The synthesis and evaluation of new carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside analogs

Article abstract of DOI:10.3987/COM-14-12999

New carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside analogs were synthesized with the key intermediate, 4-amino-6-bromo-5-cyanopyrrolo[2,3- d]pyrimidine (2), by SN2 reaction. One of the products, 4-amino-6-bromo-1- cyclopentyl-¹H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (9), showed significant anti-proliferative activity to the human ovarian cancer PA-1 cells (IC50: 3.9 μM). Based on the biological effects and the functional group characteristics of the compound 9, other carbocyclic nucleoside analogs related to the compound 9 were synthesized with key intermediate 2 by a Pd(0)-catalyzed coupling reaction. As expected, syn-4-amino-6-bromo-7-[4-(methoxymethyl)-2- cyclopenten-1-yl]- 7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (15) showed very similar antiproliferative activity (IC50: 2.6 μM) when compared to compound 9.

Full text of DOI:10.3987/COM-14-12999

HETEROCYCLES, Vol. 89, No. 7, 2014, pp. -. © 2014 The Japan Institute of Heterocyclic Chemistry
Received, 7th April, 2014, Accepted, 2nd May, 2014, Published online, 12th May, 2014
DOI: 10.3987/COM-14-12999
THE SYNTHESIS AND EVALUATION OF NEW CARBOCYCLIC
PYRROLO[2,3-d]PYRIMIDINE NUCLEOSIDE ANALOGS
Jongbok Lee,a Hyewon Seo,b Sangeun Yoon,a Kowoon Choi,b Chul-Hoon
Lee,b* and Hakjune Rheea,c*
aDepartment of Bionanotechnology, Hanyang University, Sa 3-Dong 1271,
Ansan, Kyunggi-Do, 426-791, Korea. E-mail: hrhee@hanyang.ac.kr
bDepartment of Pharmacy, Hanyang University, Sa 3-Dong 1271, Ansan,
Kyunggi-Do, 426-791, Korea. E-mail: chhlee@hanyang.ac.kr
cDepartment of Chemistry and Applied Chemistry, Hanyang University, Sa 3-
Dong 1271, Ansan, Kyunggi-Do, 426-791, Korea.
Abstract - New carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside analogs were
synthesized with the key intermediate, 4-amino-6-bromo-5-cyanopyrrolo[2,3-
d]pyrimidine (2), by SN2 reaction. One of the products, 4-amino-6-bromo-1-
cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (9), showed significant
anti-proliferative activity to the human ovarian cancer PA-1 cells (IC50: 3.9 μM).
Based on the biological effects and the functional group characteristics of the
compound 9, other carbocyclic nucleoside analogs related to the compound 9
were synthesized with key intermediate 2 by a Pd(0)-catalyzed coupling reaction.
As expected, syn-4-amino-6-bromo-7-[4-(methoxymethyl)-2-cyclopenten-1-yl]-
7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (15) showed very similar anti-
proliferative activity (IC50: 2.6 μM) when compared to compound 9.
INTRODUCTION
Carbocyclic nucleosides are analogs of naturally occurring nucleosides in which a methylene group has
replaced the oxygen atom of the furanose ring. These analogs have good metabolic stability because
they are unaffected by hydrolases and phosphorylases that cleave the glycosidic bond of natural
nucleosides.1 Interestingly, the analogs are recognized by the same enzymes that recognize the natural
nucleosides.2 Carbocyclic nucleosides have received considerable attention as potential antiviral and
antitumor agents, especially those with purine or pyrimidine bases.2-4 For example, carbovir, the
carbocyclic analog of 2',3'-didehydro-2',3'-dideoxyguanosine, has been the subject of intensive research

due to its selective anti-HIV-1 activity and hydrolytic stability.5-8 Moreover, its congener abacavir, (±)-
cis-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol] demonstrates the
importance of structure-activity relationship (SAR) studies because it has a higher oral bioavailability
than carbovir and has been used in cocktail therapy for the treatment of AIDS.9-12
OH
NH
N
N
N
N
N
N
HO
HO
N
NH2
N
NH2
Carbovir
Abacavir
Figure 1. The structures of carbovir and abacavir
Pyrrolo[2,3-d]pyrimidine analogs have also attracted attention since toyocamycin and sangivamycin
were
isolated.13-17
Sangivamycin,
(4-amino-5-carboxamido-7-(D-ribofuranosyl)pyrrolo[2,3-
d]pyrimidine), isolated from Streptomyces rimosus, has antiviral and antitumor activity.18,19 A number
of its analogs have also been synthesized and evaluated as potential anticancer agents against a variety
of human cancers such as mammary carcinoma and leukemia.20-22 Although such analogs can inhibit
the growth of various human cancer cells, none of them are used in a clinical setting because of their
toxicity to normal human cells.23 In order to overcome the weakness of these analogs, the selective
inhibition of cancer cell growth must be achieved.
O
NH2
NH2
NH2
CN
N
N
N
N
N
O
N
O
HO
HO
OH
OH
HO
HO
toyocamycin
sangivamycin
Figure 2. The structures of toyocamycin and sangivamycin
In the current study, we describe the synthesis of carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside
analogs and its biological activity.
RESULTS AND DISCUSSION
To synthesize carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside analogs, the key intermediate, 4-amino-
6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (2), was synthesized by previously reported methods.24,25 2-
Amino-5-bromo-3,4-dicyanopyrrole (1) was prepared by the reaction of tetracyanoethylene in the
presence of an excess amount of hydrogen bromide (Scheme 1). Compound 1 was reacted with
foramidine acetate in 2-ethoxyethanol to give key intermediate 2 as a pale yellow solid which was used

without further purification for the next steps.13,26 Although an improved method for the synthesis of
compound 2 has been reported, the reaction was carried out under harsh conditions.27
CN
NH2
CN
CN
CN
NC
a
b
NC
N
Br
Br
N
H2N
N
CN
N
H
H
1
2
Scheme 1. Synthesis of key intermediate 2. Reagents and conditions:
(a) HBr in AcOH (33 wt%), acetone, EtOAc, 0 oC, 1.5 h, 53%;
(b) formamidine acetate, 2-ethoxyethanol, reflux, 36 h, 63%.
The reaction of key intermediate 2 with cyclopentyl bromide in the presence of sodium hydride as a
base produced the two regioisomers 3 and 4 (Scheme 2). To analyze the two regioisomers (3 and 4), the
nuclear Overhauser effect (NOE) between hydrogen on the 2-position of the pyrrolo[2,3-d]pyrimidine
and hydrogens on the cyclopentyl ring was examined by 2-D NMR spectroscopy. The aromatic proton
and protons on the cyclopentyl ring in the 1-substituted pyrrolo[2,3-d]pyrimidine (3) appeared as cross
peaks in the NOESY spectrum, whereas the cross peaks in the 7-substituted pyrrolo[2,3-d]pyrimidine
(4) were not detected because the two protons are spatially too far away. The structure of 1-substituted
pyrrolo[2,3-d]pyrimidine (3) was also confirmed by X-ray crystallography (Figure 3).
Figure 3. ORTEP representation of the X-ray structure of 4-amino-6-bromo-
1-cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (3).28
Compounds 7 and 8 were synthesized by debromination of isolated compounds 3 and 4 and the
subsequent oxidative hydrolysis of compounds 5 and 6.29,30 Compound 9 and 10 were obtained in
excellent yields by oxidative hydrolysis of compounds 3 and 4, respectively (Scheme 2).
To determine whether the synthesized carbocyclic pyrrolo[2.3-d]pyrimidine nucleoside analogs
(compounds 3-10) can selectively inhibit the cell proliferation, we performed the MTT assay using a
human ovarian cancer PA-1 cells. As a result, compound 9 significantly demonstrated anti-proliferative

NH2
N
NH2
N
NH2
N
NH2
N
NH2
N
CN
CN
CN
CN
CN
a
b
N
N
N
N
N
Br
Br
Br
+
N
+
N
N
N
N
H
2
4
3
5
6
c
c
O
O
O
O
NH2
N
NH2
N
NH2
NH2
Br
NH2
N
NH2
NH2
Br
NH2
4
5
3 N
N
N
N
6
2
+
N
+
N
N
7
N
1N
9
10
7
8
Scheme 2. Synthesis of carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside compounds (3-10).
Reagents and conditions: (a) i) NaH, dimethylacetamide, 60 oC, 30 min. ii) cyclopentyl bromide,
60 oC, 24 h, 37% for 3, 20% for 4; (b) HCO2NH4, Pd/C, EtOH, reflux, 1h, 94% for 5, 94% for 6;
(c) H2O2 (30 wt%), K2CO3, DMSO, rt, 1 h, 83% for 7, 76% for 8, 97% for 9, 95% for 10.
effect in a dose-dependent manner (IC50: 3.9 μM for 24 h). However, the other compounds 3-8, and 10
did not successfully inhibit the cancer cell proliferation or were shown to have no effect (Figure 4).
Thus, we recognized that the amide group in compound 9 is much more effective in terms of cell
growth inhibition, while the nitrile group in compound 3 has no effect. In contrast to other reported
analogs of sangivamycin, which are usually debrominated compounds and 7-substituted compounds,
compound 9, brominated and 1-substituted carbocyclic pyrrolo[2,3-d]pyrimidine, demonstrated
excellent anti-proliferative activity in PA-1 cells.
Figure 4. Anti-proliferative effect of compounds 3-10 in human ovarian cancer PA-1 cells. PA-1 cells
were incubated with 1 - 50 μM of each compound for 24 h. The cell viability was determined by MTT
assay. The results represent the mean ± SD (standard deviation) of three independent experiments.
(*p<0.05, **p<0.01)

With this interesting result, we focused on the position of the cyclopentyl ring, which has hydrophobic
character. In order to determine the effect of the hydrophobic character on the anti-cancer activity, we
decided to select and examine 7-substituted compounds which are well known carbocyclic and normal
nucleoside analogs as carbovir, abacavir, toyocamycin, and sangivamycin. Our group has previously
reported on the synthesis of carbocyclic nucleosides by a Pd(0)-catalyzed coupling reaction. The syn-
dicarbonate (11) was prepared by the known procedure.31 The Pd(0)-catalyst, which produced π-
allylpalladium complex with the dicarbonate (11), was prepared in situ from Pd(OAc)2 with
triisopropylphosphite in the presence of n-BuLi in anhydrous THF under argon. As several research
groups reported,32-34 the π-allylpalladium complex was coupled with intermediate 2 by nucleophilic
attack to give the syn-coupling product (12) (Scheme 3).31-36 The 1,4-substitutied product was
favorably obtained over the 1,2-substituted product due to the steric hindrance of the nonbonding
interaction with the carbonate moiety in the cyclopentene ring.32-34,37
NH2
CN
MeO2CO
NH2
N
CN
N
a
Br
N
N
+
N
Br
OCO2Me
N
MeO2CO
H
11
12
2
Scheme 3. Synthesis of compound 12. Reagents and conditions: (a) i) Pd(OAc)2, (i-PrO)3P,
THF, rt, 10 min. ii) n-BuLi, rt, 5 min. iii) NaH, 2, DMSO, rt. iv) 11, DMSO, rt, 2 h, 50%.
Oxidative hydrolysis of compound 12, followed by the hydrolysis of the carbonate moiety, produced
the carbocyclic nucleoside (13) (Scheme 4).
Scheme 4. Synthesis of compound 13. Reagents and conditions:
(a) i) H2O2 (30 wt%), K2CO3, DMSO, rt, 2 h. ii) aq. MeOH, rt, 2 h. 75%.
In order to provide hydrophobic characters, the hydroxyl group of compound 13 was treated with acetic
anhydride to give acetylated compound 14. Compound 13 was also reacted with dimethyl sulfate to
produce the etherificated compound 15 (Scheme 5).

Scheme 5. Synthesis of compound 14 and 15. Reagents and conditions:
(a) Ac2O, pyridine, DMAP, DMSO, rt, 6 h, 90%; (b) Me2SO4, NaH, DMSO, rt, 1 h, 75%.
We expected that the C-N bond at 7-position can freely rotates to provide the hydrophobic environment
around the 1-position as shown in compound 14 and 15. To examine the cytotoxicity of the synthesized
carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside analogs (compounds 12-15) in PA-1 cells, we also
performed the MTT assay. In contrast to compound 12, which did not inhibit the proliferation of PA-1
cells, compound 15 showed significant cell growth inhibition with 1 - 50 μM in a dose-dependent
manner and very similar biological activity when compared to compound 9. The IC50 value of
compound 15 for growth inhibition for 24 h was 2.6 μM. Compounds 13 and 14 showed very similar
biological activity. The IC50 values for compounds 13 and 14 were 10.5 μM and 8.8 μM, respectively
(Figure 5). Such similarities were observed because compound 14 would undergo in vitro hydrolysis to
compound 13. With this result, we realized that the hydrophobic character adjacent to the 1-position of
carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside contributes considerably to the biological activity.
Figure 5. Anti-proliferative effect of compounds 12-15 in human ovarian cancer PA-1 cells. PA-1 cells
were incubated with 1 - 50 μM of each compound for 24 h. The cell viability was determined by MTT
assay. The results represent the mean ± SD (standard deviation) of three independent experiments.
(*p<0.05, **p<0.01)

CONCLUSIONS
We synthesized new carbocyclic pyrrolo[2,3-d]pyrimidine nucleoside analogs and evaluated the in vitro
cytotoxicity of the compounds. With key intermediate 2, 1-substituted carbocyclic nucleosides were
synthesized by SN2 reaction. Compound 9 significantly inhibited the growth of human ovarian cancer
cells (IC50: 3.9 μM). We also synthesized and examined other analogs, namely 7-substituted carbocyclic
nucleosides that are related to compound 9, by a Pd(0)-catalyzed coupling reaction. Similar to
compound 9, compound 15 showed excellent anti-proliferative activity (IC50: 2.6 μM). According to the
biological activity data, the amide functional group, the existence of the bromine atom, and the position
of the hydrophobic character would have an effect on the anti-cancer activity. We believed that our
synthesized compounds would be potent anti-cancer agents and provide basic data of SAR study for
human ovarian cancer cell, PA-1.
EXPERIMENTAL
1
13
General. H and C NMR spectra were obtained in DMSO-d6 with Bruker and Varian spectrometer
1
13
operating at 400 MHz and 500 MHz for H and 100 MHz and 125 MHz for C with TMS as an
internal standard. Infrared spectra were recorded on a Bruker Alpha FT-IR spectrometer. Melting points
were determined with a Sanyo Gallenkamp melting point apparatus. High-resolution mass spectra
(HRMS) were obtained on a JMS 700 mass spectrometer with double-focusing mass analyzers.
Analytical thin layer chromatography (TLC) was conducted on E. Merck 60 F254 aluminum-backed
silica gel plates (0.2 mm) with a fluorescent indicator. Flash column chromatography was performed
using Merck silica gel 60 (230-400 mesh) under positive pressure. All starting materials, reagents, and
the solvents were of reagent grade, and solvents were purified by a known procedure before use.38
Synthesis of 2-amino-5-bromo-3,4-dicyanopyrrole (1).24,25 To a stirred solution of tetracyanoethylene
o
(10.24 g, 80.0 mmol) in the mixture of acetone (60 mL) and EtOAc (120 mL) at 0 C was added 33
wt% HBr in AcOH (59 mL, 240.0 mmol) over a period of 1 h. The reaction mixture was stirred for an
additional 30 min. The resulting pale yellow solid was collected by vacuum filtration and then washed
with EtOAc (100 mL). The solid was air dried on filter, and the suspended in ice water (100 mL). The
pH of the suspension was adjusted to 11 with 50% aq. NaOH at which point, all of the solid was
dissolved. AcOH was then added to cooled solution until the pH 5, and precipitate was filtered by
vacuum filtration. The tan powder was dried under vacuum to afford crude product 1 (8.95 g, 53%)
which was used for next step without further purification. 1H NMR (500 MHz, DMSO-d6): δ 12.31 (bs,
1H), 6.49 (bs, 2H).

Synthesis of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (2).13,26-27 To a stirred solution of
compound 1 (2.1 g, 10.0 mmol) in 2-ethoxyethanol (50 mL) was added formamidine acetate (4.16 g,
40.0 mmol). The reaction mixture was then refluxed for 36 h. The solvent was evaporated, and the solid
was dissolved in NH4OH/EtOH then treated with charcoal. The solution was filtered by vacuum
filtration and added AcOH until pH was near 7. The precipitate was filtered by vacuum filtration and
dried under vacuum to afford crude product 2 (1.50 g, 63%) as a yellow powder, which was used for
1
next step without further purification. H NMR (500 MHz, DMSO-d6): δ 13.81 (bs, 1H), 8.21 (s, 1H),
7.17 (bs, 2H).
Synthesis of 4-amino-6-bromo-1-cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (3) and
4-amino-6-bromo-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (4). Compound 2
(0.26 g, 1.1 mmol) and NaH (0.05 g, 1.2 mmol) were dissolved in dimethylacetamide (5 mL). The
o
reaction mixture was stirred for 30 min at 60 C. Cyclopentyl bromide (0.24 mL, 1.3 mmol) was then
o
added to the reaction mixture and stirred at 60 C for 24 h. The solvent was evaporated and crude
mixture was purified by flash column chromatography eluting with 30 to 50% EtOAc/Hex to afford
product 3 (0.12 g, 37%) as a white powder, Rf = 0.22 (50% EtOAc/Hex) and product 4 (0.07 g, 20%) as
o
1
a white powder, Rf = 0.45 (50% EtOAc/Hex). For product 3: mp 275-277 C (dec.); H NMR (500
MHz, DMSO-d6): δ 8.48 (s, 1H), 8.36 (bs, 1H), 7.10 (bs, 1H), 5.12 (quintet, J = 8.0 Hz, 1H), 2.19-2.12
(m, 2H), 2.10-2.02 (m, 2H), 1.93-1.85 (m, 2H), 1.72-1.65 (m, 2H); 13C NMR (125 MHz, DMSO-d6): δ
155.9, 146.6, 144.0, 132.6, 116.4, 105.3, 83.6, 61.1, 31.6, 24.2; IR (KBr): 3541, 3302, 3119, 2958, 2924,
2206, 1662, 1611, 1413, 1245 cm-1; HRMS(EI+): m/z [M]+ calcd for C12H12BrN5: 305.0276; found:
o
1
305.0276. For product 4: mp 262-264 C (dec.); H NMR (500 MHz, DMSO-d6): δ 8.20 (s, 1H), 6.88
(bs, 2H), 5.07 (quintet, J = 8.8 Hz, 1H), 2.35-2.29 (m, 2H), 2.03-1.99 (m, 4H), 1.70-1.68 (m, 2H); 13C
NMR (125 MHz, DMSO-d6): δ 166.1, 156.9, 152.1, 149.8, 112.0, 110.8, 102.0, 57.3, 29.9, 24.5; IR
(KBr): 3482, 3301, 3105, 2938, 2920, 2218, 1684, 1594, 1482, 1285 cm-1; HRMS(EI+): m/z [M]+ calcd
for C12H12BrN5: 305.0276; found: 305.0276.
Synthesis of 4-amino-1-cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (5) and 4-amino-
7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine-5-carbonitrile (6). To a stirred solution of compound 3
(0,06 g, 0.2 mmol) and ammonium formate (0.13 g, 2.0 mmol) in 20 mL of EtOH was added 10% Pd/C
(10 mg). The mixture was then refluxed for 1 h. The hot reaction mixture was filtered through Celite
and washed with hot EtOH (20 mL). The solution was evaporated and the residue was diluted with
EtOAc (20 mL), washed with water (2 X 10 mL), dried over anhydrous MgSO4, and evaporated to

provide crude mixture. The crude mixture was purified by flash column chromatography eluting with
25% Hex/EtOAc to afford product 5 (0.04 g, 94%) as a white solid, Rf = 0.20 (25% Hex/EtOAc).
Product 6 (0.04 g, 94%) was obtained from compound 4 as a white solid in the same manner, Rf = 0.30
(25% Hex/EtOAc). For compound 5: mp 252 oC (dec.); 1H NMR (400 MHz, DMSO-d6): δ 8.49 (s, 1H),
7.84 (s, 1H), 7.40-6.60 (bs, 2H), 5.18 (quintet, J = 8.0 Hz, 1H), 2.17-2.13 (m, 4H), 2.00-1.87 (m, 2H),
13
1.74-1.63 (m, 2H); C NMR (100 MHz, DMSO-d6): δ 156.7, 147.1, 146.2, 143.4, 117.4, 103.7, 81.2,
60.9, 30.9, 23.8; IR (KBr): 3444, 3314, 3112, 2954, 2204, 1662, 1616, 1431, 1161 cm-1;
HRMS(FAB+): m/z [M+H]+ calcd for C12H14N5: 228.1249; found: 228.1248. For compound 6: mp 198
oC; 1H NMR (400 MHz, DMSO-d6): δ 8.34 (s, 1H), 8.21 (s, 1H), 6.79 (bs, 2H), 5.03 (quintet, J = 8Hz,
13
1H), 2.15-2.09(m, 2H), 1.90-1.85 (m, 4H), 1.72-1.64 (m, 2H); C NMR (100 MHz, DMSO-d6): δ
156.9, 153.2, 149.9, 132.4, 115.7, 101.3, 81.5, 55.9, 32.1, 23.5; IR (KBr): 3466, 3306, 3084, 2964,
2220, 1654, 1592, 1309, 1222 cm-1; HRMS(FAB+): m/z [M+H]+ calcd for C12H14N5: 228.1249; found:
228.1250.
Synthesis of 4-amino-1-cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (7) and 4-amino-
1-cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (8): To a stirred solution of compound 5
(0.14 g, 0.6 mmol) in 5 mL of DMSO, cooled in an ice bath, were added 30 wt% H2O2 (0.5 mL) and
K2CO3 (0.2 g). The mixture was then stirred at room temperature for 1 h. 50 mL of water was added to
precipitate the product. The precipitate was filtered off and dried over under vacuum to afford product 7
(0.12 g, 83%) as a white solid. Product 8 (0.11 g, 76%) was obtained from compound 6 as a white solid
in the same manner. For product 7: mp 243 oC (dec.); 1H NMR (400 MHz, DMSO-d6): δ 9.90 (bs, 1H),
8.33 (s, 1H), 7.90 (s, 1H), 7.82 (bs, 1H), 7.64 (bs, 1H), 6.91 (bs, 1H), 5.17 (quintet, J = 8.0 Hz, 1H),
13
2.15-2.09 (m, 4H), 1.96-1.86 (m, 2H), 1.74-1.63 (m, 2H); C NMR (100 MHz, DMSO-d6): δ 167.8,
157.7, 147.7, 142.5, 140.9, 110.7, 102.9, 60.0, 31.0, 23.8; IR (KBr): 3468, 3320, 3148, 2962, 1634,
1584, 1442, 1181 cm-1; HRMS(FAB+): m/z [M+H]+ calcd for C12H16N5O: 246.1355; found: 246.1356.
o
1
For product 8: mp 229 C (dec.); H NMR (400 MHz, DMSO-d6): δ 9.20-8.60 (bs, 2H), 8.09 (s, 1H),
8.07 (s, 1H), 7.84 (bs, 1H), 7.27 (bs, 1H), 5.04 (quintet, J = 7.0 Hz, 1H), 2.16-2.09 (m, 2H), 1.92-1.85
(m, 2H), 1.83-1.74 (m, 2H), 1.72-1.68 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 166.5, 158.0, 152.5,
150.5, 125.3, 109.7, 100.9, 54.7, 32.4, 23.7; IR (KBr): 3548, 3127, 2966, 1655, 1605, 1445, 1221 cm-1;
HRMS(FAB+): m/z [M+H]+ calcd for C12H16N5O: 246.1355; found: 246.1356.
Synthesis of 4-amino-6-bromo-1-cyclopentyl-1H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (9) and
4-amino-6-bromo-7-cyclopentyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide (10). To a stirred

solution of compound 3 (0.18 g, 0.6 mmol) in 5 mL of DMSO, cooled in an ice bath, were added 30
wt% H2O2 (0.5 mL) and K2CO3 (0.2 g). The mixture was then stirred at room temperature for 1 h. 50
mL of water was added to precipitate the product. The precipitate was filtered off and dried over under
vacuum to afford product 9 (0.19 g, 97%) as a white solid. Product 10 (0.18 g, 95%) was obtained from
compound 4 as a white solid in the same manner. For product 9: mp 233-235 oC (dec.); 1H NMR (400
MHz, DMSO-d6): δ 9.89 (bs, 1H), 8.36 (s, 1H), 8.07 (bs, 1H); 7.57 (bs, 1H), 7.08 (bs, 1H), 5.12
(quintet, J = 8.0 Hz, 1H), 2.19-2.11 (m, 2H), 2.09-2.00 (m, 2H), 1.94-1.84 (m, 2H), 1.74-1.65 (m, 2H);
13C NMR (100 MHz, DMSO-d6): δ 166.3, 156.7, 145.7, 142.7, 125.5, 107.9, 104.1, 59.6, 31.0, 23.6; IR
(KBr): 3541, 3304, 3148, 2960, 1668, 1648, 1450, 1238 cm-1; HRMS(EI+): m/z [M]+ calcd for
o
1
C12H14BrN5O: 323.0382; found: 323.0380. For product 10: mp 242 C (dec.); H NMR (500 MHz,
DMSO-d6): δ 8.09 (s, 1H), 7.90 (bs, 1H), 7.62 (bs, 1H), 7.74-7.34 (bs, 2H), 5.09 (quintet, J = 8.7 Hz,
13
1H), 2.39-2.36 (m, 2H), 2.01-1.95 (m, 4H), 1.68-1.67 (m, 2H); C NMR (125 MHz, DMSO-d6): δ
167.8, 157.7, 147.7, 142.5, 140.9, 110.7, 102.9, 60.0, 31.0, 23.8; IR (KBr): 3476, 3264, 3118, 2954,
1643, 1603, 1474, 1275 cm-1; HRMS(EI+): m/z [M]+ calcd for C12H14BrN5O: 323.0382; found:
323.0384.
Synthesis of syn-[4-(4-amino-6-bromo-5-cyano-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-cyclopenten-1-
yl]methyl methyl carbonate (12). To a stirred solution of Pd(OAc)2 (0.046 g, 0.2 mmol) and (i-PrO)3P
(0.15 mL, 0.61 mmol) in 5 mL of THF under argon (air and moisture free-condition) was slowly added
n-BuLi (1.6 N in hexane, 0.38 mL, 0.61 mmol) at room temperature. After 10 min, the solution of
compound 2 (0.48 g, 2.0 mmol) and NaH (0.088 g, 2.2 mmol) in DMSO (5 mL) was added into the
reaction flask by using double-tipped needle. The solution of the dicarbonate (11) (0.41 g, 1.8 mmol) in
DMSO (3 mL) was added into the reaction flask by using double-tipped needle. After 2 h, the reaction
mixture was diluted with CH2Cl2 (30 mL), washed with water (2 X 10 mL), dried over anhydrous
MgSO4, and evaporated to provide crude mixture. The crude mixture was purified by flash column
chromatography eluting with 50% EtOAc/Hex to afford product 12 (0.35 g, 50%) as a white solid, Rf =
o
0.15 (35% Hex/EtOAc). mp 222-224 C (dec.); 1H NMR (400 MHz, DMSO-d6): δ 8.48 (bs, 1H), 8.19
(s, 1H), 7.19 (bs, 1H), 6.22-6.21(m, 1H), 6.04-6.03 (m, 1H), 5.92-5.91 (m, 1H), 4.18-4.16 (m, 2H), 3.67
(s, 3H), 3.16-3.14 (m, 1H), 2.88-2.80 (m, 1H), 1.70-1.63 (m, 1H); 13C NMR (100 MHz, DMSO-d6): δ
155.6, 155.4, 146.3, 142.9, 138.9, 132.6, 129.2, 116.3, 104.8, 83.6, 69.6, 64.4, 54.9, 44.4, 34.2; IR
(KBr): 3315, 3197, 2955, 2207, 1746, 1639, 1440, 1255 cm-1; HRMS(EI+): m/z [M]+ calcd for
C15H14BrN5O3: 391.0280; found: 391.0276.

Synthesis of syn-4-amino-6-bromo-7-[4-(hydroxyl methyl)-2-cyclopenten-1-yl]-7H-pyrrolo[2,3-
d]pyrimidine-5-carboxamide (13). To a stirred solution of compound 12 (0.45 g, 1.1 mmol) in 5 mL
of DMSO, cooled in an ice bath, were added 30 wt% H2O2 (0.5 mL) and K2CO3 (0.1 g). The mixture
was then stirred at room temperature until starting material was completely consumed by TLC
monitoring. 3 mL of 20% MeOH in water was added and stirred for 2 h. The solvent was evaporated,
and the resulting solid was washed with 10 mL of water and 10 mL of Et2O ether on filter and dried
over under vacuum to afford product 13 (0.29 g, 75%) as a white solid, Rf = 0.35 (10% MeOH/CH2Cl2).
o
1
mp 245-250 C (dec.); H NMR (400 MHz, DMSO-d6): δ 9.89 (bs, 1H), 8.16 (s, 1H), 8.09 (bs, 1H),
7.58 (bs, 1H), 7.08 (bs, 1H), 6.24-6.23 (m, 1H), 5.93-5.91 (m, 2H), 4.71 (t, J = 5.2 Hz, 1H), 3.52-3.47
(m, 1H), 3.43-3.38 (m, 1H), 2.92-2.90 (m, 1H), 2.78-2.70 (m, 1H), 1.64-1.58 (m, 1H); 13C NMR (100
MHz, DMSO-d6): δ 166.3, 156.8, 145.7, 142.2, 140.5, 128.1, 125.5, 108.0, 103.9, 63.3, 63.1, 47.6,
34.0; IR (KBr): 3543, 3410, 3296, 3177, 2881, 1633, 1456, 1238, 1094, 764 cm-1; HRMS(EI+): m/z
[M]+ calcd for C13H14BrN5O2: 351.0331; found: 351.0327.
Synthesis
of
syn-[4-(4-amino-6-bromo-5-carbamoyl-7H-pyrrolo[2,3-d]pyrimidin-7-yl)-2-
cyclopenten-1-yl]methyl acetate (14). To a stirred solution of compound 13 (0.035 g, 0.1 mmol) in
DMSO (1 mL) were added Ac2O (0.011 mL, 0.11 mmol), pyridine (0.010 mL, 0.12 mmol), and
dimethylaminopyridine (0.001 g, 0.01 mmol). The reaction mixture was stirred for 6 h at room
temperature, diluted with CH2Cl2 (20 mL), washed with water (2 X 10 mL), dried over anhydrous
MgSO4, and evaporated to give the crude material. The crude material was purified by flash column
chromatography eluting with 15% Hex/EtOAc to afford product 14 (0.035 g, 90%) as a white solid, Rf
o
1
= 0.15 (15% Hex/EtOAc). mp 172-174 C (dec.); H NMR (400 MHz, DMSO-d6): δ 9.92 (bs, 1H),
8.16 (bs, 1H), 8.11 (s, 1H), 7.62 (bs, 1H), 7.09 (bs, 1H), 6.23-6.21 (m, 1H), 6.03-6.02 (m, 1H), 5.91-
5.90 (m, 1H), 4.12-4.03 (m, 2H), 3.14-3.12 (m, 1H), 2.88-2.80 (m, 1H), 1.98 (s, 3H), 1.64-1.58 (m,
13
1H); C NMR (100 MHz, DMSO-d6): δ 170.3, 166.3, 156.9, 145.6, 142.0, 138.7, 129.1, 125.6, 108.1,
104.0, 65.9, 63.1, 44.0, 34.3, 20.6; IR (KBr): 3448, 3285, 3157, 2925, 1726, 1616, 1441, 1236, 1038,
769 cm-1; HRMS(EI+): m/z [M]+ calcd for C15H16BrN5O3: 393.0437; found: 393.0435.
Synthesis of syn-4-amino-6-bromo-7-[4-(methoxymethyl)-2-cyclopenten-1-yl]-7H-pyrrolo[2,3-
d]pyrimidine-5-carboxamide (15): To a stirred solution of compound 13 (0.030 g, 0.09 mmol) in
DMSO (1 mL) was added NaH (0.004 g, 0.11 mmol). After 5 min, dimethyl sulfate (0.01 mL, 0.1
mmol) was added into the reaction mixture and stirred for 1 h at room temperature. The solvent was
evaporated to afford the crude mixture. The crude mixture was purified by HPLC on XterraTM RP187μm

column (19 X 300 mm) eluting with 30% MeOH/H2O (flow rate: 5.0 mL/min; λ = 220 nm; tmajor = 58
o
1
min; tminor = 31 min) to afford product 15 (0.025 g, 75%) as a white solid. mp 212-214 C (dec.); H
NMR (400 MHz, DMSO-d6): δ 10.65 (bs, 1H), 8.27 (s, 1H), 7.65 (bs, 1H), 7.12 (bs, 1H), 6.24-6.23 (m,
1H), 5.93-5.92 (m, 2H), 4.73 (bs, 1H), 3.51-3.48 (m, 1H), 3.42-3.38 (m, 1H), 3.05 (s, 3H), 2.92-2.90 (m,
13
1H), 2.79-2.71 (m, 1H), 1.64-1.58 (m, 1H); C NMR (100 MHz, DMSO-d6): δ 166.8, 155.8, 145.2,
142.7, 141.0, 128.3, 125.2, 108.1, 104.8, 63.6, 63.5, 47.8, 34.4, 27.6; IR (KBr): 3461, 3314, 3219, 2934,
1649, 1456, 1245, 1088 cm-1; HRMS(EI+): m/z [M]+ calcd for C14H16BrN5O2: 365.0487; found:
365.0484.
SUPPLEMENTARY DATA
1H NMR and 13C NMR spectra for products, 1H-1H NOESY spectra for 3 and 4, X-ray crystallographic
data for 3, and anti-proliferative effect of compounds 3-10, 12-15 in human ovarian cancer PA-1 cells
are provided as supplementary data.
ACKNOWLEDGEMENTS
This research was supported by the Basic Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-
0070920 and 2010-0012838). J. Lee and S. Yoon acknowledge financial support from the Korean
Ministry of Education through the second stage of the BK21 and the BK21-Plus project for the
Hanyang University graduate program. We thank Prof. H. Chun for X-ray crystallography.
REFERENCES AND NOTES
1. C. Desgranges, G. Razaka, M. Rabaud, H. Bricaud, J. Balzarini, and E. De Clercq, Biochem.
Pharmacol., 1983, 32, 3583.
2. V. E. Marquez and M.-I. Lim, Med. Res. Rev., 1986, 6, 1.
3. S. D. Patil, M. Hosoya, R. Snoeck, G. Andrei, J. Balzarini, and E. De Clercq, J. Med. Chem., 1992,
35, 3372.
4. M. D. Migliore, N. Zonta, C. McGuigan, G. Henson, G. Andrei, R. Snoeck, and J. Balzarini, J. Med.
Chem., 2007, 50, 6485.
5. R. Vince and M. Hua, J. Med. Chem., 1990, 33, 17.
6. N. Katagiri, M. Takebayashi, H. Kokufuda, C. Kaneko, K. Kanehira, and M. Torihara, J. Org.
Chem., 1997, 62, 1580.
7. M. E. Jung and H. Rhee, Tetrahedron Lett., 1993, 34, 4449.

8. B. M. Trost, L. Li, and S. D. Guile, J. Am. Chem. Soc., 1992, 114, 8745.
9. M. S. Daluge, S. S. Goog, B. M. Faletto, H. W. Miller, H. M. Clair, R. L. Boone, M. Tisdale, R. N.
Parry, E. J. Reardon, E. R. Dornsife, R. D. Averett, and A. T. Krenitsky, Antimicrob. Agents
Chemother., 1997, 41, 1082.
10. D. W. Kimberlin, D. M. Coen, K. K. Biron, J. I. Cohen, R. A. Lamb, M. Mckinlay, E. A. Emini, and
R. J. Whitley, Antiviral Res., 1995, 26, 369.
11. S. S. Good, S. M. Daluge, S. V. Ching, K. M. Ayers, W. B. Mahony, M. B. Faletto, B. A. Domin, B.
S. Owens, R. E. Dornsife, J. A. McDowell, S. W. LaFon, and W. T. Symond, Antiviral Res., 1995,
26, A229.
12. S. M. Daluge, M. T. Martin, B. R. Sickles, and D. A. Livingston, Nucleosides Nucleotides Nucleic
Acids, 2000, 19, 297.
13. R. L. Tolman, R. K. Robins, and L. B. Townsend, J. Am. Chem. Soc., 1968, 90, 524.
14. E. C. Taylor and R. W. Hendess, J. Am. Chem. Soc., 1965, 87, 1995.
15. P. Leonard, S. A. Ingale, P. Ding, X. Ming, and F. Seela, Nucleosides Nucleotides Nucleic Acids,
2009, 28, 678.
16. P. K. Gupta, S. Daunert, M. R. Nassiri, L. L. Wotring, J. C. Drach, and L. B. Townsend, J. Med.
Chem., 1989, 32, 402.
17. P. K. Gupta, M. R. Nassiri, L. A. Coleman, L. L. Wotring, J. C. Drach, and L. B. Townsend, J. Med.
Chem., 1989, 32, 1420.
18. D. E. Bergstrom, A. J. Brattesani, M. K. Ogawa, P. A. Reddy, M. J. Schweickert, J. Balzarini, and E.
De Clercq, J. Med. Chem., 1984, 27, 285.
19. S. R. Turk, C. Shipman, M. R. Nassiri, G. Genzlinger, S. H. Krawczyk, L. B. Townsend, and J. C.
Drach, Antimicrob. Agents Chemother., 1987, 31, 544.
20. R. K. Robins and G. R. Revankar, Med. Res. Rev., 1985, 5, 273.
21. M. T. Migawa, J. C. Drach, and L. B. Townsend, J. Med. Chem., 2005, 48, 3840.
22. Y. Ding, H. An, Z. Hong, and J.-L. Girardet, Bioorg. Med. Chem. Lett., 2005, 15, 725.
23. E. Gunic, J.-L. Girardet, Z. Pietrzkowski, C. Esler, and G. Wang, Bioorg. Med. Chem., 2001, 9, 163.
24. E. E. Swayze, J. M. Hinkley, and L. B. Townsend, ‘Nucleic Acid Chemistry,’ Vol. 4, ed. by L. B.
Townsend and R. S. Tipson, Wiely, New York, 1991, pp. 16-18.
25. W. J. Middleton, V. A. Engelhardt, and B. S. Fisher, J. Am. Chem. Soc., 1958, 80, 2822.
26. R. L. Tolman, R. K. Robins, and L. B. Townsend, J. Am. Chem. Soc., 1969, 91, 2102.
27. A. R. Porcari and L. B. Townsend, Synth. Commun., 1998, 28, 3835.
28. CCDC-853547 contains the supplementary crystallographic data for this paper. These data can be
obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk

/data_request/cif.
29. A. R. Porcari and L. B. Townsend, Nucleosides Nucleotides Nucleic Acids, 2004, 23, 31.
30. A. R. Katritzky, B. Pilarski, and L. Urogdi, Synthesis, 1989, 949.
31. G.-i. An and H. Rhee, Nucleosides Nucleotides Nucleic Acids, 2003, 22, 437.
32. B. M. Trost, L. Li, and S. D. Guile, J. Am. Chem. Soc., 1992, 114, 8745.
33. M. E. Jung and H. Rhee, J. Org. Chem., 1994, 59, 4719.
34. N. Katagiri, M. Takebayashi, H. Kokufuda, C. Kaneko, K. Kanehira, and M. Torihara, J. Org.
Chem., 1997, 62, 1580.
35. B. M. Trost, Angew. Chem., Int. Ed. Engl., 1986, 25, 1.
36. J. Tsuji, Tetrahedron, 1986, 42, 4361.
37. H. Rhee, D.-O. Yoon, and M. E. Jung, Nucleosides Nucleotides Nucleic Acids, 2000, 19, 619.
38. W. L. F. Armarego and C. L. L. Chai, ed. ‘Purification of Laboratory Chemicals,’ 6th ed., Elsevier,
Oxford, 2009.