Synthesis and anti-influenza activity of aminoalkyl rupestonates

Article abstract of DOI:10.1016/j.bmcl.2012.01.056

A series of aminoalkyl rupestonates were designed and synthesized by reacting rupestonic acid with 1,ω-dibromoalkanes, followed by amination. All of the new compounds were bioassayed in vitro to determine their activities against influenza A (H3N2, H1N1)

Full text of DOI:10.1016/j.bmcl.2012.01.056

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2321–2325
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and anti-influenza activity of aminoalkyl rupestonates
Jiangyu Zhao a,b, Haji Akber Aisa a,
⇑
a Key Laboratory of Xinjiang Indigenous Medicinal Plants Resource Utilization, Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Urumqi
830011, China
b Graduate School of Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of aminoalkyl rupestonates were designed and synthesized by reacting rupestonic acid with 1,
Received 7 November 2011
Revised 8 January 2012
Accepted 17 January 2012
Available online 28 January 2012
x
-dibromoalkanes, followed by amination. All of the new compounds were bioassayed in vitro to deter-
mine their activities against influenza A (H3N2, H1N1) and B viruses. The results showed that compounds
5a–5g, which each contain a 1H-1,2,4-triazolyl moiety, were found to be the most potent set of com-
pounds. Compound 5g was demonstrated to possess the highest inhibitory activity against influenza
H3N2 and H1N1, with IC50 values of 0.97 and 0.42 lM, respectively. Our results also indicated that com-
Keywords:
Rupestonic acid
Synthesis
pounds 2g, 3g, 4g and 5g, which contain ten-CH2-unit spacers between the rupestonic acid and amino
functional groups, were the most potent inhibitors of influenza H1N1 among the synthesized compounds.
Unfortunately, most of the synthesized compounds did not show an obvious activity against influenza B;
Anti-influenza activity
the only exceptions were compounds 5d and 5f, which had IC50 values of 17.3 and 3.2 lM, respectively.
Compounds 4g and 5g were potent inhibitors of influenza H1N1, and they might be potentially developed
as new lead anti-influenza virus compounds. Further studies of the mechanism of action are underway.
Ó 2012 Elsevier Ltd. All rights reserved.
Influenza is a serious global contagious disease that leads to
hundreds of thousands of deaths every year. Currently, influenza
vaccines and anti-influenza drugs are the primary means for pro-
tection against and treatment of influenza. Pharmacotherapy is
the most effective means of controlling the spread of influenza
viruses. Oseltamivir phosphate (Tamiflu), an orally administered
drug for the prophylaxis and treatment of human influenza A
and B,1,2 is the most promising drug for the treatment of the
H5N1 avian influenza virus.3,4 However, both the influenza vaccine
and anti-influenza drugs have some disadvantages. The influenza
vaccine takes effect only when it matches the subtype of the circu-
lating influenza virus, and the development of the vaccine pro-
ceeds much more slowly than the spread of the influenza virus.
In addition, the vaccine cannot effectively protect elderly people
and children. It has been reported that the H5N1 influenza virus
has shown resistant to Oseltamivir.5,6 Thus, researchers are contin-
uing to search for new inhibitors of influenza.
2-(3,8-Dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octahydroazulen-5-yl)-
acrylic acid (rupestonic acid, 1), first isolated from the dichloro-
methane extract of leaves of Decachaeta scabrella and named
pechueloic acid, is a multi-functional sesquiterpenoid compound.7
In our laboratory, more than 70 amide and ester derivatives of
rupestonic acid have been synthesized, and the in vitro activities
of these compounds against influenza viruses A and B were assayed.
Of these, 4-tert-butylphenyl rupestonate showed potent activity
against influenza A (IC50 = 0.5
M).8 Meanwhile, 2-chloroethyl
rupestonate, a by-product formed during the preparation of glyco-
syl rupestonate, was demonstrated to exhibit better in vitro inhib-
itory activity against both influenza A and B (H3N2: TC50 = 1669
l
l
M, IC50 = 17.2
l
M, SI = 96.9; H1N1: IC50 = 27.8
lM, SI = 60.0; B:
TC50 >3225 M, IC50 = 358.4
l
l
M, SI >9.0).9
The piperazinyl moiety is recognized as an important part of
many drugs, including ciprofloxacin and lomefloxacin, and has a
high enthalpy of formation and good thermal stability. In 2001,
the MDDR (MDL Drug Data Report)10 listed 2271 phenylpipera-
zines, including 65 structures that are being tested in phase II clin-
ical trials or higher. The tested activities of these compounds span
23 therapeutic areas, including antibacterials, phosphodiesterase
III inhibitors, antifungals, antivirals, anxiolytics, and antipsychot-
ics.11 In addition, the arylpiperazine scaffold is very active against
most subtypes of the serotonin receptor.12 1H-1,2,4-triazole fungi-
cides have some advantages, such as low toxicity and high efficacy,
as a class of long-term biosynthesis inhibitors. These fungicides are
still widely used in the fields of medicine and agriculture.13
Based on the above information, several series of rupestonic
acid derivatives were designed by linking rupestonic acid and
substituted piperazine or 1H-1,2,4-triazole moieties with alkyl
spacers. The length of the alkyl spacer and the type of amine moi-
ety were explored systematically.
The synthetic pathways of compounds 2a–2g, 3a–3g, 4a–4g and
5a–5g are outlined in Scheme 1. The esterification of rupestonic
⇑
Corresponding author.
E-mail address: haji@ms.xjb.ac.cn (H.A. Aisa).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.056

2322
J. Zhao, H. A. Aisa / Bioorg. Med. Chem. Lett. 22 (2012) 2321–2325
CH3
H
CH3
CH3
H
H
i
ii
O
O
O
CO2(CH2)nBr
CO2(CH2)nR
CO2H
3a~3g, 4a~4g, 5a~5g
1
2a~2g
Scheme 1. Synthesis of aminoalkyl rupestonates. Reagents and conditions: (i) Br(CH2)nBr, 5% NaOH, triethylbenzyl ammonium chloride (TEBAC), CHCl3, reflux, 4–10 h; (ii)
mono-N-substituted piperazine or 1H-1,2,4-triazole, K2CO3, anhydrous acetonitrile, reflux, 10–15 h. n = 2, 3, 4, 5, 6, 8, 10. 3a–3g: R = N-phenyl piperazinyl; 4a–4g: R = N-
methyl piperazinyl; 5a–5g: R = 1H-1,2,4-triazolyl.
acid, isolated from Artemisia rupestris L., a traditional Chinese med-
icine used in Xinjiang, China,14,15 with 1,
-dibromoalkane in the
presence of NaOH afforded -bromoalkyl rupestonates 2a–2g.
The amination of compounds 2a–2g with 1-phenyl-piperazine,
1-methyl-piperazine or 1H-1,2,4-triazole in anhydrous acetonitrile
with K2CO3 yielded three series of aminoalkyl rupestonates, 3a–3g,
4a–4g and 5a–5g, respectively.16,17 The structures of all of the
newly synthesized compounds were confirmed by spectroscopic
analysis.
Acetone or N,N-dimethylformamide (DMF) was often used as the
solvent in the ester preparation using 1, -dibromoalkanes as the
x
x
x
reactants. Acetone was initially used as the solvent in the prepara-
tion of compound 2a, but the reaction failed. DMF, which has a
higher boiling point, is more difficult to remove upon condensation.
Thus, we tried to use chloroform as the solvent for the preparation
of the derivatives 2a–2g, and the result was satisfactory. We found
that the longer the carbon chain of the 1,x-dibromoalkane was, the
Table 1
Chemical structures and inhibitory activity of the rupestonic acid derivatives 2a–2g, 3a–3g, 4a–4g and 5a–5g against influenza A (H3N2 and H1N1) and B viruses
a
Compound
n
R
TC50
(l
M)
Against influenza H3N2 virus
Against influenza H1N1 virus
Against influenza B virus
SIc
IC50
(lM)
SIc
IC50
(l
M)
SIc
b
b
b
IC50
(l
M)
e
e
1d
2a
2b
2c
2d
2e
2f
—
2
3
4
5
6
8
10
—
—
—
—
—
—
—
—
4653
25.8
180.6
>1344
—
—
—
>4032
—
—
—
e
e
e
1358.0
435.6
419.6
54.0
469.3
146.5
137.7
>313.8
>100.7
75.2
19.3
52.1
—
—
>313.8
>100.7
75.2
19.3
39.6
>313.8
>100.7
67.2
>31.2
52.1
e
e
e
5.6
2.8
9.0
10.4
8.4
5.6
2.8
11.8
11.8
20.5
6.2
e
—
9.0
e
14.1
16.4
12.4
6.7
>28.2
>26.5
—
—
e
2g
3a
3b
3c
3d
3e
3f
2
445.0
171.1
414.9
111.7
394.3
333.3
456.2
230.6
666.8
643.5
207.3
86.0
49.4
9.00
3.00
49.4
63.9
79.8
20.0
>56.5
92.6
2.4
9.0
2.7
5.2
5.6
49.4
9.0
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Ph
Ph
Ph
Ph
Ph
Ph
Ph
e
3
57.1
>11.2
>79.8
>25.8
>56.5
>160.2
>152.0
>33.0
>95.5
>92.1
20.6
—
e
e
4
>79.8
>25.7
>56.5
>160.2
94.0
—
—
e
e
5
—
—
e
e
e
6
—
—
—
e
e
8
—
3.60
188.0
1.8
—
e
3g
4a
4b
4c
4d
4e
4f
10
2
4.9
—
e
e
>33.0
>95.5
71.5
—
128.6
>95.5
10.3
17.1
>9.6
>26.9
3.7
—
e
e
e
3
—
—
—
e
4
9.00
10.1
62.8
12.1
—
5
20.6
10.1
e
e
e
6
>9.6
—
—
>9.6
—
e
e
e
8
188.3
177.4
>26.9
5.2
—
—
>26.9
>25.4
—
e
4g
10
34.0
47.6
—

J. Zhao, H. A. Aisa / Bioorg. Med. Chem. Lett. 22 (2012) 2321–2325
2323
Table 1 (continued)
a
Compound
n
R
TC50
(l
M)
Against influenza H3N2 virus
Against influenza H1N1 virus
Against influenza B virus
b
b
b
IC50
(l
M)
SIc
IC50
(lM)
SIc
IC50
(
lM)
SIc
N
N
N
N
N
N
N
e
N
N
N
N
N
N
N
5a
5b
5c
5d
5e
5f
2
224.5
311.2
386.0
672.0
119.5
201.9
27.1
74.9
3.0
83.8
2.68
>8.0
—
N
N
N
N
N
N
N
e
3
34.6
42.9
17.3
14.9
5.20
0.97
9.00
9.0
24.0
57.6
7.4
13.0
6.7
>34.6
77.5
17.3
>31.0
3.2
—
4
5.0
5
38.9
8.0
90.8
2.48
28.0
65.0
38.9
e
6
48.2
7.2
—
8
38.8
28.1
62.9
5g
10
0.42
11.3
2.40
86.9
Ribavirin
Oseltamivir
—
—
—
—
1278
1260
3.9
1.1
329.4
1135.1
1.9
15.5
665.6
81.3
14.7
>500
e
—
a
50% cytotoxic concentration.
50% virus-inhibitory concentration, determined by the CPE inhibition assay.
Selectivity Index (TC50/IC50).
b
c
d
e
Parent compound.
The SI cannot be calculated because the highest concentration tested was less than the IC50
.
shorter the reaction time became, for example, 4 h using 1,
10-dibromodecane versus 14 h using 1,2-dibromoethane.
To determine the potential activity of compounds 2a–2g, 3a–3g,
4a–4g and 5a–5g, they were preliminarily assayed in vitro against
influenza A (H3N2, H1N1) and B viruses.18 The inhibition and
selectivity of these compounds are summarized in Table 1. The
inhibition ratios against influenza H3N2 of compounds 2g, 3g, 4g
and 5g are showed in Figure 1. The IC50 values of compounds
5a–5g against influenza H3N2 and H1N1 are listed in Figure 2.
As shown in Table 1, most of the tested compounds exhibited
better anti-influenza activity against influenza A than the parent
compound. Meanwhile, the variations in the alkyl chain length
and the type of amine moiety had dramatic impacts on the inhib-
itory potency and the selectivity of the aminoalkyl rupestonates.
The modified rupestonic acid compounds all had the toxicities
greater than that of the parent compound. We further investigated
the inhibitory effect of various structural moieties on influenza A
and B.
80
70
60
50
40
30
20
10
0
2g
3g
4g
5g
0c.0o5ncen0tr.1a5tion (µg/mL)
0.46
1.37
Figure 1. Concentration-inhibition curves for 2g, 3g, 4g and 5g against influenza
H3N2.
For the
x-bromoalkyl esters 2a–2g, the order of their IC50 values
against influenza H1N1 was as follows: 2g < 2f < 2d < 2e < 2c < 2b
< 2a. Of these, compound 2g (n = 10) was the most potent, with an
90
80
70
60
50
40
30
20
10
0
IC50 value of 6.7
the parent compound. Compound 2a (n = 2) presented the lowest
toxicity, with a TC50 value of 1358 M, which was higher than that
lM (SI = 20.5); this compound was more active than
H3N2
H1N1
l
of the positive controls Ribavirin and Oseltamivir. The introduction
of bromine did not obviously improve the inhibitory activity. In the
series 3a–3g, each of which contains a phenyl piperazinyl group,
compound 3g (n = 10) showed the most promising activity against
influenza H1N1, with an IC50 value of 2.4 lM, which was smaller
than that of Oseltamivir. Meanwhile, compound 3g presented the
highest selectivity among all tested compounds, with a SI value of
187.9. Unfortunately, it exhibited higher toxicity than Oseltamivir
(TC50 value 456.2 vs 1260 lM). In the series 4a–4g, each of which
5a
5b
5c
5d
5e
5f
5g
bears a methyl piperazinyl group, the order of their IC50 values
against influenza H1N1 was as follows: 4g < 4e < 4c < 4d < 4f < 4b
< 4a. We found that compound 4g (n = 10) showed the best activity
Compound
Figure 2. IC50 values for 5a–5g against influenza H3N2 and H1N1.

2324
J. Zhao, H. A. Aisa / Bioorg. Med. Chem. Lett. 22 (2012) 2321–2325
6. Treanor, J. J.; Hayden, F. G.; Vrooman, P. S.; Barbarash, R.; Bettis, R.; Riff, D.;
Singh, S.; Kinnersley, N.; Ward, P.; Mills, R. G. J. Am. Med. Assoc. 2000, 283, 1016.
7. Miski, M.; de Luengo, D. H.; Mabry, T. J. Phytochemistry 1987, 26, 199.
8. Yong, J. P.; Aisa, H. A. Chem. Nat. Compd. 2008, 44, 311.
against influenza H3N2 and H1N1, with IC50 values of 5.2 and
3.7 M, respectively. Consequently, it could be concluded that the
l
methyl group in piperazine was very importantto the antiviral prop-
erties. The toxicity of compound 4g was higher than that of com-
pound 3g. In the series 5a–5g, each of which bears a 1H-1,2,
4-triazolyl moiety, the order of the relative potency against influ-
enza H3N2 was as follows: 5g < 5f < 5e < 5d < 5b < 5c < 5a. As
shown in Figure 2, the variations in the IC50 values of compounds
5a–5g were very similar. Of these, compound 5g (n = 10) showed
the highest inhibitory activity against influenza H3N2 and H1N1,
9. Aisa, H. A.; Zhao, J. Y., Cao, L. H. [P] C. N. CN101857613A.
10. The MDDR is a database covering the patent literature, journals, meetings and
congresses that is produced by Symyx and Prous Science (http://
www.prous.com/index.html). The MDDR contains over 180,000 biologically
relevant compounds and well-defined derivatives, with approximately 10,000
added to the database every year.
11. Nilsson, J. W.; Thorstensson, F.; Kvarnstrom, I.; Oprea, T.; Samuelsson, B.;
Nilsson, I. J. Comb. Chem. 2001, 3, 546.
12. (a) Kuipers, W.; Kruse, C. G.; van Wijngaarden, I.; Standaar, P. J.; Tulp, M. T. M.;
Veldman, N.; Spek, A. L.; Ijzerman, A. P. J. Med. Chem. 1997, 40, 300; (b) Perez,
M.; Fourrier, C.; Sigogneau, I.; Pauwels, P. J.; Palmier, C.; John, G. W.; Valentin, J.
P.; 189 Halazy, S. J. Med. Chem. 1995, 38, 3602; (c) Zhao, S. H.; Miller, A. K.;
Berger, J.; Flippin, L. A. Tetrahedron Lett. 1996, 37, 4463.
with respective IC50 values of 0.97 and 0.4
smaller than those of Ribavirin and Oseltamivir. Unfortunately, 5g
showed the highest toxicity (TC50 = 27.1 M) among all of the syn-
lM, which were even
l
13. Li, X. Z.; Si, Z. X. Agrochemicals 2003, 1, 4.
thesized compounds. Compounds 5d and 5f showed moderate
activity against influenza A (H3N2 and H1N1). As shown in Figure
1, the inhibition induced by compounds 2g, 3g, 4g and 5g obviously
increased at concentrations of 0.15–1.37 lg/mL. The inhibition
ratios of compound 4g against influenza H3N2 were 50% and 75%,
14. Aisa, H. A.;Yong, J. P.; Lv, Q. Y.; Wu, T. Acta Crystallogr., Sect. E 2008, 64, o479.
General procedure for the preparation of rupestonic acid (compound 1): Dry
Artemisia rupestris L. was extracted with 95% ethanol, the extract was
evaporated under reduced pressure. Rupestonic acid (1) was isolated by
silica gel column chromatography using petroleum ether/ethyl acetate (10:1–
1:1) as the eluent, followed by recrystallization from acetone. Compound 1:
colorless crystal; mp 130–132 °C; IR (KBr): 3230, 2970, 2860, 1720, 1680, 1635,
respectively.
1415, 1390, 1238, 958 cmÀ1 1H NMR (400 MHz, CDCl3): d 0.65 (d, J = 7.2 Hz,
;
As can been seen from Table 1, most of the synthesized com-
pounds did not show obvious activity against influenza B. Com-
pound 5f presented the best activity against influenza B among
3H, CH3), 1.56–1.68 (m, 4H, 2CH2), 1.83 (s, 3H, CH3), 2.06–2.07 (m, 1H), 2.13–
2.16 (m, 1H), 2.45–2.46 (m, 1H), 2.63–2.64 (m, 1H), 2.82–2.86 (m, 1H), 2.88–
2.90 (m, 1H), 3.16–3.18 (m, 1H), 5.73 (s, 1H), 6.36 (s, 1H); 13C NMR (100 MHz,
CDCl3): d 208.6, 174.7, 171.6, 145.8, 138.0, 125.6, 46.1, 41.5, 38.5, 37.5, 36.4,
35.4, 31.7, 12.2, 8.1; ESI-MS (m/z, %): 519 ([2M+Na]+, 80), 497 ([2M+1]+, 100),
249 ([M+H]+, 60), 247 ([MÀH]+, 30).
all of the compounds, with an IC50 value of 3.2
smaller than that of Ribavirin (IC50 = 14.7 M).
lM, which was
l
In summary, a novel series of rupestonic acid derivatives was
synthesized, and the introduction of the piperazinyl and 1H-1,2,
4-triazolyl moieties to rupestonic acid using proper alkyl spacers
increased the anti-influenza activity of rupestonic acid. Com-
pounds 5a–5g, which contained 1H-1,2,4-triazolyl moieties, were
found to be the most potent compounds, whereas the bromoalkyl
rupestonates 2a–2g did not show significant activity. This study
also indicates that compounds 2g, 3g, 4g and 5g, which had ten-
CH2-unit spacers between the rupestonic acid and the amino func-
tional group, were the most potent against influenza H1N1 among
the tested compounds. The optimal spacer distance between
rupestonic acid and the piperazine or 1H-1,2,4-triazole moiety
remains to be determined. Compounds such as 4g and 5g were
potent against influenza H1N1, and they might be developed as
new lead anti-influenza virus compounds. Further studies of the
mechanism of action are underway.
15. Ma, Y. M.; Aisa, H. A.; Liao, L. X.; Srapil, E. B.; Zhang, T. Y.; Ito, Y. J. Chromatogr., A
2005, 1076, 198.
16. Lian, Z. B.; Huang, Y.; Cao, L. H. Chin. J. Applied Chem. 2003, 20, 288.
General synthetic procedure for the preparation of compounds 2a–2g: To a
solution of rupestonic acid (1, 100 mg, 0.4 mmol) in chloroform (10 mL),
aqueous 5% NaOH (1 mL) and triethylbenzyl ammonium chloride (TEBAC)
(128 mg, 0.4 mmol) were added at room temperature with stirring. When the
solid dissolved completely,
a solution of 1,x-dibromalkane (0.4 mmol) in
chloroform (5 mL) was added. The mixture was refluxed for 4–10 h. The
progress of the reaction was monitored by TLC. When the reaction was
complete, the reaction mixture was cooled to room temperature. The organic
layer was separated and washed with aqueous 5% NaOH and distilled water
and dried over anhydrous Na2SO4. The solvent was removed at reduced
pressure, and the residue was purified by a silica gel column eluted with
petroleum ether/ethyl acetate (5:1–2:1) to give the desired compounds 2a–2g
in 63–83% yields. Compound 2a: white solid; Rf (30% ethyl acetate /petroleum
ether) 0.52; mp 75–77 °C; y: 64%; IR (KBr): 2910, 2869, 1716, 1692, 1626,
1380, 1260, 1151, 960, 662, 560 cmÀ1 1H NMR (400 MHz, CDCl3): d 0.67 (d,
;
J = 7.2 Hz, 3H, CH3), 1.63–1.69 (m, 4H, 2CH2), 1.83 (s, 3H, CH3), 2.04–2.08 (m,
1H), 2.14–2.16 (m, 1H), 2.46–2.52 (m, 1H), 2.59–2.63 (m, 1H), 2.84–2.87 (m,
1H), 2.91–2.95 (m, 1H), 3.20 (s, 1H), 3.58 (t, J = 6.0 Hz, 2H, CH2Br), 4.50 (t,
J = 6.0 Hz, 2H, OCH2), 5.70 (s, 1H), 6.31 (s, 1H); 13C NMR (100 MHz, CDCl3): d
208.1, 174.0, 166.6, 146.0, 138.0, 124.1, 71.2, 46.1, 41.6, 38.5, 38.2, 36.8, 35.6,
31.9, 27.2, 12.2, 8.2; ESI-MS (m/z, %): 355 ([M+1], 98), 357 ([M+3], 100), 377
([M+Na], 25).
Acknowledgments
This work was financially supported by the High Tech Research
and Development Program of Xinjiang (No. 200910105), the China
National Funds for Distinguished Young Scientists (No. 30925045)
and the National Natural Science Foundation of China (No.
20872174). We would like to thank the members of Institute of
Medicinal Biotechnology, Academy of Medical Sciences & Peking
Union Medical College, for testing the anti-influenza activity of
the synthesized compounds.
17. Yu, L.; Cao, R. H.; Yi, W.; Yan, Q.; Chen, Z. Y.; Ma, L.; Peng, W. L.; Song, H. C.
Bioorg. Med. Chem. Lett. 2010, 20, 3254.
General synthetic procedure for the preparation of compounds 3a–3g, 4a–4g
and 5a–5g: A mixture of compound 2a–2g (1 mmol), K2CO3 (2 mmol) and 1-
phenylpiperazine (or 1-methylpiperazine or 1H-1,2,4-triazole) (1.1 mmol) in
anhydrous acetonitrile (10 mL) was refluxed for 10–15 h. After completion of
the reaction as indicated by TLC, the solution was cooled, filtered, and then
concentrated under reduced pressure. The residue was suspended in CH2Cl2
and washed with saturated NaHCO3 and brine, dried over anhydrous Na2SO4,
and then evaporated under vacuum to dryness. The residue was purified by
silica gel column chromatography using CHCl3/MeOH (10:1) as the eluent to
afford 3a–3g, 4a–4g and 5a–5g. Compound 3f: white solid; Rf (10% MeOH/
CHCl3) 0.52; mp 85–87 °C; y: 68%; IR (KBr): 2927, 2820, 1701, 1627, 1382,
Supplementary data
1232, 1145, 957, 759, 692, 524 cmÀ1 1H NMR (400 MHz, CDCl3): d 0.68 (d,
;
J = 7.2 Hz, 3H, CH3), 1.34–1.35 (m, 8H, 4CH2), 1.53–1.55 (m, 2H), 1.61–1.66 (m,
4H, 2CH2), 1.68–1.72 (m, 2H), 1.82 (s, 3H, CH3), 2.04–2.09 (m, 1H), 2.15–2.19
(m, 1H), 2.39 (t, J = 6.8 Hz, 2H, CH2), 2.43–2.51 (m, 1H), 2.51–2.60 (m, 1H),
2.60–2.65 (m, 4H, piperazine-H), 2.83–2.88 (m, 1H), 2.91–2.96 (m, 1H), 3.21 (s,
1H), 3.23–3.24 (m, 4H, piperazine-H), 4.18 (t, J = 6.6 Hz, 2H, OCH2), 5.63 (s, 1H),
6.23 (s, 1H), 6.85–7.30 (m, 5H, Ph-H); 13C NMR (100 MHz, CDCl3): d 208.2,
174.4, 167.2, 151.3, 146.8, 138.1, 129.3, 123.0, 120.0, 116.4, 65.1, 58.4, 50.0,
46.1, 46.0, 41.7, 38.5, 38.3, 37.1, 35.5, 31.9, 30.0, 29.1, 28.6, 26.4, 25.9, 12.3, 8.0;
ESI-MS (m/z, %): 521 ([M+1], 100), 522 ([M+2], 50).
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.056.
References and notes
1. Kim, C. U.; Lew, W.; Williams, M. A.; Wu, H.; Zhang, L.; Chen, X.; Escarpe, P. A.;
Mendel, D. B.; Laver, W. G.; Stevens, R. C. J. Med. Chem. 1998, 41, 2451.
2. Schmidt, A. C. Drugs 2004, 64, 2031.
3. Moscona, A. N. Engl. J. Med. 2005, 353, 1363.
4. Russell, R. J.; Haire, L. F.; Stevens, D. J.; Collins, P. J.; Lin, Y. P.; Blackburn, G. M.;
Hay, A. J.; Gamblin, S. J.; Skehel, J. J. Nature 2006, 443, 45.
5. Le, Q. M.; Kiso, M.; Someya, K.; Sakai, Y. T.; Nguyen, T. H.; Nguyen, K. H. L.;
Pham, N. D.; Ngyn, H. H.; Yamada, S.; Muramoto, Y.; Horimoto, T.; Takada, A.;
Goto, H.; Suzuki, T.; Suzuki, Y.; Kawaoka, Y. Nature 2005, 437, 1108.
18. The procedure for the anti-influenza virus assay: (1) Each compound was
dissolved in DMSO at an initial concentration of 1000
threefold successively to obtain 8 different concentrations (333.33, 111.11,
37.04, 12.35, 4.12, 1.37, 0.46 and 0.15, 0.05 g/mL, respectively) as stock
lg/mL and then diluted
l
solutions for the following experiments. (2) Madin-Darby canine kidney cells
(MDCK) were seeded in 96-well trays and cultured at 37 °C in a humidified CO2

J. Zhao, H. A. Aisa / Bioorg. Med. Chem. Lett. 22 (2012) 2321–2325
2325
incubator (95% air, 5%CO2) for 24 h. Then, the cells were infected with influenza
A virus with 10À4 [316 times the 50% tissue culture infective dose (TCID50)] and
with influenza B virus with 1/210À2 [158 times of the 50% tissue culture
infective dose (TCID50)], respectively. All of infected tissue culture plates (96
wells) were incubated at 37 °C for 2 h, and then the medium was removed.
incubated again for 36 h at 37 °C. Then, the inhibition of the virus-induced
cytopathic effect (CPE) for each sample was recorded relative to the cell control
and the virus control. The 50% cell-inhibitory concentration (IC50) values of
active compounds were calculated accordingly. The inhibitory potentials of the
rupestonic acid derivatives were comparable to those of the parent compound
and the commercial drugs Ribavirin (RBV) and Oseltamivir.
Subsequently, 100
lL aliquots of the solutions with different concentrations of
each compound were added to the wells (one per well), and the plates were