Mucroniferanines A–G, Isoquinoline Alkaloids from Corydalis

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Article Cite This: J. Nat. Prod. 2018, 81, 364−370

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Mucroniferanines A−G, Isoquinoline Alkaloids from Corydalis mucronifera Jun Zhang,† Qing-Ying Zhang,† Peng-Fei Tu,† Fu-Chun Xu,*,‡ and Hong Liang*,† †

State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100191, People’s Republic of China ‡ Medical College of Tibet University, Lhasa 850000, People’s Republic of China S Supporting Information *

ABSTRACT: Five pairs of isoquinoline alkaloid enantiomers, mucroniferanines A−E (1−5), two inseparable epimeric pairs, mucroniferanines F and G (6, 7), and 10 known isoquinoline alkaloids (8−17) were obtained from Corydalis mucronifera. The structures were characterized using spectroscopic data analysis, and the absolute configurations were established by ECD and X-ray data analysis. The new compounds except for 3 possess a rare 9methyl group in the isoquinoline alkaloids, and compounds 2 and 3 possess rare benzo[1,2-d:3,4-d]bis[1,3]dioxole moieties. It is the first report of stereoisomerism involving the 9-methyl phthalideisoquinoline alkaloids. Compounds (−)-4, 6, and 7 exhibited acetylcholinesterase inhibitory activities with IC50 values of 28.3, 12.2, and 11.3 μM, respectively.

I

hydrogen deficiency. The UV spectrum showed characteristic absorptions for an isoquinoline alkaloid (λmax 203, 235 nm), and the IR spectrum showed absorption bands for hydroxy (3419 cm−1) and aromatic ring (1468 cm−1) functionalities. The 1H NMR spectrum displayed the presence of a 1,6,7trisubstituted isoquinoline moiety [δH 7.10 (1H, s, H-5), 7.65 (1H, s, H-8), 8.23 (1H, d, J = 5.6 Hz, H-3), and 7.42 (1H, d, J = 5.6 Hz, H-4)] and a 1,2,3,4-tetrasubstituted phenyl group [δH 6.31 (1H, d, J = 7.9 Hz, H-5′) and 6.71 (1H, d, J = 7.9 Hz, H6′)]. The 1H NMR spectrum also displayed a CH−CH3 [δH 4.79 (1H, q, J = 7.2 Hz, H-9) and 1.80 (3H, d, J = 7.2 Hz, H10)] and two methylenedioxy [δH 5.95 (2H, s), 6.14 (1H, s), and 6.15 (1H, s)] moieties. The 13C NMR data (Table 1) showed 19 carbon signals comprising 15 aromatic carbons, a methyl, a methine, and two methylenedioxy groups. The CH− CH3 moiety was attached to C-1 of the isoquinoline moiety and C-1′ of the phenyl group on the basis of the HMBC correlations of H-9/C-8a, C-2′, and C-6′ and Me-10/C-1 and C-1′. The locations of the methylenedioxy moieties were assigned to C-6, C-7 and C-3′, C-4′, respectively, by HMBC correlations of H-5/C-7 and C-8a; H-8/C-6, C-4a, and C-1; H6′/C-2′ and C-4′; and H-5′/C-1′ and C-3′. A hydroxy group was attached to C-2′ as deduced from the molecular formula and the HMBC correlations of H-9/C-2′ and C-6′. Thus, the 2D structure of 1 was determined as shown in Figure 1. Compound 1 was similar to sauvagnine, isolated from Corydalis dlaviculata,6 except for substitution of a methyl group at C-9 in place of the carbonyl functionality.

soquinoline alkaloids, the main bioactive constituents of the genus Corydalis, possess various bioactivities including acetylcholinesterase (AChE) inhibitory, antiproliferative, antiviral, and antiplasmodial activities.1,2 Corydalis mucronifera Maxim. (family Papaveraceae) is widely distributed in the high altitude regions (4200−5300 m) of China, such as Gansu, Qinghai, and Tibet.3 The whole plant of C. mucronifera has been used to treat hepatitis, hypertension, paralytic stroke, and traumatic injuries as a Tibetan medicine in China, but only four isoquinoline alkaloids have been reported.4,5 Our previous screening showed that the total alkaloid extract of C. mucronifera had strong AChE inhibitory activity at a concentration of 1.0 mg/mL. The total alkaloid extract of the whole plants of C. mucronifera was investigated and afforded five pairs of new isoquinoline alkaloid enantiomers, mucroniferanines A−E (1−5), two inseparable epimeric pairs, mucroniferanines F and G (6, 7), and 10 known isoquinoline alkaloids (8−17). Herein, the isolation, structural elucidation, and the AChE inhibitory activities are reported.

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RESULTS AND DISCUSSION

The 95% aqueous EtOH extract (390.0 g) of C. mucronifera was suspended in H2O (1.0 L) and subjected to acid−base extraction to yield the alkaloid extract (80.0 g). The extract was subjected repeatedly to various column chromatographies to afford new isoquinoline alkaloids, mucroniferanines A−G (1−7), and 10 analogues (8−17). Mucroniferanine A (1) was obtained as colorless crystals. Its molecular formula was determined as C19H15NO5 by the positive HRESIMS ion at m/z 338.1024 [M + H]+ (calcd for C19H16NO5, 338.1028), corresponding to 13 indices of © 2018 American Chemical Society and American Society of Pharmacognosy

Received: October 6, 2017 Published: February 5, 2018 364

DOI: 10.1021/acs.jnatprod.7b00847 J. Nat. Prod. 2018, 81, 364−370

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Table 1. 13C NMR Data of Compounds 1−7 position

1c

2c

3e

4c

5c

6a(7a)d

6b(7b)d

1 3 4 4a 5 6 7 8 8a 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′ OCH2O OCH2O OCH3

162.9, C 138.7, CH 120.0, CH 136.2, C 103.7, CH 149.5,a C 151.5,a C 101.0, CH 122.9, C 43.8, CH 20.5, CH3 125.4, C 142.1, C 137.3, C 148.2, C 99.8, CH 122.8, CH

154.1, C 140.3, CH 122.2, CH 136.2, C 103.3, CH 148.7,a C 150.4,a C 102.6, CH 122.9, C 119.9, C 25.4, CH3 144.2, C 129.6, C 130.2, C 144.9, C 100.2,b CH 100.6,b CH

151.2, C 141.1, CH 124.3, CH 137.8, C 104.1, CH 150.9,a C 153.0,a C 101.3, CH 125.1, C 113.1, CH

102.1, CH2 101.4, CH2

101.8, CH2 101.9, CH2

103.4, CH2 103.8, CH2

160.0, C 140.0, CH 120.2, CH 136.2, C 103.2, CH 147.7,a C 149.9,a C 104.4, CH 123.0, C 92.0, C 30.4, CH3 141.50, C 118.8, C 141.52, C 147.8,a C 109.5, CH 118.6, CH 104.1, CH 101.6, CH2 101.9, CH2 55.1, CH3

158.3, C 139.9, CH 121.0, CH 136.3, C 103.4, CH 147.8,a C 149.9,a C 103.35, CH 123.4, C 92.6, C 31.4, CH3 142.5, C 118.7, C 142.0, C 148.1, C 109.9, CH 116.4, CH 104.6, CH 101.6, CH2 102.1, CH2 55.5, CH3

158.6, C 139.9, CH 120.8, CH 136.2, C 103.26, CH 147.8,a C 149.9,a C 103.6, CH 123.3, C 92.8, C 32.3, CH3 141.87,b C 119.9, C 141.90,b C 148.2, C 109.9, CH 117.2, CH 98.4, CH 101.7, CH2 102.1, CH2

160.1, C 139.8, CH 120.5, CH 136.4, C 103.28, CH 147.9,a C 150.1,a C 104.5, CH 122.9, C 91.9, C 30.2, CH3 141.0, C 120.7, C 141.6, C 148.0, C 109.6, CH 118.2, CH 98.0, CH 101.6, CH2 102.0, CH2

145.7, C 130.9, C 131.3, C 146.7, C 101.4, CH 101.27, CH

a,b Interchangeable within the same column. cRecorded in CDCl3 at 100 MHz. dRecorded in CDCl3 at 150 MHz. eRecorded in methanol-d4 at 150 MHz.

Figure 1. Structures of compounds 1−17.

The single-crystal X-ray diffraction data showed a P1̅ space group, indicating that 1 was a racemic mixture (Figure 3), which was further supported by the lack of optical rotation. Subsequently, 1 was separated into enantiomers (+)-1 ([α]25D +72, tR = 19.0 min) and (−)-1 ([α]25D −71, tR = 25.9 min) by chiral-phase HPLC using a CHIRALPAK AD-H column with

n-hexane−2-propanol (70:30, v/v), in a 1:1 ratio (Figure S1, Supporting Information). Consequently, the absolute configurations of (+)-1 and (−)-1 were determined as (R) and (S), respectively, by comparison of the experimental and calculated electronic circular dichroism (ECD) data (Figure 5), and thus named (R)- and (S)-mucroniferanine A, respectively. 365

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and C-3′, C-4′, respectively, by HMBC correlations of H-5/C-7 and C-8a; H-8/C-6, C-4a, and C-1; H-6′/C-2′ and C-4′; and H-5′/C-1′ and C-3′. The remaining two oxygen atoms were substituted to C-1′ and C-2′, assigned by the HMBC correlations of H-6′/C-2′ and H-5′/C-1′, and formed an additional benzodioxole ring with C-9 as deduced from the remaining index of hydrogen deficiency and the downfield shift of C-9 (δC 119.9) in the 13C NMR spectrum. Thus, the 2D structure of 2 with a new benzo[1,2-d:3,4-d]bis[1,3]dioxole moiety was established as shown in Figure 1. Compound 2 was also a racemic mixture, as evidenced by the lack of optical rotation. Subsequently, 2 was separated into enantiomers (+)-2 ([α]25D +60, tR = 19.1 min) and (−)-2 ([α]25D −64, tR = 20.6 min) using a CHIRALPAK AD-H column with a mobile phase of n-hexane−2-propanol (80:20, v/v) (Figure S2, Supporting Information). The experimental and calculated ECD spectra of (R)-2 and (S)-2 formed a good match (Figure 5). Therefore, the enantiomers of (+)-2 and (−)-2 were elucidated as (R)- and (S)-mucroniferanine B, respectively. Mucroniferanine C (3) was obtained as an amorphous powder. Its molecular formula was deduced as C18H11NO6 from the positive HRESIMS ion at m/z 338.0665 [M + H]+ (calcd for C18H12NO6, 338.0665), indicating 14 indices of hydrogen deficiency. The NMR data (Tables 1 and 2) of 3 resembled those of 2 except for the absence of a methyl group at C-9 in 3. Similarly, 3 was a racemic mixture and was separated into enantiomers (−)-3 ([α]25D −47, tR = 19.7 min) and (+)-3 ([α]25D +42, tR = 21.8 min) by chiral-phase HPLC using a CHIRALPAK ID column with a mobile phase of nhexane−2-propanol (80:20, v/v) (Figure S3, Supporting Information). The structures, optical rotations, and ECD spectra of (+)-3 and (−)-3 were similar to those of (+)-2 and (−)-2. Therefore, the enantiomers of (+)-3 and (−)-3 were elucidated as (R)- and (S)-mucroniferanine C, respectively, by comparison of their ECD data with (R)-2 and (S)-2 (Figure 5). Mucroniferanine D (4) was obtained as an amorphous powder. Its molecular formula was deduced as C21H17NO6 (14 indices of hydrogen deficiency) on the basis of the positive HRESIMS ion (m/z 380.1138 [M + H]+, calcd for C21H18NO6, 380.1134). The NMR data of 4 (Tables 1 and 2) were similar to those of 1, suggesting 4 had a 1,6,7-trisubstituted isoquinoline moiety and a 1,2,3,4-tetrasubstituted phenyl group with two methylenedioxy groups assigned to C-6, C-7 and C-3′, C-4′, respectively. In addition, the 1H and 13C NMR data (Tables 1 and 2) of 4 displayed a methyl [δH 1.96 (3H, s, Me-10)], a methoxy [δH 3.19 (3H, s, OCH3)], an oxygenated methine [δH 6.34 (1H, s, H-7′)], and an oxygenated tertiary carbon (δC 92.0, C-9). The presence of a five-membered acetal moiety was deduced from the HMBC correlations of H-7′/C1′, C-3′, and C-9 and H-6′/C-9 and C-2′. The methyl and methoxy groups were assigned to C-9 and C-7′, respectively, by the HMBC correlations (Figure 2). Thus, the 2D structure of 4 was determined as shown in Figure 1. The 7′,9-trans relative configuration was established by the NOE correlation between H-7′ and Me-10 (Figure 4). Compound 4 was also a racemic mixture, as shown by the specific rotation, [α]25D 0 (c 0.1, MeOH), and was subsequently separated into enantiomers (+)-4 ([α]25D +51, tR = 8.5 min) and (−)-4 ([α]25D −54, tR = 9.1 min) by chiral-phase HPLC using a CHIRALPAK AD-H column with a mobile phase of nhexane−2-propanol (85:15, v/v) (Figure S4, Supporting

Figure 2. Key HMBC correlations of compounds 1−7.

Figure 3. Unit cell of 1 showing a 1:1 racemic mixture.

Figure 4. Selected NOE correlations for 4 and 5.

Mucroniferanine B (2) was obtained as an amorphous powder. Its molecular formula was established as C19H13NO6 (14 indices of hydrogen deficiency) from the 13C NMR data and the positive HRESIMS ion at m/z 352.0819 [M + H]+ (calcd for C19H14NO6, 352.0821). Similar to compound 1, the 1 H and 13C NMR data (Tables 1 and 2) of 2 also displayed signals of a 1,6,7-trisubstituted isoquinoline moiety [δH 7.08 (1H, s, H-5), 7.70 (1H, s, H-8), 8.39 (1H, d, J = 5.5 Hz, H-3), and 7.50 (1H, d, J = 5.5 Hz, H-4)], a 1,2,3,4-tetrasubstituted phenyl group [δH 6.34 (1H, d, J = 8.2 Hz, H-5′) and 6.42 (1H, d, J = 8.2 Hz, H-6′)], a methyl [δH 2.16 (3H, s, Me-10)], and two methylenedioxy [δH 5.94 (1H, d, J = 1.3 Hz), 5.90 (1H, d, J = 1.3 Hz), and 6.08 (2H, s)] moieties. The methyl group was attached to C-9 of the C-1 substituent of the isoquinoline moiety on the basis of the HMBC correlations of Me-10/C-1 and C-9. The methylenedioxy groups were assigned to C-6, C-7 366

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Figure 5. Experimental ECD spectra of mucroniferanines A−E (1−5) in MeOH and calculated ECD spectra of (R)-1, (S)-1, (R)-2, (S)-2, (7′R,9R)5, and (7′S,9S)-5.

Table 2. 1H NMR Data of Compounds 1−7 position

1c (J in Hz)

2c (J in Hz)

3e (J in Hz)

4c (J in Hz)

5c (J in Hz)

3 4 5 8 9 10 5′ 6′ 7′ OCH2O

8.23, 7.42, 7.10, 7.65, 4.79, 1.80, 6.31, 6.71,

8.39, 7.50, 7.08, 7.70,

8.32, 7.78, 7.31, 7.44, 7.33,

8.21, 7.34, 7.05, 8.43,

d (5.5) d (5.5) s s

8.33, 7.42, 7.05, 7.61,

d (5.5) d (5.5) s s

8.28, 7.39, 7.04, 7.84,

1.96, 6.95, 7.08, 6.34, 6.09,

s d (8.0) d (8.0) s s

2.10, 6.82, 6.75, 6.38, 6.03, 6.06, 6.05, 6.10, 3.65,

s d d s d d d d s

2.12, s 6.87, d (8.0) 6.88, d (8.0) 6.69, s 6.03,a s

OCH2O

d d s s q d d d

5.95, s 6.14, s 6.15, s

(5.6) (5.6)

(7.2) (7.2) (7.9) (7.9)

d (5.5) d (5.5) s s

d (5.6) d (5.6) s s s

2.16, s 6.34,b d (8.2) 6.42,b d (8.2)

6.43, d (8.2) 6.45, d (8.2)

5.90, d (1.3) 5.94, d (1.3) 6.08, s

5.96, d (1.0) 5.99, d (1.0) 6.17, s

OCH3 a,b

6.03, s 3.19, s

(8.0) (8.0) (1.1) (1.1) (1.4) (1.4)

6a/7ad (J in Hz) d (5.5) d (5.5) s s

6.07,a d (1.0) 6.08,a d (1.0)

6b/7bd (J in Hz) 8.21, 7.35, 7.04, 8.36,

d (5.5) d (5.5) s s

1.98, s 6.93, d (8.0) 7.07, d (8.0) 6.63, s 6.06,a d (1.0) 6.09,a d (1.0) 6.01,a d (1.4) 6.03,a d (1.4)

c

Interchangeable within the same column. Recorded in CDCl3 at 400 MHz. dRecorded in CDCl3 at 600 MHz. eRecorded in methanol-d4 at 600 MHz.

alkaloid, 9-methyldecumbenine C, reported from Corydalis hendersonii,7 but its C-9 absolute configuration was not defined. Mucroniferanine E (5) was obtained as an amorphous powder. It had the same molecular formula (C21H17NO6) as 4 based on the positive HRESIMS ion at m/z 380.1139 [M + H]+. The 2D structure was identical to that of 4 as supported by similar 1H and 13C NMR data (Tables 1 and 2) and 2D NMR data analysis. The 7′,9-cis relative configuration was established by the absence of an NOE correlation between H-7′ and Me-10 (Figure 4). Compound 5 was also a racemic

Information). The C-9 absolute configurations in (+)-4 and (−)-4 were assigned as (S) and (R) by comparison of their experimental and calculated ECD data of the (7′R, 9R) and (7′S, 9S) enantiomers (Figure 5). The C-7′ absolute configurations in (+)-4 and (−)-4 were defined as (R) and (S) based upon the relative configurations. Therefore, the absolute configurations of (+)-4 and (−)-4 were determined to be (7′R, 9S) and (7′S, 9R), respectively. It is the first report of stereoisomerism involving the 9-methylphthalideisoquinoline alkaloids. There is only one similar phthalideisoquinoline 367

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the same as those of 6a and 6b as deduced from the identical 1 H and 13C NMR data. Similarly, the 7′,9-relative configurations in 7a and 7b were defined as cis and trans, and the (9R) absolute configurations of 7a and 7b ([α]25D −68) were defined by using the same methods as above. Therefore, the absolute configurations of 7a and 7b were determined to be (7′R, 9R) and (7′S, 9R), respectively. Interestingly, the hydroxyaldehyde−hemiacetal tautomeric mixtures present in 6 and 7 were not found in the known tetrahydroisoquinoline alkaloid 17. A possible reason might be the presence of a stable hydrogen bond formed between N-2 and the hydroxy proton in 17, as is evident in the X-ray data of 17.8 Such conditions are clearly absent in compounds 6 and 7. The known compounds were identified as 8methoxydihydrosanguinarine (8),9 dihydrosanguinarine (9),10 (±)-hypecorinine (10),11 protopine (11),12 (−)-7′-O-methylegenine (12),13 oxohydrastinine (13),14 tetrahydroberberrubine (14),15 (+)-adlumine (15),16 sibiricine (16),17 and (+)-humosine A (17)18 by comparison of the observed and reported physical data. Acetylcholinesterase inhibition is considered as one of the major pharmacological means for enhancement of the central cholinergic activity.19,20 Thus, the isolates from C. mucronifera were tested for their AChE inhibitory activities. As shown in Table 3, compounds (−)-3, (−)-4, 6, 7, and 8 exhibited AChE

mixture, as evidenced by the lack of an optical rotation, and was separated into enantiomers (−)-5 ([α]25D −47, tR = 8.5 min) and (+)-5 ([α]25D +44, tR = 9.6 min) using a CHIRALPAK ADH column with a mobile phase of n-hexane−2-propanol (85:15, v/v) (Figure S5, Supporting Information). The absolute configurations of (+)-5 and (−)-5 were defined as (7′S, 9S) and (7′R, 9R), respectively, by using the same methods as described for 4. Mucroniferanine F (6) was obtained as an amorphous powder. Its molecular formula was established as C20H15NO6 on the basis of the positive HRESIMS ion (m/z 366.0974 [M + H]+, calcd for C20H16NO6, 366.0978), indicating 14 indices of hydrogen deficiency. The 1H and 13C NMR spectra of 6 displayed a mixture of two isomers 6a and 6b in a 1:1 ratio. Analysis of the 1H and 13C NMR data (Tables 1 and 2) of 6a and 6b showed close similarity to those of 4 and 5, except for the presence of a hydroxy group in 6a and 6b instead of a methoxy group in 4 and 5. Furthermore, the 7′,9-relative configurations in 6a and 6b were defined as cis and trans on the basis of the NOE correlation of Me-10 and H-7′, respectively. The (9S) absolute configurations of 6a and 6b ([α]25D +66) were defined by comparison of their experimental ECD data with the calculated ECD spectra of 6 and 7 (Figure 6). The

Table 3. AChE Inhibitory Activities of the Isolates

Figure 6. Experimental ECD spectra of 6 and 7 in MeOH and calculated ECD spectra of (7′S,9S)-6, (7′R,9S)-6, (7′R,9R)-7, and (7′S,9R)-7. a

(7′S) and (7′R) absolute configurations in 6a and 6b, respectively, were deduced from the relative configurations. Subsequent separation by chiral-phase HPLC using a CHIRALPAK AD-H column afforded two epimers (epimer 1, tR = 15.7 min; epimer 2, tR = 22.6 min). However, the preparation of purified isomers failed due to the rapid hydroxyaldehyde−hemiacetal interconversion or ring−chain tautomerism at C-7′. Epimers 1 and 2 interconverted rapidly after separation, existing as a 1:1 mixture (Figure S6, Supporting Information). Therefore, the absolute configurations of 6a and 6b were defined as (7′S, 9S) and (7′R, 9S), respectively. Mucroniferanine F (7) was obtained as an amorphous powder. Its molecular formula was determined as C20H15NO6 by the positive HRESIMS ion (m/z 366.0974 [M + H]+, calcd for C20H16NO6, 366.0978), the same as that of 6. The 1H and 13 C NMR data of 7 were identical to those of 6, suggesting that 7 was also a mixture of two epimers, 7a and 7b, that interconverted rapidly after separation by chiral-phase HPLC using a CHIRALPAK AD-H column (epimer 1, tR = 17.5 min; epimer 2, tR = 19.1 min). The 2D structures of 7a and 7b were

compound

IC50 (μM)

compound

IC50 (μM)

(+)-1 (−)-1 (+)-2 (−)-2 (+)-3 (−)-3 (+)-4 (−)-4 (+)-5 (−)-5 6 galanthaminea

>100 >100 >100 >100 >100 78.1 ± 1.0 >100 28.3 ± 0.4 >100 >100 12.2 ± 0.2 1.9 ± 0.5

7 8 9 10 11 12 13 14 15 16 17

11.3 ± 0.8 96.8 ± 1.3 >100 >100 >100 >100 >100 >100 >100 >100 >100

Positive control.

inhibitory activities with IC50 values of 78.1, 28.3, 12.2, 11.3, and 96.8 μM, respectively, whereas the others were inactive, compared with the positive control galanthamine (IC50 = 1.9 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an X-5 micromelting point apparatus. Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter. UV spectra were recorded on a Shimadzu UV-2450 UV−visible spectrophotometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter. IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrometer. NMR spectra were measured on a Bruker AV-400 or AV-600 NMR spectrometer with tetramethylsilane as reference. HRESIMS data were acquired on a Waters Xevo G2 QTOF spectrometer. X-ray data were collected using a Rigaku Micromax-003 X-ray single-crystal diffractometer with Cu Kα radiation. Semipreparative HPLC was performed on a Phenomenex Gemini C18 column (250 × 10 mm, 5 μm) using an Agilent 1260 LC system. Column chromatography (CC) was performed on silica gel (200−300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), 368

DOI: 10.1021/acs.jnatprod.7b00847 J. Nat. Prod. 2018, 81, 364−370

Journal of Natural Products

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MCI gel (75−150 μm, Mitsubishi Chemical Ltd., Tokyo, Japan), Sephadex LH-20 (Pharmacia Fine Chemical Ltd., Uppsala, Sweden), and ODS (50 μm, YMC Co. Ltd., Kyoto, Japan). HPLC grade solvents used for HPLC analysis and preparation were purchased from Fisher Scientific International. Plant Material. The whole plant of C. mucronifera was collected in Lhasa, Tibet Autonomous Region, People’s Republic of China, in October 2016. The plant material was identified by Associate Professor Ying-Tao Zhang, Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University. A voucher specimen (No. CMW20161001) was deposited at the Herbarium of the Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University. Extraction and Isolation. The dried whole plants of C. mucronifera (10.0 kg) were extracted with 95% aqueous EtOH (9 × 25 L, each 24 h) at room temperature. The solvent was removed under reduced pressure to give a residue (390.0 g), which was suspended in H2O (1.0 L) and subjected to acid−base extraction to yield the alkaloid extract (80.0 g). The extract (80.0 g) was fractionated by silica gel column chromatography (800 g) using CHCl3−MeOH (100:0, 50:1, 20:1, 5:1, 0:100, v/v) as eluent to give five fractions (A1−A5) based on TLC analysis. Fr. A1 (0.15 g) was separated by semipreparative RP-HPLC (C18, 10 mm × 250 mm, H2O−MeCN, 50:50, v/v) to give 8 (20.5 mg, tR = 17.3 min) and 9 (18.9 mg, tR = 22.4 min). Fraction A2 (28.0 g) was subjected to silica gel CC (27 g), eluting with petroleum ether− EtOAc (8:1, 4:1, 2:1, v/v), to afford three subfractions, A2−1−A2−3. Fr. A2−2 (2.0 g) was separated by Sephadex LH-20 CC eluting with CHCl3−MeOH (1:1, v/v) followed by semipreparative RP-HPLC (C18, 10 mm × 250 mm, H2O−MeCN, 45:55, v/v) to yield 1 (27.0 mg, tR = 28.1 min), 3 (0.9 mg, tR = 29.0 min), 10 (8.4 mg, tR = 33.5 min), 2 (15.5 mg, tR = 37.2 min), 11 (7.0 mg, tR = 35.0 min), and 12 (38.6 mg, tR = 43.2 min). Fr. A2−3 (10.5 g) was fractionated over an MCI gel column eluting with H2O−MeOH (4:6, 3:7, 2:8, 0:10, v/v) to afford four fractions, A2−3−1−A2−3−4. Fr. A2−3−1 (140.0 mg) was separated by semipreparative RP-HPLC (C18, 10 mm × 250 mm, H2O−MeCN, 30:70, v/v) to furnish 13 (9.2 mg, tR = 17.3 min). Fr. A2−3−2 (2.0 g) was separated by semipreparative HPLC (C18, 10 mm × 250 mm, H2O−MeCN, 53:47, v/v) to yield 5 (5.4 mg, tR = 22.2 min), 4 (6.0 mg, tR = 22.8 min), 14 (27.0 mg, tR = 17.1 min), and a mixture fraction (19.4 mg, tR = 26.3 min), which was further separated by chiral-phase HPLC (CHIRALPAK AD-H column, 5 mm × 250 mm, n-hexane−2-propanol, 85:15, v/v) to yield 6 (8.5 mg, epimer 1, tR = 15.7 min, epimer 2, tR = 22.6 min) and 7 (8.3 mg, epimer 1, tR = 17.5 min, epimer 2, tR = 19.1 min). Fr. A2−3−3 (1.5 g) was separated by semipreparative HPLC (C18, 10 mm × 250 mm, H2O−MeCN, 55:45, v/v) to yield 15 (9.5 mg, tR = 22.8 min), 16 (9.2 mg, tR = 23.5 min), and 17 (45.0 mg, tR = 32.5 min). Mucroniferanine A (1): colorless crystals (CHCl3−MeOH, 55:45, v/v); mp 183−184 °C; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.48), 235 (4.62) nm; IR (KBr) νmax 3419, 2918, 1631, 1584, 1468, 1027 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 338.1024 [M + H]+ (calcd for C19H16NO5, 338.1028). (+)-Mucroniferanine A [(+)-1]: [α]25D +72 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 236 (+45.30), 275 (−3.68), 303 (+2.67) nm. (−)-Mucroniferanine A [(−)-1]: [α]25D −71 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 236 (−37.30), 275 (+3.05), 303 (−1.34) nm. Mucroniferanine B (2): amorphous powder; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.41), 235 (4.62) nm; IR (KBr) νmax 3384, 2915, 1653, 1581, 1466, 1243, 1059 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; HRESIMS m/z 352.0819 [M + H]+ (calcd for C19H14NO6, 352.0821). (+)-Mucroniferanine B [(+)-2]: [α]25D +60 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 213 (−5.39), 243 (+6.05), 326 (+0.31) nm. (−)-Mucroniferanine B [(−)-2]: [α]25D −64 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 213 (+4.94), 243 (−6.40), 326 (−0.61) nm. Mucroniferanine C (3): amorphous powder; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.59), 235 (4.57) nm; IR (KBr) νmax 3404, 2922, 2852, 1631, 1460, 1383, 1038 cm−1; 1H and

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C NMR data, see Tables 1 and 2; HRESIMS m/z 338.0665 [M + H]+ (calcd for C18H12NO6, 338.0665). (+)-Mucroniferanine C [(+)-3]: [α]25D +42 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 213 (−4.60), 243 (+4.26), 326 (+0.38) nm. (−)-Mucroniferanine C [(−)-3]: [α]25D −47 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 213 (+5.26), 243 (−4.90), 326 (−0.43) nm. Mucroniferanine D (4): amorphous powder; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.62), 235 (4.74) nm; IR (KBr) νmax 3424, 2916, 1762, 1721, 1630, 1028, 806 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 380.1138 [M + H]+ (calcd for C21H18NO6, 380.1134). (+)-Mucroniferanine D [(+)-4]: [α]25D +51 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 212 (+9.98), 231 (+26.67), 284 (+2.27) nm. (−)-Mucroniferanine D [(−)-4]: [α]25D −54 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 212 (−6.39), 231 (−21.49), 284 (−1.95) nm. Mucroniferanine E (5): amorphous powder; [α]25D 0 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.62), 235 (4.74) nm; IR (KBr) νmax 3424, 2916, 1762, 1721, 1630, 1028, 806 cm−1; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 380.1139 [M + H]+ (calcd for C21H18NO6, 380.1134). (+)-Mucroniferanine E [(+)-5]: [α]25D +44 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 226 (+9.41), 240 (+23.27), 292 (−2.35) nm. (−)-Mucroniferanine E [(−)-5]: [α]25D −47 (c 0.1, MeOH); ECD (MeOH) λmax (Δε) 226 (−5.14), 240 (−13.12), 292 (+1.74) nm. Mucroniferanine F (6): amorphous powder; [α]25D +66 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.67), 235 (4.71) nm; IR (KBr) νmax 3422, 2963, 2918, 1462, 1245, 1037, 803, 581 cm−1; ECD (MeOH) λmax (Δε) 235 (+27.5) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 366.0974 [M + H]+ (calcd for C20H16NO6, 366.0978). Mucroniferanine F (7): amorphous powder; [α]25D −68 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 203 (4.67), 235 (4.71) nm; IR (KBr) νmax 3423, 2918, 1464, 1245, 1038 cm−1; ECD (MeOH) λmax (Δε) 235 (−25.0) nm; 1H and 13C NMR data, see Tables 1 and 2; HRESIMS m/z 366.0974 [M + H]+ (calcd for C20H16NO6, 366.0978). X-ray Crystallographic Analysis Data of Mucroniferanine A (1). C19H15NO5, M = 337.32, triclinic, space group P1̅ (no. 2), a = 6.93466(8) Å, b = 9.91657(11) Å, c = 11.54675(14) Å, α = 71.8945(10)°, β = 84.5865(10)°, γ = 75.6285(10)°, V = 730.985(16) Å3, Z = 2, T = 100 K, μ(Cu Kα) = 0.933 mm−1, Dcalc = 1.533 g/cm3, 81 430 reflections measured (8.058° ≤ 2θ ≤ 137.658°), 2648 unique (Rint = 0.0434, Rsigma = 0.0085), which were used in all calculations. The final R1 was 0.0325 (I > 2σ(I)) and wR2 was 0.0825 (all data). Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Center as supplementary publication No. CCDC 1573067. ECD Calculations. The relative configurations were initially established by using the NOESY spectra. Configuration analyses were conducted via Monte Carlo simulation with the MMFF94 molecular mechanics force field. Optimization of conformers was carried out by using the TDDFT method at the B3LYP/6-31G(d) level, and the frequencies were also calculated at the same level to confirm the stability. The ECD calculations of the stable conformers were conducted by using the TDDFT method at the B3LYP/631+G(d) level with the CPCM model in MeOH. The ECD spectra were simulated using SpecDis v1.51 with a half-bandwidth of 0.3−0.5 eV. Based on the Boltzmann-calculated contribution of different conformers, the ECD spectra were acquired. The Gaussian 09 program package was used for all calculations. Acetylcholinesterase Assay. A modified Ellman’s method was applied to measure the acetylcholinesterase inhibitory activity.21,22 Galanthamine was used as positive control.

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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.jnatprod.7b00847. X-ray crystallographic data (CIF) 369

DOI: 10.1021/acs.jnatprod.7b00847 J. Nat. Prod. 2018, 81, 364−370

Journal of Natural Products

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Article

NMR and HRESIMS spectra of compounds 1−7 (PDF)

AUTHOR INFORMATION

Corresponding Authors

*Tel (F.-C. Xu): +86-891-6832839. Fax: +86-891-6832839. Email: [email protected]. *Tel (H. Liang): +86-10-82801592. Fax: +86-10-82801592. Email: [email protected]. ORCID

Peng-Fei Tu: 0000-0003-3553-1840 Hong Liang: 0000-0001-6483-0968 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Y.-T. Zhang for plant authentication. We also thank the State Key Laboratory of Natural and Biomimetic Drugs, Peking University, for offering certain spectroscopic measurements.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.7b00847 J. Nat. Prod. 2018, 81, 364−370