AU2014100709A4 - In Vitro Anti-influenza Virus Activities of a New Lignan Glycoside from the Latex of Calotropis gigantea - Google Patents

Abstract

In Vitro Anti-influenza Virus Activities of a New Lignan Glycoside from the Latex of Calotropis gigantea The present invention provides a novel lignan glycoside compound represented by formula (I) and the use thereof as an anti-influenza agent. A method of treating influenza comprising administering an effective amount of a lignan glycoside compound to a subject in need thereof is also provided. The lignan glycoside compound comprises at least one vanilloyl moiety. Further, a method of isolating (+)-pinoresinol 4-0-[6"-0 vanilloyl]-p8-D-glucopyranosideas as represented by formula (III) is also provided. <DocRef#00125742-RM > M006.03 1.DRF 24 Spec draft v.1

Description

In Vitro Anti-influenza Virus Activities of a New Lignan Glycoside from the Latex of Calotropis gigantea CROSS-REFERENCE TO RELATED APPLICATION [0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application having Serial No. 61/923,229 filed Jan 3, 2014, which is hereby incorporated by reference herein in its entirety. FIELD OF INVENTION [0002] This invention relates to novel lignan glycoside and the use thereof as an anti influenza agent. BACKGROUND OF INVENTION [0003] Influenza virus causes many upper respiratory tract infection diseases. It is transmitted easily from person to person via air and spreads rapidly in seasonal epidemics causing numerous people death from severe complications in pandemic years worldwide as reported by World Health Organization (WHO) in 2013. The latest global pandemic caused by HINI virus, characterized by a unique triple-reassortant gene segments of bird, swine and human influenza viruses, has resulted in more than 18,000 human deaths since its appearance in April 2009. More recently, the new avian-origin influenza A (H7N9) virus has caused 111 cases of human infection in China as of May 10, 2013 (Gao H.N., et al. 2013), and some of the patients have died from severe pneumonia brought on by this virus. Moreover, the rate of emergence of viruses resistant to available antiviral drugs including M2 blockers (such as amantadine and rimantadine) and neuraminidase (NA) inhibitors (such as oseltamivir and zanamivir) has been increasing globally and greatly decreasing their effectiveness (Du J. et al., 2012; Deyde V. M. et al., 2007). It is urgently needed to develop safe and effective new antiviral drugs to combat viral infection either for therapeutic or prophylactic purposes. 1 M006.03 1.DRF Spec As Filed SUMMARY OF INVENTION [0004] In the light of the foregoing background, many researchers have tried and keep trying to find new effective antiviral remedies, especially, from the natural resources. (Yingsakmongkon S. et al., 2008; Sriwilaijareon N. et al., 2012; Kiyohara H., et al., 2012), and it is an object of the present invention to provide an alternate antiviral drug. [0005] Accordingly, the present invention, in one aspect, is a lignan glycoside compound represented by formula (I). OR HO H H OH ONe (I) [0006] In an exemplary embodiment of the present invention, in the lignan glycoside compound of formula (I), R is represented by vanilloyl group represented by formula (II), and the resulting compound is (+)-pinoresinol 4-0-[6"-O-vanilloyl]-#-D-glucopyranoside and represented by formula (III). 0OH M0. OM2 o (II) M006.031.DRF 2 Spec draft v.1 OH 4 e OM HO 5,.40 HH OH (III) [0007] In another exemplary embodiment, in the lignan glycoside compound of formula (I), R is represented by H; said compound is (+)-pinoresinol 4-O-p-D-gluco-pyranoside. [0008] According to another aspect of the present invention, a method of treating influenza comprising administering an effective amount of a lignan glycoside compound to a subject in need thereof The lignan glycoside compound comprises at least one vanilloyl moiety. [0009] In an exemplary embodiment, the lignan glycoside compound is (+)-pinoresinol 4-O-[6"-O-vanilloyl]-p8-D-glucopyranosideas as represented by formula (III). In a further exemplary embodiment, the influenza is caused by an influenza virus selected from a group consisting of influenza A strain A/Aichi/2/68, influenza A strain A/PR/8/34, influenza A strain A/FM/1/47 and influenza B strain Inf B/Lee/i 940. [0010] In a further aspect of the present invention, a method of isolating (+)-pinoresinol 4-O-[6"-O-vanilloyl]-p8-D-glucopyranosideas as represented by formula (III) comprising the steps of: [0011] (a) adding 95% ethanol to latex of Calotropis gigantea produce a filterable precipitate mixture; [0012] (b) the mixture was then sonicated at room temperature then centrifuged; M006.03 1.DRF 3 Spec draft v.1 [0013] (c) the supernatant from the centrifuge product of step (b) was evaporated under reduced pressure to obtain a light yellowish residue; [0014] (d) the residue was suspended in H 2 0 and subjected to liquid-liquid partition by adding EtOAc; [0015] (e) the residue of EtOAc layer was subjected to silica gel CC eluted with the gradient system of CHCl 3 -MeOH-H 2 0 to obtain four fractions of Fr. 1 to Fr.4; [0016] (f) the third fraction, Fr.3, from step (e) was chromatographed over MCI-gel CHP 20P CC eluted with aqueous MeOH to obtain six sub-fractions of Fr.3-1 to Fr.3-6; [0017] (g) the fourth sub-fraction, Fr.3-4, from step (f) was loaded to Bondapak Waters® ODS CC using the gradient of MeOH-H 2 0 to obtain seven sub-fractions of Fr.3-4-1 to Fr.3-4-7; [0018] (h) the fifth sub-fraction, Fr.3-4-5, from step (g) was purified by Silica gel 60 CC eluted with the gradient system of CHCl 3 -MeOH-H 2 0 to obtain eleven sub-fractions of Fr.3-4-5-1 to Fr.3-4-5-11; and [0019] (i) the fifth sub-fraction, Fr.3-4-5-5, from step (h) was further purified by MCI-gel CHP 20P CC using aqueous MeOH to obtain said compound. BRIEF DESCRIPTION OF FIGURES [0020] Fig. 1A shows the chemical structure of Compound 1 and Fig. 1B shows the key HMBC (H to C) and 1

H-

1 H COSY correlations of compound 1. [0021] Fig. 2 shows the chemical structures of Compounds 1-5. [0022] Fig. 3 shows the mild alkaline hydrolysis of Compound 1. [0023] Figs. 4A and 4B respectively illustrates the UHIPLC chromatograms and the MS spectra of Compound 1, (+)-pinoresinol-4-O-p8-D-glucopyranoside (Compound 4) and the compounds from the reaction mixture. M006.03 1.DRF 4 Spec draft v.1 [0024] Fig. 5 shows the IC 50 (tM) of Compound 1 and ribavirin against human influenza viruses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0025] As used herein and in the claims, "comprising" means including the following elements but not excluding others. [0026] Calotropis gigantea (Asclepiadaceae) is a shrub common in Eastern Asia and Southeast Asia. The barks of this plant are used traditionally in Chinese folk medicine for the treatments of neurodermatitis and syphilis while the leaves are used as a poultice (FOC., 2013). The latex of this plant has been used as an insecticidal agent (Bhaskara and Seshadri, 1943), and as a purgative and a local irritant in indigenous medicine in India (Pari K. et al., 1998). A fascinating series of bioactive secondary metabolites, e.g., cardiotonic steroids, triterpene alcohols, alkaloids and flavonoids has been isolated from different parts of Calotropis plants and showed various pharmacological properties such as analgesic, hepatoprotective, sedative, anti-inflammatory, anti-diarrheal, anti-asthmatic and anticancer activities (Pari K. et al., 1998; Lhinhatrakul and Sutthivaiyakit, 2006; Silva M. C. C. et al., 2010). In this present study, we successfully isolated a new lignan glycoside (Compound 1), together with two known phenolic compounds (Compounds 2 and 3) from the latex of Calotropis gigantea for the first time. Their isolation, structural elucidation and in vitro anti-influenza virus activities were discussed in this application. [0027] The following preparations and examples are given to enable those skilled in the art to more clearly understand and to practice the present invention. They should not be considered as limiting the scope of the invention, but merely as being illustrative and representative thereof [0028] 1. Materials and Methods [0029] 1.1 General experimental procedures [0030] Optical rotation was measured on Rudolph Autopol I automatic polarimeter (USA). UV spectrum was performed on a Beckman Coulter DU800 spectrophotometer M006.03 1.DRF 5 Spec draft v.1 (USA). IR spectrum was obtained on a PerkinElmer Spectrum One Fourier transform infrared (FTIR) spectrometer (KBr). The 1H, 1C and 2D NMR experiments were conducted on a Bruker Ascend® 600 NMR spectrometer (600 MHz for 1H and 150 MHz for 13 C) using the chemical shifts of CD 3 0D ( 1 H, 6H = 3.310; 13 C, 6C = 49.150) as the references. Chemical shifts were expressed in 6 (ppm) and coupling constants ( J ) are given in Hz. ESI-TOF-MS spectra were measured on an Agilent 6230 Accurate mass Time-of-Flight (TOF) mass spectrometer (USA). UHIPLC analyses were carried out on an Agilent Technologies 1290 Infinity liquid chromatography system using an ACQUITY UPLC BEH C 18 column (1.7 ptm, 2.1 x 100 mm, Waters*, Ireland). HPLC analysis was done on an Agilent 1100 series HPLC system using an Alltima* C 18 column (5p.im, 4.6 x 250 mm, Alltech, Grace, USA). Preparative HPLC was conducted on LabAlliance with VisionHT* C 18 Polar column (5p.m, 22 x 250 mm, Grace, USA). Medium Pressure Liquid Chromatography (MPLC, Sepacore, Buchi, Switzerland) was done using a Siliabond* C 18 ODS (40-63 ptm, Silicycle, Canada) column (36 x 460 mm). Column chromatographies (CC) were performed using silica gel (40-63 ptm, Grace, USA), silica gel 60 (50-55p.m, Merck, Germany), MCI-gel CHIP 20P (75-150p.m, Mitsubishi Chemical Co. Ltd., Japan) and Bondapak Waters® ODS (55-105 ptm, Waters, USA). Thin-layer chromatography (TLC) was conducted on pre-coated HPTLC, TLC Kieselgel 60 F 25 4 plates or TLC silica gel 60 RP-18 F 2 5 4 s (200 pim thick, Merck KGaA, Germany) and the spots were visualized under ultraviolet light (UV, wavelength 254 nm) and by spraying with 5% sulfuric acid in 95% EtOH, followed by heating at 110 C. [0031] 1.2. Reagents and compounds [0032] Absolute ethanol (AR grade) was purchased from Merck (Germany). Chloroform and ethyl acetate (AR grade) were bought from RCI Labscan Limited (Bangkok, Thailand). Methanol and acetronitrile (HPLC grade) were purchased from Tedia (USA).

CD

3 0D was purchased from Sigma Aldrich (USA). Sodium methoxide was a product of Sigma-Aldrich (USA). The purities of all isolated compounds for bioassay were more than 95% determined by UIHPLC analysis. [0033] 1.3. Plant material M006.03 1.DRF 6 Spec draft v.1 [0034] The latex of Calotropis gigantea (CGLA) was collected in Lampang, Thailand, during the period between August and October 2011. It was preserved by adding 10% EtOH and then kept in -40 C freezer. The frozen CGLA was defrozen overnight at 4'C, and was allowed to stand at room temperature for 2 h before isolation. The herbarium specimen was from a shrub. A voucher specimen (No. SKLO07) was deposited at State Key Laboratory of Quality Research in Chinese Medicine, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology. [003 5] 1.4. Extraction and isolation [0036] 95% ethanol was added to CGLA (1 L) to produce a filterable precipitate (Bhaskara and Seshadri, 1943). The mixture was sonicated at room temperature and then centrifuged. The supernatant was evaporated under reduced pressure to result in a light yellowish residue (85.5 g). The residue was suspended in H 2 0 and then subjected to liquid-liquid partition by adding EtOAc. The residue (6.3 g) of EtOAc layer was subjected to silica gel CC eluted with the gradient system of CHCl 3 -MeOH-H 2 0 (from 10:0:0 to 6:4:1) to obtain 4 fractions (Fr.1 to 4). Fr.3 (1.2 g) was chromatographed over MCI-gel CHIP 20P CC eluted with aqueous MeOH (30 % to 100%) to obtain 6 fractions (Fr.3-1 to Fr.3-6). Fr.3-4 (300 mg) was loaded to Bondapak Waters® ODS CC using the gradient of MeOH-H 2 0 (50:50 to 100:0) to give 7 fractions (Fr.3-4-1 to Fr.3-4-7). Fr.3-4 5 (76.8 mg) was purified by silica gel 60 CC eluted with the gradient system of CHCl 3 MeOH-H 2 0 (from 10:0:0 to 7:3:0.5) to obtain 11 fractions (Fr.3-4-5-1 to Fr.3-4-5-11). Finally, Fr.3-4-5-5 (20.1 mg) was further purified by MCI-gel CHiP 20P CC using aqueous MeOH (30 % - 100 %) to yield Compound 1 (5.0 mg) eluted at 80% aqueous MeOH. [0037] Fr.4 (0.7 g) was separated by MCI-gel CHP 20P CC eluted with MeOH-H 2 0 (50:50 to 100:0) to obtain 13 fractions (Fr4-1 to Fr4-13). Fr.4-3 (67mg) was subjected to silica gel 60 CC eluted with the gradient of CHCl 3 -MeOH-H 2 0 (from 10:0:0 to 7:3:0.5) to give 3 fractions (Fr.4-1 to Fr.4-3). Fr.4-2 (8 mg) was further purified by preparative HPLC using 35% aqueous MeOH isocratic as a mobile phase to afford Compound 2 (2 mg) and Compound 3 (3 mg). M006.03 1.DRF 7 Spec draft v.1 [0038] To obtain Compound 1 in a larger amount, CGLA (3 L) was isolated with similar protocols of 1L-CGLA isolation to obtain EtOH total extract and the residue from EtOAc layer 240.0 g and 20.7 g, respectively. The residue of EtOAc layer (20.7 mg) was further subjected over silica gel CC eluted with the gradient of CHCl 3 -MeOH-H 2 0 (10:0:0 to 6:4:1) to obtain 8 fractions (Fr.A to H). Fr G (3.0 g) was purified by MPLC C 18 Siliabond* ODS eluted within one hour by using 20-80% MeOH in water as a mobile phase to obtain 7 fractions (Fr.G1 to Fr.G7). Fr.G6 (2.0 g) was purified by MPLC again using the gradient system of 30-60% aqueous MeOH eluted within 60 minutes to furnish 3 fractions (Fr.G6-1 to Fr.G6-3). Fr. G6-1 (659 mg) was further purified by MPLC using 40-60% aqueous MeOH to elute within 60 minutes to furnish Compound 1 (127 mg). [0039] 1.5. Mild alkaline hydrolysis of Compound 1 [0040] A solution of Compound 1 (0.2 mg, 0.3 ptmol) in MeOH (1 ml) was hydrolyzed with sodium methoxide (NaOMe, 1.6 mg, 30 ptmol) for 48 h at room temperature (r.t.) and the hydrolysis reaction is illustrated in Fig. 3. The chemical reaction was terminated by adding formic acid after two products, Compounds la and lb (the formulae of Compounds la and lb are shown in Fig. 3) were clearly detected by TLC analysis. The reaction mixture was then subjected to UHPLC/ ESI-TOF-MS (positive mode) analysis to confirm reaction products of Compounds la and lb (Fig. 4). Compound la was identified by comparing its UHIPLC retention time (6.862 min) and ESI-TOF-MS data ([M+Na]f at m z 543.1830 and [M+NH 4 ]f at m z 538.2274) with those of the reference compound (+)-pinoresinol-4-O-p8-D-glucopyranoside (Compound 4) (retention time at 6.858 min, ESI-TOF-MS data of [M+Na]f at m z 543.1835 and [M+NH4]f at m z 538.2299, calculated for C 2 6

H

32 0 1 Na, [M+Na]f at m z 543.1837 and [M+NH4]f at m z 538.2283) while Compound lb was identified as methyl ester of 4-hydroxy-3-methoxy benzoic acid by its accurate mass ([M+H]f at m z 183.0654, calculated for C 9 HioO 4 Na, [M+H]f at m z 183.0652). [0041] 1.6. Characterization data [0042] Compound 1 : (+)-pinoresinol 4-0-[6"-O-vanilloyl]-p-D-glucopyranoside, a light brown powder; [U]2 5 D +16.48 (c 0.100, MeOH); IR (KBr): vmn. = cm 1 3405, 1705, 1598 M006.03 1.DRF 8 Spec draft v.1 and 1515 cm 1 ; UV(MeOH): mx, nm (log , ) = 221 (4.60) and 266 (4.22); for 13C and H NNMR (CD 3 0D) spectroscopic data (see Table 1 below for details of the data). ESI-TOF MS (positive mode) found [M+Na]f at m z 693.2150 (Calculated for C 34

H

38

O

14 Na at m z 693.2154). Table 1 H (600 MHz) and C (150 MVIHz) NIR spectroscopic data (in CD 3 OD) for Compound 1 Compound 1 Moiety Position oc P( Jin Hz) HMBC (H to C) COSY (H to H) Aglycone moiety 1 137.3 2 111.6 6.97(s) 1,4,6,7 6 3 150.6 4 147.1 5 117.6 6.98 (d, 8.4) 1, 3,4 6 6 119.2 6.46 (dd, 8.4,1.8) 2,4,7 2,5 7 87.0 4.70 (d, 5.4) 1, 2, 6, 8, 9' 8 8 55.4 3.01 (m) 1,8' 7, 9, 8' 9 72.8 4.27 (dd, 9.0,7.2) 7, 8 8, 9 3.83 (m) 8, 7' 8,9 3-OMe 56.7 3.86 (s) 3 1' 133.7 2' 111.0 7.00 (d, 1.8) 4', 6', 7' 6' 3' 149.1 4' 147.3 5' 116.0 6.80 (d, 7.8) 1', 3' 6' 6' 120.2 6.85 (dd, 7.8,1.8) 7, 2', 4', 7' 2', 5' 7' 87.6 4.69 (d, 5.4) 1', 2', 6' 8' 8' 55.3 3.11 (m) 7, 8, 1' 8, 7', 9' 9' 72.5 4.15 (dd, 9.0,6.6) 7' 8', 9' 3.87 (m) 7, 8' 8', 9' 3-OMe 56.4 3.90(s) 3' Sugar moiety 1" 102.4 4.88 (d, 7.2) 4 2" 2" 74.8 3.54 (m) 1", 3" 1", 3"1 3"1 77.8 3.53 (m) 2", 4" 2", 4" 4" 72.2 3.43 (t, 8.4) 3", 5" 3", 5" 5" 75.6 3.79 (m) 4", 6" 4", 6" 6" 65.0 4.65 (dd, 11.7,1.8) 7"' 5", 6" 4.49 (dd, 11.7, 7.8) 5", 7"' 5", 6" Vanilloyl moiety 1"' 122.5 2"' 113.8 7.56 (d, 1.8) 1"', 3"', 4"', 6"', 7"' 6"' 3"' 148.8 M006.03 1.DRF 9 Spec draft v.1 4"' 153.1 5"' 116.0 6.90 (d, 8.4) 1"', 3"', 4"' 6"' 6"' 125.3 7.61 (dd, 8.4,1.8) 2"', 4"', 7"' 2"', 5"' 7"' 167.7 3"'-OMe 56.5 3.86 (s) 3"' [0043 ] 1.7. Cells and virus strains [0044] Madin Darby Canine Kidney (MDCK), and HEp-2 cells were purchased from the American Tissue Culture Collection (ATCC). Influenza virus A/PR/8/34(H1N1), A/FM/1/47(H1N1), seasonal influenza A/Aichi/2/68 (H3N2) and respiratory syncytial virus (RSV; long strain) were purchased from ATCC. Influenza virus Inf B/Lee/i 940 and adenovirus type 3 (ADV3) were isolated from routine clinical specimens. Avian influenza strains of H6N2 (A/Duck/Guangdong/2009), H7N3 (A/Duck/Guangdong/1994) and H9N2 (A/Chicken/Guangdong/1996) were obtained from Dr. Chen Jianxin who is the professor of South China Agriculture University. The influenza viruses were propagated and passaged in MDCK cells. HEp-2 cells were used as the hosts for both RSV and ADV3. All the cells were grown in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS). [0045] 1.8. Cytotoxicity assay [0046] MDCK or HEp-2 cells were seeded into 96-well plate at density of 2 x 104 cells/well (100 ptL), and cultured to reach 90 % confluence at 37 'C under 5 % CO 2 for 24 h. The medium was replaced with the one containing various concentrations of the tested compounds and the cells were further incubated at 37 0 C for 48 h. The cell viability was then determined with the MTT assay as reported (Ehrhardt et al., 2007). The 50% toxic concentration (TC 5 o) was calculated by Reed-Muench analysis (Reed and Muench., 1938). The negative control was cells only and the blank control was the reagent used. [0047] 1.9. Cytopathic effect inhibition assay [0048] MDCK cells (2 x 104) were seeded into 96-well plastic plates and cultured at 37 0 C under 5 % CO 2 for 24 h. For the anti-influenza activity assay, MDCK cells were M006.03 1.DRF 10 Spec draft v.1 inoculated with serial influenza virus strains at 1 00TCID 50 at 37'C for 2 h, followed by removal of the medium and addition of the tested compounds (100 piL) with desired concentrations in serum-free Minimum Essential Medium supplemented with 2 pig/ml of TPCK-trypsin. After incubation for 48 h at 37 0 C, the CPE induced by the influenza virus was measured microscopically (Hsieh C.F. et al., 2012). The concentration required for 50% inhibition (IC 50 ) of the virus-induced CPE was calculated by the Reed-Muench analysis (Reed and Muench., 1938). The data of Compound 1 was further confirmed by MTT assay, and the resulting spectrophotometric data was used to calculate the IC 50 values (Ehrhardt et al., 2007). All data reported was an average from the values of three independent experiments. Selection index (SI) was calculated by the ratio of TC 50 / IC 50 (Yingsakmongkon et al., 2008). [0049] 1.10. Activities against respiratory syncytial virus and adenovirus type 3 [0050] The antiviral activities of Compound 1 against RSV (Graham et al., 1988) and ADV3 (Hui et al., 1994) were evaluated by using CPE inhibition assay on HEp-2 cells. A total of 100 TCID50 (50% tissue culture infective dose) of the viral infective titer was allowed to adsorb to the appropriate confluent HEp-2 cell lines for 1 h, followed by washing of virus with serum free medium. Then, the test medium containing the desired concentrations of Compound 1 was added. After appropriate periods (three to five days) of incubation, the CPE in the virus-infected cells was observed microscopically, and the

IC

50 values were determined by the method of Reed and Muench (Reed and Muench, 1938). [0051] 2. Results and discussion [0052] 2.1. Isolation, purification and structure elucidation of Compound 1 [0053] CGLA (1 L) was precipitated with 95 % EtOH at the ratio of latex: EtOH of 6:4 at room temperature to give alcoholic solution and coagulum. Alcoholic solution was evaporated under reduced pressure before subjected to liquid-liquid partition by H 2 0 EtOAc. The EtOAc layer extract was subjected to a series of separation including normal and reverse phase open column chromatographies and preparative HPLC to give one new M006.03 1.DRF 11 Spec draft v.1 lignan glycoside (Compound 1) and two known phenolic glycosides (Compounds 2 and 3). Three liters of CGLA were further purified with similar protocol of 1 L to obtain Compound 1 (127 mg). [0054] Compound 1 was obtained as a light brown powder and the chemical structure thereof was represented by formula (III) or Fig. 1A. Its molecular formula of C 34

H

38 0 14 was established by the observation of [M+Na]f at m z 693.2150 (calculated [M+Na]f at m z 693.2154) in the positive ESI-TOF-MS as shown in Fig. 4B. The IR spectrum indicated the existence of hydroxyl groups (3405 cm- 1 ), carbonyl group (1705 cm- 1 ) and aromatic rings (1598 and 1515 cm- 1 ). Its UV spectrum supported the presence of aromatic rings (221 nm, log , = 4.60 and 266 nm, log , = 4.22). [0055] The 1 H-NMR spectrum of Compound 1 (as shown in Table 1) displayed signals of three ABX benzene rings, an anomeric, three methoxyls, two oxygenated methylene and two oxygenated methines. The 1C-NMR spectrum showed existence of three benzene rings, an ester carbonyl, a $l-glucopyranosyl moiety and three methoxyl groups. [0056] The presence of characteristic proton and carbon signals of two ABX benzene rings, two methoxy groups, two oxygenated methylene, two oxygenated methines and a $l-glucopyranosyl was closely related to those previously reported for Compound 4 (Deyama T. et al., 1983) except for the presence of typical proton and carbon signals for one more set of ABX benzene ring and a methoxy group together with an ester carbonyl which was in good agreement with the reported values of a vanilloyl moiety (Yang et al., 2007). All these signals suggested the existence of a moiety of Compound 4 and a vanilloyl group in Compound 1. [0057] The 1H-1H COSY spectrum (as shown in Fig. IB) exhibited the correlations of typical proton signals of ABX benzene rings, i.e., the correlations of H-6 to H-2 and H-5 and H-6' to H-2' and H-5' , along with the correlations of characteristic resonances for two fused tetrahydrofuran moiety, i.e., the correlations of two aliphatic methines (H-8 and H-8') to two oxygenated methines (H-7 and H-7') and two oxygenated methylenes (H-9 and H-9'). Moreover, an anomeric proton (6H 4.65, d, J = 7.2 Hz, H-i") of a glucopyranosyl group showed HMBC correlations to C-4 (6c 147.1), suggesting the M006.03 1.DRF 12 Spec draft v.1 linkage of glucopyranosyl to C-4 of aglycone moiety. The large coupling constant at J = 7.2 Hz indicated the pl anomeric configuration in glucopyranosyl moiety. These evidences strongly confirmed the presence of Compound 4 within Compound 1. In addition, the correlations of H-6"' to H-2"' and H-5"' in the IH-IH COSY spectrum and HNMBC correlation between H-6"' (6H 7.61, dd, J = 8.4, 1.8 Hz) to an ester carbonyl at C-7"' (6c 167.7) confirmed the existence of a vanilloyl group in Compound 1. [0058] An alkaline hydrolysis reaction (as illustrated in Fig. 3) of Compound 1 was performed to yield two products Compound la and Compound 1b. Compound la was identified as (+)-pinoresinol 4-0-p8-D glucopyranoside by comparison of its retention time and m z with Compound 4, as illustrated in Fig. 4. Compound lb at retention time of 6.172 min showed [M+H]Tat m z 183.0654 corresponded to methyl ester of 4-hydroxy-3 methoxy benzoic acid (calculated for C 9

H

1 oO 4 Na, [M+H]f at m z 183.0652). [0059] Furthermore, the evidence of downfield shift of C-6" at glucopyranosyl moiety from 6c 60.7 in (+)-pinoresinol 4-0-p8-D-glucopyranoside, Compound 4, (Deyama T. et al., 1983) to 6c 65.0 in Compound 1 indicated the attachment of a vanilloyl group to C-6" of sugar part with similar characteristic reported for Compound 5 (Matsunami K. et al., 2009). This linkage was further confirmed by the HNBC correlations from H-6" (6H 4.65 and 4.49) to C-7"' (6c 167.7) (Table 1, Fig. 1B). Therefore, the structure of Compound 1 was assigned and was named (+)-pinoresinol 4-0-[6"-O-vanilloyl]-p-D-glucopyranoside. [0060] 2.2 Identification of Compounds 2 and 3 [0061] Two known compounds, Compounds 2 and 3, have the same molecular formula of C 2 1

H

2 4 0 11 by the evidence from positive ESI-TOF-MS ([M+Na]f at m z 475.1215 and 475.1217, respectively, calculated [M+Na]f at m z 475.1211). The 'H- and "C-NMR data of Compounds 2 and 3 (see Table 2 for detail of data) showed the presence of one pl glucopyranosyl unit, a vanilloyl moiety which were in good agreement with those reported in literature (Yang X.W. et al., 2007). Compound 2 and 3 were characterized as 6'-O-vanilloyltachioside and 6'-O-vanilloylisotachioside, respectively. Table 2 M006.03 1.DRF 13 Spec draft v.1 H (600 MHz) and C (150 MVIHz) NIR spectroscopic data (in CD 3 OD) for Compounds 2 and 3 Position Compound 2 Compound 3 P C 6H(JinHz) 6C 6H(JinHz) Aglycone moiety 1 152.7 141.0 2 104.2 6.70 (s) 152.3 a 3 149.1 101.9 6.43 (d, 2.7) 4 143.3 155.1 5 116.0 6.56 (d, 8.8) 107.6 6.11 (dd, 8.7, 2.7) 6 110.2 6.55 (d, 7.4) 120.7 6.93 (d, 8.7) 3-OMe 56.6 3.70 (s) 56.6 3.78 (s) Sugar moiety 1' 103.8 4.76 (d, 7.6) 104.3 4.71 (d, 7.6) 2' 75.1 3.45-3.50 (m) 75.2 3.46-3.48 (m) 3' 78.0 3.45-3.50 (m) 77.9 3.46-3.48 (m) 4' 72.2 3.45-3.50 (m) 72.2 3.41 (m) 5' 75.8 3.73 (m) 75.8 3.66 (m) 6' 65.3 4.70 (dd, 11.8, 1.9) 65.2 4.66 (dd, 11.7, 2.1) 4.37 (dd, 11.8, 7.4) 4.36 (dd, 11.7, 7.4) Vanilloyl moiety 1" 122.1 122.5 2" 113.7 7.53 (d, 1.7) 113.8 7.53 (d, 1.8) a 3" 149.3 148.9 4" 153.9 152.3 b 5" 116.3 6.85 (d, 8.3) 116.1 6.86 (d, 8.2) 6" 125.5 7.57 (dd, 8.3, 1.8) 125.4 7.55 (dd, 8.3, 1.8) 7" 168.1 168.0 3"-OMe 56.4 3.85 (s) 56.6 3.86 (s) a c to Assigmnents may be interchanged within each column [0062] 2.3 In vitro anti-influenza virus activity of Compounds 1-4 against A PR 8 34 (HJN]) [0063] Compounds 1, 2, 3 and 4 were first evaluated for their antiviral activities towards an influenza virus strain A/PR/8/34 (HINI) by CPE inhibition assay on MDCK cells. In the mode of treatment, Compound 1 showed strong inhibitory effect on A/PR/8/34 (HINI) virus-induced CPE in non-toxic concentration with an IC 50 of 18.7 pM and a selection index (SI) of > 11.3 (as shown in Table 3). While, the other three tested M006.03 1.DRF 14 Spec draft v.1 compounds did not exhibit any significant anti-HINI activity (SI < 1 for Compounds 2 to 4). Except for a substitution of a vanilloyl moiety at C-6" of Compound 1 (as shown in Fig. 2), Compound 1 closely resembles to Compound 4 in chemical structure. But their anti-HINI activities were notably different in that Compound 1 displayed extremely potent anti-HINI activity with SI value of > 11.3, while Compound 4 did not possess such bioactivity with SI < 1. This result indicated that a vanilloyl group at C-6" is an indispensable part for anti-HINI activity of Compound 1. Table 3 Anti-HINI virus effects of Compounds 1-4 by CPE inhibition assay TC a, IC a1 Com1pounld SI 1 >210.4 <18.7 >11.3 2 >442.5 >442.5 <1.0 3 >442.5 >442.5 <1.0 4 >384.6 >384.6 <1.0 Ribavirin >819.0 <25.6 >32.0 [0064] 2.4. In vitro anti-influenza virus activities of Compound 1 against other influenza viruses [0065] The anti-influenza effect of Compound 1 was further examined by using CPE assay against a series of both human and avian influenza viruses. As shown in Table 4, Compound 1 exhibited potent antiviral activities at different magnitudes against influenza viruses from human isolates including influenza A [A/PR/8/34 (HINI), A/FM/1/47 (HINI) and A/Aichi/2/68 (H3N2)] and influenza B (B/Lee/1940) subtypes with IC 50 values ranged from 13.4 to 39.8 pM and SI values of 3.7 - 11.4. The inhibitory effects of Compound 1 against influenza A/Aichi/2/68 and B/Lee/1940 viruses were stronger (5.3 and 6.9 folds respectively) than those of the antiviral drug ribavirin, a positive control, while showed similar efficacy towards A/PR/8/34 (HINI) and less inhibitory activity against influenza virus A/FM/1/47 (as shown in Table 4 and Fig. 5). On the contrary, Compound 1 displayed no effect towards three avian influenza virus strains including H6N2 (A/Duck/Guangdong/2009), H7N3 (A/Duck/Guangdong/1994) and H9N2 (A/Chicken/Guangdong/1996) with IC 50 values greater than 138.1 pM and SI < 1 in all M006.03 1.DRF 15 Spec draft v.1 cases. The specific inhibitory effect on human influenza viruses of Compound 1 was noted. Table 4 The effects of Compound 1 against human and avian influenza viruses Virus type and strain IC, (PM) SI 1 Ribavirin 1 Ribavirin A /PR/8/34 (HINI) 24.5+4.5 21.9+1.6 5.8+1.1 47.0±3.4 A /FIM1/47 (HINI) 39.8i11.5 23.3+2.1 3.7+0.9 44.4±4.1 A/Aichi/2/68 (H3N2) 16.3+2.3 86.6+5.0 8.6+1.3 11.9±0.7 A /Duck/Guangdong/2009 (H6N2) >138.1 81.5±3.9 <1.0 12.6+0.6 A /Duck/Guangdong/1994 (H7N3) >138.1 63.8±4.6 <1.0 16.1+1.2 A/Chicken/Guangdong/1996 (H9N2) >138.1 42.3±2.2 <1.0 24.3+1.3 Inf B/Lee/1940 13.4+4.1 92.9±5.5 11.4±3.7 11.1+0.7 ADV3 >37.3 - <1.0 RSV >37.3 - <1.0 [0066] Generally, infection of host cell with human or avian influenza viruses requires specific interaction between cell oligosaccharides containing sialic acid residues and the influenza viral hemagglutinin (Matrosovich M.N. et al., 1997, Ning Z. Y. et al., 2012 and Wang H. et al., 2012). It is widely believed that the receptors containing sialic acid with an a-2, 3-linkage to the penultimate galactose were the receptor for avian influenza viruses, while an a -2, 6-sialic acid -linked receptor was preferred by human influenza viruses (Matrosovich M.N. et al., 1997, Ning Z. Y. et al., 2012 and Wang H. et al., 2012). The specific affinity of Compound 1 to an a -2, 6-sialic acid -linked receptor rather than a -2, 3-sialic acid -linked receptor may interrupt the entry of human influenza viruses into host cells; consequently, the selective inhibiting effect on human influenza viruses of Compound 1 was observed. [0067] 2.5. Selectivity of compound 1 for influenza viruses [0068] In order to gain an insight into selectivity of Compound 1 for human influenza viruses, its antiviral activities against two non-influenza viruses, RSV and ADV3, were also investigated by CPE inhibition assay on HEp-2 cell. As a result (as shown in Table M006.03 1.DRF 16 Spec draft v.1 4), Compound 1 did not show inhibition activity with IC 50 values greater than 37.3 pM and SI < 1 in both cases. These results indicated that Compound 1 has selective and specific inhibitory effects on human influenza viruses. [0069] 3. Conclusion [0070] In this invention, a new lignan glycoside, (+)-pinoresinol 4-0-[6"-O-vanilloyl]-p3 D-glucopyranoside (Compound 1) and two known phenolic compounds, 6-0 vanilloyltachioside (Compound 2) and 6'-O-vanilloylisotachioside (Compound 3) were isolated from the latex of Calotropis gigantea (Asclepiadaceae). The structure of the new compound was elucidated by using spectroscopic and chemical methods. [0071] Compounds 1-3 and (+)-pinoresinol 4-O-p8-D-gluco-pyranoside (Compound 4) were screened for anti-HINI activity by cytopathic effect (CPE) inhibition assay, and compound 1 was further evaluated for in vitro antiviral activities by CPE inhibition assay on Madin-Darby Canine Kidney (MDCK) cell and HEp-2 cell. As demonstrated from the results, Compound 1 showed more potent inhibitory effect against influenza A strain A/Aichi/2/68 and influenza B strain B/Lee/1940 viruses (5.3 and 6.9 folds, respectively) than those of antiviral drug ribavirin, a positive control, while Compound 1 also showed similar efficacy towards A/PR/8/34 (HINI) and less inhibitory activity against A/FM/1/47 with IC 5 0 values ranged from 13.4 to 39.8 pM. However, Compound 1 was inactive towards three subtypes of avian influenza viruses, i.e., A/Duck/Guangdong/2009(H6N2), A/Duck/Guangdong/1 994(H7N3), A/Chicken/Guangdong/1996(H9N2) and two non-influenza viruses, i.e., respiratory syncytial virus (RSV) and adenovirus 3 (ADV3). [0072] In a sharp contrast, the other three compounds (Compounds 2-4) did not show inhibitory activity towards A/PR/8/34 (HINI). An analysis of structure and activity relationship between Compounds 1 and 4 revealed that the presence of a vanilloyl moiety in the sugar moiety of 1 is crucial for its anti-influenza virus activities. This novel lignan glycoside (Compound 1) represents a promising candidate for the research and development of anti-influenza agents. M006.03 1.DRF 17 Spec draft v.1 [0073] The exemplary embodiments of the present invention are thus fully described. Although the description referred to particular embodiments, it will be clear to one skilled in the art that the present invention may be practiced with variation of these specific details. Hence this invention should not be construed as limited to the embodiments set forth herein. [0074] References 1. Abe F., Yamauchi T., Lignan glycosides from Parsonsia laevigata. Phytochemistry. 28 (6), 1737 - 1741. 2. Bhaskara Rama Murti P., Seshadri T. R., 1943. Chemical composition of Calotropis gigantea Part I. wax and resin components of the latex. Proceedings - Indian Academy of Sciences, Section A. 18A, 145 - 159. 3. Deyde V. M., Xu X., Bright R. A., Shaw M., Smith C.B., Zhang Y., Shu Y.L., Gubareva L. V., Cox N. J., and Klimov A. I., 2007. Surveillance of Resistance to adamanthanes among influenza A(H3N2) and A(H1N1) viruses isolated worldwide. J. Infect. Dis. 196, 249-257. 4. Deyama T., 1983. The constituents of Eucommia ulmoides OLIV. I. Isolation of (+)-Medioresinol Di-0--D-glucopyranoside. Chem. Pharm. Bull. 31, 2993 - 2997 5. Du J., Cross T. A. and Zhou H.X., 2012. Recent progress in structure-based anti influenza drug design. Drug Discov. Today. 17(19/20), 1111-1120. 6. Ehrhardt C., Hrincius E.R., Korte V., Mazur I., Droebner K., Poetter A., Dreschers S., Schmolke M., Planz 0., Ludwig S., 2007. A polyphenol rich plant extract, CYSTUS052, exerts anti influenza virus activity in cell culture without toxic side effects or the tendency to induce viral resistance. Antiviral Res. 76, 38-47. 7. Flora of China (FOC): Calotropis gigantea, http://www.efloras.org/florataxon.aspx?flora_id=2&taxon-id=200018512, retrieved date 29 May 2013 M006.03 1.DRF 18 Spec draft v.1 8. Gao H.N., Lu, H.Z., Cao B., Du B., Shang H., Gan J.H., Lu S.H., Yang Y.D., Fang Q., Shen Y.Z., Xi X.M., Gu Q., Zhou X.M., Qu H.P., Yan Z., Li F.M., Zhao W., Gao Z.C., Wang G.F., Ruan LX, Wang W.H., Ye J., Cao H.F., Li X.W., Zhang W.H., Fang X.C., He J., Liang W.F., Xie J., Zeng M., Wu X.Z., Li J., Xia Q., Jin Z.C., Chen Q., Tang C., Zhang Z.Y., Hou B.M., Feng Z.X., Sheng J.F., Zhong N.S., Li L.J., 2013. Clinical findings in 111 cases of influenza A (H7N9) virus infection. N Engl J Med. 13; 368 (24), 2277-2285. 9. Hsieh C.F., Lo C.W., Liu C.H., Lin S., Yen H.R., Lin T.Y., Horng J.T., 2012. Mechanism by which ma-xing-shi-gan-tang inhibits the entry of influenza virus. J Ethnopharmacol 143(1): 57-67. 10. Hui M.B., Lien E.J., Trousdale M.D., 1994. Inhibition of human adenoviruses by 1-(2'-hydroxy-5'-methoxybenzylidene)amino-3-hydroxyguanidine tosylate. Antiviral Res. 24, 261-273. 11. Knox Y.M., Suzutani T., Yosida I., Azuma M., 2003. Anti-influenza virus activity of crude extract of Ribes nigrum L. Phytother Res. 17, 120-122. 12. Kiyohara H., Ichino C., Kawamura Y., Nagai T., Sato N., Yamada H., Salama M. H. and Abdel-Sattar E., 2012. In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk). Phytochemistry. 19, 111-114. 13. Lhinhatrakul T. and Sutthivaiyakit S., 2006. 19-Nor- and 18,20-Epoxy cardenolides from the Leaves of Calotropis gigantean. J. Nat. Prod. 69, 1249-1251. 14. Ludwig S, Wolff T, Ehrhardt C, et al. MEK inhibition impairs influenza B virus propagation without emergence of resistant variants. FEBS Lett. 2004, 561(1-3):37-43. 15. Matrosovich M. N., Gambaryan A. S., Teneberg S., Piskarev V. E., Yamnikova S., Lvov D. K., Robertson J. S., Karisson K. A., 1997. Avian influenza A viruses by recognition of sialyloligosaccharides and gangliosides and by a higher conservation of the HA receptor-binding site. Virology. 233, 224 - 234. M006.03 1.DRF 19 Spec draft v.1 16. Matsunami K., Otsuka H., Kondo K., Shinzato T., Kawahata M., Yamaguchi K., Takeda Y., 2009. Absolute configuration of (+)-pinoresinol 4-0-[6"-O-galloyl]-3-D glucopyranoside, macarangiosides E, and F isolated from the leaves of Macaranga tanarius. Phytochemistry. 70, 1277 - 1285. 17. Miyamoto, D., Hasegawa, S., Sriwilaijaroen, N., Yingsakmongkon, S., Hiramatsu, H., Takahashi, T., Hidari, K., Guo, C., Sakano, Y., Suzuki, T., Suzuki, Y., 2008. Clarithromycin inhibits progeny virus production from human influenza virus-infected host cells. Biol Pharm Bull. 31, 217-222. 18. Ning Z. Y., Wu X. T., Cheng Y. F., Qi W. B., An Y. F., Wang H., Zhang G. H., Li S. J., 2012. Tissue distribution of sialic acid-linked influenza virus receptors in beagle dogs. J. Vet. Sci. 13, 219 - 222. 19. Par K., Rao P. J., Devakemar C., Rastogi J. N., 1998. A novel Insect antifeedant nonprotein amino acid from Calotropis gigantea. J. Nat. Prod. 61, 102 - 104. 20. Reed L. J., Muench H., 1938. A simple method of estimating fifty percent endpoints. Am. J. Hyg. 27 (3), 493 - 497. 21. Silva M. C. C., Silva A. B., Teixeira F. M., Sousa P. C. P., Rondon T. M. M., Honorio Junior J. E. R., Sampiao L. R. L., Oliveira S. L., Holonda A. N. M. and Vasconcelos S. M. M., 2010. Therapeutic and biological activities of Calotropis procera (Ait.) R. Br. Asian. Pac. J. Trop. Med. 332-336. 22. Sriwilaijareon N., Fukumoto S., Kumagai K., Hiramatsu., Odagiri T., Tashiro M. and Suzuki Y., 2012. Antiviral effects of Psidium guajava Linn. (guava) tea on the growth of clinical isolated HINI viruses: Its role in viral hemagglutination and neuraminidase inhibition. Antiviral res. 94, 139-146. 23. Wang H., Wu X. T., Cheng Y. F., An Y. F., Ning Z. Y., 2012. Tissue distribution of human and avian type sialic acid influenza virus receptors in domestic cat. Acta Vet. Hung. Doi: 10.1556/Avet.2013030. M006.03 1.DRF 20 Spec draft v.1 24. 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Claims (6)

1. A lignan glycoside compound represented by formula (I). OR HOK (I) OMG

2. The lignan glycoside compound of claim 1 wherein R is represented by vanilloyl group represented by formula (II); OH said compound is (+)-pinoresinol 4-0-[6"-O-vanilloyl]-p-D-glucopyranoside and represented by formula (III). OMe M006.031.DRF 22 Spec draft v.1

3. A method of treating influenza comprising administering an effective amount of a lignan glycoside compound to a subject in need thereof; said lignan glycoside compound comprises at least one vanilloyl moiety.

4. The method of claim 3 wherein said lignan glycoside compound is the compound of claim 2.

5. The method of claim 3 wherein said influenza is caused by an influenza virus selected from a group consisting of influenza A strain A/Aichi/2/68, influenza A strain A/PR/8/34, influenza A strain A/FM/1/47 and influenza B strain B/Lee/i 940.

6. A method of isolating said compound of claim 2 comprising the steps of: a) adding 95% ethanol to latex of Calotropis gigantea produce a filterable precipitate mixture; b) said mixture was sonicated at room temperature then centrifuged; c) supernatant from the centrifuge product of step (b) was evaporated under reduced pressure to obtain a light yellowish residue; d) said residue was suspended in H 2 0 and subjected to liquid-liquid partition by adding EtOAc; e) said residue of EtOAc layer was subjected to silica gel CC eluted with the gradient system of CHCl 3 -MeOH-H 2 0 to obtain four fractions of Fr. Ito Fr.4; f) the third fraction, Fr.3, from step (e) was chromatographed over MCI-gel CHIP 20P CC eluted with aqueous MeOH to obtain six sub-fractions of Fr.3-1 to Fr.3-6; g) the fourth sub-fraction, Fr.3-4, from step (f) was loaded to Bondapak Waters® ODS CC using the gradient of MeOH-H 2 0 to obtain seven sub-fractions of Fr.3 4-1 to Fr.3-4-7; h) the fifth sub-fraction, Fr.3-4-5, from step (g) was purified by Silica gel 60 CC eluted with the gradient system of CHCl 3 -MeOH-H 2 0 to obtain eleven sub fractions of Fr.3-4-5-1 to Fr.3-4-5-1 1; and i) the fifth sub-fraction, Fr.3-4-5-5, from step (h) was further purified by MCI-gel CHIP 20P CC using aqueous MeOH to obtain said compound. <DocRef#00125742-RM > M006.03 1.DRF 23 Spec draft v.1