Racemic alkaloids from Macleaya cordata: structural elucidation, chiral resolution, and cytotoxic, antibacterial activities

Abstract

Three pairs of new enantiomeric natural alkaloids (±)-macleayins C–E (1–3), together with five pairs of known racemic alkaloids (4–8), were isolated from the aerial parts of Macleaya cordata. Compounds 1–5 were separated successfully by chiral-phase HPLC to yield optically pure isomers. However, chiral resolution of compounds 6–8 existing as racemers in the plant was unsuccessful. It is noteworthy that, macleayin C represents a novel type of hybrid composed of a dihydrobenzophenanthridine alkaloid and a phenylpropanoid. The structures including the absolute configurations of (±)-1–5 were established by detailed spectroscopic analyses and electronic circular dichroism calculations. All the isolates were evaluated for in vitro anti-tumor and antibacterial activities, and compounds (±)-4–5 exhibited potent cytotoxicity against HL-60 cell lines with IC₅₀ values less than 3.0 μM, and compounds (±)-1–3 showed moderate cytotoxic activity. Only compound 7 revealed inhibitory activities against Staphylococcus aureus, Bacillus subtilis, and Candida albicans with MIC values of 33.07, 8.27, and 8.27 μg mL⁻¹, respectively.

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Introduction

Macleaya cordata (Willd.) R. Br., a deciduous perennial plant in the family Papaveraceae, is a common traditional Chinese medicine, and has recently attracted much attention due to its bioactive alkaloid contents and pharmaceutical values.^1–3 Many studies have demonstrated that benzophenanthridine, dihydrobenzophenanthridine, protopine, and protoberberine type alkaloids are the major bioactive components in the plant.⁴ Experimental research showed that M. cordata has a wide spectrum of anti-microbial,⁵ anti-fungal,⁶ anti-inflammatory,⁷ pesticidal,⁸ and anti-tumor properties.^9,10 According to clinical records, M. cordata has been applied extensively as folk medicine in China, North America, and Europe, where it has been used to cure cervical cancer and thyroid cancer,³ and as traditional medicines for the treatment of diverse infectious diseases.¹¹ In addition, it was also successfully applied in veterinary medicine and agriculture. Preliminary phytochemical investigation on the ethanol extract of M. cordata afforded two pairs of novel enantiomers of benzophenanthridine–protopine hybrids,¹² which intrigued us to continue to explore the chemical diversity of the alkaloids, leading to the identification of three pairs of new enantiomers (±)-1–3, along with two pairs of known enantiomers (±)-4–5 and three racemic compounds 6–8 (Fig. 1). Herein we report the isolation, chiral resolution, structural identification, and in vitro cytotoxic and antibacterial activities of the isolated compounds.

Fig. 1 Structures of compounds 1–8.

Results and discussion

All isolated compounds 1–8 displayed strong fluorescence under UV (254 nm and 365 nm) on silica gel TLC plates, as well as positive reactions with Dragendorff's reagent. Their basic skeleton was in perfect accordance with dihydrobenzophenanthridines¹³ according to the following features: their UV absorptions at about max. 225–230, 280–285, and 318–325 nm, their IR aromatic ring absorption bands at about 1600, 1494, and 1464 cm⁻¹, their ¹H NMR spectra with the signals of two one-proton singlets at δ_H 7.11–7.42 (H-1) and 6.44–7.72 (H-4), two pairs of AB-type ortho-coupling doublets at δ_H 6.87–7.14 (d, J = 8.1–8.6 Hz, H-9) and 7.34–7.66 (d, J = 8.1–8.6 Hz, H-10), δ_H 7.70–7.85 (d, J = 8.6–8.7 Hz, H-11) and 7.48–7.56 (d, J = 8.6–8.7 Hz, H-12), as well as one N-methyl group at δ_H 2.33–2.79 (s). All of the ¹H NMR spectra also exhibited a typical signal of H-6 at δ_H 4.77–5.55, which was from a dihydrobenzophenanthridine skeleton substituted at C-6 by a directly connected methine.

Macleayin C (1) was obtained as a colourless cluster crystal (in CH₂Cl₂ : MeOH = 1 : 1), [α]²⁵_D 0 (c 0.16, CHCl₃). The molecular formula of C₃₂H₃₁NO₉ was determined by the positive HRESIMS ion at m/z 596.1892 [M + Na]⁺ (calcd 596.1891), implying eighteen indices of hydrogen deficiency. The UV spectrum showed the maximum absorptions at 227, 281, and 320 nm. The IR spectrum indicated the presence of a carbonyl (1659 cm⁻¹) and aromatic ring (1605, 1492, and 1464 cm⁻¹). The ¹H and ¹³C NMR spectra (Table 1) of 1 revealed a typical 6-substituted-dihydrochelerythrine moiety¹² with signals at δ_H 7.14 (1H, d, J = 8.6 Hz, H-9), 7.66 (1H, d, J = 8.6 Hz, H-10), 7.85 (1H, d, J = 8.6 Hz, H-11), 7.53 (1H, d, J = 8.6 Hz, H-12), 7.12 (1H, s, H-1), 6.44 (1H, s, H-4), 4.57 (1H, d, J = 11.0 Hz, H-6), 6.07, 5.81 (each d, J = 1.1 Hz, 2,3-OCH₂O), 3.84 (3H, s, 7-OCH₃), 3.90 (3H, s, 8-OCH₃), 2.33 (3H, s, N–CH₃); δ_C 103.3 (C-1), 146.8 (C-2), 146.8 (C-3), 99.6 (C-4), 126.5 (C-4a), 138.9 (C-4b), 58.4 (C-6), 125.9 (C-6a), 145.9 (C-7), 151.7 (C-8), 112.3 (C-9), 119.2 (C-10), 124.2 (C-10a), 123.0 (C-10b), 119.4 (C-11), 123.6 (C-12), 130.1 (C-12a), 100.9 (2,3-OCH₂O), 60.5 (7-OCH₃), 55.8 (8-OCH₃), 41.6 (N–CH₃). The above deduction was confirmed by the key HMBC correlations from H-1 to C-3, C-4a, and C-12, H-4 to C-2, C-4b, and C-12a, H-9 to C-7 and C-10a, H-10 to C-6a and C-10b, H-11 to C-4b, C-10a, and C-12a, H-12 to C-1, C-4a, and C-10b, N–CH₃ to C-4b and C-6, as shown in Fig. 2.

Table 1 NMR data (DMSO-d₆) of compound 1^a

Position	^13C NMR	^1H NMR (J in Hz)	HMBC (H → C)
a ^1H NMR recorded at 600 MHz, ^13C NMR recorded at 100 MHz.
1	103.3	7.12 (s)	3, 4a, 12
2	146.8
3	146.8
4	99.6	6.44 (s)	2, 4b, 12a
4a	126.5
4b	138.9
6	58.4	4.57 (d, 11.0)	4b, 7, 10a, 7′, 8′, 9′, N–CH3
6a	125.9
7	145.9
8	151.7
9	112.3	7.14 (d, 8.6)	7, 8, 10a
10	119.2	7.66 (d, 8.6)	6a, 8, 10b
10a	124.2
10b	123.0
11	119.4	7.85 (d, 8.6)	4b, 10a, 12a
12	123.6	7.53 (d, 8.6)	1, 4a, 10b
12a	130.1
1′	128.8
2′,6′	105.5	6.58 (s)	4′, 7′
3′,5′	146.7
4′	140.3
7′	201.0
8′	49.5	3.54 (ddd, 11.0, 10.6, 3.6)	6, 6a, 7′, 9′
9′	61.8	3.82 (ddd, 10.6, 10.4, 7.4)	7′, 8′
		3.16 (ddd, 10.4, 3.7, 3.6)
OCH2O	100.9	5.81, 6.07 (each d, 1.1)	2, 3
N–CH3	41.6	2.33 (s)	4b, 6
7-OCH3	60.5	3.84 (s)	7
8-OCH3	55.8	3.90 (s)	8
3′,5′-OCH3	55.3	3.41 (s)	3′, 5′
4′-OH		8.96 (br s)
9′-OH		4.21 (dd, 7.4, 3.7)	8′, 9′

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Fig. 2 Selected 2D NMR correlations of 1–3.

The remaining data in the ¹H and ¹³C NMR spectra exhibited signals for a 1,3,4,5-tetrasubstituted benzene ring [δ_H 6.58 (2H, s, H-2′,6′), 8.96 (1H, br s, 4′-OH), 3.41 (6H, s, 3′,5′-OCH₃); δ_C 128.8 (C-1′), 105.5 (C-2′,6′), 146.7 (C-3′,5′), 140.3 (C-4′), 55.3 (3′,5′-OCH₃)], a ketone group (δ_C 201.0, C-7′), a hydroxyethyl side chain [δ_H 3.54 (1H, ddd, J = 11.0, 10.6, 3.6 Hz, H-8′), 3.82 (1H, ddd, J = 10.6, 10.4, 7.4 Hz, H-9′a), 3.16 (1H, ddd, J = 10.4, 3.7, 3.6 Hz, H-9′b), 4.21 (1H, dd, J = 7.4, 3.7 Hz, 9′-OH); δ_C 49.5 (C-8′), 61.8 (C-9′)]. The above data suggested the presence of 3,5-dimethoxy-4-hydroxyphenyl 2-hydroxyethyl ketone moiety, which was confirmed by the ¹H–¹H COSY correlations of OH-9′/H-9′/H-8′ and the HMBC correlations from OH-9′ to C-8′ and C-9′, and from H-8′ and H-2′/6′ to C-7′. The ¹H–¹H COSY correlation of H-6/H-8′ and the HMBC correlations from H-6 to C-7′, C-9′, C-4b, C-10a, and N–CH₃ further established the linkage of dihydrochelerythrine and phenyl ethyl ketone via C-6 and C-8′. By detailed analyses of the 1D and 2D NMR data, the planar structure was deduced to be shown in 1. The relative configuration of macleayin C (1) was established by the coupling constant of H-6 and H-8 (11.0 Hz), and analyses of the NOESY correlations (Fig. 3) from H-6 to N–CH₃ and H-9′b, from H-8′ to H-9′a indicated that H-6 and H-8′ were located on the opposite side.

Fig. 3 NOESY correlations of 1.

The isolated sample of 1 was a racemic, due to its specific optical rotation of [α]²⁵_D 0 (c 0.16, CHCl₃). Subsequent separation by using chiral-phase HPLC yielded (−)-macleayin C (1a) (1.80 mg) and (+)-macleayin C (1b) (1.80 mg) in a ratio of 1 : 1 (Fig. 4), whose optical rotations were opposite.¹³ Furthermore, they have the similar absolute value of specific rotation, [α]²⁰_D = −85.6 (c 0.18, MeOH) and +94.4 (c 0.18, MeOH), respectively. The assignments of 1a (6R, 8′R) and 1b (6S, 8′S) were made by comparing the calculated electronic circular dichroisms (ECD) via a quantum method with the experimental data (Fig. 5).¹⁴ From the above evidence, the structures of 1a and 1b were unambiguously assigned as shown in Fig. 1.

Fig. 4 The chiral HPLC chromatograms of 1–8.

Fig. 5 Experimental and suitable calculated ECD spectra of (±)-1.

Macleayin D (2) was isolated as a colourless cluster crystal (in MeOH) with [α]²⁵_D 0 (c 0.20, CHCl₃). HRESIMS (m/z 407.1602 [M + H]⁺, calcd 407.1601) and ¹³C NMR data established the molecular formula C₂₃H₂₂N₂O₅. The UV absorption bands at 229, 283, and 318 nm indicated typical conjugated groups. The IR spectrum showed absorption bands due to amino (3382 cm⁻¹), carbonyl (1680 cm⁻¹), and aromatic ring (1602, 1494, and 1465 cm⁻¹) functionalities. The comparison of ¹H and ¹³C NMR data of 2 with 1 clearly showed that they have the same 6-substituted dihydrochelerythrine moiety (Tables 1 and 2). The remaining signals of 2 in the ¹H and ¹³C NMR as well as HSQC spectra established the presence of an acetamido group at δ_H 6.98 (1H, br s, –NH₂), 6.74 (1H, br s, –NH₂), 2.06 (1H, dd, J = 14.7, 11.3 Hz, H-1′a), 1.90 (1H, dd, J = 14.7, 3.5 Hz, H-1′b); δ_C 39.6 (C-1′), 171.5 (C-2′). The HMBC correlations (Fig. 2) of –NH₂/C-1′, C-2′ and H-1′/C-6, C-2′ further confirmed the presence of acetamido group, which was assigned to be substituted at C-6. Thus, compound 2 was elucidated as 6-acetamidyldihydrochelerythrine. Subsequent chiral resolution of 2 was performed on a chiral column (Daicel Chiralpak IC) to yield 2a (0.97 mg) and 2b (0.97 mg) in ratio of 1 : 1 (Fig. 4), of which they were virtually opposite in terms of their ECD curves and optical rotation data [α]²⁰_D (c 0.097, MeOH) −126.3 (2a), [α]²⁰_D (c 0.097, MeOH) +142.8 (2b). Their absolute configurations were determined by the quantum chemical ECD calculation method. The measured ECD curves of 2a and 2b matched with the calculated ones of (6R)-2 and (6S)-2, respectively (Fig. 6). Thus, the absolute configurations of them were established as 2a (6R) and 2b (6S) as shown in Fig. 1.

Table 2 NMR data (DMSO-d₆) of compounds 2 and 3^a

Position	2		3
	δC	δH	δC	δH
a ^1H NMR recorded at 600 MHz, ^13C NMR recorded at 150 MHz.
1	104.1	7.29 (s)	104.1	7.29 (s)
2	147.2		147.3
3	147.6		147.7
4	100.1	7.45 (s)	100.1	7.47 (s)
4a	126.8		127.0
4b	139.2		139.1
6	54.1	4.92 (dd, 11.2, 3.4)	54.0	4.77 (dd, 11.0, 3.4)
6a	128.2		116.2
7	145.0		144.0
8	151.9		146.9
9	111.8	7.08 (d, 8.6)	107.4	6.95 (d, 8.2)
10	118.7	7.64 (d, 8.6)	116.3	7.45 (d, 8.2)
10a	124.0		125.0
10b	122.8		122.8
11	119.8	7.81 (d, 8.6)	120.0	7.80 (d, 8.6)
12	123.5	7.53 (d, 8.6)	123.7	7.54 (d, 8.6)
12a	130.7		130.7
1′	39.6	2.06 (dd, 14.7, 11.3)	39.6	2.13 (dd, 14.7, 11.1)
		1.90 (dd, 14.7, 3.5)		1.97 (dd, 14.5, 2.8)
2′	171.5		171.3
NH2		6.98, 6.74 (each br s)		7.03, 6.77 (each br s)
N–CH3	42.7	2.53 (s)	43.1	2.54 (s)
2,3-OCH2O	101.1	6.13, 6.12 (each br s)	101.2	6.14, 6.13 (each br s)
7-OCH3	60.4	3.84 (s)
8-OCH3	55.7	3.87 (s)
7,8-OCH2O			101.5	6.11 (br s)

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Fig. 6 Experimental and suitable calculated ECD spectra of (±)-2.

Macleayin E (3) was obtained as a colourless cluster crystal (in MeOH) and displayed an [M + Na]⁺ ion peak at m/z 413.1110 (calcd 413.1108), corresponding to a molecular formula of C₂₂H₁₈N₂O₅. Its IR and UV spectra were similar to those of compound 2. Based on a comparison of the NMR spectroscopic data of 3 with 2 (Table 2), they had the same dihydrobenzophenanthridine skeleton and the same 6-acetamido side chain, except for the appearance of signals for one more methylenedioxy group and the absence of two methoxyl groups in 3. The additional methylenedioxy group was determined to be attached to C-7 and C-8 by HMBC correlations from the protons of the methylenedioxy [δ_H 6.11 (2H, s)] to C-7 (δ_C 144.0) and C-8 (δ_C 146.9). Accordingly, compound 3 was established as 6-acetamidyldihydrosanguinarine, which was isolated in a racemic form. The chiral HPLC separation of 3 was performed by the same method with 2 to yield 3a (0.95 mg) and 3b (0.95 mg). Subsequently, the assignment of 3a (6R) ([α]²⁰_D (c 0.095, MeOH) −143.8), and 3b (6S) ([α]²⁰_D (c 0.095, MeOH) +138.1) was accomplished by comparison of the experimental ECD spectra with those of 2a and 2b (Fig. 7).

Fig. 7 Experimental ECD spectra of (±)-2, 3, 4, and 5.

6-Acetonyldihydrosanguinarine (4) and 6-acetonyldihydrochelerythrine (5) were also isolated as known compounds. Their structures were identified by comparison of their ¹H and ¹³C NMR data with literature values.¹⁵ They were also in racemic form in M. cordata, and then subjected to chiral column chromatography respectively to obtain 4a (1.80 mg), 4b (1.80 mg), 5a (0.40 mg), and 5b (0.40 mg) (Fig. 4). Their experimental ECD spectra were compatible with those of 2a and 2b (Fig. 7), respectively, establishing their absolute configurations as 4a (6R), 4b (6S), 5a (6R), and 5b (6S). This is the first report of their chiral resolution (Table 3).

Table 3 Cytotoxic activities of compounds 1–5

Compound	IC50 (μM)
	HL-60	A-549	MCF-7
(+)-1	10.35	>100	>100
(−)-1	12.13	>100	>100
(+)-2	25.42	34.19	>100
(−)-2	26.17	35.26	>100
(+)-3	20.97	30.46	>100
(−)-3	18.46	31.57	>100
(+)-4	2.07	22.47	42.74
(−)-4	1.86	27.14	45.63
(+)-5	2.75	25.45	49.63
(−)-5	2.58	22.14	48.06
5-Fu	2.80	1.60	29.83

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The known compounds 6-methoxyldihydrosanguinarine (6),¹⁶ 6-methoxyldihydrochelerythrine (7),^17,18 and spallidamine (8)^19,20 were obtained. According to related literature and NMR data, their structures were determined. Due to their ECD curves almost in a straight line and the lack of optical activity, they existed as racemic natures. Subsequently, enantiomers of 6 were separated by chiral HPLC, while the isomers rapidly formed a racemic mixture. This unusually rapid racemization may originate from the formation of a stable iminium ion intermediate, sanguinarine.^21,22 Unfortunately, compounds 7 and 8 could not be separated using three types of chiral columns.

Considering that some benzophenanthridine alkaloids from M. cordata have been reported showing significant anti-tumor, antimicrobial, and antifungal activities, compounds (±)-1–5 were tested for cytotoxicity against three human cancer cell lines HL-60, A-549, and MCF-7, and all compounds were tested for antimicrobial activity against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Candida albicans.

Conclusions

In summary, three pairs of new enantiomers (±)-macleayins C-E (1–3), together with two pairs of known enantiomers (±)-4–5, and three racemic alkaloids (6–8) were isolated from Macleaya cordata, and the structures were elucidated by extensive spectroscopic techniques. All isolated 6-substituted dihydrobenzophenanthridine alkaloids from M. cordata are raceme. Compounds 1–5 were separated by using chiral-phase HPLC. Their absolute configurations were determined by calculated ECD method.

The cytotoxicity of compounds (±)-1–5 against three human cancer cell lines was evaluated using trypan blue and MTT methods. Compounds (±)-4 and 5 exhibited potent cytotoxicity against HL-60 cell lines, and (±)-1–3 showed moderate cytotoxicity. However, compounds (±)-1–5 showed weak or no cytotoxic activities against A-549 and MCF-7 cell lines. The results indicated that (+)-1–5 and (−)-1–5 had almost the same cytotoxicity, scilicet no selectivity, perhaps because that the enantiomer was liable to undergo racemization during incubating.

Compounds 1–8 were tested for antimicrobial activities by micro-dilution broth MIC method. However, only compound 7 showed mild effects.

Experimental section

General experimental procedures

Optical rotations were measured with Rudolph Autopol-V digital polarimeter and Perkin Elmer Model 341 Polarimeter. UV spectra were recorded on a Shimadzu UV-2201 spectrometer. IR spectra were obtained on a Bruker IFS-55 spectrometer using KBr disks. ECD spectra were acquired with a Bio-logic MOS 450 spectropolarimeter. 1D and 2D NMR were performed on Bruker ARX-300 and AV-600 NMR spectrometers using solvent signals (DMSO-d₆: δ_H 2.50/δ_C 39.52; CDCl₃: δ_H 7.26/δ_C 77.16), with tetramethylsilane (TMS) as the internal standard. HRESIMS spectra were collected using a Bruker micrOTOF-Q mass spectrometer. A Shimadzu LC-6 AD equipped with a SPD-20A (UV/VIS) detector was used for HPLC, a YMC-pack ODS-A column (250 × 20 mm, S-5 μm, 12 nm) was used for semi-preparative HPLC separation. A Shimadzu LC-20AB equipped with a SPD-M20A PDA (DIODE ARRAY) detector on a chiral column Daicel Chiralpak IC (250 × 4.6 mm) was used for chiral HPLC separation. Column chromatography (CC) were performed with silica gel (100–200 and 200–300 mesh, Qingdao Haiyang Chemical Co. Ltd., Qingdao, China), C₁₈ reversed-phase silica gel (S-50 μm, 12 nm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 gel (GE Healthcare, Sweden). Fractions obtained from CC were monitored by TLC using precoated silica gel GF₂₅₄ plates (Qingdao Haiyang Chemical Co. Ltd., Qingdao, China), which was visualized under UV light, by spraying with Dragendorff's reagent, or heating the silica gel plates after being sprayed with 10% H₂SO₄ in EtOH.

Plant material

The aerial parts of Macleaya cordata (Willd.) R. Br. was purchased from Anguo Medicines Ltd. (Hebei, China) in November 2013, and was identified by Prof. Jin-Cai Lu (School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang, China). A voucher specimen (BLH-20131108) has been deposited at the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China.

Extraction and isolation

The air-dried aerial parts of M. cordata (40.0 kg) were extracted with 95% EtOH (2 × 400 L) under reflux, and 75% EtOH (1 × 400 L). After the organic solvent was removed under reduced pressure, the crude extract was suspended in H₂O, successively partitioned with CH₂Cl₂ and n-BuOH. The dichloromethane extract (500 g) was fractionated on a silica gel column (200–300 mesh; 1000 g; Φ 10 cm × 120 cm) and eluted with petroleum ether (60–90 °C)–acetone (100 : 5, 100 : 10, 100 : 20, 100 : 50, 100 : 100 and 0 : 100, v/v) to yield six fractions (A–F). Fraction D (50 g) was subjected to Rp-C₁₈ CC (50 μm; 180 g; Φ 6 cm × 45 cm) eluting with MeOH–H₂O (50 : 50, 60 : 40, 65 : 35, 70 : 30, 80 : 20, 90 : 10 and 100 : 0, v/v) to afford seven major subfractions (D1–D7). Subfraction D2 was separated by reversed-phase preparative HPLC (MeOH–H₂O, 75 : 25, v/v; flow rate, 5 mL min⁻¹) to give four fractions (D2-1–D2-4). D2-2 was further purified by preparative HPLC procedure (MeOH–H₂O, 70 : 30, v/v; flow rate, 5 mL min⁻¹) to yield compounds 1 (8 mg, t_R = 32.6 min), 2 (6 mg, t_R = 84.5 min), and 3 (6 mg, t_R = 90.2 min). Subfraction D3 was separated by reversed-phase preparative HPLC (MeOH–H₂O, 75 : 25, v/v; flow rate, 5 mL min⁻¹), followed by purification using Sephadex LH-20 with CH₂Cl₂–MeOH (1 : 1) to afford 4 (15 mg) and 5 (16 mg). Fraction E (70 g) was loaded onto a silica gel column (200–300 mesh; 650 g; Φ 5.3 cm × 140 cm) using CH₂Cl₂–MeOH (100 : 0, 100 : 1, 100 : 2, 100 : 3, 100 : 5, and 0 : 100, v/v) as the eluting reagent to furnish six subfractions (E1–E6). Subfraction E1 (8 g) was chromatographed over Rp-C₁₈ CC (50 μm; 150 g; Φ 4 cm × 50 cm) eluted with MeOH–H₂O (30 : 70, 45 : 55, 50 : 50, 55 : 45, 65 : 35, 75 : 25, 0 : 100, v/v) to give seven fractions (E1a–E1g). Fraction E1b was purified by Sephadex LH-20 with CH₂Cl₂–MeOH (1 : 1) to afford compound 8 (4 mg). Fraction E1e was purified by Sephadex LH-20 with CH₂Cl₂–MeOH (1 : 1) to yield 6 (5 mg) and 7 (5 mg). The chiral HPLC separations of 1–5 were performed on a chiral column (Daicel Chiralpak IC) to yield 1a (1.80 mg), 1b (1.80 mg), 2a (0.97 mg), 2b (0.97 mg), 3a (0.95 mg), 3b (0.95 mg), 4a (1.80 mg), 4b (1.80 mg), 5a (0.40 mg), and 5b (0.40 mg), respectively.

Characterization of new compounds

Macleayin C (1). Colourless cluster crystal; [α]²⁵_D 0 (c 0.16, CHCl₃); UV (MeOH) λ_max (log ε) 227 (4.6), 281 (4.6), 320 (4.0) nm; IR (KBr) ν_max 3470, 2926, 1659, 1605, 1492, 1464 cm⁻¹; ¹H and ¹³C NMR data, see Table 1; positive HRESIMS m/z 596.1892 [M + Na]⁺ (calcd for C₃₂H₃₁NO₉Na, 596.1891). (−)-1a: [α]²⁰_D = −85.6 (c 0.18, MeOH), (+)-1b: [α]²⁰_D = +94.4 (c 0.18, MeOH).

Macleayin D (2). Colourless cluster crystal; [α]²⁵_D 0 (c 0.20, CHCl₃); UV (MeOH) λ_max (log ε) 229 (4.6), 283 (4.6), 318 (4.2) nm; IR (KBr) ν_max 3382, 2926, 2854, 2798, 1680, 1602, 1494, 1465 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; positive HRESIMS m/z 407.1602 [M + H]⁺ (calcd for C₂₃H₂₃N₂O₅, 407.1601). (−)-2a: [α]²⁰_D = −126.3 (c 0.097, MeOH), (+)-2b: [α]²⁰_D = +142.8 (c 0.097, MeOH).

Macleayin E (3). Colourless cluster crystal; [α]²⁵_D 0 (c 0.16, CHCl₃); UV (MeOH) λ_max (log ε) 236 (4.6), 286 (4.6), 323 (4.2) nm; IR (KBr) ν_max 3397, 2895, 1675, 1602, 1496, 1464 cm⁻¹; ¹H and ¹³C NMR data, see Table 2; positive HRESIMS m/z 413.1110 [M + Na]⁺ (calcd for C₂₂H₁₈N₂O₅Na, 413.1108). (−)-3a: [α]²⁰_D = −143.8 (c 0.095, MeOH), (+)-3b: [α]²⁰_D = +138.1 (c 0.095, MeOH).

6-Acetonyldihydrosanguinarine (4). Colourless cluster crystal; [α]²⁵_D 0 (c 0.80, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ: 7.70 (1H, d, J = 8.4 Hz, H-11), 7.60 (1H, s, H-4), 7.51 (1H, d, J = 8.4 Hz, H-12), 7.34 (1H, d, J = 8.1 Hz, H-10), 7.11 (1H, s, H-1), 6.87 (1H, d, J = 8.1 Hz, H-9), 6.05 (4H, s, –OCH₂O-2,3, –OCH₂O-7,8), 4.91 (1H, br s, H-6), 2.75 (1H, br s, Ha-13), 2.68 (3H, s, N–CH₃), 2.31 (1H, br d, J = 14.4 Hz, Hb-13), 2.07 (3H, s, 15-CH₃). ¹³C NMR (100 MHz, CDCl₃) δ: 104.5 (C-1), 147.7 (C-2), 148.4 (C-3), 100.7 (C-4), 127.5 (C-4a), 139.3 (C-4b), 43.2 (N–CH₃), 54.6 (C-6), 123.5 (C-6a), 144.4 (C-7), 147.3 (C-8), 107.7 (C-9), 116.6 (C-10), 125.7 (C-10a), 116.1 (C-10b), 120.1 (C-11), 124.2 (C-12), 131.1 (C-12a), 46.7 (C-13a,b), 207.3 (C-14), 31.4 (C-15), 101.2 (–OCH₂O-2,3), 101.7 (–OCH₂O-7,8). (−)-4a: [α]²⁰_D = −128.3 (c 0.18, MeOH), (+)-4b: [α]²⁰_D = +130.6 (c 0.18, MeOH).

6-Acetonyldihydrochelerythrine (5). ¹H NMR (400 MHz, CDCl₃) δ: 7.71 (1H, d, J = 8.6 Hz, H-11), 7.55 (1H, d, J = 8.6 Hz, H-10), 7.50 (1H, d, J = 8.5 Hz, H-12), 7.11 (1H, s, H-1), 6.97 (1H, d, J = 8.5 Hz, H-9), 6.05 (2H, s, –OCH₂O-2,3), 5.08 (1H, br d, J = 11.0 Hz, H-6), 3.96 (3H, s, 8-OCH₃), 3.93 (3H, s, 7-OCH₃), 2.67 (4H, s, N–CH₃, Ha-13), 2.26 (1H, dd, J = 14.8, 4.1 Hz, Hb-13), 2.08 (3H, s, 15-CH₃). ¹³C NMR (150 MHz, CDCl₃) δ: 104.5 (C-1), 147.7 (C-2), 148.3 (C-3), 100.7 (C-4), 128.3 (C-4a), 139.4 (C-4b), 42.9 (N–CH₃), 55.0 (C-6), 127.4 (C-6a), 145.6 (C-7), 152.3 (C-8), 111.6 (C-9), 118.9 (C-10), 124.9 (C-10a), 124.0 (C-10b), 119.9 (C-11), 123.4 (C-12), 131.2 (C-12a), 46.9 (C-13), 207.7 (C-14), 31.2 (C-15), 101.2 (–OCH₂O–), 61.1 (7-OCH₃), 55.9 (8-OCH₃). (−)-5a: [α]²⁰_D = −125.0 (c 0.04, MeOH), (+)-5b: [α]²⁰_D = +137.5 (c 0.04, MeOH).

6-Methoxyldihydrosanguinarine (6). ¹H NMR (400 MHz, CDCl₃) δ: 7.76 (1H, d, J = 8.6 Hz, H-11), 7.70 (1H, s, H-4), 7.49 (1H, d, J = 8.6 Hz, H-12), 7.42 (1H, d, J = 8.2 Hz, H-10), 7.13 (1H, s, H-1), 6.94 (1H, d, J = 8.2 Hz, H-9), 6.12, 6.07, 6.06, 6.05 (4H, each d, J = 1.5 Hz, –OCH₂O-2,3, –OCH₂O-7,8), 5.38 (1H, s, H-6), 3.46 (3H, s, 6-OCH₃), 2.79 (3H, s, N–CH₃). ¹³C NMR (100 MHz, CDCl₃) δ: 104.8 (C-1), 148.2 (C-2), 147.6 (C-3), 100.8 (C-4), 127.0 (C-4a), 138.3 (C-4b), 41.0 (N–CH₃), 86.0 (C-6), 127.0 (C-6a), 145.4 (C-7), 147.4 (C-8), 113.3 (C-9), 116.5 (C-10), 125.9 (C-10a), 122.9 (C-10b), 120.4 (C-11), 123.9 (C-12), 54.2 (6-OCH₃), 101.2 (–OCH₂O-2,3), 101.9 (–OCH₂O-7,8).

6-Methoxyldihydrochelerythrine (7). ¹H NMR (400 MHz, CDCl₃) δ: 7.78 (1H, d, J = 8.6 Hz, H-11), 7.71 (1H, s, H-4), 7.63 (1H, d, J = 8.6 Hz, H-10), 7.48 (1H, d, J = 8.6 Hz, H-12), 7.13 (1H, s, H-1), 7.05 (1H, d, J = 8.6 Hz, H-9), 6.06, 6.05 (2H, each d, J = 1.1 Hz, –OCH₂O-2,3), 5.55 (1H, s, H-6), 3.97 (3H, s, 7-OCH₃), 3.93 (3H, s, 8-OCH₃), 3.47 (3H, s, 6-OCH₃), 2.77 (3H, s, N–CH₃). ¹³C NMR (100 MHz, CDCl₃) δ: 104.8 (C-1), 148.1 (C-2), 147.5 (C-3), 100.8 (C-4), 126.9 (C-4a), 138.5 (C-4b), 40.8 (N–CH₃), 86.2 (C-6), 125.9 (C-6a), 146.8 (C-7), 152.2 (C-8), 113.1 (C-9), 119.1 (C-10), 125.0 (C-10a), 122.7 (C-10b), 120.1 (C-11), 123.6 (C-12), 131.2 (C-12a), 61.8 (7-OCH₃), 56.1 (8-OCH₃), 54.1 (6-OCH₃), 101.2 (–OCH₂O-2,3).

Spallidamine (8). ¹H NMR (400 MHz, DMSO-d₆) δ: 12.19 (1H, br s), 7.80 (1H, d, J = 8.7 Hz, H-11), 7.56 (1H, d, J = 8.7 Hz, H-12), 7.46 (1H, d, J = 8.2 Hz, H-10), 7.42 (1H, s, H-4), 7.31 (1H, s, H-1), 6.97 (1H, d, J = 8.2 Hz, H-9), 6.14, 6.13 (2H, each s, –OCH₂O-2,3), 6.12 (2H, s, –OCH₂O-7,8), 4.69 (1H, dd, J = 11.1, 3.8 Hz, H-6), 2.55 (3H, s, N–CH₃), 2.28 (1H, dd, J = 14.4, 3.3 Hz, Ha-13), 2.06 (1H, m, Hb-13). ¹³C NMR (100 MHz, DMSO-d₆) δ:104.1 (C-1), 144.1 (C-2), 146.9 (C-3), 99.9 (C-4), 126.8 (C-4a), 138.6 (C-4b), 42.9 (N–CH₃), 54.3 (C-6), 115.2 (C-6a), 147.3 (C-7), 147.8 (C-8), 107.6 (C-9), 116.5 (C-10), 124.9 (C-10a), 122.7 (C-10b), 119.9 (C-11), 123.9 (C-12), 101.2 (–OCH₂O-2,3), 101.6 (–OCH₂O-7,8), 38.6 (C-13), 171.7 (C-14).

Biological assays

Cytotoxicity assay²³. The following human cancer cell lines were used: HL-60 (human leukaemia cell lines), MCF-7 (human breast cancer cell lines), A-549 (human lung adenocarcinoma cell lines), which were purchased from America Type Culture Collection, ATCC (Rockville, MD, USA) and cultured in RPMI-1640 medium (Gibco, New York, NY, USA) supplemented with 100 U mL⁻¹ penicillin, 100 μg mL⁻¹ streptomycin, 1 mM glutamine and 10% heat-inactivated fetal bovine serum (Gibco) at 37 °C in humidified atmosphere with 5% CO₂. Cytotoxic activity was evaluated by the trypan blue method against HL-60, and MTT assay against MCF-7 and A-549.

In the trypan blue method, cells in logarithmic growth were seeded at 5 × 10⁴ cells per mL in 24-well microplates and incubated with various concentrations of the compounds under a humidified atmosphere of 5% CO₂ and 95% air at 37 °C for 3 days. The compounds were dissolved in DMSO and then diluted to the proper concentrations. Cell viability was determined after staining the cells with trypan blue. Trypan blue-stained (nonviable) cells and the total cell number were determined with a hemocytometer. The growth inhibition in cells after treatment with different concentrations was calculated comparing with control cells (5-fluorouracil was used as a positive control), and a half growth inhibitory concentration (IC₅₀) was obtained by regression analysis of the concentration response data.²⁴

In the MTT assay, briefly, cells suspensions, 200 μL, at a density of 2.5 × 10⁴ cells per mL, were seeded into 96-well microtiter plates and incubated for 24 h at 37 °C under 5% CO₂ and 95% air. Then the tested compounds with different concentrations in DMSO were placed into each microtiter plates and further incubated for 72 h. Finally, 50 μL 0.4% MTT solution was added to each well and incubated for 4 h. Then, the MTT was removed from the wells and the fromazan crystals were dissolved in DMSO (200 μL) for 10 min with shaking. Then the plate was read immediately on a microtiter plate reader (Bio-RAD) at a wavelength of 570 nm to record the optical density (OD). The IC₅₀ value was defined as the concentration of the control in the MTT assay. 5-Fluorouracil (5-Fu) was used as a positive control. All the IC₅₀ results were expressed as average of three independent experiments.²⁵

Antibacterial assay^26,27. Escherichia coli (CMCC (B) 44102), Staphylococcus aureus (CMCC (B) 26003), Bacillus subtilis (CMCC (B) 63501), Candida albicans (CMCC (F) 98001) were purchased from Liaoning Institute for Food and Drug Control, Shenyang, China. Chloramphenicol (purchased from Dalian Meilun Biology Technology Co. Ltd., Dalian, China) was induced as a positive control. The antimicrobial assay was done by micro-dilution broth MIC method. Bacterial species were cultured in Broth bouillon medium at 37 °C for 24 h; fungus was cultured in Sabouraud medium for 48 h at 28 °C. The 2 generation spore suspension was adjusted with medium to a concentration of approximately 1.0 × 10⁵ CFU mL⁻¹ for bacteria and 1.0 × 10³ CFU mL⁻¹ for fungus in a final volume of 100 μL per well. Minimum inhibitory concentration (MIC) determinations were carried out by a serial dilution technique using 96-well microtiter plates. The compounds isolated were dissolved with DMSO-hydrogenated castor oil (3 : 1) in different concentrations (0.2–500 μg mL⁻¹) and added in broth medium (bacteria)/Sabouraud (fungus) with inocula [preliminary analyses with DMSO/hydrogenated castor oil (3 : 1) do not inhibit the growth of the test organisms]. The microplates (bacteria) were incubated at 37 °C for 18–24 h, and the microplates (fungus) at 28 °C for 24–28 h. At the lowest concentration, the microorganisms did not show growth as judged by the presence of turbidity.

Acknowledgements

The work was financially supported by the National Natural Science Foundation of China (Grant No. 81172958), the Basic Research Subject of Key Laboratory Supported by Educational Commission of Liaoning Province of China (No. LZ2014044). We gratefully acknowledge Mr Yi Sha and Mrs Wen Li, Department of Analytical Testing Center, Shenyang Pharmaceutical University, for measurement of the NMR data.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: 1D and 2D NMR, HRESIMS, UV, IR, CD, and quantum chemical ECD calculations of 1–6. See DOI: 10.1039/c6ra05423d

‡ Chun-Mei Sai and Da-Hong Li contributed equally to this work.

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