Chinese Chemical Letters  2015, Vol.26 Issue (06):653-656   PDF    
Two 2-(quinonylcarboxamino)benzoates from the lateral roots of Aconitum carmichaelii
Zhi-Bo Jiang, Xian-Hua Meng, Bing-Ya Jiang, Cheng-Gen Zhu, Qing-Lan Guo, Su-Juan Wang, Sheng Lin, 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 2-(quinonylcarboxamino)benzoates, named aconicarmiquinamides A (1) and B (2), were isolated from an aqueous extract of the lateral roots of Aconitum carmichaelii. Their structures were determined by spectroscopic data analysis, and confirmed by comparison with synthetic methyl 3,6-bis(diethylamino)benzoqunonylcarboxylate (3).
Key words: Aconitum carmichaelii     Ranunculaceae     2-(Quinonylcarboxamino)benzoates     Aconicarmiquinamides A and B     Methyl 3,6-bis(diethylamino)-     benzoqunonylcarboxylate     Structure elucidation    
1. Introduction

Aconitum carmichaelii Debx. is a perennial plant of the Ranunculaceae family,and cultivated in China to meet the demands of pharmaceutical industries. In traditional Chinese medicine,the lateral roots of A. carmichaelii,“fu zi”,are indispensable drugs used in raw or prepared forms to treat cadianeuria,neuralgia,and rheumatalgia [1]. Phytochemical and pharmacological studies have shown that toxic aconitine alkaloids are the main active constituents of the raw and prepared forms of “fu zi” [2, 3, 4],and have continuously attracted the attention of chemists and pharmacologists for a long time [5, 6, 7, 8, 9, 10, 11] because of their complex structures,interesting chemistry,and noteworthy pharmacological effects [12, 13]. However,the previous studies were mainly focused on the ethanol and methanol extracts with less polarity. As part of a program to systematically study the chemical diversity of traditional Chinese medicines [14, 15, 16, 17, 18, 19],a water extract of the raw lateral roots of A. carmichaelii was investigated since decoctions of a variety of formulations containing “fu zi” are practically used. In previous paper,we reported 26 new diterpenoid alkaloids and 6 new non-alkaloid constituents from the extract [20, 21]. Investigation of the remaining fraction from the same extract led to the isolation of two rare 2- (quinonylcarboxamino)benzoates,named aconicarmiquinamides A (1) and B (2) (see Fig. 1). Herein,we report details of the isolation and structure elucidation of the two compounds.

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

UVspectra 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 NMR and 125 or 150 MHz for 13C NMR,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 λ absorbance detector on an Prevail (250 × 10 mm i.d.) semipreparative column packed with C18 (5 μm). Glass precoated silica gel GF254 plates were used for TLC. Spots were visualized under UV light or by spraying with 7% H2SO4 in 95% EtOH followed by heating.

2.2. Plant material

The lateral root of Aconitum carmichaelii Debx was collected in June 2009 from the culture field in Jiangyou,Sichuan Province, People’s Republic of China. Plant identity was verified by Dr. Yan Ren (Chengdu University of Traditional Chinese Medicine,Sichuan 610075,China). A voucher specimen (No. ID-S-2383) was deposited at the herbarium of the Department of Chemistry of Natural Products,Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Beijing 100050,China.

2.3. Extraction and isolation

For extraction and preliminary fractionation of the extract,see refs. 5 and 6. Fraction C2 (600 g) was chromatographed over MCI gel CHP 20P,with successive elution using H2O (10 L),30% EtOH (30 L),50% EtOH (20 L),and 95% EtOH (10 L),to yield corresponding subfractions C2-1-C2-4. Subfraction C2-2 (200 g) was dissolved in H2O (500 mL),basified with concentrated ammonium hydroxide (25 mL) to pH 10,and then extracted with EtOAc (500 mL × 4). The aqueous phase was acidified with 6.0 mol/L HCl (66 mL) to pH 4, and partitioned with n-butanol (500 mL × 3). The n-butanol phase was concentrated under reduced pressure to give a residue,C2-2-B (12 g),which was chromatographed over silica gel (100 g) eluting with a gradient of increasing MeOHin CHCl3 (95:5-85:15) to afford fractions C2-2-B-1-C2-2-B-6. Fraction C-2-B-3 (3.9 g) was separated by reversed-phase medium pressure liquid chromatography (RP-MPLC,30% MeCN in H2O,containing 0.1% TFA) to yield C-2-B- 3-1-C-2-B-3-6,of which C-2-B-3-6 (500 mg) was further separated by RP HPLC (60% MeOH in H2O) to afford 1 (7.3 mg) and 2 (16.3 mg).

Aconicarmiquinamide A (1): Red amorphous powder (MeOH); UV (MeOH) λmax (log ε) 202.8 (5.16),219 (5.23),298.6 (4.97),342.2 (5.08) nm; IR υmax 3232,2922,2852,1716,1640,1600 (sh),1557, 1519,1442,1377,1310,1258,1223,1137,1077,1011,828, 797 cm-1; 1H NMR (CδCl3,500 MHz) δH see Table 1; 13C NMR (CδCl3,150 MHz) δC see Table 1; (+)-ESIMS m/z 438.0 [M + Na]+; (+)-HRESIMS m/z 416.1824 [M + H]+ (calcd. for C21H26N3O6, 416.1826),438.1648 [M + Na]+ (calcd. for C21H25N3O6Na, 438.1636).

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

Aconicarmiquinamide B (2): Red amorphous powder (MeOH); UV (MeOH) λmax (log ε) 197.4 (5.63),273.3 (5.10),350 (4.43) nm; IR υmax 3229,2921,2852,1716,1640,1557,1520,1444,1377,1309, 1258,1223,1137,1077,1011,828,797 cm-1; 1H NMR (CδCl3, 600 MHz) δH see Table 1; 13C NMR (CδCl3,125 MHz) δC see Table 1; (+)-ESIMS m/z 388 [M + H]+; (+)-HR-ESIMS m/z 388.1515 [M + H]+ (calcd. for C19H22N3O6,388.1503),410.1332 [M + Na]+ (calcd. for C19H21N3O6Na,410.1323).

2.4. Synthesis of 3,6-bis(diethylamino)-benzoqunonyl carboxylate (3)

To a chloroform (50 mL) solution of methyl 2,5-dihydroxybenzoate (1.68 g) manganese dioxide (2.17 g) was added. After stirring at r.t. for 24 h,the reaction mixture was filtrated,and the filtrate was concentrated to yield a residue. The residue was dissolved in diethylamine (5.0 mL),stirred at r.t. for another 12 h, followed by evaporation under reduced pressure to remove the extra diethylamine (Scheme 1). Then the residue was chromatographed over silica gel,eluting with petroleum ether-EtOAc (3:1), to obtain a product (2.31 g),which was identified as 3,6- bis(diethylamino)benzoqunonylcarboxylate (3) by following spectroscopic data: 1H NMR [(CD3)2CO,500 MHz] δH 5.35 (s,1H,H-4), 3.74 (s,3H,OCH3),3.56 (q,4H,J = 7.0 Hz,H-1'),3.50 (q,4H, J = 7.0 Hz,H-1"),1.21 (m,12H,H-2' and H-2"); 13C NMR [(CD3)2CO, 150 MHz] δC 181.8 (C-2),180.2 (C-5),168.6 (C-7),152.6 (C-6), 151.9 (C-3),99.6 (C-4),52.1 (OCH3),47.6 (2 × CH2),46.9 (2 × CH2), 13.5 (2 × CH3),12.9 (2 × CH3); (+)ESIMS m/z 309 [M + H]+.

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Scheme 1.Synthesis of compound 3.
3. Results and discussion

Compound 1 was obtained as a red amorphous powder. Its IR spectrum showed absorption bands for amino and/or hydroxyl (3232 cm-1),carbonyl (1716 cm-1),and aromatic ring (1557 and 1519 cm-1) functionalities. The positive ESIMS of 1 exhibited pseudo molecular ion peaks at m/z 438 [M + Na]+. HR-ESIMS at m/z 416.1824 [M + H]+ (calcd. for C21H26N3O6,416.1826) and 438.1648 [M + Na]+ (calcd. for C21H25N3O6Na,438.1636) indicated the molecular formula C21H25N3O6,which was supported by the NMR data (Table 1). The 1H NMR spectrum of 1 in CδCl3 showed signals attributed to a 1,2,5-trisubstituted phenyl at δH 8.02 (d, J = 8.5 Hz,H-3),7.42 (d,J = 3.0 Hz,H-6),and 7.00 (dd,J = 8.5 and 3.0 Hz,H-4); a trisubstituted double bond at δH 5.56 (s,H-4'); and a methoxy group at δH 3.93 (s). It also showed signals assignable to an ethylamino group at δH 3.98 (q,2H,J = 6.5 Hz) and 1.33 (t,3H, J = 6.5 Hz) and a diethylamino group at δH 3.56 (m,4H) and 1.30 (m, 6H),together with signals due to hydrogen-bonded amino protons at δH 12.37 (s) and 12.47 (brs). Beside resonances corresponding to the above protonated carbons (Table 1),the 13C NMR and DEPT spectra of 1 showed resonances of 10 sp2 hybridized quaternary carbons including four carbonyl carbons at δC 179.5 (C-2'),178.7 (C-5'),168.0 (C-7'),and 166.8 (C-7) and three nitrogen-bearing or oxygen-bearing quaternary carbons at δC 157.1 (C-6'),154.6 (C-3'), and 151.5 (C-5). These spectroscopic data suggested that 1 was a rare aromatic natural product containing ethylamino and diethyl amino groups,of which the structure was further elucidated by comprehensive analysis of the 2D NMR data.

The proton and protonated carbon signals in the NMR spectra of 1 were unequivocally assigned by 1H-1H COSY and HSQC spectroscopic data interpretation. In the HMBC spectrum of 1 two- and three-bond correlations (Fig. 2,red arrows) form H-3 to C-1,C-2,C-4,and C-5; form H-4 to C-2,C-5,and C-6; from H-6 to C- 2,C-4,C-5,and C-7; from OCH3 to C-7; and from NH to C-1 and C-3; together with chemical shifts of these proton and carbon resonances,revealed unambiguously the presence of a methyl 5-hydroxy-2-iminobenzoate moiety in 1. The HMBC correlations from H-4' to C-2' ,C-3',C-5',and C-6',in combination with the two remaining quaternary carbons (C-1' and C-7') without any HMBC correlation,as well as their chemical shifts,suggested the presence of a 3',6'-disubstituted benzoquinonylcarboxylate moiety in 1. The suggestion was supported by a strong absorption band for a benzoquinone chromophore [λmax (log ε) 342.2 (5.08)] in the UV spectrum of 1. In addition,the HMBC spectrum showed the correlations from methylene protons of the ethylamino group to C- 6' ,indicating substitution of the ethylamino group at C-6' and the presence of a 3'-diethylamino-6'-ethylaminobenzoqunonyl carboxylate moiety in 1. This was confirmed by consistence of the NMR data assignable to 3'-diethylamino-6'-ethylaminobenzoqunonyl carboxylate moiety in 1 with those of a synthetic model compound methyl 3,6-bis(diethylamino)benzoqunonyl carboxylate (3). Based on the molecular formula of 1,the above two moieties must be connected by an amide bond between the only two opening sites (C-7' and 2-imino),though the expected HMBC correlations from the imino proton to C-1' and C-7' was not observed in the HMBC spectrum,possibly due to partially exchanging of the proton. Therefore,the structure of compound 1 was determined as methyl 2-(3'-diethylamino-6'-ethylaminobenzoqunonylcarboxamido)- 5-hydroxybenzoate and named aconicarmiquinamide A.

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Fig. 2.1H-1H COSY (thick lines) and main HMBC correlations (red arrows,from 1H to 13C) of 1 and 2 (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Compound 2 was obtained as a dark red amorphous powder. Its molecular formula C19H21N3O6 was indicated by HR-ESIMS at m/z 388.1515 [M + H]+ (calcd. for C19H22N3O6,388.1503),and 410.1332 [M + Na]+ (calcd. for C19H21N3O6Na,410.1323). The UV,IR,andNMRspectroscopic features of 2 were similar to those of 1. Comparison the NMR data between 2 and 1 (Table 1) indicated that the ethylamino group in 1 was replaced by an amino group in 2. In addition,as compared with those of 1,H-3 and H-4' in 2 were deshielded by DδH + 0.16 and +0.11 ppm,respectively,whereas C- 5' was shielded by DδC -3.4 ppm. This demonstrates that 2 is the 6'-amino analog of 1,which was confirmed by 2D NMR data analysis of 2,particularly by the HMBC correlations from H-4' to C- 2',C-3',C-5' ,and C-6' (Fig. 2). Therefore,the structure of compound 2 was determined as methyl 2-(3'-diethylamino-6'-aminobenzoqunonylcarboxamido)- 5-hydroxybenzoate and named aconicarmiquinamide B.

To confirm the structural assignment of compounds 1 and 2, especially that of the benzoqunonylcarboxylate moieties,a model compound methyl 3,6-bis(diethylamino)benzoqunonyl carboxylate (3) was synthesized for comparison. Oxidation of methyl 2,5- dihydroxybenzoate with manganese dioxide [22],followed by amination with diethylamine,provided the model compound,of which the NMR data (see experimental section) were consistent with those of the 3'-diethylamino-6'-ethylaminobenzoqunonylcarboxylate moiety in 1 and 3'-diethylamino-6'-aminobenzoqunonyl carboxylate moiety 2. In addition,attempts to synthesize 1 and 2 failed due to amination without selectivity.

In the preliminary in vitro assays,compounds 1 and 2 were assessed for protective activity against neurotoxicity induced by serum deprivation in PC12 cells [17],and activities against influenza virus A/Hanfang/359/95 (H3N2),Coxsackie virus B3, and HIV-1 replication [16] and against several human cancer cell lines [23],but all were inactive at a concentration of 10 μmol/L.

4. Conclusion Two 2-(quinonylcarboxamino)benzoates,aconicarmiquinamides A (1) and B (2),were isolated from the aqueous extract of the lateral roots of A. carmichaelii. Their unique structures were elucidated by spectroscopic data analysis and confirmed by synthesis of the model compound 3,6-bis(diethylamino)benzoqunonyl carboxylate. Although compounds 1 and 2 did not show activity in the preliminary in vitro assays,they are rare examples of 2-(quinonylcarboxamino)benzoates with the ethylamino and diethylamino groups in natural products. Together with cooccurring aconitine alkaloids with an N-ethyl unit [2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 24, 25], 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 N-ethyl-containing metabolites from A. carmichaelii. Acknowledgments Financial support from the National Natural Science Foundation of China (Nos. 21132009 and 30825044),the Program for Changjiang Scholars and Innovative Research Team in University (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 data associated with this article can be found, in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.04. 011.
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