Syntheses and bioactivities of songorine derivatives as novel G protein-coupled receptor antagonists

Accepted Manuscript Syntheses and bioactivities of Songorine derivatives as novel G protein-coupled receptor antagonists Jiangming Wang, Changhao Bian, Yinan Wang, Quan Shen, Bin Bao, Junting Fan, Aixue Zuo, Wenhui Wu, Ruihua Guo PII: DOI: Reference:

S0968-0896(19)30213-5 https://doi.org/10.1016/j.bmc.2019.03.045 BMC 14837

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

7 February 2019 13 March 2019 21 March 2019

Please cite this article as: Wang, J., Bian, C., Wang, Y., Shen, Q., Bao, B., Fan, J., Zuo, A., Wu, W., Guo, R., Syntheses and bioactivities of Songorine derivatives as novel G protein-coupled receptor antagonists, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.045

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Syntheses and bioactivities of Songorine derivatives as novel G protein-coupled receptor antagonists Jiangming Wang,a Changhao Bian,a Yinan Wang,a Quan Shen,a Bin Bao, a Junting Fan,e Aixue Zuo,b* Wenhui Wu,

a,c,d

* Ruihua Guo a,c,d*

a

College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China

b

College of Traditional Chinese Medicine, Yunnan University of Chinese Medicine, Kunming 650500,

China c

Shanghai Engineering Research Center of Aquatic-Product Processing & Preservation, Shanghai

201306, China d

Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation

(Shanghai), Ministry of Agriculture, Shanghai 201306, China e

School of Pharmacy, Nanjing Medical University, Nanjing 211166, China

ABSTRACT Songorine isolated from Aconitum brachypodum Diels possesses prominent activity of inhibiting G protein-coupled receptors (GPCRs) in the early screening process. In this paper, a series of Songoine derivatives were synthesized and their inhibitory activities on GPCRs were also evaluated by using the Double Antibody Sandwich ELISA (DAS-ELISA) in vitro. Among them, three derivatives (3a, 4, 7) exhibited significant inhibitory activity against GPCRs with IC50 values of 0.08-0.29 nM. Moreover, the structure-activity relationships (SARs) of songorine derivatives were discussed in detail. They have great potentials as novel GPCRs antagonists in the future.

Keywords: Songorine, Derivatives, G protein-coupled receptors, Inhibition, Synthesis 1. Introduction

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and about 800 annotated GPCRs exist in the human genome. They include various signal transduction receptors that undergo dynamic and isoform-specific membrane trafficking.1 GPCRs can bind chemicals in the surroundings of cells, which activates a series of signal pathways causing status changes in cells. GPCRs can regulate several functions of the body including vision, smell, taste, behavior, immune and

nervous systems. It is one of the main targets of drugs and about 40%-50% drugs act through GPCRs. Thus, GPCRs are considered to possess wide potential applications in new therapeutic drugs. 2 Natural products with various skeletons and their derivatives are important sources of GPCRs antagonists.3-5 In our previous works of active leads from traditional Chinese medicinal herbs, we investigated the active constituents of Aconitum brachypodum Diels.6-7 Aconitum is a Ranunculaceae plant native in China and Europe and it has been utilized as one traditional Chinese medicine in China for more than 2,000 years.8 It possesses diverse bioactivities such as anti-inflammatory, anti-tumor, analgesic effects, etc. Aconitum contains a lot of alkaloids and about 450 alkaloids were isolated from aconitum plants so far. Therefore, the high dose of Aconitum can cause arrhythmia, heart failure and even death. A. brachypodum is a perennial herb of the genus Aconitum in Ranunculaceae, distributed mainly in Yunnan, Sichuan, and Tibet. Songorine (Figure 1) is a diterpenoid alkaloid of C22 isolated from A. brachypodum with various bioactivities

involved

in

anti-arrhythmia,9

anti-anxiety,10

analgesia,11

anti-inflammation,12

anti-infection and promoting tissue regeneration.13 Our recent investigation revealed that songorine (IC50 = 3.21 nM) had inhibitory activities of GPCRs by using DAS-ELISA in vitro. Herein, a series of derivatives of songorine were synthesized via chemical modification on hydroxyl groups at 1,15-position, C16 (17) double bond, and C12 carbonyl group. Furthermore, their GPCRs inhibitory activities were also evaluated by enzyme linked immunosorbent assay (ELISA) of mouse GPCRs and their inhibition rates and IC50 values were calculated by absorbance for further study of the structure-activity relationships (SARs).

Figure 1

The chemical structure of songorine

2. Results and discussion 2.1 Chemistry The derivatives of songorine were synthesized based on the structural characteristics of songorine

including acylation, oxidation, nucleophilic addition, sulfidation and hydrogenation at positions of C-1, C-12, C-15, C-16, and C-17. The synthesis routes of songorine derivatives were shown in Schemes 1-2. The presence of free hydroxyl groups in songorine allowed us to synthesize ester derivatives for evaluating the effect of ester side chain on their GPCRs inhibitory activities (Scheme 1). 14-17 The reaction of songorine with acetyl chloride in the presence of 4-dimethylaminopyridine (DMAP) in pyridine produced derivatives 1a and 1b. Songorine reacted with propionic anhydride and butyric anhydride to achieve acyl-extended derivatives 2, 3a and 3b. In the presence of DMAP and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) in CH2Cl2, the reaction of songorine with methoxyacetic acid and ethoxyacetic acid produced derivatives 4 and 5, respectively. Moreover, large acyl groups were also considered. Interestingly, C15-OH gave priority to become ester. The treatment of songorine with benzoic acid and cinnamic acid achieved derivatives 6 and 7, respectively. As shown in Scheme 2, the treatment of songorine with NaBH4 in EtOH afforded a racemic 8a (R-isomer) and 8b (S-isomer) and its configuration was confirmed.18-20 Songorine with m-chloroperoxybenzoic acid (m-CPBA) yielded opening ring derivative 9. With Dess-Matin periodinane, the hydroxyl group of songorine could be oxidized to product oxidation derivatives 10a and 10b.21 Songorine treated with KOH and NH2OH·HCl achieved oxime derivative 11,22 which was then esterified with propionic anhydride to obtain dipropionylated derivative 12a and tripropionylated derivative 12b.23 The carbonyl group of songorine with amino group of p-toluenesulfonyl hydrazide could form a hydrazone derivative 13 in dry EtOH. Songorine reacted with Lawesson's reagent to obtain a thiocarbonyl group and sulfhydryl group-substituted derivative 14.24,25

Scheme 1

Reagents and conditions: (i) RCOCl, Pyridine, DMAP, r.t., 2 h, 20%; (ii) RCOOH, DMAP,

EDC·HCl, dry CH2Cl2, r.t., 2 h, 10%-60%; (iii) (RCOO)2O, DMAP, Pyridine, r.t., 2 h, 15%-30%.

Scheme 2

Reagents and conditions: (iv) NaBH4, dry EtOH, r.t., 2 h, 40%-60%; (v) m-CPBA, dry

CH2Cl2, r.t., 24 h, 22%; (vi) Dess–Martin periodinane, dry CH2Cl2, 0 ℃-r.t., 3 h, 20%-30%; (vii) NH2OH·HCl, 10% KOH, dry EtOH, reflux, 2-3 h, 33%; (viii) propionic anhydride, DMAP, EDC·HCl, dry CH2Cl2, r.t., 2 h, 25%-30%; (ix) p-toluene sulfonyl hydrazine, dry EtOH, reflux, 12 h, 43%; (x) Lawesson's reagent, dry xylene, 120-140 ℃, 2-4 h, 30%. 2.2 The inhibitory activities of GPCRs in vitro

The synthesized songorine derivatives were tested for their GPCRs inhibitory activities by using DAS-ELISA to assay the content of GPCRs in the samples (GPCRs standard as control). The GPCRs inhibitory activity of every compound was expressed as the concentration of compound that achieved 50% inhibition (IC50) of GPCRs standard and inhibition rates at 10 ng/L. The results were summarized in Table 1. Songorine showed moderate activity inhibition of GPCRs (IC50 = 3.21 nM). To investigate the effect of hydroxyl groups in songorine on the GPCRs inhibitory activity, derivatives 1a-7 were

synthesized by the introduction of acyl group into C1-OH or (and) C15-OH and tested for their inhibitory activities on GPCRs (Table 1). Derivatives 1a, 2, 3a, 4, and 7 showed a significant inhibitory activity on GPCRs (IC50 = 0.51 nM, 0.59 nM, 0.14 nM, 0.08 nM, 0.29 nM, respectively); however, derivatives 1b, 3b and 6 with acylation of hydroxyl group at C-15 position displayed weak inhibitory activities on GPCRs. Diacetyl derivatives 1a and dibutyryl derivatives 3a with acylation of hydroxyl group at C-1 and C-15 position exhibited more significantly potent activities inhibition against GPCRs (IC50 = 0.51 nM, 0.14 nM, respectively) than corresponding monoacyl derivatives (1a vs. 1b, 3a vs. 3b), meaning that diacylation of hydroxyl groups at C-1 and C-15 position resulted in the enhancement of the GPCRs inhibitory activity. Acetylated derivative 1a (IC50 = 0.51 nM) and propionylated derivative 2 (IC50 = 0.59 nM) showed moderate inhibitory activity on GPCRs. In addition, the replacement of acyl moiety with butyryl yielded derivative 3a (IC50 = 0.14 nM) possessing strong inhibitory activity, which demonstrated that the inhibitory activity of the derivatives increases with the increase of the acyl chain length. Methoxyacetyl derivative 4 exhibited highly inhibitory potency on GPCRs than butyryl derivative 3a (IC50 = 0.08 nM vs. IC50 = 0.14 nM). To further study the activity of songorine’s oxygen atoms in ester carbon chain derivatives, acetoxyacetyl derivative 5 was obtained. Unfortunately, derivatives 5 showed a significant decrease in inhibition on GPCRs compared with derivatives 4 (IC50 = 0.08 nM vs. IC50 = 5.97 nM). For the derivatives of aromatic acyl 6 (IC50 = 4.50 nM) and 7 (IC50 = 0.29 nM), derivatives 7 showed high potency to GPCRs, which indicated that the extension of conjugate system causes the increase of suppressant property on GPCRs. Both C1-OH and C15-OH in songorine are replaced by short-chain acyl group at the same time (n = 2, 3, 4), which displayed an excellent inhibitory activity (n = 4 is the best). C15-OH is replaced by long conjugate acyl group, which exhibits a prominent inhibitory activity. With an effort to gain more information on SARs of songorine, we observed the additional structure changes in carbonyl group (Figure 2). Derivatives 8a (R-isomer) and 8b (S-isomer) were inactive in inhibiting GPCRs, suggesting that C12 hydroxyl group may not be preferred for inhibitory activities on GPCRs. Derivative 9, which was obtained by oxidation of the double bond of songorine followed by opening the ring in the acidic surroundings, resulted in a tenfold increase in inhibition

effect on GPCRs (IC50 = 0.23 nM vs. IC50 = 3.21 nM). This suggested that the replacement of C16 (17) double bond by a large group might increase the inhibitory potency to GPCRs. Derivative 10a with one hydroxyl group oxidized and derivative 10b with both hydroxyl groups oxidized, derived from oxidation of songorine, decreased inhibition to GPCRs (IC50 = 9.99 nM, >10 nM, respectively) compared to songorine (IC50 = 3.21 nM), which indicated that carbonyl group at C-1 or (and) C-15 led to the decrease of suppressant property on GPCRs. Derivative 11 had good inhibitory activity against GPCRs. For the tripropionyl derivative 12a and dipropionyl derivative 12b from derivative 11, derivative 12a resulted in decreased inhibition on GPCRs and derivative 12b led to loss of inhibition on GPCRs compared to songorine. Derivative 13 was inactive in inhibiting GPCRs. Meanwhile, derivative 14, which resulted from songorine by bioisosteric replacements, showed loss of inhibition to GPCRs. In summary, the carbonyl group of songorine has an effect on the inhibitory activity of GPCRs. If the polarity of carbonyl group is enhanced slightly such as derivative 11, IC50 value could decrease obviously, meaning that it has prominent bioactivities. However, the polarity should not be very strong. For instance, derivatives 8a and 8b are completely replaced by hydroxyl group, and their IC50 value will increase. If the lipophilic group is attached, IC50 value will increase either.

Table 1 Inhibition ratesa and IC50 (nM) of GPCRs inhibitory activitives in vitro % GPCRs No.

b

R1

R2

R3

inhibition rates

IC50

(10 ng/L) Songorine

OH

1a

1b

OH

OH

62.8

3.21

65.2

0.51

48.6

2.62

2

65.9

0.59

3a

77.4

0.14

49.8

8.68

4

85.3

0.08

5

55.0

5.91

OH

3b

6

OH

37.7

4.50

7

OH

57.1

0.29

8a

OH

OH

46.3

＞10

8b

OH

OH

39.4

＞10

9

/

/

68.7

0.23

10a

OH

54.6

9.99

34.6

＞10

73.3

0.55

36.4

＞10

50.3

3.65

10b OH

11

OH

12a

/

12b

OH

13

OH

OH

37.7

＞10

14

SH

SH

39.3

＞10

a

Inhibition rate = [OD (control)-OD (test compound)] / OD (control) × 100%

b

The derivatives numbering corresponds to that given in Schemes 1 and 2.

3. Conclusions In summary, a series of derivatives were synthesized based on songorine and tested for their

GPCRs inhibitory activity in vitro. Most derivatives had inhibitory activities of GPCRs by using DAS-ELISA in vitro. In addition, the most promising derivatives 3a, 4 and 7 exhibited high activities against GPCRs (IC50 = 0.14 nM, 0.08 nM, 0.29 nM, respectively). Interestingly, the ester group in songorine had a great effect on its activity. Based on the data in Table 1, SARs of Songorine derivatives in GPCRs antagonism are summarized in Figure 2.

Figure 2

The SARs of songorine derivatives in GPCRs inhibitory activity

4. Experiment 4.1 General

All solvents were distilled and dried prior to use. Reagents and materials were obtained from commercial suppliers and were used without further purification. Songorine was isolated from A. brachypodum and had the purity of >98.0%. Thin-layer chromatography (TLC) was carried out on precoated silica gel plates GF254 for the analysis of reactions. Spots were visualized by UV light or by spraying with bismuth potassium iodide reagents. 1H NMR and 13C NMR spectra were recorded on a Bruker DRX 500 MHz NMR spectrometer. ESIMS were recorded on an Advantage Max LCQ Thermo-Finnigan mass spectrometer. Column Chromatography was performed using silica gel

(300-400 mesh). The Elisa kit was obtained from Shanghai Fusheng Industry Co., Ltd., and the standard in the kit was Class B GPCRs.

4.2 Chemistry

4.2.1 General procedure for preparation of derivatives 1a-3b To a solution of songorine (1 equiv.), DMAP (1 equiv.), and corresponding acid anhydride or acyl chloride (2.4 equiv.) in anhydrous CH2Cl2 (1 mL) and pyridine (1 mL) at room temperature. Pyridine was added as an acid-binding agent. The resulting mixture was stirred at room temperature until the starting material was not observed by TLC. The reaction mixture was filtered, and the residue was washed with CH2Cl2 (3 × 30 mL). Then, the CH2Cl2 solution was washed with saturated NaHCO3 (3 × 30 mL) and saturated NaCl (3 × 30 mL), respectively. Subsequently, the organic layer was dried over anhydrous Na2SO4, filtered and filtrate evaporated under reduced pressure. Finally, the residue was purified by column chromatography over the silica gel to yield the pure target derivatives.

4.2.1.1 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-3-methyl-10-methylene-8-oxotetradecahydro3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,11-diyl dia-cetate (1a) White amorphous power, yield 18% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 15:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.25 (1H, s), 5.03 (1H, s), 5.01 (1H, s), 4.96 (1H, t, J = 7.6 Hz), 3.09 (1H, m), 2.51 (2H, m, J = 6.4 Hz), 2.11 (3H, s), 2.03 (3H, s), 2.01 (2H, m), 1.96 (2H, d, J = 12.4 Hz), 1.95 (1H, d, J = 2.1 Hz), 1.56 (2H, m), 1.69 (3H, t, J = 6.2 Hz), 1.58-1.24 (6H, m), 1.22 (1H, t, J = 8.8 Hz), 1.09 (1H, m), 0.98 (1H, m), 0.75 (3H, s);

13

C NMR

(CDCl3, 125 MHz) δ: 208.9, 170.6, 170.3, 145.2, 112.9, 74.4, 65.7, 56.9, 54.3, 50.7, 50.4, 49.8, 49.0, 44.1, 37.8, 37.3, 36.7, 34.3, 32.3, 29.7, 26.7, 25.8, 23.1, 21.9, 21.5, 13.5; ESI-MS: m/z 464.5 [M + Na]+.

4.2.1.2 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-6-hydroxy-3-methyl-10-methylene-8-oxotetra decahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-11-yl acetate (1b) White amorphous power, yield 19% (after chromatography with petroleum ether/ethyl

acetate/diethylamine, 15:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.64 (1H, s), 5.24 (1H, s), 4.94 (1H, s), 3.37 (1H, m, J = 7.4 Hz), 3.01 (2H, m), 2.40 (2H, m, J = 6.3 Hz), 2.21 (2H, d, J = 12.2), 2.11 (3H, s), 1.94 (1H, m), 1.55 (2H, m, J = 3.3 Hz, 9.2 Hz), 1.43 (2H, m), 1.42 (2H, m), 1.40-1.23 (6H, m), 1.11 (3H, t, J = 6.2 Hz), 0.87 (1H, m), 0.76 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 209.6, 170.6, 145.3,

112.6, 77.3, 70.2, 65.7, 57.2, 54.0, 52.2, 50.9, 49.3, 43.4, 38.0, 37.4, 36.5, 34.2, 32.2, 32.1, 29.7, 26.1, 23.5, 21.5, 13.6; ESI-MS: m/z 422.2 [M + Na]+.

4.2.1.3 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-3-methyl-10-methylene-8-oxotetradecahyd-ro3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,11-diyl di-propionate (2) White amorphous power, yield 16% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 15:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.35 (1H, s), 5.23 (1H, s), 4.93 (1H, s), 3.68 (1H, t, J = 7.51Hz), 2.53 (1H, m), 2.49 (2H, m, J = 6.7Hz), 2.42-2.43 (4H, m), 2.21 (2H, d, J = 12.4Hz), 2.04 (2H, m), 2.01 (1H, d, J = 1.7Hz), 1.83-1.38 (4H, m), 1.27-1.26 (6H, m), 1.26-1.20 (6H, m), 1.17 (3H, t, J = 6.4Hz), 1.16 (3H, s), 0.94 (1H, m); 13C NMR (CDCl3, 125 MHz) δ: 209.5, 173.9, 172.5, 149.8, 148.1, 123.1, 112.0, 76.7, 76.4, 74.0, 65.5, 50.6, 50.4, 44.0, 42.9, 36.7, 34.2, 32.3, 31.4, 30.2, 29.7, 28.4, 28.3. 27.5, 27.4, 24.0, 23.8, 13.5; ESI-MS: m/z 470.1 [M + H]+.

4.2.1.4 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-3-methyl-10-methylene-8-oxotetradecahydro3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,11-diyl dibutyrate (3a) White amorphous power, yield 27% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 10:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.38 (1H, s), 5.32 (1H, s), 5.05 (1H, s), 4.32 (1H, t, J = 7.1 Hz), 3.52 (1H, m), 2.40 (2H, m, J = 6.5 Hz), 2.35-2.32 (4H, m), 2.20-1.88 (4H, m), 1.97 (1H, d, J = 4.5 Hz), 1.80-1.53 (8H, m), 1.44-1.32 (4H, m), 1.28 (1H, t, J = 9.1 Hz), 1.16 (1H, m), 1.14 (3H, t, J = 6.5 Hz), 1.04-1.01 (6H, m), 0.92 (1H, m), 0.75 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 209.3, 173.1, 172.8, 150.7, 112.9, 76.1, 74.2, 69.6, 57.1, 56.8, 52.4, 50.2, 48.9, 48.6, 44.0, 37.8, 37.7, 36.8, 35.3, 35.2, 34.1, 27.1, 25.7, 23.5, 18.5, 18.3, 13.7, 13.6, 12.5; ESI-MS: m/z 498.1 [M + H]+.

4.2.1.5 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-6-hydroxy-3-methyl-10-methylene-8-oxotetra decahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-11-yl butyrate (3

b) White amorphous power, yield 62% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 10:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.24 (1H, s), 5.16 (1H, s), 5.05 (1H, s), 2.94 (1H, m), 2.45 (2H, t, J = 6.5 Hz), 2.36 (2H, t, J = 8.2 Hz) 2.32 (1H, t, J = 2.5 Hz), 2.26-2.19 (6H, m), 1.94 (1H, d, J = 1.5 Hz), 1.58 (1H, s), 1.57-1.42 (8H, m), 1.32 (1H, t, J = 8.5 Hz), 1.19 (1H, m), 1.07 (6H, m, J = 6.5 Hz), 0.93 (1H, m), 0.61 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 208.9, 173.1,

145.2, 112.9, 73.9, 65.6, 65.5, 57.0, 50.5, 50.4, 48.7, 43.8, 42.3, 37.7, 37.0, 36.8, 36.7, 34.2, 32.3, 29.7, 26.7, 25.7, 25.5, 18.4, 18.3, 13.5; ESI-MS: m/z 428.1 [M + H]+.

4.2.2 General procedure for preparation of derivatives 4-7 EDC·HCl (30 equiv.) was added to the solution of songorine (1 equiv.), DMAP ( 1 equiv.), and corresponding carboxylic acid (2.4 equiv.) in anhydrous CH 2Cl2 (2 mL) at room temperature. The resulting mixture was stirred at room temperature until the starting material was not observed by TLC. The reaction mixture was filtered, and the residue was washed with CH 2Cl2 (3 × 30 mL). Then, the CH2Cl2 solution was washed with saturated NaHCO 3 (3 × 30 mL) and saturated NaCl (3 × 30 mL), respectively. Subsequently, the organic layer was dried over anhydrous Na2SO4, filtered and filtrate evaporated under reduced pressure. Finally, the residue was purified by column chromatography over the silica gel to yield the pure target derivatives.

4.2.2.1 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-3-methyl-10-methylene-8-oxotetradecahydro3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,11-diyl bis(2-methoxyaceta te) (4) Brown amorphous power, yield 8% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 40:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.72 (1H, s), 5.26 (1H, s), 5.12 (1H, s), 4.98 (1H, t, J = 7.9Hz), 3.88 (2H, s), 3.42 (2H, s), 3.29 (3H, s), 3.09 (3H, m), 2.45 (2H, m, J = 6.4Hz), 2.29-2.01 (5H, m), 1.99 (1H, d, J = 1.4Hz), 1.98-1.57 (4H, m), 1.39-1.24 (6H, m), 1.07 (3H, t, J = 6.5Hz), 0.87 (1H, m), 0.74 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 208.4, 169.9, 169.6, 144.8, 113.4, 77.3, 75.1, 70.4, 70.2, 65.7, 59.4, 56.9, 54.2, 50.9, 50.6, 50.3, 49.8, 49.2, 44.0, 37.6, 37.3, 36.6, 34.2, 32.3, 29.3, 26.7, 25.7, 13.5; ESI-MS: m/z 524.1 [M + Na]+.

4.2.2.2 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-3-methyl-10-methylene-8-oxotetradecahydro3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,11-diyl bis(2-ethoxyacetate) (5) Brown amorphous power, yield 66% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 15:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.69 (1H, s), 5.24 (1H, s), 4.96 (1H, s), 4.41 (3H, t, J = 7.8Hz), 4.09 (2H, s), 3.55-3.51 (4H, m), 3.06 (1H, s), 2.31 (2H, m, J = 6.2Hz), 2.30-1.97 (4H, m), 1.97 (1H, d, J = 2.01Hz), 1.96-1.55 (4H, m), 1.55-1.20 (12H, m), 1.05 (3H, t, J = 5.8Hz), 1.02 (1H, m), 0.72 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 208.4, 170.1, 169.8, 144.8, 113.2, 77.3, 68.8, 68.4, 67.3, 67.1, 65.8, 56.8, 54.2, 50.6, 50.2, 49.8, 49.2, 43.9, 37.6, 37.2, 36.6, 34.1, 32.2, 31.4, 30.1, 29.6, 26.6, 25.7, 14.9, 13.4; ESI-MS: m/z 552.3 [M + Na]+.

4.2.2.3 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-6-hydroxy-3-methyl-10-methylene-8-oxotetra decahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-11-yl benzoate (6) White amorphous power, yield 17% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 20:1:1); 1H NMR (CDCl3, 500 MHz) δ: 7.83 (2H, m), 7.55 (2H, m), 7.22 (2H, m), 5.32 (1H, s), 5.16(1H, s), 5.09 (1H, s), 2.88 (1H, m), 2.38 (2H, m, J = 6.3 Hz), 2.30-2.21 (5H, m), 2.15 (2H, d, J = 3.5 Hz), 1.89-1.63 (5H, m), 1.61 (1H, m, J = 3.4 Hz, 4.2 Hz), 1.48-1.12 (5H, m), 1.11 (2H, t, J = 6.3 Hz), 0.74 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 209.1, 177.4, 155.3, 138.2, 138.1,

129.6, 129.6, 129.0, 129.0, 124.9, 118.3, 78.3, 71.5, 69.0, 59.1, 58.2, 51.8, 50.5, 48.6, 47.3, 43.2, 35.8, 35.5, 34.7, 29.3, 24.6, 27.3, 25.9, 14.0; ESI-MS: m/z 462.2 [M + H]+.

4.2.2.4 Synthesis of (3R,6S,6aR,11R,12R)-1-ethyl-6-hydroxy-3-methyl-10-methylene-8-oxotetra decahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-11-yl cinnamate (7) Yellow amorphous power, yield 11% (after chromatography with petroleum ether/ethyl acetate/diethylamine, 30:1:1); 1H NMR (CDCl3, 500 MHz) δ: 7.55 (3H, d, J = 15.6 Hz), 7.43 (2H, d, J = 6.5 Hz), 7.14 (1H, d, J = 7.4 Hz), 6.49 (1H, d, J = 14.8 Hz), 5.36 (1H, s), 5.32 (1H, s), 5.03 (1H, s), 3.40 (2H, m), 2.90 (2H, m), 2.37 (2H, m, J = 6.1 Hz), 2.25 (2H, m, J = 9.3 Hz,3.3 Hz), 2.21 (2H, m),

2.05-1.61 (4H, m), 2.02 (1H, s), 1.61-1.25 (4H, m), 1.58 (1H, s), 1.25 (1H, t, J = 9.0 Hz), 1.15 (2H, m), 0.98 (1H, m), 0.72 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 207.7 169.2, 151.7, 137.0, 143.5, 129.6,

129.6, 129.5, 129.0, 129.0, 118.5, 113.2, 109.7, 79.2, 70.6, 67.1, 59.9, 56.2, 55.6, 52.6, 50.0, 46.2, 43.8, 42.4, 36.0, 35.5, 33.9, 29.4, 27.8, 25.6, 14.1; ESI-MS: m/z 488.1 [M + H]+.

4.2.3 General procedure for preparation of derivatives 8a and 8b To a solution of dry EtOH (1 mL), songorine (1 equiv.), and NaBH4 (1.5 equiv.) was added at room temperature. The reaction was warmed to 50-60 ℃ and stirred 4 h. The reaction mixture was diluted with cold water and extracted with CH2Cl2 (3 × 30 mL). Organic layer was dried over anhydrous Na2SO4 and concentrated to dryness under reduced pressure. The residue was chromatographed using a silica gel column to yield the pure target derivatives.

4.2.3.1 Synthesis of (3R,6S,6aR,8R,11R,12R)-1-ethyl-3-methyl-10-methylenetetradecahydro-3,6 a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,8,11-triol (8a) White

amorphous

power,

yield

60%

(after

chromatography

with

petroleum

ether/acetone/diethylamine, 10:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.32: (1H, s), 5.10 (1H, s), 4.18 (1H, s), 2.53 (1H, m, J = 3.5 Hz, 4.5 Hz), 2.40 (2H, t, J = 6.1Hz), 2.39 (1H, t, J = 7.7 Hz), 2.22 (2H, m), 2.06 (1H, m), 2.04 (1H, d, J = 2.0 Hz), 1.72 (2H, m), 1.64 (2H, m), 1.59 (2H, s), 1.36-1.26 (6H, m), 1.28 (1H, s), 1.23 (1H, m), 1.09 (3H, m, J = 6.3Hz), 0.86 (2H, m), 0.75 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 154.6, 111.8, 76.8, 69.8, 67.2, 58.3, 52.6, 51.0, 48.4, 43.7, 37.1, 35.9, 33.8, 32.6, 31.4, 29.7, 27.2, 26.4, 23.5, 22.7, 14.1, 13.4; ESI-MS: m/z 360.1 [M + H]+.

4.2.3.2 Synthesis of (3R,6S,6aR,8S,11R,12R)-1-ethyl-3-methyl-10-methylenetetradecahydro-3,6 a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,8,11-triol (8b) White

amorphous

power,

yield

47%

(after

chromatography

with

petroleum

ether/acetone/diethylamine, 10:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.16 (1H, s), 5.13 (1H, s), 4.17 (1H, s), 2.53 (1H, m, J = 3.6 Hz, 4.5 Hz), 2.45 (2H, t, J = 6.3Hz), 2.25 (2H, m), 2.11 (1H, m), 2.01 (2H, m), 1.76 (2H, m), 1.63 (2H, m), 1.60 (2H, s), 1.36-1.32 (6H, m), 1.31 (1H, s), 1.24 (1H, t, J = 7.1 Hz), 1.09 (3H, m, J = 6.0 Hz), 0.85 (2H, m), 0.77 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 159.1, 108.7,

76.8, 69.7, 66.0, 58.2, 52.9, 50.2, 48.2, 43.9, 36.5, 36.1, 34.1, 31.4, 30.7, 29.7, 28.6, 26.4, 23.4, 22.7, 14.1, 13.1; ESI-MS: m/z 360.1 [M + H]+.

4.2.4 Synthesis of (3R,6S,6aR,11S,12R)-1-ethyl-6,11-dihydroxy-10-(hydroxymethyl)-3-methyl-8oxotetradecahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-10-yl 3-ch lorobenzoate (9) To a solution of dry CH2Cl2 (2 mL), songorine (25 mg, 0.07 mmol), and m-CPBA (14.49 mg, 0.08 mmol) was added at room temperature. The reaction was warmed to 50-60 ℃ and stirred 4 h. The reaction mixture was diluted with cold water and extracted with CH 2Cl2 (3 × 30 mL). Then the CH2Cl2 solution was washed with saturated NaHCO3 (3 × 30 mL), respectively. Organic layer was dried over anhydrous Na2SO4, filtered and filtrate evaporated under reduced pressure. The crude material was subjected to column chromatography by using petroleum ether/ethyl acetate/diethylamine (30:1:1) as an eluent, and afforded pure derivative 9 as the brown amorphous powder. Yield: 22%; 1H NMR (CDCl3, 500 MHz) δ: 8.56 (1H, m), 7.75 (1H, m), 7.55 (1H, m), 7.49 (1H, t, J = 7.9 Hz), 3.89 (1H, s), 3.77 (2H, m), 3.34 (1H, t, J = 7.1 Hz), 2.90 (1H, t, J = 2.7 Hz), 2.42 (2H, m, J = 6.3 Hz), 2.29 (2H, m), 1.85 (2H, m), 1.82 (2H, m), 1.74 (2H, m), 1.4 (1H, s), 1.45 (1H, s), 1.30 (1H, s), 1.25(1H, t, J = 8.7 Hz), 1.22 (4H, m), 1.17 (1H, s), 1.15 (1H, m), 1.12 (3H, t, J = 6.5 Hz), 0.91 (1H, m), 0.72 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 206.4, 159.8, 134.1, 133.2, 130.6, 129.9, 129.0, 124.5, 94.1, 70.6, 70.3, 67.8, 61.4, 59.1, 52.3, 52.2, 48.8, 47.6, 45.2, 43.8, 37.2, 36.0, 35.9, 34.5, 29.6, 29.5, 27.2, 25.5, 14.1; ESI-MS: m/z 530.2 [M + H]+.

4.2.5 General procedure for preparation of derivatives 10a and 10b To a solution of dry CH2Cl2 (2 mL), songorine (1 equiv.), and Dess-Matin periodinane (2 equiv.) was added at 0 ℃. The reaction was warmed to room temperature and stirred 3 h. The reaction mixture was diluted with cold water and extracted with CH2Cl2 (3 × 30 mL). Then the CH2Cl2 solution was washed with saturated NaHSO3 (3 × 30 mL). Organic layer was dried over anhydrous Na 2SO4, filtered and filtrate evaporated under reduced pressure. The residue was chromatographed using a silica gel column to yield the pure target derivatives.

4.2.5.1 Synthesis of (3R,6S,6aR,12R)-1-ethyl-6-hydroxy-3-methyl-10-methylenedodecahydro-3, 6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-8,11-dione (10a) White

amorphous

power,

yield

22%

(after

chromatography

with

petroleum

ether/acetone/diethylamine, 7:1:1); 1H NMR (CDCl3, 500 MHz) δ: 6.00 (1H, s), 5.75 (1H, s), 3.47 (1H, t, J = 7.3 Hz), 2.90 (1H, m), 2.42 (2H, m), 2.30-1.96 (4H, m), 1.92 (1H, d, J = 2.5 Hz), 1.90-1.74 (4H, m), 1.56 (1H, s), 1.54 (1H, t, J = 9.0 Hz), 1.51-1.21 (5H, s), 1.14 (3H, t, J = 6.4 Hz), 0.98 (1H, t, J = 6.5 Hz), 0.66 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 208.4, 205..2, 142.3, 118.9, 69.8, 66.0, 65.8,

59.6, 56.8, 52.5, 49.7, 46.4, 46.3, 42.6, 40.2, 38.5, 37.4, 33.9, 30.8, 27.0, 25.7, 13.4; ESI-MS: m/z 356.5 [M + H]+.

4.2.5.2 Synthesis of (3R,6aR,12R)-1-ethyl-3-methyl-10-methylenedecahydro-3,6a,12-(epiethane [1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,8,11(1H)-trione (10b) White

amorphous

power,

yield

31%

(after

chromatography

with

petroleum

ether/acetone/diethylamine, 7:1:1); 1H NMR (CDCl3, 500 MHz) δ: 6.03 (1H, s), 5.56 (1H, s), 2.92 (1H, m), 2.41 (2H, m), 2.37-2.31 (2H, m), 2.29-2.00 (5H, m), 1.93-1.55 (5H, m), 1.53-1.41 (3H, m), 1.26 (1H, t, J = 6.9 Hz), 1.10 (3H, t, J = 6.1 Hz), 0.72 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 214.0, 208.3, 204.4, 142.0, 119.5, 69.8, 62.5, 61.4, 57.0, 54.8, 53.4, 50.6, 42.2, 41.1, 41.0, 39.5, 39.0, 38.8, 34.3, 29.3, 24.8, 13.3; ESI-MS: m/z 354.5 [M + H]+.

4.2.6 Synthesis of (3R,6S,6aR,11R,12R,Z)-1-ethyl-6,11-dihydroxy-3-methyl-10-methylenedodeca hydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-8(9H)-one oxime (11) To a solution of dry EtOH (1 mL), songorine (50 mg, 0.14 mmol), KOH (11.8 mg, 0.21 mmol) and NH2OH·HCl (14.6 mg, 0.21 mmol) was added at room temperature. The reaction was warmed to 50-60 ℃ and stirred 2-3 h. The reaction mixture was diluted with cold water and extracted with CH 2Cl2 (3 × 30 mL). Organic layer was dried over anhydrous Na 2SO4, filtered and filtrate evaporated under reduced pressure. The crude material was subjected to column chromatography by using petroleum ether/ethyl acetate/diethylamine (7:1:1) as an eluent, and afforded pure derivative 11 as the white amorphous powder. Yield: 33 %; 1H NMR (CDCl3, 500 MHz) δ: 7.92 (1H, s), 5.27 (1H, s), 5.16 (1H, s), 4.07 (1H, m), 3.91 (1H, s), 3.31 (1H, t, J = 7.1 Hz), 2.52 (2H, m, J = 6.2 Hz), 2.21-1.87 (5H, m),

1.71-1.46 (4H, m), 1.56 (1H, m), 1.45-1.19 (4H, m), 1.37 (1H, t, J = 8.6 Hz), 1.32 (1H, s), 1.19 (1H, m), 1.11 (3H, s), 0.92 (1H, m), 0.88 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 153.9, 149.3. 129.5, 76.7, 69.1, 59.1, 52.3, 51.0, 49.5, 48.4, 48.2, 45.0, 42.1, 41.2, 36.7, 35.2, 32.6, 29.9, 29.5, 25.4, 27.6, 13.8; ESI-MS: m/z 373.0 [M + H]+.

4.2.7 General procedure for preparation of derivatives 12a and 12b Derivative 11 (1 equiv.), DMAP (1 equiv), EDC·HCl (30 equiv.) and (CH3CH2CH2O)2O (4 equiv.) was dissolved in 2 mL of dry CH2Cl2, stirred at room temperature for 2-3 h. The reaction mixture was diluted with cold water and extracted with CH2Cl2 (3 × 30 mL). Organic layer was dried over anhydrous Na2SO4, filtered and filtrate evaporated under reduced pressure. The residue was chromatographed using a silica gel column to yield the pure target derivatives.

4.2.7.1 Synthesis of (3R,6S,6aR,11R,12R,Z)-1-ethyl-3-methyl-10-methylene-8-((propionyloxy)im ino)tetradecahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-6,11-diyl dipropionate (12a) White

amorphous

power,

yield

26%

(after

chromatography

with

petroleum

ether/acetone/diethylamine, 10:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.49 (1H, s), 5.15 (1H, s), 5.05 (1H, s), 4.31 (1H, t, J = 5.0 Hz), 3.91 (1H, m), 2.66 (2H, m, J = 7.0 Hz), 2.56 (2H, m, J = 7.0 Hz), 2.41 (4H, m), 2.31-1.89 (5H, m), 1.51 (2H, m), 1.45 (2H, m), 1.31 (2H, m), 1.28 (3H, t, J = 7.5 Hz), 1.26-1.24 (4H, m), 1.22 (3H, m, J = 7.0 Hz), 1.20 (2H, m), 1.18 (1H, m), 1.08 (3H, t, J = 6.5 Hz), 1.01 (1H, m), 0.7(3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 174.0, 173.3, 171.6, 167.4, 148.0, 113.5, 75.7,

73.9, 65.5, 56.9, 50.5, 50.1,49.8, 49.5, 44.2, 41.9, 38.3, 37.6, 35.8, 34.0, 29.0, 7.2, 28.1, 27.8, 27.1, 24.5, 26.3, 13.5, 9.1, 8.9, 8.8; ESI-MS: m/z 541.3 [M + H]+.

4.2.7.2 Synthesis of (3R,6S,6aR,11R,12R,Z)-1-ethyl-6-hydroxy-3-methyl-10-methylene-8-((propi onyloxy)imino)tetradecahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azoci n-11-yl propionate (12b) White

amorphous

power,

yield

31%

(after

chromatography

with

petroleum

ether/acetone/diethylamine, 10:1:1); 1H NMR (CDCl3, 500 MHz) δ: 5.47 (1H, s), 5.17 (1H, s), 5.12

(1H, s), 4.10 (1H, m), 3.44 (1H, t, J = 7.0 Hz), 3.01 (2H, m, J = 6.5 Hz), 2.75 (2H, m, J = 6.5 Hz), 2.55 (2H, m, J = 6.5 Hz), 2.29-1.98 (4H, m), 1.88 (1H, m), 1.72-1.25 (15H, m), 1.27 (3H, m), 1.24 (1H, t, J = 8.5 Hz), 1.21 (1H, s), 0.92 (1H, m), 0.70 (3H, s);

13

C NMR (CDCl3, 125 MHz) δ: 175.2, 174.0,

156.9, 150.1, 112.7, 78.9, 68.9, 66.3, 58.9, 53.4, 51.5, 51.4, 47.0, 45.6, 42.7, 42.2, 35.9, 35.8, 35.1, 29.6, 28.4, 28.1, 27.1, 26.8, 25.4, 11.1, 6.1, 5.8; ESI-MS: m/z 522.7 [M + K]+.

4.2.8 Synthesis of N'-((3R,6S,6aR,11R,12R,Z)-1-ethyl-6,11-dihydroxy-3-methyl-10-methylenedo decahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocin-8(9H)-ylidene)-4methylbenzenesulfonohydrazide (13) To a solution of dry EtOH (2 mL), songorine (50 mg, 0.14 mmol), and p-toluene sulfonyl hydrazine (52.1 mg, 0.28 mmol) was added at room temperature. The reaction was warmed to 50-60 ℃ and stirred 2-3 h. The reaction mixture was diluted with cold water and extracted with CH2Cl2 (3 × 30 mL). Organic layer was dried over anhydrous Na2SO4, filtered and filtrate evaporated under reduced pressure. The crude material was subjected to column chromatography by using DCM/methyl alcohol/diethylamine (300:1:1) as an eluent, and afforded pure derivative 13 as the brown amorphous powder. Yield: 43%; 1H NMR (CDCl3, 500 MHz) δ: 7.83 (2H, d, J = 7.3 Hz), 7.31 (2H, d, J = 7.2 Hz), 5.18 (1H, s), 5.09 (1H, s), 3.99 (1H, s), 3.66 (1H, m), 3.23 (1H, t, J = 7.5 Hz), 2.47 (3H, s), 2.38 (2H, m, J = 6.5 Hz), 2.21 (2H, m), 2.20-1.90 (5H, m), 1.60 (1H, s), 1.55(2H, m), 1.46 (1H, m), 1.46 (1H, s), 1.41 (1H, s), 1.17 (2H, m), 1.09 (3H, t, J = 6.4 Hz), 1.07 (2H, m), 0.98 (1H, m), 0.89 (1H, m), 0.75 (3H, s); 13C NMR (CDCl3, 125 MHz) δ: 162.0, 144.0, 143.4, 135.8, 129.4, 129.4, 128.0, 128.0, 112.4, 83.2, 70.3, 65.9, 57.7, 53.4, 52.4, 49.0, 48.7, 44.9, 42.7, 42.2, 36.5, 36.2, 33.0, 29.3, 27.1, 26.1, 24.5, 21.6, 13.3; ESI-MS: m/z 526.0 [M + H]+.

4.2.9 Synthesis of (3R,6aS,11R,12R)-1-ethyl-11-hydroxy-3-methyl-10-methylene-1,2,3,4,6b,7,10, 11,12,12a-decahydro-3,6a,12-(epiethane[1,1,2]triyl)-9,11a-methanoazuleno[2,1-b]azocine-8(9H)-t hione (14) To a solution of dry xylene (2 mL), songorine (50 mg, 0.14 mmol), and Lawesson's reagent (68 mg, 0.17 mmol) was added at room temperature. The reaction was warmed to 120-140 ℃ and stirred 5-6 h. The reaction mixture was diluted with cold water and extracted with CH 2Cl2 (3 × 30 mL). Then

the CH2Cl2 solution was washed with saturated NaHCO3 (3 × 30 mL), respectively. Organic layer was dried over anhydrous Na2SO4, filtered and filtrate evaporated under reduced pressure. The crude material was subjected to column chromatography by using DCM/methyl alcohol/diethylamine (50:1:1) as an eluent, and afforded pure derivative 14 (31.4 mg) as the brown amorphous powder. Yield: 30%; 1H NMR (CDCl3, 500 MHz) δ: 5.66 (1H, m), 5.51 (1H, d, J = 12.7 Hz), 3.86 (1H, s), 2.87 (1H, m), 2.60-2.26 (4H, m), 2.20-2.05 (4H, m), 2.04 (2H, m), 2.03-1.57 (4H, m), 1.45 (1H, s), 1.44 (1H, s), 1.27 (1H, t, J = 8.8 Hz), 1.23 (1H, m), 1.18 (2H, m), 1.11-1.04 (4H, m), 0.71 (3H, s);

13

C

NMR (CDCl3, 125 MHz) δ: 256.5, 163.4, 105.7, 72.8, 63.8, 59.1, 58.9, 51.4, 51.3, 51.2, 50.8, 46.9, 43.5, 42.8, 42.2, 36.0, 33.7, 29.7, 27.3, 25.5, 26.6, 14.1; ESI-MS: m/z 428.8 [M + Na]+.

4.3 Sample pretreatment

All the derivatives were completely dissolved in 300 μL DMSO, diluted to 1 mL by distilled water, and obtain the original solution containing 1 mg/mL of derivatives. The original solution was diluted to 0.1 μg/mL, 1 ng/mL and 10 ng/L in turn and stored in a refrigerator at 4 ℃ for reserve.

4.4 ELISA26,27

The inhibitory activity of test derivatives on GPCRs was studied by incubating these with mouse GPCRs provided in the kit. Other components of the kit include water solution (30 times concentrated), HRP conjunction reagent, sample diluent, standard diluent, standard, chromogenic reagent A, chromogenic reagent B and stop solution. 15 μL of the original solution and 15 μL of the diluted standard solution were completely mixed and incubate at 37 ℃ for 10 min to prepare reaction solution. Assay was done according to the manufacturer’s protocol. In brief, the reaction was carried out in 96-well enzyme-labeled plate containing solid-phase antibodies by the addition of 25 μL reaction solution. Enzyme reaction was initiated by addition of 25 μL HRP conjunction reagent for per well then incubated the plate at 37 ℃ for 30 min. After incubation, 25 μL chromogenic reagent A and 25 μL chromogenic reagent B for per well were added to react chromogenic reaction. Ultimately, reaction was terminated by addition of 25 μL stop solution for per well and mixed thoroughly. The derivatives

were tested at four different concentrations 10 ng/L, 1 ng/mL, 0.1 μg/mL, 10 μg/mL, so dilutions were prepared accordingly, other components of the kit were dissolved and diluted as instructed in the kit. After adding stop solution to reaction wells, absorbance was recorded at OD 450 nm on a Enzyme-Labeled Instrument in 5 min. The percentage inhibition by test derivatives on GPCRs was calculated based on activity in the control tube (without inhibitor) as 100% from three independent set of experiments. The concentration of DMSO in the test well (30%) had no demonstrable effect on GPCRs activity.

Acknowledgment The work was supported by the National Natural Science Foundation of China (No. 81502955), the Doctoral Scientific Research Foundation of Shanghai Ocean University (No. A2030214300077), the Young Teachers Training Program of Shanghai (No. A12056160002), the Plan of Innovation Action in Shanghai (No. 14431906000), and the Project Funded by Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening.

References and notes 1.

Eichel, K.; Zastrow, M. V. Trends Pharmacol. Sci. 2018, 39, 200-208.

2.

Garland, S. L. J. Biomol. Screen. 2013, 18, 947-966.

3.

Marchant, J. S.; Harding, W. W.; Chan, J. D. Int. J. Parasitol.-Drugs Drug Resist. 2018, 8, 550-558.

4.

Jacobson, K. A. Biochem. Pharmacol. 2015, 98, 541-555.

5.

Kitajima, M.; Iwai, M.; Kikura-Hanajiri, R.; Goda, Y.; Iida, M.; Yabushita, H.; Takayama, H. Bioorg. Med. Chem. Lett. 2011, 21, 1962-1964.

6.

Shen, Y.; Zuo, A.-X.; Jiang, Z.-Y.; Zhang, X.-M.; Wang, H.-L.; Chen, J.-J. Bull. Korean Chem. Soc. 2010, 31, 3301-3303.

7.

Shen, Y.; Zuo, A.-X.; Jiang, Z.-Y.; Zhang, X.-M.; Wang, H.-L.; Chen, J.-J. Helv. Chim. Acta 2010, 93, 863-869.

8.

Singhuber, J.; Zhu, M.; Prinz, S.; Kopp, B. J. Ethnopharmacol. 2009, 126, 18-30.

9.

Dzhakhangirov, F.; Sultankhodzhaev, M.; Tashkhodzhaev, B.; Salimov, B. Chem. Nat. Compd. 1997, 33, 190-202.

10. Shakhidoyatova, N.; Dzhakhangirov, F.; Sultankhodzhaev, M. Pharm. Chem. J. 2001, 35, 266-267. 11. Nesterova, Y. V.; Povet'eva, T.; Suslov, N.; Shults, E.; Ziuz'kov, G.; Aksinenko, S.; Afanas' eva, O.; Krapivin, A.; Kharina, T. Bull. Exp. Biol. Med. 2015, 159, 620-622. 12. Nesterova, Y. V.; Povet'yeva, T.; Suslov, N.; Zyuz'kov, G.; Pushkarskii, S.; Aksinenko, S.; Schultz, E.; Kravtsova, S.; Krapivin, A. Bull. Exp. Biol. Med. 2014, 157, 488.

13. Nesterova, Y. V.; Povetieva, T.; Suslov, N.; Zyuz'kov, G.; Aksinenko, S.; Pushkarskii, S.; Krapivin, A. Bull. Exp. Biol. Med. 2014, 156, 665-668. 14. Khan, H.; Nabavi, S. M.; Sureda, A.; Mehterov, N.; Gulei, D.; Berindan-Neagoe, I.; Taniguchi, H.; Atanasov, A. G. Eur. J. Med. Chem. 2017, 153, 29-33. 15. Guo, R.-H.; Zhang, Q.; Ma, Y.-B.; Luo, J.; Geng, C.-A.; Wang, L.-J.; Zhang, X.-M.; Zhou, J.; Jiang, Z.-Y.; Chen, J.-J. Eur. J. Med. Chem. 2011, 46, 307. 16. Guo, R. H.; Geng, C. A.; Huang, X. Y.; Ma, Y. B.; Zhang, Q.; Wang, L. J.; Zhang, X. M.; Zhang, R. P.; Chen, J. J. Bioorg. Med. Chem. Lett. 2013, 23, 1201-1205. 17. Zhang, Q.; Jiang, Z. Y.; Luo, J.; Ma, Y. B.; Liu, J. F.; Guo, R. H.; Zhang, X. M.; Zhou; J.; Niu, W.; Du, F. F.; Li, L.; Li, C.; Chen, J. J. Bioorg. Med. Chem. Lett. 2009, 19, 6659-6665. 18. Hong, S. H.; Chen, Z. G.; Lao, A. N.; Wang, H. C. Heterocycles 1987, 26, 1455-1460. 19. Takayama, H.; Wu, F. E.; Eda, H.; Oda, K.; Aimi, N.; Sakai, S. I. Chem. Pharm. Bull. 1991, 39, 1644-1646. 20. Kiss, T.; Orvos, P.; Bánsághi, S.; Forgo, P.; Jedlinszki, N.; Tálosi, L.; Hohmann, J.; Dezső Csupor. Fitoterapia 2013, 90, 85-93. 21. Boyd, M. J.; Bandarage, U. K.; Bennett, H.; Byrn, R. R.; Davies, I.; Gu, W.; Jacobs, M.; Ledeboer, M. W.; Ledford, B.; Leeman, J. R.; Perola, E.; Wang, T.; Bennani, Y.; Clark, M. P.; Charifson, P. S. Bioorg. Med. Chem. Lett. 2015, 25, 1990-1994. 22. Huang, G.; Dong, J.-Y.; Zhang, Q.-J.; Meng, Q.-Q.; Zhao, H.-R.; Zhu, B.-Q.; Li, S.-S. Eur. J. Med. Chem. 2019, 165, 160-171. 23. Bindu, P. J.; Mahadevan, K. M.; Satyanarayan, N. D.; T. R. Ravikumar Naik. Bioorg. Med. Chem. Lett. 2012, 22, 898-900 24. Eugenia, A.; Lashmanova; Anastasiya, I.; Kirdyashkina; Pavel, A.; Slepukhin; Andrey K.; Shiryaev. Tetrahedron Lett. 2018, 59, 1099-1103. 25. Mlostoń, G.; Hamera-Fałdyga, R.; Celeda, M.; Gębicki, K.; Heimgartner, H. J. Fluorine Chem. 2016, 188, 147-152. 26. Khan, M. F.; Mishra, D. P.; Ramakrishna, E.; Rawat, A. K.; Mishra, A.; Srivastava, A. K.; Maurya, R. Med. Chem. Res. 2014, 23, 4156-4166. 27. Roberti, M.; Schipani, F.; Bagnolini, G.; Milano, D.; Giacomini, E.; Falchi, F.; Balboni, A.; Manerba, M.; Farabegoli, F.; Franco, F. D.; Robertson, J.; Minucci, S.; Pallavicini, I.; Stefano, G. D.; Girotto, S.; Pellicciari, R.; Cavalli, A. Eur. J. Med. Chem. 2019, 165, 80-92.

28.