
important subject of research in the field of organic and phar-
maceutical chemistry due to the successful treatment of many
infectious diseases [14], in particular for the therapy of AIDS
[15,16]. The synthesis of analogues of natural nucleoside and
oligonucleosides has been a growing research topic over the
last few years, and several pseudonucleosides with imidazoli-
dine or thioimidazole groups [17] as well as thiourea-linked
oligonucleoside analogues [10] changing negatively charged
phosphodiester linkage by non-ionic isosteric spacers have
been synthesized [18].
In view of the advantage conferred by glycosyl isothiocya-
nate synthesis that allows rapid, convenient access to a wide
array of thioureido carbohydrates [19], together with the nota-
ble biological activities of nucleoside analogues, a great deal
of work has focused on the development of novel thiourea-
linked glycoconjugate or nucleoside [20]. However, there are
little reports on the synthesis and bioactivity of thiourea-linked
pseudonucleoside, in which the base and sugar moieties are
bound by an acylthiosemicarbazide. In this paper, a very short
and efficient synthetic route to novel acylthiosemicarbazide
derivatives and their cytotoxicity effects are reported.
Isothiocyanates are versatile synthetic intermediates in or-
ganic chemistry due to their availability and their tendency
to undergo nucleophilic additions and cycloadditions [1e3].
Particularly in carbohydrate field, sugar isothiocyanates play
a pivotal role in the preparation of a broad spectrum of carbo-
hydrate derivatives, mostly having thiourea structure, for bio-
logical and pharmaceutical interests [4,5]. Numerous antiviral,
antibacterial and antitumor agents have been prepared by reac-
tion of glycosyl isothiocyanates with biologically active
amines [4,6,7]. Recently, glycosyl isothiocyanates are being
used to prepare thiourea-linked symmetrical and unsymmetri-
cal carbohydrate mimics, such as pseudooligosaccharides
[8,9], thioureylene-di-nucleosides [10] and other glycosyl
thioureas [11], for molecular recognition studies.
At the same time, nucleosides are compounds of antiviral
and antitumor interests [12,13], so they have become an
* Corresponding author. Tel./fax: þ86 532 84022750.
E-mail address: shushzhang@126.com (Y. Bo).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.011

Z. Shusheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2778e2783
2779
2. Chemistry
i
ii
RCH2COOCH2CH3
RCH2CONHNH2
RH
10, 11, 12
13, 14, 15
16, 17, 18
The synthetic strategy involved the coupling of the glycosyl
isothiocyanates to the heterocyclic hydrazide to obtain acety-
lated N-neoglycoconjugates that could be subjected to hydro-
lysis to the deacetylated thiourea-linked pseudonucleoside.
Accordingly, the first task was to prepare the glycosyl isothio-
cyanates (Scheme 1). The reaction of per-O-acetylated sugars
(D-galactose 1, D-glucose 2, and D-xylose 3) with brominating
agent (added Br2 in a suspension of P4 in AcOH) [21] at room
temperature led to the corresponding acetylated glycosyl bro-
mides 4e6, respectively. Treatment of 4e6 with thiocyanate in
refluxing dry dimethylbenzene for 3.5 h gives the glycosyl iso-
thiocyanates 7e9 [22] according to the classical Fischer’s
method [23], The hexopyranosyl isothiocyanates of D-galacto
and D-gluco configuration 7 and 8 were obtained in about
8 h, while formation of the pentopyranosyl isothiocyanate 9
took about 8.5 h.
The heterocyclic hydrazides were prepared from amines
10e12 (Scheme 2). Adenine 10 was alkylated with ethyl
chloroacetate in DMF by first generating the anion with so-
dium hydride. This procedure resulted in only one isolable
compound which was recrystallized from methanol and iden-
tified as the expected light yellow solid adenine-9-yl-ethylace-
tate 13 [24] in a 70% yield. Then the reduction of 13 with
hydrazine hydrate in methanol gave corresponding adenine
hydrazide derivative 16 as white solid in good yield of 81%.
By the same procedure, heterocyclic hydrazide derivatives
17 and 18 were also efficiently prepared from the correspond-
ing heterocyclic compounds 11 and 12 within two steps in 52
and 47% yields, respectively.
NH2
O
N
R =
R =
R =
N
N
N
H3CO
C
NH
N
N
N
N
10, 13, 16
11, 14, 17
12, 15, 18
Scheme 2. Reagents and conditions. (i) NaH, ClCH2COOC2H5, DMF and (ii)
80% H2NNH2$H2O, MeOH.
It is interesting to note that the coupling between benzoimida-
zole-1-yl-acetyl hydrazide 17 with per-O-acetylated sugar iso-
thiocyanates 7e9 gave the per-O-protected adducts 22e24 in
good yield (see Table 1, entry 4e6, yields were more than
85%). On the other hand, the reaction of 2-(4-methoxybenza-
mide)-benzoimidazole-1-yl-acetyl hydrazide 18 was accom-
plished to afford the compounds 25e27 in 64e76% yield.
These results can be explained that the weaker steric hindrance
of the compound 17 makes its reaction smooth.
3. Biological activity
The newly synthesized compounds 28e36 were evaluated
for their in vitro cytotoxicity by growth-inhibition studies
NH2
N
N
R2
R3
AcO
H
The coupling of the glycosyl isothiocyanates to the hetero-
cyclic hydrazide afforded acetylated N-neoglycoconjugates
which finally led to the deacetylated thiourea-linked pseudo-
nucleosides (N-glycoconjugates) (Scheme 3). Nucleophilic
addition of the adenine-9-yl-acetyl hydrazide 16 to per-O-
acetylated sugar isothiocyanates 7e9 in pyridine under Ar at
room temperature afforded the corresponding per-O-protected
adducts 19e21, respectively. Then, these adducts 19e21 were
subjected to sodium methoxide-catalyzed in methanol
(pH ¼ 8.0) deacetylation furnished the corresponding unpro-
tected N-glycoconjugates 28e30, respectively. Following the
same protocols, the other deacetylated N-glycoconjugates
31e36 were prepared in two steps from the corresponding
starting materials. All purification was achieved by column
chromatography and possible recrystallization from methanol.
O
N
N
i
i
i
H
N
R1
7, 8, 9
7, 8, 9
7, 8, 9
+
+
+
16
17
18
O
N
N
H
OAc
S
19, 20, 21
ii
28, 29, 30
N
N
O
R2
H
N
H
R1
N
O
R3
AcO
N
H
S
OAc
22, 23, 24
ii
31, 32, 33
O
N
H3CO
C
NH
O
N
R2
R1
H
H
N
O
N
N
R3
AcO
H
S
OAc
R2
R2
R1
R2
R1
R1
O
O
O
i
ii
25, 26, 27
34, 35, 36
NCS
R3
R3
AcO
R3
ii
19, 22, 25: R1 = CH2OAc, R2 = OAc, R3 = H
20, 23, 26: R1 = CH2OAc, R2 = H, R3 = OAc
21, 24, 27: R1 = R2 = H, R3 = OAc
AcO
AcO
OAc
OAc
OAc
OAc
Br
28, 31, 34: R1 = CH2OH, R2 = OH, R3 = H
29, 32, 35: R1 = CH2OH, R2 = H, R3 = OH
30, 33, 36: R1 = R2 = H, R3 = OH
1, 4, 7: R1 = CH2OAc, R2 = OAc, R3 = H
2, 5, 8: R1 = CH2OAc, R2 = H, R3 = OAc
3, 6, 9: R1 = R2 = H, R3 = OAc
Scheme 1. Reagents and conditions. (i) AcOH, P, dropwise Br2, 30 min and (ii)
Pb(SCN)2, dry (CH3)2C6H4, 3.5e8.5 h.
Scheme 3. Reagents and conditions. (i) Ar, DMF, room temperature and (ii)
MeOH, MeONa, pH ¼ 8.0.

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Z. Shusheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2778e2783
Table 1
Synthesis of the per-O-protected glycoconjugates 19e27a
best of our knowledge, this type of N-neoglycoconjugates is
not known in the literature and some of them showed moderate
inhibitory effects on cancer cells. This convenience of our strat-
egy lies in the possibility of preparing a number of different an-
alogues by the reaction of a simple glycosyl isothiocyanate with
several heterocyclic hydrazides.
Entry
Sugar
isothiocyanates
Hydrazide
Product
Yieldb (%)
1
2
3
4
5
6
7
8
9
a
7
8
9
7
8
9
7
8
9
16
16
16
17
17
17
18
18
18
19
20
21
22
23
24
25
26
27
76
83
77
86
91
88
64
76
71
5. Experimental
5.1. General procedures
Solvents were pretreated, when necessary, according to
appropriate standard procedures before used. Column chro-
matography (CC) was performed on silica gel (200e
300 mesh), and precoated silica gel plates (F254: Qingdao
Marine Chemical Co., Ltd., 20 ꢀ 10 cm, 0.5 mm thick)
were used for TLC; the spots were examined with UV light,
iodine vapor and sulfuric acid spray. And reaction mixtures
were stirred magnetically. Melting points were determined
on an RY-1 apparatus in capillaries and are uncorrected. Nu-
clear magnetic resonance (NMR) spectra were recorded on
a BRUCK 500 MHz spectrometer in DMSO-d6 and chemi-
cal shifts are given in d values (ppm) relative to tetrame-
thylsilane as the internal standard. Splitting patterns are
designated as follows: s ¼ singlet, d ¼ doublet, dd ¼ double
doublet, t ¼ triplet, br s ¼ broad singlet, and m ¼ multiplet.
UV spectra were arrived on a VARIAN spectrometer using
a cell path length of 1 cm. Fourier transform infrared spec-
tra were recorded using an Nicolet 501P FTIR spectrometer
with KBr plates. Mass spectra were recorded on an ABI-
API4000 mass spectrometer in m/z. Elemental analysis
was reported with a Perkin Elmer 240C apparatus. Optical
rotations were measured on a Perkin Elmer 241 polarimeter
at 25ꢁ.
All reactions conducted at room temperature; see Section 5 for details.
Isolated yields based on sugar isothiocyanates after possible purification.
b
using two human cancer-cell lines: human liver (BEL-7402
cell) and human lung (PG cell).
As shown in Table 2, the total deacetylated 2-(4-methoxy-
benzamide)-benzoimidazole-1-yl-acetyl
pseudonucleosides
34e36 exhibited inhibitory activity against human liver can-
cer-cell lines (BEL-7402) with EC50 from 22.8 to 76.4 mM
and showed much less activity against human lung cancer-
cell lines (PG) with EC50 from 54.9 to 82.4 mM. Additionally,
only compound 29 in benzoimidazole-1-yl-acetyl glycoconju-
gates showed effect on human liver cancer-cells (BEL-7402)
with EC50 of 86.2. However, the compounds 28 and 31 dem-
onstrated inhibitory effects on human liver cancer-cell lines
(BEL-7402) with EC50 of 94.5 and 82.2 mM, respectively.
And the other compounds did not indicate any significant in-
hibition against these two human cancer-cell lines up to
200 mM.
4. Conclusions
5.2. General procedure for synthesis of per-O-acetylated
glycosyl isothiocyanates 7e9
In conclusion, a new type of sugareheterocyclic compounds
linked by thiourea derivative could be obtained from the readily
available per-O-acetylated sugars under mild condition. To the
To a suspension of P4 (4.5 g, 3.63 mmol) in AcOH (60 mL)
was added Br2 (12 mL, 234.10 mmol) dropwise. The mixture
was stirred for 30 min at room temperature, and then P4 was
filtrated. Per-O-acetylated sugar 1 (39 g, 100 mmol) was
added and the mixture was stirred at the same temperature un-
til TLC showed disappearance of 1. The bulk of AcOH was
removed under reduced pressure, and the residue was parti-
tioned between saturated Na2CO3 solution and CHCl3. The or-
ganic extracts were washed with water and brine, and dried
(Na2SO4). The organic phase was concentrated under reduced
pressure to afford 4 as a white solid. Without further purifica-
tion, per-O-acetylated bromide 4 (7.9 g, 20 mmol) was added
dropwise to a refluxed suspension of Pb(SCN)2 (9.69 g,
30 mmol) in 50 mL anhydrous dimethylbenzene. The mixture
refluxed for 3.5e8.5 h, then Pb(SCN)2 was filtrated. The sol-
vent was removed under reduced pressure and the residue was
recrystalized to afford 7 as white solid from the mixture of tolu-
ene and petroleum ether. The corresponding compounds 8 and 9
were prepared by this method. Physical data of per-O-acetylated
Table 2
Cytotoxicity of the final compounds 28e36 in two cancer-cell lines (PG, BEL-
7402)
a
EC50 (mM)
Compound
PGb
BEL-7402c
28
29
94.5
>100
>200
82.2
ꢂ200
86.2
30
31
>100
>100
>200
>200
22.8
32
33
>100
>200
54.9
34
35
82.4
73.7
76.4
36.7
36
Zidovudine
0.58
4.62
a
50% Effective concentration.
Human lung cells.
Human liver cells.
b
c

Z. Shusheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2778e2783
2781
glycosyl isothiocyanates 7e9 matched those described in the
literature [21].
5.4.1. Compound 20
Light yellow solid, 495 mg, yield 83%; mp: 171e173 ꢁC;
IR (KBr, nmax): 3340 (NH), 1643, 1574 (C]O), 1369 (Me),
1232 (gly-H), 1041 (C]S) cmꢃ1 1H NMR (500 MHz,
;
5.3. General procedure for synthesis of acetylhydrazine
heterocycle compounds 16e18
DMSO-d6): d 9.81 (1H, s, NH), 8.90 (1H, s, NH), 8.68 (1H,
s, NH), 8.25 (1H, s, adenine ring-H), 8.07 (1H, s, adenine
ring-H), 7.27 (2H, s, NH2), 3.97e5.91 (7H, m, gly-H, CH2),
4.29 (2H, s, COCH2), 1.75e2.03 (12H, m, CH3CO). Anal.
Calcd for C22H28N8O10S: C, 44.29; H, 4.73; N, 18.78. Found:
C, 44.24; H, 4.66; N, 18.54. ESI-MS: (MHþ) 597.6.
To a cooled suspension of adenine 10 (5.0 g, 37 mmol) in
dry DMF (100 mL) was added NaH (6.1 g, 0.15 mol, 60%
dispersion in oil, was washed with hexane) proportionally.
The mixture was stirred for 2 h at room temperature. Subse-
quently ethyl chloroacetate (150 mL, 140 mmol) was added
dropwise within 2 h at room temperature. After another 2 h,
the solvent was removed by evaporate, in vacuo. The resi-
due was dropped into water (250 mL) and stirring. The
solids were filtered off and washed with water, followed
by recrystallization from methanol to give 13 (5.56 g,
70%) as light yellow crystalline [25]. Without further puri-
fication, 13 (5.0 g, 23 mmol) was dissolved in 30 mL MeOH
and the mixture was heated until becoming homogeneous
solution. Then 80% hydrazine hydrate (2.8 mL, 48 mmol)
was added and the mixture was continuously stirred at the
same temperature. The mixture was stirred for an additional
8 h after white solid appeared. The resulting aqueous sus-
pension was filtered under reduced pressure, and residue
was washed with anhydrous EtOH to neutral. Then residue
was dried in vacuo to afford 16 (3.60 g, 81%) as white
solid. The corresponding compounds 17 and 18 were pre-
pared by this method. Physical data of acetylhydrazine het-
erocycle compounds 16e18 matched those described in the
literature [24].
5.4.2. Compound 21
White solid 404 mg, yield 77%; mp: 183e184 ꢁC; 1H NMR
(500 MHz, DMSO-d6): d 9.83 (1H, s, NH), 8.53 (1H, s, NH),
7.95 (1H, s, NH), 8.14 (1H, s, adenine ring-H), 8.04 (1H, s, ad-
enine ring-H), 3.50e5.75 (7H, m, gly-H), 7.26 (2H, s, NH2),
4.24 (2H, s, COCH2), 1.86e2.00 (9H, m, CH3CO). Anal.
Calcd for C19H24N8O8S: C, 43.51; H, 4.61; N, 21.36. Found:
C, 43.57; H, 4.65; N, 31.44. ESI-MS: (MHþ) 525.5.
5.4.3. Compound 22
White solid 499 mg, yield 86%; 1H NMR (500 MHz,
DMSO-d6): d 10.25 (1 H, br s, NH), 9.84 (1H, br s, NH),
8.68 (1H, br s, NH), 7.16e8.19 (5H, m, benzimidazole ring-
H), 3.90e5.88 (9H, m, gly-H and COCH2), 1.85e2.01 (12H,
3s, CH3CO). Anal. Calcd for C24H29N5O10S: C, 49.74; H,
5.04; N, 12.08. Found: C, 49.86; H, 5.08; N, 12.01. ESI-MS:
(MHþ) 580.6.
5.4.4. Compound 23
Light yellow solid 527 mg, yield 91%; 1H NMR (500 MHz,
DMSO-d6): d 10.22 (1H, br s, NH), 9.85 (1H, br s, NH), 8.54
(1H, br s, NH), 7.20e8.15 (5H, m, benzimidazole ring-H),
3.94e5.32 (9H, m, gly-H and COCH2), 1.80e2.09 (12H, 3s,
CH3CO). Anal. Calcd for C24H29N5O10S: C, 49.74; H, 5.04;
N, 12.08. Found: C, 49.67; H, 5.06; N, 12.14. ESI-MS:
(MHþ) 580.6.
5.4. General procedure for synthesis of per-O-acetylated
thiourea-linked pseudonucleosides 19e27
A stirred suspension of 16 (248 mg, 1.2 mmol) in anhy-
drous DMF (15 mL) was heated to 140 ꢁC, the mixture con-
tinuously reacted for additional 5 min under the same
conditions after solids were dissolved completely. Then
the mixture was cooled slowly to 70 ꢁC and was kept as
5.4.5. Compound 24
a
clear solution. Glycosyl isothiocyanate 7 (373 mg,
White solid 447 mg, yield 88%; 1H NMR (500 MHz,
DMSO-d6): d 10.21 (1H, br s, NH), 9.83 (1H, br s, NH),
8.72 (1H, br s, NH), 7.15e8.16 (5H, m, benzimidazole ring-
H), 3.98e5.87 (8H, m, gly-H and COCH2), 1.83e2.15 (9H,
3s, CH3CO). Anal. Calcd for C21H25N5O8S: C, 49.70; H,
4.97; N, 13.80. Found: C, 49.62; H, 5.03; N, 13.71. ESI-MS:
(MHþ) 508.5.
1 mmol) in 5 mL dry DMF was added dropwise. The reac-
tion mixture was cool to room temperature and stirred vig-
orously under Ar for 8 h. After removal of DMF in vacuo,
the resulting brown residue was diluted with anhydrous
MeOH. Silica gel was added, and the mixture was evapo-
rated to dryness. The dry powder was applied to silica gel
CC (EtOAc/MeOH) to afford 19 (441 mg, 76%) as a light
yellow powder. Mp: 168e169 ꢁC; IR (KBr, nmax): 3433
(NH), 1749, 1643 (C]O), 1369 (Me), 1231 (gly-H), 1081
5.4.6. Compound 25
(C]S) cmꢃ1
;
1H NMR (500 MHz, DMSO-d6): d 9.75
White solid 466 mg, yield 64%; 1H NMR (500 MHz,
DMSO-d6): d 12.72 (1H, br s, NH), 10.37 (1H, br s, NH),
9.98 (1H, br s, NH), 8.64 (1H, br s, NH), 7.01e8.28 (8H,
m, benzene ring-H), 3.95e5.95 (9H, m, gly-H and COCH2),
3.84 (3H, s, OeCH3), 1.79e2.01(12H, 3s, CH3CO). Anal.
Calcd for C32H36N6O12S: C, 52.74; H, 4.98; N, 11.53. Found:
C, 52.68; H, 5.03; N, 11.59. ESI-MS: (MHþ) 729.7.
(1H, s, NH), 9.75 (1H, s, NH), 8.73 (1H, s, NH), 8.23
(1H, s, adenine ring-H), 8.14 (1H, s, adenine ring-H),
7.26 (2H, s, NH2), 4.04e5.32 (7H, m, gly-H, CH2), 4.30
(2H, s, COCH2),1.91e2.16 (12H, m, CH3CO). Anal. Calcd
for C22H28N8O10S: C, 44.29; H, 4.73; N, 18.78. Found: C,
44.18; H, 4.69; N, 18.28. ESI-MS: (MHþ) 597.6.

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Z. Shusheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2778e2783
5.4.7. Compound 26
5.5.3. Compound 31
White solid 554 mg, yield 76%; 1H NMR (500 MHz,
DMSO-d6): d 12.71 (1H, br s, NH), 10.38 (1H, br s, NH),
9.98 (1H, br s, NH), 8.45 (1H, br s, NH), 7.16e8.23 (8H,
m, benzene ring-H), 3.92e5.72 (9H, m, gly-H and COCH2),
3.85 (3H, s, OeCH3), 1.78e2.01(12H, 3s, CH3CO). Anal.
Calcd for C32H36N6O12S: C, 52.74; H, 4.98; N, 11.53. Found:
C, 52.71; H, 4.94; N, 11.48. ESI-MS: (MHþ) 729.7.
White powder 382 mg, yield 93%; 1H NMR (500 MHz,
DMSO-d6): d 10.37 (1H, br s, NH), 9.71 (1H, br s, NH), 8.68
(1H, br s, NH), 7.20e8.49 (5H, m, benzimidazole ring-H),
3.15e5.65 (13H, m, gly-H and COCH2 overlapped with H of
HeO). Anal. Calcd for C16H21N5O6S: C, 46.71; H, 5.14; N,
17.02.Found:C,46.67;H,5.08;N,17.10.ESI-MS:(MHþ)412.4.
5.5.4. Compound 32
White powder 370 mg, yield 90%; H NMR (500 MHz,
1
5.4.8. Compound 27
White solid 466 mg, yield 71%; 1H NMR (500 MHz,
DMSO-d6): d 12.70 (1H, br s, NH), 10.29 (1H, br s, NH),
9.95 (1H, br s, NH), 8.68 (1H, br s, NH), 7.15e8.16 (8H,
m, benzene ring-H), 3.95e5.85 (8H, m, gly-H and COCH2),
3.85 (3H, s, OeCH3), 1.76e2.15 (9H, 3s, CH3CO). Anal.
Calcd for C29H32N6O10S: C, 53.04; H, 4.91; N, 12.80. Found:
C, 52.97; H, 4.96; N, 12.74. ESI-MS: (MHþ) 657.7.
DMSO-d6): d 10.41 (1H, br s, NH), 9.90 (1H, br s, NH),
8.58 (1H, br s, NH), 7.28e8.48 (5H, m, benzimidazole ring-
H), 3.30e5.78 (13H, m, gly-H and COCH2 overlapped with
H of HeO). Anal. Calcd for C16H21N5O6S: C, 46.71; H,
5.14; N, 17.02. Found: C, 46.75; H, 5.11; N, 17.09. ESI-MS:
(MHþ) 412.4.
5.5.5. Compound 33
White powder 332 mg, yield 87%; H NMR (500 MHz,
1
5.5. General procedure for synthesis of deacetylated
thiourea-linked pseudonucleosides 28e36
DMSO-d6): d 10.33 (1H, br s, NH), 9.45 (1H, br s, NH),
8.47 (1H, br s, NH), 7.18e8.39 (5H, m, benzimidazole ring-
H), 3.12e5.28 (11H, m, gly-H and COCH2 overlapped with
H of HeO). Anal. Calcd for C15H19N5O5S: C, 47.24; H,
5.02; N, 18.36. Found: C, 47.31; H, 4.97; N, 18.31. ESI-MS:
(MHþ) 382.4.
A solution of per-O-acetylated thiourea-linked carbody-
drate 19 (596 mg, 1 mmol) in 15 mL of anhydrous MeOH
was treated with sodium methanol (dissolved in methanol)
to pH ¼ 8.0. The mixture was stirred at room temperature
for 10e15 min until compound 19 disappeared monitored by
TLC. The reaction solution was neutralized with cation-ex-
change resin and white floccule formed. The resulting suspen-
sion was filtered under reduced pressure, and residue was dried
in vacuo to afford 28 (377 mg, 88%) as white powder. IR
(KBr, nmax): 3325 (OH), 1649 (C]O), 1255 (gly-H), 1082
5.5.6. Compound 34
White powder 510 mg, yield 91%; H NMR (500 MHz,
1
DMSO-d6): d 12.24 (1H, br s, NH), 10.18 (1H, br s, NH),
9.86 (1H, br s, NH), 8.73 (1H, br s, NH), 7.02e8.22 (8H,
m, benzene ring-H), 3.20e5.78 (13H, m, gly-H and COCH2
overlapped with H of HeO), 3.78 (3H, s, OeCH3). Anal.
Calcd for C24H28N6O8S: C, 51.42; H, 5.03; N, 14.99. Found:
C, 51.47; H, 4.96; N, 15.04. ESI-MS: (MHþ) 561.6.
1
(C]S) cmꢃ1; H NMR (500 MHz, DMSO-d6): d 9.69 (1H,
s, NH), 8.26 (1H, s, NH), 8.14 (1H, s, NH), 8.20 (1H, s, ade-
nine ring-H), 8.04 (1H, s, adenine ring-H), 7.25 (s, 2H, NH2),
5.59e3.39 (11H, m, gly-H, overlapped with H of OH), 3.53
(2H, m, COCH2). Anal. Calcd for C14H20N8O6S: C, 39.25;
H, 4.71; N, 26.15. Found: C, 39.02; H, 4.69; N, 25.99. ESI-
MS: (MHþ) 429.4.
5.5.7. Compound 35
White powder 533 mg, yield 95%; H NMR (500 MHz,
1
DMSO-d6): d 12.72 (1H, br s, NH), 10.35 (1H, br s, NH),
9.78 (1H, br s, NH), 9.14 (1H, br s, NH), 7.20e8.29 (8H,
m, benzene ring-H), 3.25e5.32 (13H, m, gly-H and COCH2
overlapped with H of HeO), 3.85 (3H, s, OeCH3). Anal.
Calcd for C24H28N6O8S: C, 51.42; H, 5.03; N, 14.99. Found:
C, 51.37; H, 4.96; N, 14.92. ESI-MS: (MHþ) 561.6.
5.5.1. Compound 29
White powder 390 mg, yield 91%; H NMR (500 MHz,
1
DMSO-d6): d 9.71 (1H, br s, NH), 8.35 (1H, br s, NH), 8.10
(1H, br s, NH), 8.15 (1H, s, adenine ring-H), 8.09 (1H, s, ad-
enine ring-H), 7.28 (2H, s, NH2), 5.31e4.95 (11H, m, gly-H
overlapped with H of HeO), 3.13e3.69 (2H, m, COCH2).
Anal. Calcd for C14H20N8O6S: C, 39.25; H, 4.71; N, 26.15.
Found: C, 39.02; H, 4.69; N, 25.99. ESI-MS: (MHþ) 429.4.
5.5.8. Compound 36
White powder 472 mg, yield 89%; H NMR (500 MHz,
1
DMSO-d6): d 12.72 (1H, br s, NH), 10.30 (1H, br s, NH),
9.75 (1H, br s, NH), 9.09 (1H, br s, NH), 7.28e8.47 (8H,
m, benzene ring-H), 3.32e5.38 (11H, m, gly-H and COCH2
overlapped with H of HeO), 3.86 (3H, s, OeCH3). Anal.
Calcd for C23H26N6O7S: C, 52.07; H, 4.94; N, 15.84. Found:
C, 51.98; H, 4.96; N, 15.90. ESI-MS: (MHþ) 531.6.
5.5.2. Compound 30
White powder 347 mg, yield 87%; H NMR (500 MHz,
1
DMSO-d6): d 10.41 (1H, br s, NH), 9.75 (1H, br s, NH),
8.27 (1H, br s, NH), 7.71 (s, 2H, NH2), 8.13 (1H, s, adenine
ring-H), 7.97 (1H, s, adenine ring-H), 3.07e5.23 (9 H, m,
gly-H overlapped with H of HeO), 3.60 (2H, m, COCH2).
Anal. Calcd for C13H18N8O5S: C, 39.19; H, 4.55; N, 12.80.
Found: C, 39.26; H, 4.51; N, 12.87. ESI-MS: (MHþ) 399.4.
5.6. Cytotoxicity assays
Growth inhibition of the synthetic compounds to various tu-
mor cells was determined by MTT assays [26]. Briefly, tumor

Z. Shusheng et al. / European Journal of Medicinal Chemistry 43 (2008) 2778e2783
2783
cells (3e5 ꢀ 104 cells mLꢃ1) were inoculated in 96-well culture
plates (100 mL/well). After 24 h culture, 50 mL of culture me-
dium containing synthetic compound of various concentrations
was added to the wells, and BCS-1640 medium in control cells,
then the cells were incubated for 48 h. The absorbance of each
well was measured using a microculture plate reader at 490 nm.
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This work was supported by the Natural Science Founda-
tion of Shandong Province (No. Z2006B01) and the National
Natural Science Foundation of China (No. 20775038) and
Doctoral Foundation of Qingdao University of Science and
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