Synthesis of rupestonic acid amide derivatives and their in vitro activity against type A3 and B flu virus and herpes simplex I and II

Article abstract of DOI:10.1007/s10600-008-9050-y

Derivatives of rupestonic acid (5a-e) were synthesized and evaluated preliminarily at the National Center for Drug Screening (PRC) for antiviral activity against type A₃ and B flu virus and HSV-I and HSV-II in order to improve the biological ac

Full text of DOI:10.1007/s10600-008-9050-y

Chemistry of Natural Compounds, Vol. 44, No. 3, 2008
SYNTHESIS OF RUPESTONIC ACID AMIDE DERIVATIVES
AND THEIR
ACTIVITY AGAINST TYPE A AND
3
in vitro
B FLU VIRUS AND HERPES SIMPLEX I AND II
J.-P. Yong1,2 and H. A. Aisa1*
UDC 547.972
Derivatives of rupestonic acid (5a-e) were synthesized and evaluated preliminarily at the National Center
for Drug Screening (PRC)for antiviral activity against type A and B flu virus and HSV-I and HSV-II in order
3
to improve the biological activity of rupestonic acid. It was found that compound 5b was more active than
rupestonic acid against type A flu virus.
3
Key words: synthesis, rupestonic acid, amide derivatives, flu virus, herpes simplex virus, activity.
The plant
L. (Chinese name Yizhihao) is a traditional Chinese medicinal herb that has been used
Artemisia rupestris
since antiquity in Uigur folk medicine. It is known for its effective antiallergy [1], antitumor [2], anti-inflammatory, and
antibacterial activities and as an antitoxin [3, 4].
(5 ,8 )-2-(3,8-Dimethyl-2-oxo-1,2,4,5,6,7,8,8α-octahydroazulen-5-yl)acrylic acid (called rupestonic acid) is a
R R
multifunctional sesquiterpene that was isolated from A. rupestris. It was noted [5] that certain sesquiterpenes exhibit a powerful
cytotoxic activity toward cancer cell lines P-388, A549, and HT-26. It was reported [6] that a sesquiterpene hydroquinone and
its acetate derivative exhibit high activity against tumor cell line P-388 and flu strain PR-8. Investigations of the activity of
rupestonic acid against viruses HSV-1 and HSV-2 and flu viruses A and B showed that it is highly active against type B flu
3
virus (TC = 258.7 μg/mL, IC = 28.7 μg/mL). Due to its unique structure, we attempted to modify it in order to prepare
50
50
compounds with higher biological activity.
Herein we describe the synthesis of intermediates 3a-e through the reaction of amines 2 with N-Fmoc-L-phenylalanine
and N-Fmoc-L-Leu-OH in the presence of POCl and imidazole.
3
R
NH
2
R
OH
imidazole,POCl / Py
3
HN
R
1
FmocHN
FmocHN
+
O
0°C - r.t.
O
1a, b
2a, b
3a - e
EtNH /DCM
R
1
2
r.t.
H
H
R
O
R
O
DCC, HOBt/DMAP
THF, 0°C~ r.t.
HN
R
1
+
H N
2
NH
R
1
COHN
5a - e
O
COOH
O
4a - e
1a: R = C6H5CH2-; 1b: R = CH2CH(CH3)2; 2a: R1 = CH3; 2b: R1 = OCH3
3, 4, 5a: R = C6H5-CH2; R1 = CH3; 3, 4, 5b: R = C6H5-CH2, R1 = OCH3; 3, 4, 5c: R = C6H5CH2, R1 = H
3, 4, 5d: R = CH2CH(CH3)2, R1 = CH3; 3, 4, 5e: R = CH2CH(CH3)2, R1 = OCH3
Scheme 1
1) Xinjiang Technical Institute of Physics and Chemistry, Academy of Sciences of the People's Republic of China,
830011, Urumchi, PRC, e-mail: haji@ms.xjb.ac.cn; 2) Graduate School of the Academyof Sciences of the PRC, 100039, PRC,
Beijing. Translated from Khimiya Prirodnykh Soedinenii, No. 3, pp. 247-249, May-June, 2008. Original article submitted
March 7, 2008.
0009-3130/08/4403-0311 ©2008 Springer Science+Business Media, Inc.
311

TABLE 1. Antiviral Activity of 5b, Oseltamivir, and Ribavirin Against Flu Viruses A3 and B
Against flu virus A3
bIC50 (μg/mL)
Against flu virus B
Compound
aTC50 (μg/mL)
cSI
bIC50 (μg/mL)
cSI
c
f
Rupestonic acid
5b
Oseltamivir
Ribavirin
259
53.4
>500
384
-
-
28.7
9.0
c
f
28.7
2.1
0.4
1.9
>242.7
936.6
-
-
c
f
-
-
2.9
133.8
______
a50% cytotoxic concentration; b50% virus-inhibiting concentration determined by CPE of the inhibiting sample; cselectivity
d
e
f
index (TC50/IC50); starting material (rupestonic acid, RA); inactive at 50% cytostatic concentration; SI cannot be calculated
because the highest tested concentration was <IC50.
Then, removal of the Fmoc protecting group from 3a-e in the presence of ethanolamine and CH Cl (DCM) produced
2
2
intermediates 4a-e. The target compounds 5a-e were synthesized by reaction of 4a-e with rupestonic acid in the presence of
dicyclohexylcarbodiimide (DCC), 1-hydroxybenzotriazole (HOBt), and 4-dimethylaminopyridine (DMAP). Scheme 1 shows
the synthetic route.
EXPERIMENTAL
Meltingpointsweredeterminedon a YanacoMP-300 apparatus equipped with amicroscopeandareuncorrected. PMR
spectra in CDCl were recorded on a Varian Inova-400 spectrometer using tetramethylsilane (TMS) as an internal standard.
3
ESI-MS spectra were obtained in an HP1100LC/MS spectrometer. Rupestonic acid was isolated from A. rupestris L. (HPLC
purity >98%). DCC, HOBt, and DMAP were purchased from ShangHai reagent company (PRC); N-Fmoc-L-Leu-OH and
N-Fmoc-L-phenylalanine, from ShangHai Qiude Biochemical Engineering Co. Ltd., PRC. Other commercially available
reagents were used without further purification. We used THF that was freshlydistilled over sodium and benzophenone; DCM,
over CaH . Viruses HSV-1 (VR733) and HSV-II (SAV) were obtained from ATCC; flu viruses, innoculated into chicken
2
embryos in 2006 and stored at 80°C at Beijing Institute of Virology immediately before use in this test.
General Method for Synthesizing Intermediates 4. L-Phenylalanine-p-methoxyanilide (4b). A solution of
p-methoxyaniline (2.1 mmol), N-Fmoc-L-phenylalanine (2.1 mmol), and imidazole (0.29 g, 3.4 mmol) in pyridine (10 mL) was
treated dropwise with POCl (3.4 mmol) at 0°C under N2 over 1 h [7]. The mixture was stirred at room temperature for 4 h,
3
poured into NaHCO solution (100 mL), and extracted with ethylacetate (3 × 50 mL). The combined organic extract was
3
washed with CuSO solution (30 mL) and brine and dried over MgSO . Solvent was removed. The crude product was purified
4
4
by flash chromatography over a column of silica gel with elution by petroleum ether:ethylacetate (5:1-2:1). Fractions with
similar R values were combined to afford N-Fmoc-L-phenylalanine-p-methoxyanilide, 3b, white solid, yield 76%, mp 170-
f
172°C.
PMR spectrum (600 MHz, CDCl , δ, ppm, J/Hz): 0.99-1.01 (6H, d, J = 8, 2 × CH ), 1.64 (1H, m), 1.73-1.82 (2H, m,
3
3
CH ), 3.82 (3H, s, OCH ), 4.23-4.26 (2H, m, O–CH –C=O), 4.49 (1H, m), 5.24-5.26 (1H, m), 6.87-6.89 (2H, d, J = 8, Ph–H),
2
3
2
7.29-7.43 (6H, m, Ph–H), 7.59-7.61 (2H, d, J = 8, Ph–H), 7.79-7.81 (2H, m, Ph–H), 7.83-7.85 (2H, br.s, NH–C=O). Other
intermediates 3a and 3c-e were synthesized analogously to 3b and were used without further purification. N-Fmoc-
L-phenylalanine-p-methoxyaniline (3b) (0.6 g) was dissolved in DCM containing ethanolamine (10 mL, 30%), stirred for 4 h
at room temperature, diluted with DCM (20 mL), and washed with water (3 × 10 mL) and brine (20 mL). The organic layer
was dried over MgSO . Solvent was removed. The crude product was purified byflash chromatographyover a column of silica
4
gel with elution by petroluem ether:ethylacetate (5:1-2:1). Fractions with similar R values were combined to afford
f
L-phenylalanine-p-methoxyanilide (4b), yellow oil. Products 4a and 4c-e were synthesized by the same method as 4b.
L-Phenylalanine-p-methoxyanilide (4b). Light-yellow oil, yield 65.8%. PMR spectrum (CDCl , δ, ppm): 1.91 (2H,
3
br.s, NH ), 2.76-2.82 (1H, m), 3.23 (H, s, CH ), 3.35-3.38 (1H, m), 7.04-7.25 (7H, m), 7.52-7.55 (2H, m), 9.26 (1H, s, NHCO).
2
3
+
IR spectrum (ν, cm−1): 3296, 2954, 1674, 1605, 1512, 1246, 829. ESI-MS (m/z, %): 255 (100) [M + 1] .
312

L-Phenylalanine-p-methylanilide (4a). Light-yellow oil, yield 75.6%. PMR spectrum (CDCl , δ, ppm): 1.65 (2H,
3
br.s, NH ), 2.76-2.82 (1H, m), 3.35-3.40 (1H, m), 3.73-3.76 (1H, m), 3.80 (3H, s, OCH ), 6.86-6.89 (2H, m), 7.25-7.27 (3H,
2
3
m), 7.32-7.36 (2H, m), 7.49-7.52 (2H, m), 9.24 (1H, s, NHCO). IR spectrum (ν, cm−1): 3290, 2956, 1666, 1605, 1512, 1246,
+
829. ESI-MS (m/z, %): 271 (100) [M + 1] .
L-Phenylalanineanilide (4c). Light-yellow oil, yield 78.8%. PMR spectrum (CDCl , δ, ppm): 1.86 (2H, br.s, NH ),
3
2
2.74-2.80 (1H, m), 3.33-3.38 (1H, m), 3.74 (1H, m), 7.08-7.62 (10H, m), 9.42 (1H, s, NHCO). IR spectrum (ν, cm−1): 3296,
2954, 1674, 1605, 1512, 1246, 768. ESI-MS (m/z, %): 241 (100) [M + 1] .
+
L-Leucine-p-methylanilide (4d). Light-yellow oil, yield 70.8%. PMR spectrum (CDCl , δ, ppm): 0.98-1.00 (6H, m,
3
2CH ), 1.42-1.47 (1H, m), 1.76-1.78 (2H, br.s, NH ), 1.79-1.86 (2H, m, CH ), 2.33 (3H, s, CH ), 3.53-3.55 (1H, m), 7.13-7.15
3
2
2
3
(2H, m), 7.49-7.50 (2H, m), 9.43 (1H, s, NHCO). IR spectrum (ν, cm−1): 3296, 2954, 1674, 1605, 1512, 1246, 831.
+
ESI-MS (m/z, %): 221 (100) [M + 1] .
L-Leucine-p-methoxyanilide (4e). Light-yellow oil, yield 76.6%. PMR spectrum (CDCl , δ, ppm): 0.91-0.94 (6H,
3
m, 2CH ), 1.43-1.46 (1H, m), 1.75-1.81 (2H, m, CH ), 1.86-2.01 (2H, br.s, NH ), 3.43 (1H, m), 3.73 (3H, s, CH ), 6.81-
3
2
2
3
6.83 (2H, m), 7.46-7.49 (2H, m), 9.37 (1H, s, NHCO). IR spectrum (ν, cm−1): 3296, 3954, 1668, 1605, 1512, 1246, 829.
+
ESI-MS (m/z, %): 237 (100) [M + 1] .
General Method for Synthesizing Rupestonic Acid Derivatives (5) [8]. Rupestonic acid (0.124 g, 0.5 mmol), DCC
(0.11 g, 0.55 mmol), and THF (dry, 5 mL) were placed in a 25-mL one-necked round-bottomed flask and stirred on an ice bath
for ~10 min. HOBt (0.08 g, 0.6 mmol) and DMAP (0.07 g, 0.55 mmol) in THF (2 mL) were added using a syringe. The
mixture was stirred on an ice bath for 30 min, treated with 4 (0.6 mmol), and stirred at 0°C for 30 min and then at room
temperature for 8 h. The completion of the reaction was monitored using TLC. The solvent was removed in vacuo. The solid
was chromatographed over a column of silica gel with elution bypetroleum ether:ethylacetate (5:1-2:1). Fractions with similar
R values were combined to afford 5a-e.
f
Amide 5a: white compound, yield 55.8%, mp 50-52°C. PMR spectrum (600 MHz, CDCl , δ, ppm, J/Hz): 0.62 (3H,
3
d, J = 7.2, CH ), 1.58 (3H, s, CH ), 1.72-1.75 (1H, s), 1.78-1.82 (2H, m), 2.01-2.05 (1H, d, J = 16), 2.11-2.12 (1H, m), 2.27-2.29
3
3
(1H, m), 2.38-2.43 (1H, m), 2.54-2.56 (1H, m), 2.76 (1H, m), 2.86-2.89 (1H, m), 3.12-3.13 (1H, m), 3.15-3.27 (2H, m), 4.09
(3H, s, OCH ), 4.76-4.82 (1H, m), 5.36 (1H, s), 5.52 (1H, s), 6.63-6.65 (1H, d, J = 8, NHCO), 7.07-7.33 (9H, m, Ph-H), 7.52
3
(1H, s, CO–NH–Ph). IR spectrum (ν, cm−1): 3431, 3304, 3066, 2957, 1697, 1647, 1608, 1541, 1514, 1249, 817. ESI-MS (m/z,
+
%): 501 (100) [M + 1] .
Amide 5b: white compound, yield 65.8%, mp 78-80°C. PMR spectrum (CDCl , δ, ppm, J/Hz): 0.62 (3H, d, J = 7.2,
3
CH ), 1.58 (3H, s, CH ), 1.72-1.75 (1H, s), 1.78-1.82 (2H, m), 2.01-2.05 (1H, d, J = 16), 2.11-2.12 (1H, m), 2.27-2.29 (1H, m),
3
3
2.30 (3H, s, CH ), 2.38-2.43 (1H, m), 2.54-2.56 (1H, m), 2.76 (1H, m), 2.86-2.89 (1H, m), 3.12-3.13 (1H, m), 3.15-3.27 (2H,
3
m), 4.76-4.82 (1H, m), 5.36 (1H, s), 5.52 (1H, s), 6.63-6.65 (1H, d, J = 8, NHCO), 7.07-7.33 (9H, m, Ph–H), 7.52 (1H, s,
CO–NH–Ph). IR spectrum (ν, cm−1): 3431, 3304, 3066, 2957, 1697, 1647, 1608, 1541, 1514, 1249, 817. ESI-MS (m/z %):
+
507 (100) [M + 23] .
Amide 5c: white compound, yield 68.8%, mp 89-90°C. PMR spectrum (600 MHz, CDCl , δ, ppm, J/Hz): 0.62 (3H,
3
d, J = 7.1, CH ), 1.58 (3H, s, CH ), 1.72-1.75 (1H, s), 1.78-1.82 (2H, m), 2.01-2.05 (1H, d, J = 8), 2.11-2.12 (1H, m), 2.27-2.32
3
3
(1H, m), 2.38-2.43 (1H, m), 2.54-2.56 (1H, m), 2.76 (1H, m), 2.86-2.89 (1H, m), 3.12-3.13 (1H, m), 3.15-3.27 (2H, m), 4.82-
4.85 (1H, m), 5.36 (1H, s), 5.52 (1H, s), 6.66-6.68 (1H, d, J = 8, NHCO), 7.07-7.35 (10H, m, Ph–H), 7.84 (1H, s, CO–NH–Ph).
IR spectrum (ν, cm−1): 3429, 3138, 3080, 2957, 1695, 1647, 1609, 1545, 1510, 1246, 1172, 829. ESI-MS (m/z, %): 493 (100)
+
+
[M + 23] , 471 (12) [M + 1] .
Amide 5d: white compound, yield 76.8%, mp 84-86°C. PMR spectrum (600 MHz, CDCl , δ, ppm, J/Hz): 0.61-0.63
3
(3H, d, J = 7.2, CH ), 0.81-0.87 (2H, m, CH ), 0.89-0.98 (6H, m, 2CH ), 1.26-1.30 (1H, m), 1.57 (3H, s, CH ), 1.72-1.82 (4H,
3
2
3
3
m), 1.99-2.01 (1H, d, J = 12), 2.09 (1H, m), 2.27 (3H, s, Ph–CH ), 2.38-2.45 (1H, m), 2.51-2.57 (1H, m), 2.75-2.80 (1H, m),
3
2.88-2.90 (1H, m), 3.06 (1H, br.s), 4.71-4.72 (1H, m), 5.37 (1H, s), 5.62 (1H, s), 6.62 (1H, d, J = 7.8, NH–C=O), 7.05 (2H, d,
J = 7.8, Ph–H), 7.36 (2H, d, J = 8.2, Ph–H), 8.59 (1H, s, O=C–NH–Ph). IR spectrum (ν, cm−1): 3431, 3304, 3066, 2957, 1697,
+
+
1647, 1608, 1541, 1514, 1249, 817. ESI-MS (m/z, %): 451 (42) [M + 1] , 473 (100) [M + 23] .
Amide 5e: white compound, yield 72.4%, mp 89-91°C. PMR spectrum (600 MHz, CDCl , δ, ppm, J/Hz): 0.62 (3H,
3
d, J = 7.2, CH ), 0.63-0.79 (2H, m), 0.91-1.02 (6H, m, 2CH ), 1.24-1.28 (1H, m), 1.54 (3H, s, CH ), 1.62-1.81 (4H, m, 2CH ),
3
3
3
2
1.99-2.09 (2H, m), 2.42-2.58 (2H, m), 2.75-2.90 (2H, m), 3.07 (1H, m), 3.75 (3H, s, CH ), 4.69-4.74 (1H, m), 5.40 (1H, s), 5.64
3
(1H, s), 6.61-6.63 (1H, d, J = 8, NH–C=O), 6.76-6.78 (2H, d, J = 8, Ph–H), 7.36-7.39 (2H, d, J = 12, Ph–H), 8.57 (1H, s,
313

O=C–NH–Ph). IR spectrum (ν, cm−1): 3293, 3138, 3080, 2957, 1695, 1647, 1609, 1545, 1510, 1246, 1172, 829. ESI-MS (m/z,
+
%): 489 (100) [M + 23] .
Biological Studies. Theantiviral activityofrupestonic acid derivatives 5a-e against virusesA3/Beijing/90/15(H3N2),
B/Beijing/97/13, HSV-1 (VR733), and HSV-2 (SAV) was evaluated preliminarily in vitro at the National Center for Drug
Screening (PRC). The inhibiting properties of the compounds were comparable with those of rupestonic acid and commercial
preparations such as ribavirin (RBV), acyclovir (ACV), and oseltamivir. The results showed that not one ofthe compounds 5a-e
was an inhibitor of HSV-I and HSV-II. However, 5b showed higher activity against flu virus A than rupestonic acid
3
(Table 1).
ACKNOWLEDGMENT
The work was supported financially by the Scientific-Research Plan of the Xinjiang High Technologies (PRC)
(No. 200515123) and the Xinjiang Foundation for Key Laboratories of Plant Resources and the Chemistry of Natural
Compounds. We thank staff members of the National Center for Drug Screening (PRC) for investigating the biological activity
of the synthesized compounds.
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