Synthesis of 6-(het) ary Xylocydine analogues and evaluating their inhibitory activities of CDK1 and CDK2 in vitro

Article abstract of DOI:10.1016/j.bmc.2011.10.003

A series of purine nucleoside analogues bearing an aryl and hetaryl group in position 6 were prepared and their biological activities were assessed by in vitro CDK1/Cyclin B1 and CDK2/Cyclin A2 kinase assay. From the synthesized chemicals, three Xylocydine derivatives 3h, 3i, and 3j exhibited specific inhibitory activities on CDK2/Cyclin A2 with IC₅₀ values of 4.6, 4.8, and 55 μM, respectively. Those three compounds all induced G1/S phase arrest in Human epithelial carcinoma cell line (HeLa), and the results suggested they may inhibit CDK2 activity in vitro. Furthermore, molecular modeling study, their docking into Cyclin Dependant Kinase 2 (CDK2) active site showed high docking scores. Taken together, these data suggest that, those three compounds are good inhibitors of CDK2 for studying this kinase signal transduction pathway in cell system.

Full text of DOI:10.1016/j.bmc.2011.10.003

Bioorganic & Medicinal Chemistry 19 (2011) 7100–7110
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of 6-(het) ary Xylocydine analogues and evaluating
their inhibitory activities of CDK1 and CDK2 in vitro
Chuan Xiao a, , Chao Sun b, , Weiwei Han b, Feng Pan a, Zhu Dan b, Yu Li a, Zhi-Guang Song a, , Ying-Hua Jin b,
⇑
⇑
a College of Chemistry, Jilin University, Changchun 130012, China
b Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, Changchun 130012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of purine nucleoside analogues bearing an aryl and hetaryl group in position 6 were prepared and
their biological activities were assessed by in vitro CDK1/Cyclin B1 and CDK2/Cyclin A2 kinase assay.
From the synthesized chemicals, three Xylocydine derivatives 3h, 3i, and 3j exhibited specific inhibitory
Received 9 August 2011
Revised 30 September 2011
Accepted 1 October 2011
Available online 7 October 2011
activities on CDK2/Cyclin A2 with IC50 values of 4.6, 4.8, and 55 lM, respectively. Those three compounds
all induced G1/S phase arrest in Human epithelial carcinoma cell line (HeLa), and the results suggested
they may inhibit CDK2 activity in vitro. Furthermore, molecular modeling study, their docking into Cyclin
Dependant Kinase 2 (CDK2) active site showed high docking scores. Taken together, these data suggest
that, those three compounds are good inhibitors of CDK2 for studying this kinase signal transduction
pathway in cell system.
Keywords:
Purine nucleoside
CDK2
Xylocydine
Inhibitory effect
Molecular docking
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
inhibitors.17–20 Purine nucleoside analogues Xylocydine (2,
Fig. 1C), as a novel inhibitor of CDKs, can selectively inhibit CDK1
Cyclin-dependent kinases (CDKs), which belong to this Ser/Thr
kinase family,1 play an important role in cell-cycle regulation.2,3
Specific CDKs are sequentially activated during different phases
of the cell cycle by their association with Cyclins and the activity
of several CDK/Cyclins has been identified.4,5 The mammalian cell
cycle is governed by the sequential activation of CDKs.6 Progres-
sion through G1 and entry into S phase are regulated by Cdk2 com-
plexed with Cyclin A and Cyclin E, and the G2/M transition is
driven by Cdc2 complexed with Cyclin B.7 CDK activity is fre-
quently upregulated in proliferative disease,8 and CDKs have been
to be proved important targets for treating hyperproliferating dis-
eases such as cancer.9,10 Currently, much effort has focused on the
discovery of new chemical inhibitors of CDKs that could act as anti-
cancer agents. Purines, pyrimidines, flavonoids, indolocarbazole
analogues, and other heterocyclic compounds have been reported
as potent CDK inhibitors.11,12 Particularly, most of the already
known CDK inhibitors (natural products or/and synthetic mole-
cules) are flat, small heterocycles that act by competing with ATP
for the kinase ATP-binding site. However, inhibitor specificity for
CDKs over other kinases and selectivity for a specific target CDK
within the CDK family of enzymes remain major challenges.13
As CDK2 is an important target in cancer therapy,13–16 much
effort has been devoted to find more specific and potent CDK2
and CDK2,21 and has been implicated to be useful for treatment
of various CDK activity-related diseases.22 Purines ring system
was used in a number of CDK inhibitors, such as Olomoucine
(Fig. 1A) which was identified as an active lead compound through
the screening of substituted purines with modest potency.23–25
Purine nucleosides and their analogues (Fig. 1B) represent an
important class of biologically active compounds that have been
extensively explored in the last five decades for their antitumor
properties.26–30 On this basis, our work was to explore new purine
nucleosides to achieve better specificity inhibition to CDK2 kinase
activity. We were interested whether the replacement of the Br by
aryl or hetaryl substituent in position 6 would result in a specific
inhibition to CDK2/CyclinA2 kianses. Here we report on the syn-
thesis and evaluation of inhibitory activities of these 6-substituted
Xylocydine analogues 3, 4 (Fig. 1C).
2. Result and discussion
2.1. Chemistry
Our synthesis of 6-substituted Xylocydine analogues 3a–j, 4a–j
relied on palladium catalyzed Suzuki reactions of protected
4-amino-6-bromo-5-cyano-7-(2,3,5-tri-O-benzoyl-b-
osyl-) pyrrolo[2,3-d]pyrimidine 11.
L-xylofuran-
As shown in Scheme 1, the first half of the convergent synthesis
required preparation of the 4-amino-6-bromo-5-cyanopyrrolo[2,
3-d]pyrimidine 7. Initially, tetracyanoethylene 5 was charged with
⇑
Corresponding authors. Tel./fax: +86 431 85155221 (Y.-H.J.).
E-mail addresses: szg@jlu.edu.cn (Z.-G. Song), yhjin@jlu.edu.cn (Y.-H. Jin).
These authors made equal contributions.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.10.003

C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
7101
Figure 1. (A) Chemical structures of some potent purine CDK2 inhibitors. (B) Chemical structures of some potent purine nucleosides. (C) Modification position and structure
of target product.
Scheme 1. Reagents and conditions: (a) HBr/HOAc, 0 °C, 2 h; (b) HN@CHNH2ÁCH3COOH, CH3CH2OCH2CH2OH; (c) CH3OH/H2SO4; (d) BzCl, pyridine; AcOH, Ac2O; (e) BSA,
CH3CN, then 1-O-acetyl-2,3,5-tri-O-benzoyl-b-L-ribofuranose, TMSOTf, 80 °C.
33% HBr in acetic acid at 0 °C, that gave 2-amino-5-bromo-3,4-
dicyanopyrrole 6.31 Treatment of 6 with fomamidine acetate in
2-ethoxyethanol at reflux temperature gave 4-amino-6-bromo-5-
cyanopyrrolo[2,3-d]pyrimidine 7. Synthesis of the compound 10
Palladium catalyzed cross-coupling reactions of compound 11
(Scheme 2) with the corresponding arylboronic acids were per-
formed to provide protected 6-substituted Xylocydine analogues
12a–i in good yield.35–38 According to the stability and activity of
these arylboronic acids, we had tried several palladium catalyst sys-
tems and determined their own optimal reaction conditions. For
arylboronic acid with an electrondonating group (entries b and c)
and electronically neutral arylboronic acid (entries a and d), we
applied Pd(OAc)2/PPh3-dioxane system as an efficient catalytic
medium for the Suzuki coupling reaction. As to 4-bromophenyl
boronic acid (entry e) and 3-bromophenyl boronic acid (entry f),
it was very possible to generate multi-coupling products.
Therefore, we applied Pd(PPh3)4-toluene system with the
bromophenyl boronic acid being 1.5 equiv of material 11.
Compound 12g was obtained from 1-N-(Boc)-pyrrole-2-boronic
began from
L
(À)-xylose 8. Compound 8 was easily converted into
9 by methylation. It should be noted that the operation of this
method is simple than reference reported.32 Then compound 10
was prepared from 9 by benzoylation and acetylation. Finally, gly-
cosylation of compound 7 and 10 gave compound 11, which was
accomplished in a similar procedure as previously described for
the synthesis of toyocamycin.33,34
Cross-coupling reactions conditions: (A) Pd(OAc)2, K2CO3, diox-
ane, 100 °C; (B) Pd(PPh3)4, K2CO3, toluene, 105 °C; (C) PdCl2(PPh3)2,
K2CO3, THF, reflux; (D) Pd(PPh3)4, K2CO3, DMF, 110 °C; (E) Pd(PPh 3)4,
Na2CO3, DME/H2O, 80 °C.

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C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
12, 3, 4
a
R
Product (yiled)
p-tolyl
12a (82
12b (86
12c (83
12d (81
12e (78
12f (76
12g (75
12h (70
12i (72
)
)
)
)
)
)
)
)
)
3a (85
3b (80
3c (80
3d (86
3e (82
3f (83
3g (76
3h (75
3i (73
3j (81
)
)
)
)
)
)
)
)
)
)
4a (89
4b (90
4c (84
4d (88
4e (85
4f (84
4g (70
4h (68
4i (79
4j (89
)
)
)
)
)
)
)
)
)
)
b
c
d
e
f
4-methoxyphenyl
3-methoxyphenyl
4-(tert-butyl)phenyl
4-bromophenyl
3-bromophenyl
1H-pyrrol-2-yl
g
h
i
thiophen-2-yl
furan-2-yl
j
5-bromothiophen-2-yl
—
Scheme 2. Synthesis of compounds 12, 3, and 4.
dine 13 (Scheme 2). Unfortunately, the reaction was multi-
product and difficult to purify, therefore this route is unreason-
able. In accordance with literature reports, conversion of the cya-
no group of compounds 3a–j to an carboxamide group afforded
O
O
HO
HO
HO
HO
Br
OH
S
OH
S
N
N
N
N
Xylocydine analogues 4-amino-6-aryl-7-(b-L-xylofuranosyl)pyr-
rolo[2,3-d] pyrimidine-5-carboxamide 4a–j, using 30% H2O2 in
concentrated ammonia.40 But the reaction carried uneasily, be-
cause of poor solubility of reactants and products in 30% H2O2-
concentrated ammonia system. After adding a certain amount of
methanol to improve solubility of reactants and products and a
certain amount of aqueous sodium hydroxide as a catalyst, the
reaction time decreased from an average of more than three days
to a few hours.
N
N
CN
CN
3g
NH
2
3j
NH2
Scheme 3. Reagents and conditions: NBS, CH3CN, 0 °C to rt, 16 h.
acid in Pd(PPh3)4-DMF-Na2CO3 system.39 As to instability of the ac-
tive hetaryl boronic acid (entries h and i), the reaction was accom-
plished in PdCl2(PPh3)2–THF system.
The deprotection step was performed by treatment with meth-
anolic sodium methoxide to afford the desired 6-substituted Xyl-
ocydine analogues 3a–3i. Alternative 3j was prepared from 3g,
using NBS bromination (Scheme 3). Compound 3a–i also had
been experimented to prepared directly from unprotected 4-ami-
2.2. Biological profiling
The inhibitory actions of all prepared compounds were evalu-
ated by kinase assays in vitro with CDK1/Cyclin B1 and CDK2/Cy-
clin A2 kianses originating in HeLa cells as well as the cell cycle
study against the HeLa cell line.
no-6-bromo-5-cyano-7-(b-L-xylofuranosyl) pyrrolo[2,3-d] pyrimi-

C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
7103
Table 1
good resolution 2.2 Å and this co-crystallized structure allowed a
good understanding of the necessary selectivity requirements.
The prepared protein was used in the determination of the impor-
tant amino in the predicted binding pocket. The docking process
was carried out for compounds (Xylocydine 2, 3h, 3i, 3j) using
the affinity energy as a scoring function. The docking results are re-
corded in the Table 1. The docking of Xylocydine 2 and active mol-
ecule 3h–j into the active site of CDK2 was performed (Fig. 3).
The docking results revealed that: Compound 3h and 3i formed
four hydrogen bonds, three with Gln131, Asn132 and Glu12
through theirs –OH and the other hydrogen bond with Leu83
through theirs –NH2 moiety and the docking energy of them were
À8.8 kcal/mol and À8.9 kcal/mol respectively. While compound 3j
formed only two hydrogen bonds with two Glu12 through its –OH
and had a higher docking energy (À8.2 kcal/mol). In addition, 3h
and 3i have the same spatial orientation while 3j taken place a
great change to lead to the hydrogen bonding between –NH2 with
Leu83 was absolutely destroyed. The in vitro CDK2 inhibitory
activity of the designed three compounds was found to approxi-
mately match with the docking values, as 3h and 3i had close
CDK2 inhibition and docking result of newly synthesized compounds
Compound
CDK inhibition in vitro, IC50
(
l
M)
Docking result
CDK2/Cyclin A2 CDK1/Cyclin B Docking value H-bonds
(kcal/mol)
3h
3i
3j
2
4.6
4.8
>100
>100
À8.8
À8.9
À8.2
À8.6
—
4
4
2
5
—
55
>100
0.061a
0.0014a
b
b
1, 3a–g, 4a–j
—
—
a
The biological value of Xylocydine 2 as reported in lit.21
No significant activity.
b
2.2.1. In vitro CDK1/Cyclin B1 and CDK2/Cyclin A2 inhibition
activity
In general, 5-carboxamide substituted purine nucleosides mod-
ified with a (het) aryl ring either with or without substitution
(compounds 4a–j) showed no significant activity against the tested
kianses at the micro molar level (Table 1). In contrast, three 5-cya-
no substituted purine nucleosides containing 5-member heterocy-
clic aryl in 6-position (compounds 3h, 3i, and 3j) exhibited
inhibitory activity. We found that the three modified compounds
inhibit CDK2 kinase activity to a higher degree than CDK1 kinase
activity, though the IC50 values are at the micro molar level (from
inhibitory effect, IC50 4.6
docking score meanwhile 3j had lower docking score with IC50
55 M. Therefore, this result suggested that hydrogen bond be-
lM and 4.8 lM respectively, with higher
l
tween –NH2 and Leu83 may play a very important role in the reg-
ulation of inhibitory activity for CDK2 inhibitors. Alignment study
of docked compound 3h, 3i, and Xylocydine 2 with the binding
pocket of CDK2 protein (Fig. 3E) suggested that newly synthesized
compound 3h and 3i was perfectly aligned with Xylocydine 2. Both
the Xylocydine 2 and compound 3h and 3i make the same hydro-
gen bonding interaction with Leu83, Gln131 and Glu12. All of the
three compounds (Xylocydine 2, 3h, 3i) make the same hydrogen
bonding interaction with Leu83, Gln131, and Glu12. This result
indicated that spatial orientation of purine ring should be fully
considered in optimizing the lead compound.
Analyzing the docking data, Xylocydine 2 forms five hydrogen
bonds in the CDK2 binding pocket: two with two Leu83 through
its –NH2 and –CONH2 moiety, three with Gln131, Asp145, and
Glu12 through its –OH (Fig. 3A). However, we found that 6-Br is
in front of the triad (Lys33, Glu51, and Asp145) of CDK2 which is
involved in ATP phosphate orientation and magnesium coordina-
tion, and is thought to be critical for catalysis.41 It was noteworthy
that the 6-Br occupies a pocket composed by Lys33, Glu51, Asp145,
Phe80, and Leu55. This area is thought to confer a very important
opportunity for selectivity.42 Subsequent in vitro kinase assay
showed excellent selectivity to CDK2/Cyclin A2 (Table 1), that ap-
peared coherent with the theories above.
4.6 lM to 55 l
M). Comparing to Xylocydine 2,21 we have synthe-
sized three new inhibitors (3h–j) specificity for CDK2/Cyclin A2.
In order to get CDK1/Cyclin B1 and CDK2/Cyclin A2 kianses, HeLa
cells were treated by Paclitaxel with 80nM and Etoptoside with
50
for 24 h, 200
itated for 6 h ant 4 °C with an antibody specific for CDK1 and CDK2
respectly. The kinase was treated with 0–200 M inhibitor in a final
volume of 25 l for 30 min. Then kinase assay were performed at
30 °C for 15 min in reaction buffer that contains 5 g Histone H1
as a specific substrate for each kinase together with 10 Ci of
32P] ATP and 15
M ATP in a final volume of 50 l. The reaction
lg/ml in DMEM with 5% newborn calf serum afer cell seeding
l
g of protein extract of Hela cells were immunoprecip-
l
l
l
l
[
c-
l
l
was stopped by adding 5Â SDS–PAGE Sample Loading Buffer. The
kinase activity was detected by autoradiograph.
2.3. Measuring cell analysis by flow cytometry
Progression through G1 and entry into S phase are regulated by
Cdk2 complexed with Cyclin A and Cyclin E, and the G2/M transi-
tion is driven by Cdc2 complexed with Cyclin B. To evaluate the
inhibitory activity of CDK2 of compound 3h, 3i, and 3j in vivo, HeLa
cell were treated with indicated concentration of compound 3h, 3i,
and 3j for 72 h. The cell number of different phase was determined
by flow cytometry. As shown in Figure 2, at the concentration of
The molecular docking study provides useful information for
understanding the structural features of CDK2 inhibitors. Three
new purine nucleoside analogues 3h–j showed good docking
scores values, and this was worth expanding to design potent
activity and selectivity purine analogues.
6.25
including 3i and 3j. But after increasing the compound concentra-
tion to 12.5 M, the amount of G1/S phase fraction were increased
lM, compound 3h shows obvious G1/S phase arrest not
l
3. Experimental section
3.1. Chemistry: General
13.6% (3h), 6.47% (3i) and 13.52% (3j) respectively contrast to un-
treated cell, while G2 phase cells were only 0.13%, 7.26% and 0.22%
of total cells number in the same time. These results indicated that
compound 3h, 3i, and 3j may inhibit CDK2 activity and thus arrest
cell cycle in G1/S phase in HeLa cells.
The 1H and 13C NMR data were recorded with a Varian Mercury
300 NMR spectrometer, using TMS as an internal standard.
Chemical shifts (d) are given in parts per million and coupling con-
stants are given as absolute values expressed in Hertz. Mass spec-
tra were obtained using LC/MS 1100 of Agilent Technology
Corporation and Alltech ELSD 2000 instrument. Melting points
were determined by a X-4 digital microscope. Column chromatog-
raphy was generally performed on silica gel (300–400 mesh) and
TLC inspections on silica gel GF254 plates.
2.4. Molecular docking studies of CDK2 inhibitors and binding
conformation
All dock runs were conducted using Autodock Vina software. An
automated docking study was carried out using the crystal struc-
ture of Olomoucine/CDK2 complex (PDB code: 1W0X, available
from the Protein Data Bank) which was successfully obtained with

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C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
3h
3i
3j
Phase
Control
6.25µM
96.3
12.5µM
99.87
0.13
6.25µM
85.13
12.5µM
92.74
7.26
6.25µM
86.83
12.5µM
99.79
0.22
G1/S
G2
86.27
13.73
3.7
14.87
13.17
Figure 2. The cell cycle was blocked at G1/S phase by compound 3h, 3i, and 3j. HeLa cells were treated with indicated concentrations of compound 3h, 3i, and 3j for 72 h.
Cells were collected, stained with DAPI, analyzed by flow cytometry and calculated by measuring the area of G1, S and G2 peak.
3.1.1. 2-Amino-5-bromo-3,4-dicyanopyrrole (6)
3.1.2. 4-Amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (7)
2-Amino-5-bromo-3,4-dicyanopyrrole (21.0 g, 0.20 mol) and
formamidine acetate (21.0 g, 0.20 mol) were dissolved in ethylene-
glycolmonoethyl ether (380 mL). The mixture was heated at reflux
temperature for 24 h, the dark solution treated with charcoal, and
the charcoal removed from the hot solution by filtration. After wash-
ing the filter cake several times with 30 mL hot ethanol, the filtrate
was cooled to room temperature to yield a brown precipitate, which
was washed with water and collected by filtration. Recrystallization
of the crude product afforded 12.14 g (50%) of pale yellow product 7.
mp >300 °C. MS (ESI): m/z 240.0 [M+H+]. 1H NMR (300 MHz,
DMSO-d6): 13.84(s, 1H), 8.21(s, 1H), 7.20(s, 2H, NH2).
A solution of tetracyanoethylene (29.0 g, 226.0 mmol) in ace-
tone (160 mL) and ethyl acetate (340 mL) was treated with a solu-
tion of 33% HBr in acetic acid (162 mL) while maintaining the
internal temperature at 0 °C. The reaction mixture was stirred for
an additional 1 h and the resulting yellow solid was filtered. The
solid was then suspended in water and the pH of the suspension
was adjusted to 11 with 50% NaOH to achieve solution, stirred
for 15 min and then treated with glacial acetic acid to pH 5 to yield
a precipitate, which was filtered and dried in vacuo over KOH to
yield 37.2 g (78%) of 6. Analytical data of compound 6 is in agree-
ment with published data.31

C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
7105
Figure 3. (A–D) The proposed binding mode of compound Xylocydine, 3h, 3i, and 3j inside the active site of CDK2 kinase resulting from docking respectively. The most
important amino acids are shown together with their respective numbers. Xylocydine (A) forms five hydrogen bonds, two with two Leu83, three with Gln131, Asp145 and
Glu12. Both 3h (B) and 3i (C) form four hydrogen bonds, three with Gln131, Asn132 and Glu12 and the other with Leu83. Compound 3j (D) forms two hydrogen bonds with
two Glu12. (E) Alignment of docked compound 3h (green color), 3i (blue color) and Xylocydine (magenta color) with the binding pocket of CDK2. (F) The alignment was
shown as surface colored gray.
3.1.3. 1-O-Acetyl-2,3,5-tri-O-benzoyl-b-
L
-ribofuranose (10)
obtained as a white solid (91.0 g, yield 71%). mp 128–130 °C.
A suspension of the
L
(À)-xylose 8 (30.0 g, 0.20 mol) in methanol
Analytical data are identical to those reported earlier.43
(500 mL) was slowly added concentrated sulfuric acid (2 mL). The
mixture was stirred for 5 h at room temperature and then anhy-
drous potassium carbonate (5.2 g, 0.037 mol) was added to adjust
the pH to 7. The solid was filtered and washed well with methanol.
The filtrate was evaporated to dryness, witch was removed water
by azeotropic distillation with toluene to afford crud compound 9.
To a solution of above-mentioned crud compound 9 in pyridine
(200 mL) at ice bath was added dropwise Benzoyl chloride (90 mL,
0.773 mol). The reaction was warmed to ambient temperature, and
stirred overnight. The reaction mixture was poured into water and
extracted with ethyl acetate (3 Â 30 mL). The combined organic
layer was washed with brine, dried over Na2SO4, and evaporated
to give a fulvous oil.
This fulvous oil compound was dissolved in acetic acid (160 mL)
and acetic anhydride (40.0 mL, 0.411 mol). The mixture was cooled
to 0 °C before slowly adding concentrated sulfuric acid (25.0 mL,
0.469 mol). After dropping, the reaction was stirred at ambient
temperature overnight. The reaction mixture was poured into
water (200 mL) and neutralized with sodium acetate (40 g) and
then extracted with chloroform (4 Â 50 mL). The combined organic
layer was washed with brine, dried over Na2SO4 and evaporated to
dryness. The residue was chromatographed using petroleum ether
and ethyl acetate (5:1, v/v) as an eluent. The product 10 was
3.1.4. 4-Amino-6-bromo-5-cyano-7-(2,3,5-tri-O-benzoyl-
b-L
-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine(11)
stirred suspension of 4-amino-6-bromo-5-cyanopyrrolo*
A
[2,3-d] pyrimidine 7 (4.8 g, 20.0 mmol) in 200 mL dry acetonitrile
was added N,O-Bis(trimethylsily1)acetamide (BSA, 8.2 g, 40.0 mmol)
at room temperature under N2. After 30 min, 1-O-acetyl-2,3,5-tri-
O-benzoyl-b-L-ribofuranose 10 (15.0 g, 30.0 mmol) was added
along with trimethylsilyl trifluoromethanesulfonate (TMSOTf,
6.66 g, 30.0 mmol). The suspension was stirred at room tempera-
ture for 10 min, heated at 80 °C for 3 h and then cooled to room
temperature. The solution was diluted with ethyl acetate
(200 mL) and water (100 ml). The aqueous layer was separated
and discarded and the organic layer was washed with aqueous so-
dium bicarbonate, brine, and dried over magnesium sulfate. The
solvent was removed and the residue was chromatographed using
a solvent system of ethyl acetate and chloroform (1:9, v/v). The
product 11 was obtained as a white solid (10.2 g, 73% yield). mp
150–152 °C. MS (ESI): m/z 683.1 [M+H+]. 1H NMR (300 MHz,
CDCl3): 8.11–8.14 (m, 2H), 7.92–7.03 (m, 5H), 7.40–7.62 (m, 9H),
6.82–6.84 (m, 1H), 6.23 (d, 1H), 5.98–6.00 (m, 1H), 5.60 (s, 2H,
NH2), 4.83–4.90 (m, 3H). Analytical data are identical to those
reported earlier.33

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C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
3.1.5. 4-Amino-6-(p-tolyl)-5-cyano-7-(2,3,5-tri-O-benzoyl-
chromatography, using a solvent system of ethylacetate and chlo-
roform (1:9, v/v). The product 12e was obtained as a white solid
(0.118 g, 78% yield). Mp 182–184 °C. IR (KBr): 3358(NH2), 2924,
2219(CN), 1722, 1628. MS (ESI): m/z 758.0 [M+H+]. 1H NMR
(300 MHz, CDCl3): 8.11–8.13 (d, 2H), 7.92–8.00 (m, 5H),
7.39–7.64 (m, 13H), 6.89–6.90 (m, 1H), 5.73–5.90 (m, 4H),
4.77–4.85 (m, 3H). Anal. Calcd for C39H28BrN5O7: C, 61.75; H,
3.72; N, 9.23. Found: C, 61.71; H, 3.66; N, 9.28.
b-L
-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12a)
4-Amino-6-bromo-5-cyano-7-(2,3,5-tri-O-benzoyl-b-
L
-xylofura
nosyl-)pyrrolo[2,3-d]pyrimidine 11 (0.136 g, 0.20 mmol) was sus-
pended in dioxane (2.0 mL). The corresponding 4-methylphenylbo-
ronic acid (0.054 mg, 0.40 mmol), K2CO3 (0.082 g, 0.60 mmol) was
added and the solution was degassed with N2 for 5 min. Then,
Pd(OAc)2 (5 mol %, 2.5 mg,0.01 mmol) was added along with tri-
phenylphosphine (15.5 mg, 0.06 mmol) and the mixture was fur-
ther degassed. The reaction was heated at 100 °C for 12 h or until
completion of the reaction as shown by TLC. The reaction mixture
was then cooled to room temperature and extracted with ethyl ace-
tate (3 Â 10 ml). The organic layer was washed with brine (30 mL)
and dried over magnesium sulfate, filtered and concentrated under
reduced pressure. The residue was chromatographed using a
solvent system of ethyl acetate and chloroform (1:9, v/v), and the
product 12a was obtained as a white solid (0.133 g, 82% yield). mp
196–198 °C. IR (KBr): 3363 (NH2), 2220 (CN), 1720, 1632. 1H NMR
(300 MHz, CDCl3): 812–8.15 (m, 2H), 7.92–8.01 (m, 5H), 7.38–7.62
(m, 13H), 6.95–6.97 (m, 1H), 5.86–5.88 (m, 2H), 5.68 (s, 2H, NH2),
4.96–4.90 (m, 3H), 2.38 (s, 3H). Anal. Calcd for C40H31N5O7: C,
69.26; H, 4.50; N, 10.10. Found: C, 69.29; H, 4.46; N, 10.07.
3.1.10. 4-Amino-6-(3-bromophenyl)-5-cyano-7-(2,3,5-tri-O-
benzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12f)
The reaction was conducted according to the procedure de-
scribed for 12e to afford 12f as a white solid (0.117 g, 76% yield).
Mp 180–183 °C. IR (KBr): 3361(NH2), 2924, 2221(CN), 1723,
1628. MS (ESI): m/z 758.0 [M+H+]. 1H NMR (300 MHz, CDCl3):
8.11–8.13 (m, 2H), 7.95–8.02 (m, 5H), 7.78–7.79 (m, 1H),
7.41–7.61 (m, 12H), 6.84–6.87 (m, 1H), 5.85–5.88 (m, 1H), 5.83
(d, 1H), 5.69 (s, 2H, NH2), 4.79–4.89 (m, 3H). Anal. Calcd for
C39H28BrN5O7: C, 61.75; H, 3.72; N, 9.23. Found: C, 61.72; H,
3.68; N, 9.26.
3.1.11. 4-Amino-6-(1H-pyrrol-2-yl)-5-cyano-7-(2,3,5-tri-O-
benzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12g)
3.1.6. 4-Amino-6-(4-methoxyphenyl)-5-cyano-7-(2,3,5-tri-O-
benzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12b)
A solution of 11 (0.272 g, 0.40 mmol) in DMF (2.5 ml) was
added sequentially 1-N-(Boc)-pyrrole-2-boronic acid (0.126 g,
0.60 mmol) and PdCl2(PPh3)2 (5 mol %, 0.02 mmol, 24.0 mg). The
mixture was heated to 70 °C and 0.675 g (3.60 mmol) of sodium
carbonate dissolved in minimal water was added. The reaction
was then heated to 110 °C for 14 h. After cooling to room temper-
ature, the reaction was diluted with water (15 mL) and extracted
with ethyl acetate (3 Â 15 mL). The organic layers were dried with
magnesium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by flash chromatography, using a solvent sys-
tem of ethylacetate and chloroform (1:6, v/v). The product 12g was
obtained as a white solid (0.123 g, 75% yield). Mp 176–178 °C. IR
(KBr): 3425 (NH2), 2923, 2218 (CN), 1726, 1635. 1H NMR(300 MHz,
CDCl3): 9.76 (s, 1H), 7.91–8.07 (m, 16 H), 7.41–7.63 (m, 9H), 7.07 (s,
1H), 6.94 (s, 1H), 6.71 (d, 1H), 6.41 (d, 2H), 6.33 (d, 1H), 5.90 (d,
1H), 5.54 (s, 2H, NH2), 5.04–5.11 (m, 1H), 4.87–4.92 (m, 1H),
4.65–4.70 (m, 1H). Anal. Calcd for C37H28N6O7: C, 66.46; H, 4.22;
N, 12.57. Found: C, 66.49; H, 4.27; N, 12.52.
The reaction was conducted according to theprocedure described
for 12a to afford 12b as a white solid (0.139 g, 86% yield). Mp
226–228 °C. IR (KBr): 3354 (NH2), 2218 (CN), 1726, 1629. 1H NMR
(300 MHz, CDCl3): 8.11–8.14 (m, 2H), 7.92–8.01 (m, 5H), 7.40–7.61
(m, 11H), 6.94–6.99 (m, 3H), 5.87–5.90 (m, 2H), 5.58 (s, 1H, NH2),
4.76–4.86 (m, 3H), 3.81 (s, 3H). Anal. Calcd for C40H31N5O8: C,
67.69; H, 4.40; N, 9.87. Found: C, 67.72; H, 4.46; N, 9.80.
3.1.7. 4-Amino-6-(3-methoxyphenyl)-5-cyano-7-(2,3,5-tri-O-
benzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12c)
The reaction was conducted according to the procedure de-
scribed for 12a to afford 12c as a white solid (0.134 g, 83% yield).
Mp 218–220 °C. IR (KBr): 3336 (NH2), 2221 (CN), 1723, 1628. 1H
NMR (300 MHz, CDCl3): 8.15 (d, 2H), 7.93–8.00 (m, 5H), 7.38–
7.62 (m, 10H), 7.12–7.19 (m, 2H), 7.01–7.03 (m, 2H), 5.84–5.89
(m, 2H), 5.56 (s, 2H, NH2), 4.76–7.87 (m, 3H), 3.71 (s, 3H). Anal.
Calcd for C40H31N5O8: C, 67.69; H, 4.40; N, 9.87. Found: C, 67.74;
H, 4.44; N, 9.91.
3.1.12. 4-Amino-6-(thiophen-2-yl)-5-cyano-7-(2,3,5-tri-Obe-
nzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12h).
3.1.8. 4-Amino-6-(4-(tert-butyl)phenyl)-5-cyano-7-(2,3,5-tri-O-
benzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12d)
To a 25 mL round-bottom flask were charged 11 (0.272 g,
0.40 mmol) and thiophen-2-ylboronic acid (0.115 g, 0.90 mmol)
together with PdCl2 (PPh3)2 (13.8 mg, 0.02 mmol) and K2CO3
(0.082 g, 0.6 mmol) in 5 mL of tetrahydrofuran under an atmo-
sphere of N2. The reaction was heated at reflux for 24 h and cooled
to room temperature. The mixture was diluted with water and ex-
tracted with ethyl acetate. The combined organic extracts were
washed with brine, dried over MgSO4 and evaporated in vacuo.
The crude product was purified by flash chromatography using a
solvent system of ethylacetate and chloroform (1:9, v/v). The prod-
uct 12h was obtained as a white solid (0.205 g, 75% yield). Mp 165–
167 °C. IR (KBr): 3345 (NH2), 2926, 2219 (CN), 1720, 1634. MS
(ESI): m/z 685.9 [M+H+]. 1H NMR (300 MHz, CDCl3): 8.13–8.16 (d,
2H), 7.96–8.13 (m, 5H), 7.38–7.62 (m, 11), 7.15–7.18 (m, 1H),
7.03–7.06 (m, 1H), 6.03 (d, 1H), 5.90–5.93 (m, 1H), 5.58 (s, 2H),
4.81–4.88 (m, 3H). Anal. Calcd for C37H27N5O7S: C, 64.81; H, 3.97;
N, 10.21; S, 4.68. Found: C, 64.84; H, 3.96; N, 10.23; S, 4.72.
The reaction was conducted according to the procedure de-
scribed for 12a to afford 12c as a white solid (0.131 g, 81% yield).
Mp 201–202 °C. IR (KBr): 3348 (NH2), 2220 (CN), 1724, 1626.1H
NMR (300 MHz, CDCl3): 8.15–8.18 (m, 2H), 7.92–8.00 (m, 5H),
7.40–7.61 (m, 13H), 7.09–7.11 (m, 1H), 5.90–5.93 (m, 1H), 5.81(d,
1H), 5.62 (s, 2H, NH2), 4.78–4.88 (m, 3H), 1.33(s, 9H). Anal. Calcd
for C43H37N5O7: C, 70.19; H, 5.07; N, 9.52. Found: C, 70.12; H,
5.06; N, 9.60.
3.1.9. 4-Amino-6-(4-bromophenyl)-5-cyano-7-(2,3,5-tri-O-
benzoyl-b-L-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12e)
To a 25 ml round-bottom flask were charged 11 (0.136 g,
0.20 mmol) and 4-bromophenyl boronic acid (0.083 g, 0.4 mmol)
together with palladium tetrakis-triphenylphosphine (11.6 mg,
0.01 mmol) and K2CO3 (0.082 g, 0.6 mmol) in toluene (5.0 mL)
under an atmosphere of N2. The reaction was heated at reflux for
24 h and cooled to room temperature. The mixture was diluted
with water (10 mL) and extracted with ethyl acetate. The com-
bined organic extracts were washed with brine, dried over MgSO4
and evaporated in vacuo. The crude product was purified by flash
3.1.13. 4-Amino-6-(furan-2-yl)-5-cyano-7-(2,3,5-tri-O-benz-oyl-
b-L
-xylofuranosyl-)pyrrolo[2,3-d]pyrimidine (12i)
The reaction was conducted according to the procedure
described for 12g to afford 12i as a white solid (0.123 g, 76% yield).

C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
7107
Mp 162–164 °C. IR (KBr): 3358 (NH2), 2220 (CN), 1720, 1632. 1H
NMR (300 MHz, CDCl3): 8.13–8.16 (m, 2H), 7.96–8.02 (m, 5H),
7.38–7.61 (m, 10H), 7.11–7.13 (m, 2H), 6.56–6.57 (m, 1H), 6.32
(d, 1H), 5.99–6.00 (d, 1H), 5.59 (s, 2H, NH2), 4.81–4.90 (m, 3H).
Anal. Calcd for C37H27N5O8: C, 66.36; H, 4.06; N, 10.46. Found: C,
66.31; H, 4.11; N, 10.43.
2H, J = 3.0 Hz), 3.91–3.93 (m, 2H), 3.84 (s, 3H), 3.32–3.73 (m, 2H).
13C NMR (75 MHz, DMSO-d6): 159.49, 157.27, 152.98, 148.75,
146.04, 130.66, 128.32, 122.30, 116.52, 115.67, 115.38, 101.82,
91.31, 82.46, 79.71, 76.70, 59.66, 55.51. Anal. Calcd for
C19H19N5O5: C, 57.43; H, 4.82; N, 17.62. Found: C, 57.54; H, 4.86;
N, 17.53.
3.2. General procedure A for the deprotection of compounds 11,
12
3.2.5. 4-Amino-6-(4-(tert-butyl)phenyl)-5-cyano-7-(b-
ofuranosyl)pyrrolo[2,3-d]pyrimidine (3d)
L-xyl-
The reaction was conducted according to the general procedure
A and the compound 3d was obtained as a white solid. Mp 144–
146 °C. IR (KBr): 3427 (OH), 3335 (NH2), 2902, 2220(CN), 1648,
1611. MS (ESI): m/z 424.1 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.29 (s, 1H), 7.57–7.71 (m, 4H), 7.16 (s, 2H, NH2), 6.72 (d, 1H,
J = 9.3 Hz), 5.86 (d, 1H, J = 4.5 Hz), 5.43 (d, 1H, J = 3.3 Hz), 4.80 (s,
2H), 3.94–4.10 (m, 2H), 3.61–3.75 (m, 2H), 1.37(s, 9H). 13C NMR
(75 MHz, DMSO-d6): 157.11, 152.77, 148.60, 146.18, 129.80,
126.14, 115.45, 101.72, 91.21, 82.41, 79.56, 76.63, 59.54, 34.79,
30.93. Anal. Calcd for C22H22N5O4: C, 62.40; H, 5.95; N, 16.54.
Found: C, 62.34; H, 5.86; N, 16.61.
Compound 11, 12 (0.20 mmol) was treated with NaOMe
(32.00 mg, 0.60 mmol) in MeOH (10 mL) for 12 h at room temper-
ature or until completion of the reaction as shown by TLC. The mix-
ture was co-evaporated with silica and chromatographed using a
solvent system of methanol and chloroform (1:9, v/v) on a column
of silica to afford 1, 3.
3.2.1. 4-Amino-6-bromo-5-cyano-7-(b-
[2,3-d]pyrimidine (1)
L-xylofuranosyl)pyrrolo
The reaction was conducted according to the general procedure
A and the compound 1 was obtained as a white solid. IR (KBr):
3460 (OH), 3332 (NH2), 3162, 2921, 2217 (CN), 16437, 1596. 1H
NMR (300 MHz, DMSO-d6): 8.24 (s, 1H), 7.25 (s, 2H, NH2), 6.47
(d, 1H, J = 9.0 Hz), 5.91 (d, 1H, J = 4.8 Hz), 5.74 (d, 1H, J = 3.6 Hz),
4.74–4.82 (m, 2H), 4.02–4.06 (m, 2H), 3.59–3.74 (m, 2H). 13C
NMR (75 MHz, DMSO-d6): 156.22, 153.00, 148.81, 142.73, 121.70,
114.09, 102.11, 92.31, 82.49, 79.27, 76.31, 59.64. Anal. Calcd for
3.2.6. 4-Amino-6-(4-bromophenyl)-5-cyano-7-(b-
anosyl) pyrrolo[2,3-d]pyrimidine (3e)
L-xylofur-
The reaction was conducted according to the general procedure
A and the compound 3e was obtained as a white solid. Mp 160 °C.
IR (KBr): 3439 (OH), 3320 (NH2), 2923, 2221 (CN), 1637, 1597. MS
(ESI): m/z 445.9 [M+H+]. 1H NMR (300 MHz, DMSO-d6): 8.30 (s,
1H), 7.90 (d, 2H), 7.58 (d, 2H), 7.19 (s, 2H, NH2), 6.61 (d, 1H,
J = 9.3 Hz), 5.82 (d, 1H, J = 4.8 Hz), 5.36 (d, 1H, J = 3.6 Hz),
4.75–4.82 (m, 2H), 3.91–3.99 (m, 2H), 3.58–3.74 (m, 2H). 13C
NMR (75 MHz, DMSO-d6): 157.15, 152.96, 148.81, 144.84, 132.25,
132.07, 126.34, 124.49, 115.00, 101.68, 91.11, 82.27, 79.37, 76.39,
59.53. Anal. Calcd for C18H16BrN5O4: C, 48.45; H, 3.61; N, 15.69.
Found: C, 48.51; H, 3.66; N, 17.63.
C12H12BrN5O4: C, 38.94; H, 3.27; N, 18.92. Found: C, 39.00; H,
3.28; N, 18.88.
3.2.2. 4-Amino-6-(p-tolyl)-5-cyano-7-(b-
[2,3-d]pyrimidine (3a)
L-xylofuranosyl)pyrrolo
The reaction was conducted according to the general procedure
A and the compound 3a was obtained as a white solid (85% yield).
Mp 142–144 °C. IR (KBr): 3416 (OH), 3311 (NH2), 2918, 2221 (CN),
1649, 1595. 1H NMR (300 MHz, DMSO-d6): 8.28 (s, 1H), 7.45–7.53
(m, 4H), 7.15 (s, 2H, NH2), 6.72 (d, 1H, J = 9.6 Hz), 5.83 (d, 1H,
J = 4.8 Hz), 5.39 (d, 1H, J = 3.3 Hz), 4.76–4.81 (m, 2H), 3.88–3.97
(m, 2H), 3.57–3.73 (m, 2H), 2.43 (s, 3H). 13C NMR (75 MHz,
DMSO-d6): 157.12, 152.77, 148.02, 146.39, 140.72, 130.18,
129.99, 120.82, 124.18, 115.37, 101.71, 91.24, 82.31, 79.55, 76.61,
55.56, 21.02. Anal. Calcd for C19H19N5O4: C, 59.84; H, 5.02; N,
18.36. Found: C, 59.96; H, 5.08; N, 18.28.
3.2.7. 4-Amino-6-(3-bromophenyl)-5-cyano-7-(b-
anosyl)pyrrolo[2,3-d]pyrimidine (3f)
L-xylofur-
The reaction was conducted according to the general procedure
A and the compound 3f was obtained as a white solid. Mp
156–157 °C. IR (KBr): 3435(OH), 3331 (NH2) 2913, 2218 (CN),
1633, 1599. MS (ESI): m/z 445.9 [M+H+]. 1H NMR (300 MHz,
DMSO-d6): 8.30 (s, 1H), 7.85–7.88 (m, 2H), 7.62–7.66 (m, 2H),
7.02 (s, 2H, NH2), 6.55 (d, 1H, J = 1.8 Hz), 5.84 (d, 1H, J = 4.5 Hz),
5.37 (d, 1H, J = 3.6 Hz), 4.74 (s, 2H), 3.94–3.96 (m, 2H), 3.58–3.73
(m, 2H). Anal. Calcd for C18H16BrN5O4: C, 48.45; H, 3.61; N,
15.69. Found: C, 48.49; H, 3.66; N, 17.59.
3.2.3. 4-Amino-6-(4-methoxyphenyl)-5-cyano-7-(b-
L-xylof-
uranosyl)pyrrolo[2,3-d]pyrimidine (3b)
The reaction was conducted according to the general procedure
A and the compound 3b was obtained as a white solid. Mp 170–
172 °C. IR (KBr): 3426 (OH), 3337 (NH2), 2895, 2221(CN), 1638,
1606. MS (ESI): m/z 398.0 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.28 (s, 1H), 7.57 (d, 2H), 7.22 (d, 2H), 7.12 (s, 2H, NH2), 6.71 (d,
1H, J = 9.6 Hz), 5.83 (d, 1H, J = 4.5 Hz), 5.41 (d, 1H, J = 3.3 Hz),
4.76–4.80 (m, 2H), 3.90–3.95 (m, 2H), 3.88 (s, 3H), 3.58–3.71 (m,
2H). 13C NMR (75 MHz, DMSO-d6): 161.00, 157.03, 152.67,
148.56, 146.32, 131.64, 119.02, 115.49, 114.76, 101.66, 91.18,
82.35, 79.52, 76.60, 59.54, 55.51. Anal. Calcd for C19H19N5O5: C,
57.43; H, 4.82; N, 17.62. Found: C, 57.50; H, 4.87; N, 17.54.
3.2.8. 4-Amino-6-(1H-pyrrol-2-yl)-5-cyano-7-(b-
anosyl)pyrrolo[2,3-d]pyrimidine (3g)
L-xylofur-
The reaction was conducted according to the general procedure
A and the compound 3g was obtained as a white solid. Mp
146–147 °C. IR (KBr): 3425 (OH), 3324 (NH2), 2923, 2216 (CN),
1635, 1599. MS (ESI): m/z 356.8 [M+H+]. 1H NMR (300 MHz,
DMSO-d6): 11.71 (s, 1H), 8.25 (s, 1H), 7.18 (d, 1H), 7.08 (s, 2H,
NH2), 6.71–6.73 (m, 1H), 6.55 (s, 1H), 6.35 (d, 1H, J = 3.0 Hz), 5.89
(d, 1H, J = 4.5 Hz), 5.73 (d, 1H, J = 3.3 Hz), 4.73 (s, 2H), 3.96 (d,
2H, J = 3.6 Hz), 3.60–3.70 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
156.80, 152.53, 148.52, 139.33, 122.77, 116.91, 115.56, 113.16,
109.81, 101.75, 90.93, 82.25, 79.67, 76.65, 59.49. Anal. Calcd for
3.2.4. 4-Amino-6-(3-methoxyphenyl)-5-cyano-7-(b-
L-xylofu-
ranosyl)pyrrolo[2,3-d]pyrimidine (3c)
C16H16N6O4: C, 53.95; H, 4.53; N, 23.85. Found: C, 53.87; H, 4.50;
The reaction was conducted according to the general procedure
A and the compound 3c was obtained as a white solid. Mp 167–
168 °C. IR (KBr): 3434 (OH), 3329 (NH2), 2898, 2219(CN), 1642,
1613. MS (ESI): m/z 398.0 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.29 (s, 1H), 7.55–7.60 (m, 1H), 7.19–7.22 (m, 5H), 6.68 (d, 1H,
J = 9.3 Hz), 5.85 (d, 1H, J = 4.8 Hz), 5.44 (d, 1H, J = 3.6 Hz), 4.79 (d,
N, 23.92.
3.2.9. 4-Amino-6-(thiophen-2-yl)-5-cyano-7-(b-
osyl)pyrrolo[2,3-d]pyrimidine (3h)
The reaction was conducted according to the general procedure
A and the compound 3h was obtained as a white solid. Mp 180 °C.
L-xylofuran-

7108
C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
IR (KBr): 3434 (OH), 3332 (NH2), 2926, 2855, 2219 (CN), 1646,
1600. MS (ESI): m/z 373.9 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.29 (s, 1H), 8.02–8.04 (m, 1H), 7.52–7.53 (m, 1H), 7.37–7.40 (m,
1H), 7.21 (s, 2H, NH2), 6.63 (d, 1H, J = 9.3 Hz), 5.86 (d, 1H,
J = 4.5 Hz), 5.61 (d, 1H, J = 3.3 Hz), 4.78–4.82 (m, 2H), 3.94–3.99
(m, 2H), 3.59–3.72 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
157.08, 153.12, 148.85, 138.79, 132.07, 131.34, 128.48, 126.38,
114.99, 101.75, 90.98, 82.37, 79.34, 76.49, 59.53. Anal. Calcd for
80.04, 76.86, 59.48, 21.02. Anal. Calcd for C19H21N5O5: C, 57.14;
H, 5.30; N, 17.53. Found: C, 57.21; H, 5.28; N, 17.48.
3.3.2. 4-Amino-6-(4-methoxy)-7-(b-L-xylofuranosyl)
pyrrolo[2,3-d]pyrimidine-5-carboxamide (4b)
The reaction was conducted according to the general procedure
B and the compound 4b was obtained as a white solid. Mp 234–
235 °C. IR (KBr): 3473 (OH), 3327 (NH2), 3169, 2892, 2839, 1626,
1596. MS (ESI): m/z 415.9 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.14 (s, 1H), 7.52 (s, 2H, NH2), 7.41–7.52 (m, 2H), 7.15–7.19 (m,
2H), 7.07 (d, 1H, J = 10.8 Hz), 5.80 (s, 1H, NH2), 5.70 (d, 1H,
J = 4.5 Hz), 5.08 (d, 1H, J = 3.0), 4.76–4.82 (m, 1H), 4.62–4.65 (m,
1H), 3.86 (s, 3H), 3.70–3.82 (m, 2H), 3.50–3.67 (m, 2H). 13C NMR
(75 MHz, DMSO-d6): 166.76, 160.55, 158.24, 151.81, 147.89,
138.42, 132.60, 120.46, 114.70, 109.15, 90.94, 82.13, 80.00, 76.85,
59.47, 55.39. Anal. Calcd for C19H21N5O6: C, 54.94; H, 5.10; N,
16.86. Found: C, 54.91; H, 5.03; N, 16.95.
C16H15N5O4S: C, 51.47; H, 4.05; N, 18.76; S, 8.59. Found: C,
51.41; H, 4.06; N, 18.63; S, 8.65.
3.2.10. 4-Amino-6-(furan-2-yl)-5-cyano-7-(b-L-xylofuranosy-l)
pyrrolo[2,3-d]pyrimidine (3i)
The reaction was conducted according to the general procedure
A and the compound 3i was obtained as a white solid. Mp 165–
167 °C. IR (KBr): 3445 (OH), 3339 (NH2), 2937, 2872, 2223 (CN),
1640, 1612. MS (ESI): m/z 357.9 [M+H+]. 1H NMR (300 MHz,
DMSO-d6): 8.28 (s, 1H), 8.12 (d, 1H), 7.22 (s, 1H, NH2), 7.14 (d,
1H), 6.85–6.86 (m, 1H), 6.64 (d, 1H, J = 9.3 Hz), 5.87 (d, 1H,
J = 4.5 Hz), 5.77 (d, 1H, J = 3.3 Hz), 4.75–4.81 (m, 2H), 3.99–4.02
(m, 2H), 3.62–3.75 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 157.27,
153.24, 148.97, 146.42, 140.90, 134.84, 115.06, 112.57, 102.04,
91.70, 82.48, 79.69, 76.56, 59.55. Anal. Calcd for C16H15N5O5: C,
53.78; H, 4.23; N, 19.60. Found: C, 53.85; H, 4.26; N, 19.53.
3.3.3. 4-Amino-6-(3-methoxy)-7-(b-L-xylofuranosyl)
pyrrolo[2,3-d]pyrimidine-5-carboxamide (4c)
The reaction was conducted according to the general procedure
B and the compound 4c was obtained as a white solid. Mp 230–
232 °C. IR (KBr): 3468 (OH), 3326 (NH2), 3168, 2902, 2845, 1628,
1594. MS (ESI): m/z 415.9 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.15 (s, 1H), 7.50–7.55 (m, 3H), 7.16–7.20 (m, 1H), 7.02–7.08 (m,
3H), 5.92 (s, 1H, NH2), 5.73 (d, 1H, J = 4.5 Hz), 5.10 (d, 1H,
J = 2.7 Hz), 4.79–4.82 (m, 1H), 4.65(s, 1H), 3.8–3.89 (m, 5H),
3.53–3.70 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 166.68, 158.32,
151.96, 147.95, 137.98, 130.40, 123.24, 116.75, 115.84, 109.25,
101.53, 91.01, 82.17, 80.08, 76.82, 59.48, 55.32. Anal. Calcd for
3.2.11. 4-Amino-6-(5-bromothiophen-2-yl)-5-cyano-7-(b-
ylofuranosyl)pyrrolo[2,3-d]py rimidine (3j)
L-x-
A solution of 3g (45.1 mg, 0.10 mmol) in acetonitrile (20 mL)
was added NBS (42.8 mg, 0.20 mmol) in small portions under N2
at 0 °C. The resulting mixture was then warmed to room tempera-
ture and stirred for 16 h. The solvent was removed under reduced
pressure. The residue was chromatographed using a solvent sys-
tem of methanol and chloroform (1:9, v/v) and the product 3i
was obtained as a white solid. Mp 185–186 °C. IR (KBr): 3435
(OH), 3331 (NH2), 2960, 2221 (CN), 1638, 1595. MS (ESI): m/z
453.7 [M+H+]. 1H NMR (300 MHz, DMSO-d6): 8.29 (s, 1H), 7.53
(d, 1H, J = 3.9 Hz), 7.37 (d, 1H, J = 3.6 Hz), 7.24 (s, 1H, NH2), 6.51
(d, 1H, J = 9.9 Hz), 5.83 (d, 1H, J = 4.8 Hz), 5.58 (d, 1H, J = 3.9 Hz),
4.77–4.81 (m, 2H), 3.99 (d, 2H, J = 6.3 Hz), 3.61–3.71 (m, 2H). 13C
NMR (75 MHz, DMSO-d6):157.16, 153.38, 149.02, 137.27, 133.14,
131.80, 128.11, 116.45, 114.70, 101.74, 90.87, 82.34, 79.21, 76.36,
59.58. Anal. Calcd for C16H14BrN5O4S: C, 42.49; H, 3.12; N, 15.48;
S, 7.09. Found: C, 42.45; H, 3.16; N, 15.53; S, 7.12.
C19H21N5O6: C, 54.94; H, 5.10; N, 16.86. Found: C, 54.89; H, 5.04;
N, 16.93.
3.3.4. 4-Amino-6-(4-(tert-butyl)phenyl)-7-(b-L-xylofuranos-yl)
pyrrolo[2,3-d]pyrimidine-5-carboxamide (4d)
The reaction was conducted according to the general procedure
B and the compound 4d was obtained as a white solid. Mp 254–
256 °C. IR (KBr): 3456 (OH), 3332 (NH2), 3171, 2894, 1629, 1600.
MS (ESI): m/z 441.9 [M+H+]. 1H NMR (300 MHz, DMSO-d6): 8.15
(s, 1H), 7.64 (d, 2H, J = 8.1 Hz), 7.51 (s, 1H, NH2), 7.44 (d, 2H,
J = 8.1 Hz), 7.11 (d, 1H, J = 10.2 Hz), 5.87 (s, 1H, NH2), 5.76 (d, 1H,
J = 4.5 Hz), 5.10 (d, 1H, 2.4 Hz), 4.77–4.80 (m, 1H), 4.66 (s, 1H),
3.79–3.88 (m, 2H), 3.52–3.70 (m, 2H). 13C NMR (75 MHz, DMSO-
d6): 166.77, 158.24, 152.76, 151.83, 147.91, 138.30, 130.91,
125.94, 125.82, 109.36, 101.59, 90.99, 82.21, 80.11, 76.93, 59.51,
34.71, 31.02. Anal. Calcd for C22H27N5O5: C, 59.85; H, 6.16; N,
15.86. Found: C, 59.90; H, 6.13; N, 15.74.
3.3. General procedure B for the synthesis of compound 4
A solution of 3 (0.2 mmol) in concentrated ammonium hydrox-
ide (5 ml) was added 30% H2O2 (0.5 mL) and methanol (0.3 mL), the
mixture was stirred at room temperature until completion of the
reaction as shown by TLC. The solvent was removed under reduced
pressure. The residue was chromatographed using a solvent sys-
tem of methanol and chloroform (1:8, v/v) and the product 4 were
obtained as white solid.
3.3.5. 4-Amino-6-(4-bromophenyl)-7-(b-L-xylofuranosyl)
pyrrolo[2,3-d]pyrimidine-5-carboxamide (4e)
The reaction was conducted according to the general procedure
B and the compound 4e was obtained as a white solid. Mp 241–
243 °C. IR (KBr): 3461 (OH), 3327 (NH2), 3171, 2925, 2851, 1629,
1602. 1H NMR (300 MHz, DMSO-d6): 8.16 (s, 1H), 7.81 (d, 2H),
7.52 (s, 1H, NH2), 7.44 (d, 2H), 6.99 (d, 1H, J = 9.9 Hz), 6.22 (s, 1H,
NH2), 5.73 (d, 1H, J = 4.8 Hz), 5.08 (d, 1H, J = 3.0 Hz), 4.78–4.82
(m, 1H), 4.65 (s, 1H), 3.80–3.90 (m, 2H), 3.51–3.72 (m, 2H). 13C
NMR (75 MHz, DMSO-d6): 166.71, 158.18, 151.95, 148.03, 136.66,
133.29, 131.88, 127.91, 123.77, 109.90, 101.52, 90.91, 82.08,
79.86, 76.68, 59.44. Anal. Calcd for C18H18BrN5O5: C, 46.57; H,
3.91; N, 15.08. Found: C, 46.51; H, 3.89; N, 15.13.
3.3.1. 4-Amino-6-(p-tolyl)-7-(b-L-xylofuranosyl)pyrrolo[2,3-d]
pyrimidine-5-carboxamide (4a)
The reaction was conducted according to the general procedure
B and the compound 4a was obtained as a white solid. Mp 238–
240 °C. IR (KBr): 3463(OH), 3325(NH2), 3168, 2919, 2846, 1634,
1598. MS (ESI): m/z 399.9 [M+H+]. 1H NMR (300 MHz, DMSO-d6):
8.17 (s, 1H), 7.53 (s, 2H, NH2),7.37–7.44 (m, 4H), 7.08 (d, 1H,
J = 10.2 Hz), 5.80(s,1H, NH2), 5.71 (d, 1H, J = 3.8 Hz), 5.06 (d, 1H,
J = 3.0 Hz), 4.77–4.81 (m, 1H), 4.63–465 (m, 1H), 3.76–3.89 (m,
2H), 3.52–3.66 (m, 2H), 2.42(s, 3H). 13C NMR (75 MHz, DMSO-
d6): 166.73, 158.29, 151.88, 147.97, 140.07, 138.51, 131.22,
131.17, 131.05, 129.81, 125.83, 105.18, 101.59, 91.02, 82.17,
3.3.6. 4-Amino-6-(3-bromophenyl)-7-(b-L-xylofuranosyl)
pyrrolo[2,3-d]pyrimidine-5-carboxamide (4f)
The reaction was conducted according to the general procedure
B and the compound 4f was obtained as a white solid. Mp 220 °C. IR

C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
7109
(KBr): 3458 (OH), 3328 (NH2), 2942, 2864, 1630, 1612. 1H NMR
(300 MHz, DMSO-d6): 8.17 (s, 1H), 7.78–7.81 (m, 1H), 7.71 (s, 2H,
NH2), 7.51–7.58 (m, 3H), 6.98 (d, 1H, J = 9.3 Hz), 6.32 (s, 1H, NH2),
5.76 (d, 1H, J = 4.8 Hz), 5.09 (d, 1H, J = 3.0 Hz), 4.80–4.81 (m, 1H),
4.64–4.66 (m, 1H), 3.82–3.89 (m, 2H), 3.56–3.71 (m, 2H). 13C
NMR (75 MHz, DMSO-d6): 166.79, 158.28, 152.10, 148.06, 136.12,
132.90, 131.02, 130.89, 130.49, 121.88, 110.27, 101.53, 91.01,
82.21, 80.01, 76.75, 59.48. Anal. Calcd for C18H18BrN5O5: C, 46.57;
H, 3.91; N, 15.08. Found: C, 46.54; H, 3.90; N, 15.15.
C16H16BrN5O5S: C, 40.86; H, 3.43; N, 14.89; S, 6.82. Found: C,
40.89; H, 3.41; N, 14.83; S, 6.92.
3.4. Biology
3.4.1. Materials and reagents
Histone H1 were purchased from Calbiochem, dissolved in
water, and stored at À80 °C as 1
lg/ll stock. Hela cells were main-
tained at 37 °C in 5% CO2 in Dulbecco’s modified Eagle’s medium
(Invitrogen) supplemented with 10% newborn calf serum (Inbitro-
gen) and aitibiotics/antimycotics (Invitrogen).
3.3.7. 4-Amino-6-(1H-pyrrol-2-yl)-7-(b-L-xylofuranosyl)pyrrolo
[2,3-d]pyrimidine-5-carboxamide (4g)
The reaction was conducted according to the general procedure
B and the compound 4g was obtained as a white solid. Mp 225–
226 °C. IR (KBr): 3439 (OH), 3345(NH2), 2922, 1636, 1445. 1H
NMR (300 MHz, DMSO-d6): 11.56 (s, 1H), 8.13 (s, 1H), 7.59 (s,
2H, NH2), 7.10–7.14 (m, 2H), 6.43–6.45 (m, 1H), 6.31–6.35 (m,
1H), 5.68 (d, 1H, J = 4.5 Hz), 5.57 (s, 1H), 5.23 (d, 1H, J = 2.7 Hz),
4.72–4.75 (m, 1H), 4.58–4.60 (m, 1H), 3.80–3.90 (m, 2H), 3.66–
3.71 (m, 1H), 3.51–3.57 (m, 1H). 13C NMR (75 MHz, DMSO-d6):
166.41, 158.32, 152.44, 148.30, 133.09, 131.51, 130.08, 128.29,
127.89, 111.99, 101.65, 90.99, 82.28, 80.06, 76.90, 59.59. Anal.
Calcd for C16H18N6O5: C, 51.33; H, 4.85; N, 22.45. Found: C,
51.37; H, 4.87; N, 22.32.
3.4.2. In vitro kinase assay
The kinase assays were performed at 30 °C for 15 min in reaction
buffer with a final volume of 50 ll that contains Histone H1 as a spe-
cific substrate for each kinase. In order to get CDK1/Cyclin B1 and
CDK2/Cyclin A2 kianses, after cell seeding for 24 h, HeLa cells were
treated by Paclitaxel with 80 nM and Etoptoside with 50
DMEM with 5% newborn calf serum for 12 h, 200 g of protein
extract of HeLa cells were immunoprecipitated for 6 h at 4 °C with
an antibody specific for CDK1 and CDK2 respectively. Then the pro-
tein was washed for several times as kinase for experiment.
lg/ml in
l
3.4.3. Cell cycle assay
In brief, after treatment with indicated concentrations of com-
3.3.8. 4-Amino-6-(thiophen-2-yl)-7-(b-
L
-xylofuranosyl)pyrrolo
pound 3h, 3i, and 3j (6.25 lM, 12.5 lM) for 72 h, HeLa cells were
[2,3-d] pyrimidine-5-carboxamide (4h)
pelleted and washed with PBS. Then cells were fixed in 75% ethanol
at 4 °C overnight. After being washed twice with PBS, the cells
were stained with 1.0 mL DAPI, 1 mg/mL RNase A (Sigma), and Tri-
ton X-100 in 3.8 mmol/L sodium citrate, and incubated on ice for
30 min in the dark. Analysis was carried out using a Becton Dickin-
son FACSCalibur cytometer, and cells were quantitated by mea-
surement the area of G1, S and G2 peak.
The reaction was conducted according to the general procedure
B and the compound 4h was obtained as a white solid. Mp
228–230 °C. IR (KBr): 3455 (OH), 3324(NH2), 2879, 1638, 1600.
1H NMR (300 MHz, DMSO-d6): 8.16 (s, 1H), 7.98 (d, 1H), 7.69
(s, 1H, NH2), 7.42–7.44 (m, 1H), 7.31–7.34 (m, 1H), 6.98 (d,
1H, J = 9.9 Hz), 6.15 (s, 1H, NH2), 5.72 (d, 1H, J = 4.8 Hz), 5.18(d,
1H, J = 3.0 Hz), 4.75–4.79 (m, 1H), 4.63–4.66 (m, 1H), 3.82–3.89
(m, 2H), 3.55–3.72 (m, 2H). 13C NMR (75 MHz, DMSO-d6):
166.27, 158.37, 152.13, 147.97, 131.68, 121.89, 116.48, 113.11,
110.52, 109.35, 101.63, 91.00, 82.16, 80.29, 76.91, 59.46. Anal.
Calcd for C16H17N5O5S: C, 49.10; H, 4.38; N, 17.89; S, 8.19. Found:
C, 49.11; H, 4.36; N, 17.83; S, 8.25.
3.4.4. Molecular docking studies of CDK2 inhibitors
Autodock vina was chosen as docking program.44 The 3D struc-
ture of the test compound was constructed by InsightII/Builder and
the geometry of the inhibitor was further optimized using AM1
method. After determining the binding site based on the crystal
structure of CDK2/Cyclin A (PDB code: 1W0X), the inhibitor (Xylo-
cydine, 3h, 3i, and 3j) was docked into the protein CDK2/Cyclin A.
Finally, the docked complex was selected by the criterion of inter-
acting energy combined with the geometrical matching.
3.3.9. 4-Amino-6-(furan-2-yl)-7-(b-L-xylofuranosyl)pyrrolo-
[2,3-d]pyrimidine-5-carboxamide (4i)
The reaction was conducted according to the general procedure B
and the compound 4i was obtained as a white solid. Mp 208–209 °C.
IR (KBr): 3461 (OH), 3332(NH2), 2929, 2832, 1634, 1596. 1H NMR
(300 MHz, DMSO-d6): 8.18 (s, 1H), 8.02–8.03 (m, 1H), 7.71 (s, 1H,
NH2), 6.99–7.02 (m, 1H), 6.95 (d, 1H, J = 3.6 Hz), 6.76–6.78 (m, 1H),
6.56 (s, 1H, NH2), 5.78 (d, 1H, J = 4.5 Hz), 5.25 (d, 1H, J = 3.0 Hz),
4.74–4.77 (m, 1H), 4.61–4.64 (m, 1H), 3.88 (s, 2H), 3.53–3.72 (m,
2H). 13C NMR (75 MHz, DMSO-d6):166.32, 158.40, 152.54, 148.15,
145.85, 140.62, 126.77, 115.83, 112.29, 111.78, 101.58, 91.36,
82.26, 80.16, 76.81, 59.46. Anal. Calcd for C16H17N5O6: C, 51.20; H,
4.57; N, 18.66. Found: C, 51.25; H, 4.53; N, 18.63.
4. Conclusion
A novel and simple route to syntheses 6-substituted purine
nucleoside analogues with tetracyanoethylene and
L
(À)-xylose as
starting molecules has been developed. Three synthesized com-
pounds (3h–j), which have specific CDK2 inhibitory activity, were
identified by in vitro CDK1/Cyclin B1 and CDK2/Cyclin A2 kinase as-
say. Those three new compounds induce cell cycle arrest obviously
at G1/S phase in HeLa cells. Molecular docking study provides use-
ful information for understanding the structural features of CDK2
inhibitors and appears coherent with the obtained biological data.
3.3.10. 4-Amino-6-(5-bromothiophen-2-yl)-7-(b-L-xylofuran-
osyl)pyrrolo[2,3-d]pyrimidine-5-carboxamide (4j)
The reaction was conducted according to the general procedure
B and the compound 4j was obtained as a white solid. Mp 232–
234 °C. IR (KBr): 3455 (OH), 3338 (NH2), 2925, 1633, 1598. 1H
NMR (300 MHz, DMSO-d6): 8.18 (s, 1H), 7.73 (s, 2H, NH2), 7.46
(d, 1H, J = 3.9 Hz), 7.26 (d, 1H, J = 3.9 Hz), 6.91 (d, 1H, J = 9.6 Hz),
6.58 (s, 1H, NH2), 5.76 (d, 1H, J = 4.5 Hz), 5.24 (d, 1H, J = 3.0 Hz),
4.80–4.83 (m, 1H), 4.64–4.66 (m, 1H), 3.85–3.93 (m, 2H), 3.31–
3.71 (m, 2H). 13C NMR (75 MHz, DMSO-d6): 166.30, 158.24,
152.61, 148.24, 133.99, 131.42, 129.73, 128.23, 115.78, 112.70,
101.47, 90.81, 82.23, 79.89, 76.71, 59.51. Anal. Calcd for
Acknowledgment
This work was supported by Grants from the National Nature
Science Foundation of China (Projects 90813003 and 31070670).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.10.003.

7110
C. Xiao et al. / Bioorg. Med. Chem. 19 (2011) 7100–7110
24. Krystof, V.; McNae, I. W.; Walkinshaw, M. D.; Fischer, P. M.; Muller, P.;
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