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Synthesis and acetylcholinesterase inhibitory activities of tabersonine derivatives
Phytochemistry Letters 14 (2015) 17–22
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Synthesis and acetylcholinesterase inhibitory activities of tabersonine derivatives Xiong Liua,b , Dongliang Yanga , Jiajia Liua,* , Na Rena,c a
Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, PR China Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, Hunan, PR China c Hunan Vocational College of Science and Technology, Changsha 410004, Hunan, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2015 Received in revised form 21 August 2015 Accepted 24 August 2015 Available online xxx
Tabersonine, the main alkaloid in Voacanga seeds, was used as a lead compound to semi-synthesize tabersonine derivatives. In total, 13 compounds, containing 10 novel tabersonine derivatives, were synthesized by introducing substituent groups R1–R5. The acetylcholinesterase (AChE) inhibitory activities of tabersnonine derivatives were evaluated using Ellman’s method. Among them, compound (7) showed the highest AChE inhibitory activity with the IC50 value was 5.32 mM. The substituent groups R1–R5 showed different influences on the AChE inhibitory activities of tabersonine derivatives. The AChE inhibitory activities of tabersonine derivatives increased with the introduction of group R1 and/or combined groups R3,R4, while decreased with the introduction of group R5. And the group R2 showed no significant influence on the AChE inhibitory activities of tabersonine derivatives. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Voacanga seeds Tabersonine derivatives AChE inhibitory activities
1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease (González-Domínguez et al., 2012). The cholinergic hypothesis is the most widely accepted biochemical theory of this disease. It is believed that the decline in cognitive and mental functions associated with AD is related to the loss of cortical cholinergic neurotransmission (Lai et al., 2013). AChE plays a significant role in hydrolysis of acetylcholine (ACh) (Yang et al., 2012). The use of AChE inhibitors is an effective clinical approach to maintain the levels of ACh and enhance cholinergic function. The inhibition of the AChE has become the standard approach in the symptomatic treatment of AD (Howes and Houghton, 2003; Pendota et al., 2013). Recently, some secondary metabolites from natural plants such as essential oils, flavonoids and alkaloids have been proven to possess AChE inhibitory activities (Iannello et al., 2014; Qin et al., 2011; da Silva et al., 2014; Vats et al., 2015). These findings suggest that natural plants were good resources for seeking compounds with high AChE inhibitory activities. Voacanga Africana is an evergreen tree, belonging to the family Apocynaceae. Its seeds are
* Corresponding author. E-mail address: [email protected] (J. Liu).
traditionally used against leprosy, diarrhea, generalized edema, convulsions in children, mental disorders and diuretic (Marnewick, 2009). Voacangine, an alkaloid in Voacanga seeds, has been proved to possess considerable AChE inhibitory activity (Zhan et al., 2010). Based on this result, we attempted to seek components with high AChE inhibitory activities from Voacanga seeds. In our previous work, we have obtained some secondary metabolites (tabersonine, essential oils, crude extracts) from Voacanga seeds and semi-synthesized new eburnamine derivative using tabersonine as a lead compound. (Liu et al., 2014a,b; Liu et al., 2015). Tabersonine is the main alkaloid in Voacanga seeds (Manske, 1965; Maurice, 1993; Rolland et al., 1976), which plays a significant role in lowering blood pressure and is used to semi-synthesize the cerebrally active eburnamine derivatives (Liu et al., 2014b; Nemes et al., 2007; Vas and Gulyas, 2005). In the biological evaluation, we found that tabersonine exhibited a certain AChE inhibitory activity while eburnamine derivatives showed no activities. This result revealed that structural modification of tabersonine is a promising approach to obtain compounds with high AChE inhibitory activities. However, to date, researches about synthesis and evaluation of AChE inhibitory activities of tabersonine derivatives have rarely been carried out. Thus, in the current investigational study, several derivatives of tabersonine were synthesized using tabersonine as lead compound and their AChE inhibitory activities were also tested.
http://dx.doi.org/10.1016/j.phytol.2015.08.015 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
2. Experimental 2.1. Chemical reagents and samples The reagents and chemicals (analytic grade unless stated otherwise) were purchased from Sinopharm Chemical Reagent Co., Ltd. and Tianjin Damao Chemical Reagent Co., Ltd. (China). Acetylthiocholine iodide (ATCI), acetylcholinesterase (AChE) and 5,50 -dithiobis [2-nitrobenzoic acid] (DTNB) purchased from J & K Chemical Technology. The Voacanga seeds were collected from Ghana, in 2012, and the sample was authenticated by professor Liu at the Central South University. A voucher specimen (CSPHA120923) has been deposited in College of Chemistry and Chemical Engineering of Central South University. The Voacanga seeds were grounded by a universal grinder. The grounded material was stored at –10 C in a domestic refrigerator before the extraction procedures were performed. Melting points were determined with a Hotstage microscope Boetius apparatus and are uncorrected. The molecular weight was measured by LC-MS 2010 instrument (Shimadzu, Japan) operating in ESI model. 1H NMR spectra was recorded on an AVANCE III (400/500 MHz) spectrometer in CDCl3 using TMS as internal standard. 2.2. Synthesis of tabersonine derivatives The derivatives of tabersonine were synthesized according to Fig. 1. The substituent groups of R1 are Br or Cl, which could be obtained by reacting tabersonine (1) with N-bromobutanimide (NBS) or N-Chlorosuccinimide (NCS) in trifluoroacetic acid. The substituent groups of R2 are also Br or Cl. However, these groups were obtained by reacting tabersonine (1) with NBS or NCS in dry CH2Cl2 solution using azodiisobutyronitrile (ABIN) as radical initiator. The combined groups of R3, R4 are carbon–carbon double bond (shown as 4) or 14, 15-dibromide (shown as Br2). And the 14, 15-dibromide group could be obtained by reacting tabersonine with NBS in CH2Cl2. The substituent groups of R5 are acetyl or Cl, which could be obtained by reacting tabersonine with acetic anhydride or NCS using Bronsted acid as catalysis. The detailed reaction processes were listed as follows.
2.2.1. Aspidospermidine-16-carboxylic acid, 2,16,14,15-tetradehydro(7R, 20a, 21a)-, methyl ester (1, C21H24N2O2) Tabersonine was isolated from Voacanga seeds according to the procedure reported before (Liu et al., 2014a). Briefly, the powered seeds were extracted with 90% methanol. The extracts were concentrated to remove methanol under reduced pressure. The residue was dissolved in 1.0% HCl solution. After filtering, the acidic solution was purified by amino acid-modified adsorption resins (PS-valine) which was synthesized in our previous work (Liu et al., 2014a). The obtained crude product (yield 2.13%) was recrystallized from methanol to afford tabersonine with purity above 98%, yield 1.75%. 2.2.2. Aspidospermidine-16-carboxylic acid, 1-acetyl-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (2, C23H26N2O3) Tabersonine 1 (1.2 g, 3.567 mmol) was dissolved in 20 mL acetic anhydride, then, 1.1 g p-toluene sulfonic acid was added into the mixture (Luo et al., 2012). After stirring at room temperature for 24 h, 50 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. The separated crystals was filtered and recrystallized from methanol to obtain product (2) 1.15 g as white crystals. Yield: 85%. mp: 141–143 C. 1H NMR (400 MHz, CDCl3): d 7.96 (dd, J = 8.0, 1.2 Hz, 1H, H-12), 7.27–7.21 (m, 2H, H-9, 11), 7.08 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H, H-10), 5.83 (ddd, J = 9.6, 4.8, 1.2 Hz, 1H, H-14), 5.66 (dt, J = 9.6, 2 Hz, 1H, H-15), 3.78 (s, 1H, –OCH3), 3.52 (ddd, J = 15.6, 4.4, 1.2 Hz, 1H, H-3), 3.07 (m, 2H, H3, 5), 2.68 (m, 2H, H-17, 21), 2.57 (d, J = 14.8 Hz, 1H, H-17), 2.50 (ddd, J = 11.6, 8.4, 5.2 Hz, 1H, H-5), 2.26 (s, 3H, COCH3), 2.16 (ddd, J = 12, 12, 6.8 Hz, 1H, H-6), 1.81 (ddd, J = 12, 5.2, 0.8 Hz, 1H, H-6), 1.09–0.90 (m, 2H, H-19), 0.63(t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 379 [M + H]+ (calcd for C23H27N2O3, 379.2022). 2.2.3. Aspidospermidine-16-carboxylic acid, 1-chlorine-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (3, C21H23N2O2Cl) and Aspidospermidine-16-carboxylic acid, 1,10-dichlorine-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (4, C21H22N2O2Cl2) Tabersonine 1 (1.2 g, 3.567 mmol) and NCS (0.5 g, 3.745 mmol) were dissolved in 20 mL trifluoroacetic acid. After stirring at room temperature for 4 h, 20 mL ice water was added and the pH of the
Fig. 1. The reaction scheme for the preparation of tabersonine derivatives.
X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
mixture was adjusted to 8.0 with 5% NaOH solution. Then, the mixture was extracted with CH2Cl2 (2 20 mL). The organic solvent was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel (Qingdao Haiyang Chemical, China) with a mixture of petroleum ether-EtOAc to afford 0.9 g product (3) and 0.11 g product (4) as pale yellow crystals; Compound (3): Yield: 68%. mp: 129–130 C. 1H NMR (400 MHz, CDCl3): d 7.17 (dd, J = 7.6, 2 Hz, 1H, ArH-9), 7.11 (ddd, J = 8, 7.6, 1.2 Hz, 1H, ArH-11), 6.73 (ddd, J = 8, 7.6, 1.2 Hz, 1H, ArH-10), 6.63 (dd, J = 8, 1.2 Hz, 1H, ArH-12), 5.63 (ddd, J = 10, 4.8, 1.2 Hz, 1H, H-14), 5.44 (d, J = 10 Hz, 1H, H-15), 5.29 (s, 1H, H-21), 3.88 (s, 3H, –COOCH3), 3.43–3.35 (m, 2H, H-3), 2.86 (d, J = 14.0 Hz, 1H, H-17), 2.77 (d, J = 14 Hz, 1H, H-17), 2.68 (m, 1H, H-5), 2.44 (m, 1H, H-5), 2.20 (dd, J = 14, 1.6 Hz, 1H, H-6), 2.18 (m, 1H, H-6), 1.68 (dq, J = 14, 7.6 Hz, 1H, H-19), 1.46 (dq, J = 14, 7.6 Hz, 1H, H-19), 0.71 (t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 371 [M + H]+ (calcd for C21H24N2O2Cl, 371.1448). Compound (4): Yield: 7%. mp: 172– 175 C. 1H NMR (400 MHz, CDCl3): d 7.55 (d, J = 2 Hz, 1H, H-9), 7.37 (d, J = 8.4 Hz, 1H, H-12), 7.18 (dd, J = 8.4, 2 Hz, 1H, H-11), 5.47 (ddd, J = 10.4, 4, 2 Hz, 1H, H-14), 5.43 (dt, J = 10.4, 2.4 Hz, 1H, H-15), 3.80 (s, 1H, H-21), 3.49 (s, 3H, –OCH3), 3.39 (m, 1H, H-3), 3.26 (m, 1H, H-5), 3.10-2.90 (m, 3H, H-3, 5, 6), 2.62 (d, J = 14 Hz, 1H, H-17), 2.50 (m, 1H, H-6), 2.04 (d, J = 14 Hz, 1H, H-17), 1.83 (dq, J = 14, 7.2 Hz, 1H, H-19), 1.47 (dq, J = 14, 7.2 Hz, 1H, H-19), 0.95 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 405 [M + H]+ (calcd for C21H23N2O2Cl2, 405.1137). 2.2.4. Aspidospermidine-16-carboxylic acid, 10-bromine-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (5, C21H23N2O2Br) Tabersonine (1.2 g, 3.567 mmol) and NBS (0.7 g, 3.933 mmol) were dissolved in 30 mL acetic acid. After stirring at room temperature for 4 h, 20 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution (Hannart and Alphonse, 1980). The mixture was extracted with CH2Cl2 (2 20 mL). The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum etherEtOAc to afford 1.34 g product (5) as oil. Yield: 91%. 1H NMR (400 MHz, CDCl3): d 9.02 (s, 1H, –NH), 7.37 (d, J = 1.6 Hz, 1H, H-9), 7.25 (dd, J = 6.8, 1.6 Hz, 1H, H-11), 6.7 (d, J = 6.8 Hz, 1H, H-12), 5.81 (ddd, 1H, H-14), 5.74 (m, 1H, H-15), 3.78 (s, 3H, –OCH3), 3.51 (s, 1H, H-21), 3.27 (m, 1H, H-3), 3.11 (m, 1H, H-5), 2.90–2.35 (m, 4H, H-3, 5, 17, 17), 2.12 (m, 1H, H-6), 1.87 (m, 1H, H-6), 0.99 (m, 2H, H-19), 0.67 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 415 [M + H]+ (calcd for C21H24N2O2Br, 415.1021). 2.2.5. Aspidospermidine-16-carboxylic acid, 3-bromine-2,16,14,15tetradehydro-(3a, 7R, 20a, 21a)-, methyl ester (6, C21H23N2O2Br) Tabersonine (1.2 g, 3.567 mmol) was dissolved in dry CH2Cl2 (20 mL). AIBN (0.1 g, 0.6 mmol) and NBS (0.7 g, 3.933 mmol) were added into the mixture. The mixture was refluxed for 5 h under the protection of nitrogen. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure (Luo et al., 2012). The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 1.23 g product (6) as oil. Yield: 81%. 2.2.6. Aspidospermidine-16-carboxylic acid, 14,15-dibromine-2,16didehydro-(7R, 14b, 15a, 20a, 21a)-, methyl ester (7, C21H24N2O2Br2) Tabersonine (1.2 g, 3.567 mmol) and NBS (0.7 g, 3.933 mmol) were dissolved in CH2Cl2 (20 mL). The mixture was refluxed for 5 h. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.34 g product (7) as pale yellow crystals. Yield: 19%. mp: 85–87 C. 1H NMR
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(400 MHz, CDCl3): d 9.0 (s, 1H, –NH), 7.27 (m, 1H, H-9), 7.17 (m, 1H, H-11), 6.91 (m, 1H, H-10), 6.83 (m, 1H, H-12), 6.06 (s, 1H, H-21), 3.78 (s, 3H, –OCH3), 3.53 (m, 2H, H-14, 15), 3.09 (m, 1H, H-3), 2.80 (m, 2H, H-3, 5), 2.60 (d, J = 14 Hz, 1H, H-17), 2.38 (m, 1H, H-5), 2.10 (m, 1H, H-6), 1.91 (d, J = 14 Hz, 1H, H-17), 1.56 (m, 1H, H-6), 0.96 (m, 2H, H-19), 0.67 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 495 [M + H]+ (calcd for C21H25N2O2Br2, 495.0283). 2.2.7. Aspidospermidine-16-carboxylic acid, 1-acetyl-10-bromine2,16,14,15-tetradehydro-(7R, 20a, 21a)-, methyl ester (8, C23H25N2O3Br) Compound 5 (1.0 g, 2.415 mmol) was dissolved in 20 mL acetic anhydride, then, 1.1 g p-toluene sulfonic acid was added into the mixture. After stirring at room temperature for 24 h, 50 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. The mixture was extracted with CH2Cl2 (2 20 mL), and the organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum etherEtOAc to afford 0.9 g product (8) as oil. Yield: 82%. 1H NMR (400 MHz, CDCl3): d 7.85 (d, J = 8.4 Hz, 1H, H-12), 7.34 (dd, J = 8.4, 2 Hz, 1H, H-11), 7.3 (d, J = 2 Hz, H-9), 5.81 (ddd, J = 10, 4.8, 1.6 Hz, 1H, H-14), 5.65 (m, 1H, H-15), 3.76 (s, 3H, –OCH3), 3.50 (ddd, J = 16, 4.4, 0.8 Hz, 1H, H-3), 3.07 (m, 2H, H-3,5), 2.66–2,54 (m, 3H, H-17, H-21), 2.48 (m, 1H, H-5), 2.22(s, 3H, –COCH3), 2.14(m, 1H, H-6), 1.80 (m, 1H, H-6), 1.02 (m, 1H, H-19), 0.92 (m, 1H, H-19), 0.63 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 457 [M + H]+ (calcd for C23H26N2O3Br, 457.1127). 2.2.8. Aspidospermidine-16-carboxylic acid, 2,16-didehydro-10,14,15tribromine-(7R, 14b,15a,20a, 21a)-, methyl ester (9, C21H23N2O2Br3) Compound 5 (3.0 g, 7.245 mmol) and NBS (2.7 g, 15.168 mmol) were dissolved in CH2Cl2 (60 mL). The mixture was refluxed for 5 h. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.87 g product (9) as oil. Yield: 21%. 1H NMR (400 MHz, CDCl3): d 9.00 (s, 1H, –NH), 7.41 (d, J = 2 Hz, 1H, H-9), 7.27 (dd, J = 8.4, 2 Hz, 1H, H-11), 6.71 (d, J = 8.4 Hz, 1H, H-12), 6.07 (s, 1H, H-21), 3.78 (s, 3H, –OCH3), 3.63 (d, J = 1.6 Hz, 1H, H-14), 3.58 (d, J = 1.6 Hz, 1H, H-15), 3.18–2.67 (m, 4H, H-3, 3, 5, 5), 2.63 (d, J = 15.6 Hz, 1H, H-17), 2.37 (d, J = 15.6 Hz, 1H, H17), 2.12 (m, 1H, H-6), 1.95 (m, 1H, H-6), 0.96 (m, 2H, H-19), 0.68 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 573 [M + H]+ (calcd for C21H24N2O2Br3, 572.9388). 2.2.9. Aspidospermidine-16- carboxylic acid, 1-acetyl-2, 16didehydro-10,14,15-tribromine-(7R, 14b,15a,20a, 21a)-, methyl ester (10,C23H25N2O3Br3) Compound 9 (0.40 g, 0.699 mmol) was dissolved in 10 mL acetic anhydride, then, 0.3 g p-toluene sulfonic acid was added into the mixture. After stirring at room temperature for 24 h, 15 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. The mixture was extracted with CH2Cl2 (2 10 mL), and the organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum etherEtOAc to afford 0.37 g product (10). Yield: 86%. mp: 167–169 C. 1H NMR (400 MHz, CDCl3): d 7.87 (d, J = 8.4 Hz, 1H, H-12), 7.38 (dd, J = 8.4, 2 Hz, 1H, H-11), 7.32 (d, J = 2 Hz, 1H, H-9), 6.04 (s, 1H, H-21), 3.79 (s, 3H, –OCH3), 3.75 (m, 1H, H-14), 3.38 (d, J = 1.2 Hz, 1H, H-15), 3.12 (m, 1H, H-5), 2.80-2.49 (m, 4H, H-3, 5, 17, 17), 2.24 (s, 3H, –COCH3), 1.87 (dd, J = 14, 4.4 Hz, 1H, H-3), 1.72 (m, 2H, H-6), 1.04 (m, 2H, H-19), 0.67 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 615 [M + H]+ (calcd for C23H26N2O3Br3, 614.9494).
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2.2.10. Aspidospermidine-16-carboxylic acid, 10- bromine-1-chlorine2, 16, 14, 15-tetradehydro-(7R, 20a, 21a)-, methyl ester (11, C21H22N2O2BrCl) Compound 5 (1.0 g, 2.415 mmol) and NCS (0.35 g, 2.62 mmol) were dissolved in 20 mL trifluoroacetic acid. After stirring at room temperature for 4 h, 20 mL ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. Then, the mixture was extracted with CH2Cl2 (2 20 mL). The organic solvent was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum ether-EtOAc to afford 0.53 g of product (11) as oil. Yield: 49%. 1H NMR (400 MHz, CDCl3): d 7.24 (d, J = 2 Hz, 1H, H-9), 7.17 (dd, J = 8.8, 2 Hz, 1H, H-11), 6.5 (d, J = 8.8 Hz, 1H, H-12), 5.61 (ddd, J = 9.6, 4.4, 1.2 Hz, 1H, H-14), 5.43 (d, J = 9.6 Hz, 1H, H-15), 5.32 (s, 1H, H-21), 3.87 (s, 3H, –OCH3), 3.35 (m, 2H, H-3, 5), 2.83 (d, J = 14 Hz, 1H, H-17), 2.67 (m, 3H, H-3, 5, 6), 2.18 (d, J = 14 Hz, 1H, H-17), 2.08 (m, 1H, H-6), 1.64 (dq, J = 14, 7.6 Hz, 1H, H19), 1.47 (dq, J = 14, 7.6 Hz, 1H, H-19), 0.73 (t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 449 [M + H]+ (calcd for C21H23N2O2BrCl, 449.0631). 2.2.11. Aspidospermidine-16-carboxylic acid, 3-bromine-1,10dichlorine-2,16,14,15-tetradehydro-(7R, 20a, 21a)-, methyl ester (12, C21H21N2O2Cl2Br) Compound 4 (0.2 g, 0.495 mmol) was dissolved in dry CH2Cl2 (10 mL). AIBN (0.01 g, 0.061 mmol) and NBS (0.12 g, 0.67 mmol) were added into the mixture. The mixture was refluxed for 5 h under the protection of nitrogen. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.11 g product (12) as oil. Yield: 46%. 1H NMR (400 MHz, CDCl3): d 7.57–7.46 (m, 3H, H-9, 11, 12), 5.78 (dd, J = 9.2, 3.6 Hz, 1H, H-14), 5.43 (d, J = 9.2 Hz, 1H, H-15), 3.98 (s, 1H, –OCH3), 3.63 (d, J = 3.6 Hz,1H, H-3), 3.44 (m, 1H, H-5), 3.34–3.23 (m, 2H, H-21, 5), 3.06 (d, J = 14.4 Hz,1H, H-17), 2.97(d, J = 14.4 Hz,1H, H-17), 2.85–2.73 (m, 2H, H-6, 6), 0.79–0.71 (m, 2H, H-19), 0.51 (t, J = 7.6 Hz, 3H, H18). ESIMS m/z: 483 [M + H]+ (calcd for C21H22N2O2Cl2Br, 483.0242). 2.2.12. Aspidospermidine-16-carboxylic acid, 14,15-dibromine-1,10dichlorine-2,16-didehydro-(7R, 14b, 15a, 20a, 21a)-, methyl ester (13, C21H22N2O2Br2Cl2) Compound 4 (0.2 g, 0.495 mmol) and NBS (0.2 g, 1.123 mmol) were dissolved in CH2Cl2 (10 mL). The mixture was refluxed for 5 h. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.09 g product (13) as pale yellow crystals. Yield: 32%. mp: 139–141 C. 1H NMR (400 MHz, CDCl3): d 7.67 (d, J = 1.2 Hz, 1H, H-9), 7.44 (dd, J = 8.4, 1.2 Hz, 1H, H-11), 7.10 (d, J = 8.4 Hz, 1H, H-12), 3.87 (s, 1H, H-21), 3.51
(s, 3H, –OCH3), 3.49 (m, 1H, H-14), 3.11 (m, 2H, H-3, 15), 2.66 (d, J = 14 Hz, 1H, H-17), 2.42–2.04 (m, 6H, H-3, 5, 5, 6, 6, 17), 1.59 (m, 2H, H-19), 0.96 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 563 [M + H]+ (calcd for C21H23N2O2Br2Cl2, 562.9503). 2.2.13. Aspidospermidine-16-carboxylic acid, 1,3- dichlorine2,16,14,15-tetradehydro-(7R, 20a, 21a)-, methyl ester (14, C21H22N2O2Cl2) Compound 3 (0.5 g, 1.351 mmol) was dissolved in dry CH2Cl2 (15 mL). AIBN (0.1 g, 0.609 mmol) and NCS (0.25 g, 1.872 mmol) were added into the mixture. The mixture was refluxed for 5 h under the protection of nitrogen. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.31 g product (14) as oil. Yield: 57%. 1H NMR (500 MHz, CDCl3): d 7.53 (dd, J = 7.5, 1 Hz, 1H, H-9), 7.34 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H, H-11), 7.24 (m, 2H, H-10, 12), 5.62 (d, J = 11 Hz, 1H, H-15), 5.58 (dd, J = 11, 3.5 Hz, 1H, H-14), 3.90 (s, 1H, H-21), 3.48 (s, 1H, –OCH3), 3.10 (d, J = 3.5 Hz, 1H, H-3), 2.66 (d, J = 12.5 Hz, 1H, H-17), 2.35 (m, 1H, H-5), 2.28 (d, J = 12.5 Hz, 1H, H-17), 2.20–2.10 (m, 3H, H-5, 6, 6), 1.62 (m, 2H, H-19), 0.98 (t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 405 [M + H]+ (calcd for C21H23N2O2Cl2, 405.1137). 2.3. AChE inhibitory assays The assay for measuring AChE activity was modified from the Ellman’s method (Ellman et al., 1961). Galanthamine hydrobromide was used as a positive control. In brief, 200 mL potassium phosphate buffer (PBS, pH 8.0), 300 mL DTNB (3 mmol mL1 in PBS), 100 mL enzyme (0.24 U mL1 in PBS) and 100 mL sample solution were mixed and pre-incubated for 20 min at 37 C, then 300 mL substrate (ATCI, 3 mmol mL1 in PBS) were added. The change in absorbance was measured at 412 nm after 15 min. The IC50 values were determined graphically from inhibition curves. The IC50 values were determined in triplicate and the values are presented as mean standard deviation. 3. Results and discussion 3.1. Synthesis of tabersonine derivatives The derivatives of tabersonine were synthesized according to Fig. 1. The H-10 in the structure of tabersonine was activated by indole ring. Thus, it could be substituted by Br or Cl through electrophilic substitution reaction. The H-3 in the structure of tabersonine was activated by the carbon–carbon double bond C14,15. Thus, H-3 could be substituted by Br or Cl through free radical reaction. The double bond C-14,15 in the structure of tabersonine was unsaturated bond, which could react with NBS by
Fig. 2. The stereostructure of 14,15- dibromine and 3-Br in tabersonine derivatives.
X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
addition reaction. The N-H in indole ring was a active site in the structure of tabersonine, which could react with anhydride or NCS to produce amide or N-Cl derivatives. By combining the above reactions, there were 13 compounds synthesized using tabersonine as a lead compound. Among them, compounds (3), (4), (7)–(14) were novel tabersonine derivatives. The chemical structures of tabersonine derivatives were determined by 1H NMR and MS. In general, the d value at 9.0 was the chemical shift of N–H in indole ring. The values of d ranged from 7.9 to 6.5 were the chemical shifts of hydrogen atoms in the benzene ring. The coupling constants between them were adopted to identify their relative positions (ortho, meta or para). The values of d ranged from 5.9 to 5.4 were the chemical shifts of hydrogen atoms on carbon–carbon double bond C-14,15. The H-14 was coupled by H-15, H-3ax and H-3eq. Thus, the peak shape of H-14 was octet (ddd). The H-15 was coupled by H-14 and H-3. Thus, the peak shape of H-15 was sextet (dt). The d around 3.8 was the chemical shifts of –COOCH3, appearing as singlet. The values of d ranged from 3.6 to 1.8 were the chemical shifts of H-3,4,5,6,17. The peak shapes and coupling constants were employed to identify them. For instance, the peak shape of H-17 was double peaks (d), with the coupling constant Jgem was about 14 Hz. The d around 1.0 was the chemical shifts of H-19, appearing as octet (dq, Jgem = 14 Hz, J18,19 = 7 Hz). The d around 0.6 was the chemical shifts of H-18, appearing as triplet (t). The stereoposition of the 14,15-dibromine group was verified by the JH,H coupling constant between H-14 and H-15. The values of J14,15 were about 1.2 Hz, which indicated that the hydrogen atoms on positions 14 and 15 were both equatorial bonds (Jax,ax range from 8 to 12 Hz, Fig. 2a). The stereostructure of 14,15-dibromine in tabersonine derivatives was shown in Fig.2b. The stereoposition of the 3-Br was verified by the JH,H coupling constant between H-3 and H-14. As shown in Fig. 2c, the dihedral between H-14 and H-3eq, H-14 and H-3ax were about 38 and 77 degrees, respectively. And the coupling constants of J14,3eq and J14,3ax were about 4.4 Hz and 1.2 Hz, respectively. After substitution of H-3 by bromine, the coupling constant of J14,3 was about 3.6 Hz. This result suggested that the hydrogen atom on position C-3 was equatorial bond (Fig. 2d). 3.2. AChE inhibitory assays The AChE inhibitory activities of tabersonine derivatives were tabulated in Table 1. The lead compound tabersonine (1) showed a certain activity with the value of IC50 was 63.47 mM. Among tabersonine derivatives, compounds (7) and (9) exhibited pronounced AChE inhibitory activities in comparison to the positive control galanthamine hydrobromide (1.79 mM). Derivatives (7) and (9) could be used as potential AChE inhibitors in the following
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investigational study with IC50 values were 5.32 mM and 6.93 mM, respectively. Besides that some derivatives exhibited considerable activities, the structure-activity relationships between tabersonine derivatives and their AChE inhibitory activities were analyzed. Compounds (2) and (3) were the derivatives of tabersonine with substituent groups of R5 were acetyl or Cl. And their IC50 values were both above 100 mM. This result indicated that the introduction of groups R5 would decrease the AChE inhibitory activity of tabersonine derivatives. For the same reason, the IC50 values of compounds (4), (8), (10), (11), (12) and (14) were all above 100 mM. Compound (5) was the tabersonine derivatives with group R1 was Br and its value of IC50 was 11.48 mM. This result revealed that the introduction of group R1 increased the AChE inhibitory activity of tabersonine derivatives. The IC50 value for compound (6) was 64.12 mM, which has no significant difference with the IC50 value of the lead compound tabersonine (1). This result indicated that the introduction of group R2 showed no significant influence on the AChE inhibitory activity of tabersonine derivatives. Compound (7) showed the highest AChE inhibitory activity with the IC50 value was 5.32 mM. The introduction of 14, 15-dibromide increased the activities of tabersonine derivatives against AChE. Among tabersonine derivatives (1)–(14), compounds (7) and (9) exhibited pronounced AChE inhibitory activities in comparison to the positive control galanthamine hydrobromide (1.79 mM). The AChE inhibitory ratios of tabersonine derivatives at concentration of 100 mM were shown in Figure 3. At this concentration, the influences of substituent groups on the AChE inhibitory activity of tabersonine derivatives are similar to the results discussed above. Overall, the results of Table 1 and Figure 3 revealed that the introduction of group R1 and/or combined groups R3, R4 could increase the AChE inhibitory activity of tabersonine derivatives. Thus, compounds (5), (7) and (9) showed considerable AChE inhibitory activities with IC50 values were 11.48 mM, 5.32 mM and 6.93 mM, respectively. The introduction of group R5 decreased the AChE inhibitory activities of tabersonine derivatives. Thus, the IC50 values of compounds (2), (3), (4), (8), (10), (11), (12) and (14) were all above 100 mM. However, the IC50 value of compound (13) was 78.26 mM. This is probably because the group R1 and combined groups R3, R4 in compound (13) increased its AChE inhibitory activity. The group R2 showed no significant influence on the AChE inhibitory activity of tabersonine derivatives. Thus, the IC50 values between tabersonine (1) and compound (6) have no significant difference. The above results indicated that the AChE inhibitory
Table 1 The IC50 values of tabersonine derivatives. Compounds
R1
R2
H H 1 2 H H 3 H H 4 –Cl H 5 –Br H 6 H –Br 7 H H 8 –Br H 9 –Br H 10 –Br H 11 –Br H 12 –Cl -Br 13 –Cl H 14 H –Cl Galanthamine hydrobromide
R3, R4
R5
Yield (%)
IC50 SD (mM)
4 4 4 4 4 4
H –COCH3 –Cl –Cl H H H –COCH3 H –COCH3 –Cl –Cl –Cl –Cl
1.95 85 68 7 91 81 19 82 21 86 49 46 32 57
63.47 4.12 >100 >100 >100 11.48 1.32 64.12 5.21 5.32 1.78 >100 6.93 2.34 >100 >100 >100 78.26 3.23 >100 1.79 0.23
Br2
4
Br2 Br2
4 4
Br2
4
Fig. 3. The AChE inhibitory ratios of tabersonine derivatives at the concentration of 100 mM.
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activity of tabersonine derivatives could be improved by structural modification. All these conclusions could be used as a structureactivity relationship to semi-synthesize more tabersonine derivatives with high AChE inhibitory activities in further investigations. 4. Conclusion In summary, there were 13 compounds, containing 10 novel tabersonine derivatives, synthesized using tabersonine as a lead compound. The AChE inhibitory activities of tabersonine and its derivatives were also tested. The AChE inhibitory activities of tabersonine derivatives increased with the introduction of group R1 and/or combined groups R3, R4, while decreased with the introduction of group R5. And the group R2 showed no significant influence on the AChE inhibitory activities of tabersonine derivatives. All these results could be used as a structure-activity relationship to semi-synthesize more tabersonine derivatives with high AChE inhibitory activities in further investigations. References Ellman, G.L., Courtney, K.D., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. pharmacol. 7, 88–95. González-Domínguez, R., García-Barrera, T., Gómez-Ariza, J.L., 2012. Metabolomic approach to Alzheimer’s disease diagnosis based on mass spectrometry. Chem. Pap.66 829–835. Howes, M.J.R., Houghton, P.J., 2003. Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function. Pharmacol. Biochem. Behav. 75, 513–527. Iannello, C., Pigni, N.B., Antognoni, F., Poli, F., Maxia, A., de Andrade, J.P., Bastida, J., 2014. A potent acetylcholinesterase inhibitor from Pancratium illyricum L. Fitoterapia 92, 163–167. Lai, D.H., Yang, Z.D., Xue, W.W., Sheng, J., Shi, Y., Yao, X.J., 2013. Isolation, characterization and acetylcholinesterase inhibitory activity of alkaloids from roots of Stemona sessilifolia. Fitoterapia 89, 257–264. Liu, X., Yang, D.L., Liu, J.J., Zhou, J., Zhao, N.N., 2014a. Synthesis of new eburnaminetype alkaloid via direct hydroalkoxylation. Chem. Pap. 68, 816–822. Liu, X., Yang, D.L., Liu, J.J., Ren, N., 2014b. Synthesis and characterization of amino acid-modified adsorption resins and their adsorption properties in the
purification of tabersonine from Voacanga africana seeds. RSC Adv. 4, 64336– 64346. Liu, X., Yang, D.L., Liu, J.J., Ren, N., 2015. Analysis of essential oils from Voacanga africana seeds at different hydrodistillation extraction stages: Chemical composition, Antioxidant activity and Antimicrobial activity. Nat. Prod. Res. 29, 1950–1953. Luo, X.D., Cai, X.H., Liu, Y.P., Li, Y., Xia, C.F., Shi, H.K., 2012. China Pat., CN 102558178 A. Marnewick, J.L., 2009. African natural plant products: new discoveries and challenges in chemistry and quality. ACS Symposium Series. Chapter 20, Voacanga africana: Chemistry, Quality and Pharmacological Activity. American Chemical Society, Washington, DC, pp. 363–380. Manske, R.H.F., 1965. The Alkaloids: Chemistry and Physiology. The Indole Alkaloids. Academic Press, Inc., New York. Maurice, M.I., 1993. Handbook of African Medicinal Plants. CRC Press, Inc., Florida. Nemes, A., Szantay Jr., C., Czibula, L., Greiner, I., 2007. Synthesis of 18hydroxyvincamines and epoxy-1,14-secovincamines; a new proof for the aspidospermane-eburnane rearrangement. Heterocycles 71, 2347–2362. Pendota, S.C., Aderogba, M.A., Ndhlala, A.R., 2013. Antimicrobial and acetylcholinesterase inhibitory activities of Buddleja salviifolia (L.) Lam. leaf extracts and isolated compounds. J. Ethnopharmacol. 148 (2), 515–520. Qin, Y.H., Zhang, J., Cui, J.T., Guo, Z.K., Jiang, N., Tan, R.X., Ge, H.M., 2011. Oligostilbenes from Vatica mangachapoi with xanthine oxidase and acetylcholinesterase inhibitory activities. RSC Adv. 1, 135–141. Rolland, Y., Kunesch, N., Poisson, J., 1976. Carbon-13 nuclear magnetic resonance spectroscopy of naturally occurring substances. 43. Carbon-13 nuclear magnetic resonance analysis of bis-indoline alkaloids of two Voacanga species. J. Org. Chem. 41, 3270–3275. da Silva, J.K.R., Pinto, L.C., Burbano, R.M.R., Montenegro, R.C., Guimarães, E.F., Andrade, E.H.A., Maia, J.G.S., 2014. Essential oils of Amazon Piper species and their cytotoxic, antifungal, antioxidant and anti-cholinesterase activities. Ind. Crop. Prod. 58, 55–60. Vas, A., Gulyas, B., 2005. Eburnamine derivatives and the brain. Med. Res. Rev. 25, 737–757. Vats, C., Dhanjal, J.K., Goyal, S., Bharadvaja, N., Grover, A., 2015. Computational design of novel flavonoid analogues as potential AChE inhibitors: analysis using group-based QSAR, molecular docking and molecular dynamics simulations. Struct. Chem. 26, 467–476. Yang, Z.D., Zhang, X., Du, J., Ma, Z.J., Guo, F., Li, S., Yao, X.J., 2012. An aporphine alkaloid from Nelumbo nucifera as an acetylcholinesterase inhibitor and the primary investigation for structure–activity correlations. Nat. Prod. Res. 26, 387–392. Zhan, Z.J., Yu, Q., Wang, Z.L., Shan, W.G., 2010. Indole alkaloids from Ervatamia hainanensis with potent acetylcholinesterase inhibition activities. Bioorg. Med. Chem. Lett. 20, 6185–6187.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Synthesis and acetylcholinesterase inhibitory activities of tabersonine derivatives Xiong Liua,b , Dongliang Yanga , Jiajia Liua,* , Na Rena,c a
Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, PR China Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan 411201, Hunan, PR China c Hunan Vocational College of Science and Technology, Changsha 410004, Hunan, PR China b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2015 Received in revised form 21 August 2015 Accepted 24 August 2015 Available online xxx
Tabersonine, the main alkaloid in Voacanga seeds, was used as a lead compound to semi-synthesize tabersonine derivatives. In total, 13 compounds, containing 10 novel tabersonine derivatives, were synthesized by introducing substituent groups R1–R5. The acetylcholinesterase (AChE) inhibitory activities of tabersnonine derivatives were evaluated using Ellman’s method. Among them, compound (7) showed the highest AChE inhibitory activity with the IC50 value was 5.32 mM. The substituent groups R1–R5 showed different influences on the AChE inhibitory activities of tabersonine derivatives. The AChE inhibitory activities of tabersonine derivatives increased with the introduction of group R1 and/or combined groups R3,R4, while decreased with the introduction of group R5. And the group R2 showed no significant influence on the AChE inhibitory activities of tabersonine derivatives. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Voacanga seeds Tabersonine derivatives AChE inhibitory activities
1. Introduction Alzheimer’s disease (AD) is the most common neurodegenerative disease (González-Domínguez et al., 2012). The cholinergic hypothesis is the most widely accepted biochemical theory of this disease. It is believed that the decline in cognitive and mental functions associated with AD is related to the loss of cortical cholinergic neurotransmission (Lai et al., 2013). AChE plays a significant role in hydrolysis of acetylcholine (ACh) (Yang et al., 2012). The use of AChE inhibitors is an effective clinical approach to maintain the levels of ACh and enhance cholinergic function. The inhibition of the AChE has become the standard approach in the symptomatic treatment of AD (Howes and Houghton, 2003; Pendota et al., 2013). Recently, some secondary metabolites from natural plants such as essential oils, flavonoids and alkaloids have been proven to possess AChE inhibitory activities (Iannello et al., 2014; Qin et al., 2011; da Silva et al., 2014; Vats et al., 2015). These findings suggest that natural plants were good resources for seeking compounds with high AChE inhibitory activities. Voacanga Africana is an evergreen tree, belonging to the family Apocynaceae. Its seeds are
* Corresponding author. E-mail address: [email protected] (J. Liu).
traditionally used against leprosy, diarrhea, generalized edema, convulsions in children, mental disorders and diuretic (Marnewick, 2009). Voacangine, an alkaloid in Voacanga seeds, has been proved to possess considerable AChE inhibitory activity (Zhan et al., 2010). Based on this result, we attempted to seek components with high AChE inhibitory activities from Voacanga seeds. In our previous work, we have obtained some secondary metabolites (tabersonine, essential oils, crude extracts) from Voacanga seeds and semi-synthesized new eburnamine derivative using tabersonine as a lead compound. (Liu et al., 2014a,b; Liu et al., 2015). Tabersonine is the main alkaloid in Voacanga seeds (Manske, 1965; Maurice, 1993; Rolland et al., 1976), which plays a significant role in lowering blood pressure and is used to semi-synthesize the cerebrally active eburnamine derivatives (Liu et al., 2014b; Nemes et al., 2007; Vas and Gulyas, 2005). In the biological evaluation, we found that tabersonine exhibited a certain AChE inhibitory activity while eburnamine derivatives showed no activities. This result revealed that structural modification of tabersonine is a promising approach to obtain compounds with high AChE inhibitory activities. However, to date, researches about synthesis and evaluation of AChE inhibitory activities of tabersonine derivatives have rarely been carried out. Thus, in the current investigational study, several derivatives of tabersonine were synthesized using tabersonine as lead compound and their AChE inhibitory activities were also tested.
http://dx.doi.org/10.1016/j.phytol.2015.08.015 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
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X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
2. Experimental 2.1. Chemical reagents and samples The reagents and chemicals (analytic grade unless stated otherwise) were purchased from Sinopharm Chemical Reagent Co., Ltd. and Tianjin Damao Chemical Reagent Co., Ltd. (China). Acetylthiocholine iodide (ATCI), acetylcholinesterase (AChE) and 5,50 -dithiobis [2-nitrobenzoic acid] (DTNB) purchased from J & K Chemical Technology. The Voacanga seeds were collected from Ghana, in 2012, and the sample was authenticated by professor Liu at the Central South University. A voucher specimen (CSPHA120923) has been deposited in College of Chemistry and Chemical Engineering of Central South University. The Voacanga seeds were grounded by a universal grinder. The grounded material was stored at –10 C in a domestic refrigerator before the extraction procedures were performed. Melting points were determined with a Hotstage microscope Boetius apparatus and are uncorrected. The molecular weight was measured by LC-MS 2010 instrument (Shimadzu, Japan) operating in ESI model. 1H NMR spectra was recorded on an AVANCE III (400/500 MHz) spectrometer in CDCl3 using TMS as internal standard. 2.2. Synthesis of tabersonine derivatives The derivatives of tabersonine were synthesized according to Fig. 1. The substituent groups of R1 are Br or Cl, which could be obtained by reacting tabersonine (1) with N-bromobutanimide (NBS) or N-Chlorosuccinimide (NCS) in trifluoroacetic acid. The substituent groups of R2 are also Br or Cl. However, these groups were obtained by reacting tabersonine (1) with NBS or NCS in dry CH2Cl2 solution using azodiisobutyronitrile (ABIN) as radical initiator. The combined groups of R3, R4 are carbon–carbon double bond (shown as 4) or 14, 15-dibromide (shown as Br2). And the 14, 15-dibromide group could be obtained by reacting tabersonine with NBS in CH2Cl2. The substituent groups of R5 are acetyl or Cl, which could be obtained by reacting tabersonine with acetic anhydride or NCS using Bronsted acid as catalysis. The detailed reaction processes were listed as follows.
2.2.1. Aspidospermidine-16-carboxylic acid, 2,16,14,15-tetradehydro(7R, 20a, 21a)-, methyl ester (1, C21H24N2O2) Tabersonine was isolated from Voacanga seeds according to the procedure reported before (Liu et al., 2014a). Briefly, the powered seeds were extracted with 90% methanol. The extracts were concentrated to remove methanol under reduced pressure. The residue was dissolved in 1.0% HCl solution. After filtering, the acidic solution was purified by amino acid-modified adsorption resins (PS-valine) which was synthesized in our previous work (Liu et al., 2014a). The obtained crude product (yield 2.13%) was recrystallized from methanol to afford tabersonine with purity above 98%, yield 1.75%. 2.2.2. Aspidospermidine-16-carboxylic acid, 1-acetyl-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (2, C23H26N2O3) Tabersonine 1 (1.2 g, 3.567 mmol) was dissolved in 20 mL acetic anhydride, then, 1.1 g p-toluene sulfonic acid was added into the mixture (Luo et al., 2012). After stirring at room temperature for 24 h, 50 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. The separated crystals was filtered and recrystallized from methanol to obtain product (2) 1.15 g as white crystals. Yield: 85%. mp: 141–143 C. 1H NMR (400 MHz, CDCl3): d 7.96 (dd, J = 8.0, 1.2 Hz, 1H, H-12), 7.27–7.21 (m, 2H, H-9, 11), 7.08 (ddd, J = 8.0, 8.0, 1.2 Hz, 1H, H-10), 5.83 (ddd, J = 9.6, 4.8, 1.2 Hz, 1H, H-14), 5.66 (dt, J = 9.6, 2 Hz, 1H, H-15), 3.78 (s, 1H, –OCH3), 3.52 (ddd, J = 15.6, 4.4, 1.2 Hz, 1H, H-3), 3.07 (m, 2H, H3, 5), 2.68 (m, 2H, H-17, 21), 2.57 (d, J = 14.8 Hz, 1H, H-17), 2.50 (ddd, J = 11.6, 8.4, 5.2 Hz, 1H, H-5), 2.26 (s, 3H, COCH3), 2.16 (ddd, J = 12, 12, 6.8 Hz, 1H, H-6), 1.81 (ddd, J = 12, 5.2, 0.8 Hz, 1H, H-6), 1.09–0.90 (m, 2H, H-19), 0.63(t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 379 [M + H]+ (calcd for C23H27N2O3, 379.2022). 2.2.3. Aspidospermidine-16-carboxylic acid, 1-chlorine-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (3, C21H23N2O2Cl) and Aspidospermidine-16-carboxylic acid, 1,10-dichlorine-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (4, C21H22N2O2Cl2) Tabersonine 1 (1.2 g, 3.567 mmol) and NCS (0.5 g, 3.745 mmol) were dissolved in 20 mL trifluoroacetic acid. After stirring at room temperature for 4 h, 20 mL ice water was added and the pH of the
Fig. 1. The reaction scheme for the preparation of tabersonine derivatives.
X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
mixture was adjusted to 8.0 with 5% NaOH solution. Then, the mixture was extracted with CH2Cl2 (2 20 mL). The organic solvent was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel (Qingdao Haiyang Chemical, China) with a mixture of petroleum ether-EtOAc to afford 0.9 g product (3) and 0.11 g product (4) as pale yellow crystals; Compound (3): Yield: 68%. mp: 129–130 C. 1H NMR (400 MHz, CDCl3): d 7.17 (dd, J = 7.6, 2 Hz, 1H, ArH-9), 7.11 (ddd, J = 8, 7.6, 1.2 Hz, 1H, ArH-11), 6.73 (ddd, J = 8, 7.6, 1.2 Hz, 1H, ArH-10), 6.63 (dd, J = 8, 1.2 Hz, 1H, ArH-12), 5.63 (ddd, J = 10, 4.8, 1.2 Hz, 1H, H-14), 5.44 (d, J = 10 Hz, 1H, H-15), 5.29 (s, 1H, H-21), 3.88 (s, 3H, –COOCH3), 3.43–3.35 (m, 2H, H-3), 2.86 (d, J = 14.0 Hz, 1H, H-17), 2.77 (d, J = 14 Hz, 1H, H-17), 2.68 (m, 1H, H-5), 2.44 (m, 1H, H-5), 2.20 (dd, J = 14, 1.6 Hz, 1H, H-6), 2.18 (m, 1H, H-6), 1.68 (dq, J = 14, 7.6 Hz, 1H, H-19), 1.46 (dq, J = 14, 7.6 Hz, 1H, H-19), 0.71 (t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 371 [M + H]+ (calcd for C21H24N2O2Cl, 371.1448). Compound (4): Yield: 7%. mp: 172– 175 C. 1H NMR (400 MHz, CDCl3): d 7.55 (d, J = 2 Hz, 1H, H-9), 7.37 (d, J = 8.4 Hz, 1H, H-12), 7.18 (dd, J = 8.4, 2 Hz, 1H, H-11), 5.47 (ddd, J = 10.4, 4, 2 Hz, 1H, H-14), 5.43 (dt, J = 10.4, 2.4 Hz, 1H, H-15), 3.80 (s, 1H, H-21), 3.49 (s, 3H, –OCH3), 3.39 (m, 1H, H-3), 3.26 (m, 1H, H-5), 3.10-2.90 (m, 3H, H-3, 5, 6), 2.62 (d, J = 14 Hz, 1H, H-17), 2.50 (m, 1H, H-6), 2.04 (d, J = 14 Hz, 1H, H-17), 1.83 (dq, J = 14, 7.2 Hz, 1H, H-19), 1.47 (dq, J = 14, 7.2 Hz, 1H, H-19), 0.95 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 405 [M + H]+ (calcd for C21H23N2O2Cl2, 405.1137). 2.2.4. Aspidospermidine-16-carboxylic acid, 10-bromine-2,16,14,15tetradehydro-(7R, 20a, 21a)-, methyl ester (5, C21H23N2O2Br) Tabersonine (1.2 g, 3.567 mmol) and NBS (0.7 g, 3.933 mmol) were dissolved in 30 mL acetic acid. After stirring at room temperature for 4 h, 20 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution (Hannart and Alphonse, 1980). The mixture was extracted with CH2Cl2 (2 20 mL). The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum etherEtOAc to afford 1.34 g product (5) as oil. Yield: 91%. 1H NMR (400 MHz, CDCl3): d 9.02 (s, 1H, –NH), 7.37 (d, J = 1.6 Hz, 1H, H-9), 7.25 (dd, J = 6.8, 1.6 Hz, 1H, H-11), 6.7 (d, J = 6.8 Hz, 1H, H-12), 5.81 (ddd, 1H, H-14), 5.74 (m, 1H, H-15), 3.78 (s, 3H, –OCH3), 3.51 (s, 1H, H-21), 3.27 (m, 1H, H-3), 3.11 (m, 1H, H-5), 2.90–2.35 (m, 4H, H-3, 5, 17, 17), 2.12 (m, 1H, H-6), 1.87 (m, 1H, H-6), 0.99 (m, 2H, H-19), 0.67 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 415 [M + H]+ (calcd for C21H24N2O2Br, 415.1021). 2.2.5. Aspidospermidine-16-carboxylic acid, 3-bromine-2,16,14,15tetradehydro-(3a, 7R, 20a, 21a)-, methyl ester (6, C21H23N2O2Br) Tabersonine (1.2 g, 3.567 mmol) was dissolved in dry CH2Cl2 (20 mL). AIBN (0.1 g, 0.6 mmol) and NBS (0.7 g, 3.933 mmol) were added into the mixture. The mixture was refluxed for 5 h under the protection of nitrogen. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure (Luo et al., 2012). The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 1.23 g product (6) as oil. Yield: 81%. 2.2.6. Aspidospermidine-16-carboxylic acid, 14,15-dibromine-2,16didehydro-(7R, 14b, 15a, 20a, 21a)-, methyl ester (7, C21H24N2O2Br2) Tabersonine (1.2 g, 3.567 mmol) and NBS (0.7 g, 3.933 mmol) were dissolved in CH2Cl2 (20 mL). The mixture was refluxed for 5 h. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.34 g product (7) as pale yellow crystals. Yield: 19%. mp: 85–87 C. 1H NMR
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(400 MHz, CDCl3): d 9.0 (s, 1H, –NH), 7.27 (m, 1H, H-9), 7.17 (m, 1H, H-11), 6.91 (m, 1H, H-10), 6.83 (m, 1H, H-12), 6.06 (s, 1H, H-21), 3.78 (s, 3H, –OCH3), 3.53 (m, 2H, H-14, 15), 3.09 (m, 1H, H-3), 2.80 (m, 2H, H-3, 5), 2.60 (d, J = 14 Hz, 1H, H-17), 2.38 (m, 1H, H-5), 2.10 (m, 1H, H-6), 1.91 (d, J = 14 Hz, 1H, H-17), 1.56 (m, 1H, H-6), 0.96 (m, 2H, H-19), 0.67 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 495 [M + H]+ (calcd for C21H25N2O2Br2, 495.0283). 2.2.7. Aspidospermidine-16-carboxylic acid, 1-acetyl-10-bromine2,16,14,15-tetradehydro-(7R, 20a, 21a)-, methyl ester (8, C23H25N2O3Br) Compound 5 (1.0 g, 2.415 mmol) was dissolved in 20 mL acetic anhydride, then, 1.1 g p-toluene sulfonic acid was added into the mixture. After stirring at room temperature for 24 h, 50 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. The mixture was extracted with CH2Cl2 (2 20 mL), and the organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum etherEtOAc to afford 0.9 g product (8) as oil. Yield: 82%. 1H NMR (400 MHz, CDCl3): d 7.85 (d, J = 8.4 Hz, 1H, H-12), 7.34 (dd, J = 8.4, 2 Hz, 1H, H-11), 7.3 (d, J = 2 Hz, H-9), 5.81 (ddd, J = 10, 4.8, 1.6 Hz, 1H, H-14), 5.65 (m, 1H, H-15), 3.76 (s, 3H, –OCH3), 3.50 (ddd, J = 16, 4.4, 0.8 Hz, 1H, H-3), 3.07 (m, 2H, H-3,5), 2.66–2,54 (m, 3H, H-17, H-21), 2.48 (m, 1H, H-5), 2.22(s, 3H, –COCH3), 2.14(m, 1H, H-6), 1.80 (m, 1H, H-6), 1.02 (m, 1H, H-19), 0.92 (m, 1H, H-19), 0.63 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 457 [M + H]+ (calcd for C23H26N2O3Br, 457.1127). 2.2.8. Aspidospermidine-16-carboxylic acid, 2,16-didehydro-10,14,15tribromine-(7R, 14b,15a,20a, 21a)-, methyl ester (9, C21H23N2O2Br3) Compound 5 (3.0 g, 7.245 mmol) and NBS (2.7 g, 15.168 mmol) were dissolved in CH2Cl2 (60 mL). The mixture was refluxed for 5 h. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.87 g product (9) as oil. Yield: 21%. 1H NMR (400 MHz, CDCl3): d 9.00 (s, 1H, –NH), 7.41 (d, J = 2 Hz, 1H, H-9), 7.27 (dd, J = 8.4, 2 Hz, 1H, H-11), 6.71 (d, J = 8.4 Hz, 1H, H-12), 6.07 (s, 1H, H-21), 3.78 (s, 3H, –OCH3), 3.63 (d, J = 1.6 Hz, 1H, H-14), 3.58 (d, J = 1.6 Hz, 1H, H-15), 3.18–2.67 (m, 4H, H-3, 3, 5, 5), 2.63 (d, J = 15.6 Hz, 1H, H-17), 2.37 (d, J = 15.6 Hz, 1H, H17), 2.12 (m, 1H, H-6), 1.95 (m, 1H, H-6), 0.96 (m, 2H, H-19), 0.68 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 573 [M + H]+ (calcd for C21H24N2O2Br3, 572.9388). 2.2.9. Aspidospermidine-16- carboxylic acid, 1-acetyl-2, 16didehydro-10,14,15-tribromine-(7R, 14b,15a,20a, 21a)-, methyl ester (10,C23H25N2O3Br3) Compound 9 (0.40 g, 0.699 mmol) was dissolved in 10 mL acetic anhydride, then, 0.3 g p-toluene sulfonic acid was added into the mixture. After stirring at room temperature for 24 h, 15 mL of ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. The mixture was extracted with CH2Cl2 (2 10 mL), and the organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum etherEtOAc to afford 0.37 g product (10). Yield: 86%. mp: 167–169 C. 1H NMR (400 MHz, CDCl3): d 7.87 (d, J = 8.4 Hz, 1H, H-12), 7.38 (dd, J = 8.4, 2 Hz, 1H, H-11), 7.32 (d, J = 2 Hz, 1H, H-9), 6.04 (s, 1H, H-21), 3.79 (s, 3H, –OCH3), 3.75 (m, 1H, H-14), 3.38 (d, J = 1.2 Hz, 1H, H-15), 3.12 (m, 1H, H-5), 2.80-2.49 (m, 4H, H-3, 5, 17, 17), 2.24 (s, 3H, –COCH3), 1.87 (dd, J = 14, 4.4 Hz, 1H, H-3), 1.72 (m, 2H, H-6), 1.04 (m, 2H, H-19), 0.67 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 615 [M + H]+ (calcd for C23H26N2O3Br3, 614.9494).
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X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
2.2.10. Aspidospermidine-16-carboxylic acid, 10- bromine-1-chlorine2, 16, 14, 15-tetradehydro-(7R, 20a, 21a)-, methyl ester (11, C21H22N2O2BrCl) Compound 5 (1.0 g, 2.415 mmol) and NCS (0.35 g, 2.62 mmol) were dissolved in 20 mL trifluoroacetic acid. After stirring at room temperature for 4 h, 20 mL ice water was added and the pH of the mixture was adjusted to 8.0 with 5% NaOH solution. Then, the mixture was extracted with CH2Cl2 (2 20 mL). The organic solvent was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with a mixture of petroleum ether-EtOAc to afford 0.53 g of product (11) as oil. Yield: 49%. 1H NMR (400 MHz, CDCl3): d 7.24 (d, J = 2 Hz, 1H, H-9), 7.17 (dd, J = 8.8, 2 Hz, 1H, H-11), 6.5 (d, J = 8.8 Hz, 1H, H-12), 5.61 (ddd, J = 9.6, 4.4, 1.2 Hz, 1H, H-14), 5.43 (d, J = 9.6 Hz, 1H, H-15), 5.32 (s, 1H, H-21), 3.87 (s, 3H, –OCH3), 3.35 (m, 2H, H-3, 5), 2.83 (d, J = 14 Hz, 1H, H-17), 2.67 (m, 3H, H-3, 5, 6), 2.18 (d, J = 14 Hz, 1H, H-17), 2.08 (m, 1H, H-6), 1.64 (dq, J = 14, 7.6 Hz, 1H, H19), 1.47 (dq, J = 14, 7.6 Hz, 1H, H-19), 0.73 (t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 449 [M + H]+ (calcd for C21H23N2O2BrCl, 449.0631). 2.2.11. Aspidospermidine-16-carboxylic acid, 3-bromine-1,10dichlorine-2,16,14,15-tetradehydro-(7R, 20a, 21a)-, methyl ester (12, C21H21N2O2Cl2Br) Compound 4 (0.2 g, 0.495 mmol) was dissolved in dry CH2Cl2 (10 mL). AIBN (0.01 g, 0.061 mmol) and NBS (0.12 g, 0.67 mmol) were added into the mixture. The mixture was refluxed for 5 h under the protection of nitrogen. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.11 g product (12) as oil. Yield: 46%. 1H NMR (400 MHz, CDCl3): d 7.57–7.46 (m, 3H, H-9, 11, 12), 5.78 (dd, J = 9.2, 3.6 Hz, 1H, H-14), 5.43 (d, J = 9.2 Hz, 1H, H-15), 3.98 (s, 1H, –OCH3), 3.63 (d, J = 3.6 Hz,1H, H-3), 3.44 (m, 1H, H-5), 3.34–3.23 (m, 2H, H-21, 5), 3.06 (d, J = 14.4 Hz,1H, H-17), 2.97(d, J = 14.4 Hz,1H, H-17), 2.85–2.73 (m, 2H, H-6, 6), 0.79–0.71 (m, 2H, H-19), 0.51 (t, J = 7.6 Hz, 3H, H18). ESIMS m/z: 483 [M + H]+ (calcd for C21H22N2O2Cl2Br, 483.0242). 2.2.12. Aspidospermidine-16-carboxylic acid, 14,15-dibromine-1,10dichlorine-2,16-didehydro-(7R, 14b, 15a, 20a, 21a)-, methyl ester (13, C21H22N2O2Br2Cl2) Compound 4 (0.2 g, 0.495 mmol) and NBS (0.2 g, 1.123 mmol) were dissolved in CH2Cl2 (10 mL). The mixture was refluxed for 5 h. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.09 g product (13) as pale yellow crystals. Yield: 32%. mp: 139–141 C. 1H NMR (400 MHz, CDCl3): d 7.67 (d, J = 1.2 Hz, 1H, H-9), 7.44 (dd, J = 8.4, 1.2 Hz, 1H, H-11), 7.10 (d, J = 8.4 Hz, 1H, H-12), 3.87 (s, 1H, H-21), 3.51
(s, 3H, –OCH3), 3.49 (m, 1H, H-14), 3.11 (m, 2H, H-3, 15), 2.66 (d, J = 14 Hz, 1H, H-17), 2.42–2.04 (m, 6H, H-3, 5, 5, 6, 6, 17), 1.59 (m, 2H, H-19), 0.96 (t, J = 7.2 Hz, 3H, H-18). ESIMS m/z: 563 [M + H]+ (calcd for C21H23N2O2Br2Cl2, 562.9503). 2.2.13. Aspidospermidine-16-carboxylic acid, 1,3- dichlorine2,16,14,15-tetradehydro-(7R, 20a, 21a)-, methyl ester (14, C21H22N2O2Cl2) Compound 3 (0.5 g, 1.351 mmol) was dissolved in dry CH2Cl2 (15 mL). AIBN (0.1 g, 0.609 mmol) and NCS (0.25 g, 1.872 mmol) were added into the mixture. The mixture was refluxed for 5 h under the protection of nitrogen. After cooling, the mixture was washed by saturated NaHCO3 solution. The organic layer was concentrated to dryness under reduced pressure. The residue was then purified by column chromatography on silica gel with CH2Cl2 to afford 0.31 g product (14) as oil. Yield: 57%. 1H NMR (500 MHz, CDCl3): d 7.53 (dd, J = 7.5, 1 Hz, 1H, H-9), 7.34 (ddd, J = 7.5, 7.5, 1.5 Hz, 1H, H-11), 7.24 (m, 2H, H-10, 12), 5.62 (d, J = 11 Hz, 1H, H-15), 5.58 (dd, J = 11, 3.5 Hz, 1H, H-14), 3.90 (s, 1H, H-21), 3.48 (s, 1H, –OCH3), 3.10 (d, J = 3.5 Hz, 1H, H-3), 2.66 (d, J = 12.5 Hz, 1H, H-17), 2.35 (m, 1H, H-5), 2.28 (d, J = 12.5 Hz, 1H, H-17), 2.20–2.10 (m, 3H, H-5, 6, 6), 1.62 (m, 2H, H-19), 0.98 (t, J = 7.6 Hz, 3H, H-18). ESIMS m/z: 405 [M + H]+ (calcd for C21H23N2O2Cl2, 405.1137). 2.3. AChE inhibitory assays The assay for measuring AChE activity was modified from the Ellman’s method (Ellman et al., 1961). Galanthamine hydrobromide was used as a positive control. In brief, 200 mL potassium phosphate buffer (PBS, pH 8.0), 300 mL DTNB (3 mmol mL1 in PBS), 100 mL enzyme (0.24 U mL1 in PBS) and 100 mL sample solution were mixed and pre-incubated for 20 min at 37 C, then 300 mL substrate (ATCI, 3 mmol mL1 in PBS) were added. The change in absorbance was measured at 412 nm after 15 min. The IC50 values were determined graphically from inhibition curves. The IC50 values were determined in triplicate and the values are presented as mean standard deviation. 3. Results and discussion 3.1. Synthesis of tabersonine derivatives The derivatives of tabersonine were synthesized according to Fig. 1. The H-10 in the structure of tabersonine was activated by indole ring. Thus, it could be substituted by Br or Cl through electrophilic substitution reaction. The H-3 in the structure of tabersonine was activated by the carbon–carbon double bond C14,15. Thus, H-3 could be substituted by Br or Cl through free radical reaction. The double bond C-14,15 in the structure of tabersonine was unsaturated bond, which could react with NBS by
Fig. 2. The stereostructure of 14,15- dibromine and 3-Br in tabersonine derivatives.
X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
addition reaction. The N-H in indole ring was a active site in the structure of tabersonine, which could react with anhydride or NCS to produce amide or N-Cl derivatives. By combining the above reactions, there were 13 compounds synthesized using tabersonine as a lead compound. Among them, compounds (3), (4), (7)–(14) were novel tabersonine derivatives. The chemical structures of tabersonine derivatives were determined by 1H NMR and MS. In general, the d value at 9.0 was the chemical shift of N–H in indole ring. The values of d ranged from 7.9 to 6.5 were the chemical shifts of hydrogen atoms in the benzene ring. The coupling constants between them were adopted to identify their relative positions (ortho, meta or para). The values of d ranged from 5.9 to 5.4 were the chemical shifts of hydrogen atoms on carbon–carbon double bond C-14,15. The H-14 was coupled by H-15, H-3ax and H-3eq. Thus, the peak shape of H-14 was octet (ddd). The H-15 was coupled by H-14 and H-3. Thus, the peak shape of H-15 was sextet (dt). The d around 3.8 was the chemical shifts of –COOCH3, appearing as singlet. The values of d ranged from 3.6 to 1.8 were the chemical shifts of H-3,4,5,6,17. The peak shapes and coupling constants were employed to identify them. For instance, the peak shape of H-17 was double peaks (d), with the coupling constant Jgem was about 14 Hz. The d around 1.0 was the chemical shifts of H-19, appearing as octet (dq, Jgem = 14 Hz, J18,19 = 7 Hz). The d around 0.6 was the chemical shifts of H-18, appearing as triplet (t). The stereoposition of the 14,15-dibromine group was verified by the JH,H coupling constant between H-14 and H-15. The values of J14,15 were about 1.2 Hz, which indicated that the hydrogen atoms on positions 14 and 15 were both equatorial bonds (Jax,ax range from 8 to 12 Hz, Fig. 2a). The stereostructure of 14,15-dibromine in tabersonine derivatives was shown in Fig.2b. The stereoposition of the 3-Br was verified by the JH,H coupling constant between H-3 and H-14. As shown in Fig. 2c, the dihedral between H-14 and H-3eq, H-14 and H-3ax were about 38 and 77 degrees, respectively. And the coupling constants of J14,3eq and J14,3ax were about 4.4 Hz and 1.2 Hz, respectively. After substitution of H-3 by bromine, the coupling constant of J14,3 was about 3.6 Hz. This result suggested that the hydrogen atom on position C-3 was equatorial bond (Fig. 2d). 3.2. AChE inhibitory assays The AChE inhibitory activities of tabersonine derivatives were tabulated in Table 1. The lead compound tabersonine (1) showed a certain activity with the value of IC50 was 63.47 mM. Among tabersonine derivatives, compounds (7) and (9) exhibited pronounced AChE inhibitory activities in comparison to the positive control galanthamine hydrobromide (1.79 mM). Derivatives (7) and (9) could be used as potential AChE inhibitors in the following
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investigational study with IC50 values were 5.32 mM and 6.93 mM, respectively. Besides that some derivatives exhibited considerable activities, the structure-activity relationships between tabersonine derivatives and their AChE inhibitory activities were analyzed. Compounds (2) and (3) were the derivatives of tabersonine with substituent groups of R5 were acetyl or Cl. And their IC50 values were both above 100 mM. This result indicated that the introduction of groups R5 would decrease the AChE inhibitory activity of tabersonine derivatives. For the same reason, the IC50 values of compounds (4), (8), (10), (11), (12) and (14) were all above 100 mM. Compound (5) was the tabersonine derivatives with group R1 was Br and its value of IC50 was 11.48 mM. This result revealed that the introduction of group R1 increased the AChE inhibitory activity of tabersonine derivatives. The IC50 value for compound (6) was 64.12 mM, which has no significant difference with the IC50 value of the lead compound tabersonine (1). This result indicated that the introduction of group R2 showed no significant influence on the AChE inhibitory activity of tabersonine derivatives. Compound (7) showed the highest AChE inhibitory activity with the IC50 value was 5.32 mM. The introduction of 14, 15-dibromide increased the activities of tabersonine derivatives against AChE. Among tabersonine derivatives (1)–(14), compounds (7) and (9) exhibited pronounced AChE inhibitory activities in comparison to the positive control galanthamine hydrobromide (1.79 mM). The AChE inhibitory ratios of tabersonine derivatives at concentration of 100 mM were shown in Figure 3. At this concentration, the influences of substituent groups on the AChE inhibitory activity of tabersonine derivatives are similar to the results discussed above. Overall, the results of Table 1 and Figure 3 revealed that the introduction of group R1 and/or combined groups R3, R4 could increase the AChE inhibitory activity of tabersonine derivatives. Thus, compounds (5), (7) and (9) showed considerable AChE inhibitory activities with IC50 values were 11.48 mM, 5.32 mM and 6.93 mM, respectively. The introduction of group R5 decreased the AChE inhibitory activities of tabersonine derivatives. Thus, the IC50 values of compounds (2), (3), (4), (8), (10), (11), (12) and (14) were all above 100 mM. However, the IC50 value of compound (13) was 78.26 mM. This is probably because the group R1 and combined groups R3, R4 in compound (13) increased its AChE inhibitory activity. The group R2 showed no significant influence on the AChE inhibitory activity of tabersonine derivatives. Thus, the IC50 values between tabersonine (1) and compound (6) have no significant difference. The above results indicated that the AChE inhibitory
Table 1 The IC50 values of tabersonine derivatives. Compounds
R1
R2
H H 1 2 H H 3 H H 4 –Cl H 5 –Br H 6 H –Br 7 H H 8 –Br H 9 –Br H 10 –Br H 11 –Br H 12 –Cl -Br 13 –Cl H 14 H –Cl Galanthamine hydrobromide
R3, R4
R5
Yield (%)
IC50 SD (mM)
4 4 4 4 4 4
H –COCH3 –Cl –Cl H H H –COCH3 H –COCH3 –Cl –Cl –Cl –Cl
1.95 85 68 7 91 81 19 82 21 86 49 46 32 57
63.47 4.12 >100 >100 >100 11.48 1.32 64.12 5.21 5.32 1.78 >100 6.93 2.34 >100 >100 >100 78.26 3.23 >100 1.79 0.23
Br2
4
Br2 Br2
4 4
Br2
4
Fig. 3. The AChE inhibitory ratios of tabersonine derivatives at the concentration of 100 mM.
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X. Liu et al. / Phytochemistry Letters 14 (2015) 17–22
activity of tabersonine derivatives could be improved by structural modification. All these conclusions could be used as a structureactivity relationship to semi-synthesize more tabersonine derivatives with high AChE inhibitory activities in further investigations. 4. Conclusion In summary, there were 13 compounds, containing 10 novel tabersonine derivatives, synthesized using tabersonine as a lead compound. The AChE inhibitory activities of tabersonine and its derivatives were also tested. The AChE inhibitory activities of tabersonine derivatives increased with the introduction of group R1 and/or combined groups R3, R4, while decreased with the introduction of group R5. And the group R2 showed no significant influence on the AChE inhibitory activities of tabersonine derivatives. All these results could be used as a structure-activity relationship to semi-synthesize more tabersonine derivatives with high AChE inhibitory activities in further investigations. References Ellman, G.L., Courtney, K.D., Featherstone, R.M., 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. pharmacol. 7, 88–95. González-Domínguez, R., García-Barrera, T., Gómez-Ariza, J.L., 2012. Metabolomic approach to Alzheimer’s disease diagnosis based on mass spectrometry. Chem. Pap.66 829–835. Howes, M.J.R., Houghton, P.J., 2003. Plants used in Chinese and Indian traditional medicine for improvement of memory and cognitive function. Pharmacol. Biochem. Behav. 75, 513–527. Iannello, C., Pigni, N.B., Antognoni, F., Poli, F., Maxia, A., de Andrade, J.P., Bastida, J., 2014. A potent acetylcholinesterase inhibitor from Pancratium illyricum L. Fitoterapia 92, 163–167. Lai, D.H., Yang, Z.D., Xue, W.W., Sheng, J., Shi, Y., Yao, X.J., 2013. Isolation, characterization and acetylcholinesterase inhibitory activity of alkaloids from roots of Stemona sessilifolia. Fitoterapia 89, 257–264. Liu, X., Yang, D.L., Liu, J.J., Zhou, J., Zhao, N.N., 2014a. Synthesis of new eburnaminetype alkaloid via direct hydroalkoxylation. Chem. Pap. 68, 816–822. Liu, X., Yang, D.L., Liu, J.J., Ren, N., 2014b. Synthesis and characterization of amino acid-modified adsorption resins and their adsorption properties in the
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