Caulophyine A, a Rare Azapyrene Alkaloid from the Roots of Caulophyllum robustum

Abstract

A novel alkaloid caulophyine A (1) was isolated from the roots of Caulophyllum robustum Maxim., along with six known alkaloids 2–7. The structure of 1 was elucidated by extensive NMR and high resolution-time-of-flight (HR-TOF)-MS analyses, it is a rare nitrogen containing polycyclic aromatic hydrocarbon. The in vitro bioassays revealed that 2 presented remarkable cytotoxicity against A549 with an IC₅₀ value of 3.83 µM in comparison with the positive control etoposide (IC₅₀ = 11.63 µM). Compounds 1 and 2 also displayed weak Acetylcholinesterase (AChE) inhibitory activity with IC₅₀ values of 123.03 and 80.74 µM respectively.

Introduction

Caulophyllum robustum Maxim. (Berberidaceae) is a perennial plant distributed widely throughout the Qingling mountain region of Shaanxi province, China.¹⁾ The genus Caulophyllum contains two species, C. robustum from eastern Asia and C. thalictroides from eastern North America. C. robustum is used as a traditional Chinese medicine to treat external injuries and irregular menses. Phytochemical studies on its chemical constituents have led to the identification of many types of secondary metabolites including alkaloids and triterpenoid saponins.^2–4) Previous chemical studies on C. robustum have afforded a number of structurally diverse alkaloids such as caulophine, caulophyllines A–E, magnoflorine, taspine, N-methylcytisine and lupanine.^5–9)

In the course of our continuing research for bioactive constituents from C. robustum,⁹⁾ one rare azapyrene alkaloid named caulophyine A (1) along with six known compounds were identified (Fig. 1). The known compounds were identified as caulophyllines A–C (2–4),⁹⁾ matrine (5),¹⁰⁾ caulophyllumine A (6),⁶⁾ and baptifoline (7).¹¹⁾ Compound 1 is a nitrogen containing azapyrene derivative, possess a naphtho[2,1,8-def]isoquinoline fragment. This is the first report of the nitrogen containing azapyrene alkaloid identified from plant. Herein, we report the structure elucidation of 1 and the cytotoxicity and acetylcholinesterase (AChE) inhibitory activities of the 1–7.

Fig. 1. Chemical Structures of Compounds 1–7

Results and Discussion

Caulophyine A (1) was isolated as red powder; its molecular formula was determined to be C₁₉H₁₈NO₄⁺ by the high resolution-electrospray ionization-time-of-flight (HR-ESI-TOF-MS) at m/z 324.1241 [M]⁺ (Calcd. 324.1230), indicating 12 degrees of unsaturation. The ¹H-NMR (MeOH-d₄, 400 MHz) spectrum showed signals of δ_H 7.25 (1H, d, J = 6.3 Hz, H-1), 7.65 (1H, d, J = 6.3 Hz, H-2); 6.75 (1H, d, J = 8.6 Hz, H-5), 7.49 (1H, d, J = 8.6 Hz, H-4) for two AB systems. The ¹³C-NMR spectrum (MeOH-d₄, 100 MHz) spectrum with DEPT-135 together revealed 19 carbon signals including five methines (δ_C 102.3, 109.5, 116.9, 122.7, 133.4), four methyl groups, of which three were methoxyls (δ_C 56.7, 56.9, 63.4) and one was nitrogen methyl (δ_C 45.3), and ten quaternary carbons (δ_C 111.5, 122.9, 127.4, 130.9, 136.6, 141.3, 144.8, 157.2, 165.3, 167.1). All of the ¹H- and ¹³C-NMR signals were assigned by heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond connectivity (HMBC) experiments, ¹H–¹H correlation spectroscopy (COSY) correlations of H-1/H-2, CH₃-3/H-5, H-4/H-5, H-10/9-OCH₃ indicated the partial structures demarked by bold lines. The HMBC experiments revealed the long-range correlation of H-2 to C-1, C-11, C-13, CH₃-3, which in conjunction with information from the ¹H-NMR spectrum showed the existence of a 2,3,4-position substituted pyridine ring. The correlations of H-1 to C-2, C-10, C-12 and H-10 to C-11, C-1, C-9, C-8, C-12 combined with the proposed 2,3,4-position substituted pyridine ring confirm the existence of an isoquinoline fragment. The correlations of H-5 to C-6, C-7, C-14, C-16 and H-4 to C-6, C-11, C-12, C-16 lead to the elucidation of the existence of the 1,2,3,4-position substituted benzene ring (Fig. 2). The nuclear Overhauser effect spectroscopy (NOESY) correlations often used to assign the position of –OCH₃ in polysubstituted aromatic compounds. δ_C 167.1 as C-9 position in compound 1 was determined based on the strong NOESY correlation of δ_H 6.68 (H-10) with δ_H 3.97 (9-OCH₃). One dimensional (1D)- and 2D-NMR analysis further showed this compound to be a fused heterocyclic aromatic alkaloid with planar geometry, thus concluding the structure determination of 1. The hypothetical biogenetic pathways of 1 is proposed in Chart 1.

Fig. 2. COSY (Bold Lines) Correlations and Key HMBCs (Arrows) of Compound 1

Chart 1. Hypothetical Biogenetic Pathway for 1

All the isolates were evaluated in vitro for cytotoxicity against three cancer cell lines A549, HeLa and SMMC-7721, in which 2 exhibited potent cytotoxicity against all three cancer cell lines with IC₅₀ values of 3.83, 6.36 and 11.48 µM, respectively, which were comparable or exceeded the positive control etoposide (IC₅₀ = 11.63, 5.07, and 4.65 µM, respectively), whereas the remaining compounds were inactive at 200 µM. Compounds 1–7 were also evaluated in vitro AChE inhibition activity, only compound 1 and 2 displayed weak inhibition with IC₅₀ values of 123.03 and 80.74 µM compared with the positive control galanthamine (IC₅₀ = 2.01 µM).

Experimental

General

The NMR spectra were taken on an Agilent DD2 400-MR NMR spectrometer (¹H-NMR: 400 MHz, ¹³C-NMR: 100 MHz). HR-ESI-MS data were obtained from an AB Sciex Triple TOF^® 4600 system. IR spectra were recorded on a Fourier Transform Infrared Spectrometer (Perkin-Elmer, U.S.A.). UV spectra were recorded on a UV-2550 UV/Vis spectrophotometer (Shimadzu, Japan). Centrifugal partition chromatography (CPC) was carried out on a Fast Centrifugal Partition Chromatograph FCPC^® A200 (Kromaton, France) and a fraction collector (CHF-122SC, ADVANTEC, Japan). The medium-pressure liquid chromatography (MPLC) apparatus used were equipped with a dual-pump gradient system, and a UV preparative detector (Buchi Inc., Swiss Confederation). Silica gel (100–200, 200–300 and 300–400 mesh) were used for column chromatography from Qingdao Haiyang Chemical Company Ltd., Qingdao, China, and from Merck, Darmstadt, Germany. Sephadex LH-20 used for column chromatography was from GE Healthcare Life Sciences, U.S.A.

Plant Material

The roots of Caulophyllum robustum Maxim. were collected on Taibai mountain, the highest peak of the Qinling mountains, Shaanxi province, China, in May 2015, identified by Prof. Xiao-Mei Wang from Baoji University of Arts and Sciences. A voucher specimen was deposited in the herbarium of the University.

Extraction and Isolation

The powder of air-dried roots of Caulophyllum robustum Maxim. (13.3 kg, wet weight) was refluxed three times with 95% MeOH. The solvent was evaporated under reduced pressure to afford MeOH extract, a thick pulp (4 L). The MeOH extract was diluted with 1% HCl and then filtrated to remove the acid-insoluble residue. The acid-soluble liquid was adjusted to pH = 9 with ammonium solution, and then extracted with chloroform (CHCl₃) to give total alkaloids (87.0 g). The alkaloids were dissolved in 0.1 N HCl and partitioned with CHCl₃ to give the weak alkaloid fraction Fr.B (8.0 g), then the acidic solution was then adjusted to pH = 9 with ammonia solution and extracted with CHCl₃ and n-BuOH to give CHCl₃ extract (27.0 g) and n-BuOH fraction Fr.F (8.0 g), respectively. The CHCl₃ extract was dissolved in 2% NaOH and partitioned with CHCl₃ to obtain nonphenolic alkaloids Fr.D (18.0 g). The remaining liquid was adjusted to pH = 7 with HCl and then extracted with CHCl₃ to obtain phenolic alkaloids Fr.E (8.0 g). Fr.B (8.0 g) was fractionated by CPC, under CHCl₃/MeOH/H₂O (with 3% acetic acid in H₂O) (1.0/1.0/0.6) conditions, upper phase being the mobile phase yielding seven fractions, Fr.B-1 to Fr.B-7. Fr.B-4 (4.6 g) was subjected to Vacuum Liquid Chromatography (VLC) on silica gel eluting with CHCl₃/MeOH (40 : 1–0 : 1) to afford compounds 2 (1.04 g) and 3 (20.2 mg). Fr.D (18.0 g) was fractionated by CPC, under CHCl₃/MeOH/H₂O (with 1% acetic acid in H₂O) (1.0/1.0/0.6) condition to give five fractions, Fr.D-1 to Fr.D-5. Fr.D-2 (6.9 g) was subjected to VLC on silica gel eluting with CHCl₃/MeOH (50 : 1–0 : 1), then followed with silica gel column eluting with CHCl₃/MeOH (50 : 1–0 : 1) to obtain compounds 5 (178.8 mg) and 7 (216.7 mg). Fr.D-3 (3.84 g) was purified by silica gel column eluting with CHCl₃/MeOH (40 : 1–0 : 1) to give compound 1 (12.8 mg). Fr.E (7.53 g) was fractionated by CPC under CHCl₃/MeOH/H₂O (with 1% acetic acid in H₂O) (1.0/1.0/0.6) condition, and then subjected to silica gel column eluting with CHCl₃/MeOH (20 : 1–0 : 1) to give compound 6 (624.3 mg). Fr.F (7.75 g) was subjected to silica gel column eluting with CHCl₃/MeOH (5 : 1–0 : 1), followed by Sephadex LH-20 column chromatography, using MeOH as an eluent, to give compound 4 (12.6 mg).

Caulophyine A (1) Red powder, UV (MeOH) absorption peak at 243, 275 and 417 nm; IR (KBr) ʋ_max 3233, 1982, 1600, 1515, 1243, and 809 cm⁻¹; ¹H-NMR (MeOH-d₄, 400 MHz) and ¹³C-NMR (MeOH-d₄, 100 MHz) data, see Table 1; HR-ESI-TOF-MS m/z 324.1242 [M + H]⁺ (Calcd for C₁₉H₁₈NO₄, 324.12303).

Table 1. NMR Data for Compound 1 in Methanol-d₄

Position	δC, mult.	δH, (J in Hz)	HMBC
1	116.9, CH	7.25 (d, 6.3)	2, 10, 12
2	133.4, CH	7.65 (d, 6.3)	1, 11, 13, 17
4	122.7, CH	7.49 (d, 8.6)	6, 11, 16
5	109.5, CH	6.75 (d, 8.6)	4, 6, 7
6	157.2, C
7	144.8, C
8	165.3, C
9	167.1, C
10	102.9, CH	6.68 (s)	1, 8, 9, 12
11	141.3, C
12	127.4, C
13	130.9, C
14	122.9, C
15	111.5, C
16	136.6, C
6-OCH3	56.7, CH3	3.94 (s)	6
7-OCH3	63.4, CH3	3.90 (s)	7
9-OCH3	56.9, CH3	3.97 (s)	9
17	45.3, CH3	4.25 (s)	2, 11

Cytotoxicity Assay

The cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Eight ×10³ cells were incubated with the tested compounds in triplicate in 96-well plates for 72 h at 37 °C in a final volume of 100 mL. Cells treated with dimethyl sulfoxide (DMSO) were set as blank, and etoposide was used as positive control. At the end of the treatment, 10 mL of MTT (5 mg/mL) was added to each well and incubated for an additional 4 h. An extraction buffer (100 mL, 10% sodium dodecyl sulfate (SDS), 5% iso-butanol, 0.1% HCl) was added, and the cells were incubated overnight at 37 °C. The viability was calculated by measuring the absorbance at 570 nm using a microplate reader (BioTek Powerwave XS2, U.S.A.).

AChE Inhibitory Assay

The classic Ellman photometric method was used to evaluate the acetylcholinesterase inhibitory activity of 1–7.¹²⁾ Galanthamine was used as the positive control.

Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 82073727), Innovation Capability Support Program of Shaanxi (No. 2018KJXX-044), Education Department of Shaanxi Provincial Government (No. 11JS009), Guangxi Key Laboratory of Marine Natural Products and Combinatorial Biosynthesis Chemistry (No. 17-259-74), Science and Technology Program of Baoji (2018JH-08).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials. Electronic supplementary information (ESI) available: HR-ESI-MS, UV, IR, and 1D and 2D-NMR spectra for 1.

References

• 1) Flom M. S., Doskotch R. W., Beal J. L., J. Pharm. Sci-US., 56, 1515–1517 (1967).
• 2) Murakami T., Nagasawa M., Urayama S., Satake T., Yakugaku Zasshi, 88, 321–324 (1968).
• 3) Strigina L. I., Chetyrina N. S., Isakov V. V., Dzizenko A. K., Elyakov G. B., Phytochemistry, 13, 479–480 (1974).
• 4) Kennelly E. J., Flynn T. J., Mazzola E. P., Roach J. A., Mccloud T. G., Danford D. E., Betz J. M., J. Nat. Prod., 62, 1385–1389 (1999).
• 5) Jhoo J. W., Sang S., He K., Cheng X., Zhu N., Stark R. E., Zheng Q. Y., Rosen R. T., Ho C. T., J. Agric. Food Chem., 49, 5969–5974 (2001).
• 6) Ali Z., Khan I. A., Phytochemistry, 69, 1037–1042 (2008).
• 7) Perry N. S., Houghton P. J., Theobald A., Jenner P., Perry E. K., J. Pharm. Pharmacol., 52, 895–902 (2010).
• 8) Wang S. C., Wen B. Y., Wang N., Liu J. T., He L. C., Arch. Pharm. Res., 32, 521–526 (2009).
• 9) Wang X. L., Liu B. R., Chen C. K., Wang J. R., Lee S. S., Fitoterapia, 82, 793–797 (2011).
• 10) Xiao P., Kubo H., Komiya H., Higashiyama K., Yan Y. N., Li J. S., Ohmiya S., Chem. Pharm. Bull., 47, 448–450 (1999).
• 11) Goller J., Hübschle C. B., Breuning M., Eur. J. Org. Chem., 2019, 895–899 (2019).
• 12) Ellman G. L., Courtney K. D., Andres V. Jr., Featherstone R. M., Biochem. Pharmacol., 7, 88–95 (1961).