Secoyanhusamine A, an Oxidatively Ring-Opened Isoquinoline Inner Salt From Corydalis yanhusuo

Secoyanhusamine A (1), a rare rearranged seco-isoquinoline alkaloid derived from ring oxidative cleavage, was isolated from an aqueous extract of Corydalis yanhusuo tubers, together with its biosynthetic precursor dehydrocorybulbine (2). Secoyanhusamine A (1) was the first example of a highly oxidized isoquinoline inner salt resulting in a 5-(2-azanylethyl)-2-carboxylate-4-oxo-4H-pyran ring system. The biosynthetic pathway of 1 was also postulated. Secoyanhusamine A (1) exhibited potent inhibition against acetylcholinesterase (AChE) with an IC50 value of 0.81 ± 0.13 μM. Molecular simulation docking demonstrated that 1 created a strong interaction with the Asp-74 residue of AChE via attractive charge of the quaternary nitrogen.


INTRODUCTION
Acetylcholinesterase (AChE) has been regarded as an attractive target in the treatment of Alzheimer's disease (AD) (Amat-ur-Rasool et al., 2021), the most common neurodegenerative disease in old age that is characterized clinically by progressive memory loss, cognitive dysfunction, language disorders, and personality changes (Dong et al., 2012;Kepp, 2012). The maintenance of acetylcholine (ACh) levels via inhibition of AChE has shown to be an effective therapy in the amelioration of the AD symptoms. Indeed, several drugs such as donepezil, galantamine, and rivastigmine have been approved defined upon this approach (Tumiatti et al., 2008).
Previous studies have shown that natural products are the great resources of candidate drugs for the treatment of AD (Irajia et al., 2020;Tang et al., 2021). One of the interesting resources of potential active compounds, such as isoquinoline alkaloids, is the plant of the Papaveraceae family (Adsersen et al., 2007;Hu et al., 2009;Hung et al., 2010;Zhou et al., 2012;Hostalkova et al., 2019;Zhang et al., 2019). The most studied isoquinoline alkaloid is berberine, which could improve cognitive impairment by promoting autophagic clearance in the mouse model of AD Ye et al., 2021). In addition, other isoquinoline alkaloids with AChE inhibitory activity have the potential to act against AD, for instance, palmatine, jatrorrhizine, and coptisine (Ingkaninan et al., 2006;Jung et al., 2009;Hung et al., 2010;Tsai and Lee, 2010;Xiao et al., 2011;Brunhofer et al., 2012;Huang et al., 2012;Mollataghi et al., 2012;Cao et al., 2018;Liu et al., 2019).
The dried tuber of Corydalis yanhusuo W.T. Wang (Papaveraceae), also known as "Yuan-Hu", is an important traditional Chinese medicine for the treatment of spasms and menstrual and abdominal pain (He et al., 2007;Chinese Pharmacopoeia, 2020). Recent study illustrated that a Chinese medicine prescription Yuan-Hu Zhi Tong (YZT) comprising of C. yanhusuo tubers could alleviate the AD symptoms in the P301S tau and 3XTg-AD mice model, of which the major bioactive constituents are the isoquinoline alkaloids originated from C. yanhusuo (Iyaswamy et al., 2020). In addition, during our preliminary AChE inhibition activity screening, the YH-D fraction of the C. yanhusuo tubers aqueous extract was hit, with an IC 50 value of 8.16 ± 1.06 μg/ml. Inspired by this finding, a bioactivity-guided isolation strategy was used to explore promising AChE inhibitors from this bioactive fraction, which resulted in the isolation of a rare rearranged seco-isoquinoline alkaloid with potent AChE inhibition, named secoyanhusamine A (1) (Figure 1), together with a biosynthetic precursor dehydrocorybulbine (2). We report details of the isolation, structure elucidation, possible biogenetic pathway, and AChE inhibitory activity of 1.

General Experimental Procedures
The UV spectrum was acquired on a Cary 300 spectrometer. The IR spectrum was obtained on a Nicolet Impact 400 FT-IR spectrophotometer. 1D-and 2D-NMR spectra were recorded using Bruker 600 MHz spectrometers (600 MHz for 1 H and 150 MHz for 13 C), and the chemical shifts were reported as δ values using internal standard TMS (measured in MeOH-d 4 ). HR-ESIMS data were obtained on Agilent 1100 Series LC-MSD-Trap-SL and Agilent 6520 Accurate-Mass Q-TOFL CMS spectrometers (Agilent Technologies, Ltd., Santa Clara, CA, United States). Column chromatography (CC) was performed with a macroporous resin (HP20, Mitsubishi Group, Japan), Sephadex LH-20 (Amersham Biosciences, Sweden), and silica gel (200-300 mesh, Qingdao Marine Chemical Inc., People's Republic of China). Analytical HPLC was performed with an Agilent 1260Ⅱ using a Titank column (Guangzhou FLM Scientific Instrument Co., Ltd.) packed with C 18 (250 × 4.6 mm, 5 μm). HPLC separation was performed on a system consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 Dual λ absorbance detector (Waters Corporation, Milford, MA, United States), with a Shiseido MGII ODS C 18 column (250 × 10 mm or 250 × 20 mm, 5 μm). TLC was conducted on precoated silica gel GF 254 plates. Spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H 2 SO 4 in 95% EtOH followed by heating or with a Dragendorff's reagent. All chemicals were obtained from commercially available sources and were used without further purification.

Bioassay for Anti-Cholinesterase Activity
Inhibition of AChE was assessed by a modified version of the colorimetric method of Ellman (Oh et al., 2004). A mixture of 196 μl phosphate buffer (PBS) and acetylcholinesterase (final concentration 0.01 μg/ml) and 2 µl inhibitor (YH-D, compound 1, and positive control donepezil) was added to the 96-well plate, and then the plate was incubated at 4°C for 20 min; 2 µl DTNB and ATCI (final concentration of 116 μM) was added, and the solution was incubated for 20 min at 37°C on a shaker. The optical density was measured at 405 nm immediately, and the percentage inhibition was calculated. Donepezil was used as a positive control.
The initial biological screening of the YH-D fraction encouraged further AChE inhibitory evaluation of secoyanhusamine A (1). As a result, secoyanhusamine A (1) showed potent inhibition with an IC 50 value of 0.81 ± 0.13 μM, compared to the positive control (donepezil,IC 50 0.15 ± 0.01 μM). Furthermore, molecular docking simulation was performed on the basis of the previously reported crystal structure of AChE (Cheung et al., 2012). As shown in Figure 3, secoyanhusamine A (1) could be docked perfectively into the catalytic cavity of AChE, while the N-atom and the pyrone ring could strongly interact with the Asp-74 residue of AChE via attractive charge and with the Tyr-341 residue of AChE via Pi-Pi T-shaped, respectively.

CONCLUSION
In summary, secoyanhusamine A (1), a rare rearranged secoisoquinoline alkaloid derived from ring oxidative cleavage, was isolated from an aqueous extract of Corydalis yanhusuo tubers and showed significant inhibitory activity against acetylcholinesterase (AChE). The structures of 1 were determined via extensive NMR spectroscopic analysis. Our study provides a new structural architecture of the natural product that can be used in followup studies relevant to the development of anti-Alzheimer's agents.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

AUTHOR CONTRIBUTIONS
LW, PL, and SL contributed to the conception and design of this research. LW carried out the study and collected important background information. HX and YW provided assistance for compound characterization and data analysis. YW carried out the 1D-and 2D-NMR spectra data. PL and SL performed manuscript revision. All authors contributed to manuscript revision and read and approved the submitted version.