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Scholarisines H–O, novel indole alkaloid derivatives from long-term stored Alstonia scholaris
Tetrahedron xxx (2014) 1e5
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Scholarisines HeO, novel indole alkaloid derivatives from long-term stored Alstonia scholaris Xing-Wei Yang a, b, Xiao-Dong Luo a, *, Paul K. Lunga a, Yun-Li Zhao a, Xu-Jie Qin a, b, Ying-Ying Chen a, b, Lu Liu a, b, Xiao-Nian Li a, Ya-Ping Liu a, * a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 16 September 2014 Accepted 17 September 2014 Available online xxx
Eight new monoterpenoid indole and quinoline alkaloids, scholarisines HeO (1e8), together with six known analogues, were isolated from seven-year stored leaves of Alstonia scholaris. Their structures were elucidated on the basis of comprehensive spectroscopic data and X-ray diffraction. The obtained compounds were presumably derived from the known precursors by mild oxidation during the post-harvest storage. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Indole alkaloid Alstonia scholaris Scholarisines HeO
1. Introduction Alstonia plants (Apocynaceae) are known to be rich sources of monoterpenoid indole alkaloids (MIAs) with diverse structures and significant bioactivities, some of which have attracted attention as new drug leads as well as challenging targets for total synthesis.1 Alstonia scholaris is extensively used as traditional medicine for the treatment of various diseases in different countries of Asia. Different organs of this plant can be used as medicine of both codified and non-codified drug system of India for the treatment of malaria, jaundice, gastrointestinal troubles, cancer, etc. Its medicinal importance has been recorded in the ancient Ayurvedic text ‘Bhavaprakasha’ in India.2 The leaves of A. scholaris have been historically used in ‘dai’ ethnopharmacy to treat chronic respiratory diseases in Yunnan province, China.3 Our previous chemical study on A. scholaris led to the isolation of a series of MIAs from different parts of this plant,4 of which (E/Z)-alstoscholarine, scholarisine A, and picrinine have been synthesized elaborately by other outstanding chemists after we reported the structures.5 Moreover, a defined mixture of alkaloids from A. scholaris leaf,6 registered as investigational new botanical drug (No. 2011L01436) has been approved for clinical trials (phase I and II) by China Food and Drug Administration (CFDA).
* Corresponding authors. Tel.: þ86 871 65223177; e-mail addresses: xdluo@mail. kib.ac.cn (X.-D. Luo), [email protected] (Y.-P. Liu).
Interestingly, during our quality control of this medicinal plant, more compounds appeared in the HPLC fingerprint profile of the seven-year stored leaves of A. scholaris. Furthermore, the extracted total alkaloids exhibited antimicrobial activities, which motivated us to trace the antimicrobial MIAs from it. As a result, eight new indole and quinoline alkaloids, scholarisines HeO (1e8, Fig. 1), together with six known analogues: nareline (9);7 19E-vallesamine;8 picrinine;9 strictamine;10 5a-methoxylstrictamine;11 and 16formyl-5a-methoxylstrictamine,12 were isolated. Their structures were elucidated on the basis of comprehensive spectroscopic data and X-ray diffraction, and compounds 1e3 possessed rare cage carbon skeletons. The new MIAs might be derived from the known precursors by N-methylation and/or mild oxidation during the storage, and they showed weak antibacterial activities against five bacterial strains.
2. Results and discussions Scholarisine H (1) was obtained as colorless gum. Its molecular formula C21H22N2O3 was established by 13C NMR and HR-EIMS data (m/z 350.1636, [M]þ) indicating 12 indices of hydrogen deficiency. The UV spectrum showed the characteristic maximal absorptions of indolenine alkaloids at 212 and 258 nm,4b while the FTIR spectrum exhibited absorption bands due to lactone group (1738 cm1) and aromatic ring (1631, 1439 cm1). The 1H and 13C NMR data of 1 indicated an ortho-disubstituted phenyl ring (Tables 1 and 2).
http://dx.doi.org/10.1016/j.tet.2014.09.052 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.
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X.-W. Yang et al. / Tetrahedron xxx (2014) 1e5
Fig. 1. Structures of compounds 1e8. Table 1 13 C (100 MHz) NMR spectral data (d in ppm) of compounds 1e8 Position 1a
2a
3a,c
4b
5b
6b
7a,d
C-2 C-3 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-18 C-19 C-20 C-21 C-22 -COOMe
186.5 s 59.3 d 81.7 d 36.5 t 53.5 s 144.0 s 123.4 d 127.5 d 129.9 d 121.3 d 154.5 s 28.4 t 34.2 d 51.0 d 7.8 q 33.1 t 42.5 s 53.5 t 45.2 q 174.9 s
189.5 s 59.7 d 74.6 d 34.8 t 54.6 s 144.8 s 123.8 d 126.7 d 129.0 d 121.0 d 154.5 s 28.6 t 28.1 d 48.5 d 7.9 q 32.5 t 42.1 s 54.3 t 45.6 q
106.2 s 57.5 t 175.5 s 39.1 t 51.1 s 135.3 s 125.1 d 120.6 d 128.8 d 110.5 d 145.9 s 25.8 t 27.1 d 51.7 d 11.2 q 58.1 d 62.2 s 56.3 t 45.6 q 171.3 s 51.3 q
106.5 s 57.7 t 175.6 s 39.0 t 51.0 s 135.5 s 125.5 d 120.6 d 128.7 d 110.4 d 145.9 s 25.0 t 31.4 d 51.1 d 13.4 q 62.0 d 61.9 s 52.1 t 46.0 q 171.5 s 51.3 q
106.3 s 57.6 t 174.5 s 39.2 t 51.9 s 134.6 s 124.6 d 120.9 d 129.1 d 111.1 d 145.8 s 25.6 t 35.6 d 52.1 d 13.5 q 57.5 d 60.9 s 48.7 t 45.9 q 172.3 s 51.9 q
140.2 s 91.7 s 47.1 t 75.0 d 150.6 d 180.3 s 119.5 d 111.7 s 152.1 s 127.0 s 128.0 s 120.3 d 128.6 d 121.8 d 127.4 d 123.8 d 130.6 d 112.6 d 129.9 d 136.9 s 149.7 s 29.6 t 32.2 t 39.4 d 41.4 d 60.6 s 60.8 d 14.4 q 13.7 q 124.7 d 121.0 d 134.4 s 136.9 s 56.2 t 54.2 t 89.9 t 174.3 s 171.2 s 53.1 q 51.8 q
a b c d
192.0 s 58.7 d 69.6 t 46.5 d 59.4 s 141.7 s 126.4 d 126.3 d 129.9 d 121.3 d 156.9 s 38.5 t 33.7 d 57.2 d 12.4 q 120.3 d 137.0 s 63.6 d 86.0 t 172.5 s 52.3 q
8a
Recorded in methanol-d4. Recorded in CDCl3. The hemiacetal carbon of 3: 92.4, d. The C-17 of 7: 71.2, t.
Besides, two quaternary carbons (dC 59.4, C-7; and 192.0, C-2), four methines (dC 58.7, C-3; 33.7, C-15; 57.2, C-16; and 63.6, C-21), one methylene (dC 38.5, C-14), five other signals assignable to a carboxymethyl group (dC 172.5 and 52.3), and an exocyclic substituted propylene group (dC 12.4, C-18; 120.3, C-19; and 137.0, C-20) were present in the 13C and DEPT NMR spectra. These data suggested that 1 was very similar to nareline (9), a monoterpenoid indole alkaloid isolated from the same plant in 1977.7 Analysis of the NMR spectroscopic data of 1 and 9 suggested that a hemiacetal carbon at dC 100.5 (C-5) in 9 was replaced by an oxygenated methylene at dC 69.6 and an additional down-field methylene at dC 86.0 (C-22) in 1.
In the 1He1H COSY spectrum, the correlation of dH 3.65, 3.24 (2H, H-5) with dH 3.30 assigned H-6. Furthermore, the correlations from dH 4.53, 4.42 (2H, H-22) to dC 58.7 (C-3), 63.6 (C-21), and 69.6 (C-5) in the HMBC spectrum suggested the linkage of C-5/O/C-22/N4, which established a new six-membered heterocycle in 1 to meet the indices of hydrogen deficiency. Other parts of 1 were identical to those of 9 by detailed analysis of 1He1H COSY and HMBC spectra (Fig. 2). Due to the rigid cage skeleton comprising a diamond-like core as shown in Fig. 2, the relative configurations of the bridgehead carbons C-3, C-6, C-7, C-15, and C-21 were obviously indicated. In the ROESY spectrum, the cross peaks between dH 2.25 (H-16) and dH 2.05 (H-14), and between dH 1.66 (Me-18) and dH 3.47 (H-15) defined the H-16a and E-configuration of C-19/20 double bond. Therefore, the structure of 1 was elucidated as shown, and a hypothetical biosynthetic pathway from 9 to 1 was presented in Scheme 1. The molecular formula of scholarisine I (2) was determined as C20H22N2O2 according to its 13C NMR and HR-EIMS (m/z 322.1684, [M]þ) analysis. The 1H and 13C NMR data of 2 (Tables 1 and 2) were similar to those of scholarisine A,4b except that the imine carbon at dC 169.8 (C-21) in scholarisine A was absent, while an up-field methylene at dC 53.5 and an extra N-methyl (dC 45.2, C-22) were instead present in 2. The difference implied that 2 was a derivative of scholarisine A with reduction at C-21 and methylation at N4. This assumption was further supported by the correlations of dH 2.49 (NMe) with dC 59.3 (C-3) and 53.5 (C-21) in the HMBC spectrum. Other partial structure and the relative configuration of 2 was determined to be the same as those of scholarisine A by detailed analysis of 2D NMR spectroscopic data of 2, coupled with the rigid cage scaffold established previously (Fig. 3).4b Scholarisine J (3) was assigned the molecular formula C20H24N2O2 by its HR-EIMS (m/z 324.1833, [M]þ) data, two mass units more than that of 2. The lactone carbonyl (dC 174.9) in 2 was replaced by a hemiacetal carbon (dC 92.4) and the corresponding proton (dH 5.79, 5.35) in 3 from detailed comparison of the 1D and 2D NMR data of two compounds. A ratio of 5:1 for a (NOE correlation of dH 5.79 with dH 2.22 (H-6)) and b (NOE correlation of dH 5.35 with dH 1.46 (H-16)) of the hemiacetal proton observed in the 1 H and ROESY spectra indicated that 3 was a tautomer with preferential b-configuration of the hemiacetal hydroxyl. Scholarisine K (4) was attributed the molecular formula C21H24N2O5 by its 13C NMR and HR-EIMS data. The UV spectrum showed maximal absorptions at 236 and 290 nm, which was characteristic of a dihydroindole chromophore.4d Comparison of the NMR spectroscopic data (Tables 1 and 2) of 4 with those of scholarisine E4d indicated that instead of the acetal group (dC 104.8, C-5; and 54.8, OMe) and the imine carbon (dC 162.5, C-21) in scholarisine E, an ester carbonyl (dC 175.5), an up-field methylene (dC 56.3), and a methyl (dC 45.6, N-Me) appeared in 4. The correlations of dH 3.40, 3.18 (2H, H-6) with dC 174.5 (C-5), and of dH 2.38 (Me-22) with dC 57.5 (C-3) and 56.3 (C-21) in the HMBC spectrum further assigned the difference. The NOE correlation of dH 2.81 (H-19) with dH 2.62 (H-15) in its ROESY spectrum placed H-19 and H-15 on the same side. Furthermore, the final refinement on the Cu Ka data of the crystal of 4 (CCDC 1022010) indicated the unambiguous assignment of its absolute configuration as 2R,3S,7R,15R,16R,19S,20S (Fig. 4).13 Scholarisine L (5) shared the same planar scaffold as 4 by analyzing its HR-EIMS and NMR spectroscopic data. The 13C NMR data of 5 (Table 1) were nearly the same to those of 4 except for the carbon resonances of C-15, C-19, and C-21, which supposed 5 as 19epimer of 4. The assumption was supported by the NOE correlation of dH 1.23 (Me-18) with dH 2.25 (H-21), and of dH 2.89 (H-19) with dH 2.72 (H-15) in ROESY spectrum of 5. Considering co-occurrence of 4e6 from picrinine-type alkaloids,14 19R and 20S for 5 could be assigned by absolute configuration of 4.
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Table 2 1 H (600 MHz) NMR spectral data (d in ppm, J in Hz) of 1e8 No.
1a
2a
3a,c
4b
5b
6b
7a,d
8a
3
4.22 (t, 3.0)
3.84 (t, 3.5)
3.77 (br s)
3.00 (br d, 4.5)
2.99 (br d, 4.5)
3.03 (br d, 4.9)
4.05 (m) 2.97 (m)
4.24 (br d, 3.8)
5
3.65 (dd, 11.7, 2.3) 3.24 (br d, 11.7) 3.30 (overlap)
4.75 (t, 2.5)
3.90 (t, 2.3)
3.12 2.22 7.24 7.28 7.39 7.55 2.60 1.61 1.94 2.12 0.99 1.66 1.61 2.75 2.68 2.49
2.79 2.22 7.50 7.25 7.34 7.51 2.52 1.40 2.27 1.29 0.92 1.96 1.72 2.64 2.45 2.45
6 9 10 11 12 14 15 16 18 19
7.65 7.21 7.40 7.58 2.41 2.05 3.47 2.25 1.66 5.66
21
3.75 (br s)
22
4.53 (d, 10.9) 4.42 (d, 10.9) 3.67 (s)
OMe a b c d
(d, 7.8) (t, 7.8) (t, 7.8) (d, 7.8) (dt, 13.2, 3.4) (dt, 13.2, 3.0) (m) (d, 2.6) (d, 6.8) (q, 6.8)
(dd, 14.7, 2.5) (dd, 14.7, 2.5) (d, 7.5) (t, 7.5) (t, 7.5) (d, 7.5) (dt, 14.3, 3.5) (m) (m) (d, 3.0) (t, 7.5) (m) (m) (d, 12.0) (d, 12.0) (s)
(d, 14.6) (dd, 14.6, 2.3) (d, 7.5) (t, 7.5) (t, 7.5) (d, 7.5) (dt, 14.0, 3.5) (br d, 14.0) (br s) (br s) (t, 7.5) (m) (m) (d, 12.0) (overlap) (s)
8.78 (d, 4.9) 3.75 3.43 7.34 6.78 7.08 6.68 2.16 2.02 2.62 2.48 1.29 2.81
(d, 18.4) (d, 18.4) (d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (dt, 14.3, 4.5) (dt, 14.3, 2.2) (m) (d, 4.5) (d, 5.6) (q, 5.6)
3.77 3.40 7.46 6.79 7.07 6.66 2.17 1.95 2.72 2.38 1.23 2.89
(d, 18.4) (d, 18.4) (d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (dt, 14.3, 4.5) (dt, 14.3, 2.2) (m) (d, 4.9) (d, 5.6) (q, 5.6)
3.40 3.18 7.22 6.80 7.11 6.70 2.52 1.98 2.36 2.55 1.31 2.96
(d, 17.8) (d, 17.8) (d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (m) (dt, 14.1, 2.4) (dd, 7.5, 4.2) (d, 4.2) (d, 5.3) (q, 5.3)
7.51 (d, 4.9) 7.58 7.11 7.16 7.41 2.13 1.93 3.69
(d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (m) (m) (m)
1.77 (d, 7.1) 5.62 (q, 7.1)
3.14 (d, 13.6) 2.12 (d, 13.6) 2.38 (s)
3.06 (d, 12.8) 2.25 (d, 12.8) 2.42 (s)
2.81 (d, 12.8) 2.49 (d, 12.8) 2.44 (s)
4.48 (d, 16.2) 3.78 (d, 16.2)
3.61 (s)
3.64 (s)
3.66 (s)
3.67 (s)
8.80 7.54 7.72 8.02 2.46 2.32 3.66 3.01 1.53 5.35
(d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (d, 13.6) (dt, 13.6, 3.8) (br s) (d, 4.9) (dd, 6.8, 2.6) (q, 6.8)
3.86 3.15 4.66 4.22 3.41
(dt, 16.6, 2.6) (d, 16.6) (d, 6.8) (d, 6.8) (s)
Recorded in methanol-d4. Recorded in CDCl3. The hemiacetal proton of 3: 5.79 (br s). The H-17 protons of 7: 4.25 (d, 10.5), and 4.01 (d, 10.5).
Fig. 2. Key 1He1H COSY, HMBC, and ROESY correlations of 1.
Fig. 4. X-ray crystallographic structure of 4 (displacement ellipsoids are drawn at the 30% probability level).
Scheme 1. Putative biosynthesis from 9 to 1.
Fig. 5. Key 1He1H COSY, HMBC, and ROESY correlations of 6.
Fig. 3. Key 1He1H COSY and HMBC correlations of 2.
Scholarisine M (6) possessed the same backbone as 4 based on its HR-EIMS, 1D and 2D NMR spectroscopic data (Fig. 5). The NOE correlations of dH 1.31 (Me-18) with dH 3.18 (H-6) and 2.49 (H-21), and of dH 2.96 (H-19) with dH 2.36 (H-15) in its ROESY spectrum could only place the endo-substitution of C-19, and H-19 and H-15 on the same side, by a molecular model indication (Fig. 5). Then, 19S and 20R for 6 could be deduced consequently.
The molecular formula of scholarisine N (7) was determined as C20H22N2O4 by analysis of its 13C NMR and HR-EIMS (m/z 354.1577, [M]þ) data. The UV maximal absorptions at 217 and 271 nm indicated an indole chromophore.8 Compound 7 shared the same planer scaffold as 19E-vallesamine8 by detailed comparison of 1D and 2D NMR spectral data of the two compounds. The methylene (dC 51.2, C-6) in 19E-vallesamine was hypothetically oxidized to a carbonyl at dC 180.3 in 7, which was further confirmed by the correlations from dH 4.05, 2.97 (2H, H-3), and 4.48, 3.78 (2H, H-21) to dC 180.3 in the HMBC spectrum of 7 (Fig. 6). In the ROESY spectrum, the NOE correlation of dH 5.62 (H-19) with dH 3.78 (H-21) assigned E-configuration of C-19/20 double bond. Besides, the NOE
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correlation of dH 1.77 (Me-18) with dH 4.25 (H-17) indicated a borientation of eCH2OH at C-16 and an a-orientation of H-15.
NMR spectra were recorded on Bruker AM-400, Bruker DRX-500, and DRX-600 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts (d) were expressed in parts per million (ppm) with reference to the solvent signals. ESIMS and HR-EIMS analysis were carried out on Waters Xevo TQS and Waters AutoSpec Premier P776 mass spectrometers, respectively. Semi-preparative HPLC was performed on an Agilent 1100 HPLC with a ZORBAX SB-C18 (9.4250 mm) column. Silica gel (100e200 and 200e300 mesh, Qingdao Marine Chemical Co., Ltd., PR China) and MCI gel (75e150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography. Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by Dragendorff’s reagent.
Fig. 6. Key 1He1H COSY and HMBC correlations of 7 and 8.
3.2. The antibacterial assays The molecular formula of scholarisine O (8) was established as C21H22N2O3 by HR-EIMS (m/z 350.1620, Mþ), which was 60 mass units less than that of corialstonine,15 a quinoline alkaloid from this plant. The UV spectrum exhibited maximal absorptions for a quinoline chromophore at 228, 290, and 317 nm.15 Its 1H and 13C NMR spectral data (Tables 1 and 2) indicated an unsubstituted quinoline derivative of corialstonine. Except for the quinoline core, other planar structure of 8 was elucidated to be identical to that of corialstonine by detailed analysis of its 1He1H COSY and HMBC spectra (Fig. 6). The bridge-head carbons C-2, C-3, and C-15 at rigid ring systems indicated by molecular model fixed their relative configurations. In the ROESY spectrum, the NOE contact between dH 1.53 (Me-18) and dH 3.66 (H-15) supported E-configuration of C-19/20 double bond. Besides, the correlation of Me-18 with dH 3.41 (eOMe) suggested the relative configuration of C-16, which had not been determined in literature.15,16 Structurally, 8 possessed a quinoline ring, but biogenetically, it may be derived from ring expansion of monoterpenoid indole alkaloid.15,17 As shown in Table 3, the total alkaloids from seven-year stored plant and the tested isolates exhibited weak activities against five bacterial strains (Providencia smartii ATCC 29916, Staphylococcus aureus ATCC 25922, Enterococcus faecalis ATCC 10541, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 11775).
Table 3 Antibacterial activities (MIC, mg/mL) of the tested samples against five bacterial strains
Total alkaloids 1 2 3 4 5 6 7 8 9 Gentamycin
P. smartii
S. aureus
E. faecalis
P. aeruginosa
E. coli
50 100 100 100 NA NA NA NA NA NA 0.20
25 100 100 100 NA 100 100 NA 100 100 0.20
100 50 100 25 NA 100 NA NA 100 50 1.56
100 100 100 100 100 50 NA NA 100 100 0.78
NA 100 NA NA NA 100 NA NA 100 25 1.56
3. Experiment section 3.1. General experimental procedures X-ray data was determined using a Broker APEX DUO instrument. Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured on a JASCO P1020 polarimeter. UV spectra were detected on a SHIMADZU UV2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. 1D and 2D
The antibacterial assay of the total alkaloids and compounds 1e9 was evaluated against P. smartii (ATCC 29916), S. aureus (ATCC 25922), E. faecalis (ATCC 10541), P. aeruginosa (ATCC 27853), and E. coli (ATCC 11775). All the bacteria were obtained from the American Type Culture Collection (Rockville, USA). The preparation of bacterial inocula was done by using 18 h old overnight bacterial cultures prepared in Nutrient Agar. A few colonies of bacteria were collected aseptically with a sterile loop and introduced into 10 mL of sterile 0.90% saline solution. The concentration of the suspension was then standardized by adjusting the optical density to 0.10 at 600 nm, corresponding to bacterial cell suspension of about 108 colony-forming units/mL (CFU/mL). This cell suspension was diluted 100 times to obtain 106 CFU/mL before use. The compounds were dissolved in DMSO and then added to bacteria suspension to obtain the final concentration of 5% (v/v) DMSO or less. Serial twofold dilutions from 200 mg/mL of the compounds were performed in 96-well micro-titer plates. Each well contained 100 mL of sample of each concentration. Into each well was then introduced 100 mL of the bacterial suspension. The final concentration range of the test samples was 100e0.781 mg/mL, and the plates were incubated at 37 C for 24 h. After incubation, the wells were examined for growth of microorganisms and the MICs were determined upon addition of 50 mL of INT (p-iodonitrotetrazolium chloride). Viable bacteria turn the yellow dye of INT into pink. Each experiment was repeated three times and gentamycin was used as a positive control. MIC was defined as the lowest concentration of the compounds at which the bacterium did not demonstrate visible growth (no color change) and MIC >100 mg/mL was considered to be inactive. 3.3. Extraction and isolation The leaves of A. scholaris were collected in Xishuangbanna, Yunnan Province, PR China, in October 2006, and it has been stored for 7 years. This material (18 kg) was extracted with EtOH (40 L3) under reflux conditions, and the solvent was evaporated in vacuum. The residue was dissolved in 0.37% HCl, and the solution was subsequently basified using ammonia water to pH 9e10. The basic solution was partitioned with EtOAc, affording a two-phase mixture including the aqueous phase and the EtOAc/organic phase. The organic fraction (180 g) was collected and then dissolved in MeOH, and the resulting solution was subjected to column chromatography over silica gel eluting with CHCl3/MeOH (from 1:0 to 2:8) to afford six fractions (Fr. AeF). Fr. A (25 g) was further chromatographed using CHCl3/Me2CO (from 9:1 to 7:3) as eluant to give five fractions (Fr. A1eA5). Picrinine (3.8 g), strictamine (130 mg), 4 (56 mg), 5 (53 mg), and 6 (18 mg) were obtained from Fr. A1 (5.2 g) by using silica gel column chromatography eluted with CHCl3/ MeOH (95: 5). Fr. A2 (4.6 g) was further purified by preparative HPLC (CH3CN/H2O, 1:1) to afford 5a-methoxylstrictamine (83 mg),
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16-formyl-5a-methoxylstrictamine (43 mg), nareline (126 mg), 19E-vallesamine (2.6 g), 1 (5 mg), 2 (32 mg), 3 (21 mg), 7 (21 mg), and 8 (38 mg).
5
(3.52), 317 (3.34) nm; IR (KBr) nmax 2924, 2853, 1745, 1629, 1581, 1436, 1384, 1281, 1157, 1114, 1020, 965, 768 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 351 [MþH]þ; HREIMS m/z 350.1620 [M]þ (calcd for C21H22N2O3, 350.1630).
3.4. The physical data of the new compounds 3.4.1. Scholarisine H (1). Colorless gum; [a]26 D 160.5 (c 0.18, MeOH); UV (MeOH) lmax (log 3 )¼212 (4.34), 258 (3.80) nm; IR (KBr) nmax 2951, 2855, 1738, 1688, 1631, 1595, 1439, 1384, 1348, 1194, 1170, 1124, 1068, 773 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 351 [MþH]þ; HREIMS m/z 350.1636 [M]þ (calcd for C21H22N2O3, 350.1630). 3.4.2. Scholarisine I (2). White powder; [a]24 D þ215.9 (c 0.11, MeOH); UV (MeOH) lmax (log 3 )¼217 (4.28), 257 (3.71) nm; IR (KBr) nmax 3426, 2964, 2933, 2856, 1755, 1633, 1590, 1457, 1384, 1357, 1217, 1057, 1009, 769, 751 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 323 [MþH]þ; HREIMS m/z 322.1684 [M]þ (calcd for C20H22N2O2, 322.1681). 3.4.3. Scholarisine J (3). White powder; [a]25 D þ225.1 (c 0.14, MeOH); UV (MeOH) lmax (log 3 )¼208 (4.37), 248 (3.82) nm; IR (KBr) nmax 3426, 2932, 2857, 1639, 1592, 1456, 1348, 1260, 1115, 1077, 1048, 749 cm1; 1 H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 325 [MþH]þ; HREIMS m/z 324.1833 [M]þ (calcd for C20H22N2O2, 324.1838). 3.4.4. Scholarisine K (4). Colorless flaky crystal from MeOH; mp 251e252 C; [a]26 D þ16.3 (c 0.13, MeOH); UV (MeOH) lmax (log 3 )¼ 203 (4.56), 236 (3.94), 290 (3.54) nm; IR (KBr) nmax 3370, 2946, 1739, 1608, 1469, 1236, 1174, 1002, 945, 917, 769 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385 [MþH]þ; HREIMS m/z 384.1687 [M]þ (calcd for C21H24N2O5, 384.1685). 3.4.5. Scholarisine L (5). White powder; [a]26 D þ34.1 (c 0.21, MeOH); UV (MeOH) lmax (log 3 )¼203 (4.48), 236 (3.85), 290 (3.45) nm; IR (KBr) nmax 3426, 2952, 2856, 1764, 1641, 1613, 1470, 1273, 1229, 1191, 1096, 1004, 948, 751 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385 [MþH]þ; HREIMS m/z 384.1683 [M]þ (calcd for C21H24N2O5, 384.1685). 3.4.6. Scholarisine M (6). Colorless needle crystal from MeOH; mp 240e242 C; [a]26 D 4.6 (c 0.09, MeOH); UV (MeOH) lmax (log 3 )¼ 203 (4.23), 236 (3.65), 290 (3.25) nm; IR (KBr) nmax 3441, 3283, 2954, 1746, 1737, 1613, 1470, 1436, 1270, 1233, 1196, 1177, 1094, 1020, 766 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 407 [MþNa]þ; HREIMS m/z 384.1681 [M]þ (calcd for C21H24N2O5, 384.1685). 3.4.7. Scholarisine N (7). white powder; [a]25 D þ178.5 (c 0.10, MeOH); UV (MeOH) lmax (log 3 )¼217 (4.85), 271 (4.24), 290 (4.20) nm; IR (KBr) nmax 3294, 3231, 2938, 1730, 1609, 1475, 1442, 1384, 1228, 1196, 1103, 884, 750 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 355 [MþH]þ; HREIMS m/z 354.1577 [M]þ (calcd for C20H22N2O4, 354.1580). 3.4.8. Scholarisine O (8). colorless gum; [a]26 D þ55.4 (c 0.21, MeOH); UV (MeOH) lmax (log 3 )¼204 (4.40), 228 (4.35), 290 (3.55), 305
Acknowledgements We are grateful to the National Natural Science Foundation of China (81225024, 31170334), the Ministry of Science and Technology of the People’s Republic of China (2014ZX09301307-003, 2013BAI11B02), and the Chinese Academy of Sciences (KSZD-EWZ-004-03) for partial financial support. Supplementary data Detailed MS, 1D and 2D NMR spectra for 1e8, X-ray crystallographic file of 4 in CIF format. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/ 10.1016/j.tet.2014.09.052. References and notes 1. Ishikura, M.; Abe, T.; Choshi, T.; Hibino, S. Nat. Prod. Rep. 2013, 30, 694e752. 2. Khyade, M. S.; Kasote, D. M.; Vaikos, N. P. J. Ethnopharmacol. 2014, 153, 1e18. 3. Ministry of Public Health, People’s Republic of China. Drug Specifications Promulgated by the Ministry of Public Health, 1997, Vol. 14, pp 49e50. 4. (a) Cai, X. H.; Du, Z. Z.; Luo, X. D. Org. Lett. 2007, 9, 1817e1820; (b) Cai, X. H.; Tan, Q. G.; Liu, Y. P.; Feng, T.; Du, Z. Z.; Li, W. Q.; Luo, X. D. Org. Lett. 2008, 10, 577e580; (c) Cai, X. H.; Liu, Y. P.; Feng, T.; Luo, X. D. Chin. J. Nat. Med. 2008, 6, 20e22; (d) Feng, T.; Cai, X. H.; Zhao, P. J.; Du, Z. Z.; Li, W. Q.; Luo, X. D. Planta Med. 2009, 75, 1537e1541. 5. (a) Gerfaud, T.; Xie, C.; Neuville, L.; Zhu, J. Angew. Chem., Int. Ed. 2011, 50, 3954e3957; (b) Adams, G. L.; Carroll, P. J.; Smith, A. B., III. J. Am. Chem. Soc. 2012, 134, 4037e4040; (c) Adams, G. L.; Carroll, P. J.; Smith, A. B., III. J. Am. Chem. Soc. 2013, 135, 519e528; (d) Smith, M. W.; Snyder, S. A. J. Am. Chem. Soc. 2013, 135, 12964e12967; (e) Smith, J. M.; Moreno, J.; Boal, B. W.; Garg, N. K. J. Am. Chem. Soc. 2014, 136, 4504e4507. 6. (a) Shang, J. H.; Cai, X. H.; Feng, T.; Zhao, Y. L.; Wang, J. K.; Zhang, L. Y.; Yan, M.; Luo, X. D. J. Ethnopharmacol. 2010, 129, 174e181; (b) Shang, J. H.; Cai, X. H.; Zhao, Y. L.; Feng, T.; Luo, X. D. J. Ethnopharmacol. 2010, 129, 293e298. 7. Morita, Y.; Hesse, M.; Schmid, H. Helv. Chim. Acta 1977, 60, 1419e1434. 8. Atta-Ur-Rahman; Alvi, K. A.; Abbas, S. A.; Voelter, W. Heterocycles 1987, 26, 413e419. 9. Abe, F.; Chen, R. F.; Yamauchi, T.; Marubayashi, N.; Ueda, I. Chem. Pharm. Bull. 1989, 37, 887e890. 10. Atta-Ur-Rahman; Habib-ur-Rehman. Planta Med. 1986, 230e231. 11. Zhou, H.; He, H. P.; Luo, X. D.; Wang, Y. H.; Yang, X. W.; Di, Y. T.; Hao, X. J. Helv. Chim. Acta 2005, 88, 2508e2512. 12. Abe, F.; Yamauchi, T.; Shibuya, H.; Kitagawa, I.; Yamashita, M. Chem. Pharm. Bull. 1998, 46, 1235e1238. 13. Crystal data for 4: C21H24N2O5, M¼384.42, orthorhombic, a¼7.6813(2) A, b¼8. 9350(2) A, c¼27.1302(7) A, a¼90.00 , b¼90.00 , g¼90.00 , V¼1862.01(8) A3, 1 T¼100(2) K, space group P212121, Z¼4, m(CuKa)¼0.810 mm , 8579 reflections measured, 3180 independent reflections (Rint¼0.0585). The final R1 values were 0.0958 (I>2s(I)). The final wR(F2) values were 0.2541 (I>2s(I)). The final R1 values were 0.0967 (all data). The final wR(F2) values were 0.2568 (all data). The goodness of fit on F2 was 1.260. Flack parameter¼0.2(3). The Hooft parameter is 0.06(10) for 1216 Bijvoet pairs. Crystallographic data for 4 have been deposited at the Cambridge Crystallographic Data Center (deposition number CCDC 1022010). 14. Yang, X. W.; Qin, X. J.; Zhao, Y. L.; Lunga, P. K.; Li, X. N.; Jiang, S. Z.; Cheng, G. G.; Liu, Y. P.; Luo, X. D. Tetrahedron Lett. 2014, 55, 4593e4596. 15. Cherif, A.; Massiot, G.; Men-Olivier, L. L. Heterocycles 1987, 26, 3055e3058. 16. Tasic, G.; Randjelovic, J.; Vusurovic, N.; Maslak, V.; Husinec, S.; Savic, V. Tetrahedron Lett. 2013, 54, 2243e2246. 17. Cai, X. H.; Shang, J. H.; Feng, T.; Luo, X. D. Z. Naturforsch., B 2010, 65, 1164e1168.
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Scholarisines HeO, novel indole alkaloid derivatives from long-term stored Alstonia scholaris Xing-Wei Yang a, b, Xiao-Dong Luo a, *, Paul K. Lunga a, Yun-Li Zhao a, Xu-Jie Qin a, b, Ying-Ying Chen a, b, Lu Liu a, b, Xiao-Nian Li a, Ya-Ping Liu a, * a State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, PR China b University of Chinese Academy of Sciences, Beijing 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 16 September 2014 Accepted 17 September 2014 Available online xxx
Eight new monoterpenoid indole and quinoline alkaloids, scholarisines HeO (1e8), together with six known analogues, were isolated from seven-year stored leaves of Alstonia scholaris. Their structures were elucidated on the basis of comprehensive spectroscopic data and X-ray diffraction. The obtained compounds were presumably derived from the known precursors by mild oxidation during the post-harvest storage. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Indole alkaloid Alstonia scholaris Scholarisines HeO
1. Introduction Alstonia plants (Apocynaceae) are known to be rich sources of monoterpenoid indole alkaloids (MIAs) with diverse structures and significant bioactivities, some of which have attracted attention as new drug leads as well as challenging targets for total synthesis.1 Alstonia scholaris is extensively used as traditional medicine for the treatment of various diseases in different countries of Asia. Different organs of this plant can be used as medicine of both codified and non-codified drug system of India for the treatment of malaria, jaundice, gastrointestinal troubles, cancer, etc. Its medicinal importance has been recorded in the ancient Ayurvedic text ‘Bhavaprakasha’ in India.2 The leaves of A. scholaris have been historically used in ‘dai’ ethnopharmacy to treat chronic respiratory diseases in Yunnan province, China.3 Our previous chemical study on A. scholaris led to the isolation of a series of MIAs from different parts of this plant,4 of which (E/Z)-alstoscholarine, scholarisine A, and picrinine have been synthesized elaborately by other outstanding chemists after we reported the structures.5 Moreover, a defined mixture of alkaloids from A. scholaris leaf,6 registered as investigational new botanical drug (No. 2011L01436) has been approved for clinical trials (phase I and II) by China Food and Drug Administration (CFDA).
* Corresponding authors. Tel.: þ86 871 65223177; e-mail addresses: xdluo@mail. kib.ac.cn (X.-D. Luo), [email protected] (Y.-P. Liu).
Interestingly, during our quality control of this medicinal plant, more compounds appeared in the HPLC fingerprint profile of the seven-year stored leaves of A. scholaris. Furthermore, the extracted total alkaloids exhibited antimicrobial activities, which motivated us to trace the antimicrobial MIAs from it. As a result, eight new indole and quinoline alkaloids, scholarisines HeO (1e8, Fig. 1), together with six known analogues: nareline (9);7 19E-vallesamine;8 picrinine;9 strictamine;10 5a-methoxylstrictamine;11 and 16formyl-5a-methoxylstrictamine,12 were isolated. Their structures were elucidated on the basis of comprehensive spectroscopic data and X-ray diffraction, and compounds 1e3 possessed rare cage carbon skeletons. The new MIAs might be derived from the known precursors by N-methylation and/or mild oxidation during the storage, and they showed weak antibacterial activities against five bacterial strains.
2. Results and discussions Scholarisine H (1) was obtained as colorless gum. Its molecular formula C21H22N2O3 was established by 13C NMR and HR-EIMS data (m/z 350.1636, [M]þ) indicating 12 indices of hydrogen deficiency. The UV spectrum showed the characteristic maximal absorptions of indolenine alkaloids at 212 and 258 nm,4b while the FTIR spectrum exhibited absorption bands due to lactone group (1738 cm1) and aromatic ring (1631, 1439 cm1). The 1H and 13C NMR data of 1 indicated an ortho-disubstituted phenyl ring (Tables 1 and 2).
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2
X.-W. Yang et al. / Tetrahedron xxx (2014) 1e5
Fig. 1. Structures of compounds 1e8. Table 1 13 C (100 MHz) NMR spectral data (d in ppm) of compounds 1e8 Position 1a
2a
3a,c
4b
5b
6b
7a,d
C-2 C-3 C-5 C-6 C-7 C-8 C-9 C-10 C-11 C-12 C-13 C-14 C-15 C-16 C-18 C-19 C-20 C-21 C-22 -COOMe
186.5 s 59.3 d 81.7 d 36.5 t 53.5 s 144.0 s 123.4 d 127.5 d 129.9 d 121.3 d 154.5 s 28.4 t 34.2 d 51.0 d 7.8 q 33.1 t 42.5 s 53.5 t 45.2 q 174.9 s
189.5 s 59.7 d 74.6 d 34.8 t 54.6 s 144.8 s 123.8 d 126.7 d 129.0 d 121.0 d 154.5 s 28.6 t 28.1 d 48.5 d 7.9 q 32.5 t 42.1 s 54.3 t 45.6 q
106.2 s 57.5 t 175.5 s 39.1 t 51.1 s 135.3 s 125.1 d 120.6 d 128.8 d 110.5 d 145.9 s 25.8 t 27.1 d 51.7 d 11.2 q 58.1 d 62.2 s 56.3 t 45.6 q 171.3 s 51.3 q
106.5 s 57.7 t 175.6 s 39.0 t 51.0 s 135.5 s 125.5 d 120.6 d 128.7 d 110.4 d 145.9 s 25.0 t 31.4 d 51.1 d 13.4 q 62.0 d 61.9 s 52.1 t 46.0 q 171.5 s 51.3 q
106.3 s 57.6 t 174.5 s 39.2 t 51.9 s 134.6 s 124.6 d 120.9 d 129.1 d 111.1 d 145.8 s 25.6 t 35.6 d 52.1 d 13.5 q 57.5 d 60.9 s 48.7 t 45.9 q 172.3 s 51.9 q
140.2 s 91.7 s 47.1 t 75.0 d 150.6 d 180.3 s 119.5 d 111.7 s 152.1 s 127.0 s 128.0 s 120.3 d 128.6 d 121.8 d 127.4 d 123.8 d 130.6 d 112.6 d 129.9 d 136.9 s 149.7 s 29.6 t 32.2 t 39.4 d 41.4 d 60.6 s 60.8 d 14.4 q 13.7 q 124.7 d 121.0 d 134.4 s 136.9 s 56.2 t 54.2 t 89.9 t 174.3 s 171.2 s 53.1 q 51.8 q
a b c d
192.0 s 58.7 d 69.6 t 46.5 d 59.4 s 141.7 s 126.4 d 126.3 d 129.9 d 121.3 d 156.9 s 38.5 t 33.7 d 57.2 d 12.4 q 120.3 d 137.0 s 63.6 d 86.0 t 172.5 s 52.3 q
8a
Recorded in methanol-d4. Recorded in CDCl3. The hemiacetal carbon of 3: 92.4, d. The C-17 of 7: 71.2, t.
Besides, two quaternary carbons (dC 59.4, C-7; and 192.0, C-2), four methines (dC 58.7, C-3; 33.7, C-15; 57.2, C-16; and 63.6, C-21), one methylene (dC 38.5, C-14), five other signals assignable to a carboxymethyl group (dC 172.5 and 52.3), and an exocyclic substituted propylene group (dC 12.4, C-18; 120.3, C-19; and 137.0, C-20) were present in the 13C and DEPT NMR spectra. These data suggested that 1 was very similar to nareline (9), a monoterpenoid indole alkaloid isolated from the same plant in 1977.7 Analysis of the NMR spectroscopic data of 1 and 9 suggested that a hemiacetal carbon at dC 100.5 (C-5) in 9 was replaced by an oxygenated methylene at dC 69.6 and an additional down-field methylene at dC 86.0 (C-22) in 1.
In the 1He1H COSY spectrum, the correlation of dH 3.65, 3.24 (2H, H-5) with dH 3.30 assigned H-6. Furthermore, the correlations from dH 4.53, 4.42 (2H, H-22) to dC 58.7 (C-3), 63.6 (C-21), and 69.6 (C-5) in the HMBC spectrum suggested the linkage of C-5/O/C-22/N4, which established a new six-membered heterocycle in 1 to meet the indices of hydrogen deficiency. Other parts of 1 were identical to those of 9 by detailed analysis of 1He1H COSY and HMBC spectra (Fig. 2). Due to the rigid cage skeleton comprising a diamond-like core as shown in Fig. 2, the relative configurations of the bridgehead carbons C-3, C-6, C-7, C-15, and C-21 were obviously indicated. In the ROESY spectrum, the cross peaks between dH 2.25 (H-16) and dH 2.05 (H-14), and between dH 1.66 (Me-18) and dH 3.47 (H-15) defined the H-16a and E-configuration of C-19/20 double bond. Therefore, the structure of 1 was elucidated as shown, and a hypothetical biosynthetic pathway from 9 to 1 was presented in Scheme 1. The molecular formula of scholarisine I (2) was determined as C20H22N2O2 according to its 13C NMR and HR-EIMS (m/z 322.1684, [M]þ) analysis. The 1H and 13C NMR data of 2 (Tables 1 and 2) were similar to those of scholarisine A,4b except that the imine carbon at dC 169.8 (C-21) in scholarisine A was absent, while an up-field methylene at dC 53.5 and an extra N-methyl (dC 45.2, C-22) were instead present in 2. The difference implied that 2 was a derivative of scholarisine A with reduction at C-21 and methylation at N4. This assumption was further supported by the correlations of dH 2.49 (NMe) with dC 59.3 (C-3) and 53.5 (C-21) in the HMBC spectrum. Other partial structure and the relative configuration of 2 was determined to be the same as those of scholarisine A by detailed analysis of 2D NMR spectroscopic data of 2, coupled with the rigid cage scaffold established previously (Fig. 3).4b Scholarisine J (3) was assigned the molecular formula C20H24N2O2 by its HR-EIMS (m/z 324.1833, [M]þ) data, two mass units more than that of 2. The lactone carbonyl (dC 174.9) in 2 was replaced by a hemiacetal carbon (dC 92.4) and the corresponding proton (dH 5.79, 5.35) in 3 from detailed comparison of the 1D and 2D NMR data of two compounds. A ratio of 5:1 for a (NOE correlation of dH 5.79 with dH 2.22 (H-6)) and b (NOE correlation of dH 5.35 with dH 1.46 (H-16)) of the hemiacetal proton observed in the 1 H and ROESY spectra indicated that 3 was a tautomer with preferential b-configuration of the hemiacetal hydroxyl. Scholarisine K (4) was attributed the molecular formula C21H24N2O5 by its 13C NMR and HR-EIMS data. The UV spectrum showed maximal absorptions at 236 and 290 nm, which was characteristic of a dihydroindole chromophore.4d Comparison of the NMR spectroscopic data (Tables 1 and 2) of 4 with those of scholarisine E4d indicated that instead of the acetal group (dC 104.8, C-5; and 54.8, OMe) and the imine carbon (dC 162.5, C-21) in scholarisine E, an ester carbonyl (dC 175.5), an up-field methylene (dC 56.3), and a methyl (dC 45.6, N-Me) appeared in 4. The correlations of dH 3.40, 3.18 (2H, H-6) with dC 174.5 (C-5), and of dH 2.38 (Me-22) with dC 57.5 (C-3) and 56.3 (C-21) in the HMBC spectrum further assigned the difference. The NOE correlation of dH 2.81 (H-19) with dH 2.62 (H-15) in its ROESY spectrum placed H-19 and H-15 on the same side. Furthermore, the final refinement on the Cu Ka data of the crystal of 4 (CCDC 1022010) indicated the unambiguous assignment of its absolute configuration as 2R,3S,7R,15R,16R,19S,20S (Fig. 4).13 Scholarisine L (5) shared the same planar scaffold as 4 by analyzing its HR-EIMS and NMR spectroscopic data. The 13C NMR data of 5 (Table 1) were nearly the same to those of 4 except for the carbon resonances of C-15, C-19, and C-21, which supposed 5 as 19epimer of 4. The assumption was supported by the NOE correlation of dH 1.23 (Me-18) with dH 2.25 (H-21), and of dH 2.89 (H-19) with dH 2.72 (H-15) in ROESY spectrum of 5. Considering co-occurrence of 4e6 from picrinine-type alkaloids,14 19R and 20S for 5 could be assigned by absolute configuration of 4.
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X.-W. Yang et al. / Tetrahedron xxx (2014) 1e5
3
Table 2 1 H (600 MHz) NMR spectral data (d in ppm, J in Hz) of 1e8 No.
1a
2a
3a,c
4b
5b
6b
7a,d
8a
3
4.22 (t, 3.0)
3.84 (t, 3.5)
3.77 (br s)
3.00 (br d, 4.5)
2.99 (br d, 4.5)
3.03 (br d, 4.9)
4.05 (m) 2.97 (m)
4.24 (br d, 3.8)
5
3.65 (dd, 11.7, 2.3) 3.24 (br d, 11.7) 3.30 (overlap)
4.75 (t, 2.5)
3.90 (t, 2.3)
3.12 2.22 7.24 7.28 7.39 7.55 2.60 1.61 1.94 2.12 0.99 1.66 1.61 2.75 2.68 2.49
2.79 2.22 7.50 7.25 7.34 7.51 2.52 1.40 2.27 1.29 0.92 1.96 1.72 2.64 2.45 2.45
6 9 10 11 12 14 15 16 18 19
7.65 7.21 7.40 7.58 2.41 2.05 3.47 2.25 1.66 5.66
21
3.75 (br s)
22
4.53 (d, 10.9) 4.42 (d, 10.9) 3.67 (s)
OMe a b c d
(d, 7.8) (t, 7.8) (t, 7.8) (d, 7.8) (dt, 13.2, 3.4) (dt, 13.2, 3.0) (m) (d, 2.6) (d, 6.8) (q, 6.8)
(dd, 14.7, 2.5) (dd, 14.7, 2.5) (d, 7.5) (t, 7.5) (t, 7.5) (d, 7.5) (dt, 14.3, 3.5) (m) (m) (d, 3.0) (t, 7.5) (m) (m) (d, 12.0) (d, 12.0) (s)
(d, 14.6) (dd, 14.6, 2.3) (d, 7.5) (t, 7.5) (t, 7.5) (d, 7.5) (dt, 14.0, 3.5) (br d, 14.0) (br s) (br s) (t, 7.5) (m) (m) (d, 12.0) (overlap) (s)
8.78 (d, 4.9) 3.75 3.43 7.34 6.78 7.08 6.68 2.16 2.02 2.62 2.48 1.29 2.81
(d, 18.4) (d, 18.4) (d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (dt, 14.3, 4.5) (dt, 14.3, 2.2) (m) (d, 4.5) (d, 5.6) (q, 5.6)
3.77 3.40 7.46 6.79 7.07 6.66 2.17 1.95 2.72 2.38 1.23 2.89
(d, 18.4) (d, 18.4) (d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (dt, 14.3, 4.5) (dt, 14.3, 2.2) (m) (d, 4.9) (d, 5.6) (q, 5.6)
3.40 3.18 7.22 6.80 7.11 6.70 2.52 1.98 2.36 2.55 1.31 2.96
(d, 17.8) (d, 17.8) (d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (m) (dt, 14.1, 2.4) (dd, 7.5, 4.2) (d, 4.2) (d, 5.3) (q, 5.3)
7.51 (d, 4.9) 7.58 7.11 7.16 7.41 2.13 1.93 3.69
(d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (m) (m) (m)
1.77 (d, 7.1) 5.62 (q, 7.1)
3.14 (d, 13.6) 2.12 (d, 13.6) 2.38 (s)
3.06 (d, 12.8) 2.25 (d, 12.8) 2.42 (s)
2.81 (d, 12.8) 2.49 (d, 12.8) 2.44 (s)
4.48 (d, 16.2) 3.78 (d, 16.2)
3.61 (s)
3.64 (s)
3.66 (s)
3.67 (s)
8.80 7.54 7.72 8.02 2.46 2.32 3.66 3.01 1.53 5.35
(d, 7.9) (t, 7.9) (t, 7.9) (d, 7.9) (d, 13.6) (dt, 13.6, 3.8) (br s) (d, 4.9) (dd, 6.8, 2.6) (q, 6.8)
3.86 3.15 4.66 4.22 3.41
(dt, 16.6, 2.6) (d, 16.6) (d, 6.8) (d, 6.8) (s)
Recorded in methanol-d4. Recorded in CDCl3. The hemiacetal proton of 3: 5.79 (br s). The H-17 protons of 7: 4.25 (d, 10.5), and 4.01 (d, 10.5).
Fig. 2. Key 1He1H COSY, HMBC, and ROESY correlations of 1.
Fig. 4. X-ray crystallographic structure of 4 (displacement ellipsoids are drawn at the 30% probability level).
Scheme 1. Putative biosynthesis from 9 to 1.
Fig. 5. Key 1He1H COSY, HMBC, and ROESY correlations of 6.
Fig. 3. Key 1He1H COSY and HMBC correlations of 2.
Scholarisine M (6) possessed the same backbone as 4 based on its HR-EIMS, 1D and 2D NMR spectroscopic data (Fig. 5). The NOE correlations of dH 1.31 (Me-18) with dH 3.18 (H-6) and 2.49 (H-21), and of dH 2.96 (H-19) with dH 2.36 (H-15) in its ROESY spectrum could only place the endo-substitution of C-19, and H-19 and H-15 on the same side, by a molecular model indication (Fig. 5). Then, 19S and 20R for 6 could be deduced consequently.
The molecular formula of scholarisine N (7) was determined as C20H22N2O4 by analysis of its 13C NMR and HR-EIMS (m/z 354.1577, [M]þ) data. The UV maximal absorptions at 217 and 271 nm indicated an indole chromophore.8 Compound 7 shared the same planer scaffold as 19E-vallesamine8 by detailed comparison of 1D and 2D NMR spectral data of the two compounds. The methylene (dC 51.2, C-6) in 19E-vallesamine was hypothetically oxidized to a carbonyl at dC 180.3 in 7, which was further confirmed by the correlations from dH 4.05, 2.97 (2H, H-3), and 4.48, 3.78 (2H, H-21) to dC 180.3 in the HMBC spectrum of 7 (Fig. 6). In the ROESY spectrum, the NOE correlation of dH 5.62 (H-19) with dH 3.78 (H-21) assigned E-configuration of C-19/20 double bond. Besides, the NOE
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X.-W. Yang et al. / Tetrahedron xxx (2014) 1e5
correlation of dH 1.77 (Me-18) with dH 4.25 (H-17) indicated a borientation of eCH2OH at C-16 and an a-orientation of H-15.
NMR spectra were recorded on Bruker AM-400, Bruker DRX-500, and DRX-600 spectrometers using TMS as an internal standard. Unless otherwise specified, chemical shifts (d) were expressed in parts per million (ppm) with reference to the solvent signals. ESIMS and HR-EIMS analysis were carried out on Waters Xevo TQS and Waters AutoSpec Premier P776 mass spectrometers, respectively. Semi-preparative HPLC was performed on an Agilent 1100 HPLC with a ZORBAX SB-C18 (9.4250 mm) column. Silica gel (100e200 and 200e300 mesh, Qingdao Marine Chemical Co., Ltd., PR China) and MCI gel (75e150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan) were used for column chromatography. Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by Dragendorff’s reagent.
Fig. 6. Key 1He1H COSY and HMBC correlations of 7 and 8.
3.2. The antibacterial assays The molecular formula of scholarisine O (8) was established as C21H22N2O3 by HR-EIMS (m/z 350.1620, Mþ), which was 60 mass units less than that of corialstonine,15 a quinoline alkaloid from this plant. The UV spectrum exhibited maximal absorptions for a quinoline chromophore at 228, 290, and 317 nm.15 Its 1H and 13C NMR spectral data (Tables 1 and 2) indicated an unsubstituted quinoline derivative of corialstonine. Except for the quinoline core, other planar structure of 8 was elucidated to be identical to that of corialstonine by detailed analysis of its 1He1H COSY and HMBC spectra (Fig. 6). The bridge-head carbons C-2, C-3, and C-15 at rigid ring systems indicated by molecular model fixed their relative configurations. In the ROESY spectrum, the NOE contact between dH 1.53 (Me-18) and dH 3.66 (H-15) supported E-configuration of C-19/20 double bond. Besides, the correlation of Me-18 with dH 3.41 (eOMe) suggested the relative configuration of C-16, which had not been determined in literature.15,16 Structurally, 8 possessed a quinoline ring, but biogenetically, it may be derived from ring expansion of monoterpenoid indole alkaloid.15,17 As shown in Table 3, the total alkaloids from seven-year stored plant and the tested isolates exhibited weak activities against five bacterial strains (Providencia smartii ATCC 29916, Staphylococcus aureus ATCC 25922, Enterococcus faecalis ATCC 10541, Pseudomonas aeruginosa ATCC 27853, and Escherichia coli ATCC 11775).
Table 3 Antibacterial activities (MIC, mg/mL) of the tested samples against five bacterial strains
Total alkaloids 1 2 3 4 5 6 7 8 9 Gentamycin
P. smartii
S. aureus
E. faecalis
P. aeruginosa
E. coli
50 100 100 100 NA NA NA NA NA NA 0.20
25 100 100 100 NA 100 100 NA 100 100 0.20
100 50 100 25 NA 100 NA NA 100 50 1.56
100 100 100 100 100 50 NA NA 100 100 0.78
NA 100 NA NA NA 100 NA NA 100 25 1.56
3. Experiment section 3.1. General experimental procedures X-ray data was determined using a Broker APEX DUO instrument. Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured on a JASCO P1020 polarimeter. UV spectra were detected on a SHIMADZU UV2401PC spectrometer. IR spectra were determined on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. 1D and 2D
The antibacterial assay of the total alkaloids and compounds 1e9 was evaluated against P. smartii (ATCC 29916), S. aureus (ATCC 25922), E. faecalis (ATCC 10541), P. aeruginosa (ATCC 27853), and E. coli (ATCC 11775). All the bacteria were obtained from the American Type Culture Collection (Rockville, USA). The preparation of bacterial inocula was done by using 18 h old overnight bacterial cultures prepared in Nutrient Agar. A few colonies of bacteria were collected aseptically with a sterile loop and introduced into 10 mL of sterile 0.90% saline solution. The concentration of the suspension was then standardized by adjusting the optical density to 0.10 at 600 nm, corresponding to bacterial cell suspension of about 108 colony-forming units/mL (CFU/mL). This cell suspension was diluted 100 times to obtain 106 CFU/mL before use. The compounds were dissolved in DMSO and then added to bacteria suspension to obtain the final concentration of 5% (v/v) DMSO or less. Serial twofold dilutions from 200 mg/mL of the compounds were performed in 96-well micro-titer plates. Each well contained 100 mL of sample of each concentration. Into each well was then introduced 100 mL of the bacterial suspension. The final concentration range of the test samples was 100e0.781 mg/mL, and the plates were incubated at 37 C for 24 h. After incubation, the wells were examined for growth of microorganisms and the MICs were determined upon addition of 50 mL of INT (p-iodonitrotetrazolium chloride). Viable bacteria turn the yellow dye of INT into pink. Each experiment was repeated three times and gentamycin was used as a positive control. MIC was defined as the lowest concentration of the compounds at which the bacterium did not demonstrate visible growth (no color change) and MIC >100 mg/mL was considered to be inactive. 3.3. Extraction and isolation The leaves of A. scholaris were collected in Xishuangbanna, Yunnan Province, PR China, in October 2006, and it has been stored for 7 years. This material (18 kg) was extracted with EtOH (40 L3) under reflux conditions, and the solvent was evaporated in vacuum. The residue was dissolved in 0.37% HCl, and the solution was subsequently basified using ammonia water to pH 9e10. The basic solution was partitioned with EtOAc, affording a two-phase mixture including the aqueous phase and the EtOAc/organic phase. The organic fraction (180 g) was collected and then dissolved in MeOH, and the resulting solution was subjected to column chromatography over silica gel eluting with CHCl3/MeOH (from 1:0 to 2:8) to afford six fractions (Fr. AeF). Fr. A (25 g) was further chromatographed using CHCl3/Me2CO (from 9:1 to 7:3) as eluant to give five fractions (Fr. A1eA5). Picrinine (3.8 g), strictamine (130 mg), 4 (56 mg), 5 (53 mg), and 6 (18 mg) were obtained from Fr. A1 (5.2 g) by using silica gel column chromatography eluted with CHCl3/ MeOH (95: 5). Fr. A2 (4.6 g) was further purified by preparative HPLC (CH3CN/H2O, 1:1) to afford 5a-methoxylstrictamine (83 mg),
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16-formyl-5a-methoxylstrictamine (43 mg), nareline (126 mg), 19E-vallesamine (2.6 g), 1 (5 mg), 2 (32 mg), 3 (21 mg), 7 (21 mg), and 8 (38 mg).
5
(3.52), 317 (3.34) nm; IR (KBr) nmax 2924, 2853, 1745, 1629, 1581, 1436, 1384, 1281, 1157, 1114, 1020, 965, 768 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 351 [MþH]þ; HREIMS m/z 350.1620 [M]þ (calcd for C21H22N2O3, 350.1630).
3.4. The physical data of the new compounds 3.4.1. Scholarisine H (1). Colorless gum; [a]26 D 160.5 (c 0.18, MeOH); UV (MeOH) lmax (log 3 )¼212 (4.34), 258 (3.80) nm; IR (KBr) nmax 2951, 2855, 1738, 1688, 1631, 1595, 1439, 1384, 1348, 1194, 1170, 1124, 1068, 773 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 351 [MþH]þ; HREIMS m/z 350.1636 [M]þ (calcd for C21H22N2O3, 350.1630). 3.4.2. Scholarisine I (2). White powder; [a]24 D þ215.9 (c 0.11, MeOH); UV (MeOH) lmax (log 3 )¼217 (4.28), 257 (3.71) nm; IR (KBr) nmax 3426, 2964, 2933, 2856, 1755, 1633, 1590, 1457, 1384, 1357, 1217, 1057, 1009, 769, 751 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 323 [MþH]þ; HREIMS m/z 322.1684 [M]þ (calcd for C20H22N2O2, 322.1681). 3.4.3. Scholarisine J (3). White powder; [a]25 D þ225.1 (c 0.14, MeOH); UV (MeOH) lmax (log 3 )¼208 (4.37), 248 (3.82) nm; IR (KBr) nmax 3426, 2932, 2857, 1639, 1592, 1456, 1348, 1260, 1115, 1077, 1048, 749 cm1; 1 H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 325 [MþH]þ; HREIMS m/z 324.1833 [M]þ (calcd for C20H22N2O2, 324.1838). 3.4.4. Scholarisine K (4). Colorless flaky crystal from MeOH; mp 251e252 C; [a]26 D þ16.3 (c 0.13, MeOH); UV (MeOH) lmax (log 3 )¼ 203 (4.56), 236 (3.94), 290 (3.54) nm; IR (KBr) nmax 3370, 2946, 1739, 1608, 1469, 1236, 1174, 1002, 945, 917, 769 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385 [MþH]þ; HREIMS m/z 384.1687 [M]þ (calcd for C21H24N2O5, 384.1685). 3.4.5. Scholarisine L (5). White powder; [a]26 D þ34.1 (c 0.21, MeOH); UV (MeOH) lmax (log 3 )¼203 (4.48), 236 (3.85), 290 (3.45) nm; IR (KBr) nmax 3426, 2952, 2856, 1764, 1641, 1613, 1470, 1273, 1229, 1191, 1096, 1004, 948, 751 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 385 [MþH]þ; HREIMS m/z 384.1683 [M]þ (calcd for C21H24N2O5, 384.1685). 3.4.6. Scholarisine M (6). Colorless needle crystal from MeOH; mp 240e242 C; [a]26 D 4.6 (c 0.09, MeOH); UV (MeOH) lmax (log 3 )¼ 203 (4.23), 236 (3.65), 290 (3.25) nm; IR (KBr) nmax 3441, 3283, 2954, 1746, 1737, 1613, 1470, 1436, 1270, 1233, 1196, 1177, 1094, 1020, 766 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 407 [MþNa]þ; HREIMS m/z 384.1681 [M]þ (calcd for C21H24N2O5, 384.1685). 3.4.7. Scholarisine N (7). white powder; [a]25 D þ178.5 (c 0.10, MeOH); UV (MeOH) lmax (log 3 )¼217 (4.85), 271 (4.24), 290 (4.20) nm; IR (KBr) nmax 3294, 3231, 2938, 1730, 1609, 1475, 1442, 1384, 1228, 1196, 1103, 884, 750 cm1; 1H and 13C NMR data, see Tables 1 and 2; ESIMS m/z 355 [MþH]þ; HREIMS m/z 354.1577 [M]þ (calcd for C20H22N2O4, 354.1580). 3.4.8. Scholarisine O (8). colorless gum; [a]26 D þ55.4 (c 0.21, MeOH); UV (MeOH) lmax (log 3 )¼204 (4.40), 228 (4.35), 290 (3.55), 305
Acknowledgements We are grateful to the National Natural Science Foundation of China (81225024, 31170334), the Ministry of Science and Technology of the People’s Republic of China (2014ZX09301307-003, 2013BAI11B02), and the Chinese Academy of Sciences (KSZD-EWZ-004-03) for partial financial support. Supplementary data Detailed MS, 1D and 2D NMR spectra for 1e8, X-ray crystallographic file of 4 in CIF format. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/ 10.1016/j.tet.2014.09.052. References and notes 1. Ishikura, M.; Abe, T.; Choshi, T.; Hibino, S. Nat. Prod. Rep. 2013, 30, 694e752. 2. Khyade, M. S.; Kasote, D. M.; Vaikos, N. P. J. Ethnopharmacol. 2014, 153, 1e18. 3. Ministry of Public Health, People’s Republic of China. Drug Specifications Promulgated by the Ministry of Public Health, 1997, Vol. 14, pp 49e50. 4. (a) Cai, X. H.; Du, Z. Z.; Luo, X. D. Org. Lett. 2007, 9, 1817e1820; (b) Cai, X. H.; Tan, Q. G.; Liu, Y. P.; Feng, T.; Du, Z. Z.; Li, W. Q.; Luo, X. D. Org. Lett. 2008, 10, 577e580; (c) Cai, X. H.; Liu, Y. P.; Feng, T.; Luo, X. D. Chin. J. Nat. Med. 2008, 6, 20e22; (d) Feng, T.; Cai, X. H.; Zhao, P. J.; Du, Z. Z.; Li, W. Q.; Luo, X. D. Planta Med. 2009, 75, 1537e1541. 5. (a) Gerfaud, T.; Xie, C.; Neuville, L.; Zhu, J. Angew. Chem., Int. Ed. 2011, 50, 3954e3957; (b) Adams, G. L.; Carroll, P. J.; Smith, A. B., III. J. Am. Chem. Soc. 2012, 134, 4037e4040; (c) Adams, G. L.; Carroll, P. J.; Smith, A. B., III. J. Am. Chem. Soc. 2013, 135, 519e528; (d) Smith, M. W.; Snyder, S. A. J. Am. Chem. Soc. 2013, 135, 12964e12967; (e) Smith, J. M.; Moreno, J.; Boal, B. W.; Garg, N. K. J. Am. Chem. Soc. 2014, 136, 4504e4507. 6. (a) Shang, J. H.; Cai, X. H.; Feng, T.; Zhao, Y. L.; Wang, J. K.; Zhang, L. Y.; Yan, M.; Luo, X. D. J. Ethnopharmacol. 2010, 129, 174e181; (b) Shang, J. H.; Cai, X. H.; Zhao, Y. L.; Feng, T.; Luo, X. D. J. Ethnopharmacol. 2010, 129, 293e298. 7. Morita, Y.; Hesse, M.; Schmid, H. Helv. Chim. Acta 1977, 60, 1419e1434. 8. Atta-Ur-Rahman; Alvi, K. A.; Abbas, S. A.; Voelter, W. Heterocycles 1987, 26, 413e419. 9. Abe, F.; Chen, R. F.; Yamauchi, T.; Marubayashi, N.; Ueda, I. Chem. Pharm. Bull. 1989, 37, 887e890. 10. Atta-Ur-Rahman; Habib-ur-Rehman. Planta Med. 1986, 230e231. 11. Zhou, H.; He, H. P.; Luo, X. D.; Wang, Y. H.; Yang, X. W.; Di, Y. T.; Hao, X. J. Helv. Chim. Acta 2005, 88, 2508e2512. 12. Abe, F.; Yamauchi, T.; Shibuya, H.; Kitagawa, I.; Yamashita, M. Chem. Pharm. Bull. 1998, 46, 1235e1238. 13. Crystal data for 4: C21H24N2O5, M¼384.42, orthorhombic, a¼7.6813(2) A, b¼8. 9350(2) A, c¼27.1302(7) A, a¼90.00 , b¼90.00 , g¼90.00 , V¼1862.01(8) A3, 1 T¼100(2) K, space group P212121, Z¼4, m(CuKa)¼0.810 mm , 8579 reflections measured, 3180 independent reflections (Rint¼0.0585). The final R1 values were 0.0958 (I>2s(I)). The final wR(F2) values were 0.2541 (I>2s(I)). The final R1 values were 0.0967 (all data). The final wR(F2) values were 0.2568 (all data). The goodness of fit on F2 was 1.260. Flack parameter¼0.2(3). The Hooft parameter is 0.06(10) for 1216 Bijvoet pairs. Crystallographic data for 4 have been deposited at the Cambridge Crystallographic Data Center (deposition number CCDC 1022010). 14. Yang, X. W.; Qin, X. J.; Zhao, Y. L.; Lunga, P. K.; Li, X. N.; Jiang, S. Z.; Cheng, G. G.; Liu, Y. P.; Luo, X. D. Tetrahedron Lett. 2014, 55, 4593e4596. 15. Cherif, A.; Massiot, G.; Men-Olivier, L. L. Heterocycles 1987, 26, 3055e3058. 16. Tasic, G.; Randjelovic, J.; Vusurovic, N.; Maslak, V.; Husinec, S.; Savic, V. Tetrahedron Lett. 2013, 54, 2243e2246. 17. Cai, X. H.; Shang, J. H.; Feng, T.; Luo, X. D. Z. Naturforsch., B 2010, 65, 1164e1168.
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