Development of new sulfur-containing conjugated compounds as anti-HCV agents

Article abstract of DOI:10.1080/10426507.2010.520284

Various conjugated compounds containing a coumarin moiety and a heterocyclic nucleus were synthesized. Their activity against hepatitis C virus was tested on subgenomic replicon replication in Huh 5-2 cells. Some heterobicycle-coumarin conjugates with the - S - CH₂linker were found to possess appealing antiviral activities. The sulfur atom in these conjugated compounds was found to be an essential element to their antiviral activity. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/10426507.2010.520284

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Phosphorus, Sulfur, and Silicon and the
Related Elements
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Development of New Sulfur-Containing
Conjugated Compounds as Anti-HCV
Agents
a b
a
a
Jih Ru Hwu , Dr Shu-Yu Lin , Professor Shwu-Chen Tsay , Dr
a
a
c
Raghunath Singha , Dr Benoy Kumar Pal , Dr Pieter Leyssen
&
c
Professor Johan Neyts
a
Department of Chemistry , National Tsing Hua University , Hsinchu,
Taiwan, R.O.C.
b
Department of Chemistry , National Central University , Jhongli
City, Taiwan, R.O.C.
c
Rega Institute for Medical Research , Katholieke Universiteit
Leuven , Leuven, Belgium
Published online: 12 Jul 2011.
To cite this article: Jih Ru Hwu , Dr Shu-Yu Lin , Professor Shwu-Chen Tsay , Dr Raghunath Singha ,
Dr Benoy Kumar Pal , Dr Pieter Leyssen & Professor Johan Neyts (2011) Development of New Sulfur-
Containing Conjugated Compounds as Anti-HCV Agents, Phosphorus, Sulfur, and Silicon and the
Related Elements, 186:5, 1144-1152, DOI: 10.1080/10426507.2010.520284
To link to this article: http://dx.doi.org/10.1080/10426507.2010.520284
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Phosphorus, Sulfur, and Silicon, 186:1144–1152, 2011
Copyright ꢀ
Taylor & Francis Group, LLC
C
ISSN: 1042-6507 print / 1563-5325 online
DOI: 10.1080/10426507.2010.520284
DEVELOPMENT OF NEW SULFUR-CONTAINING
CONJUGATED COMPOUNDS AS ANTI-HCV AGENTS
Jih Ru Hwu,1,2 Shu-Yu Lin, Shwu-Chen Tsay,
1
1
1
1
3
Raghunath Singha, Benoy K. Pal, Pieter Leyssen,
3
and Johan Neyts
1
Department of Chemistry, National Tsing Hua University, Hsinchu,
Taiwan, R.O.C.
Department of Chemistry, National Central University, Jhongli City,
2
Taiwan, R.O.C.
3
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Leuven, Belgium
Abstract Various conjugated compounds containing a coumarin moiety and a heterocyclic nu-
cleus were synthesized. Their activity against hepatitis C virus was tested on subgenomic repli-
con replication in Huh 5-2 cells. Some heterobicycle–coumarin conjugates with the
S CH2
linker were found to possess appealing antiviral activities. The sulfur atom in these conjugated
compounds was found to be an essential element to their antiviral activity.
Keywords Coumarin; guanosine; heterocycles; methylenethio linker
INTRODUCTION
In the beginning of the 21st century, many medicinal scientists have been devoting
1
their efforts to the invention of new drugs against hepatitis C virus (HCV). With a size
of around 50 nm, the HCV is an enveloped, single-stranded, positive sense RNA virus.
Infection by HCV is often asymptomatic, but once established, chronic infection can
progress to scarring of the liver and cause fibrosis. Even worse is cirrhosis, which is
generally apparent after many years. In some cases, those with cirrhosis will go on to
develop liver failure or cancer.2
HCV infections occur worldwide; the World Health Organization estimated that
3
about 3% of the world’s population has been infected with HCV. Currently the only ap-
proved treatment for HCV infection is weekly injection of polyethylene glycol-conjugated
interferon-α plus daily oral ribavirin. Interferon is thought to work by stimulating the body’s
natural defenses against virus infection. The 48-week treatment course is often poorly toler-
ated, so new approaches are needed that enhance the effectiveness and shorten the duration
4
of therapy.
Received 30 June 2010; accepted 30 August 2010.
Address correspondence to Jih Ru Hwu, Department of Chemistry, National Tsing Hua University, Hsinchu,
Taiwan 30013, R.O.C. E-mail: jrhwu@mx.nthu.edu.tw
1
144

ANTI-HCV COMPOUNDS CONTAINING SULFUR ATOM
1145
Different types of compounds have been synthesized and found to possess activity
5
against HCV. For examples, Hirashima et al. found that 2-arylbenzimidazole-5-carboxylic
6
acids possess inhibitory activity against HCV NS5B RNA polymerase. Goulet et al.
synthesized benzimidazole–diamide derivatives that exhibit potency against HCV NS5B
7
polymerase. Stankiewicz-Drogo n´ et al. prepared new acridone derivatives that inhibit
8
HCV NS3 helicase. McCauley et al. obtained a new class of macrocyclic isoindolines that
show inhibitory activity against hepatitis C virus NS3/4A protease. It can also be inhibited
9
by five- and six-membered cyclic sulfones as reported by Vel a´ zquez et al. Recently,
our laboratory found that various benzimidazole moieties conjugated with a coumarin
10
moiety by a methylenethio linker (i.e., 1) exhibit potent inhibitory effects on HCV.
Furthermore, the structure–activity relationship toward HCV is established for various
coumarins conjugated with imidazopyridine, purine, benzoxazole, and benzothiazole (i.e.,
11
2
). Herein we report our findings on the essential role of the sulfur atom in the conjugated
compounds heterocycle S CH2 coumarins for their activity against HCV.
RESULTS AND DISCUSSION
10
11
To compare with conjugated compounds 3 and 4 containing an S CH2 linker,
we synthesized two closely related “hinged” heterobicycle–coumarin conjugates as shown
12
in Scheme 1. Treatment of arylenediamines 5 with 3-(ethoxycarbonyl)coumarins 6 in
◦
8
5% o-phosphoric acid at 165 C provided heterobicycle–coumarin conjugates 7 in 53–65%
yields.

1
146
J. R. HWU ET AL.
Recently, several research groups have reported their efforts on the syntheses of
13
guanosine analogues, which exhibit antiviral activities. Accordingly, we synthesized a
series of guanosine SCH2 coumarin conjugates 11 and performed their bioassays. Treat-
ment of the commercially available 8-bromoguanosine (8) with thiourea in ethanol gave
1
4
8
1
-mercaptoguanosine (9). We then coupled 9 with different 3-(chloromethyl)coumarins
15
0 in the presence of 35% NH4OH in water and acetonitrile (Scheme 2). Upon workup
and purification by chromatography, the desired guanosine SCH2 coumarin conjugates
1a–e were produced in 74–91% yields.
1
Scheme 1 Synthesis of hinged heterobicycle–coumarin derivatives.
We confirmed the structures of conjugates 11 on the basis of their spectroscopic
characteristics. For example, the mass spectrum of 11a in the positive ion mode by fast
atom bombardment–mass spectrometry (FAB-MS) technique exhibited at 474.1091 for
+
+
species [M + H] , which indicates the molecular formula to be C20H19N5O7S + H
with the theoretical value of 474.1083. Its infrared (IR) spectrum showed one strong
−1
absorption band at 1723 cm , which was attributed to the carbonyl stretching vibration
of the coumarin moiety.16 Its C NMR spectrum had resonance at 32.67 and 160.12 ppm
for the SCH2 and the O C O carbons, respectively, in the coumarin moiety. On the other
hand, two diastereotopic SCH2 protons appeared at 4.25 and 4.20 ppm as two doublets with
13
1
J = 13.8 Hz in its H NMR spectrum. The glycosidic proton resonated at the 5.70 ppm as
a doublet with J = 6.4 Hz.
Evaluation on the Antiviral Activities in the HCV Genotype 1b
Subgenomic Replicon
All compounds were evaluated in the HCV subgenomic replicon system in Huh 5–2
cells.17 The antiviral assays and cytostatic determination assays have been described in
18
detail before. The 50% inhibitory concentrations for virus replication (EC50) and host cell
growth (CC50) of conjugates 3, 4, and 7a,b are shown in Table 1.

ANTI-HCV COMPOUNDS CONTAINING SULFUR ATOM
1147
Table 1 Inhibitory effects of conjugated compounds on HCV subgenomic replicon replication in Huh 5–2 cells
Compounda
CC50b (µM)
7a/3
7b/4
EC50 (µM)
c
7a/3
7b/4
3
45
21
11
131
11
0
.47
—
—
7
4
7
a
12
30
—
5.7
7.4
b
>170
81
—
aInterferon α-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.
bMinimum cytotoxic concentration required to cause a microscopically detectable alteration of normal cell
morphology.
cMinimum inhibitory concentration required to reduce virus-induced cytopathogenicity by 50%.
Scheme 2 Synthesis of guanosine SCH2 coumarin derivatives.
For the conjugates 11a–e, we found that their HCV replication occurred at EC50
values above 91 µM without apparent cytostatic activity. Therefore, the guanosine moiety
made a limited contribution to its HCV inhibition.
Structure–Activity Relationship
We deduce the following structure–activity relationship (SAR) by scrutinizing their
EC50 and CC50 values shown in Table 1.

1
148
J. R. HWU ET AL.
1
2
3
. Attachment of a coumarin moiety to the benzimidazole nucleus (e.g., 3) with an
S CH2 linker is the key to gain appealing anti-HCV activity. Addition of the
S CH2 linker between the coumarin moiety and the benzimidazole nucleus en-
hanced the HCV inhibitory activity by 12-fold (cf. 3 versus 7a).
. Attachment of an imidazopyridine (e.g., 4) nucleus to the thio terminal of the coumarin
conjugates may offer a convenient approach to enhance the HCV inhibition by a factor
of 7.4-fold (cf. 4 versus 7b). The S CH2 linker therein is essential and should not
be removed.
. Replacement of the benzimidazole moiety with a guanosine moiety in conjugated
coumarins (cf. 3 versus 11a) displayed no notable activity on HCV.
EXPERIMENTAL
General Experimental
◦
All reactions were carried out in oven-dried glassware (120 C) under an at-
mosphere of nitrogen unless as indicated otherwise. Dichloromethane and methanol
were purchased from Mallinckrodt Chemical Co. (Phillipsburg, NJ). Acetonitrile was
purchased from Fisher Scientific Co. (Fairlawn, NJ). Ethyl acetate (EtOAc) and hex-
anes from Mallinckrodt Chemical Co. were dried and distilled from CaH2. Aqueous
ammonium hydroxide was purchased from J. T. Baker Chemical Co. (Phillipsburg, NJ).
1
,2-Diamino-4-fluorobenzene and 2,3-diaminopyridine were purchased from Sigma-
ꢁ
Aldrich Chemical Co. (Steinheim, Germany). 2-(Coumarin-3 -yl)methylthio-5-
fluorobenzimidazole (3), 2-(8 -methoxycoumarin-3 -yl)methylthio-1H-imidazo[4,5-b]
10
ꢁ
ꢁ
11
12
14
pyridine (4), 3-(ethoxycarbonyl)coumarins 6a,b, 8-mercaptoguanosine (9), and
3
-(chloromethyl)coumarins15 10a–e were prepared according to the reported methods.
Analytical thin-layer chromatography (TLC) was performed on precoated plates
(silica gel 60 F-254) purchased from Merck Inc. (Darmstadt, Germany). Purification
by gravity column chromatography was carried out using Silicycle ultra pure silica gel
(particle size 40–63 µm, 230–400 mesh). High-performance liquid chromatography
(
HPLC) was performed on two Waters (Qu e´ bec City, Qu e´ bec) 515 HPLC pumps
equipped with a Waters 2489 UV-Visible Detector and a Thermo 5 µm Hypersil
ODS (250 × 4.6 mm D.I.). Purity of all compounds was >98.0%, as checked by
HPLC.
IR spectra were measured on a Perkin-Elmer model spectrum one B spectrophotome-
ter and a Bomem Michelson Fourier transform infrared spectrometer (FTIR). Absorption
intensities are recorded by the following abbreviations: s, strong; m, medium; w, weak.
High-resolution mass spectra were obtained by means of a JEOL JMS-700 mass spec-
trometer. Proton nuclear magnetic resonance (NMR) spectra were obtained on a Varian
Mercury-400 (400 MHz) spectrometer or Bruker AC-400 (400 MHz) spectrometer by use
of chloroform-d, dimethylsulfoxide-d6, and methanol-d4 as solvents. Proton NMR chemi-
13
cal shifts are referenced to the center of DMSO-d6 quintet (δ 2.49 ppm). C NMR spectra
were performed on a Varian Mercury-400 (100 MHz) spectrometer or Bruker AC-400 (400
13
MHz) spectrometer by use of dimethylsulfoxide-d6 as solvents. C chemical shifts are
referenced to the center of the DMSO-d6 septet (δ 39.5 ppm). Multiplicities are recorded
by the following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; J,
coupling constant (Hertz).

ANTI-HCV COMPOUNDS CONTAINING SULFUR ATOM
1149
Standard Procedure 1 for the Preparation of
ꢀ
3
-(Benzimidazol-2 -yl)coumarins (7)
To a solution containing a diamine 5 (1.0 equiv.) and 3-(ethoxycarbonyl)coumarins
6
(1.0 equiv.) was added o-phosphoric acid (85%, 15 equiv.). The reaction mixture was
◦
stirred at room temperature for 5.0 min and at 165 C for another 8.0 h. Then the solution
was permitted to cool down to 100 C and poured in a large volume of stirred water (500
◦
mL). The solution was neutralized with saturated aqueous NaHCO3 solution and extracted
with EtOAc (3 × 15 mL). The combined organic layers were washed with brine (15 mL),
dried over MgSO4 (s), filtered, and concentrated under reduced pressure to provide crude
solids. The resultant solids were recrystallized with EtOH to give the desired products with
purity >99.6%, as checked by gas chromatography (GC).
ꢀ
ꢀ
3
-(5 -Fluorobenzimidazol-2 -yl)coumarin (7a). Standard procedure 1 was fol-
lowed by the use of 1,2-diamino-4-fluorobenzene (5a, 750.1 mg, 5.948 mmol, 1.0 equiv.),
-(ethoxycarbonyl)coumarin (6a, 1.29 g, 5.91 mmol, 1.0 equiv.), and o-phosphoric acid
9.09 g, 92.8 mmol, 16 equiv.). After workup, the solids were recrystallized with EtOH to
3
(
1
give 7a (1.076 g, 3.839 mmol) in 65% yield as greenish yellow solids: H NMR (DMSO-d6,
4
00 MHz) δ: 9.09 (s, 1 H, CH C COO), 7.98 (d, J = 8.0 Hz, 1 H, ArH), 7.72–7.65 (m, 2
13
H, 2 × ArH), 7.52–7.43 (m, 4 H, 4 × ArH), 7.07 (bs, 1 H, NH); C NMR (DMSO-d6, 100
MHz) δ: 164.00 (C O), 159.23, 156.71, 154.48, 153.29, 148.38, 134.28, 133.11, 130.28,
1
30.19, 125.13, 124.82, 118.99, 118.35, 117.99, 116.18; IR (KBr) 3354 (br, NH), 1725
−1
(
s, C O), 1607 (m), 1483 (m), 1437 (m), 1412 (m), 1314 (m), 1279 (m), 1042 (s) cm ;
+
MS (LC): 280.00 (M) ; high-resolution mass spectrometry (HRMS) m/z Anal. Calcd. for
C16H9FN2O2: 280.0648; Found: 280.0646.
ꢀ
3
-(Imidazopyridin-2 -yl)-8-methoxycoumarin (7b). Standardprocedure1was
followed by the use of 2,3-diaminopyridine (5b, 217.9 mg, 1.997 mmol, 1.0 equiv.),
3-ethoxycarbonyl-8-methoxycoumarin (6b, 494.5 mg, 1.994 mmol, 1.0 equiv.), and o-
phosphoric acid (2.99 g, 30.5 mmol, 15 equiv.). After workup, the solids were recrystallized
1
with EtOH to give 7b (308.8 mg, 1.053 mmol) in 53% yield as light brown solids: H NMR
(
1
DMSO-d6, 400 MHz) δ: 9.98 (bs, 1 H, NH), 8.89 (s, 1 H, CH C COO), 8.46 (d, J =
.6 Hz, 1 H, ArH), 8.16–8.12 (m, 1 H, ArH), 7.39–7.32 (m, 4 H, 4 × ArH), 3.94 (s, 3 H,
13
OCH3); C NMR (DMSO-d6, 100 MHz) 160.45 (C O), 160.16, 153.70, 147.47, 146.33,
44.90, 143.16, 131.88, 125.27, 121.21, 119.91, 119.00, 117.96, 116.25, 112.68, 56.29
1
−1
(OCH3); IR (KBr) 3379 (br, NH), 1705 (s, C O), 1543 (m), 1466 (m) cm ; HRMS m/z
Anal. Calcd. for C16H11N3O3: 293.0800; Found: 293.0798.
Standard Procedure 2 for the Preparation of Conjugated Compounds
To a solution containing 8-mercaptoguanosine (9; 1.0 equiv.) in water (5.0 mL)
and acetonitrile (3.0 mL) was added aqueous ammonium hydroxide (0.25 mL). After the
solution was stirred at room temperature for 10 min, a 3-(chloromethyl)coumarin (10, 1.2
equiv.) was added and stirring was continued at room temperature for 15 min to 1.0 h.
Acetonitrile therein was removed under reduced pressure and water was further removed
under reduced pressure with K u¨ gelrohr GKR-51 containing P2O5. The residue was purified
by use of column chromatography packed with silica gel to give the desired products with
purity >98.0%, as determined by HPLC.
ꢀ
8
-[(Coumarin-3 -yl)methylthio]guanosine (11a). Standard procedure 2 was
followed by use of 9 (65.6 mg, 0.208 mmol, 1.0 equiv.) and 3-(chloromethyl)coumarin

1
150
J. R. HWU ET AL.
(
10a, 48.7 mg, 0.250 mmol, 1.2 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
15% methanol in CH2Cl2 as the eluant) to give 11a (81.4 mg, 0.172 mmol) in 83% yield
(
1
as white solids: H NMR (DMSO-d6, 400 MHz) δ: 10.63 (br, 1 H, NH), 7.95 (s, 1 H,
CH C COO), 7.62–7.56 (m, 2 H, 2 × ArH), 7.40 (d, J = 8.0 Hz, 1 H, ArH), 7.32 (dd,
ꢁꢁ
J = 7.6, 7.2 Hz, 1 H, ArH), 6.40 (s, 2 H, NH2), 5.70 (d, J = 6.4 Hz, 1 H, H-1 ), 5.35 (d,
J = 6.4 Hz, 1 H, OH), 5.05 (d, J = 4.8 Hz, 1 H, OH), 4.93 (dd, J = 6.4, 5.2 Hz, 1 H, OH),
ꢁ
ꢁ
ꢁ
4
4
(
.90–4.84 (m, 1 H, H-2 ), 4.25 (d, J = 13.8 Hz, 1 H, SCH), 4.20 (d, J = 13.8 Hz, 1 H, SCH),
ꢁ
ꢁꢁ
ꢁꢁ
.09–4.06 (m, 1 H, H-3 ), 3.80–3.77 (m, 1 H, H-4 ), 3.62–3.57 (m, 1 H, H-5 ), 3.50–3.44
m, 1 H, H-5 ); C NMR (DMSO-d6, 100 MHz) δ 160.12 (C O), 155.62, 153.11, 152.91,
ꢁꢁ
13
1
8
1
52.52, 141.69, 140.97, 131.74, 128.31, 124.69, 124.16, 118.85, 117.37, 116.09, 88.23,
5.75, 70.56, 70.54, 61.99, 32.67 (SCH2); IR (ATR) 3140 (br, OH), 2924 (w), 1711 (m),
−1
+
+
688 (s), 1608 (s), 1073 (s), 923 (m) cm ; MS (FAB ) m/z 474 (MH , 16), 342 (32), 219
+
(29), 105 (18), 97 (100); HRMS (FAB) Anal. Calcd. for (C20H19N5O7S + H) : 474.1083;
Found 474.1091.
ꢀ
ꢀ
8
-[(6 -Fluorocoumarin-3 -yl)methylthio]guanosine (11b). Standard proce-
dure 2 was followed by use of 9 (65.2 mg, 0.207 mmol, 1.0 equiv.) and 3-chloromethyl-
6-fluorocoumarin (10b, 52.8 mg, 0.248 mmol, 1.2 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 (15% methanol in CH2Cl2 as the eluant) to give 11b (92.4 mg,
0.188 mmol) in 91% yield as white solids: 1H NMR (DMSO-d6, 400 MHz) δ: 10.68 (br, 1
H, NH), 7.90 (s, 1 H, CH C COO), 7.53–7.42 (m, 3 H, 3 × ArH), 6.41 (s, 2 H, NH2),
ꢁ
ꢁ
5
1
.70 (d, J = 6.8 Hz, 1 H, H-1 ), 5.35 (d, J = 6.0 Hz, 1 H, OH), 5.07 (d, J = 4.8 Hz,
ꢁꢁ
H, OH), 4.94 (dd, J = 6.8, 5.2 Hz, 1 H, OH), 4.89–4.85 (m, 1 H, H-2 ), 4.24 (d, J = 14.0
ꢁꢁ
Hz, 1 H, SCH), 4.19 (d, J = 14.0 Hz, 1 H, SCH), 4.10–4.06 (m, 1 H, H-3 ), 3.80–3.76 (m,
ꢁꢁ
ꢁꢁ
ꢁꢁ 13
1
1
1
6
1
H, H-4 ), 3.62–3.56 (m, 1 H, H-5 ), 3.49–3.43 (m, 1 H, H-5 ); C NMR (DMSO-d6,
00 MHz) δ 159.83 (C O), 158.07 (d), 155.60, 153.10, 152.47, 149.25, 141.48, 139.84,
25.44, 119.81 (d), 118.83 (d), 118.00 (d), 117.42, 113.47 (d), 88.27, 85.74, 70.53, 70.50,
1.96, 32.77 (SCH2); IR (ATR) 3146 (br, OH), 2922 (w), 1723 (m), 1681 (s), 1626 (s),
−1
+
+
071(s) cm ; MS (FAB ) m/z 492 (MH , 35), 360 (62), 307 (98), 219 (23), 156 (100), 107
+
(
100); HRMS (FAB) Anal. Calcd. for (C20H18FN5O7S + H) : 492.0989; Found 492.0996.
ꢀ
ꢀ
8
-[(6 -Bromocoumarin-3 -yl)methylthio]guanosine (11c). Standard proce-
dure 2 was followed by use of 9 (62.2 mg, 0.197 mmol, 1.0 equiv.) and 6-bromo-3-
chloromethyl)coumarin (10c, 64.7 mg, 0.237 mmol, 1.2 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 (15% methanol in CH2Cl2 as the eluant) to give 11c (85.7 mg,
0.155 mmol) in 79% yield as white solids: 1H NMR (DMSO-d6, 400 MHz) δ: 10.66 (br, 1
H, NH), 7.89 (s, 1 H, CH C COO), 7.88 (d, J = 2.4 Hz, 1 H, ArH), 7.73 (dd, J = 8.8,
2
1
6
.4 Hz, 1 H, ArH), 7.38 (d, J = 8.8 Hz, 1 H, ArH), 6.40 (s, 2 H, NH2), 5.70 (d, J = 6.8 Hz,
ꢁꢁ
H, H-1 ), 5.34 (d, J = 6.4 Hz, 1 H, OH), 5.06 (d, J = 4.8 Hz, 1 H, OH), 4.93 (dd, J =
ꢁꢁ
.8, 5.2 Hz, 1 H, OH), 4.89–4.85 (m, 1 H, H-2 ), 4.23 (d, J = 14.0 Hz, 1 H, SCH), 4.19 (d,
ꢁꢁ
ꢁꢁ
J = 14.0 Hz, 1 H, SCH), 4.10–4.06 (m, 1 H, H-3 ), 3.80–3.76 (m, 1 H, H-4 ), 3.62–3.56 (m,
ꢁꢁ
ꢁꢁ 13
1
1
1
2
3
H, H-5 ), 3.49–3.43 (m, 1 H, H-5 ); C NMR (DMSO-d6, 100 MHz) δ: 159.65 (C O),
55.70, 153.18, 152.52, 151.95, 141.46, 139.52, 134.03, 130.29, 125.50, 120.78, 118.39,
17.43, 116.24, 88.23, 85.77, 70.57, 70.54, 61.99, 32.80 (SCH2); IR (ATR) 3334 (br, OH),
920 (w), 1709 (s), 1692 (s), 1599 (s), 1066 (s) cm ; MS (FAB ) m/z 552 (MH , 12),
07 (36), 289 (25), 154 (100), 136 (74); HRMS (FAB) Anal. Calcd. for (C20H18BrN5O7S
−1
+
+
+
+
H) : 552.0189; Found 552.0180.

ANTI-HCV COMPOUNDS CONTAINING SULFUR ATOM
1151
ꢀ
ꢀ
8
-[(6 -Methylcoumarin-3 -yl)methylthio]guanosine (11d). Standard proce-
dure 2 was followed by use of 9 (52.3 mg, 0.166 mmol, 1.0 equiv.) and 3-chloromethyl-
6-methylcoumarin (10d, 41.5 mg, 0.199 mmol, 1.2 equiv.). After the solution was stirred
at room temperature for 30 min and then worked up, the residue was purified by use of
column chromatography (15% methanol in CH2Cl2 as the eluant) to give 11d (59.6 mg,
0.122 mmol) in 74% yield as white solids: 1H NMR (DMSO-d6, 400 MHz) δ: 10.72 (br, 1
H, NH), 7.90 (s, 1 H, CH C COO), 7.40–7.38 (m, 2 H, 2 × ArH), 7.30 (d, J = 8.8 Hz,
ꢁ
ꢁ
1
H, ArH), 6.46 (s, 2 H, NH2), 5.69 (d, J = 6.4 Hz, 1 H, H-1 ), 5.38 (d, J = 6.4 Hz, 1 H,
OH), 5.09 (d, J = 4.8 Hz, 1 H, OH), 4.95 (dd, J = 6.0, 6.0 Hz, 1 H, OH), 4.88–4.84 (m, 1
ꢁꢁ
H, H-2 ), 4.24 (d, J = 13.8 Hz, 1 H, SCH), 4.19 (d, J = 13.8 Hz, 1 H, SCH), 4.11–4.06 (m,
ꢁꢁ
ꢁꢁ
ꢁꢁ
ꢁꢁ
1
2
1
8
2
H, H-3 ), 3.81–3.78 (m, 1 H, H-4 ), 3.63–3.57 (m, 1 H, H-5 ), 3.50–3.43 (m, 1 H, H-5 ),
.33 (s, 3 H, ArCH3); 13C NMR (DMSO-d6, 100 MHz) δ: 160.31 (C O), 155.64, 153.25,
52.53, 151.08, 141.71, 140.95, 134.00, 132.61, 127.96, 124.08, 118.61, 117.38, 115.87,
8.29, 85.77, 70.60, 70.58, 62.03, 32.65 (SCH2), 20.23 (ArCH3); IR (ATR) 3301 (br, OH),
−1
+
+
924 (w), 1720 (m), 1644 (s), 1566 (m), 1081 (s) cm ; MS (FAB ) m/z 487 (M , 1), 185
9), 97 (31), 55 (100); HRMS (FAB) Anal. Calcd. for (C21H21N5O7S) : 487.1162; Found
+
(
4
87.1164.
ꢀ
ꢀ
8
-[(8 -Methoxycoumarin-3 -yl)methylthio]guanosine (11e). Standard pro-
cedure 2 was followed by use of 9 (63.7 mg, 0.202 mmol, 1.0 equiv.) and 3-chloromethyl-
-methoxycoumarin (10e, 54.0 mg, 0.243 mmol, 1.2 equiv.) was added. After the solution
8
was stirred at room temperature for 1.0 h and then worked up, the residue was purified by
use of column chromatography (10% methanol in CH2Cl2 as the eluant) to give 11e (84.3
1
mg, 0.167 mmol) in 83% yield as white solids: H NMR (DMSO-d6, 400 MHz) δ: 10.66
(
br, 1 H, NH), 7.93 (s, 1 H, CH C COO), 7.28–7.23 (m, 2 H, 2 × ArH), 7.16–7.14 (m, 1
ꢁꢁ
H, ArH), 6.42 (s, 2 H, NH2), 5.69 (d, J = 6.8 Hz, 1 H, H-1 ), 5.38 (d, J = 6.4 Hz, 1 H, OH),
.09 (d, J = 5.2 Hz, 1 H, OH), 4.95 (dd, J = 6.0, 5.6 Hz, 1 H, OH), 4.88–4.84 (m, 1 H,
5
ꢁ
ꢁ
H-2 ), 4.24 (d, J = 13.8 Hz, 1 H, SCH), 4.19 (d, J = 13.8 Hz, 1 H, SCH), 4.09–4.05 (m, 1 H,
ꢁꢁ
ꢁꢁ
ꢁꢁ
H-3 ), 3.89 (s, 3 H, OCH3), 3.80–3.77 (m, 1 H, H-4 ), 3.61–3.56 (m, 1 H, H-5 ), 3.49–3.43
ꢁꢁ
13
(m, 1 H, H-5 ); C NMR (DMSO-d6, 100 MHz) δ: 159.87 (C O), 155.68, 153.14, 152.55,
1
8
46.41, 142.25, 141.73, 141.24, 124.66, 124.34, 119.47, 119.41, 117.38, 114.05, 88.26,
5.78, 70.55, 70.52, 62.01, 56.16 (OCH3), 32.63 (SCH2); IR (ATR) 3142 (br, OH), 2923
−1
+
+
(w), 1714 (m), 1682 (s), 1274 (m), 1092 (m) cm ; MS (FAB ) m/z 503 (M , 2), 372 (10),
+
232 (11), 154 (45), 79 (100); HRMS (FAB) Anal. Calcd. for (C21H21N5O8S) : 503.1107;
Found 503.1111.
CONCLUSIONS
New substituted heterobicycle–coumarin conjugates were synthesized by chemical
methods and their biological activities were evaluated by the HCV subgenomic replicon
system in Huh 5–2 cells. Through analysis of their anti-HCV data, structure–activity re-
lationships including three guidelines were illustrated. The most important finding is that
the lack of the sulfur atom–containing linker in these conjugated compounds would lead to
poor biological activities. These new findings provide a rational approach for development
of new anti-HCV drugs in the future.
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