Coumarin-purine ribofuranoside conjugates as new agents against hepatitis c virus

Article abstract of DOI:10.1021/jm101337v

About 3% of world's population is infected by the hepatitis C virus (HCV), for which prophylactic vaccine is not available yet. Nowadays, pegylated interferon-α and ribavirin are commonly used to treat HCV; unfortunately these drugs often produce significant side effects. Upon the desperate need of anti-HCV drugs, a plan to establish a new compound library was set that included leads with high antiviral activity, good hydrophilicity, yet low toxicity. Accordingly, 26 new conjugated compounds were synthesized through the chemical coupling of various 9-(β-d-ribofuranosyl)purine-8-thiones with 3-(chloromethyl)coumarins bearing various substituents. A SCH₂ unit was used to link the coumarin and the purine moieties. The three hydroxyl groups at the 2′-, 3′-, and 5′-positions were selectively protected with an acyl or acetal group in these coumarin-purine ribofuranosides. Their anti-HCV and cytostatic determination assays were performed, and the structure-activity relationship was established. Three conjugates in the family of 8-(coumarin-3′-yl)methylthio-9-(β-d-ribofuranos-1′-yl)purine possessed an appealing ability to inhibit HCV replication with EC₅₀ between 5.5 and 6.6 μM and EC₉₀ of 20 μM. These data in the new compound library provide clues for the future in the development of anti-HCV leads for viral eradication.

Full text of DOI:10.1021/jm101337v

ARTICLE
pubs.acs.org/jmc
Coumarin-Purine Ribofuranoside Conjugates as New Agents against
Hepatitis C Virus
,
†,‡
†
†
§
§
§
Jih Ru Hwu,* Shu-Yu Lin, Shwu-Chen Tsay, Erik De Clercq, Pieter Leyssen, and Johan Neyts
†
Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 30013, R.O.C.
‡
Department of Chemistry, National Central University, Jhongli City, Taiwan 32001, R.O.C.
§
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
S Supporting Information
b
ABSTRACT: About 3% of world’s population is infected by the
hepatitis C virus (HCV), for which prophylactic vaccine is not
available yet. Nowadays, pegylated interferon-R and ribavirin
are commonly used to treat HCV; unfortunately these drugs
often produce significant side effects. Upon the desperate need
of anti-HCV drugs, a plan to establish a new compound library
was set that included leads with high antiviral activity, good
hydrophilicity, yet low toxicity. Accordingly, 26 new conjugated
compounds were synthesized through the chemical coupling of
various 9-(β-D-ribofuranosyl)purine-8-thiones with 3-(chloro-
methyl)coumarins bearing various substituents. A -SCH -
2
unit was used to link the coumarin and the purine moieties. The
0
0
0
three hydroxyl groups at the 2 -, 3 -, and 5 -positions were selectively protected with an acyl or acetal group in these coumarin-
purine ribofuranosides. Their anti-HCV and cytostatic determination assays were performed, and the structure-activity relation-
0
00
ship was established. Three conjugates in the family of 8-(coumarin-3 -yl)methylthio-9-(β-D-ribofuranos-1 -yl)purine possessed an
appealing ability to inhibit HCV replication with EC50 between 5.5 and 6.6 μM and EC90 of ∼20 μM. These data in the new
compound library provide clues for the future in the development of anti-HCV leads for viral eradication.
0
’
INTRODUCTION
overcomes the problem that 4 -azidoadenosine does not inhibit
7
HCV replication in cell culture. A different prodrug approach is
the incorporation of two S-(acylthio)ethyl groups into 9-(2 -β-C-
methyl-β-D-ribofuranosyl)-6-substituted purines. It improves the
potency of the parent compounds by increasing the intracellular
levels of 5 -monophosphate metabolites. These nucleotide pro-
drugs show much enhanced inhibitory activities on HCV RNA
replication. Moreover, 2 -deoxyadenosine analogues with a 5 -
O-masked hydroxyl group is found to exhibit higher potency than
More than 100 000 patients die annually, which is attributable
0
to hepatitis C directly or with the intervention of cofactors such
as hepatitis B virus, HIV, or alcohol abuse. The hepatitis C virus
1
2
(HCV) was first discovered in the United States in 1989. Soon it
0
was revealed to be the cause of many hepatic diseases of
previously unknown origin. Unfortunately, more than 50% of
people at the highest risk for HCV nowadays are infected and yet
unaware of their disease. The spread of its infection and loss of
3
8
0
0
1
9
the unmasked analogues. On the other hand, modification
the treatment opportunities become a recipe for disaster.
of the adenosine moiety by attachment of a five-membered
heterocycle in the C-6 position leads to a ribonucleoside with
appealing 90% inhibitory concentration for virus replication in
the submicromolar range. Unfortunately it is accompanied by a
side effect on cellular rRNA and thus lacks for specificity.
Ribavirin is the first synthetic nucleoside with an unnatural
triazole moiety and exhibits a broad spectrum of antiviral activity
against RNA and DNA viruses. Its derivatives in the family of
arylethynyltriazole ribonucleosides can also inhibit HCV replica-
tion efficiently. Use of unnatural sugar moiety is another
Many efforts have been devoted to the discovery of drugs
4
,5
against HCV. Results from the development of nucleoside
analogues offer encouragement, which may improve therapies to
treat HCV infection. Recent reports include adenosine 5 -
phosphonates by Girardet et al., phosphoramidates of 4 -sub-
stituted purine nucleoside by McGuigan et al., 9-(2-β-C-methyl-
β-D-ribofuranosyl)-6-substituted purine derivatives by Ding
et al., 5 -O-masked 2 -deoxyadenosine analogues by Maruyama
et al., purine ribonucleosides bearing a pyrrol-3-yl or 2-furyl
0
10
6
0
7
13
8
0
0
9
11
1
0
group by Hocek et al., arylethynyltriazole ribonucleosides by
Peng et al., and 7-deazaneplanocin A analogues by Chu et al.
approach to develop new leads in the family of nucleosides.
For example, the ribofuranose moiety in ribosides is modified to
1
1
12
Adenosine phosphonate analogues constitute a class of pro-
6
mising anti-HCV leads, yet their activity is relatively weak. Use
of the phosphoramidate pronucleotide approach successfully
Received: October 15, 2010
Published: March 04, 2011
r 2011 American Chemical Society
2114
dx.doi.org/10.1021/jm101337v
|J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
ARTICLE
give the corresponding 7-deazaneplanocins, which exhibit sig-
Scheme 1. Coupling of Purine-8-thione (2) with a Polyacetyl
β-D-Ribofuranose To Give Nucleosides 3 and 4
12
nificant anti-HCV activity.
In addition to ribonucleosides, some recent inventions pro-
14
vide synthetic methods to give 3,4-disubstituted, 4-thio, 4,
1
5
16
5
-dithio, and 7,8-fused coumarins, which show activity
against HCV. Furthermore, synthetic coumestans bearing differ-
ent substituents in their A and D rings display interference at the
step of NS5B-RNA binary complex formation and is confirmed
17
by cross-linking experiments. Recently, coumarins conjugated
18
19
with various benzimidazoles and heterobicycles, respectively,
have been synthesized. These conjugates exhibit appealing anti-
HCV activity and become promising anti-HCV leads. Present-
day coumarins and their derivatives still have plenty of room for
their improvement as anti-HCV drugs.
To advance the development of new anti-HCV drugs, we
synthesized 26 new conjugated compounds through the concept
20
of molecular hybridization for drug design. In this new
compound library, we came up with their structure-activity
relationship (SAR) and planned to produce several new con-
jugated compounds with improved efficiency for viral eradication
and less toxicity to the host. We attached a coumarin onto a
purine ribofuranoside by covalent bonds for generating innova-
tive leads of anti-HCV. We herein report our synthetic strategies
on successfully obtaining the targets 1, which were constituted by
three heterocyclic components. The essential functional groups
and moieties therein are revealed.
use of the Lewis acid Me SiOTf as the catalyst and appropriate
3
conditions that favored the formation of thermodynamically
more stable N-9 regioisomers. Somewhat surprisingly, these
two compounds (i.e., 3 and 4) did not show significant anti-
HCV activity.
Syntheses of Conjugated Coumarin-Thiopurine Nucleo-
sides 6, 7, and 9. Often the coumarin moiety brings biological
activities into chemical entities. Hence, we treated 3 with 3-
(
chloromethyl)coumarins 5 bearing various substitutents, in-
cluding F, Cl, Br, Me, and OMe, at room temperature to
give O-polyacyl-N-ribofuranosides 6 in 77-93% yields (see
0
0
Scheme 2). The acyl groups at the 2 and 3 positions therein
were removable by ammonia in methanol for 45 min at 0 °C to
give diols 7. Performance of these reactions at a longer time or
under harsher conditions did not lead to the desired conjugates 9,
in which all of the hydroxyl groups were deprotected. Instead, we
’
RESULTS
To establish the scaffold of targets 1, we were facing the
found that the S-CH bond in 7 was cleaved.
2
following three challenges during their syntheses. First, the
pivotal purine nucleus possessed three nucleophilic centers as
shown by the structure 2. They included two NH and one thione
groups. A coumarin or a ribofuranose moiety would then have to
be incorporated at the right site in the right step. Second, the
reaction conditions applied during the synthesis of the targets 1
should be mild enough so that the frail allylic S-C bond and
N-glycosidic C-N bond therein would remain intact. Third, the
reagents to be used in the synthetic steps should not add to the
R,β-unsaturated ester group through a Michael addition or
induce a ring-opening of the δ-lactone moiety in 1. Given these
unique concerns, we developed sequential synthetic routes
shown in Schemes 1-4.
To pursue a way to obtain the desired conjugates 9, we first
removed all of the acyl groups in 3 successfully by methanolic
ammonia to give triol 8 in 92% yield. Coupling of this triol with
coumarins 5 with 35% ammonium hydroxide in aqueous aceto-
nitrile led to the targets 9. The yields ranged from 51-81% and
their purity was >98.0%, as determined by HPLC.
Identification of Structures and Analysis of Conforma-
tions. We confirmed the structures of conjugates 9 on the basis
of their spectroscopic characteristics. For example, the mass
spectrum of 9a in the positive ion mode by the FABMS
þ
technique exhibited 443.1016 for the species [M þ H] , which
indicates the molecular formula to be C20H N O S with a
18 4 6
theoretical value of 442.0947. Its IR spectrum showed one strong
-
1
Synthesis of Purine-8-thione-Ribofuranose Conjugates.
absorption band at 1713 cm , which was attributed to the
23 13
4,6-12
Some purine ribofuranosides possess anti-HCV activity.
carbonyl stretching vibration of the coumarin moiety. Its
NMR spectrum had resonance at 31.84 and 156.90 ppm for the
SCH carbon and the -NdC(-N)(-S) carbon, respectively.
C
Therefore, we intended to prepare mono- and bisribofuranoside
derivatives shown in Scheme 1 for biological assays. Silylation of
purine-8-thione (2) with bis(trimethylsilyl)acetamide (BTSA)
followed by addition of 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose
at 80 °C provided the desired mono N-9 nucleoside 3 and the
byproduct bis(ribofuranosyl)purine-8-thione 4 (Scheme 1).
2
On the other hand, the two characteristic singlets occurred at
8.83 and 9.07 ppm for the protons in the purine nucleus and a
doublet with J = 6.4 Hz occurred at 5.78 ppm for the glycosidic
22
1
proton in its H NMR spectrum.
21
For the monoglycosylation product, we isolated the N-9
We ensured the regioselectivity and that the glycosidic bonds
of conjugates 9 connected the purine moiety at the N-9 position
on the basis of the X-ray structure of their starting material 3. The
(
instead of the N-7) isomer only. Successful preparation of 3 was
22
attributed to the application of the Vorbr €u ggen procedure by
2
115
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Journal of Medicinal Chemistry
ARTICLE
Scheme 2. Syntheses of Conjugated Coumarin-Thiopurine Nucleosides with the Three Hydroxyl Groups Therein Fully,
Partially, or Nonprotected
Figure 1. Molecular frameworks: (A) ORTEP diagram of 3 obtained by X-ray diffraction analysis; (B) molecular modeling structure of 9a obtained
through energy minimization by CVFF calculations.
2
4
molecular framework of 3 was unequivocally confirmed by
single crystal X-ray diffraction analysis (see Figure 1A). Its
orthorhombic crystals possessed the space group P2 2 2 with
the N-9 nitrogen atom with dihedral angle of 71.9° for O-
0
C(1 )-N(9)-C(4).
As shown in Figure 1A, the crystals of 3 existed in the syn
conformation with the thione moiety sticking outward from the
ribofuranose unit. Through studies by circular dichroism, Ikehara
1
1 1
a = 9.4553(2) Å, b = 10.9763(3) Å, c = 18.5134(6) Å, and R = β =
γ = 90°. These results indicate that the glycosylation took place at
2
116
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Journal of Medicinal Chemistry
ARTICLE
2
5
0
0
et al. conclude that purine nucleosides with a bulky substituent
at the C(8) position would exist in the syn conformation.
Therefore, we believe that the noncrystal compound 9a also
held the syn conformation as shown in Figure 1B. Being obtained
by our CVFF calculations for energy minimization, this thermo-
dynamically most stable conformation would avoid unfavorable
steric and electrostatic interactions between the C(8)-coumarin
at both the 2 - and the 3 -positions (see Scheme 3). Accord-
ingly, treatment of purine-8-thione ribofuranoside 8 with 2,2-
dimethoxypropane in the presence of PTSA and acetone gave
0
0
the corresponding 2 ,3 -acetals 10. The thione moiety therein
reacted with 3-(chloromethyl)coumarins 5 to produce the
desired targets 11a,d,g with a free hydroxyl group at the 5 -
position in 52-64% yields with purity of >98.5%. Their
structures were fully confirmed by spectroscopic methods.
The characteristic peak for the gem-dimethyl groups exhib-
ited two singlets at 1.63 and ∼1.33 ppm in their H NMR
0
26
moiety and the ribofuranose unit.
Selective Protection of the Hydroxyl Groups in Ribofur-
anosides. To obtain fruitful information for establishing the
SAR, we planned to synthesize conjugates of coumarin-purine
ribofuranosides 1 with hydroxyl groups protected at different
positions. The synthetic strategies shown in Scheme 2 illustrate
our efforts on obtaining conjugated compounds with the hydro-
xyl groups in ribofuranose being fully protected (i.e., 6), pro-
tected at the 5 -position only (i.e., 7), and fully nonprotected (i.e.,
). We further performed the synthesis of coumarin-purine
ribofuranosides 11, in which the hydroxyl groups were protected
1
spectra.
Synthesis of Conjugated Coumarin-Adenosines 15. Ad-
dition of an amino group at the C(6) position provides a simple
way to modify the purine moiety. Conversion of the commer-
cially available adenosine (12) to the corresponding thione
0
27
nucleoside 14 can be accomplished by two steps through the
2
8
9
bromide intermediate 13 (see Scheme 4). Under the same
conditions as described before, we coupled 14 with coumarins 5
Scheme 3. Synthesis of Acetalized Coumarin-Purine
Ribofuranosides
Scheme 4. Synthesis of Conjugated Compounds of Cou-
marin-Adenosine Ribofuranosides
Table 1. Solubility in Water of Conjugates 6g, 7g, 9g, 11g, and 16
2
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Journal of Medicinal Chemistry
ARTICLE
Table 2. Inhibitory Effects of Conjugated Compounds on
HCV Subgenomic Replicon Replication in Huh 5-2 Cells
26 new conjugated compounds, 6a, 9a, and 9f were found to
inhibit HCV subgenomic replicon replication with potency and
exhibited low toxicity. Their EC50 values were 6.6, 5.5, and
a
b
c
d
compd
CC50 (μM)
EC50 (μM)
SI
5
.9 μM, individually. Furthermore, we obtained the 90% inhibi-
2
4
3
4
6
6
6
6
6
6
6
7
7
7
7
7
7
8
9
9
9
9
9
9
1
1
1
1
1
1
1
1
>122
>75
39
>122
66
1.0
tory concentrations for virus replication (EC ) for nine con-
90
jugated compounds, 6a, 7d, 9a, 9b, 9c, 9e, 9f, 9g, and 11d. Their
EC90 values were measured as 22, 16, 18, 18, 16, 19, 14, 20, and
>1.1
5.9
1.2
2.4
1.4
2.1
1.2
3.3
0.98
2.3
3.8
3.5
1.0
1.9
1.0
9.1
4.9
8.5
5.1
6.1
5.0
3.2
3.6
5.2
1.7
1.5
1.4
2.4
4.8
a
b
c
d
e
f
6.6
6.1
3.2
4.9
7.7
4.9
2.3
>8.9
3.8
8.3
4.6
6.8
5.8
>176
5.5
7.4
4.3
9.2
5.9
9.5
2.8
2.1
1.6
28
2
.9 μM, individually. This value could not be obtained for the
7.6
7.5
6.7
16
conjugated compound 11g; at the most, we obtained its EC70
value as 6.1 μM.
In the near future, we will evaluate the antiviral activities on a
broad sense by applying these new conjugates in the compound
library against various essential, emerging, and neglected RNA
viruses. The antiviral assays include hepatitis viruses in Huh 6 or
Huh 9-13 cells, HIV-1IIIB and HIV-2ROD in MT-4 cells, vesicular
6.0
7.5
8.7
8.6
31
g
a
b
d
e
f
stomatitis virus in E SM cells, respiratory syncytial virus in HeLa
6
cells, dengue virus in Vero Fast cells, human rhinovirus and
poliovirus in HeLa H1 cells, Chikungunya virus in Vero A cells,
and parainfluenza-3 virus, reovirus-1, Sindbis virus, Coxsackie B4
virus, and Punta Toro virus in Vero cell cultures. The results will
be reported in due course.
16
7.0
11
g
e
>176
50
a
’
DISCUSSION
b
36
c
37
Essential Moieties and Functional Groups: Structure-
Activity Relationship. The conjugated compounds in the new
library primarily contain three components with structural
e
46
f
36
g
47
modification: coumarin, purine, and ribofuranose. A -SCH -
2
1a
1d
1g
5a
5d
5f
5g
8.8
7.5
8.4
49
unit was used to link the coumarin and the purine moieties.
Through analysis of their biological activities shown in Table 2
and the solubility shown in Table 1, we deduce the following SAR
by scrutinizing their EC , CC , and SI values.
50
50
(
1) Attachment of a coumarin moiety to the purine ribofur-
anoside is the key to gain appealing anti-HCV activity.
Incorporation of this moiety greatly increased the HCV
inhibition (cf. 3 versus 6a-g and 8 versus 9a-g).
2) It is allowed to introduce various substitutents with
electron-withdrawing or donating ability, including F, Cl,
Br, Me, and OMe, onto the coumarin moiety while the
resultant conjugates were kept with the same order of the
anti-HCV activity (cf. 9a-g with EC50 = 4.3-9.5 μM).
41
27
36
26
>103
94
43
1
9
6
20
(
a
Interferon R-2b was used as a (positive) reference compound at 10 000
units/well and reduced the signal in the viral RNA (luciferase) assay to
background levels without any cytotoxic activity. The values were
obtained as the average of triplicate determinations. Minimum cyto-
b
toxic concentration required to cause a microscopically detectable
c
(3) The two-component conjugate 16 contained only the
coumarin and purine moieties, as shown in Table 2.
Addition of the third polyacyl ribofuranose moiety onto
it increased both the HCV inhibition and cytotoxicity. A
better choice was to add the parent ribofuranose moiety
to give the conjugates 9. As a result, it increased the HCV
inhibition.
alteration of normal cell morphology. Minimum inhibitory concentra-
tion required to reduce virus-induced cytopathogenicity by 50%.
d
e
Selectivity index (ratio of CC50 t4o EC50). Reported to be toxic to
2
HEp-2 and L1210 cells in culture.
to give the desired coumarin-adenosine conjugates 15a,d,f,g in
good yields for biological assays.
0
0
Solubility and Evaluation of the Anti-HCV Activity. Solu-
bility of chemical entities in water plays an important role in their
(4) In comparison with the protected forms at the 2 -, 3 -, and
0
5 -positions of the purine ribofuranosides, the presence of
29
development as a lead for new drugs and is related to the
free -OH groups at all of these positions allowed
reduction of their cytotoxicity and increment of the SI
values (cf. 9a-g versus 6a-g, 7a-g, and 11a,g,
respectively).
structure-activity relationship. Consequently, we applied the
3
0
Bookser’s method by using HPLC with a UV detector to obtain
19
the water solubility of conjugates 6g, 7g, 9g, 11g, and 16. Their
data and comparison are listed in Table 1.
(5) As the order shown in Table 1 for hydrophilicity, the most
hydrophilic compound was conjugate 9g. The least
hydrophilic compound was 6g. Given these concerns,
9a was considered as an ideal lead.
(6) Modification of the purine moiety of the conjugates 1 by
addition of an amino group at the C(6)-position gave the
coumarin-adenosine conjugates 15. In comparison with
the corresponding coumarin-purine conjugates 9, it
We evaluated the antiviral activities of conjugated compounds
31
in the HCV subgenomic replicon system in Huh 5-2 cells. The
antiviral assays and cytostatic determination assays were per-
formed on the basis of the procedures reported by us
3
2
previously. In Table 2, we list their 50% inhibitory concentra-
tions for virus replication (EC ), host cell growth (CC ), and
50
50
the selectivity index (SI = CC /EC ). Among the synthesized
50
50
2
118
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Journal of Medicinal Chemistry
ARTICLE
resulted in the abatement of both HCV inhibition and the
SI values (cf. 15a,f,g versus 9a,f,g, respectively).
(δ 2.49 ppm), and the center of methanol-d
Carbon-13 NMR spectra were performed on a Varian Mercury-400 (100
MHz) spectrometer or Bruker AC-400 (400 MHz) spectrometer by use
4
quintet (δ 3.30 ppm).
of chloroform-d and dimethylsulfoxide-d
mical shifts are referenced to the center of the CDCl triplet (δ 77.0
6
as solvents. Carbon-13 che-
’
CONCLUSIONS
3
A new compound library was established by chemical synthe-
6
ppm) and DMSO-d septet (δ 39.5 ppm). Multiplicities are recorded
with the following abbreviations: s, singlet; d, doublet; t, triplet; q,
ses; it contained 26 conjugates of coumarin-purine ribofurano-
quartet; m, multiplet; J, coupling constant (hertz).
sides with a -SCH - linker. For establishment of the structure-
2
0 0 0 0
-(2 ,3 ,5 -Tri-O-acetyl-β-D-ribofuranos-1 -yl)purine-8-thione
9
activity relationship on their anti-HCV activity, the coumarin
moiety was allowed to possess various substituents, including F,
Cl, Br, Me, and OMe. The purine moiety may contain an amino
group. The hydroxyl groups in ribofuranosides may be protected
with an acyl or acetal group.
0 0 0 0
3) and 7,9-Bis(2 ,3 ,5 -tri-O-acetyl- β-D-ribofuranos-1 -yl)-
(
purine-8-thione (4). To a solution of purine-8-thione (2, 417.9
mg, 2.746 mmol, 1.0 equiv) in dry acetonitrile (30 mL) was added
BTSA (1.01 mL, 4.12 mmol, 1.5 equiv) under nitrogen atmosphere.
After the solution was stirred at 60 °C for 30 min, 1,2,3,5-tetra-O-acetyl-
Seven among the 26 new compounds were found to inhibit
HCV subgenomic replicon replication in the Huh 5-2 cell line.
The most appealing results were associated with conjugates 6a,
β-D-ribofuranose (961.4 mg, 3.021 mmol, 1.0 equiv) and Me
3
SiOTf
(0.744 mL, 4.12 mmol, 1.5 equiv) were added in sequence. The reaction
mixture was heated to 80 °C and stirred for 18 h. Excess solvent was
removed under reduced pressure, and the residue was treated with 20%
9
5
a, and 9f, which inhibited HCV replication at EC values of 6.6,
50
.5, and 5.9 μM, individually. Moreover, guidelines were deduced
aqueous NaHCO
with ethyl acetate (3 ꢀ 25 mL). The combined organic layers were
washed with brine (30 mL), dried over MgSO (s), filtered, and
3
solution (30 mL). The aqueous phase was extracted
from the analysis of their structures and anti-HCV activity. It is an
essential for the coumarin moiety present in the conjugated
compounds to exhibit antiviral activity. This moiety bearing
various substituents can be attached to purine ribofuranosides
4
concentrated under reduced pressure to afford a residue. The residue
was purified by use of column chromatography (20% hexanes in EtOAc
as the eluant) to give 3 (717.3 mg, 1.748 mmol) in 64% and 4 (288.9 mg,
at the C(8) position through a -SCH - linker. The presence of
2
a ribofuranose moiety led to increment of the HCV inhibition,
0
.4321 mmol) in 16% as white solid foam for both.
: mp (recrystallized from methanol/ether) 185.4-186.5 °C; H
NMR (CDCl 400 MHz) δ 10.42 (bs, 1 H, NH), 8.82 (s, 1 H, H-2), 8.48
0
0
wherein the -OH groups kept unmasked at all of the 2 -, 3 -, and
1
3
0
5
-positions led to reduction of their cytotoxicity. These guide-
3
,
lines will be of value for medicinal scientists to design and
synthesize new conjugated compounds in the future.
0
(s, 1 H, H-6), 6.67 (d, J = 4.0 Hz, 1 H, H-1 ), 6.26 (dd, J = 6.0, 4.0 Hz, 1
0
0
0
H, H-2 ), 5.88 (dd, J = 6.0, 6.0 Hz, 1 H, H-3 ), 4.55-4.51 (m, 1 H, H-4 ),
0
0
4.41-4.37 (m, 1 H, H-5 ), 4.34-4.30 (m, 1 H, H-5 ), 2.14 (s, 3 H,
13
3 3 3 3
CH ), 2.06 (s, 3 H, CH ), 2.03 (s, 3 H, CH ); C NMR (CDCl , 100
’
EXPERIMENTAL SECTION
MHz) δ 172.38 (CdS), 170.89 (CdO), 169.91 (CdO), 169.79
(CdO), 151.99, 150.44, 135.50, 123.42, 87.01, 79.42, 71.61, 70.20,
General. All reactions were carried out in oven-dried glassware
(
120 °C) under an atmosphere of nitrogen unless indicated otherwise.
62.89, 20.75 (CH
3 3 3
), 20.50 (CH ), 20.48 (CH ); IR (neat) 3026 (w),
-
1
Dichloromethane, acetone, and methanol were purchased from Mal-
linckrodt Chemical Co. Acetonitrile was purchased from Fischer Scien-
tific Co. Ethyl acetate (EtOAc) and hexanes from Mallinckrodt
Chemical Co. were dried and distilled from CaH
trifluoromethanesulfonate (Me SiOTf) was purchased from Fluka
Chemika. N,O-Bistrimethylsilylacetamide (BTSA) was purchased from
Merck Chemical Co. Aqueous ammonium hydroxide was purchased
from J. T. Baker Chemical Co. p-Toluenesulfonic acid monohydrate
2941 (w), 2839 (w), 1747 (m), 1465 (s), 1234 (s), 1047 (m) cm ; MS
þ
þ
(FAB ) m/z 411 (MH , 56), 329 (21), 260 (12), 259 (80), 139 (63),
þ
77 (18). HRMS (FAB) calcd for (C16
H
N
19
O
4
S þ H) : 411.0974;
7
. Trimethylsilyl
found 411.0978. For its X-ray crystallographic data, CCDC issued a
deposition number 776792. These data can be obtained free of charge
from The Cambridge Crystallographic Data Centre via www.ccdc.cam.
ac.uk/data_request/cif.
2
3
1
4: mp (recrystallized from methanol/ether) 69.0-70.3 °C; H NMR
(PTSA) and 2,2-dimethoxypropane were purchased from Sigma-Aldrich
(CDCl3, 400 MHz) δ 8.82 (s, 2 H, H-2 þ H-6), 6.83 (d, J = 6.8 Hz, 1 H,
0
0
0
Chemical Co. 1,2,3,5-Tetra-O-acetyl-β-D-ribofuranose was purchased
from Alfa Aesar Chemical Co. Purine-8-thione (2) and 3-(chloromethyl)-
coumarins 5 were prepared according to the reported methods.
H-1 ), 6.74 (d, J = 4.2 Hz, 1 H, H-1 ), 6.20 (dd, J = 6.0, 4.2 Hz, 1 H,
33
0
0
H-2 ), 5.83 (dd, J = 6.0, 6.0 Hz, 1 H, H-3 ), 5.52 (dd, J = 6.8, 6.7 Hz, 1 H,
34
00
00
0
H-2 ), 5.40 (dd, J = 6.7, 4.2 Hz, 1 H, H-3 ), 4.51-4.48 (m, 2 H, H-4 þ
0
0
0
0
0
Melting points were obtained with a Fargo MP-2D melting point
apparatus. Analytical thin layer chromatography (TLC) was performed
on precoated plates (silica gel 60 F-254) purchased from Merck Inc.
Purification by gravity column chromatography was carried out by use of
Silicycle ultra pure silica gel (particle size 40-63 μm, 230-400 mesh).
High performance liquid chromatography (HPLC) was performed on
two Waters 515 HPLC pumps equipped with a Waters 2489 UV/visible
detector and a Thermo 5 μm Hypersil ODS (250 mm ꢀ 4.6 mm i.d.).
Purity of all compounds was >98.0%, as checked by HPLC.
H-4 ), 4.39-4.29 (m, 4 H, 2 ꢀ H-5 þ 2 ꢀ H-5 ), 2.25 (s, 3 H, CH
2.22 (s, 3 H, CH ), 2.13 (s, 3 H, CH ), 2.09 (s, 3 H, CH ), 2.06 (s, 3 H,
CH ), 2.04 (s, 3 H, CH ); C NMR (CDCl , 100 MHz) δ 172.22
3
),
3
3
3
1
3
3
3
3
(CdS), 170.49 (CdO), 170.09 (CdO), 169.57 (CdO), 169.50
(CdO), 169.39 (CdO), 169.34 (CdO), 152.63, 149.98, 136.81,
122.64, 87.52, 87.50, 80.22, 79.62, 71.28, 71.09, 70.19, 69.26, 62.92,
62.65, 20.77 (CH
(CH ), 20.29 (CH
3
), 20.65 (CH
3 3 3
) 20.45 (CH ), 20.36 (CH ), 20.33
3
3
); IR (neat) 3022 (w), 2940 (w), 1749 (s), 1606 (w),
-1
þ
þ
1474 (s), 1238 (s), 1047 (s) cm ; MS (FAB ) m/z 669 (MH , 43),
Infrared (IR) spectra were measured on a Perkin-Elmer model
Spectrum One B spectrophotometer. Absorption intensities are re-
corded with the following abbreviations: s, strong; m, medium; w, weak.
High-resolution mass spectra were obtained by means of a JEOL JMS-
411 (23), 259 (100), 154 (63), 97 (53). HRMS (FAB) calcd for
þ
(C27
H
32
N
4
O
14S þ H) : 669.1714; found 669.1716.
Standard Procedure 1 for the Preparation of Conjugated
Compounds 6 and 11. To a solution containing a thione (3 or 10,
1.0 equiv) in water and acetonitrile was added aqueous ammonium
hydroxide (0.15 mL). After the solution was stirred at room temperature
for 30 min, 3-(chloromethyl)coumarin 5 (1.5 equiv) was added. The
solution was then stirred at room temperature for 40 min to 3.0 h, and
acetonitrile was removed under reduced pressure. To the residue was
7
00 mass spectrometer. Proton NMR spectra were obtained on a Varian
Mercury-400 (400 MHz) spectrometer or Bruker AC-400 (400 MHz)
spectrometer with use of chloroform-d, dimethylsulfoxide-d , and
methanol-d as solvents. Proton NMR chemical shifts are referenced
to the CHCl singlet (δ 7.24 ppm), the center of DMSO-d quintet
6
4
3
6
2
119
dx.doi.org/10.1021/jm101337v |J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
ARTICLE
1 H, ArH), 6.21 (dd, J = 6.0, 4.8 Hz, 1 H, H-2 ), 5.97 (d, J = 4.8 Hz, 1 H,
0
0
4
added saturated aqueous NH Cl (10 mL), which was extracted with
00 00
H-1 ), 5.79 (dd, J = 6.0, 5.6 Hz, 1 H, H-3 ), 4.51 (s, 2 H, SCH
2
CHCl
3
(3 ꢀ 15 mL). The combined organic layers were washed with
), 4.46 (dd,
J = 12.0, 3.2 Hz, 1 H, H-5 ), 4.36-4.32 (m, 1 H, H-4 ), 4.27 (dd, J = 12.0,
0
0
00
brine (10 mL), dried over MgSO (s), filtered, and concentrated under
4
0
0
reduced pressure. The residue was purified by use of column chroma-
tography packed with silica gel to give the desired products with purity of
6.4 Hz, 1 H, H-5 ), 2.13 (s, 3 H, CH
3
), 2.02 (s, 3 H, CH
, 100 MHz) δ 170.45 (CdO), 169.49 (CdO),
169.35 (CdO), 160.50, 155.63, 153.08, 151.87, 151.46, 145.44, 140.46,
34.52, 131.59, 129.88, 127.24, 125.20, 120.00, 117.95, 86.80, 80.02,
71.73, 70.18, 62.72, 31.23 (SCH ), 20.63 (CH ), 20.47 (CH ), 20.32
(CH ); IR (neat) 3061 (w), 2937 (w), 1745 (s), 1723 (s), 1462 (m),
3
), 2.02 (s, 3 H,
1
3
3 3
CH ); C NMR (CDCl
>
98.0%, as determined by HPLC.
0
00 00 00
1
8
-(Coumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-O-acetyl-β-D-
0
0
ribofuranos-1 -yl)purine (6a). Standard procedure 1 was followed
by use of 3 (96.8 mg, 0.236 mmol, 1.0 equiv), water (5.0 mL),
acetonitrile (3.0 mL), and 3-(chloromethyl)coumarin (5a, 68.9 mg,
2
3
3
3
-
1
þ
þ
1371 (m), 1230 (s) cm ; MS (FAB ) m/z 603 (MH , 69), 345 (51),
0
.354 mmol, 1.5 equiv). After the solution was stirred at room
289 (11), 259 (99), 154 (77), 139 (100). HRMS (FAB) calcd for
þ
temperature for 2.0 h and then worked up, the residue was purified by
use of column chromatography (25% hexanes in EtOAc as the eluant) to
give 6a (121.1 mg, 0.1972 mmol) in 90% yield as white solids: mp
(C26
H
23ClN
4
O
9
S þ H) : 603.0953, found 603.0945.
0
0
00 00 00
8-(6 -Bromocoumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-O-
0
0
acetyl-β-D-ribofuranos-1 -yl)purine (6d). Standard procedure 1
was followed by use of 3 (21.2 mg, 51.7 μmol, 1.0 equiv), water (2.5 mL),
acetonitrile (1.5 mL), and 6-bromo-3-(chloromethyl)coumarin (5d, 21.2
mg, 77.5 μmol, 1.5 equiv). After the solution was stirred at room tempe-
rature for 1.0 h and then worked up, the residue was purified by use of
column chromatography (25% hexanes in EtOAc as the eluant) to give6d
1
(
recrystallized from dichloromethane/diethyl ether) 75.2-76.6 °C; H
3
NMR (CDCl , 400 MHz) δ 8.96 (s, 1 H, H-6), 8.85 (s, 1 H, H-2), 8.07
(
2
s, 1 H, CHdC-COO), 7.52-7.47 (m, 2 H, 2 ꢀ ArH), 7.26-7.32 (m,
0
0
H, 2 ꢀ ArH), 6.21 (dd, J = 6.0, 5.2 Hz, 1 H, H-2 ), 5.99 (d, J = 5.2 Hz, 1
0
0
0
0
H, H-1 ), 5.77 (dd, J = 6.0, 5.6 Hz, 1 H, H-3 ), 4.55 (s, 2 H, SCH
2
), 4.46
0
0
00
(dd, J = 11.9, 3.2 Hz, 1 H, H-5 ), 4.37-4.34 (m, 1 H, H-4 ), 4.29 (dd, J
(29.3 mg, 45.3 μmol) in 87% yield as white solids: mp (recrystallized from
0
0
1
=
11.9, 5.6 Hz, 1 H, H-5 ), 2.14 (s, 3 H, CH ), 2.02 (s, 6 H, 2 ꢀ CH );
dichloromethane/diethyl ether) 83.1-84.4 °C; H NMR (CDCl
3
, 400
3
3
1
3
C NMR (CDCl
3
, 100 MHz) δ 170.37 (CdO), 169.41 (CdO),
MHz) δ 8.94 (s, 1 H, H-6), 8.82 (s, 1 H, H-2), 7.98 (s, 1 H, CHdC-
COO), 7.60 (d, J = 2.2 Hz, 1 H, ArH), 7.55 (dd, J = 8.8, 2.2 Hz, 1 H, ArH),
7.17 (d, J = 8.8 Hz, 1 H, ArH), 6.20 (dd, J = 6.0, 4.6 Hz, 1 H, H-2 ), 5.97
1
1
7
69.23 (CdO), 161.02, 156.36, 153.63, 153.30, 151.09, 144.88, 141,80,
34.60, 131.73, 128.03, 124.60, 123.90, 119.00, 116.56, 86.85, 80.15,
1.67, 70.30, 62.74, 31.55 (SCH ), 20.56 (CH ), 20.41 (CH ), 20.23
0
0
0
0
00
(d, J = 4.6 Hz, 1 H, H-1 ), 5.79 (dd, J = 6.0, 5.6 Hz, 1 H, H-3 ), 4.51 (s, 2
2
3
3
0
0
(CH
3
); IR (neat) 3064 (w), 2915(w), 2850 (w), 1746 (m), 1717 (s),
462 (m), 1228 (s), 1049 (m) cm ; MS (FAB ) m/z 569 (MH , 80),
H, SCH
2
), 4.46 (dd, J = 11.8, 3.4 Hz, 1 H, H-5 ), 4.35-4.32 (m, 1 H,
-1
þ
þ
00
00
1
3
H-4 ), 4.27 (dd, J = 11.8, 5.4 Hz, 1 H, H-5 ), 2.12 (s, 3 H, CH
3
), 2.01 (s, 3
1
3
11 (100), 259 (96), 159 (92), 139 (99), 97 (80). HRMS (FAB) calcd
H, CH
3
), 2.01 (s, 3 H, CH
3
); C NMR (CDCl
3
, 100 MHz) δ 170.52
þ
for (C H N O S þ H) : 569.1342, found 569.1348.
(CdO), 169.56 (CdO), 169.41 (CdO), 160.50, 155.80, 153.19, 152.41,
151.44, 145.38, 140.41, 134.56, 134.48, 130.35, 125.24, 120.56, 118.30,
2
6 24 4 9
0
0
00 00 00
8
-(6 -Fluorocoumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-O-
0
0
acetyl-β-D-ribofuranos-1 -yl)purine (6b). Standard procedure 1
was followed by use of 3 (20.4 mg, 49.7 μmol, 1.0 equiv), water
2 3
117.24, 86.87, 80.10, 71.78, 70.25, 62.78, 31.30 (SCH ), 20.67 (CH ),
20.51 (CH ), 20.36 (CH ); IR (neat) 3061 (w), 2939 (w), 1745 (s), 1725
3
3
-1
þ
þ
(
(
2.5 mL), acetonitrile (1.5 mL), and 3-chloromethyl-6-fluorocoumarin
5b, 15.8 mg, 74.3 μmol, 1.5 equiv). After the solution was stirred at
(s), 1462 (m), 1370 (m), 1230 (s) cm ; MS (FAB ) m/z 647 (MH ,
22), 391 (46), 259 (77), 154 (89), 139 (100), 107 (40). HRMS (FAB)
þ
room temperature for 1.0 h and then worked up, the residue was purified
by use of column chromatography (25% hexanes in EtOAc as the eluant)
to give 6b (27.1 mg, 46.2 μmol) in 93% yield as white solids: mp
calcd for (C26
H
23BrN
4
O
9
S þ H) : 647.0447, found 647.0444.
0
0
00 00 00
8-(6 -Methylcoumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-O-
0
0
acetyl-β-D-ribofuranos-1 -yl)purine (6e). Standard procedure 1
was followed by use of 3 (152.9 mg, 0.3726 mmol, 1.0 equiv), water
(5.0 mL), acetonitrile (3.0 mL), and 3-chloromethyl-6-methylcoumarin
(5e, 116.6 mg, 0.5589 mmol, 1.5 equiv). After the solution was stirred at
room temperature for 40 min and then worked up, the residue was
purified by use of column chromatography (25% hexanes in EtOAc as
the eluant) to give 6e (201.4 mg, 0.3457 mmol) in 93% yield as white
1
(
recrystallized from dichloromethane/diethyl ether) 80.2-81.5 °C; H
NMR (CDCl
3
, 400 MHz) δ 8.93 (s, 1 H, H-6), 8.82 (s, 1 H, H-2), 8.00
(s, 1 H, CHdC-COO), 7.29-7.26 (m, 1 H, ArH), 7.21-7.14 (m, 2 H,
0
0
2
ꢀ ArH), 6.20 (dd, J = 6.0, 4.8 Hz, 1 H, H-2 ), 5.97 (d, J = 4.8 Hz, 1 H,
0
0
0
0
H-1 ), 5.79 (dd, J = 6.0, 6.0 Hz, 1 H, H-3 ), 4.52 (s, 2 H, SCH
2
), 4.46
0
0
00
(dd, J = 11.8, 3.2 Hz, 1 H, H-5 ), 4.36-4.32 (m, 1 H, H-4 ), 4.27 (dd, J
0
0
=
11.8, 5.4 Hz, 1 H, H-5 ), 2.12 (s, 3 H, CH ), 2.02 (s, 3 H, CH ), 2.00
solids: mp (recrystallized from dichloromethane/diethyl ether) 81.8-
3
3
13
1
(s, 3 H, CH ); C NMR (CDCl , 100 MHz) δ 170.48 (CdO), 169.52
83.2 °C; H NMR (CDCl
3
, 400 MHz) δ 8.95 (s, 1 H, H-6), 8.82 (s, 1 H,
3
3
(CdO), 169.38 (CdO), 160.73, 158.77 (d), 155.89, 153.16, 151.30,
H-2), 8.00 (s, 1 H, CHdC-COO), 7.30-7.19 (m, 3 H, 3 ꢀ ArH), 6.25
0
0
0
0
1
1
2
1
49.68, 145.19, 140.79 (d), 134.52, 125.13, 119.69 (d), 119.14 (d),
18.10 (d), 113.33 (d), 86.81, 80.04, 71.73, 70.18, 62.73, 31.28 (SCH2),
(dd, J = 5.8, 5.0 Hz, 1 H, H-2 ), 5.97 (d, J = 5.0 Hz, 1 H, H-1 ), 5.81 (dd,
0
0
J = 5.8, 5.2 Hz, 1 H, H-3 ), 4.53 (s, 2 H, SCH ), 4.46 (dd, J = 11.8, 3.4
Hz, 1 H, H-5 ), 4.35-4.32 (m, 1 H, H-4 ), 4.28 (dd, J = 11.8, 5.4 Hz, 1
H, H-5 ), 2.36 (s, 3 H, ArCH ), 2.13 (s, 3 H, CH ), 2.02 (s, 3 H, CH ),
2.02 (s, 3 H, CH ); C NMR (CDCl , 100 MHz) δ 170.50 (CdO),
2
0
0
00
0.63 (CH
3
), 20.48 (CH
3
), 20.33 (CH
3
); IR (neat) 3068 (w), 2923 (w),
-1
00
747 (m), 1723 (s), 1584 (m), 1463 (m), 1230 (s), 1049 (m) cm ; MS
3
3
3
þ
þ
13
3 3
(FAB ) m/z 587 (MH , 100), 329 (84), 289 (15), 259 (96), 139 (87),
þ
9
7 (39). HRMS (FAB) calcd for (C H FN O S þ H) : 587.1248,
169.53 (CdO), 169.34 (CdO), 161.34, 156.11, 153.14, 151.72, 151.38,
145.33, 141.91, 134.63, 134.38, 132.79, 127.84, 123.68, 118.71, 116.26,
26
23
4 9
found 587.1252.
0
0
00 00 00
8
-(6 -Chlorocoumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-O-
86.76, 80.04, 71.66, 70.24, 62.78, 31.51 (SCH
(CH ), 20.50 (CH ), 20.32 (CH ); IR (neat) 3001 (w), 2930 (w), 2851
(w), 1748 (m), 1717 (s), 1463 (s), 1231 (s), 1050 (m) cm ; MS
2 3
), 20.67 (CH ), 20.65
0
0
acetyl-β-D-ribofuranos-1 -yl)purine (6c). Standard procedure 1
was followed by use of 3 (30.6 mg, 74.6 μmol, 1.0 equiv), water (2.5 mL),
acetonitrile (1.5 mL), and 6-chloro-3-(chloromethyl)coumarin (5c, 25.6
mg, 112 μmol, 1.5 equiv). After the solution was stirred at room
temperature for 2.0 h and then worked up, the residue was purified by
use of column chromatography (25% hexanes in EtOAc as the eluant) to
3
3
3
-
1
þ
þ
(FAB ) m/z 583 (MH , 72), 325 (100), 258 (81), 154 (48), 139 (85).
þ
HRMS (FAB) calcd for (C27
583.1505.
8-(6 ,8 -Dibromocoumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-
O-acetyl-β-D-ribofuranos-1 -yl)purine (6f). Standard procedure
1 was followed by use of 3 (35.6 mg, 86.7 μmol, 1.0 equiv), water
(2.5 mL), acetonitrile (1.5 mL), and 3-chloromethyl-6,8-dibromocou-
marin (5f, 45.9 mg, 130 μmol, 1.5 equiv). After the solution was stirred at
H
N
O
S þ H) : 583.1499, found
26
4
9
0
0
0
00 00 00
0
0
give 6c (38.8 mg, 64.3 μmol) in 86% yield as white solids: mp
1
(
recrystallized from dichloromethane/diethyl ether) 82.6-84.1 °C; H
NMR (CDCl
3
, 400 MHz) δ 8.95 (s, 1 H, H-6), 8.81 (s, 1 H, H-2), 7.98 (s,
H, CHdC-COO), 7.44-7.39 (m, 2 H, 2 ꢀ ArH), 7.23 (d, J = 8.4 Hz,
1
2
120
dx.doi.org/10.1021/jm101337v |J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
ARTICLE
0
0
00
room temperature for 3.0 h and then worked up, the residue was purified
by use of column chromatography (25% hexanes in EtOAc as the eluant)
H-2 ), 4.70 (dd, J = 9.7, 5.0 Hz, 1 H, H-3 ), 4.52 (s, 2 H, SCH
2
), 4.41
0
0
00
00
(dd, J = 12.0, 3.6 Hz, 1 H, H-5 ), 4.30-4.22 (m, 2 H, H-4 þ H-5 ),
13
to give 6f (48.6 mg, 66.9 μmol) in 77% yield as white solids: mp
3.71 (br, 1 H, OH), 2.91 (br, 1 H, OH), 1.99 (s, 3 H, CH
(CDCl , 100 MHz) δ 170.93(CdO), 161.21, 156.73, 153.52, 153.25
150.98, 144.97, 141.96, 134.53, 131.85, 128.03, 124.72, 123.93, 118.97,
116.64, 89.34, 82.15, 72.34, 70.67, 65.57, 31.42 (SCH ), 20.74 (CH );
IR (neat) 3406 (br, OH), 3054 (w), 2930 (w), 1741 (m), 1716 (s), 1609
3
); C NMR
1
(
recrystallized from dichloromethane/diethyl ether) 89.4-90.8 °C; H
3
NMR (CDCl
3
, 400 MHz) δ 8.95 (s, 1 H, H-6), 8.84 (s, 1 H, H-2), 7.93
(
s, 1 H, CHdC-COO), 7.82 (d, J = 2.2 Hz, 1 H, ArH), 7.54 (d, J = 2.2
2
3
0
0
Hz, 1 H, ArH), 6.16 (dd, J = 6.0, 4.8 Hz, 1 H, H-2 ), 5.96 (d, J = 4.8 Hz, 1
H, H-1 ), 5.79 (dd, J = 6.0, 6.0 Hz, 1 H, H-3 ), 4.52 (s, 2 H, SCH
(
J = 12.0, 5.6 Hz, 1 H, H-5 ), 2.13 (s, 3 H, CH
(
(
0
0
00
-1
þ
þ
2
), 4.48
(m), 1463 (m), 1238 (m) cm ; MS (FAB ) m/z 485 (MH , 67), 311
(86), 307 (45), 282 (56), 136 (100). HRMS (FAB) calcd for
(C22
0
0
00
dd, J = 12.0, 3.2 Hz, 1 H, H-5 ), 4.37-4.33 (m, 1 H, H-4 ), 4.28 (dd,
0
0
þ
3
), 2.03 (s, 3 H, CH
, 100 MHz) δ 170.49 (CdO), 169.54
CdO), 169.47 (CdO), 159.56, 155.68, 153.17, 151.30, 149.36,
3
), 2.02
H
20
N
00
4
O
7
S þ H) : 485.1131, found 485.1133.
13
00
0
s, 3 H, CH
3
); C NMR (CDCl
3
9-(5 -O-Acetyl-β-D-ribofuranos-1 -yl)-8-[(6 -fluorocoumarin-
0
3 -yl)methylthio]purine (7b). Standard procedure 2 was followed
by use of 6b (104.1 mg, 0.1775 mmol). After the solution was stirred for
45 min and then worked up, the residue was purified by use of column
chromatography (0-2% methanol in EtOAc as the eluant) to give 7b
(49.9 mg, 99.3 μmol) in 56% yield as white solids: mp (recrystallized
from dichloromethane/methanol) 105.6-107.0 °C; H NMR (CDCl
1
1
2
1
45.16, 139.94, 137.13, 134.46, 129.56, 125.95, 121.28, 117.11,
11.02, 87.00, 80.02, 71.66, 70.15, 62.71, 31.13 (SCH ), 20.66 (CH ),
2
3
3 3
0.48 (CH ), 20.33 (CH ); IR (neat) 3068 (w), 2925 (w), 1741 (m),
-
1
þ
733 (s), 1462 (m), 1366 (m), 1225 (s), 1047 (s) cm ; MS (FAB ) m/
þ
1
z 725 (MH , 31), 543 (12), 469 (68), 307 (25), 259 (100), 154 (98),
3
,
þ
139 (98). HRMS (FAB) calcd for (C26
H
22Br
2
N
4
O
9
S þ H) : 724.9552,
400 MHz) δ 8.89 (s, 1 H, H-6), 8.75 (s, 1 H, H-2), 8.02 (s, 1 H,
CHdC-COO), 7.30-7.26 (m, 1 H, ArH), 7.22-7.17 (m, 2 H, 2 ꢀ
found 724.9542.
0
0
00 00 00
00
8
-(8 -Methoxycoumarin-3 -yl)methylthio-9-(2 ,3 ,5 -tri-
ArH), 5.93 (d, J = 4.6 Hz, 1 H, H-1 ), 5.10 (dd, J = 5.2, 4.6 Hz, 1 H,
0
0
00
00
O-acetyl-β-D-ribofuranos-1 -yl)purine (6g). Standard proce-
dure 1 was followed by use of 3 (97.4 mg, 0.237 mmol, 1.0 equiv),
water (5.0 mL), acetonitrile (3.0 mL), and 3-chloromethyl-8-methox-
ycoumarin (5g, 79.9 mg, 0.356 mmol, 1.5 equiv). After the solution was
stirred at room temperature for 2.0 h and then worked up, the residue
was purified by use of column chromatography (20% hexanes in EtOAc
as the eluant) to give 6g (113.8 mg, 0.1901 mmol) in 80% yield as white
H-2 ), 4.66 (dd, J = 5.2, 5.2 Hz, 1 H, H-3 ), 4.51 (s, 2 H, SCH
2
), 4.43
0
0
00
00
(dd, J = 11.4, 3.0 Hz, 1 H, H-5 ), 4.31-4.24 (m, 2 H, H-4 þ H-5 ),
1
3
3 3
1.99 (s, 3 H, CH ); C NMR (CDCl , 100 MHz) δ 170.95 (CdO),
160.85, 158.85 (d), 156.66, 153.31 150.97, 149.65, 144.88, 140.90,
134.49, 125.26, 119.72 (d), 119.25 (d), 118.21 (d), 113.32 (d), 89.38,
82.25, 72.22, 70.69, 63.65, 31.27 (SCH
(br, OH), 3068 (w), 2926(w), 2849 (w), 1716 (s), 1440 (s), 1238 (s),
1044 (m) cm ; MS (FAB ) m/z 503 (MH , 41), 329 (100), 177 (44),
), 20.75 (CH ); IR (neat) 3418
2
3
-
1
þ
þ
solids: mp (recrystallized from dichloromethane/diethyl ether) 79.8-
1
þ
8
1.3 °C; H NMR (CDCl
3
, 400 MHz) δ 8.94 (s, 1 H, H-6), 8.81 (s, 1 H,
154 (97), 136 (75). HRMS (FAB) calcd for (C H FN O S þ H) :
2
2
20
4 7
H-2), 8.03 (s, 1 H, CHdC-COO), 7.17 (dd, J = 8.0, 8.0 Hz, 1 H, ArH),
503.1037; found 503.1031.
0
0
00
00
0
7
5
(
.03 (d, J = 8.0 Hz, 2 H, 2 ꢀ ArH), 6.26 (dd, J = 5.6, 5.0 Hz, 1 H, H-2 ),
9-(5 -O-Acetyl-β-D-ribofuranos-1 -yl)-8-[(6 -bromocoumarin-
0
0
00
0
.97 (d, J = 5.0 Hz, 1 H, H-1 ), 5.80 (dd, J = 5.6, 5.6 Hz, 1 H, H-3 ), 4.53
3 -yl)methylthio]purine (7d). Standard procedure 2 was followed
by use of 6d (94.4 mg, 0.146 mmol). After the solution was stirred for 45
min and then worked up, the residue was purified by use of column
chromatography (0-2% methanol in EtOAc as the eluant) to give 7d
(47.3 mg, 84.0 μmol) in 57% yield as white solids: mp (recrystallized
0
0
s, 2 H, SCH ), 4.46 (dd, J = 11.6, 3.2 Hz, 1 H, H-5 ), 4.36-4.32 (m, 1
2
0
0
00
H, H-4 ), 4.26 (dd, J = 11.6, 5.2 Hz, 1 H, H-5 ), 3.93 (s, 3 H, ArOCH
3
),
); C NMR
3
, 100 MHz) δ 170.50(CdO), 169.53 (CdO), 169.35 (CdO),
1
3
2
.13 (s, 3 H, CH
3 3 3
), 2.03 (s, 3 H, CH ), 2.02 (s, 3 H, CH
(CDCl
1
1
1
5
60.59, 156.28, 153.22, 151.25 147.11, 145.13, 143.29, 142.03, 134.65,
24.52, 124.15, 119.65, 119.47, 113.71, 86.84, 80.10, 71.68, 70.29, 62.78,
from dichloromethane/methanol) 132.1-133.7 °C; H NMR (CDCl ,
3
400 MHz) δ 8.92 (s, 1 H, H-6), 8.77 (s, 1 H, H-2), 7.99 (s, 1 H,
CHdC-COO), 7.60 (d, J = 2.0 Hz, 1 H, ArH), 7.56 (dd, J = 8.7, 2.0 Hz,
6.27 (OCH
3 2 3 3
), 31.53 (SCH ), 20.66 (CH ), 20.51 (CH ), 20.33
00
1 H, ArH), 7.19 (d, J = 8.7 Hz, 1 H, ArH), 5.91 (d, J = 4.6 Hz, 1 H, H-1 ),
(
1
3
CH ); IR (neat) 3016 (w), 2936 (w), 2851 (w), 1748 (m), 1720 (s),
463 (m), 1232 (s), 1106 (s) cm ; MS (FAB ) m/z 599 (MH , 71),
3
-
1
þ
þ
00 00
5.08 (dd, J = 5.2, 4.6 Hz, 1 H, H-2 ), 4.70 (dd, J = 5.2, 5.2 Hz, 1 H, H-3 ),
0
0
41 (84), 259 (73), 154 (100), 136 (97), 97 (58). HRMS (FAB) calcd
4.50 (s, 2 H, SCH ), 4.41 (dd, J = 12.2, 3.4 Hz, 1 H, H-5 ), 4.31-4.22
2
þ
00
00
for (C27
H
26
N
4
O
Standard Procedure 2 for the Preparation of the 9-(5 -O-
10S þ H) : 599.1448, found 599.1439.
(m, 2 H, H-4 þ H-5 ), 3.66 (br, 1 H, OH), 2.92 (br, 1 H, OH), 1.98 (s,
13
0
0
3 3
3 H, CH ); C NMR (CDCl , 100 MHz) δ 170.91 (CdO), 160.52,
0
0
0
Acetyl-β-D-ribofuranos-1 -yl)-8-[(coumarin-3 -yl)methylthio]-
purines 7. To a solution containing 6 (1.0 equiv) in methanol (10 mL)
was added saturated methanolic ammonia (15 mL) at 0 °C. After the
solution was stirred at 0 °C for 45 min, it was concentrated under
156.59, 153.27, 152.31, 150.97, 144.96, 140.43, 134.53, 134.51, 130.27,
125.29, 120.50, 118.33, 117.29, 89.36, 82.23, 72.29, 70.70, 63.58, 31.21
(SCH ), 20.74 (CH ); IR (neat) 3418 (br, OH), 3019 (w), 2925 (w),
2853 (w), 1723 (s), 1463 (m), 1240 (s), 1044 (m) cm ; MS (FAB )
m/z 563 (MH , 12), 391 (19), 307 (45), 291 (50), 213 (90), 154 (100).
2
3
-
1
þ
þ
reduced pressure. To the residue was added saturated aqueous NH
4
Cl
þ
(
20 mL), which was extracted with CHCl (3 ꢀ 20 mL). The combined
HRMS (FAB) calcd for (C H BrN O S þ H) : 563.0236, found
3
22 19
4 7
organic layers were washed with brine (20 mL), dried over MgSO
4
(s),
563.0227.
00 00 0
filtered, and concentrated under reduced pressure. The residue was
purified by use of column chromatography packed with silica gel to give
the desired products with purity of >98.0%, as determined by HPLC.
9-(5 -O-Acetyl-β-D-ribofuranos-1 -yl)-8-[(6 -methylcoumarin-
0
3 -yl)methylthio]purine (7e). Standard procedure 2 was followed
by use of 6e (122.8 mg, 0.2108 mmol). After the solution was stirred for
45 min and then worked up, the residue was purified by use of column
chromatography (0-4% methanol in EtOAc as the eluant) to give 7e
(71.1 mg, 0.143 mmol) in 68% yield as light yellow solids: mp
00 00 0
9
-(5 -O-Acetyl-β-D-ribofuranos-1 -yl)-8-[(coumarin-3 -yl)-
methylthio]purine (7a). Standard procedure 2 was followed by use
of 6a (89.3 mg, 0.157 mmol). After the solution was stirred for 45 min
and then worked up, the residue was purified by use of column
chromatography (0-3% methanol in EtOAc as the eluant) to give 7a
1
(recrystallized from dichloromethane/methanol) 131.2-132.4 °C; H
NMR (CDCl , 400 MHz) δ 8.91 (s, 1 H, H-6), 8.76 (s, 1 H, H-2), 7.99
3
(
39.5 mg, 81.5 μmol) in 52% yield as white solids: mp (recrystallized
(s, 1 H, CHdC-COO), 7.31-7.28 (m, 2 H, 2 ꢀ ArH), 7.19 (d, J = 8.4
1
00
from dichloromethane/methanol) 91.2-92.7 °C; H NMR (CDCl ,
4
Hz, 1 H, ArH), 5.93 (d, J = 4.6 Hz, 1 H, H-1 ), 5.09 (dd, J = 5.4, 4.6 Hz, 1
3
0
0
00
00 MHz) δ 8.90 (s, 1 H, H-6), 8.75 (s, 1 H, H-2), 8.06 (s, 1 H,
H, H-2 ), 4.70 (dd, J = 5.6, 5.4 Hz, 1 H, H-3 ), 4.51 (s, 2 H, SCH
2
), 4.41
0
0
00
00
CHdC-COO), 7.51-7.46 (m, 2 H, 2 ꢀ ArH), 7.32-7.25 (m, 2 H, 2
(dd, J = 12.0, 3.6 Hz, 1 H, H-5 ), 4.31-4.21 (m, 2 H, H-4 þ H-5 ),
0
0
ꢀ
ArH), 5.92 (d, J = 4.0 Hz, 1 H, H-1 ), 5.10 (dd, J = 9.7, 4.0 Hz, 1 H,
3
3.66 (br, 1 H, OH), 2.89 (br, 1 H, OH), 2.36 (s, 3 H, ArCH ), 1.99 (s,
2
121
dx.doi.org/10.1021/jm101337v |J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
ARTICLE
S þ H) : 285.0658, found
1
3
þ
3
1
1
H, CH
3
); C NMR (CDCl
3
, 100 MHz) δ 170.88 (CdO), 161.42,
(100). HRMS (FAB) calcd for (C10
285.0650.
12 4 4
H N O
56.83, 153.29, 151.71, 150.99, 144.99, 141.95, 134.58, 134.48, 132.87,
27.80, 123.79, 118.75, 116.35, 89.39, 82.20, 72.35, 70.76, 63.59, 31.54
Standard Procedure 3 for the Preparation of Conjugated
Compounds 9 and 15. To a solution containing a thione (8 or 14,
1.0 equiv) in water (2.5 mL) and acetonitrile (1.5 mL) was added
aqueous ammonium hydroxide. After the solution was stirred at room
temperature for 30 min, a 3-(chloromethyl)coumarin (5, 1.5 equiv) was
added and stirring was continued at room temperature for 15 min to
2.0 h. Acetonitrile therein was removed under reduced pressure, and
water was further removed under reduced pressure with K €u gelrohr
GKR-51 containing P O . The residue was purified by use of column
(
(
SCH ), 20.72 (CH ), 20.70 (CH ); IR (neat) 3407 (br, OH), 3067
2 3 3
-
1
w), 2924 (w), 2849 (w), 1737 (m), 1714 (s), 1463 (m), 1235 (s) cm
;
þ
þ
MS (FAB ) m/z 499 (MH , 34), 307 (43), 289 (24), 173 (35), 154
þ
(
4
100), 107 (34). HRMS (FAB) calcd for (C H N O S þ H) :
2
3 22 4 7
99.1297, found 499.1287.
0
0
00
0
0
9
-(5 -O-Acetyl-β-D-ribofuranos-1 -yl)-8-[(6 ,8 -dibromo-
0
coumarin-3 -yl)methylthio]purine (7f). Standard procedure 2
was followed by use of 6f (84.8 mg, 0.117 mmol). After the solution
was stirred for 45 min and then worked up, the residue was purified by
use of column chromatography (0-3% methanol in EtOAc as the
eluant) to give 7f (38.8 mg, 60.4 μmol) in 52% yield as light yellow
2
5
chromatography packed with silica gel to give the desired products with
purity of >98.0%, as determined by HPLC.
0
00
8-(Coumarin-3 -yl)methylthio-9-(β-D-ribofuranos-1 -yl)-
purine (9a). Standard procedure 3 was followed by use of 8 (43.3 mg,
0.153 mmol, 1.0 equiv), aqueous ammonium hydroxide (0.25 mL), and
3-(chloromethyl)coumarin (5a, 44.8 mg, 0.230 mmol, 1.5 equiv). After
the solution was stirred at room temperature for 2.0 h and then worked
up, the residue was purified by use of column chromatography (3-5%
methanol in EtOAc as the eluant) to give 9a (54.9 mg, 0.124 mmol) in
81% yield as white solids: mp (recrystallized from dichloromethane/
solids: mp (recrystallized from dichloromethane/methanol) 107.9-
1
1
09.2 °C; H NMR (CDCl
3
, 400 MHz) δ 8.90 (s, 1 H, H-6), 8.76 (s, 1
H, H-2), 7.96 (s, 1 H, CHdC-COO), 7.82 (d, J = 2.2 Hz, 1 H, ArH),
00
.55 (d, J = 2.2 Hz, 1 H, ArH), 5.89 (d, J = 4.4 Hz, 1 H, H-1 ), 5.08 (br, 1
7
0
0
00
H, H-2 ), 4.69 (br, 1 H, H-3 ), 4.50 (s, 2 H, SCH
2
), 4.41 (dd, J = 11.4,
0
0
00
00
3
.0 Hz, 1 H, H-5 ), 4.31-4.24 (m, 2 H, H-4 þ H-5 ), 3.75 (br, 1 H,
1
3
3 3
OH), 2.94 (br, 1 H, OH), 1.99 (s, 3 H, CH ); C NMR (CDCl , 100
1
MHz) δ 170.93 (CdO), 159.65, 156.42, 153.26, 150.97, 149.25, 144.91,
methanol) 170.7-172.1 °C; H NMR (DMSO-d , 400 MHz) δ 9.07 (s,
6
1
8
40.08, 137.17, 134.40, 129.51, 126.07, 121.24, 117.19, 111.08, 89.32,
2.25, 72.23, 70.69, 63.58, 30.94 (SCH ), 20.77 (CH ); IR (neat) 3418
1 H, H-6), 8.83 (s, 1 H, H-2), 8.26 (s, 1 H, CHdC-COO), 7.68 (d, J =
7.6 Hz, 1 H, ArH), 7.60 (dd, J = 8.2, 7.4 Hz, 1 H, ArH), 7.41 (d, J = 8.2
Hz, 1 H, ArH), 7.34 (dd, J = 7.6, 7.4 Hz, 1 H, ArH), 5.78 (d, J = 6.4 Hz, 1
2
3
(
1
3
br, OH), 3078 (m), 2932 (w), 1734 (s), 1710 (s), 1464 (m), 1365 (m),
236 (m), 1046 (m) cm- ; MS (FAB ) m/z 641 (MH , 6), 469 (14),
1
þ
þ
00
H, H-1 ), 5.47 (d, J = 6.0 Hz, 1 H, OH), 5.29 (d, J = 5.2 Hz, 1 H, OH),
0
0
07 (55), 213 (23), 154 (100), 136 (95). HRMS (FAB) calcd for
5.11-5.06 (m, 1 H, H-2 ), 4.96 (dd, J = 6.4, 5.2 Hz, 1 H, OH), 4.54 (d,
J = 14.6 Hz, 1 H, SCH), 4.52 (d, J = 14.6 Hz, 1 H, SCH), 4.22-4.18 (m,
þ
(C
22
H
18Br
2
N
4
O
7
S þ H) : 640.9341, found 640.9354.
0
0
00
0
00
00
00
9
-(5 -O-Acetyl-β-D-ribofuranos-1 -yl)-8-[(8 -methoxycou-
1 H, H-3 ), 3.95-3.91 (m, 1 H, H-4 ), 3.68-3.63 (m, 1 H, H-5 ),
0
00 13
marin-3 -yl)methylthio]purine (7g). Standard procedure 2 was
followed by use of 6g (110.7 mg, 0.1849 mmol). After the solution was
stirred for 45 min and then worked up, the residue was purified by use of
column chromatography (0-4% methanol in EtOAc as the eluant) to
3.54-3.48 (m, 1 H, H-5 ); C NMR (DMSO-d , 100 MHz) δ 160.92
6
(CdO), 156.90, 153.40, 153.28, 151.29, 145.77, 142.55, 134.76, 132.61,
129.02, 125.39, 123.78, 119.20, 116.60, 89.13, 86.68, 71.38, 70.93, 62.25,
2
31.84 (SCH ); IR (KBr) 3335 (br, OH), 3161 (w), 2946 (w), 1713 (s),
-
1
þ
þ
give 7g (47.5 mg, 94.7 μmol) in 51% yield as white solids: mp
1594 (m), 1470 (m), 1073 (m) cm ; MS (FAB ) m/z 443 (MH , 12),
1
(
recrystallized from dichloromethane/methanol) 92.3-93.8 °C; H
307 (69), 289 (38), 154 (100), 136 (93), 89 (40). HRMS (FAB) calcd
þ
NMR (CDCl
3
, 400 MHz) δ 8.89 (s, 1 H, H-6), 8.75 (s, 1 H, H-2),
for (C20
H
18
N
4
O
6
S þ H) : 443.1025, found 443.1016.
0
0
8.02 (s, 1 H, CHdC-COO), 7.17 (dd, J = 8.0, 8.0 Hz, 1 H, ArH), 7.03
8-(6 -Fluorocoumarin-3 -yl)methylthio-9-(β-D-ribofuranos-
0
0
00
(
5
d, J = 8.0 Hz, 1 H, ArH), 5.93 (d, J = 4.4 Hz, 1 H, H-1 ), 5.07 (dd, J =
.4, 4.4 Hz, 1 H, H-2 ), 4.69 (dd, J = 5.6, 5.4 Hz, 1 H, H-3 ), 4.50 (s, 2 H,
1 -yl)purine (9b). Standard procedure 3 was followed by use of 8
(49.7 mg, 0.175 mmol, 1.0 equiv), aqueous ammonium hydroxide
(0.25 mL), and 3-chloromethyl-6-fluorocoumarin (5b, 55.8 mg, 0.262
mmol, 1.5 equiv). After the solution was stirred at room temperature for
2.0 h and then worked up, the residue was purified by use of column
chromatography (3-5% methanol in EtOAc as the eluant) to give 9b
(52.4 mg, 0.114 mmol) in 65% yield as white solids: mp (recrystallized
0
0
0
0
0
0
00
2
SCH ), 4.41 (dd, J = 12.0, 3.6 Hz, 1 H, H-5 ), 4.31-4.22 (m, 2 H, H-4
13
0
0
þ H-5 ), 3.92 (s, 3 H, ArOCH
3 3
), 1.99 (s, 3 H, CH ); C NMR
(
CDCl , 100 MHz) δ 170.93 (CdO), 160.72, 156.68, 153.24, 150.98,
3
1
1
2
1
3
47.05, 144.94, 143.13, 142.12, 134.52, 124.61, 124.18, 119.61, 119.40,
13.67, 89.34, 82.13, 72.29, 70.66, 63.60, 56.25 (OCH
3 2
), 31.54 (SCH ),
1
0.76 (CH ); IR (neat) 3442 (br, OH), 3012 (w), 2924 (w), 1715 (s),
3
from dichloromethane/methanol) 183.2-184.3 °C; H NMR (DMSO-
-1
þ
þ
464 (m), 1274 (m), 1107 (s) cm ; MS (FAB ) m/z 515 (MH , 39),
6
d , 400 MHz) δ 9.06 (s, 1 H, H-6), 8.83 (s, 1 H, H-2), 8.22 (s, 1 H,
41 (63), 289 (20), 189 (45), 154 (100), 136 (88). HRMS (FAB) calcd
CHdC-COO), 7.66-7.63 (m, 1 H, ArH), 7.48-7.46 (m, 2 H, 2 ꢀ
þ
00
for (C23
H
22
N
4
O
8
S þ H) : 515.1237, found 515.1238.
ArH), 5.78 (d, J = 6.4 Hz, 1 H, H-1 ), 5.47 (d, J = 6.4 Hz, 1 H, OH), 5.28
0
00
9
-(β-D-Ribofuranos-1 -yl)purine-8-thione (8). To a solution
(d, J = 4.8 Hz, 1 H, OH), 5.10-5.06 (m, 1 H, H-2 ), 4.96 (dd, J = 6.4, 5.6
24
of 3 (210.6 mg, 0.5132 mmol, 1.0 equiv) in methanol (10 mL) was
added saturated methanolic ammonia (20 mL) under nitrogen atmo-
sphere. After the reaction mixture was stirred at room temperature for
Hz, 1 H, OH), 4.56 (d, J = 13.8 Hz, 1 H, SCH), 4.52 (d, J = 13.8 Hz, 1 H,
0
0
00
SCH), 4.22-4.19 (m, 1 H, H-3 ), 3.95-3.91 (m, 1 H, H-4 ), 3.69-
0
0
00 13
6
3.63 (m, 1 H, H-5 ), 3.54-3.48 (m, 1 H, H-5 ); C NMR (DMSO-d ,
36 h, it was concentrated under reduced pressure to afford a residue. The
100 MHz) δ 160.22, 158.31 (d), 156.37, 152.99, 151.03, 149.46, 145.56,
residue was purified by use of column chromatography (10% methanol
141.04, 134.43, 124.84, 119.89 (d), 119.33 (d), 118.30 (d), 113.91 (d),
in EtOAc as the eluant) to give 8 (134.2 mg, 0.4721 mmol) in 92% yield
88.75, 86.39, 70.98, 70.61, 61.93, 31.33 (SCH
2
); IR (KBr) 3367 (br,
1
as white solids: mp (recrystallized from methanol) 222.6-223.7 °C; H
OH), 3154 (m), 2924 (w), 1713 (s), 1582 (m), 1441 (m), 1093
-1
þ
þ
NMR (MeOD-d , 400 MHz) δ 8.68 (s, 1 H, H-2), 8.46 (s, 1 H, H-6),
(m) cm ; MS (FAB ) m/z 461 (MH , 31), 329 (97), 177 (71),
4
0
0
0
00
6
(
1
.66 (d, J = 5.6 Hz, 1 H, H-1 ), 5.11 (dd, J = 5.6, 5.6 Hz, 1 H, H-2 ), 4.48
dd, J = 5.6, 3.8 Hz, 1 H, H-3 ), 4.11-4.08 (m, 1 H, H-4 ), 3.86 (dd, J =
2.3, 3.0 Hz, 1 H, H-5 ), 3.74 (dd, J = 12.3, 4.4 Hz, 1 H, H-5 );
154 (100), 107 (58), 91 (94). HRMS (FAB) calcd for (C20
H
17FN
4
O
S
6
0
00
þ
þ H) : 461.0931, found 461.0938.
0
0
00 13
0
0
C
8-(6 -Chlorocoumarin-3 -yl)methylthio-9-(β-D-ribofuranos-
0
0
NMR (DMSO-d , 100 MHz) δ 172.62 (CdS), 151.06, 150.28, 136.04,
1 -yl)purine (9c). Standard procedure 3 was followed by use of 8
(38.5 mg, 0.135 mmol, 1.0 equiv), aqueous ammonium hydroxide
(0.25 mL), and 6-chloro-3-(chloromethyl)coumarin (5c, 46.4 mg, 0.203
mmol, 1.5 equiv). After the solution was stirred at room temperature for
6
1
24.39, 88.68, 85.28, 70.51, 70.32, 61.99; IR (KBr) 3326 (br, OH), 3067
-
1
(s), 2954 (s), 1626 (m), 1501 (s), 1472 (s), 1266 (s), 1118 (m) cm ;
þ
þ
MS (FAB ) m/z 285 (MH , 20), 235 (39), 219 (25), 165 (35), 136
2
122
dx.doi.org/10.1021/jm101337v |J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
.0 h and then worked up, the residue was purified by use of column
ARTICLE
0 0
8-(8 -Methoxycoumarin-3 -yl)methylthio-9-(β-D-ribofura-
2
0
0
chromatography (0-5% methanol in EtOAc as the eluant) to give 9c (39.6
nos-1 -yl)purine (9g). Standard procedure 3 was followed by use of
8 (39.8 mg, 0.140 mmol, 1.0 equiv), aqueous ammonium hydroxide
(0.25 mL), and 3-chloromethyl-8-methoxycoumarin (5g, 47.2 mg, 0.210
mmol, 1.5 equiv). After the solution was stirred at room temperature for
2.0 h and then worked up, the residue was purified by use of column
chromatography (5% methanol in EtOAc as the eluant) to give 9g (33.5
mg, 83.0 μmol) in 62% yield as white solids: mp (recrystallized from
1
dichloromethane/methanol) 189.9-191.3 °C; H NMR (DMSO-d
6
, 400
MHz) δ 9.07 (s, 1 H, H-6), 8.83 (s, 1 H, H-2), 8.22 (s, 1 H, CHdC-
COO), 7.88 (d, J = 1.6 Hz, 1 H, ArH), 7.62 (dd, J = 9.0, 1.6 Hz, 1 H, ArH),
0
0
7
6
.60 (d, J = 9.0 Hz, 1 H, ArH), 5.78 (d, J = 6.0 Hz, 1 H, H-1 ), 5.47 (d, J =
.4 Hz, 1 H, OH), 5.28 (d, J = 4.8 Hz, 1 H, OH), 5.10-5.05 (m, 1 H,
mg, 69.4 μmol) in 51% yield as white solids: mp (recrystallized from
dichloromethane/methanol) 169.8-171.2 °C; H NMR (DMSO-d
0
0
1
H-2 ), 4.95 (dd, J = 6.0, 5.2 Hz, 1 H, OH), 4.55 (d, J = 14.4 Hz, 1 H, SCH),
.51(d, J=14.4Hz, 1H,SCH), 4.21-4.19 (m, 1H,H-3 ), 3.94-3.92 (m,
H, H-4 ), 3.68-3.63 (m, 1 H, H-5 ), 3.54-3.48 (m, 1 H, H-5 );
6
,
0
0
4
1
400 MHz) δ 9.06 (s, 1 H, H-6), 8.82 (s, 1 H, H-2), 8.22 (s, 1 H,
CHdC-COO), 7.29-7.25 (m, 3 H, 3 ꢀ ArH), 5.78 (d, J = 6.4 Hz, 1 H,
0
0
00
00 13
C
00
H-1 ), 5.46 (d, J = 6.0 Hz, 1 H, OH), 5.27 (d, J = 5.2 Hz, 1 H, OH),
NMR (DMSO-d , 100 MHz) δ 159.82, 156.11, 152.84, 151.57, 150.89,
6
0
0
1
8
3
45.44, 140.48, 134.27, 131.46, 128.48, 127.58, 124.84, 120.19, 118.15,
8.58, 86.23, 70.77, 70.43, 61.75, 31.11 (SCH ); IR (KBr) 3390 (br, OH),
168 (m), 2936 (w), 1716 (s), 1596 (m), 1472 (m), 1092 (m) cm ; MS
5.10-5.05 (m, 1 H, H-2 ), 4.95 (dd, J = 6.4, 5.6 Hz, 1 H, OH), 4.56 (d,
J = 14.0 Hz, 1 H, SCH), 4.51 (d, J = 14.0 Hz, 1 H, SCH), 4.22-4.18 (m,
2
-1
00
00
1 H, H-3 ), 3.94-3.92 (m, 1 H, H-4 ), 3.91 (s, 3H, OCH
3
), 3.68-3.63
þ
þ
00
00 13
(FAB ) m/z 477 (MH , 18), 307 (56), 235 (16), 154 (100), 136 (92),
(m, 1 H, H-5 ), 3.54-3.50 (m, 1 H, H-5 ); C NMR (DMSO-d , 100
6
þ
120 (28). HRMS (FAB) calcd for (C20
H17ClN
4
O
6
S þ H) : 477.0636,
MHz) δ 159.99, 156.32, 152.87, 150.92, 146.42, 145.41, 142.26, 142.05,
134.33, 124.80, 123.67, 119.67, 119.35, 114.24, 88.57, 86.26, 70.74,
found 477.0625.
0
0
8
-(6 -Methylcoumarin-3 -yl)methylthio-9-(β-D-ribofuranos-
70.47, 61.78, 56.13 (OCH
3 2
), 31.23 (SCH ); IR (KBr) 3400 (br, OH),
0
0
-1
1
-yl)purine (9e). Standard procedure 3 was followed by use of 8
3351 (br, OH), 3075 (w), 2938 (w), 1710 (s), 1467 (m), 1103 (m) cm ;
þ
þ
(
(
40.6 mg, 0.143 mmol, 1.0 equiv), aqueous ammonium hydroxide
0.25 mL), and 3-chloromethyl-6-methylcoumarin (5e, 44.7 mg, 0.214
MS (FAB ) m/z 473 (MH , 8), 307 (25), 154 (100), 136 (75), 107
þ
(22). HRMS (FAB) calcd for (C21
473.1120.
H
20
N
4
O
7
S þ H) : 473.1131, found
mmol, 1.5 equiv). After the solution was stirred at room temperature for
0 min and then worked up, the residue was purified by use of column
chromatography (0-5% methanol in EtOAc as the eluant) to give 9e
48.1 mg, 0.105 mmol) in 74% yield as white solids: mp (recrystallized
0
0
0
3
9-(2 ,3 -O-Isopropylidene-β-D-ribofuranos-1 -yl)purine-8-
thione (10). To a solution of 8 (68.5 mg, 0.241 mmol, 1.0 equiv) in 2,2-
dimethoxypropane (6.0 mL) and acetone (12 mL) was added PTSA
(45.8 mg, 0.241 mmol, 1.0 equiv) under nitrogen atmosphere. The
reaction mixture was stirred at room temperature for 24 h. Excess solvent
was removed under reduced pressure, and the residue was treated with
(
1
from dichloromethane/methanol) 184.7-186.1 °C; H NMR (DMSO-
, 400 MHz) δ 9.07 (s, 1 H, H-6), 8.83 (s, 1 H, H-2), 8.18 (s, 1 H,
CHdC-COO), 7.50 (s, 1 H, ArH), 7.40 (d, J = 8.6 Hz, 1 H, ArH), 7.30
d
6
0
0
(
d, J = 8.6 Hz, 1 H, ArH), 5.78 (d, J = 6.4 Hz, 1 H, H-1 ), 5.48 (d, J = 6.0
aqueous NaHCO
extracted with ethyl acetate (3 ꢀ 20 mL). The combined organic layers
were washed with brine (25 mL), dried over MgSO (s), filtered, and
3
solution (10%, 20 mL). The aqueous phase was
0
0
Hz, 1 H, OH), 5.27 (d, J = 4.4 Hz, 1 H, OH), 5.10-5.06 (m, 1 H, H-2 ),
.97 (dd, J = 6.0, 5.6 Hz, 1 H, OH), 4.55 (d, J = 14.2 Hz, 1 H, SCH), 4.51
4
4
0
0
(d, J = 14.2 Hz, 1 H, SCH), 4.21-4.19 (m, 1 H, H-3 ), 3.94-3.92 (m, 1
concentrated under reduced pressure. The residue was purified by use of
column chromatography (5% methanol in EtOAc as the eluant) to give
10 (57.6 mg, 0.178 mmol) in 74% yield as white solids: mp
0
0
00
00
H, H-4 ), 3.68-3.63 (m, 1 H, H-5 ), 3.54-3.48 (m, 1 H, H-5 ), 2.32
(
13
s, 3H, ArCH
52.84, 151.10, 150.86, 145.36, 141.73, 134.29, 134.05, 132.80, 128.13,
23.36, 118.49, 115.90, 88.57, 86.23, 70.75, 70.44, 61.76, 31.24 (SCH ),
0.20 (CH ); IR (KBr) 3371 (br, OH), 3029 (w), 2924 (m), 1712 (s),
3 6
); C NMR (DMSO-d , 100 MHz) δ 160.36, 156.30,
1
1
1
2
1
3
(recrystallized from dichloromethane/methanol) 142.6-144.1 °C; H
6
NMR (DMSO-d , 400 MHz) δ 8.78 (s, 1 H, H-2), 8.56 (s, 1 H, H-6),
2
0
0
00
6.59 (d, J = 2.4 Hz, 1 H, H-1 ), 5.53 (dd, J = 6.4, 2.4 Hz, 1 H, H-2 ), 5.02
3
-1
þ
þ
00
470 (m), 1232 (m), 1094 (m) cm ; MS (FAB ) m/z 457 (MH , 14),
07 (48), 289 (26), 154 (100), 136 (93). HRMS (FAB) calcd for
(dd, J = 6.4, 3.6 Hz, 1 H, H-3 ), 4.91 (dd, J = 6.0, 6.0 Hz, 1 H, OH),
0
0
00
4.10-4.06 (m, 1 H, H-4 ), 3.62-3.51 (m, 1 H, H-5 ), 3.50-3.46 (m, 1
13
þ
00
(
C
21
H
20
N
4
O
6
S þ H) : 457.1182, found 457.1174.
H, H-5 ), 1.53 (s, 3H, CH
100 MHz) δ 171.59 (CdS), 151.38, 149.75, 136.96, 123.79, 113.25,
88.50, 87.13, 82.01, 81.59, 61.59, 27.15 (CH ), 25.28 (CH ); IR (neat)
3329 (br, OH), 3180 (m), 2988 (m), 1657 (s), 1455 (s), 1217 (m), 1101
3 3 6
), 1.30 (s, 3H, CH ); C NMR (DMSO-d ,
0
0
0
8
-(6 ,8 -Dibromocoumarin-3 -yl)methylthio-9-(β-D-ribo-
0
0
furanos-1 -yl)purine (9f). Standard procedure 3 was followed by
use of 8 (40.2 mg, 0.141 mmol, 1.0 equiv), aqueous ammonium
hydroxide (0.25 mL), and 3-chloromethyl-6,8-dibromocoumarin (5f,
3
3
-
1
þ
þ
(m) cm . MS (FAB ) m/z 325 (MH , 2), 307 (39), 289 (21), 154
7
4.7 mg, 0.212 mmol, 1.5 equiv). After the solution was stirred at room
(100), 136 (90), 107 (33). HRMS (FAB) calcd for (C13
H
26
N
4
O
4
S þ
þ
temperature for 2.0 h and then worked up, the residue was purified by
use of column chromatography (3-5% methanol in EtOAc as the
eluant) to give 9f (62.6 mg, 0.104 mmol) in 74% yield as white solids: mp
H) : 325.0971, found 325.0961.
0 00 00
8-(Coumarin-3 -yl)methylthio-9-(2 ,3 -O-isopropylidene-
0
0
β-D-ribofuranos-1 -yl)purine (11a). Standard procedure 1 was
followed by use of 10 (42.7 mg, 0.132 mmol, 1.0 equiv), water (2.5 mL),
acetonitrile (1.5 mL), and 3-(chloromethyl)coumarin (5a, 38.5 mg,
0.198 mmol, 1.5 equiv). After the solution was stirred at room
temperature for 1.0 h and then worked up, the residue was purified by
use of column chromatography (100% EtOAc as the eluant) to give 11a
1
(
recrystallized from dichloromethane/methanol) 180.7-182.2 °C; H
NMR (DMSO-d
6
, 400 MHz) δ 9.06 (s, 1 H, H-6), 8.83 (s, 1 H, H-2),
8
2
.20 (s, 1 H, CHdC-COO), 8.11 (d, J = 2.2 Hz, 1 H, ArH), 8.03 (d, J =
0
0
.2 Hz, 1 H, ArH), 5.79 (d, J = 6.4 Hz, 1 H, H-1 ), 5.45 (d, J = 6.4 Hz, 1
0
0
H, OH), 5.27 (d, J = 5.2 Hz, 1 H, OH), 5.09-5.04 (m, 1 H, H-2 ), 4.95
dd, J = 6.4, 5.2 Hz, 1 H, OH), 4.57 (d, J = 14.2 Hz, 1 H, SCH), 4.52 (d,
(
(33.1 mg, 68.6 μmol) in 52% yield as white solids: mp (recrystallized
0
0
1
J = 14.2 Hz, 1 H, SCH), 4.22-4.19 (m, 1 H, H-3 ), 3.95-3.92 (m, 1 H,
from dichloromethane/hexanes) 109.1-110.8 °C; H NMR (CDCl
3
,
0
0
00
00 13
C
H-4 ), 3.69-3.63 (m, 1 H, H-5 ), 3.55-3.49 (m, 1 H, H-5 );
NMR (DMSO-d , 100 MHz) δ159.17, 155.99, 152.84, 150.91, 148.88,
45.47, 140.19, 136.27, 134.25, 130.27, 125.54, 121.66, 116.42, 110.12,
8.59, 86.24, 70.81, 70.44, 61.76, 30.94 (SCH ); IR (KBr) 3351 (br,
400 MHz) δ 8.97 (s, 1 H, H-6), 8.77 (s, 1 H, H-2), 8.06 (s, 1 H,
CHdC-COO), 7.50-7.44 (m, 2 H, 2 ꢀ ArH), 7.31-7.22 (m, 2 H, 2
6
0
0
1
8
ꢀ ArH), 5.93 (d, J = 5.2 Hz, 1 H, H-1 ), 5.63 (d, J = 10.8 Hz, 1 H, OH),
0 00
2
5.19 (dd, J = 5.6, 5.2 Hz, 1 H, H-2 ), 5.03 (d, J = 5.2 Hz, 1 H, H-3 ), 4.60
(d, J = 14.0 Hz, 1 H, SCH), 4.47 (d, J = 14.0 Hz, 1 H, SCH), 4.49-4.46
OH), 3068 (w), 2924 (w), 1729 (s), 1591 (m), 1465 (m), 1245 (m),
-
1
þ
þ
00
00
00
1
1
5
053 (m) cm ; MS (FAB ) m/z 599 (MH , 2), 307 (20), 154 (100),
36 (72), 107 (22). HRMS (FAB) calcd for (C20
98.9236, found 598.9234.
(m, 1 H, H-4 ), 3.90-3.93 (m, 1 H, H-5 ), 3.78-3.72 (m, 1 H, H-5 ),
13
þ
H
16Br
2
N
4
O
6
S þ H) :
1.65 (s, 3 H, CH
3 3 3
), 1.33 (s, 3 H, CH ); C NMR (CDCl , 100 MHz) δ
160.01 (CdO), 156.23, 153.60, 152.44, 150.62, 145.95, 141.83, 135.36,
2
123
dx.doi.org/10.1021/jm101337v |J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
ARTICLE
1
8
31.79, 127.99, 124.62, 123.95, 118.97, 116.63, 114.32, 92.12, 85.59,
2.39, 81.42, 63.21, 31.44 (SCH ), 27.67 (CH ), 25.37 (CH ); IR
1 H, ArH), 7.34 (dd, J = 7.6, 7.2 Hz, 1 H, ArH), 5.68 (d, J = 7.2 Hz, 1 H,
0
0
H-1 ), 5.59 (dd, J = 8.8, 4.4 Hz, 1 H, OH), 5.40 (d, J = 6.8 Hz, 1 H, OH),
2
3
3
0
0
(
(
(
neat) 3324 (br, OH), 3068 (w), 2933 (m), 1717 (s), 1463 (m), 1290
5.20 (d, J = 4.4 Hz, 1 H, OH), 4.96-4.91 (m, 1 H, H-2 ), 4.39 (d, J = 13.8
Hz, 1 H, SCH), 4.35 (d, J = 13.8 Hz, 1 H, SCH), 4.13-4.09 (m, 1 H,
-
1
þ
þ
m), 1102 (m) cm ; MS (FAB ) m/z 483 (MH , 38), 311 (100), 159
43), 136 (62), 107 (17). HRMS (FAB) calcd for (C H N O S þ
0
0
00
00
H-3 ), 3.92-3.89 (m, 1 H, H-4 ), 3.64-3.59 (m, 1 H, H-5 ), 3.51-3.44
23 22 4 6
þ
00 13
H) : 483.1338, found 483.1336.
(m, 1 H, H-5 ); C NMR (DMSO-d , 100 MHz) δ160.16 (CdO),
6
0
0
00 00
8
-(6 -Bromocoumarin-3 -yl)methylthio-9-(2 ,3 -O-isopro-
0
154.60, 152.99, 151.43, 150.52, 147.87, 142.29, 131.78, 128.35, 124.71,
123.45, 119.46, 118.95, 116.11, 88.74, 86.66, 71.29, 70.90, 62.13, 31.74
0
pylidene-β-D-ribofuranos-1 -yl)purine (11d). Standard proce-
dure 1 was followed by use of 10 (40.9 mg, 0.126 mmol, 1.0 equiv), water
(SCH ); IR (KBr) 3438 (br, OH), 3223 (w), 2932 (w), 1715 (s), 1602
2
-1
þ
þ
(
(
2.5 mL), acetonitrile (1.5 mL), and 6-bromo-3-(chloromethyl)coumarin
5d, 51.7 mg, 0.189 mmol, 1.5 equiv). After the solution was stirred at
(s), 1462 (m), 1316 (m), 1226 (m) cm ; MS (FAB ) m/z 458 (MH ,
50), 326 (59), 289 (45), 154 (100), 107 (62). HRMS (FAB) calcd for
þ
room temperature for 2.0 h and then worked up, the residue was purified
by use of column chromatography (100% EtOAc as the eluant) to give
1
(C20
H
19
N
5
O
6
S þ H) : 458.1131, found 458.1139.
0
0
8-[(6 -Bromocoumarin-3 -yl)methylthio]adenosine (15d).
Standard procedure 3 was followed by use of 14 (25.3 mg, 84.5 μmol,
1.0 equiv), aqueous ammonium hydroxide (0.15 mL), and 6-bromo-
3-(chloromethyl)coumarin (5d, 34.7 mg, 0.127 mmol, 1.5 equiv). After
the solution was stirred at room temperature for 15 min and then worked
up, the residue was purified by use of column chromatography (5-10%
methanol in EtOAc as the eluant) to give 15d (32.7 mg, 61.0 μmol) in
1d (43.8 mg, 78.0 μmol) in 62% yield as white solids: mp
1
(
recrystallized from dichloromethane/hexanes) 151.4-152.9 °C; H
NMR (CDCl , 400 MHz) δ 8.99 (s, 1 H, H-6), 8.79 (s, 1 H, H-2), 8.01
s, 1 H, CHdC-COO), 7.60-7.56 (m, 2 H, ArH), 7.31-7.22 (d, J =
3
(
0
0
12.0 Hz, 1 H, ArH), 5.91 (d, J = 5.2 Hz, 1 H, H-1 ), 5.76 (d, J = 12.0 Hz,
0
1
H, OH), 5.19 (dd, J = 5.6, 5.2 Hz, 1 H, H-2 ), 5.03 (d, J = 5.6 Hz, 1 H,
0
0
H-3 ), 4.58 (d, J = 14.0 Hz, 1 H, SCH), 4.46 (d, J = 14.0 Hz, 1 H, SCH),
4
H, H-5 ), 1.65 (s, 3 H, CH
72% yield as white solids: mp (recrystallized from dichloromethane/
0
0
00
1
.49-4.44 (m, 1 H, H-4 ), 3.94-3.91 (m, 1 H, H-5 ), 3.79-3.73 (m, 1
methanol) 217.2-218.7 °C; H NMR (DMSO-d
6
, 400 MHz) δ 8.41 (s,
0
0
13
), 1.34 (s, 3 H, CH
3
); C NMR (CDCl
3
,
1 H, H-2), 8.04 (s, 1 H, CHdC-COO), 7.94 (d, J = 2.4 Hz, 1 H, ArH),
7.73 (dd, J = 8.8, 2.4 Hz, 1 H, ArH), 7.44 (s, 2 H, NH ), 7.38 (d, J = 8.8 Hz,
3
1
1
9
(
(
00 MHz) δ160.40 (CdO), 155.95, 152.40, 152.35, 150.68, 146.07,
40.38, 135.31, 134.51, 130.24, 125.25, 120.47, 118.34, 117.22, 114.31,
2.19, 85.55, 82.34, 81.44, 63.22, 31.19 (SCH
CH ); IR (neat) 3322 (br, OH), 3064 (w), 2989 (w), 1725 (s), 1716
s), 1463 (m), 1221 (m), 1102 (m) cm ; MS (FAB ) m/z 563 (MH ,
2
0
0
1 H, ArH), 5.67 (d, J = 7.2 Hz, 1 H, H-1 ), 5.59 (dd, J = 8.6, 3.4 Hz, 1 H,
OH), 5.41 (d, J = 6.4 Hz, 1 H, OH), 5.22 (d, J = 4.4 Hz, 1 H, OH), 4.95-
2 3
), 27.70 (CH ), 25.37
00
4.91 (m, 1 H, H-2 ), 4.37 (d, J = 14.6 Hz, 1 H, SCH), 4.33 (d, J = 14.6 Hz,
3
-
1
þ
þ
00
00
1 H, SCH), 4.13-4.09 (m, 1 H, H-3 ), 3.92-3.89 (m, 1 H, H-4 ), 3.64-
3.60 (m, 1 H, H-5 ), 3.51-3.45 (m, 1 H, H-5 ); C NMR (DMSO-d ,
6
0
0
00 13
16), 391 (100), 239 (35), 154 (44), 107 (15). HRMS (FAB) calcd for
þ
(C
23
H
-(8 -Methoxycoumarin-3 -yl)methylthio-9-(2 ,3 -O-iso-
21BrN
4
O
6
S þ H) : 561.0443, found 561.0441.
100 MHz) δ159.75 (CdO), 154.61, 152.09, 151.49, 150.55, 147.89,
141.12, 134.12, 130.30, 124.67, 120.90, 119.49, 118.51, 116.27, 88.75,
0
0
00 00
8
0
0
propylidene-β-D-ribofuranos-1 -yl)purine (11g). Standard
procedure 1 was followed by use of 10 (33.4 mg, 0.103 mmol, 1.0
equiv), water (2.5 mL), acetonitrile (1.5 mL), and 3-chloromethyl-8-
methoxycoumarin (5g, 34.7 mg, 0.155 mmol, 1.5 equiv). After the
solution was stirred at room temperature for 1.0 h and then worked up,
the residue was purified by use of column chromatography (0-4%
methanol in EtOAc as the eluant) to give 11g (33.9 mg, 66.1 μmol) in
86.72, 71.33, 70.94, 62.16, 31.67 (SCH
2
); IR (KBr) 3435 (br, OH), 3099
-1
(w), 2850 (w), 1717 (s), 1660(m), 1470 (m), 1232 (m), 1094 (m) cm
;
þ
þ
MS (FAB ) m/z 536 (MH , 28), 307 (66), 289 (56), 168 (38), 154
þ
(100). HRMS (FAB) calcd for (C20
H18BrN
O
S þ H) : 536.0239,
5
6
found 536.0247.
0
0
0
8-[(6 ,8 -Dibromocoumarin-3 -yl)methylthio]adenosine
(15f). Standard procedure 3 was followed by use of 14 (42.2 mg, 0.141
mmol, 1.0 equiv), aqueous ammonium hydroxide (0.15 mL), and
3-chloromethyl-6,8-dibromocoumarin (5f, 74.5 mg, 0.211 mmol, 1.5
equiv). After the solution was stirred at room temperature for 90 min
and then worked up, the residue was purified by use of column
chromatography (0-9% methanol in EtOAc as the eluant) to give 15f
(72.1 mg, 0.117 mmol) in 83% yield as white solids: mp (recrystallized
64% yield as white solids: mp (recrystallized from dichloromethane/
1
hexanes) 109.7-111.2 °C; H NMR (CDCl
H-6), 8.78 (s, 1 H, H-2), 8.03 (s, 1 H, CHdC-COO), 7.17 (dd, J = 7.8,
.6 Hz, 1 H, ArH), 7.19-7.15 (m, 2 H, 2 ꢀ ArH), 5.91 (d, J = 5.2 Hz, 1
3
, 400 MHz) δ 8.97 (s, 1 H,
7
0
0
H, H-1 ), 5.66 (d, J = 10.8 Hz, 1 H, OH), 5.18 (dd, J = 5.2, 5.2 Hz, 1 H,
0
00
H-2 ), 5.02 (d, J = 5.6 Hz, 1 H, H-3 ), 4.60 (d, J = 13.8 Hz, 1 H, SCH),
0
0
1
4.48 (d, J = 13.8 Hz, 1 H, SCH), 4.50-4.46 (m, 1 H, H-4 ), 3.93-3.90
from dichloromethane/methanol) 168.5-170.1 °C; H NMR (DMSO-
d , 400 MHz) δ 8.37 (s, 1 H, H-2), 8.10 (d, J = 2.0 Hz, 1 H, ArH), 8.04 (s,
6
0
0
00
(
CH
m, 4 H, ArOCH þ H-5 ), 3.79-3.73 (m, 1 H, H-5 ), 1.65 (s, 3 H,
3
13
3
), 1.33 (s, 3 H, CH
3
); C NMR (CDCl
3
, 100 MHz) δ 160.51
1 H, CHdC-COO), 7.95 (d, J = 2.0 Hz, 1 H, ArH), 7.44 (s, 2 H, NH ),
2
0
0
(CdO), 156.28, 152.43, 150.68, 147.12, 145.95, 143.26, 141.98, 135.38,
5.67 (d, J = 7.2 Hz, 1 H, H-1 ), 5.58 (dd, J = 8.6, 3.4 Hz, 1 H, OH), 5.40
(d, J = 6.8 Hz, 1 H, OH), 5.22 (d, J = 4.4 Hz, 1 H, OH), 4.95-4.90 (m, 1
H, H-2 ), 4.37 (d, J = 15.0 Hz, 1 H, SCH), 4.34 (d, J = 15.0 Hz, 1 H,
SCH), 4.12-4.09 (m, 1 H, H-3 ), 3.92-3.89 (m, 1 H, H-4 ), 3.63-
3.59 (m, 1 H, H-5 ), 3.50-3.44 (m, 1 H, H-5 ); C NMR (DMSO-d ,
1
8
24.51, 124.22, 119.61, 119.37, 114.33, 113.68, 92.15, 85.54, 82.38,
1.44, 63.23, 56.26 (OCH ) 31.44 (SCH ), 27.69 (CH ), 25.37 (CH );
0
0
3
2
3
3
0
0
00
IR (neat) 3356 (br, OH), 3061 (w), 2937 (m), 1716 (s), 1463 (m), 1274
m), 1220 (m), 1105 (s) cm- ; MS (FAB ) m/z 513 (MH , 25), 241
100), 189 (52), 154 (85). HRMS (FAB) calcd for (C24
1
þ
þ
00
00 13
(
(
6
H
24
N
4
O
7
S þ
100 MHz) δ159.08 (CdO), 154.61, 151.49, 150.51, 148.96, 147.69,
140.85, 136.11, 129.92, 125.35, 121.82, 119.47, 116.31, 110.20, 88.73,
þ
H) : 513.1444, found 513.1453.
0
8
-[(Coumarin-3 -yl)methylthio]adenosine (15a). Standard
86.70, 71.32, 70.89, 62.13, 31.56 (SCH
2
); IR (KBr) 3337 (br, OH), 3061
-1
procedure 3 was followed by use of 8-mercaptoadenosine (14, 32.1 mg,
(w), 1723 (s), 1633 (m), 1453 (m), 1244 (m), 1127 (m) cm ; MS
þ
þ
0
3
.107 mmol, 1.0 equiv), aqueous ammonium hydroxide (0.15 mL), and
-(chloromethyl)coumarin (5a, 31.3 mg, 0.161 mmol, 1.5 equiv). After
(FAB ) m/z 614 (MH , 6), 307 (68), 289 (45), 168 (11), 154 (100),
þ
107 (63). HRMS (FAB) calcd for (C20
found 613.9335.
H
17Br
2
N
5
O
6
S þ H) : 613.9345,
the solution was stirred atroom temperature for 1.0 h andthenworkedup,
the residue was purified by use of column chromatography (5-10%
methanol in EtOAc as the eluant) to give 15a (41.2 mg, 90.1 μmol) in
0
0
8-[(8 -Methoxycoumarin-3 -yl)methylthio]adenosine (15g).
Standard procedure 3 was followed by use of 14 (57.7 mg, 0.193 mmol,
1.0 equiv), aqueous ammonium hydroxide (0.15 mL), and 3-chloro-
methyl-8-methoxycoumarin (5g, 64.5 mg, 0.290 mmol, 1.5 equiv). After
the solution was stirred at room temperature for 90 min and then worked
up, the residue was purified by use of column chromatography (0-10%
84% yield as white solids: mp (recrystallized from dichloromethane/
1
methanol) 179.3-181.0 °C; H NMR (DMSO-d
6
, 400 MHz) δ 8.44 (s,
H, H-2), 8.04 (s, 1 H, CHdC-COO), 7.68 (d, J = 7.6 Hz, 1 H, ArH),
.60 (dd, J = 8.4, 7.2 Hz, 1 H, ArH), 7.46 (s, 2 H, NH ), 7.40 (d, J = 8.4 Hz,
1
7
2
2
124
dx.doi.org/10.1021/jm101337v |J. Med. Chem. 2011, 54, 2114–2126

Journal of Medicinal Chemistry
ARTICLE
methanol in EtOAc as the eluant) to give 15g (79.2 mg, 0.162 mmol) in
6, 1001–1018. (c) Belon, C. A.; Frick, D. N. Helicase Inhibitors as
Specifically Targeted Antiviral Therapy for Hepatitis C. Future Virol.
2009, 4, 277–293.
8
4% yield as white solids: mp (recrystallized from dichloromethane/
1
methanol) 150.3-151.9 °C; H NMR (DMSO-d
6
, 400 MHz) δ 8.39 (s,
), 7.27-7.20
m, 3 H, 3 ꢀ ArH), 5.69 (d, J = 7.2 Hz, 1 H, H-1 ), 5.53 (dd, J = 8.6, 3.8
Hz, 1 H, OH), 5.40 (d, J = 5.6 Hz, 1 H, OH), 5.18 (br, 1 H, OH), 4.93-
(5) For recent representative work, see the following: (a) Chen, C.-
1
(
H, H-2), 8.04 (s, 1 H, CHdC-COO), 7.42 (s, 2 H, NH
2
0
0
S.; Chiou, C.-T.; Chen, G. S.; Chen, S.-C.; Hu, C.-Y.; Chi, W.-K.; Chu,
Y.-D.; Hwang, L.-H.; Chen, P.-J.; Chen, D.-S.; Liaw, S.-H.; Chern, J.-W.
Structure-Based Discovery of Triphenylmethane Derivatives as Inhibi-
tors of Hepatitis C Virus Helicase. J. Med. Chem. 2009, 52, 2716–2723.
00
.90 (m, 1 H, H-2 ), 4.39 (d, J = 13.6 Hz, 1 H, SCH), 4.35 (d, J = 13.6
4
0
0
00
Hz, 1 H, SCH), 4.13-4.10 (m, 1 H, H-3 ), 3.93-3.90 (m, 1 H, H-4 ),
(b) Manfroni, G.; Paeshuyse, J.; Massari, S.; Zanoli, S.; Gatto, B.; Maga,
0
0
3
.89 (s, 3H, OCH ), 3.64-3.59 (m, 1 H, H-5 ), 3.51-3.45 (m, 1 H,
3
13
G.; Tabarrini, O.; Cecchetti, V.; Fravolini, A.; Neyts, J. Inhibition of
Subgenomic Hepatitis C Virus RNA Replication by Acridone Deriva-
tives: Identification of an NS3 Helicase Inhibitor. J. Med. Chem. 2009,
0
0
H-5 ); C NMR (DMSO-d
6
, 100 MHz) δ 159.89 (CdO), 154.61,
51.45, 150.53, 147.83, 146.40, 142.51, 142.31, 124.68, 123.61, 119.52,
19.50, 119.46, 114.09, 88.74, 86.68, 71.31, 70.91, 62.14, 56.12 (OCH ),
1.72 (SCH ); IR (KBr) 3450 (br, OH), 3143 (w), 2872 (w), 1712 (s),
1
1
3
1
3
52, 3354–3365. (c) Di Francesco, M. E.; Dessole, G.; Nizi, E.; Pace, P.;
2
Koch, U.; Fiore, F.; Pesci, S.; Di Muzio, J.; Monteagudo, E.; Rowley, M.;
Summa, V. Novel Macrocyclic Inhibitors of Hepatitis C NS3/4A
Protease Featuring a 2-Amino-1,3-thiazole as a P4 Carbamate Re-
placement. J. Med. Chem. 2009, 52, 7014–7028. (d) Popovici-Muller,
J.; Shipps, G. W., Jr.; Rosner, K. E.; Deng, Y.; Wang, T.; Curran, P. J.;
Brown, M. A.; Siddiqui, M. A.; Cooper, A. B.; Duca, J.; Cable, M.;
Girijavallabhan, V. Pyrazolo[1,5-a]pyrimidine-Based Inhibitors of HCV
Polymerase. Bioorg. Med. Chem. Lett. 2009, 19, 6331–6336. (e) Goulet,
S.; Poupart, M.-A.; Gillard, J.; Poirier, M.; Kukolj, G.; Beaulieu, P. L.
Discovery of Benzimidazole-diamide Finger Loop (Thumb Pocket I)
Allosteric Inhibitors of HCV NS5B Polymerase: Implementing Parallel
Synthesis for Rapid Linker Optimization. Bioorg. Med. Chem. Lett. 2010,
-
1
þ
641 (m), 1482 (m), 1276 (m), 1196 (m) cm ; MS (FAB ) m/z 488
þ
(MH , 13), 356 (23), 189 (45), 154 (100), 136 (94). HRMS (FAB)
þ
calcd for (C H N O S þ H) : 488.1240, found 488.1243.
21 21 5 7
’
ASSOCIATED CONTENT
1
13
S
Supporting Information. H NMR and C NMR spec-
b
tra and HPLC data for all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
’
AUTHOR INFORMATION
20, 196–200. (f) Farghaly, T. A.; Hafez, N. A. A.; Ragab, E. A.; Awad,
H. M.; Abdalla, M. M. Synthesis, Anti-HCV, Antioxidant, and Perox-
ynitrite Inhibitory Activity of Fused Benzosuberone Derivatives. Eur. J.
Med. Chem. 2010, 45, 492–500.
Corresponding Author
*Phone: 886-35-725813. Fax: 886-35-721594. E-mail: jrhwu@
mx.nthu.edu.tw.
(
6) Koh, Y.-h.; Shim, J. H.; Wu, J. Z.; Zhong, W.; Hong, Z.; Girardet,
0
J.-L. Design, Synthesis, and Antiviral Activity of Adenosine 5 -Phospho-
nate Analogues as Chain Terminators against Hepatitis C Virus. J. Med.
Chem. 2005, 48, 2867–2875.
’
ACKNOWLEDGMENT
(
7) Perrone, P.; Daverio, F.; Valente, R.; Rajyaguru, S.; Martin, J. A.;
For financial support, we thank the National Science Council
L ꢀe v ^e que, V.; Le Pogam, S.; Najera, I.; Klumpp, K.; Smith, D. B.;
McGuigan, C. First Example of Phosphoramidate Approach Applied
to a 4 -Substituted Purine Nucleoside (4 -Azidoadenosine): Conversion
of an Inactive Nucleoside to a Submicromolar Compound versus
Hepatitis C Virus. J. Med. Chem. 2007, 50, 5463–5470.
and Ministry of Education of R.O.C. The work in Leuven is
supported by the European Commission SILVER project sup-
ported within the 7th Framework Programme as Cooperation
Project Grant Agreement No. 260644. We also thank Well-Being
Biochemical Corp. for its support on instrumentation.
0
0
(8) Ding, Y.; Girardet, J.-L.; Hong, Z.; Lai, V. C. H.; An, H.; Koh, Y.-
h.; Shaw, S. Z.; Zhong, W. Synthesis of 9-(2-β-C-Methyl-β-D-
ribofuranosyl)-6-substituted Purine Derivatives as Inhibitors of HCV
RNA Replication. Bioorg. Med. Chem. Lett. 2005, 15, 709–713.
’
ABBREVIATIONS USED
BTSA, bistrimethylsilylacetamide; CC , 50% inhibitory concen-
trations for host cell growth; CCDC, Cambridge Crystallographic
Data Center; CVFF, consistent valence force field; EC , 50%
50
(
9) Ikejiri, M.; Ohshima, T.; Kato, K.; Toyama, M.; Murata, T.;
0
0
Shimotohno, K.; Maruyama, T. 5 -O-Masked 2 -Deoxyadenosine Ana-
logues as Lead Compounds for Hepatitis C Virus (HCV) Therapeutic
Agents. Bioorg. Med. Chem. 2007, 15, 6882–6892.
50
inhibitory concentrations for virus replication; EC , 90% inhibi-
90
tory concentrations for virus replication; EtOAc, ethyl acetate;
(10) Hocek, M.; Nau ꢁs , P.; Pohl, R.; Votruba, I.; Furman, P. A.;
Tharnish, P. M.; Otto, M. J. Cytostatic 6-Arylpurine Nucleosides. 6. SAR
in Anti-HCV and Cytostatic Activity of Extended Series of 6-Hetar-
ylpurine Ribonucleosides. J. Med. Chem. 2005, 48, 5869–5873.
HCV, hepatitis C virus; Me SiOTf, trimethylsilyl trifluorometha-
3
nesulfonate; PTSA, p-toluenesulfonic acid monohydrate; SI, selec-
tivity index, ratio of CC50 to EC50
(11) Wan, J.; Xia, Y.; Liu, Y.; Wang, M.; Rocchi, P.; Yao, J.; Qu, F.;
Neyts, J.; Iovanna, J. L.; Peng, L. Discovery of Novel Arylethynyltriazole
Ribonucleosides with Selective and Effective Antiviral and Antiproli-
ferative Activity. J. Med. Chem. 2009, 52, 1144–1155.
’
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