molecules Article Picrotoxane Sesquiterpene Glycosides and a Coumarin Derivative from Coriaria nepalensis and Their Neurotrophic Activity Yuan-Yuan Wang, Jun-Mian Tian, Cheng-Chen Zhang, Bo Luo and Jin-Ming Gao * Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Science, Northwest A&F University, Yangling 712100, Shaanxi, China; [email protected] (Y.-Y.W.); [email protected] (J.-M.T.); [email protected] (C.-C.Z.); [email protected] (B.L.) * Correspondence: [email protected]; Tel.: +86-29-8709-2335; Fax: +86-29-8709-2226 Academic Editor: Isabel C. F. R. Ferreira Received: 10 September 2016; Accepted: 9 October 2016; Published: 12 October 2016 Abstract: Two picrotoxane sesquiterpene lactone glycosides, nepalactones A (1) and B (2), and one new coumarin, nepalarin (3), were isolated from the root barks of the poisonous plant Coriaria nepalensis. Their structures were elucidated via HRESIMS and 1D and 2D NMR spectroscopic analyses, and further verified via transformation methods. In addition, compounds 1–3 and five semisynthetic congeners (1a–e) were assayed for the activity to induce neurite outgrowth in rat pheochromocytoma (PC12) cells. As a result, nepalactone A derivative 1c and nepalarin (3) significantly enhanced nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells. Keywords: Coriaria nepalensis; Coriariaceae; natural products; picrotoxane; sesquiterpene; neurite outgrowth-potentiating activity; neurotrophic activity 1. Introduction Coriaria nepalensis Wall (Coriaria sinica Maxim) (Coriariaceae) is a Chinese medicine herb mainly distributed in the southwest of China. Traditionally, this herb is used to treat numbness, toothache, traumatic injury, and acute conjunctivitis [1]. Sesquiterpene lactones are the characteristic bioactive constituents from family Coriariaceae [2–9]. A series of sesquiterpenes [6–9] and prenyled coumarins [10,11] have been previously identified from C. nepalensis. Among these sesquiterpene lactones, coriamytin and tutin have been suggested as main bioactive substances for the treatment of schizophrenia [8,12], and as insecticidal agents [9]. In our recent effort to search for neurotrophic natural products [13–16], we employed rat dopaminergic PC12 cells as an in vitro cell model to screen various natural compounds for the effect on nerve growth factor (NGF)-induced neurite outgrowth. The active compounds are expected to be potentially useful for the treatment of Alzheimer’s disease and other neurological disorders [17,18]. Here, two new picrotoxane-type sesquiterpene glycosides, nepalactones A (1) and B (2), and one new polyprenyled coumarin, nepalarin (3) (Figure1), were isolated from the root barks of this plant. We describe herein the isolation and structure elucidation of the three new compounds as well as the assay for the ability to stimulate NGF-mediated neurite outgrowth from PC12 cells. Molecules 2016, 21, 1344; doi:10.3390/molecules21101344 www.mdpi.com/journal/molecules Molecules 2016, 21, 1344 2 of 9 Molecules 2016, 21, 1356 2 of 9 FigureFigure 1. 1.Chemical Chemical structuresstructures of compounds 11––33 andand 1a1a–e–.e . 2. Results and Discussion 2. Results and Discussion Compound 1 was isolated as a white, amorphous powder. The molecular formula was Compound 1 was isolated as a white, amorphous powder. The molecular formula was established established as C21H32O8 via HRESIMS (m/z [M + Na]+ 435.1991; calcd. 435.1995) in combination with m/z + 13 asthe C21 H13C-NMR32O8 via spectrum, HRESIMS requiring ( [M +six Na] degrees435.1991; of unsatura calcd.tion. 435.1995) Its IR inspectrum combination indicated with the the presenceC-NMR spectrum,of hydroxy requiring (3563 cm six−1) degreesand γ-lactone of unsaturation. carbonyl (1765 Its cm IR−1 spectrum) groups. The indicated 13C and the DEPT presence NMR spectra of hydroxy of −1 −1 13 (35631 (Table cm 1)) exhibited and γ-lactone 21 carbon carbonyl signals (1765 correspondi cm )ng groups. to three ThemethylC groups, and DEPT four methylene NMR spectra groups of 1 (Table(including1) exhibited one olefinic 21 carbon carbon signals at δ 111.5 corresponding and one oxygenated to three methyl carbon groups, at δ 61.6), four 11 methylene methine groups groups (including(seven oxygenated one olefinic carbons carbon including at δ 111.5 five and carbon one oxygenateds of sugar), carbonand three at δquaternary61.6), 11methine carbons groups(one (sevenlactone oxygenated carbonyl at carbons δ 180.6 includingand one olefinic five carbons carbon at of δ sugar), 158.2). andThe three1H and quaternary 13C-NMR spectra carbons of (one1 lactonedisplayed carbonyl signals at forδ 180.6 tertiary and methyl one olefinic(δ 1.25, s, carbon 3H), isopropyl at δ 158.2). (δ 0.99, The 1.01,1H andd, each13C-NMR 3H, and spectraδ 1.83, m, of 1 displayed1H), and signals exocyclic for olefinic tertiary bond methyl (δH( δ5.21,1.25, s, s,1H, 3H), 5.42, isopropyl s, 1H) groups. (δ 0.99, A 1.01, series d, of each sugar 3H, signals and δ 1.83,were m, 1 1H),also and detected exocyclic in the olefinic H-NMR bond spectrum (δH 5.21, (Table s, 1H, 1) between 5.42, s, 1H)δH 3.22–3.87, groups. with A series an anomeric of sugar proton signals at wereδH 1 13 also4.35 detected (d, 1H, J in= 7.5 the Hz,H-NMR H-1′). The spectrum C-NMR (Table spectrum1) between indicatedδH the3.22–3.87, presence with of a anglucose anomeric unit due proton to a at 0 13 δHset4.35 of carbon (d, 1H, signalsJ= 7.5 at Hz, δC H-1101.5,). 73.5, The 76.2,C-NMR 70.2, 76.6, spectrum and 61.6. indicated These data the suggested presence the of presence a glucose of unita glucopyranosyl moiety in 1. The glucose in nepalactone A derivative 1a appeared to be tetraacetylated, due to a set of carbon signals at δC 101.5, 73.5, 76.2, 70.2, 76.6, and 61.6. These data suggested the + 1 presencebased on of aNMR glucopyranosyl spectra (Table moiety 1) and in HRESIMS1. The glucose (m/z 603.2416 in nepalactone [M+Na] A). derivative Furthermore,1a appeared the H and to be 13 tetraacetylated,C-NMR data based of 1 were on NMR similar spectra to those (Table of 1the) and known HRESIMS picrotoxane ( m/z 603.2416 sesquiterpenoid, [M + Na] corialactone+). Furthermore, D (1b), which has been previously identified from the same plant [8], except for the presence of the the 1H and 13C-NMR data of 1 were similar to those of the known picrotoxane sesquiterpenoid, glucose unit at C-12 in 1. The attachment of the glucose moiety to C-12 of the aglycone 1b was corialactone D (1b), which has been previously identified from the same plant [8], except for the deduced from the HMBC correlations from H-1′ (δH 4.35) to C-12 (δC 79.4). These spectroscopic data presence of the glucose unit at C-12 in 1. The attachment1 of1 the glucose moiety to C-12 of the aglycone suggested that 1 is a glucoside of corialactone D. The 0H- H COSY and HSQC correlations indicated 1b was deduced from the HMBC correlations from H-1 (δH 4.35) to C-12 (δC 79.4). These spectroscopic the presence of two substructures–CH2(2)–CH(3)–CH(4)–CH(5)–CH(6)–CH(11)–CH(12)–, and –CH(4)– data suggested that 1 is a glucoside of corialactone D. The 1H-1H COSY and HSQC correlations CH(8)[CH3(9)]–CH3(10)–units, whose connectivity was defined by the key HMBC correlations (Figure 2). indicatedThe the relative presence configuration of two substructures–CH of the aglycone2 (2)–CH(3)–CH(4)–CH(5)–CH(6)–CH(11)–CH(12)–,(1b) of 1 was defined by a NOESY experiment and(Figure –CH(4)–CH(8)[CH 2). The NOE correlations3(9)]–CH3(10)–units, of H3-7/H-6, whose H3-9/H-5, connectivity and H-12/H-1 was′ definedindicated by that the CH key3-7, HMBCH-5, correlationsH-6, H-12, (Figure and the2). isopropyl group were at the same orientation (α-configuration). Additionally, enzymaticThe relative hydrolysis configuration of 1 afforded of the aglycone the known (1b )intact of 1 was aglycone defined 1b by, which a NOESY was experimentidentified from (Figure 1H 2). 0 TheNMR, NOE and correlations its absolute of H 3configuration-7/H-6, H3-9/H-5, (1R,3 andS,4S,5 H-12/H-1R,6S,12S) indicatedwas previously that CH determined3-7, H-5, H-6, from H-12, CD and thespectrum isopropyl [8]. group In addition, were at thethe samecoupling orientation constant (α (-configuration).J = 7.5 Hz) of the Additionally, anomeric proton enzymatic (δH 4.35, hydrolysis H-1′) of suggested1 afforded a the β-configuration known intact for aglycone the glucose1b, which unit. wasIndeed, identified compound from 11H was NMR, hydrolyzable and its absolute via configurationβ-glucocidase. (1R GC,3S ,4analysisS,5R,6S suggested,12S) was previouslythat acid hydrolyzate determined of from1 contained CD spectrum the glucose [8]. In moiety addition, with the 0 couplingthe D-configuration. constant (J = 7.5Hence, Hz) ofthe the structure anomeric of proton1 was (determinedδH 4.35, H-1 unambiguously) suggested a b -configurationas shown, and for thenamed glucose nepalactone unit. Indeed, A. compound 1 was hydrolyzable via b-glucocidase. GC analysis suggested that acid hydrolyzate of 1 contained the glucose moiety with the D-configuration. Hence, the structure of 1 was determined unambiguously as shown, and named nepalactone A. Molecules 2016, 21, 1356 3 of 9 Molecules 2016Table, 21, 1344 1. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data for 1, 1a, 1b, and 2 (δ ppm). 3 of 9 1 a 1a b 2 a No.