Open Access

Synthesis and biological evaluation of lycorine derivatives as dual inhibitors of humanacetylcholinesterase and butyrylcholinesterase

Chemistry Central Journal20126:96

https://doi.org/10.1186/1752-153X-6-96

Received: 27 May 2012

Accepted: 24 August 2012

Published: 8 September 2012

Abstract

Background

Alzheimer’s disease (AD) is a neurologically degenerative disorder that affects more than20 million people worldwide. The selective butyrylcholinesterase (BChE) inhibitors and bivalentcholinesterase (ChE) inhibitors represent new treatments for AD.

Findings

A series of lycorine derivatives (110) were synthesized and evaluated foranti-cholinesterase activity. Result showed that the novel compound2-O-tert-butyldimethylsilyl-1-O-(methylthio)methyllycorine (7) wasa dual inhibitor of human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) withIC50 values of 11.40 ± 0.66 μM and 4.17 ± 0.29 μM, respectively. Thestructure-activity relationships indicated that (i) the 1-O-(methylthio)methyl substituentin lycorine was better than the 1-O-acetyl group for the inhibition of cholinesterase; (ii)the acylated or etherified derivatives of lycorine and lycorin-2-one were more potent against hBChEthan hAChE; and (iii) the oxidation of lycorine at C-2 decreases the activity.

Conclusion

Acylated or etherified derivatives of lycorine are potential dual inhibitors of hBChE and hAChE.Hence, further study on the modification of lycorine for ChE inhibition is necessary.

Keywords

Amaryllidaceae alkaloidsLycorineAcetylcholinesteraseButyrylcholinesterase

Findings

Alzheimer’s disease (AD) is a neurologically degenerative disorder that affects more than20 million people worldwide [1], and is the third-most costly disease after cardiovascular disease and cancer [2]. The neuropathological hallmarks of the disease include β-amyloid (Aβ) plaques,neurofibrillary tangles, and synaptic loss. Based on the cholinergic hypothesis, the symptoms of ADare the result of the reduction in brain acetylcholine (ACh) activity due to the catabolism of AChby its principal hydrolytic enzyme acetylcholinesterase (AChE). AChE inhibition is the currentapproach for AD treatment. Tacrine, donepezil, rivastigmine, and galanthamine are all examples oftypical AChE inhibitory drugs [3].

Similar to AChE, butyrylcholinesterase (BChE) can also inactivate ACh. The reduction in ACh isusually accompanied by a decrease in AChE activity. By contrast, BChE in AD remains at normal levelsor even elevated in the brain. BChE may be a significant contributor to the observed loss of ACh inAD [4]. Furthermore, BChE inhibition can lower Aβ peptide [5, 6]. BChE is essential in AD plaque maturation [7]. Selective BChE inhibition may be crucial in the mid to late stages of AD pathogenesis tocircumvent further decline in mental and cognitive ability as the depletion of cholinergic neuronspersists [3]. Hence, selective BChE inhibitors or bivalent ChE inhibitors represent a new treatmentfor AD.

Lycorine, the most frequent alkaloid in Amaryllidaceae plants, has very weak inhibitory activityagainst electric eel acetylcholinesterase (eeAChE), with an IC50 value of 213 μM [8]. Acylated or etherified derivatives of lycorine, such,as1-O-acetyllycorine and1-O-acetyl-2-O tert-butyldimethylsilyllycorine (6, Figure 1), possess potent activity against eeAChE [8, 9]. However, the inhibitory effect of analogues on BChE has not been reported. In ourcontinuing work on Amaryllidaceae alkaloids [1012], the present study reports the synthesis of lycorine derivatives (1 10), andtheir biological evaluation for inhibition of ChE.
Figure 1

Chemical structures of compounds under study.

Previous researchers considered that a hydrogen-bond acceptor at the C-1 of lycorine is necessaryfor AChE inhibitory activity, and a bulky, lipophilic substituent, such as the TBS group, at C-2increases the activity [9, 13]. Therefore, in the present study, benzoic acid or cinnamic acid were used to acylate the1-OH and/or 2-OH of lycorine and its C-1 or C-2 oxidation derivatives. Mono- or di-acylatedderivatives (1 5, and 10) of lycorine and lycorin-2-one (8) were obtainedby Steglich esterification (DCC/DMAP). Lycorine oxidation using pyridinium chlorochromate (PCC) inDMF yielded 8, and the acetylated analogue (9) of the latter was obtained by thereaction of 8 with Ac2O/pyridine. The DMSO/Ac2O system was used tooxidate C-1 of lycorine, with the protection of 1-OH by the TBS group. However, 1-O-acetyland 1-O-(methylthio)methyl derivatives (6 and 7) were obtained instead of thedesired C-1 oxidation product.

The anti-ChE activity of these prepared lycorine derivatives (1 10) was evaluated byin vitro ChE inhibition assay, modified from Ellman’s method [14]. The results were expressed as IC50 values and summarized in Table 1.
Table 1

Inhibitory effect of compounds 1–10 on human AChE and BChE

Lycorine analogue

No.

IC50(μM)

 

hAChE

hBChE

1,2-O,O'-Dibenzoyllycorine

1

> 50

7.72 ± 0.26

2-O-Benzoyllycorine

2

> 50

> 50

1,2-O,O'-Di-trans-cinnamoyllycorine

3

46.76 ± 0.95

17.45 ± 0.19

2-O-trans-Cinnamoyllycorine

4

> 50

19.74 ± 1.37

1-O-trans-Cinnamoyllycorine

5

> 50

12.13 ± 0.77

1-O-Acetyl-2-O-tert-butyldimethylsilyllycorine

6

> 50

> 50

2-O-tert-Butyldimethylsilyl-1-O-(methylthio)methyllycorine

7

11.40 ± 0.66

4.17 ± 0.29

Lycorin-2-one

8

> 50

> 50

1-O-Acetyllycorin-2-one

9

> 50

44.46 ± 0.88

1-O-trans-Cinnamoyllycorin-2-one

10

> 50

20.91 ± 0.13

Tacrine (positive control)

 

0.26 ± 0.015

0.02 ± 0.00

Galanthamine (positive control)

 

1.60 ± 0.14

18.30 ± 0.14

2-O-tert-Butyldimethylsilyl-1-O-(methylthio)methyllycorine (7)showed dual inhibitory activity against both hAChE (IC50 = 11.40 ± 0.66 μM) andhBChE (IC50 = 4.17 ± 0.29 μM). The inhibitory potency of 7 wasapproximately four-fold stronger than that of galanthamine (IC50 = 18.30 ± 0.14μM) on hBChE. Compounds 1, and 24 also showed good effects onhBChE, with IC50 values of less than 20 μM.

Table 1 shows that the acylated or etherified derivatives (1,35, 7, 9, and 10) of lycorine and lycorin-2-one are morepotent against hBChE than hAChE. The hBChE inhibitory activity of1-O-trans-cinnamoyllycorine (5, IC50 = 12.13 ± 0.77μM) is about two-fold better than that of 1-O-trans-cinnamoyllycorin-2-one(10, IC50 = 20.91 ± 0.13 μM). This result implied that lycorineoxidation at C-2 may decrease the activity.

A previous study reported that1-O-acetyl-2-O tert-butyldimethylsilyllycorine (6) showedsignificant inhibitory activity against ACh biotransformation by eeAChE (Ki =0.34 μM) [9]. However, in the current study,1-O-acetyl-2-O tert-butyldimethylsilyllycorine was inactive(IC50 > 50 μM) against both of hAChE and hBChE. 1-O-(Methylthio)methylsubstituent at C-1 of lycorine significantly increased the inhibitory activity against both of hAChEand hBChE in 7 compared with that of 6. Compound 7 was an unexpected product;its formation mechanism can be explained by a Pummerer rearrangement (Scheme 1) [15].
Scheme 1

The proposed mechanism for the formation of 7.

The bioassay result of compound 7 compared with those of other tested compounds showedthat a bulky, lipophilic substituent at C-1 or C-2 of lycorine is necessary for the human ChEinhibitory activity. In addition, the substituted group at C-1 is important in the activity.

The positive control tacrine showed a significant inhibitory effect on both hAChE and hBChE.However, tacrine is currently rarely used because of its hepatotoxicity [16]. Based on the results in the present study, modification of lycorine for the inhibitionof ChE, especially of hBChE, is necessary.

Experimental

Chemistry

NMR spectra were recorded on Bruker AM-400 and DRX-500 spectrometers with TMS as an internalstandard. ESIMS data were measured on an API-Qstar-Pulsar-1 instrument and HREIMS on a WatersAutospec Premier P776 mass spectrometer. Column chromatography was performed over silica gel G(80–100 and 300–400 mesh), silica gel H (10–40 μm), and Sephadex LH-20(40–70 μm; Amersham Pharmacia Biotech AB). TLC was conducted on precoated silica gelplates GF254. HPLC separations were performed using an Agilent 1200 series pump equipped with adiode array detector, a semi-preparative Agilent Zorbax SB-C18 (5 μm, 9.4 × 250 mm)column, and a semi-preparative Waters XBridge C-18 column (5 μm, 10 × 250 mm).

Preparation of the acylated derivatives (1–5 and 10) of lycorine and lycorin-2-one

A suspension of lycorine or lycorin-2-one (1 mmol), cinnamic acid or benzoic acid (1 eq.),dicyclohexylcarbodiimide (1 eq.), and 4-(N,N-dimethylamino)pyridine (1 eq.) in 25mL of dry DMF was stirred for 12 h at room temperature. The urea byproduct was filtered, and thefiltrate was evaporated. The resulting residue was purified by column chromatography on silica gel,using a mixture of hexane-CHCl3-Me2CO (10:2:1), CHCl3, andCHCl3-MeOH (100:1) as eluent to yield the products (15 and10).

1,2-O,O'-Dibenzoyllycorine (1)

Elution with hexane-CHCl3-Me2CO (10:2:1) and CHCl3 afforded1[17] as a colorless solid, with a yield of 2.5%; 1H-NMR (CDCl3, 400MHz): δ 8.06 (d, J = 7.7 Hz, 2H, H-2'',6''), 7.91 (d, J = 7.7 Hz,2H, H-2',6'), 7.55 (m, 2H, H-4',4''), 7.42 (m, 4H, H-3',5',3'',5''), 6.84 (s, 1H, H-8), 6.59 (s, 1H,H-12), 6.16 (br s, 1H, H-1), 5.90 and 5.86 (s, 1H each, H2-11), 5.69 (br s, 2H, H-2,3),4.09 and 3.62 (br s, 1H each, H2-7), 3.47 and 2.53 (m, 1H each, H2-5), 3.19(m, 1H, H-12c), 3.06 (m, 1H, H-12b), 2.77 (m, 2H, H2-4); ESIMS m/z: 496 [M +H]+; HREIMS for C30H25NO6 [M]+: calcd.495.1682; found: 495.1682.

2-O-Benzoyllycorine (2)

Elution with CHCl3-MeOH (100:1) afforded 2[17] as a colorless solid, with a yield of 12.0%; 1H-NMR (CDCl3, 400MHz): 1H-NMR (CDCl3, 400 MHz): δ 8.02 (d, J = 7.3 Hz,2H, H-2',6'), 7.54 (t, J = 7.3 Hz, 1H, H-4'), 7.40 (t, J = 7.3 Hz, 2H, H-3',5'),6.80 (s, 1H, H-8), 6.58 (s, 1H, H-12), 5.87 and 5.87 (s, 1H each, H2-11), 5.58 (br s, 2H,H-2,3), 4.64 (br s, 1H, H-1), 4.16 and 3.55 (d, J = 14.2 Hz, 1H each, H2-7),3.37 and 2.40 (m, 1H each, H2-5), 2.89 (d, J = 10.5 Hz, 1H, H-12b), 2.83 (d,J = 10.5 Hz, 1H, H-12c), 2.67 (m, 2H, H2-4); 13C-NMR(CDCl3, 100 MHz): δ 166.0 (O(CO)Ph), 146.5 (C-10), 146.2 × 2(C-3a,9), 133.1 (C-4'), 130.0 and 129.8 (C-7a,1'), 129.7 × 2 (C-2',6'), 128.3 × 2(C-3',5'), 127.3 (C-12a), 113.7 (C-3), 107.5 (C-8), 104.8 (C-12), 100.9 (C-11), 74.1 (C-1), 69.0(C-2), 60.7 (C-12c), 57.0 (C-7), 53.7 (C-5), 41.8 (C-12b), 28.7 (C-4); ESIMS m/z: 392 [M +H]+; HREIMS for C23H21NO5 [M]+: calcd.391.1420; found: 391.1413.

1,2-O,O'-Di-trans-cinnamoyllycorine (3)

Elution with hexane-CHCl3-Me2CO (10:2:1) and CHCl3 afforded3 as a colorless solid, with a yield of 3.5%; 1H-NMR (CDCl3, 400 MHz):δ 7.71 (d, J = 16.1 Hz, 1H, H-7''), 7.64 (d, J = 16.1 Hz, 1H, H-7'),7.52 (m, 2H, H-2'',6''), 7.46 (m, 2H, H-2',6'), 7.38 (m, 3H, H-3'',4'',5''), 7.34 (m, 3H,H-3',4',5'), 6.84 (s, 1H, H-8), 6.60 (s, 1H, H-12), 6.45 (d, J = 16.1 Hz, 1H, H-8''), 6.32(d, J = 16.1 Hz, 1H, H-8'), 5.99 (br s, 1H, H-1), 5.90 and 5.88 (s, 1H each,H2-11), 5.65 (br s, 1H, H-2), 5.51 (br s, 1H, H-3), 4.18 and 3.62 (d, J = 11.4Hz, 1H each, H2-7), 3.40 and 2.50 (m, 1H each, H2-5), 3.06 (d, J =9.6 Hz, 1H, H-12c), 2.97 (m, 1H, H-12b), 2.73 (m, 2H, H2-4); 13C-NMR(CDCl3, 100 MHz): δ 165.8 and 165.5 (C-9',9''), 146.4 (C-10), 145.8 and145.5 × 3 (C-3a,9,7',7''), 134.3 and 134.1 (C-1',1''), 130.4 and 130.4 (C-7a,12a), 128.9 ×2 and 128.8× 2 (C-3',5',3'',5''), 128.1 × 4 (C-2',6',2'',6''), 126.6 × 2 (C-4',4''),117.7 and 117.3 (C-8',8''), 114.3 (C-3), 107.4 (C-8), 105.2 (C-12), 101.0 (C-11), 70.7 (C-1), 69.1(C-2), 61.3 (C-12c), 56.7 (C-7), 53.7 (C-5), 40.4 (C-12b), 28.8 (C-4); ESIMS m/z: 548 [M +H]+; HREIMS for C34H29NO6 [M]+: calcd.547.1995; found: 547.1999.

2-O-trans-Cinnamoyllycorine (4)

Elution with CHCl3-MeOH (100:1) afforded 4 as a colorless solid, with a yieldof 16.7%; 1H-NMR (CDCl3, 400 MHz): δ 7.70 (d, J = 16.1Hz, 1H, H-7'), 7.50 (m, 2H, H-2',6'), 7.38 (m, 3H, H-3',4',5'), 6.84 (s, 1H, H-8), 6.61 (s, 1H,H-12), 6.43 (d, J = 16.1 Hz, 1H, H-8'), 5.91 and 5.90 (s, 1H each, H2-11), 5.57(br s, 1H, H-2), 5.48 (br s, 1H, H-3), 4.62 (br s, 1H, H-1), 4.14 and 3.68 (d, J = 14.1 Hz,1H each, H2-7), 3.38 and 2.59 (m, 1H each, H2-5), 3.01 (m, 1H, H-12b), 2.82(d, J = 8.7 Hz, 1H, H-12c), 2.71 (m, 2H, H2-4); 13C-NMR(CDCl3, 100 MHz): δ 166.3 (C-9'), 146.4 (C-10), 145.5 × 3 (C-3a,9,7'),134.2 (C-1'), 130.4 × 2 (C-7a,12a), 128.9 × 2 (C-3',5'), 128.1 × 2 (C-2',6'), 127.3(C-4'), 117.7 (C-8'), 113.8 (C-3), 107.6 (C-8), 104.8 (C-12), 101.0 (C-11), 73.5 (C-1), 68.9 (C-2),60.5 (C-12c), 56.5 (C-7), 53.8 (C-5), 41.3 (C-12b), 28.9 (C-4); ESIMS m/z: 418 [M +H]+; HREIMS for C25H23NO5 [M]+: calcd.417.1576; found: 417.1567.

1-O-trans-Cinnamoyllycorine (5)

Elution with CHCl3-MeOH (50:1) afforded 5 as a colorless solid, with a yield of1.4%; 1H-NMR (CDCl3, 400 MHz): 1H-NMR (CDCl3, 400 MHz):δ 7.59 (d, J = 16.0 Hz, 1H, H-7'), 7.45 (m, 2H, H-2',6'), 7.33 (m, 3H,H-3',4',5'), 6.64 (s, 1H, H-8), 6.59 (s, 1H, H-12), 6.28 (d, J = 16.0 Hz, 1H, H-8'), 5.89and 5.87 (s, 1H each, H2-11), 5.72 (br s, 1H, H-1), 5.59 (br s, 1H, H-3), 4.27 (br s, 1H,H-2), 4.19 and 3.61 (d, J = 14.0 Hz, 1H each, H2-7), 3.37 and 2.51 (m, 1H each,H2-5), 2.95 (m, 2H, H-12b,12c), 2.67 (m, 2H, H2-4); 13C-NMR(CDCl3, 100 MHz): δ 166.6 (C-9'), 146.6 (C-10), 146.3 and 145.5 × 2(C-3a,9,7'), 134.1 (C-1'), 130.4 × 2 (C-7a,12a), 128.8 × 2 (C-3',5'), 128.1 × 2(C-2',6''), 127.1 (C-4'), 117.5 (C-8'), 113.8 (C-3), 107.4 (C-8), 104.9 (C-12), 100.9 (C-11), 72.6(C-1), 69.4 (C-2), 61.5 (C-12c), 56.8 (C-7), 53.7 (C-5), 39.1 (C-12b), 28.7 (C-4); ESIMSm/z: 418 [M + H]+; HREIMS for C25H23NO5[M]+: calcd. 417.1576; found: 417.1583.

1-O-trans-Cinnamoyllycorin-2-one (10)

Elution with hexane-CHCl3-Me2CO (10:2:1), CHCl3, andCHCl3-MeOH (100:1) afforded 10 as a colorless solid, with a yield of 62.7%;1H-NMR (CDCl3, 400 MHz): δ 7.63 (d, J = 16.0 Hz, 1H,H-7'), 7.44 (m, 2H, H-2',6'), 7.34 (m, 3H, H-3',4',5'), 6.79 (s, 1H, H-8), 6.57 (s, 1H, H-12), 6.28(d, J = 16.0 Hz, 1H, H-8'), 6.14 (d, J = 1.6 Hz, 1H, H-1), 6.03 (br s, 1H, H-3),5.90 and 5.89 (s, 1H each, H2-11), 4.18 and 3.65 (d, J = 14.0 Hz, 1H each,H2-7), 3.48 and 2.59 (m, 1H each, H2-5), 3.35 (m, 2H, H-12b,12c), 2.90 (m, 2H,H2-4); 13C-NMR (CDCl3, 100 MHz): δ 204.4 (C-2), 165.5(C-9'), 146.7 × 2 (C-3a,10), 146.1 × 2 (C-9,7'), 134.1 (C-1'), 130.5 × 2 (C-7a,12a),128.8 × 2 (C-3',5'), 128.1 × 2 (C-2',6''), 125.2 (C-4'), 120.6 (C-3), 116.9 (C-8'), 107.3(C-8), 105.4 (C-12), 101.1 (C-11), 68.9 (C-1), 62.3 (C-12c), 56.1 (C-7), 53.2 (C-5), 45.4 (C-12b),30.0 (C-4); ESIMS m/z: 416 [M + H]+; HREIMS forC25H21NO5 [M]+: calcd. 415.1420; found: 415.1418.

Synthesis of 1-O-acetyl-2-O-tert-butyldimethylsilyllycorine (6) and2-O-tert-butyldimethylsilyl-1-O-(methylthio)methyllycorine (7)

A solution of 2-O tert-butyldimethylsilyllycorine [9] (60 mg, 0.150 mmol), dry dimethyl sulfoxide (0.26 mL), and acetic anhydride (0.18 mL) wasstirred overnight at room temperature. Afterward, the reaction mixture was quenched withH2O (0.7 mL) and aqueous NH4OH (0.4 mL). The resulting solution was extractedwith Et2O. The organic layer was separated, dried over Na2SO4, andthen concentrated. The residue was purified by silica gel column chromatography (petrol/EtOAc, 10:1)and HPLC with a Waters XBridge C-18 column (5 μm, 10 × 250 mm) using MeOH-H2O(95:5) as eluent to yield 6[9] (tR = 9.876 min, 23 mg, 0.0530 mmol) and 7(tR = 11.955 min, 17 mg, 0.0368 mmol).

1-O-Acetyl-2-O-tert-butyldimethylsilyllycorine (6)

A white solid, yield 35.4%; 1H-NMR (CDCl3, 400 MHz): δ 6.73(s, 1H, H-8), 6.56 (s, 1H, H-12), 5.92 and 5.91 (s, 1H each, H2-11), 5.55 (br s, 1H,H-1), 5.39 (br s, 1H, H-3), 4.17 (br s, 1H, H-2), 4.14 and 3.52 (d, J = 14.1 Hz, 1H each,H2-7), 3.36 and 2.37 (m, 1H each, H2-5), 2.94 (d, J = 8.8 Hz, 1H,H-12b), 2.74 (d, J = 8.8 Hz, 1H, H-12c), 2.63 (m, 2H, H2-4), 1.94 (s, 3H,O(CO)CH3), 0.89 (s, 9H, SiC(CH3)3), 0.19 and 0.11(s, 3H each, Si(CH3)2); ESIMS m/z: 444 [M + H]+;HREIMS for C24H33NO5Si [M]+: calcd. 443.2128; found:443.2127.

2-O-tert-Butyldimethylsilyl-1-O-(methylthio)methyllycorine (7)

A pale yellow solid, yield 24.5%; 1H-NMR (CDCl3, 400 MHz): δ7.02 (s, 1H, H-8), 6.56 (s, 1H, H-12), 5.91 and 5.91 (s, 1H each, H2-11), 5.42 (br s, 1H,H-3), 4.66 and 4.62 (d, J = 12.0 Hz, 1H each, OCH2SCH3),4.51 (br s, 1H, H-1), 4.34 (br s, 1H, H-2), 4.12 and 3.50 (d, J = 14.0 Hz, 1H each,H2-7), 3.34 and 2.33 (m, 1H each, H2-5), 2.88 (d, J = 10.4 Hz, 1H,H-12b), 2.73 (d, J = 10.4 Hz, 1H, H-12c), 2.61 (m, 2H, H2-4), 1.97 (s, 3H,SCH3), 0.89 (s, 9H, SiC(CH3)3), 0.16 and 0.12 (s,3H each, Si(CH3)2); ESIMS m/z: 462 [M + H]+;HREIMS for C24H35NO4SiS [M]+: calcd. 461.2056; found:461.2058.

Lycorin-2-one (8)

Lycorine [10, 12] (1 g, 3.481 mmol), PCC (6.657 g, 30.886 mmol) and silica gel (6.657 g) in anhydrous DMF(50 mL) were stirred at room temperature for 24 h. Afterward, the reaction mixture was filtratedthrough a pad of Celite. The filtrate was then poured into water, adjusted the pH to 9 usingammonia, and extracted with CHCl3. The solvent was evaporated under reduced pressure. Theresidue was purified by silica gel column chromatography (CHCl3-MeOH, 20:1) to yield8[18, 19] (70 mg, 0.245 mmol).

A gray powder, yield 7.0%; 1H-NMR (CDCl3, 500 MHz): δ 6.77(s, 1H, H-8), 6.60 (s, 1H, H-12), 5.97 and 5.95 (s, 1H each, H2-11), 5.93 (br s, 1H,H-3), 4.55 (d, J = 2.3 Hz, 1H, H-1), 4.16 and 3.64 (d, J = 14.0 Hz, 1H each,H2-7), 3.45 and 2.53 (m, 1H each, H2-5), 3.25 (br s, 1H, H-12b), 3.14 (d,J = 9.4 Hz, 1H, H-12c), 2.86 (br s, 2H, H2-4); ESIMS m/z: 286 [M +H]+; HREIMS for C16H15NO4 [M]+: calcd.285.1001; found: 285.1000.

1-O-Acetyllycorin-2-one (9)

A suspension of (20 mg, 0.0702 mmol) of lycorin-2-one (8) in 0.5 mL of pyridine and 0.5 mLAc2O was stirred for 12 h at room temperature and then 20 mL of water was added. Thesolution was adjusted to pH 9 using ammonia (5 mL) and extracted with CHCl3 for fourtimes before the removal of CHCl3. The resulting residue was purified by prep. TLC(CHCl3-MeOH, 30:1) to yield 9 (5 mg, 0.0153 mmol).

A gray solid, yield 21.8%; 1H-NMR (CDCl3, 400 MHz): δ 6.72(s, 1H, H-8), 6.57 (s, 1H, H-12), 6.00 and 5.99 (s, 1H each, H2-11), 5.93 and 5.92 (br s,1H each, H-1,3), 4.17 and 3.61 (d, J = 14.1 Hz, 1H each, H2-7), 3.48 and 2.52(m, 1H each, H2-5), 3.26 (br d, J = 10.0 Hz, 1H, H-12b), 3.16 (d, J =10.0 Hz, 1H, H-12c), 2.86 (m, 2H, H2-4), 1.96 (s, 3H, O(CO)CH3);ESIMS m/z: 328 [M + H]+; HREIMS for C18H17NO5[M]+: calcd. 327.1107; found: 327.1105. The NMR spectra of compounds110 were also available as a PDF file (Additional file 1).

Cholinesterase inhibitory activity

AChE/BChE inhibitory activity of compounds 1 10 (purity >95%) was assayed usingthe spectrophotometric method developed by Ellman et al. [14], with slight modification. S-Acetylthiocholine iodide,S-butyrylthiocholine iodide, 5,5'-dithio-bis-(2-nitrobenzoic) acid (DTNB, Ellman’sreagent), hAChE, and hBChE, were purchased from Sigma Chemical. The test compounds were dissolved inDMSO. The reaction mixture contained 110 μL of phosphate buffer (pH 8.0), 10 μL of testcompounds (50 μM), and 40 μL of hAChE or hBChE (0.04 U/100 μL), and the mixture wasincubated for 20 min (30 °C). Subsequently, the reaction was initiated by the addition of20 μL of DTNB (6.25 mM) and 20 μL of ACh or butyrylthiocholine (BCh) for hAChE or hBChEinhibitory activity, respectively. Hydrolysis of ACh or BCh was monitored at 405 nm after 30 min.All reactions were performed in triplicate. Inhibition percentage was calculated as follows: %inhibition = (ES)/E × 100, where E is the enzymeactivity without the test compounds and S is the enzyme activity with the test compounds.Inhibition curves were obtained for each compound by plotting the inhibition percentage versus thelogarithm of the inhibitor concentration in the assay solution. Linear regression parameters weredetermined for each curve, and the IC50 values were extrapolated. The same procedure wasapplied for the positive control tacrine (Sigma, purity 98%) and galanthamine (purity >95%) [12]. The study was approved by the ethical committee in Kunming Institute of Botany(reference number 1205) and performed according to the Helsinki Declaration.

Conclusion

A series of lycorine derivatives (110) were synthesized and evaluated foranti-cholinesterase activity. The novel compound2-O-tert-butyldimethylsilyl-1-O-(methylthio)methyllycorine (7) wasa dual hAChE and hBChE inhibitor. The structure-activity relationships indicated that (i) the1-O-(methylthio)methyl substituent in lycorine is better than the 1-O-acetyl groupfor the inhibition of cholinesterase; (ii) the acylated or etherified derivatives of lycorine andlycorin-2-one are more potent against hBChE than hAChE; and (iii) the oxidation of lycorine at C-2decreases the activity. Hence, further study on the modification of lycorine for the inhibition ofChE is necessary.

Abbreviations

Aβ: 

β-amyloid

Ach: 

Acetylcholine

AChE: 

Acetylcholinesterase

Ac2O: 

Acetic anhydride

AD: 

Alzheimer’s disease

BChE: 

Butyrylcholinesterase

ChE: 

Cholinesterase

DMAP: 

4-(N,N-dimethylamino)pyridine

DMF: 

N,N-dimethylformamide

DTNB: 

5,5'-dithio-bis-(2-nitrobenzoic) acid

DMSO: 

Dimethylsulphoxide

DCC: 

Dicyclohexylcarbodiimide

eeAChE: 

Electric eel acetylcholinesterase

ESIMS: 

Electrospray ionization mass spectrometry

EtOAc: 

Ethyl acetate

hAChE: 

Human acetylcholinesterase

hBChE: 

Human butyrylcholinesterase

HPLC: 

High pressure liquid chromatography

HREIMS: 

Highresolution electrospray ionization mass spectrometry

IC50

Concentration producing 50%inhibition

Me2CO: 

Acetone

NMR: 

Nuclear magnetic resonance

PCC: 

Pyridiniumchlorochromate

TBS: 

Tert-butyldimethylsilyl

TLC: 

Thin layer chromatography

TMS: 

Tetramethylsilane.

Declarations

Acknowledgements

This work was funded by the National Natural Science Foundation of China (Nos. 20972166,31161140345, 31070288), the Natural Science Foundation of Yunnan Province, China (No. 2011FZ205),and the Ministry of Education of China through its 111 and 985 projects (Nos. B08044, MUC985-9,MUC98506-01000101).

Authors’ Affiliations

(1)
Key Laboratory of Economic Plants and Biotechnology, Kunming Institute of Botany, Chinese Academy of Sciences
(2)
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences
(3)
School of Chemistry and Chemical Engineering, Nanjing University
(4)
College of Life and Environmental Sciences, Minzu University of China

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