Phenylpropionamides, Piperidine, and Phenolic Derivatives from the Fruit of Ailanthus altissima

Four novel compounds—two phenylpropionamides, one piperidine, and one phenolic derivatives—were isolated and identified from the fruit of a medicinal plant, Ailanthus altissima (Mill.) Swingle (Simaroubaceae), together with one known phenylpropionamide, 13 known phenols, and 10 flavonoids. The structures of the new compounds were elucidated as 2-hydroxy-N-[(2-O-β-d-glucopyranosyl)phenyl]propionamide (1), 2-hydroxy-N-[(2-O-β-d-glucopyranosyl-(1→6)-β-d-glucopyranosyl)phenyl]propionamide (2), 2β-carboxyl-piperidine-4β-acetic acid methyl ester (4), and 4-hydroxyphenyl-1-O-[6-(hydrogen-3-hydroxy-3-methylpentanedioate)]-β-d-glucopyranoside (5) based on spectroscopic analysis. All the isolated compounds were evaluated for their inhibitory activity against Tobacco mosaic virus (TMV) using the leaf-disc method. Among the compounds isolated, arbutin (6), β-d-glucopyranosyl-(1→6)-arbutin (7), 4-methoxyphenylacetic acid (10), and corilagin (18) showed moderate inhibition against TMV with IC50 values of 0.49, 0.51, 0.27, and 0.45 mM, respectively.


Introduction
Ailanthus altissima (Mill.) Swingle (Simaroubaceae), a deciduous tree (6-20 m in height), is native to Mainland China and now naturalized in many temperate regions of the world [1,2]. The stem and root bark have been used as traditional Chinese medicines for the treatment of colds, bleeding, and gastric diseases [3,4]. Phytochemical studies, especially on the stem and root bark of A. altissima, have led to the characterization of quassinoids [5,6], alkaloids [7,8], triterpenoids [9,10], coumarins [9], lignans [11], as well as sterols, lipids, and other phenolic derivatives [12]. However, little is known concerning the constituents of the fruit of A. altissima, which was also used as traditional Chinese medicine for bleeding and antibacterial. By far, previous phytochemical studies have demonstrated the identification of only four quassinoid glycosides [13], and several stigmasterols [14,15] from the fruit. We report in this paper the isolation and structure elucidation of four novel compounds-two phenylpropionamides (1 and 2), one piperidine (4) and one phenolic (5) derivatives-as well as 24 known constituents-one known phenylpropionamide (3), 13 phenols (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), and 10 flavonoids (19)(20)(21)(22)(23)(24)(25)(26)(27)(28). All compounds were investigated for their anti-Tobacco mosaic virus (TMV) activity.   (Table 1) and DEPT (distortionless enhancement by polarization transfer) spectra showed 15 carbon resonances, including one methyl, one methylene, 10 methines, and three quaternary carbons (including one carbonyl). The HMBC (heteronuclear multiple bond correlation) correlations ( Figure 2) observed from the amide proton to C-1 (δ C 173.1), C-1 (δ C 128.6), C-2 (δ C 146.5), and C-6 (δ C 119.6) and from H-2 to C-1, C-2 (δ C 67.8), and C-3 (δ C 20.8) indicated the presence of a 2-hydroxypropionamide moiety, which was attached to C-1 of the benzene ring via an NH linkage. The anomeric proton appearing as a doublet at δ H 4.85 with a diaxial coupling constant of 7.5 Hz suggested that the glucopyranosyl moiety must be a β-anomer. Additionally, it was attached to C-2 of the benzene ring through an oxygen, as indicated from the HMBC correlations from the anomeric proton to C-2 , which was further confirmed by the NOESY (nuclear Overhauser effect spectroscopy) correlation between H-3 /H-1 The acid hydrolysis of 1 afforded d-glucose, which was identified using TLC by comparison with standard sugars. Therefore, the structure of Compound 1 was established as 2-hydroxy-N-[(2-O-β-D-glucopyranosyl)phenyl]propionamide. glucopyranosyl moiety must be a β-anomer. Additionally, it was attached to C-2′ of the benzene ring through an oxygen, as indicated from the HMBC correlations from the anomeric proton to C-2′, which was further confirmed by the NOESY (nuclear Overhauser effect spectroscopy) correlation between H-3′/H-1‴. The acid hydrolysis of 1 afforded D-glucose, which was identified using TLC by comparison with standard sugars. Therefore, the structure of Compound 1 was established as 2-hydroxy-N-[(2-O-β-D-glucopyranosyl)phenyl]propionamide.     (Table 1) suggested that Compound 2 possessed the same aglycone as that of 1, which was confirmed by the observed HMBC correlations as shown in Figure 2. The 1 H-NMR spectra of 2 showed two anomeric proton signals at δ H 4.86 (1H, d, J = 7.4 Hz, H-1 ) and 4.22 (1H, d, J = 7.8 Hz, H-1 ), which correlated to the corresponding anomeric carbon signals at δ C 101.5 (C-1 ) and 103.2 (C-1 ) in the 13 C-NMR spectra, respectively, indicating the presence of two glucopyranosyl units with β-form. The HMBC correlations from an anomeric proton at δ H 4.22 (H-1 ) to δ C 68.3 (C-6 ) and from another anomeric proton at δ H 4.86 (H-1 ) to δ C 146.4 (C-2 ) indicated that two sugars were connected through 1→6 linkage, and the sugar chain was attached at C-2 of the aglycone through an oxygen. Therefore, Compound 2 was determined as Compound 3 was obtained as a white amorphous powder, with a molecular formula of C 9 H 12 NO 3 as indicated by an ion peak at m/z 182.0840 [M + Na] + (Calcd. for C 9 H 12 NO 3 Na, 182.0812) in its HR-ESI-MS. Comparison of the 1 H-and 13 C-NMR data with that of Compounds 1 and 2, as well as with those reported in the literature [16], indicated that Compound 3 was the aglycone of Compounds 1 and 2 with a known structure, 2-hydroxy-N-(2-hydroxyphenyl)propionamide.
Compound 4 was isolated as a white amorphous powder. It was assigned with a molecular formula of C 9 H 15 NO 4 by an HR-ESI-MS ion peak at m/z = 224.0907 [M + Na] + (Calcd. for C 9 H 15 NO 4 Na, 224.0899). Its IR spectrum (Supplementary Materials) showed the presence of a secondary amide (1624 cm −1 ), a carbonyl (1729 cm −1 ), and a strong peak for a hydroxyl group (3428 cm −1 ). The 1 H-NMR (Table 1) and HSQC spectrum of 4 revealed the presence of one methoxyl at δ H 3.66 (3H, s), two methines at δ H 3.47 (1H, dd, J = 12.8, 3.2 Hz) and 2.11 (1H, m), as well as four methylene protons. The 13 C-NMR spectrum and DEPT showed the presence of two methine carbons at δ C 60.6 and 32.9, one methoxyl carbon at δ C 52.1, two carbonyl carbons at δ C 173.7 and 173.8, as well as four methylene carbons. The key HMBC correlations ( Figure 2) from H-2 to C-3, C-4, and C-6 and from H-4 to C-2, C-3, C-5, and C-6 revealed the presence of a 2,4-disubstituted piperidine ring, which was confirmed by 1 H-1 H COSY (correlated spectroscopy) correlations between H-2/H-3, H-3/H-4, H-4/H-5, H-5/H-6, and H-4/H-8. An acetic acid methyl ester group was established by the HMBC correlations from the methoxyl protons to C-8 and C-9, and it was attached to C-4 of the piperidine ring as indicated by the HMBC correlations from H-4 to C-8 and C-9. The HMBC correlation from H-2 to C-7 proved that a carbonyl group was located at C-2. The NOESY correlations ( Figure 3) between H-2/H-4, H-2/H-6, and H-4/H-6 indicated that the two substituents were cis to each other. Thus, Compound 4 was determined as 2β-carboxyl-piperidine-4β-acetic acid methyl ester. Compound 2 was obtained as a white amorphous powder. Its molecular formula was deduced to be C21H31NO13 by an HR-ESI-MS ion peak at m/z 528.1726 [M + Na] + (Calcd. for C21H31NO13Na, 528.1688). The IR spectrum (Supplementary Materials) exhibited absorption bands due to the presence of hydroxyl, amide, and phenyl groups (3423, 3346, 1662, 1602, 1532, and 1454 cm −1 ). Analysis of the 1 H-and 13 C-NMR data ( Table 1) suggested that Compound 2 possessed the same aglycone as that of 1, which was confirmed by the observed HMBC correlations as shown in Figure 2. The 1 H-NMR spectra of 2 showed two anomeric proton signals at indicated that two sugars were connected through 1→6 linkage, and the sugar chain was attached at C-2′ of the aglycone through an oxygen. Therefore, Compound 2 was determined as 2-hydroxy- Compound 3 was obtained as a white amorphous powder, with a molecular formula of C9H12NO3 as indicated by an ion peak at m/z 182.0840 [M + Na] + (Calcd. for C9H12NO3Na, 182.0812) in its HR-ESI-MS. Comparison of the 1 H-and 13 C-NMR data with that of Compounds 1 and 2, as well as with those reported in the literature [16], indicated that Compound 3 was the aglycone of Compounds 1 and 2 with a known structure, 2-hydroxy-N-(2-hydroxyphenyl)propionamide.
Compound 4 was isolated as a white amorphous powder. It was assigned with a molecular formula of C9H15NO4 by an HR-ESI-MS ion peak at m/z = 224.0907 [M + Na] + (Calcd. for C9H15NO4Na, 224.0899). Its IR spectrum (Supplementary Materials) showed the presence of a secondary amide (1624 cm −1 ), a carbonyl (1729 cm −1 ), and a strong peak for a hydroxyl group (3428 cm −1 ). The 1 H-NMR (Table 1) and HSQC spectrum of 4 revealed the presence of one methoxyl at δH 3.66 (3H, s), two methines at δH 3.47 (1H, dd, J = 12.8, 3.2 Hz) and 2.11 (1H, m), as well as four methylene protons. The 13 C-NMR spectrum and DEPT showed the presence of two methine carbons at δC 60.6 and 32.9, one methoxyl carbon at δC 52.1, two carbonyl carbons at δC 173.7 and 173.8, as well as four methylene carbons. The key HMBC correlations ( Figure 2) from H-2 to C-3, C-4, and C-6 and from H-4 to C-2, C-3, C-5, and C-6 revealed the presence of a 2,4-disubstituted piperidine ring, which was confirmed by 1 H-1 H COSY (correlated spectroscopy) correlations between H-2/H-3, H-3/H-4, H-4/H-5, H-5/H-6, and H-4/H-8. An acetic acid methyl ester group was established by the HMBC correlations from the methoxyl protons to C-8 and C-9, and it was attached to C-4 of the piperidine ring as indicated by the HMBC correlations from H-4 to C-8 and C-9. The HMBC correlation from H-2 to C-7 proved that a carbonyl group was located at C-2. The NOESY correlations ( Figure 3) between H-2/H-4, H-2/H-6, and H-4/H-6 indicated that the two substituents were cis to each other. Thus, Compound 4 was determined as 2β-carboxyl-piperidine-4β-acetic acid methyl ester.   , and one methyl [δ C 27.6 (C-6 )] carbons, besides signals for a typical glucopyranosyl moiety and a p-hydroxyphenyl group. The HMBC correlations (Figure 2) observed from H 2 -2 to C-1 and C-3 and from H 2 -4 to C-3 and C-5 , as well as from H 3 -CH 3 to C-2 , C-3 , and C-4 , allowed for the establishment of a 3-hydroxy-3-methyl glutaryl (HMG) group. The HMBC correlations from the anomeric proton H-1 to C-1 (δ C 150.1) and from H 2 -6 to C-1 indicated that the p-hydroxyphenyl and HMG group were connected with C-1 and C-6 of the glucopyranosyl moiety, respectively. The anomeric proton appearing as a doublet at δ H 4.65 with a diaxial coupling constant of 7.7 Hz suggested that the glucopyranosyl moiety must be a β-anomer. Furthermore, the NOESY correlations ( Figure 3

Discussion
Continuous efforts have been made since the 1980s in the phytochemical and biological study of secondary metabolites from the Chinese medicinal plant A. altissima. Phytochemical studies have led to the characterization of quassinoids, alkaloids, lipids, coumarins, and other phenolic derivatives, of which quassinoids are the major components, with antitumor, antimalarial, antifeedant, anti-inflammatory, and other activities [2,35]. Twenty-eight compounds, including four novel structures (1, 2, 4, and 5), were obtained from the fruit extract of A. altissima in our present study. Among the known structures, 12 compounds, including 3, 6-10, 15-16, 20-21, 25, and 28, were isolated from this plant for the first time.
Previous studies have revealed the presence of alkaloids with varying structural patterns, including indole, β-carboline, as well as canthinone types [7,8,[36][37][38]. However, the nitrogenous compounds 1-4 obtained in our present study represent two novel types that have never been reported from the secondary metabolites of A. altissima. Phenylpropionamides 1-3 contain an α-hydroxyamide scaffold, which is present in a variety of compounds with confirmed biological activity, such as pantothenic acid (vitamin B5), the cholesterol-lowering drug bestatin, and the antibiotics amikacin and cefamandole [39]. 2-Hydroxy-N-(2-hydroxyphenyl)propionamide (3) has been previously reported to be isolated from the solid cultures of phytopathogen Peronophythora litchii, which is a major disease of lychee that causes twig withering, panicle shattering, fruit downfall, and rot [16]. To the best of our knowledge, this is the first report of 2-hydroxy-N-(2-hydroxyphenyl)propionamide (3) from plant secondary metabolites.
Piperidine alkaloids are among the most abundant metabolites of terrestrial plants [40]. Many of them, such as piperidine alkaloids identified from several Prosopis species, have been reported to possess diverse bioactivities, such as antibacterial, antifungal, and antiparasitic activities [41,42]. Recently, epidihydropinidine, the main piperidine alkaloid compound of Norway spruce (Picea abies), was reported to show promising antibacterial and anti-Candida activity [43]. 2β-Carboxyl-piperidine-4β-acetic acid methyl ester (4) was an unusual 2,4-disubstituted piperidine derivative of plant origin. Compounds 1-4 showed no potent anti-TMV activity in our present study, however, these unusual structures deserve further effort to evaluate their potential biological and pharmacological value.
Among the other known compounds obtained, corilagin (18) is a member of the tannin family, which has been discovered in a number of medicinal plants such as the Phyllanthus species. Corilagin was reported to possess diverse pharmacological activities such as antioxidative, anti-inflammatory, thrombolytic, antihypertensive, hepatoprotective, and antiatherogenic activities, as well as anti-tumor action in hepatocellular carcinoma, ovarian cancer, etc. [44,45]. Meanwhile, corilagin can reduce the cytotoxicity induced by human enterovirus 71 (EV71) and Coxsackie-virus A16 (CA16) on Vero cells and has been shown to protect against HSV1 encephalitis through inhibiting the TLR2 signaling pathways in vivo and in vitro [45,46]. Our present study indicated that corilagin (18) showed moderate antiviral activity against the positive single strand virus TMV, the type member of genus Tobamovirus.

Plant Material
The fruit of Ailanthus altissima was collected in Muyang City, Jiangsu Province, China, in October 2013. The plant was identified by associate Professor Chun-Mei Huang, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, China. A voucher specimen (sample MF131001) was deposited at the Key Laboratory of Bio-Pesticide and Chemistry-Biology, Ministry of Education, Fujian Agriculture and Forestry University.

Extraction, Fraction, and Isolation
The air-dried and pulverized fruit (7.5 kg) of A. altissima Swingle was extracted with MeOH (25 L, 3 day) at room temperature for three times. After being concentrated in vacuo, the extract (390.0 g) was suspended in water and successively partitioned with n-hexane, CHCl 3 , and n-BuOH.  Table 1).  Table 1).

Acid Hydrolysis
Compounds 1, 2, and 5 (2 mg each) were hydrolyzed in 1 M HCl (dioxane-H 2 O, 1:1, 2 mL) at 95 • C for 2 h, respectively. After evaporated to dryness, the reaction mixtures were diluted in H 2 O and extracted with Et 2 O (3 × 2 mL). The aqueous layer was neutralized with NaHCO 3 and evaporated under vacuum to furnish a neutral residue, from which D-glucose was identified by TLC comparison with standard sugars.

Virus and Host Plant
Purified TMV (strain U1) was obtained from Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, China, and concentration was determined as 15 mg/mL using an ultraviolet spectrophotometer method [virus concentration = (A 260 × dilution ration)/E 0.1%, 260 nm 1 cm ]. The purified virus was kept at −20 • C and diluted to 15 µg/mL with 0.01 M PBS before use. Nicotiana tabacum cv. K326, which were cultivated and grown to a 5-6-leaf stage in an insect-free greenhouse, were used for anti-TMV assay.

The Leaf-Disc Method
Pure compounds were dissolved in DMSO and diluted with 0.01 M PBS to a certain concentration for test. The final concentration of DMSO in the test solution (≤2%) showed no adverse effect on the plants. Anti-TMV assay was carried out using the leaf-disc method as described previously in our paper [47]. Growing leaves of N. tabacum cv. K326 were mechanically inoculated with TMV (15 µg/mL in 0.01 M PBS). After 6 h, three leaf discs (1 cm diameter) were punched and floated on solutions for test. Discs of healthy and inoculated leaves floated on a solution of 0.01 M PBS with 2% DMSO were used as a mock and control, respectively. Ningnanmycin and ribavirin were used as agent control. Three replicates were carried out for each sample. After incubating for 48 h at 25 • C in a culture chamber, the leaf discs were ground in a 0.01 M carbonate coating buffer (pH 9.6, 500 µL for each leaf disc) and centrifuged. The supernatant (200 µL) was transferred to a 96-well plate and used for an indirect enzyme-linked immunosorbent assay (ELISA). Indirect ELISA was performed as described in the literature [48,49]. Virus concentration was calculated from a standard curve constructed using OD 405 values of purified TMV at concentrations of 1.0, 0.5, 0.25, 0.125, and 0.0625 µg/mL. The inhibition of test solutions on TMV was calculated as follows: inhibition rate = [1 − (virus concentration of treatment)/(virus concentration of control)] × 100%.  (2), one piperidine, 2β-carboxyl-piperidine-4β-acetic acid methyl ester (4), and one phenol derivative, 4-hydroxyphenyl-1-O-[6-(hydrogen 3-hydroxy-3-methylpentanedioate)]-β-D-glucopyranoside (5), as well as one known phenylpropionamide (3), 13 phenols (6-18), and 10 flavonoids (19)(20)(21)(22)(23)(24)(25)(26)(27)(28), were identified from the n-BuOH-soluble fraction from MeOH extract of Ailanthus altissima fruit. All the compounds obtained were evaluated for their antiviral activity against TMV; however, only weak to moderate activity was observed. These results provide us with general knowledge of these diverse phenolic constituents and suggest the distribution of novel nitrogenous analogues in the metabolites of this Simaroubaceae plant.