The roots of Codonopsis pilosula (Franch.) Nannf. (Campanulaceae),named "dang shen" in Chinese,are among the most common drugs in Chinese traditional medicine,used for the treatment of body weakness,poor appetite,thirsty,indigestion,chronic diarrhea,archoptoma,chronic anemia,and leukemia,and usually used as a substitute of the Panax ginseng exhibiting similar therapeutic effects ^[1]. Pharmacological activities of the root extracts were reported in protecting against peptic ulceration and promoting its healing,enhancing immunity,and enhancing learning and memory behavior,as well as inhibiting inducible NO synthase and protein oxidation and attenuating the cardiacimpaired insulin-like growth factor II receptor pathway ^{[2, 3, 4, 5, 6]}. Meanwhile,different types of chemical principles were isolated from the extracts,including steroids,terpenoids,polyacetylenes,alkaloids,phenylpropanoids,lignanoids,and polysaccharides ^{[7, 8, 9, 10, 11, 12]}. However,a literature survey shows that the previous investigations were mainly carried out on the ethanol or methanol extracts of the C. pilosula roots,which is inconsistent with a practical usage of decocting the drug and formulations. Therefore,an aqueous decoction of the C. pilosula roots was investigated,as part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects ^{[13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23]}. We previously described the characterization of eleven new C14-polyacetylenes and a new unsaturated v-hydroxy fatty acid from the decoction,as well as preliminary bioassays of those isolates ^{[24, 25]}. A continuation of the investigation has resulted in the isolation and structural elucidation of a minor polycyclic neolignan with a novel carbon skeleton (1) (Fig. 1),and reported herein is the details.

Fig. 1

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Fig. 1.Structure of compound 1

Optical rotation was measured on a P-2000 polarimeter. UV spectrum was recorded on a V-650 spectrometer. CD spectrum was measured on a JASCO J-815 CD spectrometer. IR spectrum was recorded on a Nicolet 5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission). 1D-and 2D-NMR spectra were obtained at 600 MHz for ¹H and 150 MHz for ¹³C,respectively,on a Bruker 600 MHz spectrometer in acetone-d₆,with solvent peaks as references. ESIMS and HR-ESIMS data were obtained on an AccuToFCS JMS-T100CS spectrometer. Column chromatography (CC) was performed with HPD-110 (Cangzhou Bon Absorber Technology Co. Ltd,Cangzhou,China),MCI gel CHP 20P (Mitsubishi Chemical Inc.,Tokyo,Japan),silica gel (200-300 mesh,Qingdao Marine Chemical Inc.,China),or RP silica gel (Grace Davison Discovery Science,Deerfield,USA). HPLC separation was performed on an instrument consisting of an Agilent ChemStation for LC system,an Agilent1200 pump,and an Agilent 1100 singlewavelength absorbance detector with a Welch ultimate XB-Phenyl (250 × 10mm i.d.) semi-preparative column packed with phenylsilica gels (5 μm) (Welch Materials inc. Shanghai,China) and a Chiralpak AD-H column (250 × 10mm i.d.) packed with amylose tris(3,5-dimethylphenylphenylcarbamate) coated on silica gel (5 μm) (Daicel Corporation,Osaka,Japan). TLC was conducted on precoated silica gel GF₂₅₄ plates. Spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H₂SO₄ in 95% EtOH followed by heating.

The roots of C. pilosula (Franch.) Nannf. were collected in October 2012 from the culture field in Weiyuan,Gansu Province,China. Plant identity was verified by Mr. Lin Ma (Institute of Materia Medica,Beijing 100050,China). A voucher specimen (No. ID-S-2503) was deposited at the herbarium of the Department of Natural Medicinal Chemistry,Institute of Materia Medica,Beijing 100050,China.

The dried and powered roots of C. pilosula (50 kg) were decocted with H₂O (150 L,3 × 0.5 h). The decoction was evaporated under reduced pressure to yield a brown residue (26 kg). The residue was dissolved in H₂O (100 L),loaded on a macroporous adsorbent resin (HPD-110,20 L) column (20 × 200 cm),and eluted successively with H₂O (100 L),50% EtOH (120 L),and 95% EtOH (80 L) to yield three corresponding fractions A-C. Fraction B (270 g) was chromatographed over MCI gel CHP 20P (5.5 L),successively eluting with H₂O (20 L),30% EtOH (30 L),50% EtOH (30 L),and 95% EtOH (8 L),to give B1-B4. Fraction B3 (22 g) was subjected to flash chromatography over RP silica gel,eluting with a gradient of increasing MeOH (40%-100%) in H₂O,to yield subfractions B3-1-B3-5. Fraction B3-4 (2.5 g) was separated by CC over silica gel,eluting with a gradient of increasing MeOH concentration (0-100%) in CH₃Cl,to give subfractions B3-4-1-B3-4-10. Fraction B3-4-1 (100 mg),was purified by RP HPLC (XB-Phenyl column,31% MeOH in H₂O,flow rate 1.5 mL/min) to yield 1 (0.8 mg,0.2016011216%,t_R = 47 min).

Codonopiloneolignanin A (1): Yellowish amorphous powder; [α]²⁰ D + 19.3 (c 0.06,MeOH); UV(MeOH) λ_max (log e) 203 (4.72),288 (3.57) nm; CD (MeOH) 211 (Δε + 7.07),239 (Δε × 0.70) nm; IR v_max 3444,2926,2854,1677,1612,1491,1466,1431,1343,1300,1219,1188,1110,1011,908,843,799,721,697 cm^-1; ¹H NMR (acetone-d₆,600 MHz) data,see Table 1; 13 C NMR (acetone-d₆,150 MHz) data,see Table 1; (+)-ESIMS m/z 385 [M + H]⁺,407 [M + Na]⁺; (+)HR-ESIMS m/z 385.1647 [M + H]⁺ (calcd. for C₂₂H₂₅O₆,385.1646),407.1470 [M + Na]⁺ (calcd. for C₂₂H₂₄O₆Na,407.1465).

Table 1

Table 1 NMR spectroscopic data for compound 1a.

Table 1 NMR spectroscopic data for compound 1a.

Conformational analysis of 1 was performed by using the MMFF94 molecular mechanics force field via the MOE software package ^[26]. Only one lowest energy conformer of 1 with relative energy within 2 kcal/mol was obtained (Fig. S1 in Supporting information),which was further optimized at the B3LYP/6-31 g (d,p) level in the gas phase. The energies,oscillator strengths,and rotational strengths of the first 60 electronic excitations were calculated using the TDDFT methodology at the B3LYP/6-311G (2d,2p) level for ECD and at the B3LYP/6-311G/aug-cc-pVDZ level for optical rotation calculation. ECD spectrum of the conformer was simulated using a Gaussian function with a half-bandwidth of 0.33 eV. The corresponding theoretical ECD spectrum of the enantiomeric 1 was depicted by inverting that of 1 (Supporting information). All quantum computations were performed using Gaussian 09 program package ^[27],on an IBM cluster machine located at the High Performance Computing Center of Peking Union Medical College. In the 200-400 nm region,the theoretically calculated ECD spectrum of 1 was in good agreement with the experimental ECD (Fig. 4). This supported the assignment of the absolute configuration for 1.

Compound 1 was obtained as a white amorphous powder. The IR spectrum of 1 showed absorption bands for hydroxyl (3444 cm^-1) and aromatic ring (1612 and 1491 cm^-1) functionalities. The positive mode ESIMS of 1 exhibited quasimolecular ion peaks at m/z 385 [M + H]⁺ and 407 [M + Na]⁺. The molecular formula C₂₂H₂₄O₆,with 11 degrees of unsaturation,was determined by HR-ESIMS at m/z 385.1647 [M + H]⁺ (calcd for C₂₂H₂₅O₆,385.1646),combined with the NMR data (Table 1). The ¹H NMR spectrum of 1 in acetone-d₆ showed signals attributable to two penta-substituted benzene rings at δ_H 6.68 (s,H-6) and 6.27 (s,H-60); four aromatic methoxy groups at δ_H 3.90 (s,MeO-30),3.84 (s,MeO-3),3.74 (s,MeO-5),and 3.70 (s,MeO-50); and two exchangeable phenolic hydroxyl groups at δ_H 7.02 (s,OH-40) and 6.91 (s,OH-4),in addition to signals due to eight aliphatic methine and/or methylene protons (Table 1). Besides resonances corresponding to the two penta-substituted benzene rings and four aromatic methoxy groups,the ¹³C NMR and DEPT spectra displayed six aliphatic carbon resonances including four methines and two methylnenes (Table 1). The presence of two pentasubstituted benzene rings and six aliphatic methylene and methine units suggests that 1 is a lignanoid containing two C₆C₃ skeletal units with the phenolic hydroxyl and aromatic methoxy substituents. As compared with those of the lignanoids previously reported from the genus Codonopsis ^{[9, 28, 29, 30]},to match requirement of the 11 degrees of unsaturation,the above spectroscopic data indicate that three unusual cyclic rings must be formed between the two C₆C₃ units in 1,which was further elucidated by comprehensive analysis of 2D NMR data.

The proton and proton-bearing carbon resonances in the NMR spectra of 1 were unambiguously assigned by the HSQC experiment. In the ¹H-¹ H COSY spectrum of 1,vicinal homonuclear coupling correlations H-9/H-8/H2-9'/H2-8'/H-7'/H-7/H-8 (Fig. 2),together with their chemical shifts and coupling constants,revealed the presence of a 7,7' -disubstituted five-membered ring with substitution of the methine (CH-9) at C-8. The HMBC spectrum of 1 showed two-and three-bond heteronuclear correlations (Fig. 2,arrows) from H-6 to C-2,C-4,and C-5; from OH-4 to C-3,C-4,and C-5; from OMe-3 to C-3; and from OMe-5 to C-5. These correlations,together with chemical shifts of the proton and carbon resonances,demonstrated that there was a 1,2-disubstitued 4-hydroxy-3,5-dimethoxyphenyl ring in 1. Similarly,the HMBC correlations H-6' to C-1' ,C-2' ,C-4' ,and C-5' ; from OH-4' to C-3' ,C-4' ,and C-5' ; from OMe-3' to C-3' ; and from OMe-5' to C-5' indicated the presence of another 1' ,2' -disubstitued 4' -hydroxy-3' ,5' -dimethoxyphenyl ring. In addition,the HMBC correlations from H-6 to C-7; from H-6' to C-7' ; from H-7 to C-1,C-1' ,C-2,C-8,and C-9; and from H-7' to C-1' ,C-2' ,C-6' ,C-8,and C-9' unequivocally indicated that C-1 and C-1' of the two phenyl rings were,respectively,bonded to C-7 and C-7' of the five-membered ring. Meanwhile,the HMBC correlations from H-9 to C-1,C-1' ,C-2,C-2' ,C-3,C-3' ,C-7,and C-9' revealed that the methine (CH-9) is connected with both C-2 and C-2' of the two phenyl rings. Accordingly,a planar structure of 1 was elucidated as shown in Fig. 2.

Fig. 2

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

In the NOE difference spectrum of 1,H-6,H-7' ,H-8,and H-8' a were enhanced by irradiation of H-7; irradiation of H-8 induced enhancements of H-7,H-9,and H-9' a; and irradiation of H-9 caused enhancements of H-8,H-9' b,OMe-3,and OMe-3' (Fig. 3,dashed double arrows). These enhancements,together with restriction of the bridged and fused rings,not only indicated the relative configuration of 1 as shown in Fig. 3,but also confirmed the structural assignment. The circular dichroism (CD) spectrum of 1 showed Cotton effects at 211 (positive) and 239 (negative) nm,which are consistent with the Cotton effects at 210 and 248 in the electronic CD (ECD) spectrum of 1 predicted from quantum mechanical time dependent density functional theory (TDDFT) calculations ^[31],but opposite to those in the theoretically calculated ECD spectrum of the enantiomeric 1 (Fig. 4). This suggests that 1 possesses the 7R,70S,8R,9S-configuration,which was further supported by the calculated specific rotation of 1 {[α]_D + 104.5} having the same positive sign {^[a]20 D + 19.3 (c 0.06,MeOH)}as the measured data. Since intensity of the Cotton effects in the experimental spectrum of 1 are much weaker than that in the calculated ECD spectrum and the amplitude of the calculated specific rotation is much greater than the measured data,1 was suspected to be a mixture of enantiomers with unequal amounts. However,subsequent HPLC analysis,using chiral columns (Chiralpak AD-H) with varied mobile phases,indicated that 1 was a pure compound. The difference between the experimental and calculated Cotton effects and [α]_D values should be due to commonly existing systematic differences between the experimentation and theory ^[31]. Therefore,the structure of 1 was determined and given a trivial name codonopiloneolignanin A. According to the IUPAC suggested nomenclature of lignans and neolignans ^[32],this compound is systematically named (+)-(7R,70S,8R,9S)-4,40-dihydroxy-3,30,5,50-tetramethoxy-2,9:20,9:7,70-tricyclo-8,90-neolignane.

Fig. 3

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Fig. 3.Main irradiation-induced enhancements in NOE difference spectrum (arrows,between protons) of compound 1.

Fig. 4

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Fig. 4.The experimental CD and calculated ECD spectrum of compound 1.

Compound 1 is the first example of natural products with an unique skeleton of 2,9:209:7,70-tricyclo-8,90-neolignane. A plausible biosynthetic pathway for 1 is proposed in Scheme 1. The biosynthetic precursor is proposed to be the synapyl alcohol (2),which occurs as the glucoside form in this plant ^{[7, 9]}. Dehydration of one molecule of the precursor followed by coupling with another precursor molecule would give 3,which is an aglycone of tangshenoside III reported form Codonopsis tangshen ^[28]. The coupling reaction would be catalyzed by specific enzyme(s) existing in species of the Codonopsis genus,since tangshenosides III is one of the only two natural products with a 2,90-neolignane skeleton from C. tangshen ^[28]. Sequential or simultaneous dehydration and hydrogen transfer rearrangement of 3 would yield an intermediate (4),which would undergo intramolecular annulation through sequential and/or simultaneous 1,2-. 1,4-,1,6-,and 1,8-additions to give 1.

Scheme 1

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Scheme 1.The plausible biosynthetic pathway of compound 1 (dashed arrows represent a simultaneous procedure of dehydration,double bond rearrangement,and/or additions).

A novel neolignan codonopilolignanin A (1),which possessing an unprecedented 2,9:20,9:7,70-tricyclo-8,90-neolignane skeleton,was isolated as the minor component from the aqueous extract of the C. pilosula roots. Although biological activity of 1 was not assayed due to limitation of the sample amount,the structure adds a new skeletal entity to the neolignan natural products,and provides a new framework for synthesis and biological evaluation in future. In particular,the plausible biosynthetic pathway provides an important clue for studies of biomimetic and total synthesis,chemical transformation,structural modification,bioactivity assay,and structure-activity relationship,as well as biosynthesis of the diverse lignans and neolignans from plants of this genus.

Financial support from the National Natural Science Foundation of China (Nos. 30825044,21132009),the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT,No. IRT1007),and the National Science and Technology Project of China (Nos. 2012ZX09301002-002,2011ZX0 9307-002-01) are acknowledged.

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.11.009.