Chinese Chemical Letters  2015, Vol.26 Issue (01):69-72   PDF    
Two 1-(6'-O-acyl-β-D-glucopyranosyl)pyridinium-3-carboxylates from the flower buds of Lonicera japonica
Zhi-Bo Jiang, Wei-Xia Song, Jian-Gong Shi     
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract: Two new 1-(6'-O-acyl-β-D-glucopyranosyl)pyridinium-3-carboxylates, lonijaponinicotinosides A (1) and B (2), were isolated from an aqueous extract of the flower buds of Lonicera japonica. Their structures were determined by spectroscopic data analysis, and confirmed by comparison with synthetic 1-β-D-glucopyranosylpyridinium-3-carboxylate.
Key words: Lonicera japonica     Caprifoliaceae     1-(6'-O-Acyl-β-D-glucopyranosyl)pyridinium-3-carboxylates     1-β-D-Glucopyranosylpyridinium-3-carboxylate     Isolation     Structure elucidation    
1. Introduction

In traditional Chinese medicine,the flower buds of Lonicera japonica Thunb. (Caprifoliaceae),Jin Yin Hua,is a common ingredient of formulations used for treating influenza,cold,fever, and infections [1]. More than a hundred of chemical constituents including caffeoyl quinic acids,secoiridoids,flavonoids,saponins, cerebrosides,and polyphenols [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13] have been reported from extracts of this plant. However,the previous studies were mainly focused on the constituents with a relatively lower polarity from the alcoholic extracts,which is not consistent with the practical application of decoctions. As part of a program to assess the chemical and biological diversity of traditional Chinese medicines [14, 15, 16],we conducted detailed chemical analysis of an aqueous extract of the flower buds of L. japonica. In previous papers [17, 18, 19, 20], we have reported the isolation and biological assay of 29 homosecoiridoids with unusual structural characters of the secoiridoid nucleus coupled with N-substituted nicotinic acid or pyridine units,phenylpyruvic acid derived moieties,and β-hydroxy amino acid units,respectively. In addition,after the flower buds were extracted by water,the residue was extracted with EtOH (95%), from which six new aromatic glycosides and 48 known compounds were characterized [21, 22]. Continuing examination of the aqueous extract have resulted in isolation and structure characterization of two new 1-(6’-O-acyl-β-D-glucopyranosyl)pyridinium- 3-carboxylates,named lonijaponinicotinosides A (1) and B (2) (Fig. 1).

Fig. 1. Structures of compounds 1 and 2.
2. Experimental 2.1. General experimental procedures

Optical rotations were measured on a PE Model 343. UV spectra were measured on a JASCO J-810 spectropolarimeter. IR spectra were recorded on a Nicolet 5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission). 1D and 2D NMR spectra were obtained at 500 or 600 MHz for 1H and 125 or 150 MHz for 13C,respectively,on an Inova 500 MHz or Bruker AV III spectrometer with solvent peaks as references (unless otherwise noted). ESIMS data were measured with a Q-Trap LC/MS/MS (Turbo Ionspray source) spectrometer. HR-ESIMS data were,in turn,measured on an AccuToFCS JMS-T100CS spectrometer. Column chromatography was performed with silica gel (200-300 mesh,Qingdao Marine Chemical Inc.,Qingdao,China) and Pharmadex LH-20 (Pharmacia Biotech AB,Uppsala,Sweden). HPLC separation was performed on an instrument with a Waters 600 controller,a Waters 600 pump,and a Waters 2487 dual l absorbance detector on an Prevail (250 mm × 10 mm i.d.) semipreparative column packed with C18 (5 μm). Glass precoated silica gel GF254 plates were used for TLC. Spots were visualized underUV light or by sprayingwith 7% H2SO4 in 95% EtOHfollowed by heating. 2.2. Plant material

See Ref [14]. 2.3. Extraction and isolation

For extraction and preliminary fractionation of the extract,see Refs [14, 15]. Fraction B4 (86 g) was chromatographed over a RP silica gel column,eluting with a gradient of EtOH (0-100%) in H2O, to yield subfractions B4-1-B4-7,of which subfraction B4-7 (1.4 g) was further separated by flash chromatography over RP silica gel, eluting with a gradient of MeOH (0-50%) in H2O to give subfractions (B4-7-1-B4-7-4). B4-7-2 (326 mg) was subjected to RP-HPLC,using CH3CN-H2O (13:87) containing 0.5% HOAc as the mobile phase,to afford 1 (24.1 mg) and 2 (10.4 mg).

Lonijaponinicotinoside A (1): White amorphous powder,soluble in H2O,insoluble in MeOH,and EtOH; [α]D20 +33.0 (c 1.12,H2O); UV (H2O) λmax (log e) 265 (3.57) nm; IR nmax 3228,2963,1729, 1636,1610,1580,1492,1452,1363,1294,1193,1155,1076,1023, 946,922,818,771,682 cm-1; 1HNMR(D2O,500 MHz),see Table 1; 13C NMR (D2O,125 MHz),see Table 1; (+)-ESIMS m/z 370 [M+H]+, 392 [M+Na]+,408 [M+K]+; HR-ESIMS m/z 370.1490 [M+H]+ (calcd. for C17H24NO8 370.1502).

Table 1
NMR spectroscopic data for compounds 1 and 2.a

Lonijaponinicotinoside B (2): White amorphous powder,soluble in H2O,insoluble in MeOH,and EtOH; [α]D20 +22.1 (c 0.39,H2O); UV (H2O) λmax (log e) 230 (3.85),257 (3.63),299 (3.09) nm; IR nmax 3228,1682,1647,1612,1579,1540,1394,1285,1204,1135,1025, 838,802,772,722,676 cm-1; 1HNMR(D2O,500 MHz),see Table 1; 13C NMR (D2O,125 MHz),see Table 1; (+)-ESIMS m/z 368 [M+H]+, 390 [M+Na]+,406 [M+K]+; HR-ESIMS m/z 368.1377 [M+H]+ (calcd. for C17H22NO8 368.1345). 2.4. Synthesis of 1-β-D-glucopyranosylpyridinium-3-carboxylate

2,3,4,6-O-Tretraacetyl-a-D-glucopyranosylbromide (480 mg), which was prepared by following the procedure described in literature [23],was reacted with methyl nicotinate (163 mg, Beijing Chemical Reagent Co.,) in CH2Cl2 at room temperature for 2 h,and the reaction mixture was evaporated under reduced pressure to yield a residue. The residue was hydrolyzed with 2 mol/L NaOH (5 mL) at 40 8C for 12 h,acidified to pH 4 with 6 mol/ L HCl,and extracted with EtOAc (3 × 5 mL). The EtOAc phase was evaporated under reduced pressure to yield a white amorphous powder (270 mg),which was identified as 1-β-D-glucopyranosylpyridinium- 3-carboxylate by the optical rotation and spectroscopic data: [α]D20 +32.9 (c 2.5,H2O) {[a]D 24 +41 (c 1.5,H2O)}[24]; 1H NMR (D2O,600 MHz,TMS): d 9.35 (s,1H,H-2),9.10 (d,1H, J = 6.6 Hz,H-6),9.00 (d,1H,J = 7.8 Hz,H-4),8.22 (dd,1H,J = 7.8 and 6.6 Hz,H-5),5.86 (d,1H,J = 9.0 Hz,H-1'),3.68 (t,1H,J = 9.0 Hz,H- 20),3.80 (dd,1H,J = 9.0 and 9.6 Hz,H-3'),3.73 (t,1H,J = 9.6 Hz,H- 40),3.88 (m,1H,H-50),4.01 (dd,1H,J = 12.6 and 1.8 Hz,H-6'a),3.91 (dd,1H,J = 12.6 and 5.4 Hz,H-60b); 13C NMR (D2O,6'' MHz,TMS) d 170.2 (C-7),150.5 (C-2),145.8 (C-6),145.5 (C-4),140.0 (C-3), 130.7 (C-5),98.0 (C-1'),82.4 (C-5'),78.2 (C-3'),76.9 (C-4'),71.5 (C- 2'),63.3 (C-6'); (+)-ESIMS m/z 286 [M+H]+,308 [M+Na]+; HR-ESIMS m/z 286.0922 [M+H]+ (calcd. for C12H16NO7 286.0921). 2.5. Alkali hydrolysis of 1 and 2

Each compound (~6 mg) in H2O (~1 mL) was hydrolyzed with NaOH (25 mg) at room temperature for 3 h. The hydrolysate was neutralized with 2 mol/L HCl and evaporated under reduced pressure to yield a residue. The residue was separated by RP-HPLC, using CH3CN-H2O (10:90) containing 0.5% HOAc as the mobile phase,to afford a product,which displayed specific rotation and spectroscopic data identical to those of the synthetic 1-β-Dglucopyranosylpyridinium- 3-carboxylate. 2.6. Assays for pharmacological activities of 1 and 2

Details may be found in Refs [14, 15, 16] and the references cited therein. 3. Results and discussion

Compound 1 was obtained as a white amorphous solid,[α]D20 +33.0 (c 1.12,H2O). Its IR spectrum showed absorption bands for hydroxy (3228 cm-1),carbonyl (1729 cm-1),and aromatic ring (1636,1610,1580,and 1492 cm-1) functional groups. The positive ESIMS of 1 exhibited pseudo molecular ion peaks at m/z 370 [M+H]+,392 [M+Na]+,and 408 [M+K]+. HR-ESIMS at m/z 370.1490 [M+H]+,together with the NMR data (Table 1),indicated the molecular formula C17H23NO8 (calcd. for C17H24NO8 370.1502). The 1H NMR spectrum of 1 in D2O showed resonances characteristic for a nicotinic acid moiety at δH 9.31 (s,H-2),9.08 (d,J = 5.5 Hz, H-6),9.03 (d,J = 8.0 Hz,H-4),and 8.24 (dd,J = 8.0 and 5.5 Hz,H-5) and a β-glucopyranosyl unit at δH 5.90 (d,J = 8.5 Hz,H-1'),3.74 (dd, J = 9.0 and 8.5 Hz,H-2'),3.83 (t,J = 9.0 Hz,H-3'),3.78 (t,J = 9.0 Hz, H-4'),4.09 (m,H-5'),4.60 (d,J = 12.5 Hz,H-6'a),and 4.48 (dd, J = 12.5 and 6.0 Hz,H-6'b). In addition,the 1H NMR spectrum displayed signals assignable to a 3-methybutyryl (isopentanoyl) unit at δH 2.32 (d,J = 7.0 Hz,H2-2''),2.04 (m,H-3''),0.91 (d,6H, J = 7.0 Hz,H3-4'' and H3-5''). The 13C NMR and DEPT spectra of 1 showed carbon resonances corresponding to the above units (Table 1),including two carbonyl carbons at δC 178.6 (C-1'') and 169.8 (C-7). These spectroscopic data suggested that 1 was an unusual plant metabolite consisting of β-glycopyranosyl,nicotinic acid,and isopentanoyl moieties. The suggestion was confirmed by comprehensive analysis of the 2D NMR data,which led to an unambiguous structure assignment of 1.

The proton and proton-bearing carbon signals in the NMR spectra of 1 were assigned by the gHMQC experiment. In the 1H-1H COSY spectrum of 1,cross-peaks H-4↔H-5↔H-6,H-1' ↔H- 2' ↔H-3' ↔H-4' ↔H-5' ↔H2-6',H2-2'' ↔H-3'' ↔H3-4''/H3-5'' (Fig. 2,thick lines) demonstrated the presence of the structural moieties with vicinal coupling chain extensions. In the HMBC spectrum of 1,two- and three-bond heteronuclear correlations (Fig. 2,arrows) from H-2 to C-3,C-4,C-6,and C-7; from H-4 to C-2, C-6,and C-7; from H-5 to C-3 and C-6; and from H-6 to C-2,C-4, and C-5,together with the chemical shifts and coupling constants of these proton and carbon resonances,confirmed the occurrence of the nicotinic acid moity. The HMBC correlations form H2-2'' to C- 1'',C-3'',C-4'',and C-5''; from H-3'' to C-1'',C-2'',C-4'',and C-5''; and from H3-4'' and H3-5'' to C-2'' and C-3'' verified the presence of the isopentanoyl unit. The HMBC correlations from H-2 and H-6 to C-10 and from H-10 to C-2 and C-6 revealed that the β-glucopyranosyl was located at the nitrogen atom to give a 1-glucopyranosylpyridinium- 3-carboxyloxy (N-glucopyranosylnicotinic acid) moiety in 1. In addition,HMBC correlations from H2-6' to C-1'' demonstrated that the isopentanoyloxy was linked to C-6' of the β-glucopyranosyl moiety. This,combined with the molecular formula,as indicated from the HR-ESIMS data,suggested that compound 1 must be obtained as an inner salt. Accordingly,the gross structure of 1 was elucidated as shown. The d-configuration of the bglucopyranosyl in 1 was determined by comparison of the [α]D20 value of 1 with that of the synthetic 1-D-glucopyranosylpyridinium- 3-carboxylate derivatives [24]. Therefore,the structure of compound 1 was determined as (+)-1-(6'-O-isopentanoyl-β-Dglucopyranosyl) pyridinium-3-carboxylate and designated as lonijaponinicotinoside A.

Fig. 2. Main 1H-1H COSY (black thick lines) and HMBC correlations (red arrows,from 1H to 13C) of 1 and 2.

Compound 2 was obtained as a white amorphous solid,[α]D20 +22.1 (c 0.39,H2O). The positive ESIMS exhibited [M+H]+ at m/z 368 [M+H]+,390 [M+Na]+,and 406 [M+K]+. The molecular formula C17H21NO8 with tow less hydrogen atoms than that of 1 was indicated by HR-ESIMS at m/z 368.1377 [M+H]+ (calcd. for C17H22NO8 368.1345). Comparison of the NMR data of 2 and 1 (Table 1) indicated replacement of the isopentanoyl in 1 by an isopentenoyl in 2. This was confirmed by the HMBC experiment of 2 (Fig. 2). Particularly,the HMBC correlations from H-2'' to C-1'',C- 4'',and C-5'' and from H3-4'' and H3-5'' to C-2'' and C-3'',in combination with their shifts,proved the presence of the isopentenoyl unit in 2. Meanwile,the HMBC correlations from H2-6' to C-1'' verified that the isopentenoyloxy was located at C-60 of the β-D-glucopyranosyl,of which the D-configuration was indicated by the positive [a] value. Therefore,the structure of compound 2 was determined as (+)-1-(6'-O-isopentenoyl-β-Dglucopyranosyl) pyridinium-3-carboxylate and designated as lonijaponinicotinoside B.

To confirm the absolute configuration of the d-glucopyranosyl in 1 and 2,a model compound 1-β-D-glucopyranosylpyridinium-3- carboxylate was synthesized for comparison. Glucosylation of methyl niconate with 2,3,4,6-O-tretraacetyl-a-D-glucopyranosylbromide, flowed by alkali hydrolysis of the product,provided the model compound,of which the NMR data (see Section 2) was absent in the literature [24]. The physical-chemical properties of the synthetic compound were consistent with those of a product obtained from alkali hydrolysis of compounds 1 and 2.

In the preliminary in vitro assays,compounds 1 and 2 were assessed for inhibitory activity against the release of glucuronidase in rat polymorphonuclear leukocytes induced by the platelet-activating factor [18],protective activity against neurotoxicity induced by serum deprivation in PC12 cells,activities against influenza virus A/Hanfang/359/95 (H3N2),Coxsackie virus B3,and HIV-1 replication,and the activities against several human cancer cell lines [19],but all were inactive at a concentration of 10 μmol/L. 4. Conclusion

Two 1-(6’-O-acyl-β-D-glucopyranosyl)pyridinium-3-carboxylates, lonijaponinicotinosides A (1) and B (2),were isolated from the aqueous extract of L. japonica flower buds. Their unique structures were elucidated by spectroscopic data analysis and confirmed by synthesis of the model compound 1-D-glucopyranosylpyridinium- 3-carboxylate. Although compounds 1 and 2 did not show activity in the preliminary in vitro assays,they are rare examples of 1-glucopyranosylpyridinium-3-carboxylate derivatives in natural products. Together with co-occurring pyridiniumcontaining homosecoiridoids [17, 18, 19],this provides an important clue for further studies of biomimetic and total synthesis,chemical transformation,structural modification and structure-activity relationships,as well as biosynthesis of the diverse pyridiniumcontaining metabolites from the flower buds of L. japonica.


Financial support from the National Natural Science Foundation of China (NNSFC; Nos. 20772156 and 30825044), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT, No. IRT1007), and the National Science and Technology Project of China (Nos. 2012ZX09301002-002 and 2011ZX0 9307- 002-01) is acknowledged. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at 10.011.

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