Aconicarmisulfonine A, a Sulfonated C20-Diterpenoid Alkaloid from

Letter Cite This: Org. Lett. 2018, 20, 816−819

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Aconicarmisulfonine A, a Sulfonated C20-Diterpenoid Alkaloid from the Lateral Roots of Aconitum carmichaelii Qinglan Guo,† Huan Xia,† Gaona Shi, Tiantai Zhang,* and Jiangong 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, People’s Republic of China S Supporting Information *

ABSTRACT: A novel sulfonated C20-diterpenoid alkaloid with an unprecedented carbon skeleton and significant analgesic activity (46.7% inhibition at 0.1 mg/kg, i.p.), named aconicarmisulfonine A (1), was isolated from an aqueous extract of the lateral roots of Aconitum carmichaelii. Its structure was determined by comprehensive analysis of spectroscopic data, especially by 2D NMR spectroscopic data combined with ECD calculation and single-crystal X-ray diffraction. The plausible biosynthetic pathways of compound 1 are also discussed.

D

possesses an unprecedented and sulfonated carbon skeleton and would exist as an inner salt in neutral hydrophilic biosystems (Figure 1). Herein, we report details of the isolation, structure elucidation, plausible biogenetic pathways, and analgesic activity of this compound.18

iterpenoid alkaloids are structurally diverse natural products classified as C20, C19, and C18 categories according to the number of skeletal carbon atoms.1 Because of their complex structures and significant activities, these molecules continuously attracted the interest of chemists and pharmacologists for more than a century. So far, around a thousand diterpenoid alkaloids have been isolated from the plant families of Ranunculaceae, Rosaceae, Asteraceae, Garryaceae, Escalloniaceae, and Polygonaceae.1 Aconitum carmichaelii Debx. is an herb belonging to the Ranunculaceae family. Its parent and lateral roots, named “wu tou” and “fu zi” in Chinese, respectively, are indispensable herbal medicines for the treatment of neuralgia, rheumatalgia, and cardianeuria in Chinese medicine.2 The medicinal demand for this plant is largely satisfied by cultivation in Sichuan province. Chemical and pharmacological studies showed that alkaloids were key active constituents.3,4 From different parts of this plant, more than a hundred chemical constituents were reported, mainly from “fu zi”.4 Although these herbal medicines are practically used by decocting with water and the procedure may significantly reduce toxicity,5 a majority of the chemical studies were performed by the extraction of the medicinal materials with organic solvents (benzene, chloroform, methanol, or ethanol).6−11 Taking advantage of reversible replacement reactions, acidification and saponification with a variety of acids and bases are classic methods in extraction and separation of the diterpenoid alkaloids.6−15 Therefore, an aqueous decoction of the lateral roots of A. carmichaelii was investigated as part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects.16 Previously we reported eight new C20- and 26 new C19-diterpenoid alkaloids including four unique glycosides with isomeric arabinosyls as well as two 2-(quinonylcarboxamino)benzoates and seven new aromatic acid derivatives from relatively less polar fractions of the decoction.17 A continuation of this work on a more polar fraction has resulted in the isolation and structural characterization of a novel C20diterpenoid alkaloid, named aconicarmisulfonine A (1), which © 2018 American Chemical Society

Figure 1. Structure of compound 1.

Compound 1 was obtained as colorless prisms with [α]20D − 54.5 (c 0.84, MeOH). Its IR spectrum indicated the presence of hydroxyl and/or amino (3573 and 3445 cm−1) and carbonyl (1720 and 1680 cm−1) functionalities. The positive- and negative-mode ESIMS of 1 exhibited quasimolecular ion peaks at m/z 436 [M + H]+ and 434 [M − H]−, respectively. The molecular formula of C22H29NO6S was deduced from HRESIMS at m/z 436.1782 [M + H]+ (calcd for C22H30NO6S, 436.1788) and 434.1652 [M − H]− (calcd for C22H28NO6S, 434.1643), in combination with NMR spectroscopic data (Table 1). The 1H NMR spectrum of 1 in D2O displayed resonances assignable to a trisubstituted double bond conjugating with the carbonyl at δH 8.11 (s, H-15), a nitrogen-bearing methine at δH 4.35 (brs, H20), an oxygen-bearing methine at δH 4.09 (dd, J = 11.4 and 6.6 Hz, H-1), an isolated nitrogen-bearing methylene at δH 3.46 (d, J = 13.8 Hz, H-19a) and 3.04 (d, J = 13.8 Hz, H-19b), a tertiary methyl group at δH 0.97 (s, H3-18), and a typical N-ethyl group for diterpenoid alkaloids17a at δH 3.37 (q, J = 7.2 Hz, H2-21) and Received: December 20, 2017 Published: January 12, 2018 816

DOI: 10.1021/acs.orglett.7b03956 Org. Lett. 2018, 20, 816−819

Letter

Organic Letters Table 1. NMR Data for Compound 1 in D2Oa position

δH

δC

1 2a 2b 3a 3b 4 5 6a 6b 7 8 9 10 11a 11b 12 13 14a 14b 15 16 17 18 19a 19b 20 21 22

4.09 dd (11.4, 6.6) 2.15 m 1.60 m 1.71 m 1.47 ddd (11.8, 11.8, 4.2)

69.3 33.1

1.80 brd (7.8) 2.33 dd (16.2, 7.8) 1.92 dd (16.2, 5.4) 2.80 d (5.4) 2.25 dd (10.8, 7.2) 3.81 dd (15.0, 10.8) 2.74 dd (15.0, 7.2) 3.47 m 2.88 ddd (13.8, 3.6, 3.0) 2.56 brd (13.8) 8.11 brs

0.97 s 3.46 d (13.8) 3.04 d (13.8) 4.35 brs 3.37 q (7.2) 1.42 t (7.2)

37.7

Figure 2. Main 1H−1H COSY (blue thick lines) and three-bond HMBC correlations (red arrows, from 1H to 13C) of compound 1; the two-bond HMBC correlations are omitted for clarity.

37.7 49.0 25.4

The proton and proton-bearing carbon resonances in the NMR spectra of 1 were assigned unambiguously by comprehensive interpretation of the 1H−1H COSY and HSQC spectroscopic data. In the 1H−1H COSY spectrum, the homonuclear coupling correlations (Figure 2, thick lines) of H1/H2-2/H2-3, H-5/H2-6/H-7/H-20, H-9/H-11b, H-13/H2-14, and H2-21/H3-22 revealed the presence of five vicinal coupling systems in 1. Connections of these systems and the abovementioned units with atoms of the quaternary carbon and nitrogen were unequivocally established by analysis of the HMBC spectroscopic data of 1 (Figure 2, red arrows). The HMBC spectrum showed two- and three-bond heteronuclear correlations from H3-18 to C-3, C-4, C-5, and C-19 and from H219 to C-3, C-4, C-5, and C-18. These correlations, together with the chemical shifts of the proton and carbon resonances, demonstrated a linkage of the quaternary C-4 with C-3, C-5, C18, and C-19. The connection of the quaternary C-8 with C-7, C9, C-14, and C-15 was revealed by the HMBC correlations from H2-6 to C-8; from H-7 to C-8, C-9, C-14, and C-15; from H-9 to C-8, C-14, and C-15; from H2-14 to C-7, C-8, C-9, and C-15, from H-15 to C-7 and C-14, and from H-20 to C-8. Additionally, the HMBC spectrum showed the correlations from both H-1 and 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, C-9, and C-10; indicating that the quaternary C-10 was connected by C-1, C-5, C-9, and C-20. The HMBC correlations from both H-13 and H2-14 to C-12 and C16, in combination with their chemical shift values, indicated that the two carbonyls connected to C-13. The HMBC correlation from H-9 to C-12, together with the above-mentioned vicinal coupling of H-9 with the exchangeable H2-11, demonstrated that C-9 and C-12 must be bridged via C-11. This was supported by a weak cross-peak between H-9 and H-11b in the 1H−1H COSY spectrum as well as by the HMBC correlation from H-9 to a carbon resonance around at δC 44.1 (C-11), which was not appeared in the 13C NMR spectrum. Furthermore, the HMBC correlations from H-15 to both C-16 and C-17, along with their chemical shifts and substitution patterns, revealed the linkage between C-16 and C-17. This was confirmed by the correlation from H-13 to C-17. Meanwhile, the HMBC correlations from H2-19 to C-20 and C-21, from H-20 to C-19 and C-21, and from H2-21 to C-19, and C-20, together with their chemical shift values, indicated that C-19, C-20, and C-21 connected to the same nitrogen atom. The hydroxy group must be located at C-1 based on comparison of the chemical shifts of H-1 and C-1 between 1 and the related C20-diterpenoid alkaloids.17a,d A sulfonic acid unit must be located at C-17 to match requirement of the molecular formula as well as substitution and chemical shift of this sp2 hybrid carbon. Accordingly, the planar structure of 1 was elucidated as shown, of which the carbon skeleton is unique and unprecedented in natural and synthetic products. Because of the presence of both alkali amine and acidic sulfonic units, 1 would exist as an inner salt under a neutral condition.

46.1 44.5 51.4 54.9 44.1 210.8 60.7 33.8 158.6 191.6 141.6 26.9 59.5 66.8 57.2 12.4

a

NMR data (δ) were measured at 600 MHz for 1H and at 150 MHz for 13C. Proton coupling constants (J) in Hz are given in parentheses. The assignments were based on DEPT, 1H−1H COSY, HSQC, and HMBC experiments.

1.42 (t, J = 7.2 Hz, H3-22). In addition, the spectrum showed resonances due to four aliphatic methines and five aliphatic methylenes between δH 3.47 and 1.47. The 13C NMR and DEPT spectra of 1 showed 21 carbon resonances corresponding to the above-described units, including two sp2-hybridized quaternary carbonyl carbons at δC 210.8 (C-12) and 191.6 (C-16) as well as three additional sp3-hybridized quaternary carbons at δC 54.9 (C10), 44.5 (C-8), and 37.7 (C-4). However, the 13C NMR spectrum showed one less methylene carbon than that expected from the molecular formula and the 1H NMR spectroscopic data. Reacquirement of the 1H NMR spectrum of the same sample demonstrated that the resonances of two protons germinating to one methylene at δH 3.81 (dd, J = 15.0 and 10.8 Hz, H-11a) and 2.74 (dd, J = 15.0 and 7.2 Hz, H-11b) disappeared, while the methine proton resonance at δH 2.25 (J = 10.8 and 7.2 Hz, H-9) was changed from the double doublet to a singlet (Figures S14 and S17, Supporting Information). The chemical shifts and coupling patterns of other proton resonances were unchanged. This suggested that the methylene protons (H2-11) were deuterated so that the H2-11 and C-11 resonances and the vicinal couplings between H-9 and H2-11 were undetectable when the sample was kept in D2O for a period of time. As compared with those of the compounds previously isolated from A. carmichaelii,17 this property, together with the above spectroscopic data, indicates that 1 is an abnormal sulfurcontaining C20-diterpenoid alkaloid, for which the structure was further elucidated by 2D NMR spectroscopic analysis. 817

DOI: 10.1021/acs.orglett.7b03956 Org. Lett. 2018, 20, 816−819

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Organic Letters

Figure 3. Main NOE enhancements (pink dash lines with double arrows) of compound 1.

Figure 5. ORTEP diagram of compound 1 dihydrate.

Crystallography indicated that this compound was crystallized as an inner salt together with two water molecules. An ORTEP drawing with the atom numbering scheme indicated is shown in Figure 5. Therefore, the structure of compound 1 was determined and designated as aconicarmisulfonine A. Based on biogenetic relationship of the co-occurring C20diterpenoid alkaloids, two plausible biosynthetic pathways for 1 are postulated in Scheme 1 (shown as black and red dashed

The configuration of 1 was deduced from J-based configurational analysis and the NOESY spectrum, combined with circular dichroism (CD) data and single-crystal X-ray diffraction. In the 1 H NMR spectrum, the splitting patterns and coupling constants of H-1 and H-3b (Table 1) indicated that the hydroxyhexane ring in 1 had a typical chair conformation in the D2O solution and that the OH-1 was equatorially orientated. In the NOESY spectrum of 1, correlations between H-1 with H-2a, H-3b, H-5, and H-9; between H-5 with H-3b, H-6a, H-9, and H3-18; and between H15 with H-6a and H-9 (Figure 3) demonstrated that these protons were cofacial on the same side of the ring system. In addition, the NOESY spectrum showed correlations between both H-2b and H-3a with H-19a; between H-19b with H-6b and H2-21; and between H-20 with H-14a, H2-21 and H3-22, indicating that these protons oriented on the other side of the ring system. Accordingly, the relative configuration of 1 was assigned as shown in Figure 3. The CD spectrum of 1 displayed two Cotton effects at 258 (Δε −3.22) and 294 (Δε +1.32) nm, corresponding to overlapped π−π* and n−π* transitions of the conjugated and isolated carbonyl chromophores, respectively. Based on the octant rule for the cyclohexanones and cyclohexenones,19 in combination with the relative configuration, the Cotton effects suggested that 1 possessed the absolute configuration as depicted in Figure 3. Using quantum-mechanical time-dependent density functional theory (TDDFT) calculations,20 the theoretically calculated ECD spectrum of 1 was in good agreement with the experimental CD spectrum (Figure 4), supporting assignment of

Scheme 1. Plausible Biosynthetic Pathway of Compound 1

arrows, respectively). The biosynthetic precursor of 1 is proposed to be songorine (2), which abundantly occurs in the A. carmichaelii.4,17d An enzyme-catalyzed oxidation of the double bond in 2 would produce an epoxy derivative 3, which then reacts with a thiol group of the enzyme to generate an adduct 4. Semipinacol rearrangement (1,2-migration)21 of the adduct produces an intermediate 5, which undergoes either oxidative hydrolysis and intramolecular dehydration via 6 or the reverse sequence via 7 would afford 1. Alternatively, an enzymatic catalyzed oxidative sulfonation of 2, accompanied by the 1,2migration, gives the intermediate 6, which undergoes dehydration to produce 1. In order to exclude the possibility of artificial formation of 1, the putative precursor 2, which was isolated from the same extract in this study,17d was refluxed with sodium bisulfite in CH3OH−H2O (1:1) for 8 h. TLC and HPLC analysis of the reaction mixture indicated that 1 was not formed. This supports that 1 is not abiotically originated from 2.

Figure 4. Experimental CD spectrum of compound 1 (black) and the calculated ECD spectra of compound 1 (red line) and its enantiomer (blue line).

the absolute configuration. Fortunately a single crystal was obtained from the D2O solution that was applied for measurements of the NMR spectra of 1 and then kept at room temperature for a slow evaporation of the solvent. Subsequently a single-crystal X-ray diffraction analysis by using an anomalous scattering of Cu Kα radiation confirmed the absolute configuration of 1 with a Flack parameter of 0.010 (16). 818

DOI: 10.1021/acs.orglett.7b03956 Org. Lett. 2018, 20, 816−819

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Organic Letters According to clinical application of “fu zi”, the analgesic effect of 1 was evaluated using an acetic acid induced writhing assay in mice.22 This compound showed significant activity in a dosedependent manner. At doses of 1.0, 0.3, and 0.1 mg/kg (i.p.), as compared with the black control, 83.0%, 61.1%, and 46.7% reduction of writhes were observed for 1, respectively, while the positive control morphine gave 65.5% reduction of writhes at 0.3 mg/kg (i.p.). In conclusion, aconicarmisulfonine A (1) with significant analgesic activity was isolated from the decoction of the lateral roots of A. carmichaelii. This compound has a novel carbon skeleton and is the first example of sulfonated diterpenoid alkaloid inner salt. The unique structure and chemical and pharmacological properties of 1 are attractive for chemists and biologists. The plausible biosynthetic pathways associated with the co-occurring putative precursor provides a clue for further studies of biomimetic and total synthesis as well as biosynthesis of the diverse diterpenoid alkaloids from the genus Aconitum. In addition, the result of this study supports clinic application of the traditional herbal medicine.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03956. Experimental procedure; CD, UV, IR, ESIMS, HRESIMS, and 1D and 2D NMR spectra of compound 1 (PDF) Accession Codes

CCDC 1582161 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_ [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Jiangong Shi: 0000-0003-3701-0627 Author Contributions †

Q.G. and H.X. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the National Natural Sciences Foundation of China (81630094, 21732008, and 81373445) and CAMS Innovation Fund for Medical Science (2016-I2M-1010, 2017-I2M-3-010, and 2017-I2M-3-011) is acknowledged.

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REFERENCES

(1) (a) Wang, F. P.; Liang, X. T. In The Alkaloids: Chemistry and Pharmacology; Cordell, G. A., Ed.; Academic Press: New York, 1992; Vol. 42, pp 151−248. (b) Wang, F. P.; Liang, X. T. In The Alkaloids, Chemistry and Biology; Cordell, G. A., Ed.; Elsevier Science: Amsterdam, 2002; Vol. 59, pp 1−280. (c) Wang, F. P.; Chen, Q. H.; Liang, X. T. In The Alkaloids, Chemistry and Biology; Cordell, G. A., Ed.; Elsevier Science: Amsterdam, 2009; Vol. 67, pp 1−78. (d) Wang, F. P.; Chen, Q. 819

DOI: 10.1021/acs.orglett.7b03956 Org. Lett. 2018, 20, 816−819