Icariin derivatives, compositions and uses thereof

Abstract

This invention discloses an icariin derivative, a composition, and its application. The structural formula of the icariin derivative is as follows: wherein, R 1 Independently selected from hydrogen, hydroxyl, and C1-C3 alkoxy groups; R 2 Independently selected from hydrogen, isopentenyl, isopentyl, and Mannich bases; R 3 Independently selected from hydrogen, hydroxyl, or OR 3’ R 3’ Selected from rhamnosyl, rhamnosyl-(2→1)-glucosyl, rhamnosyl-(3→1)-glucosyl, propynyl, and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups; R 4 Independently selected from hydroxyl or OR 4’ R 4’ Selected from propynyl, glucosyl, and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups; R 5 Independently selected from hydrogen, isopentenyl, and Mannich bases; Mannich bases are selected from N(CH3)2, N(CH2CH3)2, and R. 6 Independently selected from hydroxyl, OR 6’ R 6’ The derivatives are selected from hydrogen, rhamnosyl, rhamnosyl-(2→1)-xylosyl, and rhamnosyl-(2→1)-rhamnosyl. The icariin derivatives of this invention provide a new direction for the treatment of sarcopenia.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to icariin derivatives, compositions comprising icariin derivatives, and the use of icariin derivatives or compositions thereof in the preparation of drugs for the prevention or treatment of sarcopenia. Background Technology

[0002] Epimedium, a classic medicinal herb recorded in the Shennong's Classic of Materia Medica, has been used clinically for over two thousand years. It has long been used alone or in combination with other herbs to treat various diseases, including reproductive system dysfunction, osteoporosis, cardiovascular and neuroprotective diseases.

[0003] Sarcopenia is a pathological condition characterized by a significant decline in muscle mass and strength, primarily affecting the elderly. With the accelerating aging of the global population, the incidence of sarcopenia is gradually increasing, severely impacting patients' quality of life and independence. The occurrence of sarcopenia is related to multiple factors, including age, malnutrition, lack of exercise, and chronic diseases. Current treatments mainly focus on exercise and nutritional interventions. Regarding drug therapy, most potential therapeutic targets remain in the basic research or preclinical exploration stages, and no specific therapeutic drugs have yet been marketed.

[0004] This application uses epimedium flavonoids as the parent nucleus for structural modification in order to obtain a drug with therapeutic effects on sarcopenia. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides an icariin derivative through structural modification. This application also provides compositions containing the icariin derivative and the use of the icariin derivative or its composition in the preparation of drugs for the prevention or treatment of sarcopenia.

[0006] In a first aspect, the present invention provides an icariin derivative with the following structural formula:

[0007]

[0008] in,

[0009] R 0 Independently selected from hydrogen, R 1 Independently selected from hydrogen, hydroxyl, and C1-C3 alkoxy groups;

[0010] R 2 Independently selected from hydrogen, isopentenyl, isopentyl, and Mannich bases;

[0011] R 3 Independently selected from hydrogen, hydroxyl, phenol, or OR 3’ R 3’Selected from rhamnosyl, rhamnosyl-(2→1)-glucosyl, rhamnosyl-(3→1)-glucosyl, rhamnosyl-(2→1)-xylose, rhamnosyl-(2→1)-rhamnosyl, propynyl, and benzyl or glycosyl-substituted triazole groups;

[0012] R 4 Independently selected from hydroxyl or OR 4’ R 4’ Selected from propynyl, glucosyl, and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups;

[0013] R 5 Independently selected from hydrogen and Mannich bases; Mannich bases are selected from N(CH3)2, N(CH2CH3)2, N(CH2CH2CH3)2, N[CH(CH3)2]2, N(CH3)(CH2CH3), N(CH3)(CH2CH2CH3), N(CH2CH3)(CH2CH2CH3).

[0014]

[0015] R 6 Independently selected from hydroxyl, OR 6’ R 6’ Selected from hydrogen, rhamnosyl, rhamnosyl-(2→1)-glucosyl, rhamnosyl-(3→1)-glucosyl, rhamnosyl-(2→1)-xylose, and rhamnosyl-(2→1)-rhamnosyl.

[0016] As a preferred embodiment, the structural formula is as follows:

[0017]

[0018] in,

[0019] R 1 Independently selected from hydrogen, hydroxyl, and C1-C3 alkoxy groups;

[0020] R 2 Selected from hydrogen, isopentenyl, and isopentyl;

[0021] R 3 Selected from hydrogen, hydroxyl or OR 3’ R 3’ Selected from triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups;

[0022] R 4 Independently selected from hydroxyl or OR 4’ R 4’ Selected from propynyl, glucosyl, and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups;

[0023] R6 Independently selected from hydroxyl, OR 6’ R 6’ Selected from rhamnosyl, rhamnosyl-(2→1)-glucosyl, rhamnosyl-(3→1)-glucosyl, rhamnosyl-(2→1)-xylosyl, and rhamnosyl-(2→1)-rhamnosyl.

[0024] As a more preferred embodiment, R 1 Independently selected from hydroxyl and methoxy groups; R 2 Selected from isopentyl; R 3 Selected from hydroxyl; R 4 Independently selected from hydroxyl or OR 4’ R 4’ It is glucose; R 6 Independently selected from hydroxyl, OR 6’ R 6’ Selected from rhamnosyl, rhamnosyl-(2→1)-glucosyl, rhamnosyl-(3→1)-glucosyl, rhamnosyl-(2→1)-xylosyl, and rhamnosyl-(2→1)-rhamnosyl.

[0025] As a more preferred embodiment, R 1 Selected from methoxy groups; R 2 Selected from isopentyl; R 3 Selected from hydroxyl; R 4 Selected from OR 4’ R 4’ It is glucose; R 6 Independently selected from hydroxyl, OR 6’ R 6’ Selected from rhamnosyl, rhamnosyl-(2→1)-glucosyl, rhamnosyl-(3→1)-glucosyl, rhamnosyl-(2→1)-xylosyl, and rhamnosyl-(2→1)-rhamnosyl.

[0026] As a more preferred embodiment, R 1 R 3 R 4 R 6 All are selected from hydroxyl groups; R 2 Selected from isopentyl.

[0027] As a preferred embodiment, R 1 Independently selected from hydrogen, hydroxyl, and C1-C3 alkoxy groups; R 2 Selected from hydrogen, isopentenyl, and isopentyl; R 3 Selected from hydrogen, hydroxyl or OR 3’ R 3’ Selected from triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups; R 4 Independently selected from hydroxyl or OR 4’ R 4’Selected from propynyl, glucosyl, and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups; R 6 Selected from hydroxyl groups.

[0028] As a more preferred embodiment, R 1 Independently selected from hydrogen, hydroxyl, and methoxy; R 2 Selected from hydrogen, isopentenyl, and isopentyl; R 3 Selected from hydrogen, hydroxyl or OR 3’ R 3’ Selected from glycosyl-substituted triazole groups; R 4 Selected independently from OR 4’ R 4’ Selected from propynyl, glucosyl, and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups; R 6 Selected from hydroxyl groups.

[0029] As a more preferred embodiment, R 1 Selected from methoxy groups; R 2 Selected from isopentenyl and isopentyl; R 3 Selected from hydroxyl or OR 3’ R 3’ A triazole group substituted with a glycosyl group; R 4 Selected from OR 4’ R 4’ Selected from propynyl and triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups; R 6 Selected from hydroxyl groups.

[0030] As a preferred embodiment, the structural formula is as follows:

[0031]

[0032] R 1 Independently selected from hydrogen, hydroxyl, and C1-C3 alkoxy groups;

[0033] R 2 Selected from hydrogen, isopentenyl, and isopentyl;

[0034] R 3 Selected from hydrogen, hydroxyl or OR 3’ R 3’ Selected from triazole groups substituted with alcohol hydroxyl, benzyl, or glycosyl groups;

[0035] R 6 Selected from hydroxyl groups;

[0036] R 7 It is independently selected from benzyl, alcohol hydroxyl, or sugar group.

[0037] As a more preferred embodiment, R 1 Independently selected from hydrogen and methoxy groups; R 2Selected from hydrogen, isopentenyl, and isopentyl; R 3 Selected from OR 3’ R 3’ A triazole group substituted with a glycosyl group; R 6 Selected from hydroxyl; R 7 It is independently selected from benzyl, alcohol hydroxyl, or sugar group.

[0038] As a preferred embodiment, the structural formula is as follows:

[0039]

[0040] in,

[0041] R 8 Independently selected from hydrogen, C1-C3 alkyl groups, and C1-C3 alkoxy groups;

[0042] R 9 R 10 R 11 R 12 Independently selected from N(CH3)2, N(CH2CH3)2, N(CH2CH2CH3)2, N[CH(CH3)2]2, N(CH3)(CH2CH3), N(CH3)(CH2CH2CH3), N(CH2CH3)(CH2CH2CH3),

[0043] As a more preferred embodiment, R 9 =R 10 R 11 =R 12 .

[0044] As a more preferred embodiment, the structural formula of the above-mentioned icariin derivative is as follows:

[0045]

[0046] In a second aspect, the present invention provides a composition comprising the above-described epimedium derivative.

[0047] A third aspect of the invention provides the use of the above-described epimedium derivatives or the above-described compositions in the preparation of medicaments for the prevention or treatment of sarcopenia.

[0048] Beneficial effects include:

[0049] (1) The epimedium derivative of the present invention has a protective effect on C2C12 muscle cells and can be used to develop drugs for the prevention or treatment of sarcopenia, providing a new direction for the treatment of sarcopenia.

[0050] (2) In the dexamethasone model, the cell activity of compounds L-9, L-10, L-14, L-17, L-19 and L-25 was better than that of the positive control drug EB, among which L-10 and L-25 had the most outstanding activity.

[0051] (3) Compounds L-48 and L-50, at concentrations of 5 μM and 100 μM, significantly reversed the inhibitory effect of dexamethasone sodium phosphate on the viability of C2C12 myoblasts, achieving cell viability of 66% and 69%, respectively. In the starvation model, compounds L-49 and L-50, at concentrations of 10 μM and 50 μM, significantly enhanced the starvation-induced viability of C2C12 myoblasts, achieving cell viability of 90% and 92%, respectively.

[0052] (4) Compounds L-48 and L-50 significantly improved the survival rate of C2C12 cells under dexamethasone sodium phosphate injury, and compounds L-49 and L-50 significantly improved the survival rate of C2C12 cells under starvation injury, suggesting that the compounds have potential sarcopenia treatment activity.

[0053] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0054] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0055] Figure 1 The effects of compounds L-1 to L-7 on the C2C12 activity induced by dexamethasone sodium phosphate;

[0056] Figure 2 The effect of compound L-48 on the C2C12 activity induced by dexamethasone sodium phosphate;

[0057] Figure 3 The effect of compound L-49 on starvation-induced C2C12 activity;

[0058] Figure 4 The effect of compound L-50 on the C2C12 activity induced by dexamethasone sodium phosphate;

[0059] Figure 5 The effect of compound L-50 on starvation-induced C2C12 activity. Detailed Implementation

[0060] The applicant will now provide a clear and complete description of the technical solution of the present invention, in conjunction with the embodiments of the present invention, the accompanying description, and compound data.

[0061] Unless otherwise specified, all percentages mentioned in this article refer to mass percentages.

[0062] C1-C3 alkyl groups: including methyl, ethyl, and propyl;

[0063] Alkoxy group: -OR, where R is an alkyl group;

[0064] C1-C3 alkoxy groups: including methoxy, ethoxy, and propoxy groups;

[0065] Isopentenyl: (CH3)2C=CH-CH-;

[0066] Isopentyl: CH3CH2CH(CH3)CH-

[0067] Propyne group: HC≡CCH-

[0068] Triazole: That is, 1,2,3-triazole

[0069] Substituted triazole group: The structure in which one or more hydrogen atoms of the 1,2,3-triazole ring are replaced by other atoms or groups;

[0070] Triazole group substituted with alcohol hydroxyl, benzyl, or glycosyl groups: The structure after one or more hydrogen atoms of the 1,2,3-triazole ring are substituted with alcohol hydroxyl, benzyl, or glycosyl groups;

[0071] Glucosyl group: refers to the group formed after a glucose molecule loses a hydrogen atom from its C1 hydroxyl group; its chemical formula is C6H. 11 O5-

[0072] O-Rha-(2→1)-Glc: The C1 position of rhamnose (Rha) is linked to the C2 hydroxyl group of glucose (Glc) via a glycosidic bond;

[0073] O-Rha-(3→1)-Glc: The C1 position of rhamnose (Rha) is linked to the C3 hydroxyl group of glucose (Glc) via a glycosidic bond;

[0074] O-Rha-(2→1)-Xyl: The C1 position of rhamnose (Rha) is linked to the C2 position of xylose (Xyl) via a glycosidic bond;

[0075] O-Rha-(2→1)-Rha: Two rhamnose units are linked by a C1→C2 glycosidic bond.

[0076] PE: Petroleum ether

[0077] EA, EtOAc: Ethyl acetate

[0078] DCM: Dichloromethane

[0079] MeOH: Methanol

[0080] Sodium Vitamin C: Sodium Ascorbate

[0081] DIPEA: Diisopropylethylamine

[0082] FBS: Fetal bovine serum

[0083] Dsp: Dexamethasone sodium phosphate

[0084] Yield calculation method: Yield = (Actual output / Theoretical output) × 100%

[0085] Example 1: Preparation of compounds L1-L7

[0086]

[0087] Table 1

[0088]

[0089] L-1: Icariin (10 mg, 0.02 mmol) and 10% palladium on carbon (1 mg) were dissolved in 0.5 mL of methanol under a hydrogen atmosphere and reacted overnight at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (PE / EA = 2:1, v / v) to obtain a yellow powder L-1 (5.8 mg, yield 57%).

[0090] 1 H NMR (400MHz, DMSO-d6) δ12.34(s,1H),10.65(s,1H),9.48(s,1H),8.14(d,J=8.8Hz,2H),7.11(d,J=8.8Hz,2H), 6.29(s,1H),3.84(s,3H),2.81–2.67(m,2H),1.64–1.56(m,1H),1.43–1.37(mz,2H),0.95(s,3H),0.94(s,3H).

[0091] 13 C NMR (100MHz, DMSO) δ176.3,161.4,160.5,158.1,153.5,146.0,135.9,129.0,12 3.6,114.1,106.6,103.0,97.8,55.4,40.2,40.1,40.0,38.0,27.9,22.5,20.1.

[0092] HRMS(ESI)m / z:[M+H]+Calcd for C 21 H 23 O6371.1489; Found 371.1492.

[0093] The solubility of L-1 in DMSO is 10-15 mg / mL.

[0094] L-2: Icariin (10.2 mg, 0.015 mmol) and 10% palladium on carbon (1 mg) were dissolved in 0.5 mL of methanol, purged three times with hydrogen, and reacted overnight at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 4:1, v / v) to obtain a yellow powder L-2 (5.4 mg, yield 54%).

[0095] 1 H NMR(600MHz,MeOD-d4)δ7.90(d,J=8.4Hz,2H),7.10(d,J=8.4Hz,2H),6.65(s,1H),5.43(d,J=1.8Hz,1 H),5.06(d,J=7.2Hz,1H),4.56(s,1H),4.23(t,J=3.0Hz,1H),3.92(dd,J=12.0,2.4Hz,1H),3.90(s,3H ),3.77–3.70(m,2H),3.56–3.46(m,3H),3.46–3.40(m,1H),3.34(d,J=9.0Hz,1H),2.93–2.81(m,2H), 1.63–1.58(m,1H),1.49–1.40(m,2H),1.36–1.27(m,3H),0.94(t,J=6.6Hz,6H),0.91(d,J=6.0Hz,3H).

[0096] 13 C NMR (150MHz, MeOD-d4) δ180.3,163.7,162.4,161.0,159.3,155.1,136.5,131.9,131.0,124.1,115.4,111.8,107.6,103. 6,102.0,99.5,78.51,78.47,75.1,73.3,72.2,72.0,71.3,62.5,56.2,43.7,39.8,29.6,23.21,23.17,21.8,17.8,11.7.

[0097] HRMS(ESI)m / z:[M+H] +Calcd for C 33 H 43 O 15 679.2596; Found 679.2594.

[0098] The solubility of L-2 in DMSO is 95-120 mg / mL.

[0099] L-3: Dissolve 10.1 mg of orthodoxine A (0.012 mmol) and 10% palladium on carbon (1 mg) in 0.5 mL of methanol, purge three times with hydrogen, and react overnight at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate it, and perform column chromatography (DCM / MeOH = 4:1) to obtain a yellow powder L-3 (4.9 mg, yield 49%).

[0100] 1 H NMR (600MHz, MeOD-d4) δ7.94(d,J=9.0Hz,2H),7.15(d,J=9.0Hz,2H),6.70(s,1H),5.79(d,J=1.8Hz,1H),5.09( d,J=7.8Hz,1H),4.59(s,3H),4.46(d,J=7.8Hz,1H),4.34(dd,J=3.6,1.8Hz,1H),3.96(d,J=2.4Hz,1H),3.93(s, 3H),3.84(dd,J=9.6,3.6Hz,1H),3.79–3.72(m,2H),3.72–3.66(m,2H),3.57–3.50(m,3H),3.49–3.35(m,6H),3. 28–3.23(m,2H),2.97–2.71(m,1H),2.89–2.84(m,1H),1.67–1.61(m,1H),1.51–1.43(m,2H),1.00–0.94(m,9H).

[0101] 13 C NMR(100MHz,MeOD-d4)δ180.2,163.7,162.3,160.8,159.3,155.0,136.7,131.9,123.9,115.3,107.1,102.6,101.9,99.4,82.6,78.4,78.0,7 7.9,75.3,75.0,73.4,72.0,71.8,71.2,71.0,62.4,56.1,49.6,49.4, 49.2,49.0,49.0,48.8,48.6,48.4,39.7,29.5,23.1,23.0,21.6,17.6.

[0102] HRMS(ESI)m / z:[M+H] + Calcd for C 39 H 53 O 20 841.3125; Found 841.3127.

[0103] The solubility of L-3 in DMSO is 160-190 mg / mL.

[0104] L-4: Ascorbic acid A1 (10.0 mg, 0.012 mmol) and 10% palladium on carbon (1 mg) were dissolved in 0.5 mL of methanol, purged three times with hydrogen, and reacted overnight at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 4:1, v / v) to obtain a yellow powder L-4 (6.3 mg, yield 63%).

[0105] 1 H NMR(600MHz,MeOD-d4)δ7.92(d,J=9.0Hz,1H),7.15(d,J=9.0Hz,1H),6.69(s ,1H),5.45(d,J=1.8Hz,1H),5.09(d,J=7.2Hz,1H),4.59(s,3H),4.54(d,J=7 .8Hz,1H),4.47(dd,J=3.0,1.8Hz,1H),3.95(dd,J=12.6,2.4Hz,1H),3.93(s ,3H),3.89(dd,J=12.0,2.4Hz,1H),3.85(dd,J=9.6,3.0Hz,1H),3.81–3.74(m 2H),3.60–3.49(m,5H),3.50–3.35(m,6H),2.95–2.90(m,1H),2.87–2.82(m,1H),1.6 5–1.61(m,1H),1.49–1.40(m,2H),1.01–0.96(d,J=6.6Hz,3H),0.96(t,J=6.6Hz,6H).

[0106] 13C NMR(100MHz,MeOD-d4)δ180.1,163.7,162.3,160.9,159.2,155.0,136.1 ,136.0,131.9,128.6,123.9,115.4,111.8,107.4,105.8,103.1,101.8,9 9.3,82.7,78.39,78.35,77.84,77.78,75.4,75.0,71.9,71.2,71.1,71. 0,62.4,62.3,56.1,49.6,49.4,49.2,39.6,29.5,23.1,23.0,21.6,17.8.

[0107] HRMS(ESI)m / z:[M+H] + Calcd for C 39 H 53 O 20 841.3125; Found 841.3128.

[0108] The solubility of L-4 in DMSO is 160-190 mg / mL.

[0109] L-5: Ascorbic acid B (10.0 mg, 0.012 mmol) and 10% palladium on carbon (1 mg) were dissolved in 0.5 mL of methanol, purged three times with hydrogen, and reacted overnight at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 4:1, v / v) to obtain L-5 (4.1 mg, yield 41%).

[0110] 1H NMR (600MHz, MeOD-d4) δ7.94(d,J=8.8Hz,2H),7.14(d,J=8.8Hz,2H),6.68(s,1H),5.51(d,J=1.2Hz,1H),5.09(d,J=7.2Hz,1H),4.5 9(s,3H),4.33(d,J=7.8Hz,1H),4.25–4.24(m,1H),3.96–3.93(m,1H),3.93(s,3H),3.86(dd,J=9.6,3.0Hz,1H),3.76(dd,J=12.6,5 .4Hz,1H),3.72–3.64(m,2H),3.58–3.49(m,3H),3.49–3.40(m,2H),3.37(s,2H),3.31(d,J=9.0Hz,1H),3.23–3.20(m,1H),3.10–3. 06(m,1H),2.97–2.91(m,1H),2.88–2.82(m,1H),1.67–1.61(m,1H),1.52–1.44(m,2H),1.01(d,J=6.0Hz,3H),0.97(t,J=6.6Hz,6H).

[0111] 13 C NMR(150MHz,MeOD-d4)δ180.3,163.6,162.3,159.1,155.0,136.9,131.8,123.9,115.3,107.7,107.4,103.1,101.9,9 9.4,82.6,78.4,78.3,77.8,75.2,75.0,73.6,71.9,71.2,71.0,67.1,62.4,56.1,39.7,29.5,23.1,23.0,21.6,17.7.

[0112] HRMS(ESI)m / z:[M+H] + Calcd for C 38 H 51 O 19 811.3019; Found 811.3016.

[0113] The solubility of L-5 in DMSO is 150-180 mg / mL.

[0114] L-6: Dissolve 10.0 mg of ascorbic acid C (0.012 mmol) and 10% palladium on carbon (1 mg) in 0.5 mL of methanol, purge three times with hydrogen, and react overnight at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate it, and perform column chromatography (DCM / MeOH = 4:1, v / v) to obtain a yellow powder L-6 (5.3 mg, yield 53%).

[0115] 1 H NMR (600MHz, MeOD-d4) δ7.93(d,J=9.0Hz,2H),7.13(d,J=9.0Hz,2H),6.68(s,1H),5.58(d,J=1.8Hz,1H),5.09(d,J=7.8Hz,1H),5.02 (d,J=1.8Hz,1H),4.59(s,3H),4.32–4.31(m,1H),3.97–3.96(m,1H),3.94(dd,J=12.6,2.4Hz,1H),3.92(s,3H),3.88–3.86(m,1H),3 .78–3.74(m,1H),3.65–3.60(m,2H),3.57–3.50(m,3H),3.48–3.44(m,1H),3.38(t,J=9.6Hz,1H),3.35(d,J=2.4Hz,1H),2.96–2.91( m,1H),2.89–2.84(m,1H),1.67–1.60(m,1H),1.52–1.44(m,1H),1.24(d,J=6.0Hz,3H),0.97(t,J=6.0Hz,6H),0.94(d,J=5.4Hz,3H).

[0116] 13 C NMR(100MHz,MeOD-d4)δ180.1,163.6,162.3,160.9,159.1,155.0,136.5,131.8,123.9,115.3,111.8,107.4,103.7,102.3,101.9, 99.3,78.9,78.4,78.3,75.0,74.0,73.5,72.3,72.1,72.0,71.9,71.2,70.4,62.4,56.0,39.7,29.5,23.1,23.0,21.6,17.9,17.8.

[0117] HRMS(ESI)m / z:[M+H] + Calcd for C 39 H 53 O 19825.3176; Found 825.3179.

[0118] The solubility of L-6 in DMSO is 150-180 mg / mL.

[0119] L-7: 10.3 mg of cyproheptadine I (0.02 mmol) and 10% palladium on carbon (1 mg) were dissolved in 0.5 mL of methanol. The mixture was purged with hydrogen three times and reacted overnight at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 4:1, v / v) to obtain a yellow powder L-7 (6.2 mg, 60% yield).

[0120] 1 H NMR (400MHz, MeOD-d4) δ7.91(d,J=8.4Hz,2H),7.12(d,J=8.8Hz,2H),6.28(s,1H),5.43(s,1H),4.60(s,2H),4.25(t,J=2.4Hz,1H),3.92(s, 3H),3.75(dd,J=8.8,3.2Hz,1H),2.80–2.76(m,2H),1.67–1.59(m,1H),1.48–1.43(m,2H),0.98(s,3H),0.96(s,3H),0.93(d,J=5.6Hz,3H).

[0121] 13 C NMR(150MHz,MeOD-d4)δ179.9,163.4,163.3,160.6,158.5,155.6,136.2,131.7,129.0,128.8,124.1 ,115.1,108.9,105.9,103.5,99.3,73.2,72.1,72.0,71.9,56.0,39.6,29.5,23.1,23.0,21.5,17.7.

[0122] HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 33 O 10 517.2068; Found 517.2070.

[0123] The solubility of L-7 in DMSO is 20-35 mg / mL.

[0124] Example 2: Preparation of compound L8-L25

[0125] Table 2

[0126]

[0127]

[0128] L-8: Dissolve 102 mg (0.4 mmol) of salicylic acid in 2 mL of anhydrous ethanol, add 30 μL of 37%-40% (w / v) formaldehyde aqueous solution, and slowly add piperidine (40 μL, 0.4 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate it, and perform column chromatography (DCM / MeOH = 40:1, v / v) to obtain a yellow powder L-8 (39 mg, yield 28%).

[0129] 1 H NMR(400MHz, DMSO-d6)δ8.05(d,J=7.2Hz,2H),7.59(t,J=8.0Hz,3H),6.91(s,1H),6.38(s, 1H),3.83(s,2H),2.65(t,J=5.2Hz,4H),1.58(t,J=5.6Hz,4H),1.51–1.42(t,J=6.0Hz,2H).

[0130] The solubility of L-8 in DMSO is 12-25 mg / mL.

[0131] L-9: Dissolve 102 mg (0.4 mmol) of apigenin in 2 mL of anhydrous ethanol, add 30 μL of 37%-40% (w / v) formaldehyde aqueous solution, and slowly add morpholine (131 μL, 1.5 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate it, and perform column chromatography (DCM / MeOH = 40:1, v / v) to obtain a yellow powder L-9 (163 mg, yield 41%).

[0132] 1 H NMR (400MHz, CDCl3) δ13.24 (s, 1H), 11.98 (s, 1H), 7.96–7.85 (m, 2H), 7.54 (d, J = 6. 8Hz, 3H), 6.66 (s, 1H), 3.85 (d, J = 4.4Hz, 4H), 3.80–3.71 (m, 7H), 2.76–2.52 (m, 7H).

[0133] The solubility of L-9 in DMSO is 8-15 mg / mL.

[0134] L-10: Dissolve 102 mg (0.4 mmol) of apigenin in 2 mL of anhydrous ethanol, add 30 μL of 37%-40% (w / v) formaldehyde aqueous solution, and slowly add pyrrolidine (61 μL, 0.75 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate it, and perform column chromatography (DCM / MeOH = 30:1, v / v) to obtain a yellow powder L-10 (48 mg, yield 29%).

[0135] 1 H NMR(400MHz, CDCl3)δ12.69(s,1H),10.96(s,1H),7.86–7.78(m,2H),7.58–7.49 (m,3H),6.63(s,1H),6.29(s,1H),4.16(s,2H),2.81(s,4H),2.37–1.77(m,4H).

[0136] The solubility of L-10 in DMSO is 15-25 mg / mL.

[0137] L-11: 127 mg (0.5 mmol) of apigenin was dissolved in 4 mL of isopropanol. 61 μL of a 37%-40% (w / v) formaldehyde aqueous solution was added, followed by slow addition of dimethylamine (375 μL of a 2 M THF solution containing 0.75 mmol of dimethylamine). The reaction was allowed to proceed at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 20:1, v / v) to obtain a yellow powder, L-11 (60 mg, 32% yield). 1 H NMR (400MHz, CDCl3) δ12.37(s,1H),7.85–7.78(m,2H),7.68–7.43(m,3H),6.62(s,1H),6.28(s,1H),3.95(s,2H),2.44(s,6H). 13 C NMR (100MHz, CDCl3) δ182.5,166.5,163.1,161.7,154.7,131.8,131.7,129.3,126.2,105.9,104.6,100.4,99.1,55.3,44.6.

[0138] The solubility of L-11 in DMSO is 10-25 mg / mL.

[0139] L-12: The experimental procedure for synthesizing compound L-12 was the same as that for compound L-11, yielding a yellow powder L-12 (18 mg, yield 11%).

[0140] 1H NMR (400MHz, CDCl3) δ13.16(s,1H),7.88(dd,J=7.6,2.4Hz,2H),7.56–7.50(m,3H),6.63(s,1H),6.44(s,1H),3.88(s,2H),2.45(s,6H).

[0141] 13 C NMR (100MHz, CDCl3) δ182.6,167.0,163.8,159.4,157.7,131.8,131.7,129.2,126.4,105.7,104.2,103.9,94.9,54.5,44.3.

[0142] HRMS(ESI)m / z:[M+H] + Calcd for C 18 H 18 NO4312.1230; Found 312.1234.

[0143] The solubility of L-12 in DMSO is 20-35 mg / mL.

[0144] L-13: 127 mg (0.5 mmol) of guar gum was dissolved in 4 mL of isopropanol. 61 μL of a 37%-40% (w / w) formaldehyde aqueous solution was added, followed by slow addition of diethylamine (78 μL, 0.75 mmol). The reaction was carried out at room temperature, and TLC monitoring showed that the starting material was almost completely consumed. The solution was concentrated and subjected to column chromatography (DCM / MeOH = 20:1) to obtain a yellow powder, L-13 (83 mg, yield 50%).

[0145] 1 H NMR (400MHz, CDCl3) δ13.12 (s, 1H), 7.87 (dd, J = 6.8, 2.4Hz, 2H), 7.54–7.47 (m, 3H) ,6.61(s,1H),6.39(s,1H),3.95(s,2H),2.76–2.71(m,3H),1.18(t,J=7.2Hz,6H).

[0146] The solubility of L-13 in DMSO is 15-35 mg / mL.

[0147] L-14: 127 mg (0.5 mmol) of salinomycin was dissolved in 4 mL of anhydrous ethanol. 61 μL of a 37%–40% (w / v) formaldehyde aqueous solution was added, followed by slow addition of diisopropylamine (105 μL, 0.75 mmol). The reaction was carried out at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 40:1, v / v) to obtain a yellow powder, L-14 (105 mg, yield 57%).

[0148] 1 H NMR(400MHz, CDCl3)δ13.14(s,1H),7.91–7.77(m,2H),7.52–7.43(m,2H),6.58 (s,1H),6.31(s,1H),3.99(s,2H),3.29–3.23(m,2H),1.22(s,6H),1.20(s,6H).

[0149] The solubility of L-14 in DMSO is 15-30 mg / mL.

[0150] L-15: 127 mg (0.5 mmol) of salinomycin was dissolved in 4 mL of isopropanol. 61 μL of a 37%-40% (w / v) formaldehyde aqueous solution was added, followed by slow addition of N-methylpropylamine (77 μL, 0.75 mmol). The reaction was carried out at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (DCM / MeOH = 20:1, v / v) to obtain a yellow powder, L-15 (28 mg, yield 16.5%), along with L-16.

[0151] L-15 melting point: 154-155℃; 1 H NMR(400MHz, CDCl3)δ12.71(s,1H),7.88–7.75(m,2H),7.59–7.49(m,3H),6.64(s,1H),6.31(s, 1H),4.03(s,2H),2.60(t,J=7.6Hz,2H),2.41(s,3H),1.68–1.63(m,2H),0.97(t,J=7.6Hz,3H). 13 C NMR (100MHz, CDCl3) δ182.5,166.6,163.1,161.8,154.9,131.9,129.3,126.2, 106.1,104.7,100.5,98.9,59.1,53.9,41.3,20.1,11.7.HRMS(ESI)m / z:[M+H] + Calcd for C 20 H 22NO4340.1543; Found340.1545.

[0152] The solubility of L-15 in DMSO is 5-20 mg / mL.

[0153] L-16: The experimental procedure for synthesizing compound L-16 was the same as that for compound L-15, yielding a yellow powder L-16 (31.5 mg, yield 18.6%). Melting point: 131-132℃. 1 H NMR (400MHz, CDCl3) δ13.15 (s, 1H), 7.87 (dd, J = 7.2, 3.0Hz, 2H), 7.56–7.47 (m, 3H), 6.62 (s, 1H), 6.4 3(s,1H),3.92(s,2H),2.60(t,J=7.6Hz,2H),2.41(s,3H),1.68–1.63(m,2H),0.96(t,J=7.2Hz,3H). 13 CNMR (100MHz, CDCl3) δ182.5,167.3,163.7,159.3,157.6,131.8,131.7,129.2,12 6.4,105.7,104.1,103.8,94.8,58.8,53.2,41.1,19.8,11.7.HRMS(ESI)m / z:[M+H] + Calcd for C 20 H 22 NO4340.1543; Found340.1544.

[0154] The solubility of L-16 in DMSO is 5-20 mg / mL.

[0155] L-17: Dissolve farnesin (182 mg, 0.5 mmol) in 2 mL of anhydrous ethanol, add paraformaldehyde (54 mg, 0.6 mmol), and slowly add piperidine (65 μL, 0.65 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate, and perform column chromatography (DCM / MeOH = 40:1, v / v) to obtain a yellow powder L-17 (65 mg, yield 34%). Melting point: 213-214 °C. 1 H NMR(400MHz, DMSO-d6)δ8.01(d,J=8.8Hz,2H),7.11(d,J=8.8Hz,3H),6.82(s,1H),6.39(s,1H), 3.85(s,4H),2.62(t,J=5.2Hz,4H),1.61–1.55(m,4H),1.50–1.46(m,2H).HRMS(ESI)m / z:[M+H]+ Calcd for C 22 H 24 NO5382.1649; Found 382.1645.

[0156] The solubility of L-17 in DMSO is 15-30 mg / mL.

[0157] L-18: Dissolve farnesin (142 mg, 0.5 mmol) in 2 mL of isopropanol, add paraformaldehyde (54 mg, 0.6 mmol), and slowly add morpholine (57 μL, 0.65 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate, and perform column chromatography (DCM / MeOH = 35:1, v / v) to obtain a yellow powder, L-18 (198 mg, yield 41%). Melting point: 189-190℃. 1 H NMR (400MHz, CDCl3) δ13.34(s,1H),7.86(d,J=8.8Hz,2H),7.03(d,J=8.8Hz,2H ),6.57(s,1H),3.90(s,3H),3.85(s,4H),3.78–3.72(m,8H),2.65–2.60(m,8H). 13 C NMR (100MHz, CDCl3) δ182.7,164.5,163.5,162.7,159.3,155.4,128.0,123.9,114.7,10 4.1,104.0,103.8,101.2,67.0,66.7,55.7,53.5,53.0,52.5,51.2.HRMS(ESI)m / z:[M+H] + Calcdfor C 26 H 31 N2O7483.2126;Found 483.2129.

[0158] The solubility of L-18 in DMSO is 15-30 mg / mL.

[0159] L-19: Dissolve farnesin (85.3 mg, 0.3 mmol) in 3 mL of isopropanol, add 37 μL of a 37%-40% (w / v) formaldehyde aqueous solution, and slowly add pyrrolidine (38 μL, 0.45 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate, and perform column chromatography (DCM / MeOH = 20:1, v / v) to obtain a yellow powder L-19 (8 mg, yield 7%). Melting point: 166-167℃. 1H NMR (400MHz, CDCl3) δ13.27(s,1H),7.82(d,J=8.6Hz,2H),6.99(d,J=8.6Hz,2H),6 .52(s,1H),6.42(s,1H),4.08(s,2H),3.88(s,3H),2.90(s,4H),2.02–1.80(m,4H). 13 C NMR (100MHz, CDCl3) δ182.5,163.7,162.6,159.2,157.7,128.1,124.0,114.6,104.1,103.7,94.9,55.6,53.6,50.6,23.8.HRMS(ESI)m / z:[M+H] + Calcd forC 21 H 22 NO5368.1492; Found 368.1490.

[0160] The solubility of L-19 in DMSO is 15-30 mg / mL.

[0161] L-20: Dissolve farnesin (85.3 mg, 0.5 mmol) in 4 mL of isopropanol, add 37 μL of a 37%-40% (w / v) formaldehyde aqueous solution, and slowly add dimethylamine (2M THF solution of dimethylamine, 230 μL, 0.45 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate, and perform column chromatography (DCM / MeOH = 20:1, v / v) to obtain a yellow powder L-20 (19 mg, yield 18.6%). Melting point: 126-127℃. 1 H NMR (400MHz, CDCl3) δ12.79(s,1H),7.79(d,J=8.4Hz,2H),7.04(d,J=8.8Hz,2H),6.55(s,1H),6.30(s,1H),3.96(s,2H),3.90(s,3H),2.45(s,6H). 13 C NMR (100MHz, CDCl3) δ182.5,166.2,163.2,162.7,161.8,154.7,128.0,124 .1,114.8,104.5,100.4,99.0,55.7,55.4,44.6,29.8.HRMS(ESI)m / z:[M+H] + Ca3cd for C 19 H 20 NO5342.1336; Found 342.1340.

[0162] The solubility of L-20 in DMSO is 15-25 mg / mL.

[0163] L-21: Dissolve farnesin (85.3 mg, 0.3 mmol) in 4 mL of isopropanol, add 37 μL of a 37%-40% (w / v) formaldehyde aqueous solution, and slowly add diethylamine (47 μL, 0.45 mmol). React at room temperature; TLC monitoring shows the starting material is almost completely consumed. Concentrate and perform column chromatography (DCM / MeOH = 20:1, v / v) to obtain a yellow powder, L-21 (49 mg, yield 44.7%). Melting point: 142-143 °C. 1 H NMR (400MHz, CDCl3) δ13.19(s,1H),12.32(s,1H),7.81(d,J=8.8Hz,2H),6.99(d,J=8.8Hz,2H),6.50(s,1H) ,6.35(s,1H),3.93(s,2H),3.87(s,3H),2.72(q,J=7.2Hz,4H),1.16(t,J=7.2Hz,6H).HRMS(ESI)m / z:[M+H] + Calcd for C 21 H 24 NO5370.1649; Found 370.1646.

[0164] The solubility of L-21 in H2O (25℃) is 20-40 mg / mL.

[0165] L-22: Dissolve farnesin (85.3 mg, 0.3 mmol) in 3 mL of anhydrous ethanol, add 37 μL of a 37%-40% (w / v) formaldehyde aqueous solution, and slowly add diisopropylamine (63 μL, 0.45 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate, and perform column chromatography (DCM / MeOH = 30:1, v / v) to obtain a yellow powder L-22 (20.4 mg, yield 17%). Melting point: 146-147℃. 1 H NMR (400MHz, CDCl3) δ13.22(s,1H),7.82(d,J=8.8Hz,2H),7.00(d,J=8.8Hz,2H),6.50(s,1 H),6.30(s,1H),3.99(s,2H),3.88(s,3H),3.26(p,J=6.6Hz,2H),1.21(s,6H),1.19(s,6H). 13C NMR (100MHz, CDCl3) δ182.3,168.8,163.5,162.5,158.6,157.3,128.1,124. 1,114.5,104.1,104.0,103.3,94.9,55.6,49.5,42.3.HRMS(ESI)m / z:[M+H] + Calcd for C 23 H 28 NO5398.1962; Found 398.1966.

[0166] The solubility of L-22 in DMSO is 10-25 mg / mL.

[0167] L-23: Dissolve farnesin (142 mg, 0.5 mmol) in 4 mL of isopropanol, add 61 μL of a 37%-40% (w / v) formaldehyde aqueous solution, and slowly add N-methylpropylamine (77 μL, 0.75 mmol). React at room temperature until the starting material is almost completely consumed. Filter the reaction solution, concentrate, and perform column chromatography (DCM / MeOH = 20:1, v / v) to obtain a yellow powder L-23 (20.8 mg, yield 11%), along with L-24. L-23 data: Melting point: 132-133℃. 1 H NMR (400MHz, CDCl3) δ12.79(s,1H),7.78(d,J=8.8Hz,2H),7.04(d,J=8.4Hz,2H),6.54(s,1H),6.29(s,1H ),4.00(s,2H),3.90(s,3H),2.58(t,J=7.6Hz,2H),2.39(s,3H),1.69–1.60(m,2H),0.96(t,J=7.6Hz,3H). 13 CNMR (100MHz, CDCl3) δ182.5,166.4,163.1,162.6,161.6,154.7,127.9,124.0,114.7 ,104.4,104.4,100.3,99.0,59.1,55.7,54.1,41.3,20.2,11.7.HRMS(ESI)m / z:[M+H] + Calcdfor C 21 H 24 NO5370.1649; Found370.1652.

[0168] The solubility of L-23 in DMSO is 20-45 mg / mL.

[0169] L-24: The experimental procedure for synthesizing compound L-24 was the same as that for compound L-23, yielding a yellow powder L-24 (11.4 mg, yield 6%). Melting point: 149-150℃. 1 H NMR (400MHz, CDCl3) δ13.21(s,1H),7.83(d,J=8.4Hz,2H),7.01(d,J=8.8Hz,2H),6.54(s,1H), 6.40(s,1H),3.89(s,6H),2.56(s,2H),2.37(s,3H),1.67–1.60(m,2H),0.96(t,J=7.2Hz,3H). 13 C NMR (100MHz, CDCl3) δ182.5,163.8,162.6,159.2,128.1,114.6,104.2,94.7,7 7.5,77.2,76.8,58.9,55.7,53.4,41.1,29.9,20.0,11.7.HRMS(ESI)m / z:[M+H] + Calcd for C 21 H 24 NO5370.1649; Found370.1650.

[0170] The solubility of L-24 in DMSO is 10-25 mg / mL.

[0171] L-25: Icariin (37 mg, 0.1 mmol) was dissolved in 2 mL of isopropanol, and 12 μL of a 37%-40% (w / w) formaldehyde aqueous solution was added. Piperidine (15 μL, 0.15 mmol) was slowly added, and the reaction was carried out at room temperature until the starting material was almost completely consumed. The reaction solution was filtered, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to obtain a yellow powder L-25 (42 mg, yield 89%). 1 HNMR(400MHz,DMSO-d6)δ12.81(s,1H),8.12(d,J=8.4Hz,2H),7.13(d,J=8.4Hz,2H),5.21(t,J=7.2Hz,1H)3.85(s,3H) ,3.43(d,J=6.8Hz,2H),3.16(d,J=4.4Hz,2H),2.62(s,4H),1.75(s,3H),1.62(s,3H),1.61–1.58(m,4H),1.48(s,2H).

[0172] The solubility of L-25 in DMSO is 5-20 mg / mL.

[0173] Example 3 Preparation of compounds L-26 and L-27

[0174]

[0175] Icariin (736 mg, 2.0 mmol) and anhydrous potassium carbonate (359 mg, 2.6 mmol) were dissolved in acetone and stirred at room temperature for 30 minutes. Then, 3-bromopropyne (517 μL, 6.0 mmol) was added, and the mixture was heated to 50 °C and reacted overnight. The reaction solution was filtered, concentrated, and subjected to column chromatography (PE / EtOAc = 5:1 to 3:1, v / v) to give yellow powder L-26 (208 mg, yield 23%) and yellow powder L-27 (159 mg, yield 20%).

[0176] L-26: 1 H NMR (400MHz, CDCl3) δ12.63 (s, 1H), 8.14 (d, J = 8.8Hz, 2H), 7.01

[0177] (d,J=9.2Hz,2H),6.50(s,1H),4.91(d,J=2.4Hz,2H),4.78(d,J=2.4Hz,2H),3.90(s,3H),3. 52(d,J=7.2Hz,2H),2.56(t,J=2.4Hz,1H),2.37(t,J=2.4Hz,1H),1.79(s,4H),1.69(s,4H).

[0178] The solubility of L-26 in DMSO is 30-50 mg / mL.

[0179] L-27: 1 H NMR (400MHz, CDCl3) δ12.55 (s, 1H), 8.12 (d, J = 9.2Hz, 2H), 7.02

[0180] (d,J=9.2Hz,2H),6.30(s,1H),5.29(t,J=7.2Hz,1H),4.91(d,J=2.4Hz,2H),3.9 0(s,3H),3.58(d,J=7.2Hz,2H),2.36(t,J=2.4Hz,1H),1.84(s,3H),1.77(s,3H).

[0181] The solubility of L-27 in DMSO is 30-50 mg / mL.

[0182] Example 4: Preparation of compounds L-28 and L-29

[0183]

[0184] Compound L-1 (371 mg, 1.0 mmol) and anhydrous potassium carbonate (180 mg, 1.3 mmol) were dissolved in acetone and stirred at room temperature for 30 minutes. Then, 3-bromopropyne (259 μL, 3.0 mmol) was added, and the mixture was heated to 50 °C and reacted overnight. The reaction solution was filtered, concentrated, and subjected to column chromatography (PE / EtOAc = 5:1 to 3:1, v / v) to give yellow powder L-28 (138 mg, 31% yield) and yellow powder L-29 (100 mg, 25% yield).

[0185] L-28: 1 H NMR (400MHz, CDCl3) δ12.60 (s, 1H), 8.16 (d, J = 8.8Hz, 2H), 7.02

[0186] (d,J=9.2Hz,2H),6.50(s,1H),4.92(d,J=2.4Hz,2H),4.78(d,J=2.4Hz,2H),3.90(s,2H),2.87–2.75(m,2H), 2.55(t,J=2.0Hz,1H),2.37(t,J=2.4Hz,1H),1.72–1.61(m,1H),1.51–1.40(m,2H),0.99(s,3H),0.97(s,3H). 13 C NMR (100MHz, CDCl3) δ179.1,161.9,160.8,160.0,157.0,153.6,136.0,130.7,123.4,114.1,109 .9,106.0,96.1,78.7,77.8,76.3,59.5,56.5,55.6,38.6,28.5,22.7,20.8.HRMS(ESI)m / z:[M+H] + Calcd forC 24 H 25 O6409.1646; Found 409.1642.

[0187] The solubility of L-28 in DMSO is 30-50 mg / mL.

[0188] L-29: 1 H NMR (400MHz, CDCl3) δ12.52 (s, 1H), 8.16 (d, J = 8.4Hz, 2H), 7.02

[0189] (d,J=8.8Hz,2H),6.39(s,1H),6.32(s,1H),4.87(d,J=2.4Hz,2H),3.90(s,4H),2.86–2.75 (m,2H),2.39–2.33(m,1H),1.72–1.65(m,1H),1.54–1.43(m,2H),1.00(s,3H),0.98(s,3H). 13 C NMR (100MHz, CDCl3) δ179.0,161.9,160.1,159.5,157.1,154.3,136.0,130.7,123.3,114. 1,107.8,105.9,99.1,78.6,76.4,59.6,55.6,38.5,28.6,22.7,20.8.HRMS(ESI)m / z:[M+H] + Calcd for C 27 H 27 O6447.1802; Found 447.1803.

[0190] The solubility of L-29 in DMSO is 30-50 mg / mL.

[0191] Example 5: Preparation of compounds L-30-L-47

[0192] Table 3

[0193]

[0194]

[0195]

[0196] L-30: Compound L-26 (20 mg, 0.05 mmol), benzyl azide (10 μL, 0.075 mmol), cuprous iodide (5 mg, 0.025 mmol), and sodium vitamin C (5 mg, 0.025 mmol) were dissolved in tetrahydrofuran (0.7 mL) and water (0.3 mL). DIPEA (13 μL, 0.075 mmol) was added, and the reaction was carried out at room temperature until the starting material was almost completely consumed. The solution was diluted with DCM, washed with saturated brine, dried over anhydrous Na2SO4, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to give a white solid L-30 (21 mg, 76% yield). Melting point: 194-195 °C. 1H NMR (600MHz, DMSO-d6) δ12.59(s,1H),10.83(s,1H),8.12(s,1H),7.94(d,J=9.0Hz,2H),7.33–7.29(m,3H),7.17–7.15(m,2H),7.03(d,J =8.4Hz,2H),6.32(s,1H),5.53(s,2H),5.21(s,2H),5.14(t,J=7.2Hz,1H),3.84(s,3H),3.39(d,J=6.6Hz,2H),1.70(s,3H),1.61(s,3H). 13 C NMR (100MHz, CDCl3) δ179.1,161.8,161.3,160.4,156.8,153.6,144.2,143.8,136.7,134.5,132.0,130.6,129.8,129.3,129.2,129.0,128.9 ,128.20,128.18,123.9,123.1,122.8,122.2,114.1,108.2,105.9,95. 9,65.4,62.8,55.6,54.4,54.2,25.8,21.9,18.0.HRMS(ESI)m / z:[M+H] + Calcd for C 31 H 30 N3O6540.2129; Found 540.2124.

[0197] The solubility of L-30 in DMSO is 20-40 mg / mL.

[0198] L-31: Compound L-27 (22 mg, 0.05 mmol), benzyl azide (9.4 μL, 0.075 mmol), copper sulfate pentahydrate (63 mg, 0.25 mmol), and sodium vitamin C (149 mg, 0.75 mmol) were dissolved in dichloromethane (1 mL) and water (1 mL). The reaction was carried out at room temperature until the starting material was almost completely consumed. The solution was diluted with DCM, washed with saturated brine, dried over anhydrous Na₂SO₄, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to give a yellow powder L-31 (49 mg, 58% yield). Melting point: 203-204 °C. 1H NMR (600MHz, DMSO-d6) δ12.59(s,1H),10.83(s,1H),8.12(s,1H),7.94(d,J=9.0Hz,2H),7.33–7.29(m,3H),7.17–7.15(m,2H),7.03(d,J =8.4Hz,2H),6.32(s,1H),5.53(s,2H),5.21(s,2H),5.14(t,J=7.2Hz,1H),3.84(s,3H),3.39(d,J=6.6Hz,2H),1.70(s,3H),1.61(s,3H). 13 C NMR (100MHz, CDCl3) δ179.1,161.8,161.3,160.4,156.8,153.6,144.2,143.8,136.7,134.5,132.0,130.6,129.8,129.3,129.2,129.0,128. 9,128.2,128.2,123.9,123.1,122.8,122.2,114.1,108.2,105.9,95. 9,65.4,62.8,55.6,54.4,54.2,25.8,21.9,18.0.HRMS(ESI)m / z:[M+H] + Calcd for C 41 H 39 N6O6711.2926;Found 711.2928.

[0199] The solubility of L-31 in DMSO is 10-30 mg / mL.

[0200] L-32: Compound L-28 (20 mg, 0.05 mmol), benzyl azide (10 μL, 0.075 mmol), cuprous iodide (5 mg, 0.025 mmol), and sodium vitamin C (5 mg, 0.025 mmol) were dissolved in tetrahydrofuran (0.7 mL) and water (0.3 mL). DIPEA (13 μL, 0.075 mmol) was added, and the reaction was carried out at room temperature until the starting material was almost completely consumed. The mixture was diluted with DCM, washed with saturated brine, dried over anhydrous Na2SO4, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to give a yellow powder L-32 (21 mg, 76% yield). Melting point: 217-218 °C. 1H NMR(400MHz,DMSO-d6)δ12.57(s,1H),10.75(s,1H),8.13(s,1H),7.96(d,J =8.8Hz,2H),7.36–7.29(m,3H),7.18–7.15(m,2H),7.07–6.98(m,2H),6.32 (s,1H),5.53(s,2H),5.22(s,2H),3.85(d,J=1.6Hz,3H),2.69(dd,J=9.6,6 .0Hz,2H),1.65–1.53(m,1H),1.40–1.34(m,2H),0.93(s,3H),0.92(s,3H). 13 C NMR (100MHz, DMSO) δ178.3,161.7,161.2,158.7,155.4,153.7,142.5,136.0,135.6,129.9,128.6,128.0,127.6 ,125.1,122.4,114.0,106.9,104.1,98.2,64.3,55.5,52.6,40.2,38.0,27.8,22.5,20.1.HRMS(ESI)m / z:[M+H] + Calcd for C 31 H 32 N3O6542.2286;Found 542.2283.

[0201] The solubility of L-32 in DMSO is 10-30 mg / mL.

[0202] L-33: Compound L-29 (22 mg, 0.05 mmol), benzyl azide (9.4 μL, 0.075 mmol), copper sulfate pentahydrate (63 mg, 0.25 mmol), and sodium vitamin C (149 mg, 0.75 mmol) were dissolved in dichloromethane (1 mL) and water (1 mL). The reaction was carried out at room temperature until the starting material was almost completely consumed. The solution was diluted with DCM, washed with saturated brine, dried over anhydrous Na₂SO₄, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to give a yellow powder L-33 (15 mg, 42% yield). Melting point: 228-229 °C. 1H NMR(400MHz,DMSO-d6)δ8.28(s,1H),8.13(s,1H),7.95(d,J=8.8Hz,2H),7.4 1–7.26(m,9H),7.17–7.15(m,2H),7.02(d,J=8.8Hz,2H),6.79(s,1H),5.75(s ,2H),5.64(s,2H),5.52(s,2H),5.31(s,2H),5.22(s,2H),3.84(s,3H),2.67– 2.63(m,2H),1.53–1.45(m,1H),1.35–1.21(m,3H),0.81(s,3H),0.80(s,3H). 13 C NMR(100MHz,DMSO-d6)δ178.6,161.4,161.3,159.3,155.9,152.8,142.5,142.4,136.03,135.97,135.7,130.0,128.7,128.6,128.1,128.0,1 27.8,127.6,125.2,124.7,122.3,114.0,108.6,104.9,96.4,64.3,62. 2,55.5,54.9,52.8,52.6,37.9,27.6,22.3,20.0.HRMS(ESI)m / z:[M+H] + Calcd forC 41 H 41 N6O6713.3082;Found 713.3085.

[0203] The solubility of L-33 in DMSO is 10-30 mg / mL.

[0204] L-34: Compound L-26 (31 mg, 0.05 mmol), azidoethanol (6.0 μL, 0.075 mmol), sodium vitamin C (5 mg, 0.025 mmol), and cuprous iodide (6.2 mg, 0.025 mmol) were dissolved in tetrahydrofuran (1.4 mL) and water (0.6 mL). DIPEA (13.0 μL, 0.075 mmol) was added, and the mixture was reacted at room temperature until the starting material was almost completely consumed. The mixture was diluted with EA, washed with citric acid and saturated brine, and extracted three times with EA. The organic phase was dried over anhydrous Na₂SO₄, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to obtain a yellow powder, L-34 (15 mg, 61% yield). Melting point: 203-204 °C. 1H NMR (400MHz, DMSO-d6) δ12.82(s,1H),8.25(s,1H),8.14–8.07(m,2H),7.66–7.57(m,3H),7.05(s,1H),6.97(d,J=2 .2Hz,1H),6.52(d,J=2.1Hz,1H),5.29(s,2H),5.06(t,J=5.2Hz,1H),4.43(t,J=5.3Hz,2H),3.78(t,J=5.3Hz,2H). 13 CNMR(100MHz,DMSO-d6)δ182.1,164.1,163.5,161.2,157.3,132.2,130.6,129.2,126.5, 125.5,105.4,105.1,98.7,93.6,61.9,59.8,52.3,23.0,19.2,13.5.HRMS(ESI)m / z:[M+H] + Calcd for C 26 H 28 N3O7494.1922;Found 494.1919.

[0205] The solubility of L-34 in DMSO is 30-60 mg / mL.

[0206] L-35: Compound L-27 (20.4 mg, 0.05 mmol), azidoethanol (6.0 μL, 0.075 mmol), sodium vitamin C (5 mg, 0.025 mmol), and cuprous iodide (6.2 mg, 0.025 mmol) were dissolved in tetrahydrofuran (1.4 mL) and water (0.6 mL). DIPEA (13.0 μL, 0.075 mmol) was added, and the mixture was reacted at room temperature until the starting material was almost completely consumed. The mixture was diluted with EA, washed with citric acid and saturated brine, and extracted three times with EA. The organic phase was dried over anhydrous Na₂SO₄, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to give a yellow powder, L-35 (11 mg, 45% yield). Melting point: 227-228 °C. 1H NMR (800MHz, DMSO-d6) δ12.59(s,1H),8.05(s,1H),8.00(d,J=8.8Hz,2H),7.07(d,J=8.8Hz,2H),6.32(s,1H),5.17(s,2H),5.01(s,1H),4. 34(t,J=5.6Hz,2H),3.86(s,3H),3.72(t,J=5.6Hz,2H),2.72–2.68(m,2H),1.62–1.57(m,1H),1.41–1.37(m,2H),0.93(s,3H),0.93(s,3H). 13 C NMR (200MHz, DMSO) δ178.3,161.8,161.3,158.7,155.5,153.7,142.1,135.9,129.9,125.2,122.5 ,114.0,107.0,104.2,98.2,64.6,59.8,55.5,52.0,38.0,27.8,22.5,20.1.HRMS(ESI)m / z:[M+H] + Calcd for C 26 H 30 N3O7496.2078; Found 496.2077.

[0207] The solubility of L-35 in DMSO is 30-60 mg / mL.

[0208] L-36: Propylene acetonitrile (29 mg, 0.1 mmol), azide ethanol (12 μL, 0.15 mmol), cuprous iodide (10 mg, 0.05 mmol), and sodium vitamin C (10 mg, 0.05 mmol) were dissolved in a mixed solution of tetrahydrofuran (0.7 mL) and water (0.3 mL). DIPEA (26 μL, 0.15 mmol) was added, and the reaction was carried out at room temperature until the starting material was almost completely consumed. The solution was diluted with EA, washed with saturated brine, dried over anhydrous Na₂SO₄, concentrated, and subjected to column chromatography (PE / EtOAc = 1:1, v / v) to obtain a yellow powder, L-36 (19 mg, 49% yield). Melting point: 305-306 °C. 1H NMR (400MHz, DMSO-d6) δ12.82(s,1H),8.25(s,1H),8.14–8.07(m,2H),7.66–7.57(m,3H),7.05(s,1H),6.97(d,J=2 .2Hz,1H),6.52(d,J=2.1Hz,1H),5.29(s,2H),5.06(t,J=5.2Hz,1H),4.43(t,J=5.3Hz,2H),3.78(t,J=5.3Hz,2H). 13 C NMR(100MHz,DMSO-d6)δ182.1,164.1,163.5,161.2,157.3,132.2,130.6,129.2,126.5,1 25.5,105.4,105.1,98.7,93.6,61.9,59.8,52.3,23.0,19.2,13.5.HRMS(ESI)m / z:[M+H] + Calcdfor C 20 H 18 N3O5380.1241; Found 380.1244.

[0209] The solubility of L-36 in DMSO is 25-50 mg / mL.

[0210] L-37: 5-Propyinylindole (64 mg, 0.35 mmol), azide ethanol (40 μL, 0.53 mmol), cuprous iodide (67 mg, 0.35 mmol), sodium vitamin C (69 mg, 0.53 mmol), and DIPEA (93 μL, 0.53 mmol) were dissolved in tetrahydrofuran (3.2 mL) and water (0.8 mL), and reacted at room temperature for 6 h. After the reaction was complete, as monitored by TLC (DCM:MeOH = 5:1), the mixture was diluted with EA, washed with saturated brine, dried over anhydrous Na₂SO₄, concentrated, and subjected to column chromatography (DCM / MeOH = 5:1, v / v) to give a white powder L-37 (49 mg, 54% yield). Melting point: 188-189 °C. 1 H NMR (400MHz, DMSO-d6) δ10.94(s,1H),8.16(s,1H),7.34–7.26(m,2H),7.20(d,J=2.4Hz,1H),6.79(dd,J=8. 8,2.4Hz,1H),6.35(t,J=2.0Hz,1H),5.11(s,2H),5.07(s,1H),4.42(t,J=5.2Hz,2H),3.80(t,J=5.2Hz,2H). 13C NMR(100MHz,DMSO-d6)δ152.1,143.1,131.3,128.0,125.9,124.7,112.0,111.7,103.1,100.9,61.7,59.9,52.2.HRMS(ESI)m / z:[M+H] + Calcd for C 13 H 15 N4O2259.1190; Found 259.1195.

[0211] The solubility of L-37 in DMSO is 20-40 mg / mL.

[0212] L-38: 7-Propynyl succinate (44 mg, 0.15 mmol), ribosyl azide (110 mg, 0.225 mmol), cuprous iodide (14 mg, 0.075 mmol), sodium vitamin C (15 mg, 0.075 mmol), and DIPEA (39 μL, 0.225 mmol) were dissolved in tetrahydrofuran (1 mL) and water (0.4 mL), and reacted overnight at room temperature. After the reaction was complete, TLC (PE:EA = 1:1) monitored a main spot with increased polarity (R). f =0.6) was generated. EA was diluted, washed with saturated brine, the organic phase was dried over anhydrous Na2SO4, concentrated, and subjected to rapid silica gel column chromatography (PE:EA = 1:1) to give a white foamy solid (80 mg, yield 68%). This was directly added to the next step, dissolved in 1 mL of dichloromethane, 3 mL of methanol was added, and sodium methoxide (150 μL) was added. The reaction was carried out at room temperature for 2 h. TLC (DCM:MeOH = 5:1) was used to monitor the completion of the reaction, resulting in a yellow suspension. Column chromatography (DCM / MeOH = 40:1, v / v) gave a yellow foamy solid L-38 (36 mg, yield 77%). 1 H NMR (400MHz, DMSO-d6) δ12.75(s,1H),8.54(s,0.5H),8.48(s,0.5H),8.11(d,J=7.2Hz,2 H),7.63–7.59(m,3H),7.06(s,1H),6.98(s,1H),6.52(s,1H),6.35(d,J=5.6Hz,0.5H),5. 97(d,J=4.4Hz,0.5H),5.66(s,1H),5.49(s,0.5H),5.31(s,2H),5.01(s,0.5H),4.90(s,0 .5H),4.42(d,J=16.8Hz,1H),4.12(s,1.5H),3.98(d,J=4.4Hz,0.5H),3.63–3.44(m,3H). 13C NMR (201MHz, DMSO) δ182.0,164.0,163.9,163.5,163.5,157.2,142.0,141.4,132.1,130.5,129.1,126.4,125.2,123.5,105.4,9 8.6,92.0,89.4,85.9,79.1,77.6,75.0,70.7,70.3,70.3,61.8,61.7,61.3,61.2,39.5,39.4,39.3,39.1.HRMS(ESI)m / z:[M+Na] + Calcd for C 23 H 21 N3O8Na 490.1221; Found 490.1223.

[0213] The solubility of L-38 in DMSO is 30-60 mg / mL.

[0214] L-39: 7-Propynyl succinate (117 mg, 0.4 mmol), glucose azide (249 mg, 0.6 mmol), CuI (76 mg, 0.4 mmol), and VcNa (79 mg, 0.4 mmol) were dissolved in a mixture of 3.7 mL tetrahydrofuran and 1.6 mL water. DIPEA (105 μL, 0.6 mmol) was added, and the reaction was carried out at room temperature. TLC was used to monitor complete consumption of the starting material. The mixture was extracted three times with EA and saturated citric acid solution. The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. Column chromatography (PE:EA = 4:1) yielded a yellow foamy solid (149.2 mg, 41% yield), which was directly added to the next step. 3 mL of methanol and 720 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. TLC (DCM:MeOH = 5:1, v / v) showed the reaction was complete, yielding a yellow suspension. Column chromatography (DCM / MeOH = 40:1) yielded a colored, foamy solid, L-39 (8 mg, 48% yield). [α] D 25 = -4.3(c 0.1, CDCl3). 1H NMR (600MHz, DMSO-d6) δ12.78(s,1H),8.52(s,1H),8.07(d,J=7.2Hz,2H),7.62–7.56(m,3H),7.01(s,1H),6.96(d,J=1.8Hz,1H),6.49(d, J=1.8Hz,1H),5.58(d,J=9.6Hz,1H),5.29(s,2H),3.80(t,J=9.0Hz,1H),3.68(d,J=10.8Hz,1H),3.52–3.42(m,3H),3.27(t,J=9.0Hz,1H). 13 C NMR (150MHz, DMSO) δ182.1,164.1,163.6,161.2,157.3,141.7,132.2,130.6,129.2,126.5,12 4.4,105.4,105.2,98.7,93.5,87.5,80.0,77.0,72.1,69.5,61.8,60.7.HRMS(ESI)m / z:[M+H] + Calcd for C 24 H 24 N3O9498.1507;Found 498.1509.

[0215] The solubility of L-39 in DMSO is 30-60 mg / mL.

[0216] L-40: 7-Propylene succinate (44 mg, 0.15 mmol), xylose azide (110 mg, 0.225 mmol), cuprous iodide (28 mg, 0.15 mmol), sodium vitamin C (30 mg, 0.15 mmol), and DIPEA (39 μL, 0.225 mmol) were dissolved in tetrahydrofuran (1.4 mL) and water (0.6 mL), and reacted at room temperature for 5 h. After the reaction was complete as monitored by TLC (PE:EA = 2:1), EA was diluted, washed with saturated brine, the organic phase was dried over anhydrous Na2SO4, concentrated, and column chromatography (PE / EtOAc = 1:3) yielded a yellow foamy solid (149 mg, yield 41%), which was directly added to the next step. 3 mL of methanol and 310 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (DCM:MeOH = 5:1), a yellow suspension was obtained. Column chromatography (DCM / MeOH = 40:1, v / v) yielded L-40 (43 mg, 78% yield). [α] D 25 = +21.8(c 0.1, CDCl3). 1H NMR(400MHz, DMSO-d6)δ12.81(s,1H),8.43(s,1H),8.08(d,J=7.6Hz,2H),7.63–7.56(m,3H),7.03(s,1H),6.96(s,1H),6.51(s,1H),6 .10(d,J=3.6Hz,1H),5.50(s,1H),5.42(s,2H),5.31(s,2H),4.00(t,J=6.4Hz,1H),3.92–3.82(m,1H),3.77–3.70(m,2H),3.54(s,1H). 13 C NMR (100MHz, DMSO-d6) δ182.1,164.1,163.6,161.2,157.3,141.2,132.2,130.6,129.2,126. 5,126.0,105.4,105.2,98.7,93.6,85.1,70.7,70.4,68.6,67.1,61.7.HRMS(ESI)m / z:[M+Na] + Calcd for C 23 H 21 N3O8Na490.1221; Found 490.1224.

[0217] The solubility of L-40 in DMSO is 30-60 mg / mL.

[0218] L-41: 7-Propynyl succinate (44 mg, 0.15 mmol), arabinose azide (110 mg, 0.225 mmol), CuI (29 mg, 0.15 mmol), and VcNa (30 mg, 0.15 mmol) were dissolved in tetrahydrofuran. DIPEA (39 μL, 0.225 mmol) and 0.6 mL H₂O were added, and the mixture was reacted at room temperature until the starting material was almost completely consumed. The mixture was extracted three times with EA and saturated citric acid solution. The organic phases were combined, washed with saturated NaCl solution, dried over anhydrous Na₂SO₄, and concentrated under reduced pressure by column chromatography (PE:EA = 4:1, v / v) to obtain a yellow solid (110 mg, 69%), which was directly added to the next step. 3 mL of methanol and 400 μL of sodium methoxide were added, and the mixture was reacted at room temperature for 2 h. The reaction was monitored by TLC (DCM:MeOH = 6:1) until completion, yielding a yellow suspension. Column chromatography (DCM / MeOH = 40:1, volume ratio) yielded a yellow, foamy solid, L-41 (30 mg, yield 46%). [α] D 25 = +10.3(c 0.1, CDCl3). 1H NMR (400MHz, DMSO-d6) δ12.82(s,1H),8.43(s,1H),8.10(d,J=7.2Hz,2H),7.61(s,4H),7.05(s,1H),6.98(s,1H),6.52( s,1H),5.46(d,J=9.2Hz,1H),5.31(s,3H),5.06(s,1H),4.83(s,1H),4.11–4.00(m,1H),3.83–3.75(m,3H),3.57(s,1H). 13 C NMR (100MHz, DMSO) δ182.1,164.1,163.6,161.2,157.3,141.8,132.2,130.6,129.2,126 .5,123.9,105.4,105.2,98.7,93.6,88.4,73.3,69.3,68.4,61.8.HRMS(ESI)m / z:[M+Na] + Calcd for C 23 H 21 N3O8Na 490.1221; Found490.1220.

[0219] The solubility of L-41 in DMSO is 30-60 mg / mL.

[0220] L-42: 7-Propylene succinate (32 mg, 0.1 mmol), rhamnosyl azidosyl (75 mg, 0.15 mmol), cuprous iodide (10 mg, 0.05 mmol), and sodium vitamin C (10 mg, 0.05 mmol) were dissolved in a mixed solution of tetrahydrofuran (0.7 mL) and water (0.3 mL). DIPEA (26 μL, 0.15 mmol) was added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (PE:EA = 2:1), EA was diluted, washed with saturated saline, and the organic phase was dried over anhydrous Na₂SO₄. The solution was concentrated and subjected to column chromatography (PE / EtOAc = 1:2) to obtain a yellow foamy solid (42 mg, yield 35%), which was directly added to the next step. 3 mL of methanol and 350 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (DCM:MeOH = 5:1, v / v), a yellow suspension was obtained. Column chromatography (DCM / MeOH = 40:1, volume ratio) yielded a yellow, foamy solid, L-42 (20 mg, yield 83%). [α] D 25 = -3.0(c 0.1, CDCl3). 1H NMR(400MHz,DMSO-d6)δ12.81(s,1H),8.35(s,1H),8.08(d,J=7.2Hz,2H),7 .62–7.56(m,3H),7.02(s,1H),6.96(s,1H),6.50(s,1H),6.03(s,1H),5.39( d,J=5.2Hz,1H),5.30(s,2H),5.11–5.04(m,2H),3.91(t,J=3.6Hz,1H),3.6 0–3.56(s,1H),3.50–3.46(m,1H),3.35–3.27(m,1H),1.22(d,J=6.4Hz,3H). 13 C NMR(100MHz,DMSO-d6)δ182.6,164.5,164.0,161.7,157.8,141.8,132.6,131.0,129.6,126.9,1 25.1,105.9,105.6,99.2,94.0,86.4,75.5,73.4,71.7,71.0,62.2,18.3.HRMS(ESI)m / z:[M+Na] + Calcd for C 24 H 23 N3O8Na 504.1377; Found 504.1381.

[0221] The solubility of L-42 in DMSO is 30-60 mg / mL.

[0222] L-43: Compound L-26 (41 mg, 0.1 mmol), arabinose-azidosose (73 mg, 0.15 mmol), cuprous iodide (19 mg, 0.1 mmol), sodium vitamin C (20 mg, 0.1 mmol), and DIPEA (26 μL, 0.15 mmol) were dissolved in tetrahydrofuran (1.4 mL) and water (0.6 mL), and reacted at room temperature for 2 h. After the reaction was complete as monitored by TLC (PE:EA = 2:1), EA was diluted, washed with saturated brine, the organic phase was dried over anhydrous Na2SO4, concentrated, and column chromatography (PE / EtOAc = 1:2, v / v) to give a yellow foamy solid (23 mg, yield 26%), which was directly added to the next step. 3 mL of methanol and 350 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (DCM:MeOH = 5:1), a yellow suspension was obtained. Column chromatography (DCM / MeOH = 10:1, volume ratio) yielded a yellow, foamy solid, L-43 (7 mg, 79% yield). [α] D 25 = +36.9(c 0.1, CDCl3). 1H NMR(800MHz,DMSO-d6)δ12.59(s,1H),10.91(s,1H),8.11(s,1H),7.99–7.96(m,2H),7.08–7.05(m ,2H),6.36(s,1H),5.95(d,J=1.6Hz,1H),5.61(d,J=5.6Hz,1H),5.28(d,J=4.0Hz,1H),5.18(d,J= 2.4Hz,2H),5.16–5.14(m,1H),4.83(d,J=6.4Hz,1H),3.97–3.94(m,1H),3.93–3.91(m,1H),3.85( s,3H),3.78–3.76(m,1H),3.69(d,J=7.2Hz,2H),3.40(d,J=7.2Hz,2H),1.71(s,3H),1.62(s,3H). 13 C NMR (200MHz, DMSO) δ178.2,161.3,158.8,155.7,153.7,142.0,136.1,131.1,130.0,124.7,122.4,122.3,1 14.0,105.9,104.2,98.3,84.3,70.1,69.9,66.0,64.6,63.4,55.4,25.4,21.2,17.8.HRMS(ESI)m / z:[M+Na] + Calcd for C 29 H 31 N3O 10 Na604.1902; Found604.1902.

[0223] The solubility of L-43 in DMSO is 35-70 mg / mL.

[0224] L-44: The first step, following the synthesis of compound L-32, yielded a yellow, foamy solid (81 mg, 90%), which was directly introduced to the next step. 3 mL of methanol and 380 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by TLC (DCM:MeOH = 5:1) until completion, resulting in a yellow suspension. Column chromatography (DCM / MeOH = 5:1) yielded the yellow, foamy solid L-44 (29 mg, 64% yield). [α] D 25 = +27.8(c 0.1, CDCl3). 1H NMR (800MHz, DMSO-d6) δ12.58(s,1H),8.22(s,1H),7.99(d,J=8.8Hz,2H),7.09 –7.03(m,2H),6.32(s,1H),6.02(t,J=2.4Hz,1H),5.20(s,2H),4.00–3.95(m,1 H),3.85(s,3H),3.70(s,1H),3.66(s,1H),3.53–3.51(m,1H),3.18(s,1H),2.7 0–2.68(m,2H),1.61–1.57(m,1H),1.40–1.37(m,2H),0.93(s,3H),0.92(s,3H). 13 C NMR(200MHz,DMSO-d6)δ178.1,162.0,161.2,158.6,155.4,153.6,141.7,135.9,129.8,125.6,122.4,113.9 ,106.9,104.0,98.2,84.8,70.6,70.3,68.5,66.9,64.4,55.3,37.9,27.7,22.4,20.0.HRMS(ESI)m / z:[M+Na] + Calcd for C 29 H 33 N3O 10 Na 606.2058; Found 606.2053.

[0225] The solubility of L-44 in DMSO is 35-70 mg / mL.

[0226] L-45: Compound L-27 (88 mg, 0.2 mmol), rhamnosine (150 mg, 0.3 mmol), cuprous iodide (38 mg, 0.2 mmol), sodium vitamin C (48 mg, 0.2 mmol), and DIPEA (53 μL, 0.3 mmol) were dissolved in tetrahydrofuran (2.1 mL) and water (0.3 mL), and reacted at room temperature for 10 h. After the reaction was complete as monitored by TLC (PE:EA = 2:1), EA was diluted, washed with saturated brine, the organic phase was dried over anhydrous Na2SO4, concentrated, and column chromatography (PE / EtOAc = 1:1, v / v) to give a yellow foamy solid (77 mg, yield 27%), which was directly added to the next step. 3 mL of methanol and 380 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (DCM:MeOH = 5:1), a yellow suspension was obtained. Column chromatography (DCM / MeOH = 5:1, volume ratio) yielded a yellow, foamy solid, L-45 (28 mg, yield 68%). [α] D25 =+2.3(c 0.1,CDCl3). 1 H NMR (400MHz, DMSO-d6) δ8.31(s,1H),8.19(s,1H),7.90(d,J=8.8Hz,2H),7.01(d,J =8.4Hz,2H),6.02–5.93(m,2H),5.83(s,1H),5.36(s,2H),5.19–4.99(m,9H),4.43( s,1H),4.37(s,1H),3.90–3.84(m,2H),3.81(s,3H),3.24–3.20(m,3H),1.56(s,3H ),1.50(s,3H),1.19(d,J=5.6Hz,3H),1.13(d,J=6.0Hz,3H).HRMS(ESI)m / z:[M+Na] + Calcd for C 39 H 50 N7O 14 Na 863.3302; Found 863.3307.

[0227] The solubility of L-45 in DMSO is 65-90 mg / mL.

[0228] L-46: Compound L-27 (31 mg, 0.07 mmol), xylose azide (102 mg, 0.21 mmol), cuprous iodide (28 mg, 0.14 mmol), sodium vitamin C (28 mg, 0.14 mmol), and DIPEA (37 μL, 0.21 mmol) were dissolved in tetrahydrofuran (1.4 mL) and water (0.6 mL), and reacted at room temperature for 5 h. After the reaction was complete as monitored by TLC (PE:EA = 2:1), EA was diluted, washed with saturated brine, the organic phase was dried over anhydrous Na2SO4, concentrated, and column chromatography (PE / EtOAc = 1:2, v / v) to give a yellow foamy solid (59 mg, 60% yield), which was directly added to the next step. 3 mL of methanol and 260 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (DCM:MeOH = 5:1), a yellow suspension was obtained. Column chromatography (DCM / MeOH = 5:1, volume ratio) yielded a yellow, foamy solid, L-46 (21 mg, 73% yield). [α] D 25 = +10.5(c 0.1, CDCl3). 1H NMR (800MHz, DMSO-d6) δ12.74(s,1H),8.37(s,1H),8.22(s,1H),7.97(d,J=9.6Hz,2H),7.06(d,J=8.8Hz,2H),6.82(s,1H),6.09(d,J=3.5H z,1H),6.01(d,J=3.2Hz,1H),5.51(d,J=4.8Hz,1H),5.48(d,J=4.0Hz,1H),5.45(d,J=5.6Hz,1H),5.42(d,J=5.6Hz,1H),5.38(d,J=7.2Hz,1 H),5.34(d,J=7.2Hz,3H),5.20(s,2H),5.12–5.06(m,1H),4.00–3.98(m,1H),3.97–3.95(m,1H),3.87(dd,J=11.2,3.2Hz,1H),3.84(s,3H), 3.81(d,J=4.0Hz,1H),3.76–3.66(m,2H),3.63(dd,J=12.0,6.4Hz,1H),3.56–3.48(m,3H),3.39(d,J=7.2Hz,2H),1.58(s,3H),1.56(s,3H). 13 CNMR(200MHz,DMSO)δ178.5,161.4,161.2,159.5,156.2,152.8,141.7,141.4,136.1,131.5,130.1,125.7,125.5,122.2,121.8,114.1,1 07.4,104.9,96.3,84.9,84.8,70.6,70.5,70.4,70.3,68.5,68.5,67.1,67.0,64.6,62.1,55.4,25.4,21.3,17.6.HRMS(ESI)m / z:[M+Na] + Calcd for C 37 H 42 N6O 14 Na 817.2651; Found 817.2650.

[0229] The solubility of L-46 in DMSO is 70-90 mg / mL.

[0230] L-47: Compound L-29 (31 mg, 0.07 mmol), xylose azide (102 mg, 0.21 mmol), cuprous iodide (28 mg, 0.14 mmol), sodium vitamin C (28 mg, 0.14 mmol), and DIPEA (37 μL, 0.21 mmol) were dissolved in tetrahydrofuran (1.4 mL) and water (0.6 mL), and reacted at room temperature for 5 h. After the reaction was complete as monitored by TLC (PE:EA = 2:1), EA was diluted, washed with saturated brine, the organic phase was dried over anhydrous Na2SO4, concentrated, and column chromatography (PE / EtOAc = 1:2, v / v) to give a yellow foamy solid (34 mg, yield 34%), which was directly added to the next step. 3 mL of methanol and 210 μL of sodium methoxide were added, and the reaction was carried out at room temperature for 2 h. After the reaction was complete as monitored by TLC (DCM:MeOH = 5:1), a yellow suspension was obtained. Column chromatography (DCM / MeOH = 5:1, volume ratio) yielded a yellow, foamy solid, L-47 (15 mg, 82% yield). [α] D 25 = +10.8(c 0.1, CDCl3). 1 H NMR (800MHz, DMSO-d6) δ8.36(s,1H),8.22(s,1H),7.99(d,J=8.8Hz,2H),7.06(d,J=8.8Hz,2H),6.82(s,1H),6.09(d,J=4.0Hz,1H),6 .01(d,J=4.0Hz,1H),5.49(d,J=4.0Hz,1H),5.46(d,J=4.0Hz,1H),5.43(d,J=5.6Hz,1H),5.40(d,J=5.6Hz,1H),5.36–5.35(m,4H),5. 32(d,J=6.4Hz,1H),5.21(s,2H),4.01–3.94(m,2H),3.87(dd,J=12.0,3.2Hz,1H),3.84(s,3H),3.84–3.80(m,1H),3.76–3.66(m,3H) ,3.63(dd,J=11.2,6.4Hz,1H),3.55–3.48(m,3H),2.73–2.68(m,2H),1.53–1.49(m,1H),1.38–1.32(m,2H),0.86(s,3H),0.85(s,3H). 13CNMR(200MHz,DMSO)δ178.6,161.4,161.3,159.3,156.1,152.9,141.7,141.6,136.0,130.0,125.7,125.3,122.3,114.1,108.7,1 04.9,96.3,84.9,84.8,70.7,70.4,70.3,68.5,68.5,67.1,67.0,64.5,62.1,55.5,38.0,27.6,22.4,20.0.HRMS(ESI)m / z:[M+Na] + Calcd for C 37 H 44 N6O 14 Na819.2808; Found 819.2805.

[0231] The solubility of L-47 in DMSO is 40-70 mg / mL.

[0232] Example 6 Preparation of compound L-48

[0233] The structural formula of L-48:

[0234]

[0235] The preparation method is as follows:

[0236] Step 1: Take 25 kg of dried Epimedium sagittatum Maxim. leaves and stir-fry them with 5 kg of mutton fat to obtain the processed product. After drying the processed product, pulverize it into powder and extract it with 50% ethanol under reflux at 83℃ for 1.5 h. After extracting twice, obtain the extract and concentrate it under reduced pressure to obtain 5 kg of total fluid extract.

[0237] Step 2: The fluid extract obtained in Step 1 was adsorbed using a macroporous resin column and eluted sequentially with 20%, 40%, 60%, 80%, and 95% ethanol by volume fraction to obtain a 20% fraction (200g), a 40% fraction (236.5g), a 60% fraction (305g), an 80% fraction (105g), and a 95% fraction (175g).

[0238] Step 3: Separate the 80% ethanol fraction obtained in Step 2 using 200-300 mesh normal phase silica gel, and elute sequentially with dichloromethane:methanol in volume ratios of 20:1, 15:1, 10:1, 5:1, 2:1, 1:1, and 0:1 to obtain ten fractions Fr1-10.

[0239] Step 4: The Fr4 fraction obtained in Step 3 is subjected to C18 reverse phase (ODS-AQ-HG packing material) column chromatography, eluted with methanol / water (60%-100%), to obtain two fractions. The eluent is collected with pure methanol as the mobile phase to obtain Fr4-a and Fr4-remaining fraction.

[0240] Step 5: The Fr4-a fraction obtained in Step 4 was separated by high performance liquid chromatography (using 43% acetonitrile / water as the mobile phase, 19 mL / min) to obtain compound L-48 (25.8 mg, t). R =11.2min).

[0241] Structural identification: High-resolution mass spectrometry (HR-ESI-MS) was used. 1 H NMR, 13 The compounds obtained in Example 6 were structurally identified by 1C NMR and two-dimensional NMR (HMBC, HMQC).

[0242] Compound L-48 in Example 6 was identified as a yellow powder, readily soluble in methanol, showing a positive result in the hydrochloric acid-magnesium powder reaction, and exhibiting brown fluorescence under ultraviolet light, suggesting it is a flavonoid compound. High-resolution mass spectrometry (HR-ESI-MS) determined the quasi-molecular ion peak m / z to be 531.1869 [M+H]. + (C 27 H 31 O 11 The calculated value is 531.1866), combined with 1 H and 13 The C10 NMR (Table 3.4) suggests the molecular formula is C10. 27 H 30 O 11 Its degree of unsaturation is 13. 1 In H NMR spectra, δ H 8.20 (2H, d, J = 9.0 Hz) and 7.06 (2H, d, J = 9.1 Hz) are presumed to be the AABB' coupling system of the B ring of the flavonoid core; δ H 6.62 (1H, s) is presumed to be a signal from an isolated aromatic hydrogen atom on ring A; δ H 3.88 (3H, s) and δ C 56.3 suggests the presence of a methoxy group, which, combined with the δ in the HMBC spectrum... H The strong correlation between 3.88 and C (δC163.0) suggests that the methoxy group is located at the C-4' position. H 5.22 (H,t,J=6.8Hz),δ HThe 1.82 (3H,s) and 1.67 (3H,s) values ​​are presumed to be the olefinic proton and methyl signal of the isopentenyl group, respectively. Combined with the HMBC spectrum, H-11 and C-5 (δ) signals are also considered. C 155.1) and C-7 (δ C 162.1) The correlation indicates that the isopentenyl group may be attached at the C-6 position. 1 The H NMR spectrum shows a glucose terminal hydrogen signal δ H A value of 5.06 (1H, d, J = 7.5 Hz) suggests the possible presence of a glucose molecule. This is further supported by the H-1″ and C(δ) values ​​in the HMBC spectrum. C 110.8) The related hypothesis is that the glucose linkage is located at C-6. The terminal proton coupling constant of glucose (J = 7.5 Hz) suggests that the relative configuration of glucose is β-configuration.

[0243] Compound L-48 1 H NMR (600MHz), 13 The C NMR (151MHz) and HMBC NMR data are shown in Table 4.

[0244] Table 4

[0245]

[0246]

[0247] Note: 1 H(600MHz, CD3OD), 13 C(151MHz, CD3OD)

[0248] The HMBC spectrum of compound L-48 is shown in the following formula:

[0249]

[0250] Example 7 Preparation of compound L-49

[0251] L-49 structural formula:

[0252]

[0253] Preparation method:

[0254] Step 1: The 40% fraction (236.5g) obtained in Step 2 of Example 6 was coarsely segmented with 1800g of positive silica gel (200-300 mesh), and then eluted sequentially with dichloromethane:methanol in volume ratios of 50:1, 25:1, 15:1, 5:1, and 0:1 to obtain 8 components (Fr1-Fr8);

[0255] Step 2: The Fr5 fraction obtained in Step 1 was subjected to preparative high-performance liquid chromatography (HPLC) with methanol / water (50%-85% v / v) as the mobile phase for 25 min at an elution rate of 19 mL / min, yielding compound L-49 (45 mg, t). R =18.2min).

[0256] Structural identification: using modern spectroscopic techniques 1 H NMR, 13 The structure of the compound obtained in Example 7 was identified by C NMR spectroscopy.

[0257] Upon identification, compound L-49 in Example 7 was found to be a yellow powder, and the hydrochloric acid-magnesium powder reaction was positive, suggesting it is a flavonoid compound. 1 In HNMR spectra, δ H The values ​​of 7.77 (2H, d, J = 8.3 Hz) and 6.94 (1H, d, J = 8.3 Hz) are presumed to be proton signals of the cyclic AA'BB' coupling system of the flavonoid core, δ H Signals at 6.38 (1H, s) and 6.21 (1H, s) are presumed to be isolated aromatic hydrogen signals from the A ring of the flavonoid nucleus, δ H The values ​​of 5.38 (1H, s) and 0.93 (3H, d, J = 4.8) suggest the presence of a rhamnose signal. 13 The CNMR spectrum shows 21 carbons, including 15 carbon signals from the flavonoid skeleton and 6 carbon signals from a rhamnose.

[0258] NMR data for compound L-49: yellow powder, C 39 H 50 O 19 , 1 HNMR (400MHz, CD3OD)δ H :7.77(2H,d,J=8.3Hz,H-2 / H-6'),6.94(1H,d,J=8.3Hz,H-3' / H-5'),6.38(1H,s,H-8),6.21(1H,s,H-6),5.38(1H,s,Rhamnosyl H-1"),0.933H,d,J=4.8(3H,Rhamnosyl CH3); 13CNMR(101MHz,CD3OD)δC:179.6(C-4),165.9(C-7),163.2(C-5),161.5(C-4'),159.2(C-2),158.5(C-9),136.2(C-3),131.9(C-2' / C-6'),122.6 (C-1'),116.5(C-3' / C-5'),105.9(C-10),103.5(C-1"),99.9(C-6),94. 8(C-8),73.1(C-4"),72.1(C-5"),72,0(C-3"),71.9(C2"),17.6(C-6").

[0259] Example 8 Preparation of compound L-50

[0260] L-50 structural formula:

[0261]

[0262] Preparation method:

[0263] Step 1: Take 20 kg of dried Epimedium brevicornum Maxim. leaves and stir-fry them with 5 kg of mutton fat to obtain the processed product. After drying the processed product, pulverize it into powder and extract it with 50% ethanol under reflux at 83℃ for 1.5 h. After extracting twice, obtain the extract, concentrate it under reduced pressure to obtain 8.8 kg of total fluid extract;

[0264] Step 2: The fluid extract obtained in Step 1 was adsorbed using a macroporous resin column and eluted sequentially with 40%, 80%, and 95% ethanol by volume fraction to obtain a 40% fraction (812.0 g), an 80% fraction (381.0 g), and a 95% fraction (29.0 g).

[0265] Step 3: Separate the 40% fraction obtained in Step 2 into segments using 1.0 kg of normal-phase silica gel, and elute sequentially with a dichloromethane:methanol gradient at volume ratios of 50:1, 25:1, 10:1, 5:1, and 0:1 to obtain six fractions of Fr(1-6).

[0266] Step 4: Separate the Fr1 fraction (1.347g) obtained in Step 3 using normal phase silica gel with dichloromethane:methanol = 50:1 as the mobile phase to obtain 721.0mg of FrA-1 fraction;

[0267] Step 5: Pass the FrA-1 fraction obtained in Step 4 through a normal phase column with petroleum ether:ethyl acetate ratios of 50:1, 25:1, 10:1, 5:1, and 1:1 (volume ratio) to obtain compound L-50 (4.0 mg).

[0268] Structural identification: using modern spectroscopic techniques 1 H NMR, 13 The structure of the compound obtained in Example 8 was identified by C NMR spectroscopy;

[0269] Upon identification, compound L-50 in Example 8 was found to be a colorless powder, soluble in methanol, and showed a positive result in the hydrochloric acid-magnesium powder reaction, suggesting it is a flavonoid compound. 1 In H NMR spectra, δ H 8.02 (s, 1H) suggests the presence of an isolated aromatic hydrogen atom. δ H 7.45 (d, J = 7.6 Hz, 2H) and δ H The 6.46 (d, J = 7.6 Hz, 2H) signal is presumed to be the proton signal of the AA'BB' coupling system of the B ring of the flavonoid nucleus. δ H 7.53 (d, J = 7.4 Hz, 1H) and δ H 6.55 (d, J = 7.4 Hz, 1H), coupling constant J = 7.4 Hz, suggesting possible ortho-substitution of the flavonoid A ring. Combined with the 1H spectrum, 13 In C NMR spectra, δ C 152.24 is speculated to be a characteristic signal of isoflavones, δ C 147.11 and 123.48 may correspond to the ortho carbon of the carbonyl group on the C ring of isoflavones and the carbon of olefins, δ C The values ​​of 133.23, 132.72 and 117.64, 116.29 suggest that ring B may be substituted with a para-hydroxyl group. δ C The low-field shifts of 123.37 and 113.6 suggest that there may be hydroxyl substitution in the A ring.

[0270] NMR data for compound L-50: colorless powder, C 15 H 10 O5, 1 H NMR (CD3OD, 400MHz)δ H 8.02(s,1H,H-2),7.53(d,J=7.4Hz,1H,H-6),7.45(d,J=7.6Hz,2H,H-2',6'),6.55(d,J=7.4Hz,1H,H-5),6.46(d,J=7.6Hz,2H,H-3',5'). 13 C NMR (CD3OD, 101MHz)δ C:152.24(C-2),147.11(C-9),133.23(C-2'),132.72(C-6'),130.60(C-1') ,123.48(C-10),123.37(C-6),117.64(C-3'),116.29(C-5'),113.61(C-8).

[0271] Example 9 Cell viability assay

[0272] To detect the activity of the compounds obtained in Examples 1-5 against mouse myoblast C2C12 cells, the following cell activity experiments were performed:

[0273] 1. Materials and Methods:

[0274] 1.1 Experimental Materials

[0275] 1.1.1 The C2C12 mouse myoblasts used in this experiment were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (catalog number: GNM26).

[0276] 1.1.2 Main Reagents and Instruments: DMEM culture medium (AK30795786, HyClone), fetal bovine serum (SH30406.05, HyClone), 0.25% trypsin (25200-072, Gibco), PBS buffer (28224968CJ, Biosharp), Penicillin-Streptomycin Solution 100x (P3362625, Adamas Life). 96-well white transparent flat-bottomed 96-well cell culture plates (010924240701, Beyotime), 15mL centrifuge tubes (35423124E, Labselect), 50mL centrifuge tubes (132221250, Labselect). Dexamethasone sodium phosphate (011145225, Aladdin), CellTiter-Meiluncell chemiluminescence immunoassay kit (PWL111-2, MeilunBio). SW-CJ-IFD type ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd.), Glaaxy 170S type cell culture incubator (Eppendorf New Brunswick, Germany), SPARK multi-functional microplate reader (Tecan, USA).

[0277] The following complete culture media are used: ① DMEM / high glucose version, containing L-glutamine and sodium pyruvate 450mL; ② Penicillin-Streptomycin Solution 100x 5mL; ③ FBS fetal bovine serum 50mL.

[0278] Culture media without fetal bovine serum: ① DMEM / high glucose version, containing L-glutamine and sodium pyruvate 450mL; ② Penicillin-Streptomycin Solution 100x 5mL.

[0279] 1.2 Experimental Methods

[0280] 1.2.1 Cell Culture

[0281] Step 1, Cell Resuscitation: Quickly transfer the C2C12 cell cryovials stored in liquid nitrogen to a 37°C constant temperature water bath and gently shake until completely thawed (approximately 1-2 minutes) to reduce the toxic effects of DMSO on the cells. Immediately transfer the thawed cell suspension to a centrifuge tube containing 5 mL of complete culture medium, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, add 2 mL of pre-warmed culture medium, gently pipette to resuspend, and then seed into a 10 cm⁻¹ agar tube containing 1 mL of complete culture medium. 2 Place the culture dish in a culture medium. Finally, place the culture dish in a 37℃, 5% CO2 incubator for static culture. After 24 hours, change the medium and observe the cell adhesion.

[0282] Step 2, Cell Passage: Select adherent cells with 80%-90% confluence. Discard the old culture medium and gently wash twice with 2 mL of PBS to remove residual serum. Then add 1 mL of 0.25% trypsin-EDTA and digest at 37℃ for 3 min. After confirming under a microscope that the cells have become rounded and begun to detach, immediately add 2 mL of complete culture medium to stop digestion. Repeatedly pipette to form a single-cell suspension and transfer to a 15 mL centrifuge tube. Centrifuge at 1000 rpm for 3 min, discard the supernatant, and resuspend in an appropriate amount of complete culture medium at a ratio of 1:2 to 1:5 according to the required cell volume for the experiment. Aspirate the cells into the corresponding culture dishes according to the proportion, labeling the cell passage, date, and operator. Incubate at 37℃ in a 5% CO2 incubator. Observe the adherence after passage. When the cell confluence reaches 80%, it can be passaged again.

[0283] Step 3, Cell Cryopreservation: The cryopreservation procedure is the same as after cell passage, digestion, centrifugation, and discarding the supernatant. Then, slowly add 5 mL of pre-freezing solution and gently pipette 30 times. Use a 1 mL pipette to aliquot the suspension into 2 mL cryovials, 1 mL per tube. Label each tube with the cell passage number, date, and operator information, and then perform gradient cooling. Vertically insert the cryovials into the isopropanol cryopreservation box and immediately transfer the cryopreservation box to a -80°C freezer. The isopropanol is used to control the cooling rate at -1°C / min through phase transition, continuing for 8-12 hours. The next day, transfer the cryovials to a liquid nitrogen tank for long-term storage.

[0284] 1.2.2 Viability Measurement of a Dexamethasone Sodium Phosphate-Induced C2C12 Cell Damage Model

[0285] Step 4: Seed the C2C12 cells obtained in Step 2 into 96-well plates (2 × 10⁻⁶ cells / well). 3 Cells / well), 100 μL per well, and the cells were divided into normal control group, 150 μM dexamethasone sodium phosphate group (model group), 150 μM dexamethasone sodium phosphate + compound group (experimental group), and complete culture medium (blank group), and cultured for 24 h.

[0286] Step 5: Then change the culture medium. Add complete culture medium again to the normal control group, add 150 μM dexamethasone sodium phosphate diluted with complete culture medium to the model group, add 150 μM dexamethasone sodium phosphate diluted with complete culture medium and 10, 5, 2.5, 1, 0.5, 0.25, 0.1, and 0.05 μM dilutions of the above compounds to the experimental group, and add complete culture medium to the blank group. Add 100 μL of complete culture medium to each well and incubate at 37°C and 5% CO2 for 48 h.

[0287] Step 6: Remove the cell culture plate and allow it to equilibrate at room temperature for 10 minutes. Add 100 μL of ATP solution to each well (ATP is from the CellTiter-Meiluncell luminescence assay kit, purchased from Meilun; the concentration and amount of ATP solution used here are the same as in the kit). Shake at room temperature for 2 minutes and incubate for 10 minutes to allow the luminescence signal to stabilize. Detect the luminescence signal using a multi-functional microplate reader.

[0288] The formula for calculating cell viability is: Cell viability % = (Experimental group - Blank group) Rlu / (Control group - Blank group) Rlu × 100%

[0289] 1.2.3 Viability Measurement of Serum Starvation Cell Model

[0290] Step 1: Seed C2C12 cells into 96-well plates (5×103 cells / well), 100μL per well, and divide the cells into normal control group, starvation group (model group), starvation + compound group (experimental group), and culture medium without fetal bovine serum (blank group), and culture for 24h.

[0291] Step 2: Change the culture medium. The normal control group was replenished with complete culture medium, the model group was replenished with culture medium without fetal bovine serum, the experimental group was replenished with different concentrations of monomer compound solution diluted with culture medium without fetal bovine serum, and the blank group was replenished with culture medium without fetal bovine serum. 100 μL of culture medium was added to each well and cultured at 37°C and 5% CO2 for 24 h.

[0292] Step 3: Add 10 μL CCK8 solution to each well, incubate at 37℃ and 5% CO2 for 1 h, and then use a microplate reader to detect the OD value at a wavelength of 450 nm.

[0293] The formula for calculating cell viability is: Cell viability % = (Experimental group - Blank group)OD / (Control group - Blank group)OD × 100%

[0294] 1.2.4 Statistics and Analysis

[0295] Following the method for measuring cell viability using a starvation model, C2C12 myoblasts were cultured under serum-free conditions and simultaneously treated with dehydrated icariin (AHI), L-1 (AHI*); icariin (ICA), L-2 (ICA*); cypermethrin A (EA), L-3 (EA*); cypermethrin A1 (EA1), L-4 (EA1*); cypermethrin B (EB), L-5 (EB*); cypermethrin C (EC), L-6 (EC*); and cypermethrin I (BI), L-7 (BI*) for 24 hours, and the results were detected by CCK8 assay.

[0296] Experimental data The results are shown below. One-way ANOVA was performed using GraphPadPrism 10.1.2 software. Figure 1 As shown.

[0297] from Figure 1 As can be seen from the results, compound cytotoxic bismuth substituent I (BI) exhibited significant cytotoxic effects in a serum starvation cell model, and these effects were concentration-dependent. For compound L-7, however, cytotoxic effects were significantly reduced at concentrations below 25 μM. Therefore, hydrogenation of the isopentenyl group significantly affected the activity of BI (BI), and this structure has the potential for further structural modification. This finding indicates that hydrogenation modification of the isopentenyl side chain can effectively reduce the cytotoxicity of BI while preserving its potential pharmacological activity, providing a key direction for subsequent structural optimization.

[0298] Following the method for measuring cell viability in the C2C12 cell starvation model, the effects of compounds L-8 to L-25 on the cell viability of the C2C12 cell starvation model were obtained. The cell viability corresponding to the addition of compounds L-8 to L-25 in the starvation group is shown in Table 4.

[0299] Table 4

[0300]

[0301]

[0302] The effects of compounds L-8 to L-25 on cell viability in the C2C12 cell DEX model (dexamethasone sodium phosphate-induced cell damage model) are shown in Table 5.

[0303] Table 5

[0304]

[0305]

[0306] The effects of compounds L-26 to L-47 on the viability of C2C12 starvation model cells are shown in Table 6.

[0307] Table 6

[0308]

[0309] The effects of compounds L-26 to L-47 on the viability of C2C12 cells in the DEX model are shown in Table 7.

[0310] Table 7

[0311]

[0312]

[0313] As shown in Table 4-7, in the cell starvation model, compounds L-10, L-12, L-13, and L-17 exhibited greater potential for cell protection in promoting C2C12 myocyte survival than the positive control drug EB. In the dexamethasone model, compounds L-9, L-10, L-14, L-17, L-19, and L-25 all showed better cell activity than the positive control drug EB, with L-10 and L-25 showing the most significant and statistically significant activity. This result indicates that within this concentration range, this series of derivatives has a better protective effect on myocytes than EB. In the serum starvation model, CCK-8 assay revealed that some triazole derivatives, such as compounds L-40 and L-46, showed slightly better promoting effects on C2C12 myocyte survival than the EB group, and can be used as lead compounds for structural optimization. Furthermore, through correlation analysis between triazole substituent differences and myocyte viability data, we found that glycosyl modification may significantly improve the survival and protective effects on C2C12 myocytes.

[0314] Compound BI exhibited significant cytotoxic effects in a serum starvation cell model, showing a concentration-dependent relationship. However, compound L-7 significantly reduced cytotoxicity, even losing its toxicity, at concentrations below 25 μM (P < 0.05). Therefore, hydrogenation of the isopentenyl group significantly affects the activity of BI, and this structure has potential for further structural modification. This finding indicates that hydrogenation modification of the isopentenyl side chain can effectively reduce the cytotoxicity of BI while preserving its potential pharmacological activity, providing a key direction for subsequent structural optimization.

[0315] In summary, compounds L-10 and L-25, with their significant activity advantages, have become the primary targets for subsequent structural optimization efforts. Specifically, both L-10 and L-25 introduce a piperidine ring into their chemical structures, a structural feature that may play a significant role in anti-sarcopenic activity.

[0316] The effect of compound L-48 on the C2C12 activity induced by dexamethasone sodium phosphate is as follows: Figure 2 As shown, the effect of compound L-49 on starvation-induced C2C12 activity is as follows: Figure 3 As shown, the effect of compound L-50 on the C2C12 activity induced by dexamethasone sodium phosphate is as follows: Figure 4 As shown, the effect of compound L-50 on starvation-induced C2C12 activity is as follows: Figure 5 As shown in the figure (Unfed refers to the starvation condition / model).

[0317] like Figure 2-5 As shown, compounds L-48 and L-50 both exhibited protective effects against dexamethasone sodium phosphate-damaged C2C12 cells. Specifically, compounds L-48 and L-50, at concentrations of 5 μM and 100 μM, significantly reversed the inhibitory effect of dexamethasone sodium phosphate on C2C12 myoblast viability, achieving cell viability of 66% and 69%, respectively. In a starvation model, compounds L-49 and L-50, at concentrations of 10 μM and 50 μM, significantly enhanced starvation-induced C2C12 myoblast viability, achieving cell viability of 90% and 92%, respectively.

[0318] The results showed that compounds L-48 and L-50 significantly improved the survival rate of C2C12 cells under dexamethasone sodium phosphate injury, and compounds L-49 and L-50 significantly improved the survival rate of C2C12 cells under starvation injury, suggesting that these compounds have potential sarcopenia therapeutic activity.

[0319] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. Use of an icariin derivative or a composition comprising the icariin derivative in the preparation of a medicament for preventing or treating sarcopenia, characterized in that, The structural formula of the icariin derivative is as follows: 、 、 、 、 、 、 。

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