Alzheimer's disease (AD) is the major progressive chronic neurodegenerative disease, accounting for an estimated 60–80% of cases of patients suffering from dementia worldwide.¹ It is deterioration in cognitive function affecting memory, thinking, learning, judgement, and behavior which is ultimately interfering with the ability of the person to perform daily tasks.² In 2019, over 50 million people were living with dementia worldwide. This number is expected to increase to 152 million by 2050.³ The impact of AD is not only substantial in economic terms for health care resources and medical services, but also represents the extensive human costs to countries, societies, families, and individuals. Previously reported hypotheses on the progression of AD pathologies included cholinergic dysfunction, amyloid plaques, neurofibrillary tangles, neuroinflammation, and mitochondrial dysfunction, as well as oxidative stress, which are the major hallmarks of AD.⁴ Cholinergic hypothesis is one of the theories for AD pathology. Acetylcholine (ACh) neurotransmitter used by cholinergic neurons is considered to play a critical role in the peripheral and central nervous systems.⁵ Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the biologically important enzymes that hydrolyze ACh.⁶ In addition, BuChE was associated with AD pathology, such as amyloid-beta (Aβ) plaques.⁷ It has been reported that acetylcholinesterase inhibitors (AChEIs) decrease the level of AChE in the brains of AD patients, which resists cerebral neurotransmitter acetylcholine metabolism and prolongs ACh action at the synapses.⁸ Thus, the inhibitions of AChE and BuChE to protect and enhance the ACh levels in patients have been the most attractive therapeutic strategy. US Food and Drug Administration (FDA)-approved medications are palliative and offer only temporary modulate the symptoms by changing the level of neurotransmitters in the brain. Donepezil, rivastigmine, and galanthamine are currently the only three AChEIs available in clinics while memantine is an N-methyl-d-aspartate receptor (NMDAR) antagonist.⁹ However, these AChEI drugs cannot stop or reverse disease progression and cause several side effects.¹⁰ Due to these issues, there is a crucial need to find an effective and safe disease-modifying medication to overcome AD and the research for new medications with potential clinical value seems to be necessary.

Plant-derived natural products have long been and will continue to be extremely important as the most promising source of biologically active compounds. A great potential is expected for indigenous plants to be used as a source of new drugs. Among these natural products, alkaloids are considered to be the most promising candidates for the treatment of AD due to their complex nitrogen-containing structures.¹¹ Many alkaloids that are active cholinesterase inhibitors have already been described in various families, for example, the Menispermaceae family.¹²

The genus Stephania belongs to the Menispermaceae family, a large family of approximately 65 genera and 350 species, distributed in temperate regions of the world. The plants of the genus Stephania are slender climbers with peltate and membranous leaves. The plants of this genus are commonly used in Asian folk medicine to treat a wide range of biological activities including malaria, fever, dysentery, and tuberculosis.¹³ At least 15 species in the genus Stephania have been found distributed throughout Thailand.¹⁴Stephania pierrei Diels, known in Thai as Sabu-lueat, is a medicinal plant regularly used in traditional remedies and as herbal medicine to treat body oedema, migraine, and heart disease, which are primarily distributed in Cambodia, Thailand, and Vietnam.¹⁵ The reported data showed that several types of alkaloids had been isolated from this plant species, including aporphine, tetrahydroprotoberberine, tetrahydrobenzylisoquinoline, and miscellaneous compounds.^16,17 However, the anti-cholinesterase activity of this plant has not been reported. From our preliminary investigation on phytochemicals with cholinesterase inhibitory activities from Thai medicinal plants, we found that the active crude extracts of the tubers of S. pierrei showed inhibitory activities on AChE and BuChE. Accordingly, we report herein the isolation, structure elucidation, and absolute configuration assignments of eight new alkaloids (1–8), and three new naturally occurring alkaloids (9–11), together with thirty-four known alkaloids (12–45) from the tuber extracts of S. pierrei. Most of the isolated compounds were also evaluated for their AChE and BuChE inhibitory activities. For further insight into the experimental results, in silico studies were performed.

The tubers of S. pierrei were extracted successively with n-hexane, EtOAc, and MeOH. Preliminary screening of the hexane, EtOAc and MeOH extracts of this plant revealed significant in vitro cholinesterase inhibitory activities towards AChE with IC₅₀ ranges from 1.01–20.95 ng mL⁻¹ and exhibited BuChE inhibitory activity with the IC₅₀ values of 2.76–17.46 ng mL⁻¹. These active extracts were therefore subjected to chromatographic isolation for the active principles. Based on the spectroscopic analysis and the physical properties, the chemical structures of the isolated compounds were elucidated and characterized as eight previously undescribed alkaloids, which are four new tetrahydroprotoberberine alkaloids stephapierrines A–D (1–4) and four new aporphine alkaloids stephapierrines E–H (5–8). Three new naturally occurring alkaloids, O,N-diacetylasimilobine (9),¹⁸N-acetamidesecocrebanine (10)¹⁹ and 2,3-didemethyltetrahydropalmatine (11),²⁰ together with thirty-four known alkaloids (12–45) were identified. The previously described alkaloids were identified as stepholidine (12),²¹ discretamine (13),²² tetrahydropalmatine (14),²³N-methylstepholidine (15),²¹ cyclanoline (16),²¹N-methyltetrahydropalmatine (17),²³ jatrorrhizine (18),²⁴ palmatine (19),²⁵ dehydrocorydaline (20),²⁶ pseudodehydrocorydaline (21),²⁷ roemerine (22),²⁸ (−)-stephanine (23),²⁹ (−)-isolaureline (24),³⁰ crebanine (25),²³ dicentrine (26),³¹ (−)-ushinsunine (27),³² (−)-ayuthianine (28),²⁹ sukhodianine (29),³³ (−)-N-fonnylanonaine (30),³² (−)-N-methylasimilobine (31),³² (−)-asimilobine (32),³² (−)-asimilobine-2-O-β-d-glucoside (33),^16,34 lanuginosine (34),³⁵ dicentrinone (35),³⁶ oxocrebanine (36),³⁵ 8-methoxyuvoriopsine (37),³⁷ dehydroroemerine (38),³⁸ dehydrostephanine (39),³⁹ dehydroisolaureline (40),⁴⁰ dehydrocrebanine (41),⁴⁰ dehydrodicentrine (42),⁴¹ (−)-crebanine-β-N-oxide (43),⁴² coclaurine (44)⁴³ and salutaridine (45)⁴⁴ by physical and spectroscopic data comparisons with those of the literature values (see Fig. 1). The spectroscopic (IR, ¹H and ¹³C NMR and mass) spectra of the new compounds 1–8, and the ¹H and ¹³C NMR spectra, together with the specific optical rotations and mass spectral data of compounds 9–45 are presented in the ESI.†

Stephapierrine A (1) was assigned the molecular formula C₂₁H₂₃NO₅ as deduced from the HR-ESI-TOF-MS at m/z 370.1633 [M + H]⁺ and NMR data. This alkaloid and all other isolated alkaloids gave positive orange coloration with Dragendorff's reagent. The IR spectrum indicated the presence of a hydroxy group (3380 cm⁻¹), an acetoxy group (1761 cm⁻¹) and aromatic rings (1608 and 1514 cm⁻¹). The ¹H NMR data (Table 1) showed two singlet methoxy signals at δ_H 3.79 and 3.85 (each 3H, s), and an acetoxy methyl proton signal at δ_H 2.30 (s), together with two ortho-coupled aromatic protons at δ_H 6.90 and 6.88 (each 1H, d, J = 8.8 Hz, H-11 and H-12) and two aromatic singlet protons at δ_H 6.78 and 6.58 (1H each, s, H-1 and H-4). The ¹³C NMR (Table 2) and DEPT spectra showed twenty-one carbon signals which included twelve aromatic carbons between δ_C 110.6 and 147.7, one carbonyl carbon (δ_C 169.1), three methyl carbons (δ_C 20.8, 55.9 and 60.6), four methylene carbons (δ_C 28.7, 36.0, 51.3 and 53.5), and one methine carbon (δ_C 58.8). The seventeen-carbon and one-nitrogen skeleton of 1 suggested a tetraoxygenated tetrahydroprotoberberine alkaloid core structure.^45,46 This was further confirmed by the presence of two adjacent methylenes as multiplet signals at δ_H 2.68 and 3.18 (H-5) and δ_H 2.68 and 3.21 (H-6), methylene signals at δ_H 3.59 and 4.22 (each d, J = 15.7 Hz, H-8), methylene signals at δ_H 2.90 (dd, J = 16.4, 12.2 Hz) and 3.28 (dd, J = 16.4, 3.2 Hz) (H-13), together with a partially overlapping signal at δ_H 3.64 (H-13a). The COSY correlations of these protons are shown in Fig. 2. The structure of 1 was similar to that of stepholidine (12).²¹ The main difference between the ¹H NMR spectrum of 12 and that of 1 was the presence of an acetoxy methyl signal at δ_H 2.30 (1H, s) which was proven to be at C-10 by HMBC correlations between COCH₃ (δ_H 2.30) and COCH₃ (δ_C 169.1), and C-10 (δ_C 141.0) (see Fig. 2). The HMBC correlations between the OH signal (δ_H 5.52) and C-1 (δ_C 111.2) and C-2 (δ_C 144.0) allowed the location of this phenolic function at C-2. The location of the methoxy groups at C-3 and C-9 were assigned according to the correlations of the methoxy signals at δ_H 3.85 and 3.79 with the carbons at δ_C 145.2 (C-3) and δ_C 147.7 (C-9), respectively. The structure of compound 1 was confirmed by key HMBC correlations from H-1 to C-3, C-4a and C-13a, H-4 to C-2 and C-5, H-8 to C-6, C-9, C-12a and C-13a, and H-13 to C-13b (see Fig. 2). The structure of compound 1 was further confirmed by the following key NOESY correlations: CH₃O-3/H4, H-6/H-5 and H-8, CH₃O-9/H-8, and H-13a/H-1 and H-13 (Fig. 3). The absolute configuration of C-13a of this class of alkaloids has been established, for example, by stereoselective asymmetric synthesis.⁴⁷ The negative sign of specific optical rotation of 1 is consistent with an α-orientation of hydrogen at C-13a. The absolute configuration of 1 was confirmed by the electronic circular dichroism (ECD) spectrum (Fig. S234†), which was similar to those reported for tetrahydroprotoberberine alkaloids,^48,49 with negative Cotton effects at 204 nm (Δε −10.69), 243 nm (Δε −50.55) and 287 nm (Δε −6.61). Thus, the absolute configuration of C-13a was established as S-configuration (Fig. 1). Accordingly, compound 1 was identified as 10-O-acetylstepholidine.

The molecular formula of stephapierrine B (2) was determined to be C₂₃H₂₅NO₆ on the basis of HR-ESI-TOF-MS (m/z 412.1747 [M + H]⁺) and NMR data. The IR spectrum showed absorption bands of acetoxy groups (1753 cm⁻¹) and aromatic rings (1623 and 1511 cm⁻¹). The ¹H and ¹³C NMR data (Tables 1 and 2) showed similar spectral features to those of compound 1. The significant difference was the presence of an additional acetoxy group at δ_H 2.29, and δ_C 20.8 and 169.3. The HMBC experiments showed correlations between AcO-2 (δ_H 2.29) and C-2 (δ_C 138.0), whereas H-1 (δ_H 6.89) correlated with C-2, C-3 (δ_C 149.2), C-4a (δ_C 130.0), C-13a (δ_C 58.6) and C-13b (δ_C 133.1) (see Fig. 2). It should be noted that the presence of the acetoxy group at the 2-position resulted in 0.11 and 8.5 ppm down-field shifts of H-1 and C-1 resonances, respectively, as compared with those of compound 1. These observations indicated that the acetoxy group should be located at the 2-position. Compound 2 showed the negative sign of specific optical rotation as that of compound 1, suggesting the same S-configuration or α-orientation of hydrogen at C-13a. This was confirmed by the similar ECD curve of 2 (Fig. S234†) that exhibited similar negative Cotton effects at 206 nm (Δε −14.21), 240 nm (Δε −63.97) and 286 nm (Δε −2.69), respectively. Compound 2 was therefore identified as 2,10-di-O-acetylstepholidine (see Fig. 1).

Stephapierrine C (3) was deduced to have the molecular formula C₂₃H₂₅NO₆ by combined analysis of the HR-ESI-TOF-MS m/z 412.1748 [M + H]⁺ and NMR data. The IR spectrum displayed the absorption bands of acetoxy groups (1758 cm⁻¹) and aromatic rings (1619 and 1512 cm⁻¹). The ¹H and ¹³C NMR data of 3 (Tables 1 and 2) were similar to those of compound 1. The main differences were the presence of an additional acetoxy group (δ_H 2.29, and δ_C 20.8 and 169.2) and the methoxy group should be placed at C-2 as was supported by the HMBC correlation between the CH₃O-2 and C-2 (δ_C 149.3) (see Fig. 2). This was confirmed by the NOESY correlation between the CH₃O-2 and H-1 (δ_H 6.89) (see Fig. 3). The location of the acetoxy group at C-3 was also supported by the HMBC correlation of the acetoxy methyl proton (δ_H 2.29) with C-3 (δ_C 138.1), together with the correlations of H-4 with C-2, C-3, C-4a (δ_C 127.1), C-5 (δ_C 28.6), and C-13b (δ_C 133.6). Compound 3 exhibited the same negative specific rotation and displayed a similar ECD spectrum as those of compound 1 (Fig. S234†), suggesting the same S-configuration at C-13a. Therefore, compound 3 was established as 3,10-di-O-acetyldiscretamine (see Fig. 1).

The molecular formula of stephapierrine D (4) was determined to be C₂₅H₃₁NO₉ from the HR-ESI-TOF-MS (m/z 490.2056 [M + H]⁺) and NMR data. Its IR data indicated the presence of hydroxy groups (3314 cm⁻¹) and aromatic rings (1611 and 1512 cm⁻¹). The ¹H and ¹³C NMR data of 4 (Tables 1 and 2) were similar to those of discretamine (13), which has also been isolated from S. pierrei in the present work. The significant differences were the presence of an anomeric proton signal at δ_H 4.88 (1H, d, J = 7.5 Hz, H-1′) along with signals of sugar residue at δ_H 3.38 (2H, overlapping, H-4′ and H-5′), 3.45 (1H, dd, J = 8.7 and 7.5 Hz, H-2′), 3.48 (1H, t, J = 8.7, H-3′), 3.68 (1H, dd, J = 12.1 and 5.2 Hz, H_a-6′), and 3.85 (1H, dd, J = 12.1 and 1.7 Hz, H_b-6′) in the ¹H NMR spectrum, and from the carbon resonances at δ_C 103.3 (C-1′), 78.29 (C-3′ and C-5′), 75.4 (C-2′), 71.9 (C-4′), and 63.0 (C-6′) in the ^I3C NMR spectrum. The large coupling constant of the anomeric proton signal together with the remaining characteristic ¹H and ¹³C signals of the sugar residue suggested this sugar moiety to be a β-glucoside.^16,50

The anomeric proton signal showed correlation with the anomeric carbon signal at δ_C 103.3 (C-1′) in the HMQC spectra (Figs. S66–S68†). The sequence of the glucose unit connected to C-3 of the aglycone was deduced from the HMBC correlations of the anomeric H-1′ (δ_H 4.88) with the aglycone carbon signals at C-3 (δ_C 147.1), C-3′ (δ_C 78.3) and C-5′ (δ_C 78.7), indicating the attachment of β-d-glucose at C-3 (see Fig. 2). In addition, the ¹H–¹H COSY correlations, H-1′/H-2′, H-2′/H-3′, H-3′/H-4′, H-4′/H-5′, and H-5′/H-6′, were observed (see Fig. 2). Moreover, the NOESY correlations of H-1′ and H-4 (δ_H 6.93), H-3′ (δ_H 3.48) and H-5′ (δ_H 3.38) (Fig. 3) were also key interactions to support this glucoside structure. The β-d-glucosyl nature of this sugar residue was confirmed by enzymatic hydrolysis of 4 with β-glucosidase⁵¹ and the hydrolysis products were identified to be discretamine (13)²² and d-glucose by TLC comparison with authentic 13 and d-glucose. Furthermore, the location of the methoxy group at C-2 was supported by the HMBC correlation between the CH₃O-2 signal and C-2 (δ_C 150.0) (see Fig. 2) and this was further confirmed by the NOESY correlation between the CH₃O-2 and H-1 (δ_H 6.92) (see Fig. 3). As the substituent on tetrahydroprotoberberine molecule, including a glucose moiety, does not exert a considerable effect on the optical rotation and ECD spectrum,^49,52 the same sign of optical rotation and similar ECD curve of compound 4 (Fig. S234†) when compared with those of compounds 1–3 has led to a conclusion that the absolute configuration at C-13a of 4 is S. On the basis of these findings, compound 4 is the glucoside analogue of discretamine (13).²² Compound 4 was therefore identified as discretamine 3-O-β-d-glucopyranoside (see Fig. 1).

The molecular formula of stephapierrine E (5) was deduced to be C₁₈H₁₈NO₄ from HR-ESI-TOF-MS (m/z 312.1224 [M + Na]⁺) and NMR data. The IR spectrum exhibited the absorption bands of the hydroxy group (3383 cm⁻¹) and aromatic rings (1603 and 1575 cm⁻¹). The ¹H NMR spectrum (Table 3) showed the characteristic signals of an aporphine alkaloid as a singlet N-methyl proton at δ_H 2.64 (3H, s, NCH₃), a singlet aromatic proton at δ_H 6.50 (1H, s, H-3), two ortho-coupled aromatic protons at δ_H 7.46 (1H, d, J = 8.4 Hz, H-11) and 6.71 (1H, d, J = 8.4 Hz, H-10), one methine proton at δ_H 3.20 (1H, dd, J = 14.1 and 4.5 Hz, H-6a), one coupled methylene protons at δ_H 3.09 and 2.64 (2H, overlapping signal, H-5), and another coupled methylene protons at δ_H 3.14 and 2.68 (2H, overlapping signal, H-4). Additional methylene protons at the 7-position appeared at δ_H 3.69 (1H, dd, J = 14.5 and 4.5 Hz) and δ_H 2.19 (1H, dd, J = 14.5 and 14.1 Hz). In addition, the spectrum displayed resonances due to a methylenedioxy proton at δ_H 6.02 and 5.88 (each 1H, d, J = 1.1 Hz). The ¹³C NMR and DEPT-135 spectra (Table 4) revealed the presence of 18 signals which included one methylenedioxy carbon, three methylene carbons, four methine carbons, nine quaternary carbons, and one N-methyl carbon. The sixteen-carbon and one-nitrogen skeleton of 5 suggested an aporphine alkaloid core structure.^41,53,54 The spectroscopic data for 5 was similar to those of crebanine (25),²³ especially the substitution patterns on the molecule. The significant difference is the absence of two methoxy signals in the NMR spectra of 5 (Tables 3 and 4). The structure of compound 5 was further confirmed by HMBC and NOESY experiments (see Fig. 2 and 3). The absolute configuration of compound 5 was deduced by optical rotation experiments. The negative sign of specific optical rotation suggested that the absolute configuration of C-6a was R, or the hydrogen at the C-6a position is in the β-orientation.²⁹ This was supported by the ECD spectrum (Fig. S235†). The structure of 5 was therefore elucidated as di-O-demethylcrebanine.

The molecular formula of stephapierrine F (6) was established as C₂₃H₂₇NO₇ from the HR-ESI-TOF-MS (m/z 430.1856 [M + H]⁺) and NMR data. The IR spectrum indicated the presence of hydroxy groups (3311 cm⁻¹) and aromatic rings (1593 cm⁻¹). The ¹H NMR data (Table 3) showed the presence of a methoxy signal at δ_H 3.72, four adjacent aromatic hydrogens δ_H 7.31 (1H, dd, J = 7.1, 1.2 Hz, H-8), 7.26 (1H, ddd, J = 7.5, 7.1, 1.2 Hz, H-9), 7.32 (1H, ddd, J = 7.6, 7.5, 1.2 Hz, H-10), and 8.33 (1H, brd, J = 7.6 Hz, H-11), which were ascribed to the hydrogens of the unsubstituted D ring of the aporphine alkaloid.⁵⁴ In addition, the ¹H NMR spectrum revealed the presence of a sugar unit with its anomeric proton appearing at δ_H 4.96 (1H, d, J = 7.6 Hz, H-1′). The remaining sugar proton signals H-2′ to H-6′ are in the region δ_H 3.38–3.92 exhibiting similar features to those of compound 4. The ¹³C NMR spectrum (Table 4) showed 23 carbon resonances, of which five methines and one methylene were assigned to the β-glucopyranose moiety, suggested that 6 was an aporphine glucoside. The existence of a β-d-glucosyl moiety was confirmed by enzymatic hydrolysis⁵¹ in a similar manner to compound 4. The sugar linkage was determined by the HMBC correlations of H-1′ with C-2 (δ_C 153.1), C-3′ (δ_C 78.8) and C-5′ (δ_C 78.8) (see Fig. 2).⁵⁰ The HMBC correlation between the methoxy proton at δ_H 3.72 and a carbon resonance at δ_C 147.9 (C-1) confirmed that the location of the methoxy group was placed to C-1. Additionally, the structural assignment for compound 6 was supported by the analysis of its NOESY spectrum (see Fig. 3); the H-1′ signal displayed a key interaction with H-3, H-3′ and H-5′. Moreover, H-6a (δ_H 4.11) displayed key interactions with H-7 (δ_H 2.87 and 3.03) and H-8 (δ_H 7.31), whereas CH₃O-1 (δ_H 3.72) showed correlation to H-11 (δ_H 8.33). The ¹H and ¹³C NMR spectra were consistent with those of (−)-asimilobine-2-O-β-d-glucoside (33).¹⁶ However, the sign of specific optical rotation of 6 was opposite to that of 33. The reported specific optical rotation of 33 was −107 (c = 0.1, MeOH).¹⁶ It is therefore possible that these two alkaloids are C-6a enantiomers. The ECD of 6 has therefore been determined and it was found that its ECD curve exhibited opposite Cotton effects to those of compounds 5 (see Fig. S235†). This led to a conclusion that the absolute configuration at C-6a of 6 was S. It is normal for the aporphine alkaloids to exist at different chirality at the asymmetric carbon C-6a.⁵⁷ Based on these data, the structure of compound 6 was established as (+)-asimilobine 2-O-β-d-glucopyranoside.

Stephapierrine G (7) was assigned the molecular formula C₁₈H₁₁NO₅ as determined from the HR-ESI-TOF-MS at m/z 344.0523 [M + Na]⁺ and NMR data. The IR spectrum showed absorption bands of hydroxy groups (3276 cm⁻¹) and a conjugated carbonyl group (1638 cm⁻¹). The ¹³C NMR spectrum in combination with the DEPT-135 experiment (Table 4) indicated the presence of 18 carbon atoms belonging to eleven quaternary carbons, five methine carbons, one methylene carbon, and one methyl carbon. The ¹H NMR spectrum (Table 3) revealed characteristic resonances for protons and carbons at C-4 and C-5 of an aporphine unit as a pair of doublet signals at δ_H 7.76 and 8.56 (each 1H, d, J = 5.2 Hz), which connected to δ_C 125.8 and 144.9, respectively. Two singlet signals at δ_H 7.07 (1H, s) and 6.28 (2H, s) attaching to carbons at δ_C 102.7, and 104.4, respectively, were assigned to proton signals at C-3 and methylenedioxy protons, respectively. Two doublets at δ_H 7.15 and 8.19 (each 1H, d, J = 8.8 Hz) were attributed to two ortho-coupled aromatic protons at C-10 and C-11, respectively. In addition, a singlet signal at δ_H 3.92 (3H, s) was assigned to the methoxy proton at C-8. This assignment was confirmed by the HMBC correlations of the CH₃O-8 and H-10 with C-8 (δ_C 152.0). The correlations of H-11 with C-1a (δ_C 110.4), C-7a (δ_C 126.3), C-9 (δ_C 157.9), and C-11a (δ_C 124.9) suggested the location of the hydroxy group at C-9 (see Fig. 2). The carbonyl carbon resonance at δ_C 184.1 together with a high-field resonance of its adjacent carbon (δ_C 126.3, C-7a) supported that 7 was a 7-oxoaporphine scaffold.^58,59 Furthermore, the NOESY correlations of H-3 with H-4 and H-4 with H-5 (Fig. 3) as well as additional key HMBC correlations (Fig. 2) confirmed the structure of compound 7 to be a 7-oxoaporphine analogue. The spectroscopic data suggested that 7 is closely related to oxocrebanine (36),³⁵ which has also been isolated from S. pierrei in the present work. Comparison of the NMR data of compounds 7 and 36 revealed that 7 is 9-de-O-methyloxocrebanine. Thus, compound 7 was concluded to be 1,2-methylenedioxy-8-methoxy-9-hydroxyoxoaporphine.

The sodiated molecular ion of the HR-ESI-TOF-MS of stephapierrine H (8) at m/z 388.0772 and the NMR data have led to the assignment of the molecular formula C₂₀H₁₅NO₆. The IR spectrum indicated the presence of a conjugated carbonyl group (1651 cm⁻¹). The ¹H and ¹³C NMR data (Tables 3 and 4) showed two ortho-coupled aromatic protons at δ_H 7.30 and 8.66 (each 1H, d, J = 9.0 Hz, H-10 and H-11),⁵⁸ together with two singlet protons at δ_H 7.82 and 8.03 (each 1H, s, H-4, and H-8). The remaining signals were assignable to three methoxys at δ_H 3.82 (3H, s, CH₃O-5) and 4.03 (6H, s, CH₃O-3, and CH₃O-9), together with a methylenedioxy signal at δ_H 6.40 (2H, s). The foregoing ¹H NMR data, together with the ¹³C NMR data (Table 4) suggested 8 to be an oxoaporphine type alkaloid.^46,58,59 The HMBC experiments (see Fig. 2) confirmed the structure of compound 8. The structure of 8 was also confirmed by the NOESY correlations (see Fig. 3). On the basis of these data, the structure of compound 9 was established as 1,2-methylenedioxy-3,5,9-trimethoxyoxoaporphine.

Compound 9 was assigned the molecular formula C₂₁H₂₁NO₄ as determined from the HR-ESI-TOF-MS (m/z 374.1363 [M + Na]⁺). The IR spectrum exhibited absorption bands of the acetoxy group (1761 cm⁻¹) and amide group (1636 cm⁻¹). The ¹H and ¹³C NMR spectra (Tables 3 and 4) suggested an aporphine alkaloid core structure.^41,53,54 The COSY correlations of these protons are shown in Fig. 2. The ¹H NMR data showed the typical resonance of the N-acetyl signal at δ_H 2.21. The aromatic protons showed four adjacent signals for H-8, H-9 and H-10 in the range δ_H 7.25–7.32 and at δ_H 8.30 (1H, brd, J = 7.7 Hz) for H-11, which were attributed to the hydrogens of the unsubstituted D ring of the aporphine alkaloid.⁵⁴ These observations were confirmed by ¹H–¹H COSY and HMBC experiments (see Fig. 2). The significant down-field shift of H-11 is the characteristic of this class of alkaloids.^55,56 Furthermore, an aromatic singlet signal at δ_H 6.91 (H-3), a methoxy signal at δ_H 3.53 and an acetoxy proton signal at δ_H 2.32 were observed. The ¹H and ¹³C NMR spectra of 9 were in agreement with the structure of O,N-diacetylasimilobine (9),¹⁸ which has been synthesized from asimilobine (32).³² This was confirmed by the HMBC experiments (Fig. 2) and the NOESY experiment (Fig. 3). It is noteworthy that the H-11 down-field shift in 9 (δ_H 8.30) was more pronounced than that of compound 5 (δ_H 7.46), and this was due to the vicinity of the methylenedioxy group in 5.^55,56 The negative sign of specific optical rotation of 9 suggested that the absolute configuration of C-6a was R, or the hydrogen at the C-6a position is in the β-orientation.²⁹ This was supported by the ECD spectrum (Fig. S235†). Based on these data, the structure of compound 9 was identical to (−)-O,N-diacetylasimilobine.¹⁸ Compound 9 has been reported in the present work as a naturally occurring alkaloid for the first time.

The molecular formula of compound 10 was deduced to be C₂₂H₂₃NO₅ from the HR-ESI-TOF-MS at m/z 382.1258 [M + H]⁺. The IR spectrum indicated the presence of an acetoxy group (1751 cm⁻¹). The ¹H and ¹³C NMR features of 10 (Table 5) were similar to those of 8-methoxyuvoriopsine (37),³⁷ the significant difference of which was the presence of an N-acetyl group, and the absence of an N-methyl signal. The rest of the ¹H and ¹³C NMR data were consistent with the core structure of both compounds. The structure of 10 was confirmed by HMBC and NOESY analyses (see Fig. 2 and 3, respectively). Compound 10 has been reported as a structurally modified analogue from crebanine (25) and was called N-acetamidesecocrebanine,¹⁹ though some of the reported NMR spectroscopic data do not match with our data. This compound has thus been reported in the present work as a naturally occurring alkaloid for the first time.

Compound 11 was assigned the molecular formula C₁₉H₂₁NO₄ as deduced from the HR-ESI-TOF-MS at m/z 328.1542 [M + H]⁺ and NMR data. The IR spectrum revealed the presence of hydroxy groups (3380 cm⁻¹). The ¹H and ¹³C NMR data (Tables 1 and 2) showed similar patterns to those of tetrahydropalmatine (14),²³ except that the ¹H NMR spectrum of 11 at the aromatic region was less well-defined. The NMR spectra of compound 11 showed only two methoxy signals at δ_H 3.71 (δ_C 59.2) and δ_H 3.73 (δ_C 55.5). Placement of the two methoxy groups at C-9 and C-10 was based on HMBC correlations of the CH₃O-9 proton signal with C-9 (δ_C 143.2), CH₃O-10 signal with C-10 (δ_C 146.0), H-8 and H-11 with C-9, and H-12 with C-10. This was further confirmed by NOESY correlations between CH₃O-9 and H-8, and correlation between CH₃O-10 and H-11. In addition, the locations of the hydroxy groups at C-2 and C-3 were deduced from HMBC correlations of H-1 with C-2 (δ_C 147.3) and C-3 (δ_C 144.6), and H-4 with C-2 and C-3 (Fig. 2). The positions of the hydroxy groups were confirmed by NOESY correlations (see Fig. 3). Compound 11 was therefore the 2,3-di-O-demethylated analogue of tetrahydropalmatine (14).²³ Compound 11 was previously detected as one of the demethylated metabolites of compound 14 from rat's urine by ultra high-performance liquid chromatography–tandem mass spectrometric analysis.²⁰ Thus, we report herein the spectroscopic data of 11, 2,3-didemethyltetrahydropalmatine, which was identified as a new naturally occurring alkaloid in the plant species.

In summary, eight new alkaloids (1–8), three new naturally occurring alkaloids (9–11), together with thirty-four known alkaloids (12–45), have been isolated from Stephania pierrei tubers. Most of the isolated compounds were evaluated for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities. Compound 42 exhibited the most potent AChE inhibitory effect, followed by compound 38, which were respectively slightly more active than and comparable to galanthamine. Moreover, compound 23 exhibited the highest BuChE inhibitory activity, which was 1.3-fold more active than galanthamine, followed by compounds 22, 38 and 39 which also showed high inhibitory activity. Molecular docking studies are well in agreement with these experimental results. The alkaloids 42 and 23 may respectively be regarded as lead compounds for anti-AChE and anti-BuChE drug development.

Waraluck Chaichompoo: methodology, investigation, data curation, formal analysis, visualization, and writing – original draft. Pornchai Rojsitthisak: conceptualization, resources, supervision, funding acquisition, project administration, and writing – review & editing. Wachirachai Pabuprapap: investigation, data curation, formal analysis, visualization, and writing – review & editing. Yuttana Siriwattanasathien: investigation, validation, data curation, and formal analysis. Pathumwadee Yotmanee: investigation, data curation, formal analysis, software, and writing – review & editing. Woraphot Haritakun: investigation, and data curation. Apichart Suksamrarn: conceptualization, resources, supervision, funding acquisition, project administration, and writing – review & editing.

The authors declare no conflict of interest.

The authors express gratitude to the Thailand Science Research and Innovation (TSRI) Fund (CU_FRB640001_01_33_3), the Ratchadaphiseksomphot Endowment Fund for the Natural Products for Ageing and Chronic Diseases, Chulalongkorn University (GRU 6404733002-1) (P. R.) and The Thailand Research Fund (DBG6180030) (A. S. and P. R.). This research is supported by Ratchadapisek Somphot Fund for Postdoctoral Fellowship, Chulalongkorn University (W. C.). Support from the Center of Excellence for Innovation in Chemistry, Ministry of Higher Education, Science, Research and Innovation is gratefully acknowledged.