Diterpenoid alkaloids are a large group of natural products, which are classified as C20-, C19-, and C18-categories and mainly isolated from plants of several genera belonging to the Ranuncu laceae, Rosaceae, Asteraceae, Garryaceae, Escalloniaceae, and Polygonaceae families [1-4]. Because of diversities of complex structures and biological activities, these molecules continuously attract great interests of scientists in phytochemistry, synthesis, medicinal chemistry, and pharmacology for more than a century. Taking advantage of reversible replacement reactions, acidification and saponification with a variety of acids and bases are classic procedures in extraction and separation of the diterpenoid alkaloids and application of acids and/or bases is almost unavoidable [6-11]. Although a majority of diterpenoid alkaloids were finally obtained and structurally characterized as free base forms [1-11], their hydrophobic and alkali properties suggest that, under in vivo hydrophilic conditions, the basic alkaloids have to interact with various acidic molecules to increase solubility, bioavailability, transportations, and functions, as well as to maintain a relatively stable physiological pH of aqueous biological systems. In addition, the properties suggest that there are dynamic equilibrations between different forms/species of the alkaloids in the biological systems, and the equilibrations, along with the forms/species with structural alterations, may play important biological roles. The suggestion have been demonstratedby studies on several bioactive benzo[c]phenanthridine alkaloids, including natural products sanguinarine and chelerythrine[11-19], as well as synthetic analogues . However, detailed interactions of the diterpenoid alkaloids with solvents, bases, and acids, as well as the structural characterization of the associated forms/species in solution systems, are not investigated yet.
"Fu zi", the lateral roots of Aconitum carmichaelii Debx.(Ranunculaceae), is an indispensable ingredient of formulations in traditional Chinese medicine for the treatment of cardianeuria, neuralgia, and rheumatalgia in China, Japan, and Korea [21-23].Previous studies have shown that toxic aconitine C19-diterpenoid alkaloids are the main active constituents of "fu zi", while more than a hundred of compounds were reported from various extracts of A. carmichaeliii [9, 24-32]. However, the previous investigations of the plant materials including raw and prepared "fu zi" were mainly extracted with organic solvents, such as benzene, CHCl3, methanol, and ethanol [22-31]. This is inconsistent with a practical application of decocting the formulations. the refore, an aqueous extract of the raw lateral roots of A. carmichaeliii was investigated as part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects [33-50].In previous papers, we reported four new hetisan-type C20- and twenty-one new aconitane-type C19-diterpenoid alkaloids, two new 2-(quinonylcarboxamino) benzoates, and seven new aromatic acid derivatives [51-53] from the aqueous extract. A continuation of the investigation has resulted in the isolation and structural characterization of three new napelline-type C20-diterpenoidalkaloids (1-3) (Fig. 1), which were obtained as the alcoholiminiums. Since the chemical transformation from the aza acetalcontainingnapelline-type C20-diterpenoid alkaloids to the alcoholiminium salts was reported [1, 5, 54, 55], the alcohol iminiums in 1-3 could readily be formed from the corresponding aza acetals, and the anion counterparts were obviously induced by using hydrochloride (HCl) and trifluoroacetic acid (TFA) in fractionation and HPLC separation steps of this study. However, the only alcoholiminium salt "songoramine hydrochloride" was previouslyobtained and characterized only by IR spectroscopic data with the absence of experimental details. This, together with the above mentioned multiple properties of alkaloids and equilibrationsbetween the iminium and alkanolamine forms of sanguinarineand chelerythrine in the biological systems, especiallybiological interaction variations of the different forms withproteins [11-19], inspired us to fully characterize the structures of 1-3 and to preliminarily investigate influences of protic and aprotic solvents including the alkali pyridine-d5, as well ascommon acids TFA, AcOH, and HCl, on the transformation and equilibration between the alcohol iminium and aza acetal forms of these napelline-type C20-diterpenoid alkaloids. Reported hereinare the details.
2. Experimental 2.1. General experimental procedures
Optical rotations were measured on a P-2000 polarimeter (JASCO, Tokyo, Japan). UV spectra were recorded on a V-650spectrometer (JASCO). CD spectra were measured on a JASCO J-815CD spectrometer (JASCO). IR spectra were recorded on a Nicolet5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission).1D NMR and 2D NMR spectra were obtained at 600 or500 MHz for 1H; 150 or 125 MHz for 13C; and 470 MHz for 19F, respectively, on an Inova 500 MHz, a SYS 600 MHz (VarianAssociates Inc., Palo Alto, CA, USA), a Bruker 600 MHz spectrometer (Bruker Corp., Karlsruhe, Germany), or a WNMR-I 500 MHz (Wuhan Zhongke Niujin Magnetic Resonance Technology Co., Ltd., Wuhan, China), with TMS or solvent peaks as references.ESIMS and HR-ESIMS data were obtained on Agilent 1100 SeriesLC-MSD-Trap-SL and Agilent 6520 Accurate-Mass Q-T of L CMSspectrometers (Agilent Technologies, Ltd., Santa Clara, CA, USA), respectively. Column chromatography (CC) was performed withsilica gel (200-300 mesh, Qingdao Marine Chemical Inc., China), reversed phase C-18 silica gel (W. R. Grace & Co., Maryland, USA), Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden), and MCI gel (CHP20P, 75-150 mm) (Mitsubishi Chemical Corporation, Tokyo, Japan). HPLC separation was performed on a systemconsisting of a Waters 600 controller, a Waters 600 pump, and aWaters 2487 dual absorbance detector (Waters Corporation, Milford, MA, USA) or a Smartline RI detector (KNAUER, Berlin, Germany), using an Ultimate XB-Phenyl semi-preparative column (250 mm × 10 mm i.d.) packed with phenyl-silica gel (5 mm)(Welch, shanghai, China) or a YMC-Pack Ph column (250 mm × 10 mm i.d.) packed with phenyl-silica gel (5 mm)(YMC Co., Ltd, Kyoto, Japan). The pH values were measured using aFiveEasyPlusTM FE28-Stand ard pH meter equipped with an In labscience pH electrode (Mettler-Toledo, Zurich, Switzerland) which was calibrated from pH 0 to pH 12 with stand ard solutions. When needed, the pure TFA (99.9%, J&K Chemica, Germany), AcOH (99.8%, J&K Chemica, Germany), or stand ard solutions of TFA, AcOH, or HCl (Beijing Chemical Works, Beijing, China) in MeOH-d4 (CambridgeIsotope Laboratories, Inc., MA, America) or D2O (Beijing EasobioTechnology Co., Ltd, Beijing, China) was added to the sample in the NMR tube using a 10 μL syringe or finnpipette. The maximumvolume of the stand ard solutions added to the samples is 8 μL. TLCwas conducted on precoated silica gel GF254 plates. Spots werevisualized under UV light (254 or 365 nm) or by spraying with 7%H2SO4 in 95% EtOH followed by heating or with a Dragendorff’sreagent. All other chemicals were purchased from Sigma-Aldrichor J&K Chemica.2.2. Plant material 2.3. Extraction and isolation
For extraction and preliminary fractionation of the extract, seeref 51. Fraction C2-1 (200 g) was dissolved in H2O (500 mL), basified with concentrated ammonium hydroxide (25 mL) to pH10, then extracted with EtOAc (500 mL × 4). The EtOAc phase wasconcentrated under reduced pressure to give C2-1-A. The aqueouslayer was acidified with 6 mol/L HCl (66 mL) to pH 4, and partitioned with n-butanol (500 mL × 3). Evaporation of the nbutanolphase under reduced pressure yielded C2-1-B (12 g).
Fraction C2-1-A (60.0 g) was chromatographed over basifiedsilica gel (pH 8-9), eluting with a gradient of petroleum ether-Me2CO-diethylamine (15:1:1-4:1:1) mixture to afford C2-1-A-1-C2-1-A-6. Fraction C2-1-A-6 (8.3 g) was further fractionated byreverse phase (RP) flash charomatography (20-90% MeOH in H2O, containing 0.1% TFA) to give C2-1-A-6-1-C2-1-A-6-3. Separation of C2-1-A-6-3 (1.40 g) by CC over Sephadex LH-20 (50% MeOH in H2O) yielded C2-1-A-6-3-1-C2-1-A-6-3-2, of which C2-1-A-6-3-2(112 mg) was purified by HPLC (YMC-Pack Ph column, 20% MeCNin H2O containing 0.1% TFA, 1.5 mL/min) to yielded 2 (33.4 mg, tR = 18 min).
Fraction C2-2 (200 g) was separated by CC over Sephadex LH-20(CHCl3-MeOH, 1:1) yielded C2-2-1-C2-2-8. Fraction C2-2-4 (9.5 g) was chromatographed over silica gel (150 g) eluting with agradient of petroleum ether-Me2CO-diethylamine (5:2:1-2:2:1) to give C2-2-4-1-C-2-2-4-7, of which C2-2-4-4 (420 mg) wasfurther separated by CC over silica gel, eluting with a gradient of CHCl3 (saturated with ammonia water)-MeOH (30:1-5:1), to yieldC2-2-4-4-1-C-2-2-4-4-6. Isolation of C2-2-4-4-5 (200 mg) bypreparative TLC [CHCl3 (saturated with ammonia water)-MeOH, 5:1] afforded C2-2-4-4-5-1-C-2-2-4-4-5-3. HPLC purification of C2-2-4-4-5-1 (Ultimate XB-Phenyl column, 10% MeCN in H2Ocontaining 0.1% TFA, 2 mL/min) obtained 1 (6 mg, tR = 19 min).Fraction C-2-2-4-6 (2.65 g) was further fractionated by CC oversilica gel, eluting with a gradient of CHCl3 (saturated withammonia water)-MeOH (20:1-5:1), to give C2-2-4-6-1-C2-2-4-6-11, of which fraction C2-2-4-6-6 (1.5 g) was separated by CC overreversed phase C-18 silica gel (30%-50% MeOH in H2O) to give C2-2-4-6-6-1-C2-2-4-6-6-3. Fraction C2-2-4-6-6-1 (1.1 g) was subjectedto preparative TLC [CHCl3 (saturated with ammonia water)-MeOH, 5:1] to yield C2-2-4-6-6-1-1 and C2-2-4-6-6-1-2. Furtherfractionation of C2-2-4-6-6-1-1 (0.9 g) by HPLC (Ultimate XBPhenylcolumn, 15% MeCN in H2O containing 0.1% TFA) yielded C2-2-4-6-6-1-1-1-C2-2-4-6-6-1-1-5. Fraction C2-2-4-6-6-1-1-2(350 mg) was separated by HPLC using the same column (10%MeCN in H2O containing 0.1% TFA, 2 mL/min) afforded C2-2-4-6-6-1-1-2-1-C2-2-4-6-6-1-1-2-5, of which C2-2-4-6-6-1-1-2-1 waspurified by HPLC (the same column, 20% MeOH in H2O containing0.1% TFA, 2 mL/min) to yield 3 (1 mg, tR = 35 min).
Aconicarmichinium A trifluoroacetate (1): Colorless gum; [α]D20+5.6 (c 0.31, MeOH); UV (MeOH) λmax (log ε) 203 (3.3) nm; CD (MeOH) 200 (Δ ε + 1.90) nm; IR νmax 3384, 2936, 1678, 1467, 1422, 1357, 1318, 1202, 1133, 1062, 1037, 1016, 956, 913, 888, 835, 801, 721 cm-1; 1H NMR (MeOH-d4, 600 MHz) data, see Table 1; 13CNMR (MeOH-d4, 150 MHz) data, see Table 1; 1H NMR (pyridine-d5, 600 MHz) data, see Table 2; 13C NMR (pyridine-d5, 150 MHz) data, see Table 2; (+)-ESIMSm/z 358; (+)-HR-ESIMSm/z 358.2382 (calcd.for C22H32NO3, 358.2377).
Aconicarmichinium B trifluoroacetate (2): Colorless gum; [α]D20-68.0 (c 0.09, MeOH); UV (MeOH) λmax (log ε) 203 (4.09), 245(2.98, sh), 295 (2.65) nm; CD (MeOH) 225 (Δ ε + 2.69), 297 (Δ ε- 3.22) nm; IR νmax 3357, 2923, 2852, 1683, 1467, 1423, 1351, 1205, 1137, 1062, 1014, 945, 908, 884, 840, 802, 722 cm-1; 1HNMR (MeOH-d4, 600 MHz) data, see Table 1; 13C NMR (MeOH-d4, 150 MHz) data, see Table 1; 1H NMR (pyridine-d5, 600 MHz) data, see Table 2; 13C NMR (pyridine-d5, 150 MHz) data, see Table 2; (+)-ESIMS m/z 356; (+)-HR-ESIMS m/z 356.2220 (calcd. for C22H30NO3, 356.2226).
Aconicarmichinium C chloride (3): Colorless gum; [α]D20 +23.7 (c0.08, MeOH); UV (MeOH) λmax (log ε) 203 (3.43) nm; CD (MeOH)200 (Δ ε + 5.61), 241 (Δ ε - 0.48) nm; IR νmax 3359, 2924, 2852, 1678, 1468, 1426, 1317, 1280, 1204, 1136, 1066, 1030, 963, 877, 840, 802, 723 cm-1; 1H NMR (MeOH-d4, 600 MHz) data, seeTable 1; 13C NMR (MeOH-d4, 150 MHz) data, see Table 1; (+)-ESIMSm/z 374; (+)-HR-ESIMS m/z 374.2331 (calcd. for C22H32NO4, 374.2326).
2.4. Preparation of 2a
Compound 2 (27.5 mg) was suspended in a saturated aqueoussolution of NaHCO3 (3 mL), and partitioned with EtOAc (3 mL × 3).The combined EtOAc phase was evaporated under reducedpressure at 40 8C to yield a residue, which was purified by CCover Sephadex LH-20 (MeOH) to obtain 2a (21.0 mg): colorlessprism (MeOH), [α]D20 -52.85 (c 0.77, MeOH); UV (MeOH) λmax (log ε) 206 (3.28), 251 (2.54), 292 (1.97) nm; CD (MeOH) 200(Δ ε - 5.97), 223 (Δ ε +3.36), 296 (Δ ε - 2.04) nm; IR νmax 3418, 2958, 2901, 2861, 2826, 1716, 1656, 1456, 1373, 1348, 1307, 1236, 1192, 1173, 1127, 1107, 1075, 1059, 973, 928, 881, 840, 707 cm-1;1H NMR (MeOH-d4, 600 MHz) data, see Table 2; 13C NMR (MeOH-d4, 150 MHz) data, see Table 2; (+)-ESIMS m/z 356[M+H]+, 378 [M+Na]+; (+)-HR-ESIMS m/z 356.2225 [M+H]+ (calcd.for C22H30NO3, 356.2220).
2.5. X-ray crystallography of 2a
C22H29NO3, M = 355.46, orthorhombic, a = 8.9252 (5)Å , b = 13.1639 (7)Å , c = 15.3374 (13)Å , a = b = g = 908, V = 1802.0(2)Å 3, space group P212121, Z = 4, Dcalcd = 1.310 g/cm3, μ (Cu Ka) = 0.684, 11, 880 reflections measured, 3450 reflectionsindependent, 3326 reflections observed. R1 = 0.0347, wR2 = 0.0891(w = 1/sjFj2), S = 1.039. The crystallographic data were collected onan Agilent Xcalibur Eos Gemini diffractometer with Cu Karadiation using the v scan technique to a maximum 2u value of142.388. The crystal structures were solved by direct methodsusing the SHELXS-97 program , and all non-hydrogen atomswere refined anisotropically by the least-squares method. Allhydrogen atoms were positioned by geometrical calculations and difference Fourier overlapping calculation. The absolute configurationwas determined on the basis of the Flack parameter -0.08(11). Crystallographic data for the structure of 2a have beendeposited with the Cambridge Crystallographic Data Centre assupplementary publication (CCDC 1470499). Copies of these datacan be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, CambridgeCB21EZ, UK; fax: +44 1223 336 033; or e mail: email@example.com. Results and discussion
Compound 1 was isolated as a colorless gum with [α]D20 +5.6 (c0.31, MeOH). Its IR spectrum exhibited absorption bands assignableto hydroxyl (3384 cm-1) and iminium (1678 cm-1) functionalities. The 1H NMR spectrum of 1 in MeOH-d4 showedsignals attributable to a typical exocyclic double bond at dH 5.16(brd, J = 2.4 Hz, H-17a) and 5.13 (brs, H-17b); four oxygen- and/ornitrogen-bearing methines at dH 4.44 (brs, H-20), 4.16 (t, J = 2.4 Hz, H-15), 4.04 (dd, J = 6.6 and 9.0 Hz, H-1), and 3.52 (dd, J = 6.6 and10.2 Hz, H-12); an ethyl unit at dH 4.07 and 3.99 (1H each, dq, J = 13.8, 7.2 Hz, H2-21) and 1.53 (t, J = 7.2 Hz, H3-22); a tertiarymethyl group at dH 1.30 (s, H3-18); and several aliphatic methineand methylene units between dH 3.04 and 1.14; as well as a signaldue to an isolated iminium methine at dH 8.43 (s, H-19).The 13C NMR and DEPT spectra showed carbon signals correspondingto the above units and three aliphatic quaternary carbons (Table 1). As compared with those of the previously isolatedalkaloids from "fu zi", these spectroscopic data suggest that 1 is anabnormal C20-diterpenoid alkaloid possessing the iminiummethine unit (δC 184.8) in the skeleton, of which the structurewas further elucidated by 2D NMR data analysis.
The proton resonances and corresponding proton-bearingcarbon resonances in the NMR spectra were assigned based onanalysis of the HSQC spectroscopic data. In the 1H-1H COSYspectrum of 1, cross-peaks of H-1/H2-2/H2-3, H-5/H2-6/H-7/H-20, H-9/H2-11/H-12, H-13/H2-14, H-13/H2-17/H-15, and H2-21/H3-22revealed the presence of four vicinal and one allylic homonuclearcoupling systems (Fig. 2, thick lines). In the HMBC spectrum of 1, two- and three-bond correlations (Fig. 2, arrows) from H2-3 to C-4, C-5, C-18, and C-19; from H-5 to C-3, C-18, and C-19; from H3-18 toC-3, C-4, C-5, and C-19; and from H-19 to C-4, C-5, and C-18indicated a linkage of the quaternary C-4 with C-3 and C-5 of the two coupling systems, as well as with the methyl (CH3-18) and the iminium methine (CH-19). The HMBC correlations from H-7 to C-8, C-9, and C-14; from H-9 to C-7, C-8, C-14, and C-15; from H2-14 toC-7, C-8, C-9, and C-15; and from H-15 to C-7, C-8, and C-9 revealeda connection of the quaternary C-8 with C-7, C-9, C-14 and C-15. Anattachment of the remaining quaternary C-10 with C-1, C-5, C-9, and C-20 was established by the HMBC correlations from both H-1and H-5 to C-9, C-10, and C-20; from H-9 to C-5, C-10, and C-20;and from H-20 to C-5 and C-10. Meanwhile, the HMBC correlationsfrom H2-11 to C-13; from H-12 to C-13, C-14, and C-16; and fromH2-14 to C-12 and C-13 demonstrated the connection betweenC-12 and C-13, though no coupling cross-peak between H-12 and H-13 was observed in the 1H-1H COSY spectrum possibly due to aperpendicular dihedral angle between the two protons. Additionally, the HMBC spectrum showed correlations from H-19 to C-20and C-21; from H-20 to C-19 and C-21; and from H2-21 to both C-19 and C-20. This, together with the chemical shifts and couplingpatterns of the proton and carbon resonances, suggests that the iminium methine (CH-19) must connect via the nitrogen atom toC-20 and C-21 to provide a parent structure of napelline-type C20-diterpenoid alkaloid iminium for 1. Three hydroxyl groups wereaccordingly located at C-1, C-12, and C-15 based on the chemicalshifts of these carbons and their attaching protons, together withthe formula of the alkaloid iminium cation as determined by (+)-HR-ESIMS at m/z 358.2382 (358.2377 calcd. for C22H32NO3). In the NOESY spectrum of 1, the NOE correlations (Fig. 3) between H-5with H-1, H-6a, H-9, and H3-18; between H-7 with H-6b, H-14b, and H-15; between H-15 with both H-14b and H-17b; between H-13 with H-12, H-14b, and H-17a; and between H-12 and H-17ademonstrated that the diterpenoid iminium in 1 possessed the same relative configuration as the co-occurring napelline . the absolute configuration of the parent diterpenoid alkaloid moiety in1 is proposed to be also identical to napelline, which was supportedby single-crystal X-ray crystallographic analysis of 2a (see below).
|Figure 2. Main 1H-1H COSY (thick lines) and three-bond HMBC correlations (red arrows, from 1H to 13C; two-bond correlations and negative counterparts were omitted forclarity) of 1-3.|
|Figure 3. Main NOESY correlations (pink dashed double arrows, negative counterparts were omitted) of 1-3.|
Because trifluoroacetic acid (TFA) was used in the HPLCpurification of 1 and the alkaloid iminium must be positivelycharged, Compound 1 was considered being obtained as atrifluoroacetate. Although the 13C resonances of TFA anion (TFA-) were not observed in the 13C NMR spectrum and the quasimolecularion peak for the expected iminium trifluoroacetate wasabsent in the negative mode ESI mass spectrum (Fig. S4 inSupporting information), the 19F NMR spectrum of 1 displayed a19F resonance at dF -76.5. Quantitative 19F NMR analysis of 1 inMeOH-d4, using hexafluorobenzene (C6F6) as an internal stand ard (Fig. S13 in Supporting information) confirmed the presence of anapproximate 1:1 ratio of the alkaloid iminium cation and TFA anionin the sample. the refore, the structure of 1 was determined asshown and named aconicarmichinium A trifluoroacetate. the absence of the 13C resonances of TFA- in the 13C NMR spectrummay be explained by a combination of multiple reasons, includingspin coupling-split of the 13C resonances by the 19F nucleus, conjugation between different anion species commonly consideringfor carboxylic groups, and interactions of the anion specieswith the iminium cation and solvent.
Compound 2 was isolated as a white amorphous powder with[α]D20 -68.0 (c 0.09, MeOH). The IR and NMR spectroscopic features of 2 were similar to those of 1. Comparison of the 13C NMR spectraldata between 2 and 1 (Table 1) indicated that a carbonyl group (δC 211.2, C-12) in 2 replaced the hydroxymethine (CHOH-12) in 1.Meanwhile, the C-11, C-13, C-14, and C-17 resonances in 2 weredeshielded by δC +8.0, +6.4, +3.1, and +3.0 ppm, respectively, whereas C-16 was shielded by δC -7.8 ppm. This suggests that 2is a 12-oxo derivative of 1. The suggestion was confirmed by HRESIMSat m/z 356.2220 [C22H30NO3]+ and 2D NMR data analysis, particularly by the HMBC correlations of C-12 with H2-11, H-13, and H2-14. Similarity of the coupling constants of H-1 and H-15between 2 and 1 (Table 1) indicated that the hydroxyl groups at C-1and C-15 in the two compounds had the same orientations, whichwas verified by the cross-peaks between H-5 with H-9 and H3-18and between H-7 with H-14b and H-15 in the NOESY spectrum of2. The absolute configuration was verified by converting 2 tosongoramine (2a) (see below). Compound 2 was proved to beobtained as the trifluoroacetate by quantitative 19F NMR analysisas described for 1 (Fig. S24 in Supporting information). the refore, the structure of 2 was determined as shown. The alkaloid moiety in 2 is identical to that in "songoramine hydrochloride", which wasprepared from 2a and characterized only by the IR spectral data. Since songoramine (2a) is not the free base of the chloride or 2wherein the alkaloid structures are completely different, the alkaloid iminium was named aconicarmichinium B to avoidconfusion. Accordingly, compound 2 was named aconicarmichiniumB trifluoroacetate.
Compound 3 was obtained as a colorless gum with [α]D20 +23.7 (c0.08, MeOH). Comparison of the NMR spectroscopic data between3 and 2 (Table 1) demonstrated that the two compounds differedonly in replacement of the methylene unit (CH2-11) in 1 by anoxymethine unit [δH 4.14 (dd, J = 10.8, 7.2 Hz); dC 73.0] in 3, suggesting that 3 is a 11-hydroxy derivative of 1. This wassupported by HR-ESIMS at m/z 374.2331 [C22H32NO3]+ and confirmed by 2D NMR data analysis of 3. Especially the 1H-1HCOSY cross-peaks between H-11 with both H-9 and H-12 and the HMBC correlations from H-9 to C-8, C-10, C-11, C-12, and C-14;from H-11 to C-8 and C-9; from H-12 to C-11 and C-14; and fromH-13 to C-8, C-11, and C-12, together with their chemical shifts, verified the presence of the hydroxyl group at C-11 in 3. Anequatorial b-orientation of OH-11 in 3 was indicated by the coupling constant (J9, 11 = 10.8 Hz, Table 1) of trans-orientedprotons between H-9 and H-11 and confirmed by the NOESYcorrelations (Fig. 3) between H-1 with both H-5 and H-9, betweenH-9 and H-12, between H-14a with both H-11 and H-20, and between H-15 with both H-7 and H-14b. Because 3 couldbiogenetically be derived from hydroxylation of 1, the absoluteconfiguration of the napelline nucleus in 3 was assigned to beidentical with that of 1 and 2. Interestingly, although TFA was alsoused in the HPLC isolation of 3, both the 13C and 19F NMR spectradid not show resonances attributable to TFA- (Fig. S35 inSupporting information). This suggests that the anion counterpartin 3 was not replaced by TFA in the HPLC mobile phase. Since HClwas used in fractionation step of the extract, the anion in 3 wasproposed to be Cl-, but not confirmed due to limitation of the sample amount. the refore, the structure of 3 was determined andtentatively named aconicarmichinium C chloride.
According to the reported chemical transformation from the azaacetal-containing napelline-type C20-diterpenoid alkaloids to the alcohol iminium salts [1, 5, 54, 55], compounds 1-3 could readily beformed from the corresponding aza acetals, while the counterpartswere induced by using HCl and TFA in fractionation and HPLCseparation steps of this study. However, the transformationconditions were not detailed in the literatures, and hydrophobicproperties of the aza acetals suggested that in the in vivo hydrophilic environments the occurrence of the alcohol iminiums of these diterpenoid alkaloids would not been excluded because 1-3 were actually isolated from the aqueous extract. In addition, the previous studies demonstrated that the alkaloids sanguinarine and chelerythrine [11-19] could be transformed and dynamicallyequilibrated between the different alkaloid species (alkanolamineand iminium) under the relatively stable and neutral biologicalenvironments to play and/or manipulate biological roles of the alkaloids with the structural alterations and equilibrations.Therefore, condition details become important to address whatmajor factors would possibly interfere in the transformation and equilibration between the alcohol iminium and aza acetal forms of the diterpenoid alkaloids in vitro and whether the transformationand equilibration occur in vivo.
To investigate effects of the solvents and base on the transformation and equilibration, the NMR spectra of 1 and 2were acquired using the aprotic solvents deuterium acetone-d6and pyridine-d5 (base), respectively, which were selected based onsolubility of the compounds. Although 1 and 2 were well dissolvedin acetone-d6, the 1H NMR spectra of 1 and 2 (Figs. S36 and S38 inSupporting information) showed that almost all the 1H resonanceswere severely broadened and unresolved with integrations out of proportion. Approximate 0.48 and 0.52 integrations of the specificiminium methine protons (δH 8.70 for 1 and 8.83 for 2) wereestimated when the distinguished allylic proton integrations wereused as the internal references. Especially no resonance or weakresonances were detectable in the 13C NMR spectra of 1 or 2 inacetone-d6 (Figs. S37 and S39 in Supporting information) with the same sample amounts and scanning numbers as those in MeOH-d4.These observations suggested the presence of dynamic equilibrationsbetween the diterpenoid alkaloid species and that onlyaround half amounts of the alcohol iminiums were maintained inthe acetone-d6 solutions of 1 and 2. Meanwhile, the broadened 1Hand undetectable or weak 13C signals indicate that in the NMR timescale either the structures of the alkaloid species are fluctuating orthe equilibrations are taking place in a relatively slow manner. Ascompared with those in the protic solvent MeOH-d4 (Fig. S7 and S18 in Supporting information), in the aprotic solvent acetone-d6the alcohol iminiums 1 and 2 are unstable and there areequilibrations between the alcohol iminium and aza acetalforms of which the structures are relatively non-static. Thus, the protic solvent play crucial roles to transfer and stabilize the alcohol iminiums from the aza acetals of these diterpenoidalkaloids.
The 1H NMR spectra of 1 and 2 in pyridine-d5 (Figs. S40 and S42in Supporting information) displayed sharp and resolvableresonances assignable to only one alkaloid species for each.However, the spectra showed that the diagnostic resonances forthe iminium methines (δH ∼8.43 and dC ∼184.8) of 1 and 2 inMeOH-d4 were replaced by signals assignable to the aza acetalmethines (δH ∼3.82 and dC ∼93.2) in pyridine-d5. This demonstratedthat the alkaloid cations in 1 and 2 were completelyconverted into corresponding aza acetals 1a and 2a in pyridine-d5(Scheme 1). The conversion was proved unambiguously by 2DNMR data analysis of 1 and 2 in pyridine-d5, which assistedassignments of the NMR spectroscopic data (Table 2). Especiallythe HMBC correlations from H-1 to C-19 and from H-19 to C-1, together with their chemical shifts, verified the formation of theaza acetals in the pyridine-d5 solutions of 1 and 2. Accordingly, the structures of the aza acetals 1a and 2a were assigned to be identicalto dehydronapelline  and songoramine , respectively. Afterremove of pyridine-d5 by evaporation under reduced pressure, the reacquired NMR spectra of residues in MeOH-d4 exhibitedresonances completely overlapped with those of 1 and 2 in the same solvent, indicating that the aza acetals were fully returned tothe alcohol iminiums (Figs. S44 and S45 in Supporting information).This demonstrates that the basicity of pyridine-d5 (pH10.33 ± 0.01 for the pure solvent) is the key factor for the transformation from the alcohol iminiums to the aza acetals of the diterpenoid alkaloids, suggesting the occurrence of pH-sensitive or base-dependent transformation and equilibration from the iminiumsto the aza acetals of the diterpenoid alkaloids in solution state. the suggestion was supported by further measurements of the NMRspectra of 1 and 2 in the solvent mixtures increasing pyridine-d5 inMeOH-d4. As shown in Figs. 4 and 5, with increase of pyridine-d5, the resonances due to the aza acetals 1a and 2a were gradually enhancedwhereas signals for the alkaloid iminiums in 1 and 2 werecorrespondingly diminished. When the MeOH-d4:pyridine-d5 ratiowas 1:5 (v/v, pH 9.90 ± 0.00 for the pure solvent mixture), the conversion from the alcohol iminium in 1 into 1a was completed.However, the larger pyridine-d5 ratio (MeOH-d4:pyridine-d5, 1:15, v/v, pH 10.16 ± 0.02 for the pure solvent mixture) was requiredto fully convert 2 into 2a. This indicates that 2a is more basic than 1aand that basicity of the diterpenoid alkaloid 2a is enhanced bydehydrogenation of the hydroxy group at C-12. the refore, in the protic solvent MeOH-d4 there are the base-sensitive/dependenttransformation and equilibration from the alcohol iminiums to theaza acetals.
|Figure 4. The overlaid 1H NMR spectra of 1 (600 MHz) in MeOH-d4, solvent mixtures increasing pyridine-d5 in MeOH-d4, and pyridine-d5. The same sample (2.5 mg) wasrepeatedly used after evaporation under reduced pressure and the same volume (0.6 mL) of the solvent mixtures was applied.|
|Figure 5. The overlaid 1H NMR spectra of 2 (600 MHz) in MeOH-d4, solvent mixtures increasing pyridine-d5 in MeOH-d4, and pyridine-d5. The same sample (3.5 mg) wasrepeatedly used after evaporation under reduced pressure and the same volume (0.6 mL) of the solvent mixtures was applied.|
To further explore influences of the solvents and acids on the reverse conversion from the aza acetals to the alcohol iminiums, Compound 2 (obtained in a relatively large amount) waspartitioned between EtOAc and a saturated aqueous solution of NaHCO3. Followed up evaporation of the EtOAc phase and purification by CC over Sephadex LH-20 (MeOH) afforded 2a.The structure of 2a, including the absolute configuration, wasidentified by extensive spectroscopic analysis and confirmed bysingle-crystal X-ray crystallographic analysis using anomalousscattering of Cu Ka radiation. The ORTEP drawing, with the atomnumberingindicated, is shown in Fig. 6. Then the NMR spectra of2a were measured in the aprotic deuterium solvents acetone-d6and CDCl3 and the protic deuterium solvents MeOH-d4 and D2O, respectively, as well as in MeOH-d4 and D2O with gradual additions of TFA, AcOH, or HCl in a quantitative manner calculating the acid:2a molar ratios.
In the aprotic acetone-d6 and CDCl3 and the protic MeOH-d4, the NMR spectra of 2a (Figs. S52-S57 in Supporting information) displayed sharp and resolvable signals due to the identical azaacetal structure which was confirmed by 2D NMR data analysis inCDCl3 and MeOH-d4, even though only a partial amount of the sample 2a in the NMR tube was dissolved in CDCl3. However, the 1H NMR spectrum of the turbid solution of 2a in D2O showedthe presence of equilibration between 2a and the alcohol iminiumin an approximate 3:1 ratio (Figs. S61-S63 in Supportinginformation). These results indicated that the aza acetal 2a hadrelatively high solubility and stability in the aprotic and proticorganic solvents, but less soluble, unstable, and equilibrated withthe alcohol iminium in D2O. Thus, the reverse transformation and equilibration from the aza acetal to the iminium indeed exist in the pure aqueous solution of the diterpenoid alkaloid.
By increasing the molar ratio of TFA, AcOH, or HCl, the NMRspectra of 2a in MeOH-d4 (pH 7.85 ± 0.03 for the pure solvent) showed that the resonances due to the aza acetal 2a were graduallydiminished, in contrast the resonances attributable to the alcoholiminium were correspondingly enhanced (Figs. 7-9). Although all the three acids converted 2a into the alcohol iminium in the MeOH-d4solutions, the different acid:2a molar ratios were required tomaintain the equilibration between 2a and the alcohol iminium (TFA:2a < 1.5; AcOH:2a < 20; HCl:2a < 0.6) and to completelyconvert 2a into the alcohol iminium (TFA:2a ≥ 1.5; AcOH:2a ≥ 20;HCl:2a ≥ 0.6). In addition, after 2a was fully transformed into the alcohol iminium by addition of TFA, AcOH, or HCl, the samples wereevaporated under reduced pressure at 40 8C. Re-acquirement of the NMR spectra of the residues in MeOH-d4 demonstrated that the alcohol iminium transformed by AcOH was completely returned tothe aza acetal 2a by evaporation, whereas conversions induced by TFAand HCl were unchanged. This indicated that interactions of the diterpenoid alkaloid with TFA and HCl were stronger than with AcOHand that the alcohol iminiums could capture the TFA and HCl anionsto form relative stable salts but not AcOH. the refore, the transformationand equilibration from 2a into the alcohol iminium in MeOH-d4were not only dependent on acidities of the acids, but also on the acidspecies.
|Figure 7. The overlaid 1H NMR spectra of 2a (21 mg) in MeOH-d4 with gradual additions of TFA (600 MHz). Calculated volumes of a stand ard MeOH-d4 solution of TFA (1.35mol/L) were added using a 10 mL syringe in a step by step manner.|
|Figure 8. The overlaid 1H NMR spectra of 2a (21 mg) in MeOH-d4 with gradual addition of AcOH (600 MHz). Calculated volumes of a stand ard MeOH-d4 solution of AcOH (3.55 mol/L) or AcOH (99.8%) were added using a 10 mL syringe in a step by step manner.|
In D2O (pH 7.50 ± 0.01 for the pure solvent), with the gradualincreases of TFA, AcOH, or HCl in the NMR tubes, 2a was graduallydissolved. The corresponding NMR spectra (Figs. 10 and S61-S63 inSupporting information) revealed that only the alcohol iminiumexisted in the solutions even with addition of a small amount of the weak acid AcOH (AcOH:2a molar ratio = 0.2). After evaporation of the D2O solutions, the NMR spectra of the residues in MeOH-d4 indicatedthat the conversions induced by TFA and HCl were retained but the conversion induced by AcOH was reversed. This, together with the above results, confirmed that the acidity and species of the acids, aswell as the solvents, play the crucial roles in the transformation and equilibration from the aza acetal 2a into the alcohol iminium. the confirmation supports that, in the aqueous biological fluid with pHless than 7.50, the napelline-type C20-diterpenoid alkaloids havingthe similar structure as 2a would more likely exist as the alcoholiminium forms accompanied by various anion counterparts  toincrease their solubility, bioavailability, and transportations, as wellas to play biological functions. Nevertheless, the aza acetal forms, along with the equilibration, would also be functioned in specific and local alkali (pH > 7.50) microenvironments of the bio-systems.
Because no obvious byproduct was observed in all the NMRspectra, the transformations from the iminiums into the aza acetalswere predominated by anintramolecular reactionwithanucleophilic attack at the iminium carbon. This suggests that the iminium carbonin the diterpenoid alkaloids is electron-deficient and that the positivecharge is delocalized on the iminium carbon (sharing more positivecharge) and the other nitrogen-bearing carbons (sharing relativelyless positive charge). The suggestion is supported by the chemicalshifts of the nitrogen-bearing units (CH-19, CH-20, and CH2-21, Table 1) in the NMR spectra, especially of the iminium methine (δH > 8.43; dC >184.8, Table 1). Consequently, we speculate that the iminium nitrogen atom is an electron-rich center to bond with the carbons, rather than a positive charge carrier. For the reversetransformations from the aza acetals into the alcohol iminiums, theetheric oxygen readily reacts with the minor acids to produce thealcohol iminium, instead of the nitrogen to give a tertiary amine salt.This indicates that the aza acetal tertiary amine salts of the sediterpenoid alkaloids may not exist at least under the commonconditions.
In the preliminary in vitro assays, the alcohol iminium 1inhibited lipopolysaccharide (LPS)-induced NO production incultivated murine microglial cell line BV-2 with 42.1% inhibitionat 10 mmol/L, while the positive control curcumin gave 87.3%inhibition at the same concentration . Compound 1 alsoshowed antiviral activity against the influenza virus A/Hanfang/359/95 (H3N2) with IC50 and SI values of 33.3 mmol/L and 3.0, respectively, the positive control, ribavirin, gave IC50 = 1.71mmol/L and SI [2TD$DIF]= 680.8 . Other assays, including influence onsynchronized Ca2+[1TD$DIF] oscillation in cultivated cardiac muscle cell;activity against KCNQ2 potassium channel in CHOK1 cells;protection against L-glutamic acid-induced SK-N-SH neuroblastomacell damage; inhibition against Fe2+-cysteine induced ratliver microsomal lipid peroxidation; antiviral activity againstcoxsackie virus B3 type (CVB3); and cytotoxicity against severalhuman cancer cell lines were also performed in this study.However, the alcohol iminium and aza acetal forms were inactiveat a concentration of 10 mmol/L in each assay.
|Figure 9. The overlaid 1H NMR spectra of 2a (21 mg) in MeOH-d4 with gradual additions of HCl (600 MHz). Calculated volumes of a stand ard MeOH-d4 solution of HCl (0.60 mol/L) were added using a 10 mL syringe in a step by step manner.|
|Figure 10. The overlaid 1H NMR spectra of 2a (7.0 mg) in D2O with additions of a calculated 1:0.2 molar ratio of 2a:TFA, 2a:AcOH, or 2a:HCl at 600 MHz. A stand ard D2O solution of TFA (0.40 mol/L), AcOH (0.80 mol/L), or HCl (0.40 mol/L), was added using a 10 mL finnpipette.|
From the aqueous extract of the lateral roots of A. carmichaeliii, three C20-diterpenoid alkaloids 1-3 were isolated. These compoundsrepresent the first examples of napelline-type C20-diterpenoid alkaloid iminiums with the structures characterizedby the comprehensive spectroscopic data. Although the anioncounterparts in 1-3 should be induced by using TFA and HCl in the HPLC isolation and fractionation steps of the experiments, thedetailed investigations reveal that the transformations and equilibrations between the alcohol iminiums (1-3) and the azaacetals (1a-3a) are solvent-, base-, and acid-dependent. Especially, in the hydrophilic bio-systems (pH < 7.50), these alkaloids morelikely exist as the alcohol iminiums accompanied by the in vivo anions to increase their solubility, bioavailability, and transportations, as well as to play potential functions. However, under the specific conditions of the varied bio-systems, such as the local and alkali (pH ≥ 7.50) microenvironments, the functions of the azaacetals, along with the equilibrations, would not be excluded.Based on the intramolecular transformation reactions, togetherwith the chemical shifts of the nitrogen-bearing units, the positivecharges in 1-3 are delocalized on the nitrogen-bearing unitsparticularly on the iminium methines, instead of centralized on thenitrogen atoms as usually considered. In addition, the aza acetalsalts of these diterpenoid alkaloids may not exist under thecommon conditions. Although weak inhibitory activities againstNO-production and influenza virus A/Hanfang/359/95 (H3N2) were found only for 1, the potential activities of the different forms of these C20-diterpenoid alkaloids and contributions of the diversestructures are still expected from in-deep evaluations in future. Inaddition, this study, along with our previous observation of TFAinducedconformational variations of ring A in the C19-diterpenoidalkaloids [51, 61, 62], suggests the presence of the transformationsand equilibrations between the different forms/species of thediterpenoid alkaloids under the experimental and biologicalconditions. Because the transformations and equilibrations, alongwith the structure variations, are critical for their solubility, bioavailability, transportations, and biological functions in thebiological systems, on a case by case basis, in-depth chemical and biological studies are required and of greatly interesting to seemultiple faces of the diterpenoid alkaloids .Acknowledgments
Financial support from the National Natural Science Foundation of China (NNSFC; Nos. 21132009, 30825044) and the NationalScience and Technology Project of China (Nos. 2012ZX09301002-002, 2011ZX0 9307-002-01) is acknowledged.
Appendix A. Supplementary dataSupplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.cclet.2016.05.013.
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