J. A. Kim et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2840–2844
2843
11
content of this publication does not necessarily reflect the views
or policies of the Department of Health and Human Services, nor
does mention of trade names, commercial products, or organiza-
tions imply endorsement by the US Government. This Research
was supported in part by the Intramural Research Program of the
NIH, National Cancer Institute, Center for Cancer Research.
CH3
a
H
13
H3C
H
1
3
12
OH
2
CH3
GlcO
5
10
CH3
6
8
H
7
9
4
H
H
References and notes
OH
H
1. Klumpp, K.; Mirzadegan, T. Curr. Pharm. Des. 2006, 12, 1909.
2. Himmel, D. M.; Sarafianos, S. G.; Dharmasena, S.; Hossain, M. M.; McCoy-
Simandle, K.; Ilina, T.; Clark, A. D., Jr.; Knight, J. L.; Julias, J. G.; Clark, P. K.;
Krogh-Jespersen, K.; Levy, R. M.; Hughes, S. H.; Parniak, M. A.; Arnold, E. ACS
Chem. Biol. 2006, 1, 702.
3. Tramontano, E.; Esposito, F.; Badas, R.; Di Santo, R.; Costi, R.; La Colla, P.
Antiviral Res. 2005, 65, 117.
4. Ko, R. K.; Lee, S.; Hyun, C.-G.; Lee, N. H. Bull. Korean Chem. Soc. 2009, 30, 1376.
5. Ribonuclease H inhibitory assay: Inhibition assay was performed using an RNase H
fluorescence resonance energy transfer (FRET) quenching assay previously
reported.6 In brief, the assays were conducted with solutions of the RNA/DNA
hybrid (50 mM Tris, pH 8.0, 60 mM KCl, and 5 mM MgCl2) and 1.0 nM
recombinant reverse transcriptase (each HIV-1, HIV-2, human, and E. coli).
Reactions were initiated by adding each reverse transcriptase for 30 min at 37 °C
and quenched by the addition of EDTA (0.5 M, pH 8.0). The fluorescence intensity
of 96-well microplates was carried out using a SpectraMax Gemini XS dual-
scanning microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA),
and 384-well microplates was performed using a Victor2V multilabel plate
reader (Perkin–Elmer Life Sciences, Boston, MA). To assess the effect of the
+0.81
b
H
-0.07 +0.23
H
OMTPA
+1.05
+0.2
APTMO
H
-0.07
+0.2
+0.15
H
H
+0.27
-0.01
-0.006
H
OH
+0.12
Figure 3. Diagnostic NOESY correlation of 3 (a) and results with the modified
Mosher’s methods (
D
dS ꢀ dR) of 3a (b).
inhibitors, 1 lL of inhibitor in DMSO was added to the microplate well before
adding the substrate and rt solutions. N-(3,4,5-Trihydroxybenzoyl)-1-
naphthaldehyde hydrazone (KMMP) was used as a positive control in all assays.
6. Parniak, M. A.; Min, K. L.; Budihas, S. R.; Le Grice, S. F. J.; Beutler, J. A. Anal.
Biochem. 2003, 322, 33.
7. Cardellina, J. H., II; Munro, M. H. G.; Fuller, R. W.; Manfredi, K. P.; McKee, T. C.;
Tischler, M.; Bokesch, H. R.; Gustafson, K. R.; Beutler, J. A.; Boyd, M. R. J. Nat.
Prod. 1993, 56, 1123.
Table 2
Inhibitory activity of isolated compounds (1–8) on ribonuclease H
a
Compounds
RNase H IC50
HIV-2
(lM)
HIV-1
Human
E. coli
8. Isolation: The 25% MeOH fraction (F2, 8.0 g) was separated on an ODS C-18
column successively eluting with mixtures of H2O/MeOH (1:0, 1:3, 1:1, 3:1, v/
v) to yield three fractions (F21–F23). Fraction F21 (5.9 g) was partitioned on a
Sephadex LH-20 column using pure MeOH as eluting solvent to give 4 (6.0 mg)
and five fractions (F211–F215). The fraction F211 (130.1 mg) was further
separated on an silica gel column using CH2Cl2/MeOH (10:1, v/v) as an eluent
to yield five fractions (F2111–F2115). The fraction F2112 (5.0 mg) was purified
by preparative ODS HPLC, using CH3CN/H2O (17:83, 3.0 mL/min) as solvent
system to afford 8 (tR 7.4 min, 1.2 mg). The fractions F2113 (22.7 mg) and
F2115 (8.6 mg) were purified by ODS gel column chromatography with H2O/
MeOH (8:1, 9:1, v/v) as an eluent to give new compounds 3 (6.8 mg) and 1
(2.7 mg), respectively. The fraction F212 (1.2 g) was chromatographed on an
silica gel column using a CH2Cl2/MeOH (1:1, 9:1, 8:2, v/v) as an eluent to yield
seven fractions, F2121–F2127. The fraction F2125 (20.0 mg) was purified by
preparative ODS HPLC, using CH3CN/H2O (20:80, 3.0 mL/min) as solvent
system to afford 7 (tR 9.8 min, 10.7 mg). The fraction of F2127 (68.0 mg) was
chromatographed on a silica gel column using a CH2Cl2/MeOH (9:1, v/v) as an
1
2
3
4
5
6
7
8
>20.0
>20.0
>20.0
>20.0
>20.0
17.4
>20.0
>20.0
5.0
>20.0
>20.0
>20.0
>20.0
>20.0
3.5
>20.0
>20.0
>20.0
16.4
>20.0
1.7
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
>20.0
KMMPb
a
All data are derived from triplicate tests with the variation of the mean aver-
aging 10%.
N-(3,4,5-Trihydroxybenzoyl)-1-naphthaldehyde hydrazone, positive control
(n = 8).
b
eluent to yield compound
5
(46.5 mg). Fraction F22 (1.3 g) was
remaining compounds yielded negligible effects with IC50 values
more than 20.0 lM against all four RNase H RTs. In addition, none
of the compounds inhibited the activity of E coli RNase H in our as-
say system (Table 2).
This study indicated the structures (4 and 6) bearing a pyrogal-
loyl (1,2,3-trihydroxybenzyl) group might play an important role
in inhibition of viral activity and is consistent with previous re-
ports that polyphenolic tannins formed by polymerization of phe-
chromatographed over Sephadex LH-20 column using pure MeOH as
a
eluting solvent to afford six fractions (F221–F226). Further purification of the
fractions F221 (484.0 mg) on a silica gel column using a CH2Cl2/MeOH (8:1, v/v)
as an eluent resulted in the isolation of new compound 2 (7.2 mg). The fraction
F223 (106.0 mg) was purified by preparative ODS HPLC, using CH3CN/H2O
(18:82, 2.0 mL/min) as solvent system to afford 6 (tR 7.8 min, 3.5 mg).
9. Kumar, N. S.; Rajapaksha, M. J. Chromatogr., A 2005, 1083, 223.
10. Landtag, J.; Baumert, A.; Degenkolb, T.; Schmidt, J.; Wray, V.; Scheel, D.; Strack,
D.; Rosahl, S. Phytochemistry 2002, 60, 683.
11. Kim, J. I.; Kim, H. H.; Kim, S.; Lee, K. T.; Ham, I. H.; Whang, W. K. Arch. Pharm.
Res. 2008, 31, 274.
nol, catechol, and pyrogallol precursors possess
a range of
biological activities, such as antioxidant, anti-carcinogenic, antivi-
12. Zhu, X.; Dong, X.; Wang, Y.; Peng, J.; Luo, S. Helv. Chim. Acta 2005, 88, 339.
13. Youn, U.-J.; Chen, Q. C.; Jin, W.-Y.; Lee, I.-S.; Kim, H.-J.; Lee, J.-P.; Chang, M.-J.;
Min, B.-S.; Bae, K.-H. J. Nat. Prod. 2007, 70, 1687.
ral, and anti-inflammatory properties.23
Thus, the present study suggests a potent possibility of contri-
bution on antiviral properties by compound 6 isolated from D.
racemosum.
14. Distyloside A (1), NSC# 742190: an amorphous powder; ½a D27
ꢀ31.0 (c 0.1,
ꢁ
MeOH); 1H NMR (C5D5N, 500 MHz) and 13C NMR (C5D5N, 125 MHz), see Table
1; ESIMS m/z 357.2 [M+Li]+; HRESIMS m/z 357.2131 [M+Li]+ (calcd for
C
16H30O8Li, 357.2101). Distyloside B (2), NSC# 742191: an amorphous
powder; ½a 2D7
ꢁ
ꢀ18.2 (c 0.5, MeOH); 1H NMR (CD3OD, 500 MHz) and 13C NMR
(CD3OD, 125 MHz), see Table 1; ESIMS m/z 471.2 [M+Li]+; HRESIMS m/z
471.2437 [M+Li]+ (calcd for C21H36O11Li, 471.2418). iso-Dihydrodendrant-
Acknowledgments
hemoside (3), NSC# 742189: an amorphous powder; ½a D27
ꢀ37.0 (c 0.1, MeOH);
ꢁ
This work was supported by Priority Research Centers Program
through the National Research Foundation of Korea (NRF) funded
by the Ministry of Education, Science and Technology (2009-
0093815). This project has been funded in whole or in part with
federal funds from the National Cancer Institute, National
Institutes of Health, under Contract no. HHSN26120080001E. The
1H NMR (CD3OD, 500 MHz) and 13C NMR (CD3OD, 125 MHz), see Table 1;
HRESIMS m/z 399.2586 [M+Li]+ (calcd for C19H36O8Li, 399.2570).
15. Acid hydrolysis and sugar determination of compounds 1–3: Each compound
(1–3, 0.1 mg) was dissolved in 6 N HCl (dioxane–H2O, 1:1, v/v, 200 lL) and
then heated to 110 °C in a sand bath for 4 h. The reaction mixture was then
concentrated to dryness under N2 gas overnight. The residue was dissolved in
100 lL of trimethylsilylimidazole and 100 lL of pyridine. Heating at 60 °C was