Phytochemistry 96 (2013) 389–396

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Monoterpene indole alkaloids from the stem bark of Mitragyna diversifolia and their acetylcholine esterase inhibitory effects Xing-Fen Cao, Jun-Song Wang, Xiao-Bing Wang, Jun Luo, Hong-Ying Wang, Ling-Yi Kong ⇑ State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic of China

a r t i c l e

i n f o

Article history: Received 4 March 2013 Received in revised form 1 October 2013 Available online 26 October 2013 Keywords: Mitragyna diversifolia Rubiaceae Indole alkaloids Acetylcholinesterase inhibition

a b s t r a c t Five monoterpene indole alkaloids, mitradiversifoline, with a unique rearranged skeleton, specionoxeineN(4)-oxide, 7-hydroxyisopaynantheine, 3-dehydropaynantheine, and 3-isopaynantheine-N(4)-oxide, and 10 known ones, were isolated from Mitragyna diversifolia. All the isolates were evaluated for their inhibition of acetylcholinesterase activities, and four showed moderate activities, with IC50 values of 4.1, 5.2, 10.2, and 10.3 lM, respectively. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Monoterpene indole alkaloids have attracted significant interest due to their established therapeutic importance (Zenk and Juenger, 2007), structural diversity (Leonard, 1999), and complex biosynthesis (O’ Connor and Maresh, 2006). They have been extensively investigated for a wide variety of pharmacological effects, such as cytotoxicity (Feng et al., 2010), anti-inflammatory (Feng et al., 2009), and AChE inhibitory activities (Zhan et al., 2010). The genus Mitragyna (family Rubiaceae) is a rich source of monoterpene indole alkaloids (Pandey et al., 2006; Shellard et al., 1978). The crude extract and purified alkaloids obtained from the Mitragyna species have demonstrated promising antidiarrheal (Chittrakarn et al., 2008), neuromuscular blocking (Chittrakarn et al., 2010), and analgesic activities (Takayama, 2004). As part of our program searching for bioactive alkaloids, the stem bark of Mitragyna diversifolia Korth was investigated for its alkaloidal components and afforded five new alkaloids, mitradiversifoline (1), specionoxeine-N(4)-oxide (2), 7-hydroxyisopaynantheine (3), 3-dehydropaynantheine (4), and 3-isopaynantheine-N(4)-oxide (5), respectively (Fig. 1). The new structures were elucidated by means of spectroscopic methods, and the absolute configuration of 1, an unusual rearranged monoterpene oxindole alkaloid from natural sources, was determined by ECD. In addition, 10 known alkaloids were identified as 3-isopaynantheine (6) (Kitajima et al., 2006), mitraciliatine (7) (Kitajima et al., 2006), speciociliatine N(4)-oxide (8) (Takayama

⇑ Corresponding author. Tel./fax: +86 25 83271405. E-mail address: [email protected] (L.-Y. Kong). 0031-9422/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phytochem.2013.10.002

et al., 2002), isospecionoxeine (9) (Trager et al., 1968), specionoxeine (10) (Trager et al., 1968), isocorynoxeine (11) (Yuan et al., 2008), corynoxeine (12) (Yuan et al., 2008), mitrafoline (13) (Hemingway et al., 1975), rotundifoleine (14) (Hemingway et al., 1975), and isorhynchophylline (15) (Yuan et al., 2008) by comparing experimental and reported data. These isolates were evaluated for their in vitro acetylcholinesterase (AChE) inhibitory potential.

2. Results and discussion Compound 1 was obtained as an amorphous powder and gave a positive Dragendorff’s test for alkaloids. Its HRESIMS data exhibited a pseudomolecular ion peak at m/z 429.2026 [M+H]+, corresponding to a molecular formula of C23H28N2O6 with 11° of unsaturation. The characteristic UV absorption maxima at 203, 244, and 294 nm suggested an oxindole chromophore. (Lim et al., 2009) The IR spectrum showed absorption bands due to an amino group (3443 cm1), an ester carbonyl (1709 cm1), an amide carbonyl (1621 cm1), and a vinyl group (1465 and 921 cm1). These observations, and the carbon resonances at dC 177.1 and 74.9, characteristic of an oxindole carbonyl and a spirocyclic quaternary carbon, suggested the presence of an oxindole alkaloid. The proton signals at dH 5.47 (ddd, J = 16.5, 10.0, 9.5 Hz, H-19), 4.92 (d, J = 16.5 Hz, H-18a), and 4.90 (d, J = 9.5 Hz, H-18b) were typical of a vinyl group (CH2@CH). Its 1H NMR spectrum presented two singlet methyls at dH 3.59 (CO2Me) and 3.82 (OMe), and one severely down-field shifted olefinic proton at dH 7.35 (1H, s, H-17), which were characteristic of a b-methoxyacrylate methyl ester moiety. The 1H, 13C NMR, and HMQC spectra (Table 1)

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Fig. 1. Structures of alkaloids 1–15.

indicated a substituted oxindole ring [dC 55.4 (s, 9-OCH3), 74.9 (s, C-7), 102.7 (d, C-12), 106.1 (d, C-10), 115.3 (s, C-8), 131.2 (d, C-11), 142.8 (s, C-13), 156.8 (s, C-9), and 177.1 (s, C-2); dH 7.20 (t, J = 8.0 Hz, H-11), 6.62 (d, J = 8.0 Hz, H-10), 6.41 (d, J = 8.0 Hz, H-12), 3.80 (s, 9-OCH3), and 10.30 (br s, NH)]. The HMBC correlations from H-19 to C-21 (dC 58.3), C-15 (dC 36.4), and C-20 (dC 41.5) located the vinyl group at C-20. Other cross-peaks from H-15 (dH 2.56, dt, J = 12.0, 3.0 Hz) to C-14 (dC 34.6), C-3 (dC 85.3), C-21, and C-20, and from H-21b (dH 2.0, t, J = 11.0 Hz) to C-3, C-15, and C-20, suggested that a tertiary nitrogen, C-3, C-14, C-15, C-20, and C-21 formed a piperidine ring. The methyl b-methoxyacrylate moiety was assigned to C-15 according to the HMBC correlations of H-17 with C-15 (dC 36.4), C-16 (dC 110.1), and C-22 (dC 167.1). The above evidence suggested that 1 was a Corynanthe-type monoterpene indole alkaloid related to isospecionoxeine (9), except for two severely down-field shifted carbon signals at dC 85.3 (C-3, D + 18.1 ppm) and 74.9 (C-7, D + 19.8 ppm). Deducting 10° of unsaturation associated with the above mentioned functionalities and rings, the remaining 1° of unsaturation suggested the presence of an additional ring in 1. The HMBC correlations arising from H-3, H-5, and H-6 (Fig. 2), combined with one unassigned oxygen atom in the molecular formula, and severely down-field shifted signals of C-3 and C-7 in 1 constructed an oxazine ring (Kitajima et al., 2012). Thus, the gross structure of 1 was established, representing an unusual rearranged

monoterpene oxindole alkaloid in nature. According to the biosynthetic origin, the H-15 of Corynanthe-type indole alkaloids was constantly in an a-orientation (Ma et al., 2009; Rueffer et al., 1978). Consequently, the ROESY correlations of H-15a/H-3, H-15a/H-14b, and H-14b/H-3 assigned an a-orientation to H-3, and those of H-15a/H-19 and H-20/H-14a suggested a b-orientation of H-20. The ROESY cross-peak between H-17 and 22-OCH3 suggested their proximity in space, thus indicating an (E)-configuration for the D16 (17) double bond. As a tetracyclic oxindole alkaloid, its 7R configuration was established according to a positive Cotton effect (CE) at 271 nm and a negative one at 250 nm in the ECD spectrum (Trager et al., 1968). This assertion was further verified by a computational method. The ECD spectrum of 1 was calculated with the Gaussian-09 program package using TDDFT at the B3LYP/6-31++G(d, p) level (Zhang et al., 2012). The calculated ECD spectrum for the 3R, 7R, 15S, 20R stereoisomer of 1 agreed well with the experimental one (Fig. 3), thus the absolute configuration of 1 was finally established. Compound 2 was isolated as a light yellow amorphous powder and was alkaloid-positive by the Dragendorff’s reagent. The UV absorptions at 202, 241, and 292 nm suggested the presence of an oxindole chromophore. The molecular formula of 2 was determined to be C23H28N2O6 by the HRESIMS ion at m/z 429.2028 [M+H]+ (calcd 429.2020), one more oxygen atom than that of specionoxeine (10) (Trager et al., 1968). The NMR spectroscopic data of

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X.-F. Cao et al. / Phytochemistry 96 (2013) 389–396 Table 1 H (500 MHz) and

1

13

C NMR (125 MHz) spectroscopic data of compounds 1 and 2. Compound 1a

Position

dC 2 3 5a 5b 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17 18a 18b 19 20 21a 21b 22 9-OCH3 17-OCH3 22-OCH3 NH (1) a b c

177.1 85.3 46.8 26.9 74.9 115.3 156.8 106.1 131.2 102.7 142.8 34.6 36.4 110.1 160.1 115.2 139.0 41.5 58.3 167.1 55.4 61.4 50.7

Compound 2b dH, multi (J in Hz)

dC 179.0 77.2 67.4

4.56, d (7.5) 2.98, t (11.5) 2.67c 2.84c 1.60, d (13.5)

30.8 54.3 114.6 155.1 105.8 131.1 105.2 143.2 23.4

6.62, d (8.0) 7.20, t (8.0) 6.41, d (8.0) 1.85, q (12.0) 1.36, d (12.0) 2.56, dt (12.0, 3.0) 7.35, s 4.92, d (16.5) 4.90, d (9.5) 5.47, ddd (16.5, 10.0, 9.5) 2.88c 2.73, dd (11.0, 3.5) 2.0, t (11.0) 3.80, s 3.82, s 3.59, s 10.30 br, s

36.5 109.0 160.8 118.8

dH, multi (J in Hz) 4.14c 4.41, br, s 4.12c 2.85, m 2.56, dd (14.0, 8.0)

6.66, d (8.0) 7.20, t (8.0) 6.77, d (8.0) 2.63c 1.31, m 2.73, m

135.0 37.5 66.7

7.30, s 5.06, d (18.0) 5.04, d (10.0) 5.45, ddd (18.0, 10.0,9.0) 3.58c 4.05c

167.5 55.8 62.0 51.3

3.90, s 3.78, s 3.61, s

Recorded in DMSO-d6. Recorded in CDCl3. Overlapped with other signals.

Fig. 2. Selected HMBC (a) and ROESY (b) correlations of 1.

Fig. 3. Calculated and experimental ECD spectra of 1.

2 (Table 1) resembled those of 10, except for the downfield shifted signals neighboring the sp3 nitrogen atom: H-3, H-5, and H-21; and C-3, C-5, and C-21, which suggested that 2 was an N(4)-oxide of 10, similar to ciliaphylline N-oxide (Phillipson et al., 1973), but with a vinyl functionality at C-20 instead of an ethyl group. Due to the high polarity of N-oxide, the Rf values of 2 in silica gel TLC were much lower than those of its parent compound (10) in three developing solvent systems, which also supported the N-oxide nature of 2. The absolute configuration of 2 was determined by the ROESY and ECD spectra. The ROESY correlations of H-15a/H-3, H-15a/H19, H-15a/H-14b, and H-20/H-14a indicated that the relative configurations of C-3 and C-20 in 2 were identical to those in 1. The 7R configuration was deduced based on the presence of a negative CE at 290 nm and a positive CE at 241 nm in ECD spectrum (Trager et al., 1968). Thus, the absolute configuration of 2 was established as shown and the compound was named as specionoxeine-N(4)oxide.

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The molecular formula of 3, C23H28N2O5, was determined by the HRESIMS ion at m/z 413.2073 [M+H]+ (calcd 413.2071). Its UV absorption bands at 222, 347, and 394 nm, and an IR absorption band at 3451 cm1 supported an indolenine fragment (Sakai et al., 1971). The 1H NMR spectrum showed three aromatic protons with an ABX coupling pattern: dH 6.85 (1H, d, J = 8.5 Hz, H-10), 7.30 (1H, t, J = 8.5 Hz, H-11), and 7.12 (1H, d, J = 8.5 Hz, H-12). The 1H and 13C NMR spectra displayed a methyl b-methoxyacrylate moiety, a vinyl group, and a methoxylated benzene, suggestive of a common 9-methoxylated Corynanthe-type monoterpene indole alkaloid (Takayama et al., 1998). Two characteristic quaternary carbons at dC 183.6 and 81.1 were assigned to C-2 and C-7, respectively, according to the HMBC correlations as shown (Fig. 4). In addition, the exchangeable signal at dH 5.54 (1H, s, OH) in the 1H NMR spectrum and its HMBC correlations with C-7 (dC 81.1), C-2 (dC 183.6), and C-6 (dC 35.1) allowed its assignment to C-7. The NMR spectroscopic data of 3 resembled those of 7-hydroxyspeciociliatine (Kitajima et al., 2006), except for presence of two olefinic carbons (dC 113.3 and 139.8) in 3 instead of two sp3 carbons in 7hydroxyspeciociliatine. The exo-olefinic bond was placed at C-20 on the basis of HMBC correlations from H-19 (dH 5.42, ddd, J = 17.5, 10.0, 8.5 Hz) to C-15 (dC 32.3), C-20 (dC 42.7), and C-21 (dC 51.2). The configuration of 3 was deduced from the ROESY and ECD spectra. The H-15 was consistently in a-orientation for Corynanthe-type indole alkaloids in terms of their biosynthetic origin. The ROESY correlations from H-15a to H-19, H-14b, and H-21a, and from H-20 to H-14a and H-21b established the b-orientations for H-20 and H-14a. Likewise, the ROESY crosspeaks of H-3/H-20 and H-3/H-14a assigned a b-orientation to H-3, which correlated in turn with 7-OH, suggesting a b-orientation of 7-OH. The 3R configuration was established by the negative CEs at 261 and 311 nm in the ECD spectrum (Brown and Blackstock, 1972). The structure of 3 was thus fully established as shown with the absolute configurations of 3R, 7R, 15S, 20R. Compound 4 was isolated as yellow oil and exhibited a molecular ion at m/z 395.1969 [M]+ in the HRESIMS. Careful inspection of the NMR spectroscopic data (Table 2) of 4 and 3-dehydromitragynine (Houghton and Said, 1986) suggested the resemblance of their gross structures, except for two olefinic carbons (dC 119.2 and 136.9) in 4 instead of two sp3 carbons in 3-dehydromitragynine. Two olefinic carbons were assigned to an D18 (19) double bond based on the HMBC correlations of H-19 (dH 5.61, ddd, J = 17.0, 10.0, 8.0 Hz) with C-15 (dC 32.0), C-20 (dC 41.3), and C-21 (dC 58.0), indicating a vinyl group at C-20. The a-orientation of the vinyl group was determined by strong ROESY correlation between H-15a and H-19. Hence, the structure of 4 was determined as shown. The molecular formula of 5 was deduced to be C23H28N2O5 from its HRESIMS, indicating 11° of unsaturation. The NMR spectra of 5 bear close resemblance to those of 3. The most notable differences were that resonances for the hydroxyl, the oxygenated quaternary

carbon, and the imine carbon in 3 were absent in 5. Instead, two olefinic carbon signals at dC 128.5 (C-2) and dC 106.0 (C-7), and an exchangeable proton resonance at dH 12.54 (NH), were observed, indicating the presence of an indole skeleton, which was further confirmed by HMBC correlations from H-6 (dH 3.41) to C7 and C-2, and from NH to C-2 and C-7. Deducting the atoms for the skeleton and exhibited functionalities, one oxygen atom remained unaccounted for. This fact, combined with the characteristic downfield shifted signals of C-3 (dC 70.9), C-5 (dC 68.6), and C-21 (dC 62.6), suggested the presence of an N(4)-oxide functionality in 5, similar to hirsuteine N-oxide (Sakai and Shinma, 1978), but with an additional methoxy at C-9. Co-TLC of N-oxide (5) and its parent compound (6) found that the Rf values of 5 were also much lower in three developing solvent systems, featuring a N-oxide nature of 5. ROESY correlations of H-15a/H-19, H-15a/H-14b, H-3/H-14a, and H-3/H-20 indicated the b-orientations of H-3 and H-20. The absolute configuration of 5 was identical to that of 3 according to a similar CE to 3, the structure of 5 was thus determined as shown. Mitradiversifoline (1) is a rearranged monoterpene oxindole alkaloid in nature containing an unusual oxazine ring. Two plausible biosynthesis pathways for 1 could be proposed (Fig. 5) based on our findings and the literature (Carmona et al., 2000; Gan and Kam, 2009). 3-Isopaynantheine (6), a major component of M. diversifolia, was rationalized as the precursor of 1. On pathway A, b-oxidation of the indole framework of 6 would afford 7-hydroxyisopaynantheine (3), which on subsequent oxidative cleavage followed by rearrangement would give isospecionoxeine (9). After a series of intramolecular rearrangement, intermediate (9a) was generated by breaking the C3–C7 bond, followed by epoxidation (9b) and cleavage (9c). Finally, mitradiversifoline (1) was formed by cyclization via intramolecular trapping by OH. On pathway B, the oxidative dehydrogenation of the C3–N4 bond of 6 yielded 3dehydropaynantheine (4), which was also obtained in this investigation. Intermediate 4a could be formed by an initial attack of water and the following rearrangement. Epoxidation of the D2 (3) double bond (4b) and subsequent cleavage of the epoxide ring would afford the key intermediate (4c), which proceed with electrophilic attack of H2O at C-2 of 4c leading to the bond fission between C2 and C3 (4d). A six-membered oxazine ring was built by intramolecular trapping of OH-3 and subsequent dehydration (4e), which was then tautomerized to mitradiversifoline (1). As one of the most common causes of mental deterioration in elderly people, Alzheimer’s disease is a devastating neurodegenerative disorder with an ever-increasing incidence due to the increasing average life expectancy world-wide (Brookmeyer et al., 2007). Among the multiple targets implicated in this complex disease, acetylcholinesterase (AChE) is one of the most important. Besides synthetic AChE inhibitors, plant-derived ones have also been successfully used for the treatment of Alzheimer’s disease (Zhan et al., 2010), such as galanthamine and huperzine

Fig. 4. Selected HMBC (a) and ROESY (b) correlations of 3.

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1

Position

13

C NMR (125 MHz) spectroscopic data of compounds 3–5. Compound 3a dC

2 3 5a 5b 6a 6b 7 8 9 10 11 12 13 14a 14b 15 16 17 18a 18b 19 20 21a 21b 22 9-OCH3 17-OCH3 22-OCH3 7-OH NH (1) a b c d

183.6 53.3 48.1 35.1 81.1 127.5 155.9 109.2 129.8 114.7 154.3 27.2 32.3 110.9 160.1 113.3 139.8 42.7 51.2 167.4 55.3 61.3 50.6

Compound 4b dH, multi (J in Hz)

dC 126.6 166.7 53.7

4.13, d (5.0) 3.36d 2.64, d (11.0) 2.40, d (14.0) 1.80, dt(14.0,4.0)

22.2 124.4 117.4 157.9 101.6 131.5 106.7 144.4 31.5

6.85, d (8.5) 7.30, t (8.5) 7.12, d (8.5) 2.27d 1.94, dd(12.0,3.5) 3.14, dt(12.0,4.0) 7.38, s 4.85, d (17.5) 4.83, d (8.5) 5.42, ddd (17.5, 10.0, 8.5) 2.7, m 2.30d 2.17, t (11.0) 3.81, 3.84, 3.61, 5.54,

s s s s

32.0 109.3 163.3 119.2 136.9 41.3 58.0 169.2 56.1 62.7 51.9

Compound 5c dH, multi (J in Hz)

dC

dH, multi (J in Hz)

4.06, m

128.5 70.9 68.6

5.25, s 4.14d

22.5

3.41, m

3.47, t (8.0)

6.56, d (8.0) 7.34, t (8.0) 7.01, d (8.0) 3.57, m 1.28, m 3.29d 7.58, s 5.19, d (17.0) 5.16, d (10.0) 5.61, ddd (17.0, 10.0, 8.0) 3.28d 3.85d 3.78, m 3.94, s 3.88, s 3.69, s

106.0 117.9 155.2 100.6 123.4 106.1 140.3 27.5 33.0 110.5 161.6 118.0 137.6 38.4 62.6 168.0 55.6 62.1 51.4

6.58, d (7.5) 7.20d 7.20d 3.74d 2.74, d (13.5) 3.02, t (11.0) 7.26, s 5.15, d (17.0) 4.98, d (10.0) 5.51, ddd (17.0, 10.0, 9.5) 4.14d 3.70d 3.51, dd (12.0,3.5) 3.83, s 3.59, s 3.57, s 12.54, s

Recorded in DMSO-d6. Recorded in methanol-d4. Recorded in pyridine-d5. Overlapped with other signals.

Fig. 5. Two possible biosynthesis pathways to 1.

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inhibitory abilities and compounds 3, 4, 7, and 8 showed moderate activities.

Table 3 Anti-AChE activities of compounds 1–15. Compounds

IC50 (lM)

Compounds

IC50 (lM)

1 2 3 4 5 6 7 8

NA NA 10.3 ± 1.3 4.1 ± 1.0 55.4 ± 5.8 120.8 ± 17.1 5.2 ± 1.2 10.2 ± 0.5

9 10 11 12 13 14 15 Galanthamine

NA NA NA NA NA NA NA 1.3 ± 0.2

NA, not active at the concentration of 200 lM. ‘‘±’’ Represent standard deviation of mean of these assays. Galanthamine was used as a positive control.

4. Experimental 4.1. General experimental procedures Optical rotations were measured with a JASCO P-1020 polarimeter, whereas ECD spectra were obtained on a JASCO J-810 spectropolarimeter. UV spectra were obtained on a Shimadzu UV-2450 spectropolarimeter. IR spectra (KBr disks) were recorded on a Bruker Tensor 27 spectrometer, whereas NMR spectra were acquired on a Bruker Avance III-500 NMR instrument (1H: 500 MHz, 13C: 125 MHz), with TMS as internal standard. Mass spectra were obtained on an MS Agilent 1100 series LC/MSD ion-trap mass spectrometer (ESIMS) and a Mariner ESITOF spectrometer (HRESIMS). Acetylcholinesterase (AChE) inhibitory activities were measured spectrophotometrically using a multidetection microplate reader (Molecular Devices). Silica gel (Qingdao Haiyang chemical Co., Ltd.), Sephadex LH-20 (Pharmacia), and RP-C18 (40–63 lm, Fuji) were used for column chromatography (CC). Preparative HPLC was carried out using an Agilent 1100 Series instrument with a Shim-Pak RP-C18 column (5 lm, 20  200 mm) and a 1100 Series multiple wavelength detector. 4.2. Plant material Stem bark of M. diversifolia was collected in May 2011 from Xishuangbanna, Yunnan Province, People’s Republic of China, and was identified by Prof. Shun-Cheng Zhang, Xishuangbanna Botanical Garden, Chinese Academy of Sciences, People’s Republic of China. A voucher specimen (No. MD-201105) has been deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 4.3. Extraction and isolation

Fig. 6. The dose-dependent effects of 3–8 and galanthamine.

A. Indole alkaloids have been reported with potential AChE inhibitory activities (Khorana et al., 2012). These isolates (1–15) were therefore evaluated for their potential to inhibit the activity of AChE with galanthamine (IC50 1.3 lM) as reference (Table 3, Fig. 6). Compounds 3, 4, 7, and 8 exhibited moderate inhibitory activities with IC50 values of 10.3, 4.1, 5.2, and 10.2 lM, respectively; 5 and 6 showed marginal activities with IC50 values of 55.4 and 120.8 lM. Compounds of an indole skeleton (3–8) showed more potent inhibitory activities than those of an oxindole framework (1, 2, and 9–15), which might offer a promising entry point for the development of new acetylcholinesterase inhibitors. Further investigations on the modes of action of this type of monoterpene indole alkaloids were warranted.

3. Conclusions Phytochemical investigation of the stem bark of M. diversifolia resulted in the isolation of five monoterpenoid indole alkaloids (1–5), together with 10 known ones. The structures of these alkaloids were established by extensive spectroscopic methods. Mitradiversifoline (1), containing an unusual oxazine ring, is a rearranged monoterpene oxindole alkaloid in nature. All the isolates were evaluated for their in vitro acetylcholinesterase

The stem bark (10 kg) was extracted with EtOH–H2O (95:5, v/ v  3) under condition of reflux. After removal of the solvent in vacuo, the viscous concentrate was first suspended in H2O and then partitioned with CH2Cl2. Solvent was removed to afford the CH2Cl2 residue (64 g). The CH2Cl2 extract was then applied to a D101 porous resin eluted with a gradient of EtOH–H2O (30%, 60%, and 90%, EtOH in H2O) to afford three fractions (A–C). Fraction B (20 g) was subjected to silica gel CC, eluted with CH2Cl2–MeOH in a gradient (1:0 to 0:1) to afford five fractions (B1–B5). Fraction B1 (7 g) was subjected to reversed-phase C18 silica gel CC, eluted with MeOH– H2O (30:70 to 80:20), to give four major fractions (B1a–B1d). Fraction B1b was separated over reversed-phase C18 silica gel CC, using MeOH–H2O (20:80 to 70:30) as mobile phase, to afford seven fractions (B1ba–B1bg). Fraction B1bb (690 mg) was further subjected to Sephadex LH-20 CC eluted with MeOH, and finally, preparative HPLC was carried out using MeOH–H2O (55:45) containing 0.04% Et2NH as an eluent at a flow rate of 10 mL/min to afford 9 (63 mg), 10 (28 mg), 11 (24 mg), 12 (6 mg), 13 (14 mg), 14 (23 mg), and 15 (5 mg), respectively. Fraction B1bf (85 mg) was applied to an ODS open column eluted with MeOH–H2O (50:50), and finally, to preparative HPLC using MeOH–H2O (65:35) plus 0.04% Et2NH as an eluent at a flow rate of 10 mL/min to yield 1 (19 mg), 2 (10 mg), and 3 (28 mg), respectively. Fraction B1c (2.5 g) was subjected to reversed-phase C18 silica gel CC eluted with MeOH–H2O (50:50) to give a fraction which was further isolated and purified by repeated silica gel CC (CH2Cl2–MeOH, 10:1) to yield 4 (7 mg), 5 (178 mg), 6 (900 mg), 7 (10 mg), and 8 (7 mg), respectively.

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4.3.1. Mitradiversifoline (1) Colorless amorphous powder; [a]28D + 6.0 (c 0.04, MeOH); UV (MeOH) kmax (log e) 294 (3.54), 244 (4.09), 203 (4.26) nm; ECD (MeOH) kmax (De) 222 (+2.70), 250 (0.90), 271 (+0.18), 287 (0.06) nm; IR (KBr) mmax 3443, 1709, 1621, 1465, 1244, 1095, 921, 773 cm1; For 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 429.2026 [M+H]+ (calcd for C23H29N2O6, 429.2020). 4.3.2. Specionoxeine-N(4)-oxide (2) Light yellow amorphous powder; [a]28D 91.8 (c 0.30, MeOH); UV (MeOH) kmax (log e) 292 (3.07), 241 (3.76), 202 (3.90) nm; ECD (MeOH) kmax (De) 221 (3.01), 241 (+1.41), 290 (0.74) nm; IR (KBr) mmax 3449, 2951, 1693, 1623, 1467, 1245, 1201, 1127, 782 cm1; For 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 429.2028 [M+H]+ (calcd for C23H29N2O6, 429.2020). 4.3.3. 7-Hydroxyisopaynantheine (3) Yellow amorphous powder; [a]28D 13.1 (c 0.06, MeOH); UV (MeOH) kmax (log e) 394 (2.96), 347 (3.30), 222 (3.90) nm; ECD (MeOH) kmax (De) 208 (1.08), 227 (+0.30), 261 (1.41), 311 (0.40) nm; IR (KBr) mmax 3451, 1633, 1400, 1386, 1257, 1104 cm1; For 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z 413.2073 [M+H]+ (calcd for C23H29N2O5, 413.2071). 4.3.4. 3-Dehydropaynantheine (4) Yellow oil; [a]28D + 4.8 (c 0.03, MeOH); UV (MeOH) kmax (log e) 398 (3.55), 348 (3.91), 249 (4.08), 205 (4.21) nm; IR (KBr) mmax 3447, 1685, 1635, 1206, 1136, 722 cm1; For 1H and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z 395.1969 [M]+ (calcd for C23H27N2O4, 395.1965). 4.3.5. 3-Isopaynantheine-N(4)-oxide (5) Light yellow amorphous powder; [a]28D + 51.2 (c 0.18, MeOH); UV (MeOH) kmax (log e) 282 (3.70), 240 (4.05), 223 (4.56) nm; ECD (MeOH) kmax (De) 234 (+1.09), 267 (0.39), 286 (0.18) nm; IR (KBr) mmax 3450, 1694, 1636, 1106, 986, 777 cm1; For 1H and 13 C NMR spectroscopic data, see Table 2; HRESIMS m/z 413.2071 [M+H]+ (calcd for C23H29N2O5, 413.2071). 4.4. Determination of inhibitory effect on AChE AChE inhibitory activities of 1–15 were determined using Ellman’s method with slight modification (Tsai and Lee, 2010). Each compound was initially dissolved in DMSO at a concentration of 0.25 mol/L as a stock solution, and was then serially diluted with buffer A [50 mmol/L Tris–HCl (pH 8.0) containing 0.10 mol/L NaCl and 0.02 mol/L MgCl26H2O]. To each well of the 96-well plate, a 10 lL test sample was added together with 160 lL of 1.5 mmol/L 5,50 -dithiobis (2-nitrobenzoic acid) (DTNB) in buffer A, and 50 lL of 0.25 U/mL AChE in buffer B [50 mmol/L Tris–HCl (pH 8.0) containing 0.1% bovine serum albumin]. The reaction was then initiated by addition of 30 lL 3.6 mmol/L acetylthiocholine iodide (ATCI) in buffer A. Hydrolysis of ATCI was monitored by the formation of yellow-colored 5-thio-2-nitrobenzoate anion at a wavelength of 405 nm using a 96-well microplate reader. Percentage of inhibition was calculated by comparing the rate of enzymic hydrolysis of ATCI for the sample to that of the blank (buffer A). All assays were performed in triplicate from three independent runs with galanthamine as a positive control. Based on the absorbance (A) of the blank (A0) and the test sample (A1), the inhibitory percentage for each sample was calculated with an equation

Inhibition ð%Þ ¼ ð1  A1 =A0 Þ  100: The IC50 values were calculated from the average of three independent runs by plotting a concentration–response curve.

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Acknowledgments This research work was financially supported by the National Science Foundation of China (21272275 and 81173526), the Program for Changjiang Scholars and Innovative Research Team in University (PCSIRT-IRT1193), the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for New Century Excellent Talents in University, State Education Ministry of China (#NCET-11-0738). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2013. 10.002. These data include MOL files and InChiKeys of the most important compounds described in this article. References Brookmeyer, R., Johnson, E., Ziegler-Graham, K., Arrighi, H.M., 2007. Forecasting the global burden of Alzheimer’s disease. Alzheimers Dement. 3, 186–191. Brown, R.T., Blackstock, W.P., 1972. Adina alkaloids: 10-b-D-glucosyloxyvincoside lactam. Tetrahedron Lett. 30, 3063–3065. Carmona, C., Ghanem, R., Balón, M., Guardado, P., Muñoz, M., 2000. Mechanism of the oxidation of yohimbine and two of its 7H-substituted derivatives by sodium peroxodisulfate. J. Chem. Soc. Perkin Trans. 2, 839–845. Chittrakarn, S., Sawangjaroen, K., Prasettho, S., Janchawee, B., Keawpradub, N., 2008. Inhibitory effects of kratom leaf extract (Mitragyna speciosa Korth.) on the rat gastrointestinal tract. J. Ethnopharmacol. 116, 173–178. Chittrakarn, S., Keawpradub, N., Sawangjaroen, K., Kansenalak, S., Janchawee, B., 2010. The neuromuscular blockade produced by pure alkaloid, mitragynine and methanol extract of kratom leaves (Mitragyna speciosa Korth.). J. Ethnopharmacol. 129, 344–349. Feng, T., Li, Y., Cai, X.H., Gong, X., Liu, Y.P., Zhang, R.T., Zhang, X.Y., Tan, Q.G., Luo, X.D., 2009. Monoterpenoid indole alkaloids from Alstonia yunnanensis. J. Nat. Prod. 72, 1836–1841. Feng, T., Li, Y., Liu, Y.P., Cai, X.H., Wang, Y.Y., Luo, X.D., 2010. Melotenine A, a cytotoxic monoterpenoid indole alkaloid from Melodinus tenuicaudatus. Org. Lett. 12, 968–971. Gan, C.Y., Kam, T.S., 2009. Leucolusine, a tetracyclic alkaloid with a novel ring system incorporating an oxindole moiety and fused piperidine-tetrahydrofuran rings. Tetrahedron Lett. 50, 1059–1061. Hemingway, S.R., Houghton, P.J., Phillipson, J.D., Shellard, E.J., 1975. 9Hydroxyrhynchophylline-type oxindole alkaloids. Phytochemistry 14, 557–563. Houghton, P.J., Said, M.I., 1986. 3-Dehydromitragynine: an alkaloid from Mitragyna speciosa. Phytochemistry 25, 2910–2912. Khorana, N., Changwichit, K., Ingkaninan, K., Utsintong, M., 2012. Prospective acetylcholinesterase inhibitory activity of indole and its analogs. Bioorg. Med. Chem. Lett. 22, 2885–2888. Kitajima, M., Misawa, K., Kogure, N., Said, I.M., Horie, S., Hatori, Y., Murayama, T., Takayama, H., 2006. A new indole alkaloid, 7-hydroxyspeciociliatine, from the fruits of Malaysian Mitragyna speciosa and its opioid agonistic activity. J. Nat. Med. 60, 28–35. Kitajima, M., Ohara, S., Kogure, N., Wu, Y., Zhang, R., Takayama, H., 2012. New indole alkaloids from Melodinus henryi. Heterocycles 85, 1949–1959. Leonard, J., 1999. Recent progress in the chemistry of monoterpenoid indole alkaloids derived from secologanin. Nat. Prod. Rep. 16, 319–338. Lim, K.H., Sim, K.M., Tan, G.H., Kam, T.S., 2009. Four tetracyclic oxindole alkaloids and a taberpsychine derivative from a Malayan Tabernaemontana. Phytochemistry 70, 1182–1186. Ma, B., Wu, C.F., Yang, J.Y., Wang, R., Kano, Y., Yuan, D., 2009. Three new alkaloids from the leaves of Uncaria rhynchophylla. Helv. Chim. Acta 92, 1575–1585. O0 Connor, S.E., Maresh, J.J., 2006. Chemistry and biology of monoterpene indole alkaloid biosynthesis. Nat. Prod. Rep. 23, 532–547. Pandey, R., Singh, S.C., Gupta, M.M., 2006. Heteroyohimbinoid type oxindole alkaloids from Mitragyna parvifolia. Phytochemistry 67, 2164–2169. Phillipson, J.D., Rungsiyakul, D., Shellard, E.J., 1973. N-Oxides of the oxindole alkaloids, isorhynchophylline, rhynchophylline, rhynchociline and ciliaphylline. Phytochemistry 12, 2043–2048. Rueffer, M., Nagakura, N., Zenk, M.H., 1978. Strictosidine, the common precursor for monoterpenoid indole alkaloids with 3a and 3b configuration. Tetrahedron Lett. 19, 1593–1596. Sakai, S., Shinma, N., 1978. The partial synthesis of heteroyohimbine alkaloids: akuammigine, 3-isorauniticine and corynanthe alkaloid, corynantheidine. Chem. Pharm. Bull. 26, 2596–2598. Sakai, S., Aimi, N., Kubo, A., Kitagawa, M., Shiratori, M., Haginiwa, J., 1971. Gardneria alkaloids-VI structures of gardneramine and alkaloid G (demethylgardneramine). Tetrahedron Lett. 12, 2057–2060. Shellard, E.J., Houghton, P.J., Resha, M., 1978. The Mitragyna species of Asia, 31: the alkaloids of Mitragyna speciosa Korth from Thailand. Planta Med. 34, 26–36.

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Monoterpene indole alkaloids from the stem bark of Mitragyna diversifolia and their acetylcholine esterase inhibitory effects.

Five monoterpene indole alkaloids, mitradiversifoline, with a unique rearranged skeleton, specionoxeine-N(4)-oxide, 7-hydroxyisopaynantheine, 3-dehydr...
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