key: cord-0073324-lyy30w1f
authors: Yang, Xiao-Tong; Li, Tian-Ze; Geng, Chang-An; Liu, Pei; Chen, Ji-Jun
title: Synthesis and biological evaluation of (20S,24R)-epoxy-dammarane-3β,12β,25-triol derivatives as α-glucosidase and PTP1B inhibitors
date: 2022-01-11
journal: Med Chem Res
DOI: 10.1007/s00044-021-02836-0
sha: f9176b6e57992627515042cd4a1e9990caef029c
doc_id: 73324
cord_uid: lyy30w1f

The dammarane triterpenoid (20S,24R)-epoxy-dammarane-3β,12β,25-triol obtained from Cyclocarya paliurus in our previous study showed inhibitory activity on α-glucosidase in vitro with an inhibitory ratio of 32.2% at the concentration of 200 μM. In order to reveal the structure-activity relationships (SARs) and get more active compounds, 42 derivatives of (20S,24R)-epoxy-dammarane-3β,12β,25-triol were synthesized by chemical modification on the hydroxyls (C-3 and C-12), rings A and E, and assayed for their α-glucosidase and PTP1B inhibitory activities. Two compounds (8, 26) increased activity against α-glucosidase, and four compounds (8, 15, 26, 42) significantly inhibited PTP1B. It was noted that compounds 8 and 26 could inhibit both α-glucosidase and PTP1B as dual-target inhibitors with IC(50) values of 489.8, 467.7 μM (α-glucosidase) and 319.7, 269.1 μM (PTP1B). Compound 26 was revealed to be a mix-type inhibitor on α-glucosidase and a noncompetitive-type inhibitor on PTP1B based on enzyme kinetic study. Furthermore, compound 42 could selectively inhibited PTP1B as a mix-type inhibitor with IC(50) value of 134.9 μM, which was 2.5-fold higher than the positive control, suramin sodium (IC(50) 339.0 μM), but not inhibit α-glucosidase. [Image: see text]

Diabetes mellitus is a chronic metabolic disease due to that pancreas is unable to produce sufficient insulin or body cannot take full advantage of insulin, its complications including blurred vision, cardiovascular diseases, kidney failure, and organ damage [1] [2] [3] . Diabetes mellitus is mainly divided into type 1 and type 2 diabetes mellitus (T1DM and T2DM), and~90% of diabetic patients suffers from T2DM. α-Glucosidase and protein tyrosine phosphatase 1B (PTP1B) are two enzymes close related to T2DM. α-Glucosidase is the crucial enzyme for hydrolyzing the 1,4-α-glucosidic linkages of oligosaccharides to release absorbable monosaccharides in small intestine. α-Glucosidase inhibitors can delay the absorption of carbohydrates, and reduce the effect of postprandial hyperglycaemia [4] [5] [6] . Protein tyrosine phosphatase 1B is a key negative regulator of leptin and insulin signaling pathways due to its ability to dephosphorylate and inactivate the insulin receptor [7, 8] .

The gene knockout studies indicate PTP1B acts as a major negative regulator of insulin signaling [9, 10] . α-Glucosidase inhibitors are the preferred drugs for controlling postprandial blood glucose, and PTP1B inhibitors can improve insulin sensitivity. Therefore, compounds with αglucosidase and PTP1B dual inhibition could be more effective and will provide important clues for the development of new antidiabetic candidates. Natural products are rich sources for searching new antidiabetic agents [11, 12] , and the discovery of natural products and their derivatives as potential antidiabetic lead compounds are continuing goals of our laboratory [13] [14] [15] [16] [17] [18] [19] [20] [21] . The leaves of Cyclocarya paliurus were widely used to treat obesity and diabetes in China. Dammarane-type triterpenoids were characteristic components of C. paliurus, which had shown potent α-glucosidase inhibitory and antiinflammatory activities [22, 23] . Our previous antidiabetic investigation of C. paliurus indicated (20S,24R)-epoxydammarane-3β,12β,25-triol (1) exhibited inhibitory activity on α-glucosidase in vitro with an inhibitory ratio of 32.2% at the concentration of 200 μM. Compound 1 also showed weak inhibition on PTP1B with an inhibitory ratio of 16.4% at concentration of 400 µM. Compound 1 was one of the main constituents of C. paliurus, which provides possibilities for the chemical modification to explore the structureactivity relationships (SARs) and search for new antidiabetic candidates. In current investigation, 42 derivatives of compound 1 were synthesized and assayed for their αglucosidase and PTP1B inhibitory activities.

To figure out the roles of hydroxyls at C-3 and C-12 for inhibiting α-glucosidase and PTP1B, a series of derivatives were synthesized as shown in Scheme 1. Compound 1 was treated with anhydrides or carboxylic acids in the presence of N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) to yield compounds 2-8 with acyl groups at C-3, compounds 9-15 with acyl groups at C-12 and di-esterified products 16-21. Oxidation of compound 1 using pyridinium chlorochromate (PCC) afforded ketones 22-24 with different oxidation location at C-3 and C-12, while compound 22 could be selectively obtained in 70% yield via Oppenauer oxidation [24] . When Scheme 1 Reagents and conditions: (a) DCC, DMAP, appropriate carboxylic acid, dry DCM, 0°C ̶ r.t., 59 % (3, 10, 17, 19% + 23% + 16%), 43% (4, 11, 18 , 16% + 13% + 14%), 53% (5, 12, 19 , 17% + 30% + 6%), 42% (6, 13, 20 , 13% + 23% + 6%), 37% (7, 14, 21, 13% + 16% + 8%) or o-phthalic anhydride, pyridine, DMAP, 80°C, 38% (8, 15 , 10% + 28%) or Ac 2 O, pyridine, r.t., 68% (2, 9, 16 , 20% + 24% + 24%); b Al(Oi-Pr) 3 , acetone, toluene, reflux, 70% (22) or PCC, DCM, r.t., 68% (22, 23, 24 , 18% + 20% + 30%); c NH 2 OH•HCl, CH 3 COONa•3H 2 O, EtOH-H 2 O (10:1), reflux, 93%; d ophthalic anhydride, pyridine, DMAP, 80°C, 76% compound 22 and hydroxylamine hydrochloride were heated in the presence of sodium acetate trihydrate, the oxime derivative 25 was afforded in 93% yield [25] . From compound 23, phthalic derivative 26 was provided in 76% yield by esterification with o-phthalic anhydride.

Next, different functionalities were introduced on ring A (Scheme 2). The formylation of compound 22 with ethyl formate in the presence of Na gave compound 27 [26] , and subsequent reduction with sodium borohydride delivered 2-hydroxymethyl derivative 28. Treatment of compound 27 with hydroxylamine hydrochloride provided isoxazole 29 in good yield [27] . Exposure of compound 22 to Baeyer-Villiger conditions delivered ring expansion product 30 [28, 29] , which was further transformed into ring A opening derivatives 31 and 32 by hydrolysis or reduction of ester [30] .

Oxidative cleavage of compound 16 with PCC and subsequent hydrolysis with NaOH resulted in lactone 33, and reduction of the lactone with LiAlH 4 afforded ringopening product 34. To get more ring-opening products and study the influence of substituted tetrahydrofuran side chain on the activity, nine derivatives with different oxidation location on the side chain were synthesized from naturally abundant dammarane triterpenoid, 20(S)-PPD. Alkene hydrogenation of compound 35 with H 2 and Pd/C gave derivative 36. A protection/dihydroxylation/deprotection sequence produced compounds 38 and 39 as a 1.2:1 mixture of diastereomers, and oxidative cleavage of the diol using NaIO 4 delivered hemiacetal 40. Pd/C-catalyzed regioselective hydrogenation of compound 37 followed by one pot dehydration and deacetylation afforded compounds 42 and 43 Scheme 3. Compound 44 was synthesized via Lemieux-Johnson oxidation in 61% yield.

Taking compound 26 as an example, the structural characterization of the synthesized compounds was explained as follows. Compound 

The inhibitory activities of all the synthesized derivatives on α-glucosidase and PTP1B were tested with acarbose and suramin sodium as the positive control, respectively. ( Table  1 ). The inhibitory ratio of derivatives on α-glucosidase was primarily tested at the concentration of 200 μM. For compounds 2-8 with different acyloxy group at C-3, only phthalic derivative 3 maintained activity. Among C-12 esterification derivatives 9-15, acetylation product 9 exhibited the similar inhibitory potency with that of compound 1. Di-esterification products 16-21 lost suppressant properties on α-glucosidase. When the hydroxyl group at C-3 was oxidized to be carbonyl group, compound 10 showed better inhibitory ratio than that of compound 1 (46.6% vs 32.2%). Compounds 27-32 modified on ring A were inactive, which indicated that ring A of compound 1 is essential for α-glucosidase inhibitory effects. For compounds 33-44 with different oxidation location on the side chain, compounds 34 and 35 maintained activity at the concentration of 200 μM.

For PTP1B, the phthalate compounds 8, 15 and 26 increased 3.8, 3.6 and 4.8-folds of inhibitory activity at the concentration of 400 μM, suggesting the additional carboxyl groups were favorable. For derivatives 27-44 with different functionalities on ring A and the side chain, compound 42 exhibited activity with an inhibitory ratio of 73.6%, which was 4.5 times stronger than that of compound 1. Different with its double bond positional isomers 42, derivative 43 was almost inactive, which indicated that the location of the double bond on the side chain had significant influence on PTP1B inhibitory effects.

Dose-response relationships of the active compounds were further studied to measure their IC 50 values ( Table 2) . Interestingly, compounds 8 and 26 containing a phthalic acid moiety at C-3 showed inhibitory activity on both enzymes with IC 50 values of 489.8, 467.7 μM (α-glucosidase) and 319.7, 269.1 μM (PTP1B), which were superior to compound 1 (IC 50 values higher than 800 μM). When the phthalic acid was located at C-12, the resulted compound 15 was only active against PTP1B with an IC 50 value of 341.7 μM; and all the three phthalate derivatives showed the similar inhibitory activity and compound 42 showed about 2.5-fold higher by comparison with the positive control suramin sodium. Compound 26 showed activity against both α-glucosidase and PTP1B with IC 50 values of 467.7 and 269.1 µM, respectively. Enzyme kinetic studies by Lineweaver-Burk plot and Dixon plot showed the lines of compound 26 intersected at the third quadrant and the V max and K i values were decreased with the increase of concentration (Fig. 1A) , indicating compound 26 was a mixed-type inhibitor against α-glucosidase with K i value of 414.4 μM. Meanwhile, the line of compound 26 intersected on the x-axis (Fig. 1B) , indicating it was a noncompetitive-type inhibitor against PTP1B (K i value: 110.7 μM). Compound 42 showed the highest activity against PTP1B with an IC 50 value of 134.9 µM, and enzyme kinetics study manifested it was a mixed-type inhibitor with a K i value of 139.2 μM.

In summary, 42 derivatives of (20S,24R)-epoxy-dammarane-3β,12β,25-triol were synthesized and assayed for their α-glucosidase and PTP1B inhibitory activities. Two compounds (8, 26) increased activity on α-glucosidase. Four compounds (8, 15, 26, 42) were active on PTP1B, of which compound 42 showed the highest activity with the IC 50 value superior to suramin sodium. Especially, phthalic derivatives 8 and 26 showed inhibitory activity on both αglucosidase and PTP1B. Enzyme kinetic study consolidated The tested concentrations were 200 µM (α-glucosidase) and 400 µM (PTP1B) c "-" means no activity Acarbose was used as the positive control against α-glucosidase c Suramin sodium was used as the positive control against PTP1B that compound 26 was a mix-type inhibitor against α-glucosidase and a noncompetitive-type inhibitor against PTP1B with K i values of 414.4 μM and 110.7 μM, respectively. The primary SARs were concluded as: (a) ring A is crucial for maintaining α-glucosidase and PPT1B inhibitory activity; (b) the incorporation of carboxyl groups at C-3 is favorable. These results provide valuable clues for the discovery of PTP1B and α-glucosidase dual inhibitors.

All reagents and solvents were obtained from commercial supplies and used without further purification. 1 H NMR and 13 C NMR spectra were tested on Avance III HD 400 (Bruker, Germany), Avance III 500 (Bruker, Germany) and Avance III 600 (Bruker, Germany) spectrometers with TMS as the internal standard. (20S,24R)-Epoxy-dammarane-3β,12β,25-triol was isolated from Cyclocarya paliurus. All synthetic compounds were purified by column chromatography on silica gel (200-300 mesh, Qingdao Makall Group Co., Ltd., Qingdao, China).

General procedure for the synthesis of compounds 2, 9 and 16

To a solution of (20S,24R)-epoxy-dammarane-3β,12β,25triol (56.0 mg, 0.12 mmol) in pyridine (1.2 mL) was added acetic anhydride (1.2 mL) at room temperature, and the mixture was stirred at room temperature for 12 h. The reaction mixture was then diluted with EtOAc and washed with 5% HCl aqueous solution. The aqueous phase was extracted with EtOAc, and the combined organic phases were dried over Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (EtOAc-petroleum ether, 10:90) to give compounds 2, 9, and 16. General procedure for the synthesis of compounds 3-7, 10-14, 17-21

To a solution of appropriate carboxylic acid (0.06 mmol) in anhydrous dichloromethane (2 mL) was added DCC (27.3 mg, 0.132 mmol) and DMAP (1.2 mg, 0.01 mmol) at 0°C. After stirred for 10 min, (20 S,24 R)-epoxy-dammarane-3β,12β,25-triol (25.0 mg, 0.05 mmol) was added. The mixture was then warmed to room temperature and stirred until the reaction completed (monitored by TLC). After completion of the reaction, placed the flask in the freezer at −10°C for 6 h, and the suspension was filtered, and the filtrate was concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether, 6:94) to give target compounds. 

To a solution of (20 S,24 R)-epoxy-dammarane-3β,12β,25triol (23.8 mg, 0.05 mmol) in pyridine (0.6 mL) was added phthalic anhydride (13.3 mg, 0.09 mmol) at room temperature. The solution was then heated to 80°C and stirred for 24 h. Then, the reaction mixture was diluted with EtOAc and washed with 5% HCl aqueous solution. The aqueous layer was extracted with EtOAc and the combined organic extracts were dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (chloroform-methanol-acetic acid, 98:2:0.01) to give compounds 8 and 15. (20S,24R)-epoxy-12β,25-dihydroxy-dammarane-3-one (22) The (20 S,24 R)-epoxy-dammarane-3β,12β,25-triol (100.0 mg, 0.21 mmol) was dissolved in toluene (3.0 mL) followed by the addition of aluminium isopropoxide (107.2 mg, 0.53 mmol). After stirred at room temperature for 1 h, acetone was added and the reaction was heated to reflux for 7 h. The reaction was cooled to room temperature and quenched by the addition of 5% HCl aqueous solution. The layers were separated and the aqueous phase was extracted with EtOAc. The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated to remove EtOAc. The residue was purified by column chromatography on silica gel (EtOAc-petroleum ether, 25:75, 35:65) to give compound 22 (70% yield) as a white powder, 1 General procedure for the synthesis of compounds 23 and 24

To a solution of (20 S,24 R)-epoxy-dammarane-3β,12β,25triol (30.0 mg, 0.06 mmol) in dry CH 2 Cl 2 (2 mL) was added pyridinium chlorochromate (PCC, 25.8 mg, 0.12 mmol) and the mixture was stirred at room temperature for 24 h. The suspension was then filtered by a Celite pad and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAcpetroleum ether, 35:65) to afford compounds 23 and 24. (20S,24R)-epoxy-3β-(2-carboxybenzoyl)-12-oxo-dammarane-25-ol (26) To a solution of compound 23 (12.0 mg, 0.03 mmol) in pyridine (0.6 mL) was added o-phthalic anhydride (22.2 mg, 0.15 mmol) and the mixture was refluxed for 72 h. After cooling to room temperature, the reaction mixture was diluted with EtOAc, and washed with 5% HCl aqueous solution. The aqueous phase was extracted with EtOAc, the combined organic extracts were dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether-acetic acid, 20:80:0.1, 25:75:0.1) to give compound 26 (76% yield) as a white solid, 1 (20S,24R)-epoxy-2-hydroxymethlidene-3-oxo-dammara-ne12β, 25-diol (27) To a round-bottom bottle equipped with Na (0.5 g) was added ethyl ether (5.0 mL), and the mixture was refluxed for 1 h. After cooling to 0°C, a solution of compound 22 (20.0 mg, 0.04 mmol) in ethyl formate (1.0 mL) was added dropwise to the mixture. After 30 min, the solution was allowed to warm to room temperature and stirred for 6 h before it was quenched by the addition of EtOH (2.0 mL). The mixture was poured into 5 mL water. The aqueous layer was extracted with EtOAc and the combined organic extracts were dried over anhydrous MgSO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (EtOAcpetroleum ether, 6:94) to give compound 27 (76% yield) as a white solid. 1 (20S,24R)-epoxy-2-hydroxymethyl-3-oxo-dammarane12β, 25 -diol (28) To a solution of compound 27 (20.0 mg, 0.04 mmol) in methanol (1.0 mL) was added NaBH 4 (3.0 mg, 0.08 mmol). The reaction mixture was allowed to stir at room temperature for 6 h before it was quenched by the addition of water (1.0 mL). The aqueous phase was extracted with EtOAc, the combined organic phase was dried over anhydrous MgSO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetonepetroleum ether, 15:85, 20:80) to give compound 28 (69% yield) as a white solid. 1 (20S,24R)-epoxy-3,4-lactone-dammarane-12β, 25-diol (30) To a solution of compound 22 (15.0 mg, 0.03 mmol) in 1.5 mL of DCM was added NaHCO 3 (10.8 mg, 0.12 mmol) and m-CPBA (10.4 mg, 0.06 mmol). The resulting suspension was stirred for 12 h at room temperature before it was quenched with sat. aq. Na 2 S 2 O 3 aqueous solution. Layers were separated, and the aqueous layer was extracted with DCM, and the combined organic phase was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether, 10:90) to give compound 30 (71% yield) as a white solid, 1 (20S,24R)-epoxy-3-carboxyl-dammarane-4,12β,25-triol (31) To a solution of compound 30 (24.0 mg, 0.05 mmol) in 3 mL of methanol was added 10% NaOH aqueous solution (1 mL) and the mixture was heated to reflux for 5 h. After the completion of hydrolysis, the solution was neutralized with 1 M HCl and the mixture was extracted with EtOAc. Combined organic layers were dried over Na 2 SO 4 and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (chloroform-methanol, 10:90) to give compound 31 (82% yield) as a white solid, 1 (20S,24R)-epoxy-dammarane-3,4,12β,25-tetrol (32) Under a nitrogen atmosphere, a solution of compound 30 (19.0 mg, 0.04 mmol) in THF was treated dropwise with solution of LiAlH 4 (5.2 mg, 0.12 mmol) in 1.5 mL of dried THF at 0°C. After complete addition, the solution was heated to 50°C and stirred at the same temperature for 12 h. After cooling to 0°C, water and 0.1 M NaOH aqueous solution was added slowly and filtered. The filtrate was extracted with EtOAc, and the combine organic phase was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether, 20:80) to give compound 32 (50% yield) as a white solid, 1 

To a solution of compound 16 (30.0 mg, 0.05 mmol) in dry CH 2 Cl 2 (1.0 mL) was added pyridinium chlorochromate (PCC, 46.6 mg, 0.22 mmol) at room temperature. The resultant mixture was then heated to 40°C and stirred at the same temperature for 12 h. Upon consumption of starting material, the suspension was filtered by a Celite pad and the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (EtOAc-petroleum ether, 5:95 to 10:90) to afford intermediate. To a solution of intermediate (24.0 mg, 0.05 mmol) in MeOH (3.0 mL) was added 10% NaOH aqueous solution (1.0 mL) at room temperature. Then, the mixture was heated to reflux and stirred overnight. After cooling to room temperature, the organic solvent was removed under reduced pressure. Then the crude was diluted with EtOAc, washed with 5% HCl aqueous solution, sat. aq. NaHCO 3 , and brine in sequence, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude product was purified by column chromatography on silica gel (acetone-petroleum ether, 25:75, 10:90) to give compound 33 (91% yield) as a white powder.

Under a nitrogen atmosphere, a solution of compound 33 (5.3 mg, 0.14 mmol) in THF was treated dropwise with a solution of LiAlH 4 (5.2 mg, 0.12 mmol) in 1.5 mL of dried THF at 0°C. After complete addition, the solution was heated to 50°C and stirred at the same temperature for 12 h. After cooling to 0°C, water and 0.1 M NaOH aqueous solution was added slowly and filtered. The filtrate was extracted with EtOAc, and the combine organic phase was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether, 20:80 to 35:65) to give compound 34 (35% yield) as a white solid.

suspension was filtered through a Celite pad and the pad was washed with CH 2 Cl 2 . The filtrate was concentrated and purified by column chromatography on silica gel (acetonepetroleum ether, 10:90) to give compound 36 (80% yield) as a white powder, 1 20S-3β,12β-diacetyl-protopanaxadiol (37) To a solution of compound 20(S)-PPD (150.0 mg, 0.33 mmol) in pyridine (1.0 mL) was added acetic anhydride (1.2 mL) and DMAP (1.8 mg, 0.02 mmol) at room temperature. After being stirred for 4 h, the reaction mixture was diluted with EtOAc, and washed with 5% HCl aqueous solution and brine in sequence. The organic phases were combined, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether, 10:90) to give compound 37 (86% yield) as a white powder, 1 

To the solution of compound 37 (24.0 mg, 0.04 mmol) in ethanol-water (15:1, v/v, 6.4 mL) was added KMnO 4 (8.2 mg, 0.05 mmol) at −40°C and the reaction was stirred for 6 h at the same temperature before it was quenched by the addition of 10% Na 2 S 2 O 3 aqueous solution. The mixture was extracted with EtOAc and the combined organic layers were washed with 5% HCl and brine, dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude was dissolved in methanol (3.0 mL), followed by the addition 10% NaOH aqueous solution (1.0 mL). The reaction was heated to reflux and stirred for 2 h. After cooling to room temperature, the solution was diluted with EtOAc and washed with 5% HCl aqueous solution and brine in sequence. The organic phase was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (methanol-acetone-petroleum ether, 7.5:15:85) to give compounds 38 and 39.

The mixture of compounds 38 and 39 were dissolved in methanol-water (3:2, v/v, 1.0 mL) and followed by the addition of NaIO 4 (21.4 mg, 0.10 mmol). The reaction was stirred at room temperature overnight. The reaction was quenched with 10% Na 2 S 2 O 3 and extracted with EtOAc. The combined organic phases were washed with brine and dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (acetone-petroleum ether, 10:90) to give compound 40.

20S-epoxy-3β,12β-dihydroxy-dammarane -24-one (33) White powder

6H, s), 0.98 (3H, s), 0.92 (3H, s), 0.86 (3H, s); 13 C NMR (100 MHz, CD 3 OD) δ 36.3 (CH 2 , C-1), 26.2 (CH, C-2), 76.8 (CH, C-3)

HRMS (ESI − ) m/z calcd for C 28 H 45 O 6 477.3222, found 477.3203 (M + HCOO − )

20S-protopanaxadiol-24-ol (34) White powder, 35% yield

3H, s), 1.06 (3H, s), 0.99 (3H, s), 0.96 (3H, s), 0.92 (3H, s), 0.86 (3H, s); 13 C NMR (100 MHz, CD 3 OD) δ 35.0 (CH 2 , C-1)

HRMS (ESI − ) m/z calcd for C 28 H 49 O 6 481.3535, found 481.3530 (M + HCOO − )

After being stirred for 12 h, the 20S-24,25-dihydroxy-protopanaxadiol (38) White powder, 52% yield; 1 H NMR (400 MHz

C-11), 71.2 (CH, C-12), 47.8 (CH, C-13), 51.6 (C, C-14)

HRMS (ESI − ) m/z calcd for C 31 H 55 O 7 539.3953, found 539.3934 (M + HCOO − )

3H, s), 1.18 (6H, s), 0.99 (3H, s), 0.98 (3H, s), 0.88 (6H, s), 0.78 (3H, s); 13 C NMR (100 MHz, CDCl 3 ) δ 39.1 (CH 2 , C-1)

HRMS (ESI − ) m/z calcd for C 31 H 55 O 7 539.3953, found 539.3914 (M + HCOO − )

White powder, 82% yield; 1 H NMR (400 MHz

3H, s), 0.78 (3H, s); 13 C NMR (100 MHz

HRMS (ESI + ) m/z calcd for C 27 H 46 O 4 Na 457.3288, found 457.3310 (M + Na + )

25-dihydro-3β,12β-diacetyl -protopanaxadiol (41) White powder, 87% yield; 1 H NMR (400 MHz

C-24), 28.1 (CH, C-25)

HRMS (ESI + ) m/z calcd for C 34 H 58 O 5 Na 569.4176, found 569.4189 (M + Na + )

3H, s), 0.99 (3H, s), 0.98 (3H, s), 0.88 (12H, overlap), 0.78 (3H, s); 13 C NMR (100 MHz, CDCl 3 ) δ 39.0 (CH 2 , C-1)

ESI + ) m/z calcd for C 30 H 53 O 2 445

H-22), 1.02 (3H, s), 0.98 (3H, s), 0.89 (3H, s), 0.88 (9H, overlap), 0.86 (3H, s), 0.78 (3H, s); 13 C NMR (100 MHz, CDCl 3 ) δ 38.6 (CH 2 , C-1)

20-oxo-protopanaxadiol (44) To a stirred solution of compound 43 (26.6 mg, 0.06 mmol) in 2.0 mL of methanolwater (3:2) was added NaIO 4 (29.7 mg, 0.14 mmol) and OsO 4 (35 μL, 2% aqueous solution, 0.002 mmol) at ambient temperature. After stirred for 12 h, the reaction was quenched with saturated aqueous Na 2 S 2 O 3 and extracted with EtOAc. The organic extracts were combined, dried over anhydrous sodium sulfate, filtered. After removal of the solvent under vacuum, the residue was purified by column chromatography on silica gel (acetone-petroleum ether, 10:90) to give compound 44 (61% yield) as a white powder, 1 H NMR (400 MHz

C-6)

ESI + ) m/z calcd for C 24 H 40 O 3 Na 399.2870, found 399.2832 (M + Na + ). previous researches

Test samples dissolved in methanol-PB (1:1, v/v, 20 μL) and p-Nitrophenyl-α-D-glucopyranoside (20 μL, 5.0 mM, p-NPG) dissolved in PB (in triplicate) were added to a 96-well plate and incubated at 37°C for 5 min. Then α-glucosidase dissolved in PB (2.0 U/mL, 20 μL, Shanghai yuanye Bio-Technology Co Ltd, China) was added to each well as a substrate

The system using PB replace test compounds was used as control. The mixtures' reaction without α-glucosidase was used as blank. The α-glucosidase inhibitory rate % = [(ΔOD control − ΔOD control blank ) − (ΔOD sample − ΔOD sample blank )]/(ΔOD control − ΔOD control blank ). IC 50 values were tested and calculated through nonlinear regression using Graphpad prism 8 software

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Synthesis of guggulsterone derivatives as potential anti-austerity agents against PANC-1 human pancreatic cancer cells

Scope and mechanism of a true organocatalytic Beckmann rearrangement with a boronic acid/perfluoropinacol system under ambient conditions

A novel necroptosis inhibitor-necrostatin-21 and its SAR study

A comparative study of proapoptotic potential of cyano analogues of boswellic acid and 11-keto-boswellic acid

Synthesis and biological evaluation of ring A and/or C expansion and opening echinocystic acid derivatives for anti-HCV entry inhibitors

Synthesis of 12-oxa, 12-aza and 12-thia cholanetriols

A suspension of 10% Pd/C (12.0 mg) and compound 37 (116.0 mg, 0.21 mmol) in ethanol (3.0 mL) was stirred at room temperature under hydrogen atmosphere. After being stirred for 12 h, the suspension was filtered through a Celite pad and the pad was washed with CH 2 Cl 2 . The filtrate was concentrated and purified by column chromatography on silica gel (acetone-petroleum ether, 10:90) to give compound 41.To a solution of compound 41 in pyridine (2.0 mL) was added POCl 3 (40.8 μL, 1.32 mmol) at 0°C. After 20 min, the mixture was heated to 40°C and stirred overnight. The solution was quenched with water and extracted with EtOAc. The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated. The crude was dissolved in methanol and followed by the addition of 10% NaOH aqueous solution (1.0 mL). The mixture refluxed for 2 h. After cooling to room temperature, the solution was diluted with EtOAc and washed with 5% HCl aqueous solution and brine in sequence. The organic phase was dried over anhydrous Na 2 SO 4 , filtered and concentrated. The residue was purified by column chromatography on silica gel (EtOAc-petroleum ether, 4:96) to give compounds 42 and 43.

The PTP1B inhibition assay was investigated by the method as previous reports [14, 16, 18] . Working buffer containing MOPS (34.5 mM), DTT (1.9 mM), EDTA·4Na (0.67 mM), BSA (2.0 mg/mL), and NaCl (2.1 mM) in deionized water was prepared before the assay. Suramin sodium was utilized as a positive control dissolved in DMSO. Working buffer (70 µL), test samples dissolved in DMSO (10 µL) and PTP1B dissolved in working buffer (10 µL, 4.9 mg/L) were added to a 96-well plate and incubated at 37°C for 15 min. Then the substrate in working buffer (10 µL, 100 mM, p-NPP) was added to each well. After incubation for 30 min, the reaction was stopped by adding 100 μL of Na 2 CO 3 solution (0.1 mM). The absorbance was measured at 405 nm via a Bio-Rad 680 microplate reader (Hercules, CA, USA). The system using DMSO replace test compounds was used as control. The mixtures' reaction without PTP1B was used as blank. The PTP1B inhibitory rate and IC 50 values of compounds were calculated using the same method as described above.Enzyme kinetic studies of compounds 26 and 42 for αglucosidase and PTP1BThe enzyme kinetics of α-glucosidase and PTP1B inhibition for compounds 26 and 42 were investigated according to experiments as described above. 

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