Tripterine acrylamide derivative as well as preparation method and application thereof

Abstract

The invention discloses a tripterine acrylamide derivative with a structure shown as a formula III, wherein A1 and A2 are respectively and independently selected from O and NH; l is selected from-(CH2) n-and-((CH2) 2O) m (CH2) 2-, n is an integer ranging from 2 to 6, and m is an integer ranging from 1 to 6; r1 and R2 are respectively and independently selected from H, # imgabs0 # R3 and R4 are respectively and independently selected from C1-C4 alkyl, or R3 and R4 are connected with each other to form a 5-7-membered heterocyclic ring containing one oxygen atom together with an oxygen atom, or R3 and R4 are connected with each other to form a 5-7-membered heterocyclic ring together with a nitrogen atom. Compared with tripterine, the tripterine acrylamide derivative disclosed by the invention has the advantage that the inhibition effect of the tripterine acrylamide derivative on tumor cells is obviously enhanced. The invention also discloses an application of the tripterine acrylamide derivative in preparation of antitumor drugs. # imgabs1 #

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical chemistry and pharmacotherapy, and relates to a tripterygium wilfordii acrylamide derivative and a preparation method thereof and use thereof in preparing anti-tumor drugs. Technical Background

[0002] In recent years, the incidence and mortality of cancer have shown a rapid upward trend, seriously endangering human physical and mental health. Although the prevention, diagnosis and treatment of cancer have developed rapidly, it is still one of the main causes of death.

[0003] Celastrol (CEL) is a type of pentacyclic triterpenoid compound derived from the root of the traditional Chinese medicine Tripterygium wilfordii. A large number of studies have found that celastrol has a variety of biological activities, such as anti-tumor, anti-inflammatory, anti-obesity, and anti-neurodegenerative diseases. The anti-tumor activity of celastrol is a research hotspot, and its activity still needs to be improved.

[0004] Summary of the invention

[0005] The purpose of the present invention is to use tripterygium wilfordii as a lead compound for structural modification and transformation, and to provide a series of tripterygium wilfordii derivatives by introducing Michael addition receptors such as acrylamide. Pharmacological experiments show that the anti-tumor activity of the tripterygium wilfordii derivatives of the present invention is significantly improved compared with tripterygium wilfordii.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] The structure of the tripterygium wilfordii acrylamide derivative is shown in Formula III:

[0008]

[0009] Wherein, A1 and A2 are independently selected from O and NH;

[0010] L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, n is an integer from 2 to 6, and m is an integer from 1 to 6;

[0011] R1 and R2 are independently selected from H, R3 and R4 are independently selected from C1-C4 alkyl, or R3 and R4 are connected to each other and together with the oxygen atom to form a 5-7 membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 5-7 membered heterocyclic ring.

[0012] Preferably, A1 and A2 are independently selected from O;

[0013] L is selected from -(CH2) n -, n is an integer from 3 to 5;

[0014] R1 is selected from H, R2 is selected from R3 and R4 are independently selected from C1-C4 alkyl, or R3 and R4 are connected to each other and together with the oxygen atom to form a 5-7 membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 5-7 membered heterocyclic ring;

[0015] or A1 and A2 are independently selected from NH;

[0016] L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, n is an integer from 2 to 6, and m is an integer from 1 to 3;

[0017] R1 and R2 are independently selected from H.

[0018] More preferably, A1 and A2 are independently selected from O;

[0019] L is selected from -(CH2) n -, n is an integer from 3 to 5;

[0020] R1 is selected from H, R2 is selected from R3 and R4 are independently selected from CH3, CH2CH3, CH(CH3)2, or R3 and R4 are connected to each other and together with the oxygen atom to form a 6-membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring;

[0021] or A1 and A2 are independently selected from NH;

[0022] L is selected from -(CH2) n -, -(CH2)2O(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, n is an integer from 3 to 6;

[0023] R1 and R2 are independently selected from H;

[0024] But not including: A1=A2=O, L is selected from -(CH2)5-, R1 is selected from H, R2 is selected from R3=R4=CH3, R3=R4=CH(CH3)2, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring.

[0025] More preferably, when A1 and A2 are independently selected from O, L is selected from -(CH2) n -, n is 3, R1 is selected from H, R2 is selected from R3, R4 are independently selected from CH3; L is selected from -(CH2) n -, n is an integer of 3 to 5, R1 is selected from H, R2 is selected from R3, R4 are independently selected from CH2CH3; L is selected from -(CH2) n -, n is an integer of 3 to 5, R1 is selected from H, R2 is selected from R3 and R4 are connected to each other and together with the oxygen atom form a 6-membered heterocyclic ring containing 1 oxygen atom; L is selected from -(CH2) n -, n is an integer of 3 to 4, R1 is selected from H, R2 is selected from R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring;

[0026] When A1 and A2 are independently selected from NH, L is selected from -(CH2) n -, -(CH2)2O(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, n is an integer from 4 to 6, and R1 and R2 are independently selected from H.

[0027] Specifically, it is selected from the tripterygium wilfordii acrylamide derivatives having the following structure:

[0028]

[0029]

[0030] As a further preferred technical solution of the present invention, when A1 and A2 are independently selected from O, the structure of the tripterygium wilfordii acrylamide derivative is as shown in Formula I:

[0031]

[0032] Wherein, n, R3, and R4 are as described above.

[0033] Specifically, n is an integer of 2 to 6; R3 and R4 are independently selected from C1-C4 alkyl groups, or R3 and R4 are connected to each other and form a 5-7 membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and form a 5-7 membered heterocyclic ring with a nitrogen atom.

[0034] Preferably, n is an integer of 3 to 5; R3 and R4 are independently selected from C1-C4 alkyl, or R3 and R4 are connected to each other and together with the oxygen atom to form a 5-7 membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 5-7 membered heterocyclic ring.

[0035] More preferably, n is an integer of 3 to 5; R3 and R4 are independently selected from CH3, CH2CH3, CH(CH3)2, or R3 and R4 are connected to each other and together with the oxygen atom to form a 6-membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring;

[0036] However, it does not include: n is 5, R3=R4=CH3, R3=R4=CH(CH3)2, or R3 and R4 are connected to each other and form a 6-membered heterocyclic ring with the nitrogen atom.

[0037] More preferably, n is 3, R3 and R4 are independently selected from CH3; n is an integer from 3 to 5, R3 and R4 are independently selected from CH2CH3; n is an integer from 3 to 5, R3 and R4 are connected to each other and form a 6-membered heterocyclic ring containing 1 oxygen atom together with the oxygen atom; n is an integer from 3 to 4, R3 and R4 are connected to each other and form a 6-membered heterocyclic ring together with the nitrogen atom.

[0038] As a further preferred technical solution of the present invention, when A1 and A2 are independently selected from NH, the structure of the tripterygium wilfordii acrylamide derivative is as shown in Formula II:

[0039]

[0040] Wherein, L is as described above.

[0041] Specifically, L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, n is an integer from 2 to 6, and m is an integer from 1 to 6. n -、-((CH2)2O) m (CH2)2-) increases, and the inhibitory effect on tumor cells increases accordingly.

[0042] Preferably, L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, n is an integer of 2-6, and m is an integer of 1-3.

[0043] More preferably, L is selected from -(CH2) n -, -(CH2)2O(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, where n is an integer from 3 to 6.

[0044] More preferably, L is selected from -(CH2) n -, -(CH2)2O(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, where n is an integer from 4 to 6.

[0045] Another object of the present invention is to provide a method for preparing the tripterygium wilfordii acrylamide derivative, when A1 and A2 are independently selected from O, L is selected from -(CH2) n -, R1 is selected from H, R2 is selected from The synthetic route is as follows:

[0046]

[0047] Wherein, n, R3, and R4 are as described above;

[0048] The following steps are involved:

[0049] Step (1), using anhydrous dichloromethane as a reaction solvent and triethylamine as a base catalyst, maleic anhydride and an amine compound represented by formula IV are reacted at room temperature to obtain an intermediate V; wherein the molar ratio of maleic anhydride to the amine compound is 1:1.3 to 1:1.5; and the molar ratio of maleic anhydride to triethylamine is 1:1.3 to 1:1.5;

[0050] Step (2), using N,N-dimethylformamide (DMF) as the reaction solvent and K2CO3 as the base catalyst, the intermediate V is reacted with the formula The dibromoalkane shown is reacted at room temperature to obtain intermediate VI; wherein the molar ratio of the intermediate V to the dibromoalkane is 1:3 to 1:4; the molar ratio of the intermediate V to K2CO3 is 1:2 to 1:3;

[0051] Step (3), using N,N-dimethylformamide (DMF) as a reaction solvent and NaHCO3 as a base catalyst, the intermediate VI reacts with celastrol under heating reflux to obtain a celastrol acrylamide derivative; wherein the molar ratio of the celastrol to the intermediate VI is 1:1.5 to 1:2; and the molar ratio of the celastrol to NaHCO3 is 1:3 to 1:4.

[0052] When A1 and A2 are independently selected from NH, L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, R1, R2 are independently selected from H, the synthesis route is as follows:

[0053]

[0054] Wherein, n, m, and L are as described above;

[0055] The following steps are involved:

[0056] Step (1), using anhydrous dichloromethane as the reaction solvent and triethylamine as the base catalyst, the formula The N-(tert-butoxycarbonyl) diamine compound shown in formula VII is reacted with chloroacetamide shown in formula VII at room temperature to obtain intermediate VIII; wherein the molar ratio of the N-(tert-butoxycarbonyl) diamine compound to chloroacetamide is 1:1.5 to 1:2; the molar ratio of the N-(tert-butoxycarbonyl) diamine compound to triethylamine is 1:2 to 1:3;

[0057] Step (2), using anhydrous dichloromethane as the reaction solvent, in the presence of trifluoroacetic acid, the intermediate VIII is subjected to acid hydrolysis at room temperature to remove the Boc protecting group to obtain the intermediate IX; wherein the volume ratio of trifluoroacetic acid to anhydrous dichloromethane is 1:5 to 1:10;

[0058] Step (3), using N,N-dimethylformamide (DMF) as a reaction solvent, in the presence of HATU and DIPEA, the intermediate IX undergoes an amidation reaction with the carboxyl group of celastrol at room temperature to obtain a celastrol acrylamide derivative; wherein the molar ratio of celastrol to the intermediate IX is 1:4 to 1:5; the molar ratio of celastrol to HATU is 1:1.2 to 1:1.5; and the molar ratio of celastrol to DIPEA is 1:3 to 1:4.

[0059] Compared with tripterygium wilfordii, the tripterygium wilfordii acrylamide derivatives of the present invention have significantly enhanced inhibitory effects on tumor cells. Another object of the present invention is to provide the use of the tripterygium wilfordii acrylamide derivatives in the preparation of anti-tumor drugs.

[0060] The tumors are breast cancer, gastric cancer and colon cancer.

[0061] Another object of the present invention is to provide a pharmaceutical composition, which uses the tripterygium wilfordii acrylamide derivative as the main active ingredient and is supplemented with a pharmaceutically acceptable carrier to form any pharmaceutically acceptable dosage form.

[0062] The tripterygium wilfordii acrylamide derivative can be prepared into different dosage forms alone or with one or more pharmaceutical carriers.

[0063] The dosage form is selected from tablets, capsules, pills, granules, powders, lozenges, aqueous or oily suspensions, injections, patches, and nano preparations for clinical oral, injection, or topical administration. In these different preparations, the content of the tripterygium wilfordii acrylamide derivative of the present invention can be 0.1%-99.9%.

[0064] The tripterygium wilfordii acrylamide derivative of the present invention can be used alone or in combination with commonly used clinical anti-tumor drugs such as antimetabolites, alkylating agents, anti-tumor antibiotics, anti-tumor botanicals, hormones, and can also be used in combination with radiotherapy.

[0065] Beneficial effects of the present invention:

[0066] The anti-tumor activity of the tripterygium wilfordii derivative of the present invention is significantly improved compared with that of tripterygium wilfordii.

[0067] The preparation method of the tripterygium wilfordii derivative of the present invention has mild reaction conditions, low-toxic reagents, easy-to-obtain raw materials, convenient post-processing and high yield. Specific implementation plan

[0068] To further illustrate the technical solution of the present invention, a series of embodiments are listed below. These embodiments are illustrative and should not be construed as limiting the present invention.

[0069] Example 1

[0070] Preparation of Compound I-1

[0071]

[0072] Step (1), maleic anhydride (200mg, 2.04mmol) was dissolved in 5mL of anhydrous dichloromethane, dimethylamine (155μL, 3.06mmol) and triethylamine (425.3μL, 3.06mmol) were added in sequence, and the reaction was stirred at room temperature. The reaction progress was detected by TLC (dichloromethane: methanol = 20: 1V / V). After the reaction was completed for 1h, the raw material reaction was detected to be complete. After the reaction was completed, the organic phase was spin-dried to obtain the intermediate (E)-4-(dimethylamino)-4-oxobut-2-enoic acid, which was directly used in the next step reaction.

[0073]

[0074] Step (2), all (E)-4-(dimethylamino)-4-oxobut-2-enoic acid and K2CO3 (552.84 mg, 4 mmol) obtained in step (1) were stirred and dissolved in 4 mL DMF, and then 1,3-dibromopropane (609 μL, 6 mmol) was added, and the reaction was reacted at room temperature. TLC was used to detect the reaction progress (petroleum ether: ethyl acetate = 1: 2 V / V). After reacting for 4 hours, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent is petroleum ether: ethyl acetate = 3: 2 V / V), vacuum dried to obtain compound A1 (white solid, 356.8 mg, yield of about 66.2%).

[0075]

[0076] Step (3), celastrol (90.12 mg, 0.2 mmol), compound A1 (105.65 mg, 0.4 mmol) and NaHCO3 (67.2 mg, 0.8 mmol) were dissolved in 4 mL DMF, heated to reflux at 60°C for reaction, and the reaction progress was detected by TLC (dichloromethane: methanol = 50: 1 V / V). After 8 h of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent: dichloromethane: methanol = 150: 1 V / V), vacuum dried to obtain the target compound I-1 (orange-red powder, 83.2 mg, yield 65.7%).

[0077] mp:229.4℃.

[0078] ESI-MS: 634.3722[M+H] + .

[0079] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.76 (s, 1H), 7.09 (d, J = 6.8Hz, 1H), 6.70 (d, J = 11.9Hz, 1H), 6.39 (s, 1H), 6.36 (d, J = 6.4Hz, 1H), 5.95 (d, J = 12.1Hz,1H),4.20-4.03(m,2H),4.01-3.80(m,2H),2.82(s,6H),2.10(s ,3H),1.39(s,3H),1.22(s,3H),1.13(s,3H),1.08(s,3H),0.46(s,3H).

[0080] Example 2

[0081] Preparation of Compound I-2

[0082]

[0083] 1,4-dibromobutane (716.52 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (1) of Example 1, while other conditions remained unchanged, to obtain compound A2 (white solid, 366.1 mg, yield about 65.8%).

[0084]

[0085] Referring to the preparation method of compound I-1 in step (3) of Example 1, compound A1 was replaced with an equimolar amount of compound A2, and other conditions remained unchanged to obtain the target compound I-2 (orange-red powder, 61.8 mg, yield 47.7%).

[0086] mp:227.7℃.

[0087] ESI-MS: 648.3883[M+H] + .

[0088] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 7.08 (d, J = 6.7Hz, 1H), 6.75 (d, J = 12.0Hz, 1H), 6.40 (s, 1H), 6.36 (d, J = 7.0Hz, 1H), 6.00 (d, J = 12.0Hz, 1H),4.06(t,J=6.0Hz,2H),3.97-3.80(m,2H),2.86(s,3H),2.84(s,3H),2. 10(s,3H),1.39(s,3H),1.23(s,3H),1.13(s,3H),1.08(s,3H),0.48(s,3H).

[0089] Example 3

[0090] Preparation of Compound I-3

[0091]

[0092] 1,5-dibromopentane (817 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 1, while other conditions remained unchanged, to obtain compound A3 (white solid, 262.4 mg, yield about 44.9%).

[0093]

[0094] Referring to the preparation method of compound I-1 in step (3) of Example 1, compound A1 was replaced with an equimolar amount of compound A3, and other conditions remained unchanged to obtain the target compound I-3 (orange-red powder, 89.5 mg, yield 67.7%).

[0095] mp:230.0℃.

[0096] ESI-MS: 662.4032[M+H] + .

[0097] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.72 (s, 1H), 7.08 (d, J = 6.8Hz, 1H), 6.75 (d, J = 12.0Hz, 1H), 6.38 (s, 1H), 6.35 (d, J = 7.0Hz, 1H), 6.00 (d, J = 12.0Hz, 1H),4.04(t,J=6.4Hz,2H),3.94-3.77(m,2H),2.85(s,3H),2.83(s,3H),2. 10(s,3H),1.39(s,3H),1.23(s,3H),1.13(s,3H),1.08(s,3H),0.47(s,3H).

[0098] Example 4

[0099] Preparation of Compound I-4

[0100]

[0101] Step (1), maleic anhydride (200mg, 2.04mmol) was dissolved in 5mL of anhydrous dichloromethane, diethylamine (317μL, 3.06mmol) and triethylamine (425.3μL, 3.06mmol) were added successively, and the reaction was stirred at room temperature. The reaction progress was detected by TLC (dichloromethane: methanol = 20: 1V / V). After the reaction was completed for 1h, the reaction of the raw materials was detected to be complete. After the reaction was completed, the organic phase was spin-dried to obtain (E)-4-(diethylamino)-4-oxobut-2-enoic acid, which was directly used in the next step reaction.

[0102]

[0103] Step (2), all (E)-4-(diethylamino)-4-oxobut-2-enoic acid and K2CO3 (552.84 mg, 4 mmol) obtained in step (1) were stirred and dissolved in 4 mL DMF, and then 1,3-dibromopropane (609 μL, 6 mmol) was added, and the reaction was reacted at room temperature. TLC was used to detect the reaction progress (petroleum ether: ethyl acetate = 1: 1 V / V). After 4 hours of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent is petroleum ether: ethyl acetate = 3: 1 V / V), vacuum dried to obtain compound A4 (white solid, 338.2 mg, yield of about 58.1%).

[0104]

[0105] Step (3), celastrol (90.12 mg, 0.2 mmol), compound A4 (116.42 mg, 0.4 mmol) and NaHCO3 (67.2 mg, 0.8 mmol) were dissolved in 4 mL DMF, heated to reflux at 60 ° C, and the reaction progress was detected by TLC (dichloromethane: methanol = 50: 1 V / V). After 8 hours of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent is dichloromethane: methanol = 150: 1 V / V), vacuum dried to obtain the target compound I-4 (orange-red powder, 68.9 mg, yield 52.1%).

[0106] mp:224.2℃.

[0107] ESI-MS: 662.4034[M+H] + .

[0108] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 7.09 (d, J = 6.2Hz, 1H), 6.75 (d, J =12.0Hz,1H),6.39(s,1H),6.35(d,J=6.4Hz,1H),5.96(d,J=11.9Hz,1H),4.11(t,J =6.6Hz,2H),4.01-3.82(m,2H),3.30-3.22(m,2H),3.17(q,J=7.1Hz,2H),2.10(s, 3H),1.39(s,3H),1.23(s,3H),1.13(s,3H),1.08(s,3H),1.02(m,6H),0.46(s,3H).

[0109] Example 5

[0110] Preparation of Compound I-5

[0111]

[0112] 1,4-dibromobutane (716.52 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 4, and other conditions remained unchanged to obtain compound A5 (white solid, 435.6 mg, yield about 71.4%).

[0113]

[0114] Referring to the preparation method of compound I-4 in step (3) of Example 4, compound A4 was replaced with an equimolar amount of compound A5, and other conditions remained unchanged to obtain the target compound I-5 (orange-red powder, 70 mg, yield 51.8%).

[0115] mp:229.4℃.

[0116] ESI-MS: 698.3995[M+Na] + .

[0117] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 7.08 (d, J = 6.1Hz, 1H), 6.81 (d, J = 12 .0Hz,1H),6.39(s,1H),6.36(d,J=6.7Hz,1H),5.98(d,J=11.9Hz,1H),4.05(t,J=5.9H z,2H),3.96-3.79(m,2H),3.29(q,J=7.1Hz,2H),3.21(q,J=7.1Hz,2H),2.10(s,3H),1 .39(s,3H),1.22(s,3H),1.13(s,3H),1.08(s,3H),1.04(t,J=6.8Hz,6H),0.47(s,3H).

[0118] Example 6

[0119] Preparation of Compound I-6

[0120]

[0121] 1,5-dibromopentane (817 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 4, and other conditions remained unchanged to obtain compound A6 (white solid, 499.5 mg, yield about 78.3%).

[0122]

[0123] Referring to the preparation method of compound I-4 in step (3) of Example 4, compound A4 was replaced with an equimolar amount of compound A6, and other conditions remained unchanged to obtain the target compound I-6 (orange-red powder, 77 mg, yield 55.8%).

[0124] mp:227.8℃.

[0125] ESI-MS: 690.4352[M+H] + .

[0126] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.72 (s, 1H), 7.08 (d, J = 6.7Hz, 1H), 6.81 (d, J = 12.0H z,1H),6.38(s,1H),6.34(d,J=6.4Hz,1H),5.99(d,J=12.0Hz,1H),4.03(t,J=6.4Hz,2H),3 .86 (dp, J = 22.3, 5.6 Hz, 2H), 3.28 (q, J = 7.1 Hz, 2H), 3.20 (q, J = 7.0 Hz, 2H), 2.10 (s, 3H), 1.39 (s, 3H), 1.22 (s, 3H), 1.13 (s, 3H), 1.08 (s, 3H), 1.04 (t, J = 7.2 Hz, 6H), 0.47 (s, 3H). Example 7

[0127] Preparation of Compound I-7

[0128]

[0129] Step (1), maleic anhydride (200mg, 2.04mmol) was dissolved in 5mL of anhydrous dichloromethane, diisopropylamine (429μL, 3.06mmol) and triethylamine (425.3μL, 3.06mmol) were added successively, and the reaction was stirred at room temperature. The reaction progress was detected by TLC (dichloromethane: methanol = 20: 1V / V). After the reaction was completed for 1h, the reaction of the raw materials was detected to be complete. After the reaction was completed, the organic phase was spin-dried to obtain (E)-4-(diisopropylamino)-4-oxobut-2-enoic acid, which was directly used in the next step reaction.

[0130]

[0131] Step (2), all the (E)-4-(diisopropylamino)-4-oxobut-2-enoic acid and K2CO3 (552.84 mg, 4 mmol) obtained in step (1) were stirred and dissolved in 4 mL of DMF, and then 1,3-dibromopropane (609 μL, 6 mmol) was added, and the reaction was reacted at room temperature. TLC was used to detect the reaction progress (petroleum ether: ethyl acetate = 2: 1 V / V). After reacting for 4 hours, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent is petroleum ether: ethyl acetate = 6: 1 V / V), vacuum dried to obtain compound A7 (white solid, 383.7 mg, yield of about 59.1%).

[0132]

[0133] Step (3), celastrol (90.12 mg, 0.2 mmol), compound A7 (127.6 mg, 0.4 mmol) and NaHCO3 (67.2 mg, 0.8 mmol) were dissolved in 4 mL DMF, heated to reflux at 60 ° C, and the reaction progress was detected by TLC (dichloromethane: methanol = 50: 1 V / V). After 8 hours of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent is dichloromethane: methanol = 150: 1 V / V), vacuum dried to obtain the target compound I-7 (orange-red powder, 92.5 mg, yield 67.1%).

[0134] mp:231.8℃.

[0135] ESI-MS: 690.4343[M+H] + .

[0136] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: δ8.73(s,1H),7.09(d,J=6.7Hz,1H),6.72(d,J=12.0Hz,1H) ,6.39(s,1H),6.36(d,J=7.0Hz,1H),5.85(d,J=12.0Hz,1H),4.17-4.07(m,2H),4.02-3.82(m,2H ),3.75(p,J=6.5Hz,1H),3.44(p,J=6.8Hz,1H),2.10(s,3H),1.39(s,3H),1.32(d,J=6.7Hz,6H), 1.22(s,3H),1.13(s,3H),1.08(s,3H),1.05(d,J=2.3Hz,3H),1.03(d,J=2.3Hz,3H),0.46(s,3H).

[0137] Example 8

[0138] Preparation of Compound I-8

[0139]

[0140] 1,4-dibromobutane (716.52 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 7, while other conditions remained unchanged, to obtain compound A8 (white solid, 434 mg, yield about 63.9%).

[0141]

[0142] Referring to the preparation method of compound I-7 in step (3) of Example 7, compound A7 was replaced by an equimolar amount of compound A8, and other conditions remained unchanged to obtain the target compound I-8 (orange-red powder, 100.3 mg, yield 71.3%).

[0143] mp:230.2℃.

[0144] ESI-MS: 726.4326[M+Na] + .

[0145] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 7.08 (d, J = 6.8Hz, 1H), 6.76 (d, J = 1 1.9Hz,1H),6.39(s,1H),6.35(d,J=6.4Hz,1H),5.87(d,J=11.9Hz,1H),4.10-4.04(m, 2H),3.93-3.78(m,3H),3.46(p,J=6.8Hz,1H),2.10(s,3H),1.39(s,3H),1.33(d,J=6 .7Hz,6H),1.22(s,3H),1.12(s,3H),1.09(s,3H),1.07(d,J=2.7Hz,6H),0.47(s,3H).

[0146] Example 9

[0147] Preparation of Compound I-9

[0148]

[0149] 1,5-dibromopentane (817 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 7, while other conditions remained unchanged, to obtain compound A9 (white solid, 401.6 mg, yield about 56.7%).

[0150]

[0151] Referring to the preparation method of compound I-7 in step (3) of Example 7, compound A7 was replaced with an equimolar amount of compound A9, and other conditions remained unchanged to obtain the target compound I-9 (orange-red powder, 82.1 mg, yield 57.2%).

[0152] mp:227.3℃.

[0153] ESI-MS: 718.4660 [M+H] + .

[0154] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 7.08 (d, J = 6.4Hz, 1H), 6.77 (d, J =11.9Hz,1H),6.38(s,1H),6.35(d,J=6.7Hz,1H),5.88(d,J=12.0Hz,1H),4.05(t,J =6.4Hz,2H),3.92-3.75(m,3H),3.45(p,J=6.7Hz,1H),2.10(s,3H),1.39(s,3H),1 .33(d,J=6.8Hz,6H),1.22(s,3H),1.12(s,3H),1.07(d,J=6.2Hz,9H),0.47(s,3H).

[0155] Example 10

[0156] Preparation of Compound I-10

[0157]

[0158] Step (1), maleic anhydride (200mg, 2.04mmol) was dissolved in 5mL of anhydrous dichloromethane, morpholine (267μL, 3.06mmol), triethylamine (425.3μL, 3.06mmol) were added in sequence, the reaction was stirred at room temperature, and the reaction progress was detected by TLC (dichloromethane: methanol = 20: 1V / V). After the reaction was completed for 1h, the raw material reaction was detected to be complete. After the reaction was completed, the organic phase was spin-dried to obtain the intermediate (E)-4-(morpholino)-4-oxobut-2-enoic acid, which was directly used in the next step reaction.

[0159]

[0160] Step (2), all (E)-4-(morpholino)-4-oxobut-2-enoic acid and K2CO3 (552.84 mg, 4 mmol) obtained in step (1) were stirred and dissolved in 4 mL DMF, and 1,3-dibromopropane (609 μL, 6 mmol) was added, and the reaction was reacted at room temperature. TLC was used to detect the reaction progress (petroleum ether: ethyl acetate = 1: 2 V / V). After 4 hours of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent is petroleum ether: ethyl acetate = 3: 2 V / V), vacuum dried to obtain compound A10 (white solid, 313.8 mg, yield of about 50.4%).

[0161]

[0162] Step (3), celastrol (90.12 mg, 0.2 mmol), compound A10 (122 mg, 0.4 mmol) and NaHCO3 (67.2 mg, 0.8 mmol) were dissolved in 4 mL DMF, heated under reflux at 60°C for reaction, and the reaction progress was detected by TLC (dichloromethane: methanol = 42: 1 V / V). After 8 h of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent: dichloromethane: methanol = 126: 1 V / V), vacuum dried to obtain the target compound I-10 (orange-red powder, 103.2 mg, yield 76.4%).

[0163] mp:227.9℃.

[0164] ESI-MS: 676.3831[M+H] + .

[0165] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.72 (s, 1H), 7.09 (d, J = 6.7Hz, 1H), 6.70 (d, J = 12.0Hz, 1H), 6 .39(s,1H),6.36(d,J=7.0Hz,1H),6.00(d,J=12.0Hz,1H),4.13(dq,J=15.0,5.6Hz,2H),3.92(ddt ,J=32.3,11.4,6.0Hz,2H),3.57(t,J=4.7Hz,2H),3.51(t,J=4.7Hz,2H),3.45(t,J=4.8Hz,2H),3. 28(t,J=4.9Hz,2H),2.10(s,3H),1.39(s,3H),1.23(s,3H),1.14(s,3H),1.08(s,3H),0.47(s,3H).

[0166] Embodiment 11

[0167] Preparation of Compound I-11

[0168]

[0169] Substituting 1,4-dibromobutane (716.52 μL, 6 mmol) for 1,3-dibromopropane in step (2) of Example 10, with other conditions unchanged, gave Compound A11 (white solid, 367.8 mg, yield about 56.5%).

[0170]

[0171] Referring to the preparation method of compound I-10 in step (3) of Example 10, compound A10 was replaced with an equimolar amount of compound A11, and other conditions remained unchanged to obtain the target compound I-11 (orange-red powder, 74.4 mg, yield 54%).

[0172] mp:228.0℃.

[0173] ESI-MS: 690.3998[M+H] + .

[0174] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.72 (s, 1H), 7.08 (d, J = 6.8Hz, 1H), 6.76 (d, J = 11.8Hz, 1H),6.40(s,1H),6.35(d,J=7.0Hz,1H),6.04(d,J=11.7Hz,1H),4.10(t,J=5.4Hz,2H),3.89 (dp, J = 17.5, 5.7 Hz, 2H), 3.56 (t, J = 11.0, 4.6 Hz, 4H), 3.46 (t, J = 4.6 Hz, 2H), 3.29 (t, J = 5.1 Hz, 2H), 2.10 (s, 3H), 1.39 (s, 3H), 1.24 (s, 3H), 1.13 (s, 3H), 1.08 (s, 3H), 0.48 (s, 3H). Example 12

[0175] Preparation of Compound I-12

[0176]

[0177] 1,5-dibromopentane (817 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 10, while other conditions remained unchanged, to obtain compound A12 (white solid, 384.5 mg, yield about 56.6%).

[0178]

[0179] Referring to the preparation method of compound I-10 in step (3) of Example 10, compound A10 was replaced with an equimolar amount of compound A12, and other conditions remained unchanged to obtain the target compound I-12 (orange-red powder, 100.5 mg, yield 71.5%).

[0180] mp:227.2℃.

[0181] ESI-MS: 726.3956[M+Na] + .

[0182] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.72 (s, 1H), 7.08 (d, J = 7.0Hz, 1H), 6.76 (d, J = 11.8Hz, 1H),6.38(s,1H),6.35(d,J=6.7Hz,1H),6.05(d,J=11.6Hz,1H),4.06(t,J=6.3Hz,2H),3.95 -3.77(m,2H),3.57(t,J=4.7Hz,2H),3.53(t,J=4.2Hz,2H),3.47(t,J=4.6Hz,2H),3.29(t,J =5.5Hz,2H),2.10(s,3H),1.39(s,3H),1.22(s,3H),1.13(s,3H),1.08(s,3H),0.47(s,3H).

[0183] Embodiment 13

[0184] Preparation of Compound I-13

[0185]

[0186] Step (1), maleic anhydride (200mg, 2.04mmol) was dissolved in 5mL of anhydrous dichloromethane, piperidine (280.2μL, 3.06mmol), triethylamine (425.3μL, 3.06mmol) were added successively, the reaction was stirred at room temperature, TLC detected the reaction progress (dichloromethane: methanol = 20: 1V / V), after reacting for 1h, it was detected that the raw material reaction was complete. After the reaction was completed, the organic phase was spin-dried to obtain intermediate (E)-4-oxo-4-(piperidin-1-yl) but-2-enoic acid, and the next step of reaction was continued.

[0187]

[0188] Step (2), all (E)-4-oxo-4-(piperidin-1-yl)but-2-enoic acid and K2CO3 (552.84 mg, 4 mmol) obtained in step (1) were stirred and dissolved in 4 mL DMF, and 1,3-dibromopropane (609 μL, 6 mmol) was added, and the reaction was reacted at room temperature. TLC was used to detect the reaction progress (petroleum ether: ethyl acetate = 1: 1 V / V). After 4 hours of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 mL × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (petroleum ether: ethyl acetate = 3: 1 V / V), vacuum dried to obtain compound A13 (white solid, 405.7 mg, yield of about 65.6%).

[0189]

[0190] Step (3), celastrol (90.12 mg, 0.2 mmol), compound A13 (121.2 mg, 0.4 mmol) and NaHCO3 (67.2 mg, 0.8 mmol) were dissolved in 4 mL DMF, heated to reflux at 60°C for reaction, and the reaction progress was detected by TLC (dichloromethane: methanol = 44: 1 V / V). After 8 h of reaction, the reaction was detected to be complete. After the reaction was completed, 90 mL of ethyl acetate was added to the reaction solution for dilution, washed with water (30 mL × 3), and the organic phase was taken. The organic phase was washed with saturated NaCl solution (100 mL × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated. After normal phase silica gel column chromatography (eluent was dichloromethane: methanol = 132: 1 V / V), vacuum dried to obtain the target compound I-13 (orange-red powder, 86.6 mg, yield 64.3%).

[0191] mp:226.1℃.

[0192] ESI-MS: 674.4038[M+H] + .

[0193] 1 H-NMR (400MHz, DMSO-d6, TMS), δppm: 8.75 (s, 1H), 7.09 (d, J = 6.8Hz, 1H), 6.70 ( d,J=12.0Hz,1H),6.38(s,1H),6.35(d,J=6.4Hz,1H),5.94(d,J=11.9Hz,1H),4. 17-4.06(m,2H),4.01-3.81(m,2H),3.41(t,J=5.7Hz,2H),3.21(t,J=6.4Hz,2H) ,2.10(s,3H),1.39(s,3H),1.22(s,3H),1.13(s,3H),1.07(s,3H),0.46(s,3H).

[0194] Embodiment 14

[0195] Preparation of Compound I-14

[0196]

[0197] 1,4-dibromobutane (716.52 μL, 6 mmol) was used to replace 1,3-dibromopropane in step (2) of Example 13, while other conditions remained unchanged, to obtain compound A14 (white solid, 453.1 mg, yield about 70.1%).

[0198]

[0199] Referring to the preparation method of compound I-13 in step (3) of Example 13, compound A13 was replaced with an equimolar amount of compound A14, and other conditions remained unchanged to obtain the target compound I-14 (orange-red powder, 80.8 mg, yield 58.8%).

[0200] mp:222.6℃.

[0201] ESI-MS: 688.4196[M+H] + .

[0202] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.74 (s, 1H), 7.08 (d, J = 6.9 Hz, 1H), 6.76 (d, J =11.8Hz,1H),6.40(s,1H),6.36(d,J=6.5Hz,1H),5.97(d,J=11.8Hz,1H),4.06(t,J =5.9Hz,2H),3.88(dt,J=23.8,6.6Hz,2H),3.42(t,J=5.4Hz,2H),3.26(t,J=5.5Hz, 2H),2.10(s,3H),1.39(s,3H),1.22(s,3H),1.13(s,3H),1.08(s,3H),0.48(s,3H).

[0203] Embodiment 15

[0204] Preparation of Compound I-15

[0205]

[0206] Substituting 1,5-dibromopentane (817 μL, 6 mmol) for 1,3-dibromopropane in step (2) of Example 13, with other conditions remaining unchanged, yielded compound A15 (white solid, 330.8 mg, yield about 49%).

[0207]

[0208] Referring to the preparation method of compound I-13 in step (3) of Example 13, compound A13 was replaced with an equimolar amount of compound A15, and other conditions remained unchanged to obtain the target compound I-15 (orange-red powder, 69.3 mg, yield 49.4%).

[0209] mp:224.9℃.

[0210] ESI-MS: 724.4163[M+Na]+ .

[0211] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.74 (s, 1H), 7.08 (d, J = 6.7Hz, 1H), 6.76 (d ,J=11.8Hz,1H),6.37(s,1H),6.34(d,J=7.0Hz,1H),5.98(d,J=11.8Hz,1H),4.04 (t,J=6.2Hz,2H),3.95-3.76(m,2H),3.42(t,J=5.4Hz,2H),3.25(t,J=6.5Hz,2H ),2.09(s,3H),1.39(s,3H),1.23(s,3H),1.13(s,3H),1.07(s,3H),0.47(s,3H).

[0212] Example 16

[0213] Preparation of compound II-1

[0214]

[0215] Step (1), dissolve N-(tert-butyloxycarbonyl)-1,2-ethylenediamine (296.4 μL, 1.87 mmol) in 5 mL of anhydrous dichloromethane, add triethylamine (780.8 μL, 5.62 mmol), stir at 0 ° C for 5 min; then add acryloyl chloride (304.3 μL, 3.75 mmol), stir at room temperature, and detect the reaction progress by TLC (petroleum ether: ethyl acetate = 1: 2 V / V). After reacting for 1 hour, the reaction is detected to be complete. After the reaction is completed, spin dry the organic phase, add 90 mL of ethyl acetate, wash with water (30 mL × 3), take the organic phase, wash the organic phase with saturated NaCl solution (100 ml × 3), dry it with anhydrous Na2SO4 overnight, filter, concentrate the filtrate, and chromatograph on a normal phase silica gel column (eluent is petroleum ether: ethyl acetate = 3: 2 V / V), and vacuum dry to obtain A16-1 (white solid, 227.1 mg, yield 56.7%);

[0216] Step (2), 179.7 mg of compound A16-1 was dissolved in 4 mL of anhydrous CH2Cl2, 0.4 mL of trifluoroacetic acid was added dropwise, and the mixture was reacted at room temperature for 1.5 h. TLC monitoring indicated that the reaction was complete. After the reaction was complete, the organic phase was dried to obtain compound A16 (white viscous substance, 95.8 mg, yield about 79.2%).

[0217]

[0218] Step (3), dissolve celastrol (90.12 mg, 0.2 mmol) and HATU (114.1 mg, 0.3 mmol) in 4 mL DMF, add DIPEA (139.3 μL, 0.8 mmol) dropwise, stir at room temperature until TLC detection shows that celastrol is activated, add compound A16 (91.2 mg, 0.8 mmol), continue to react at room temperature, and detect the reaction progress by TLC (dichloromethane: methanol = 25:1 V / V). After 4 h of reaction, the reaction is detected to be complete. After the reaction, 90 mL of ethyl acetate was added to the reaction solution to dilute it, and the mixture was washed with water (30 mL × 3). The organic phase was taken and washed with saturated NaCl solution (100 ml × 3), dried over anhydrous Na2SO4 overnight, filtered, and the filtrate was concentrated and chromatographed on a normal phase silica gel column (eluent: dichloromethane: methanol = 75:1 V / V), and dried in vacuo to obtain the target compound II-1 (orange-red powder, 32.2 mg, yield 29.5%).

[0219] mp:200.6℃.

[0220] ESI-MS: 547.3523[M+H] + .

[0221] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 8.11 (t, J = 5.3Hz, 1H), 7.65 (t, J = 4 .9Hz,1H),7.07(d,J=6.7Hz,1H),6.39(s,1H),6.34(d,J=7.0Hz,1H),6.14(dd,J=17. 1,9.4Hz,1H),6.04(dd,J=17.0,2.9Hz,1H),5.56(dd,J=9.4,2.9Hz,1H),3.15-2.92( m,4H),2.09(s,3H),1.37(s,3H),1.20(s,3H),1.07(s,3H),1.04(s,3H),0.51(s,3H).

[0222] Embodiment 17

[0223] Preparation of compound II-2

[0224]

[0225] N-(tert-butoxycarbonyl)-1,3-propylenediamine (300.6 μL, 1.72 mmol) was used to replace N-(tert-butoxycarbonyl)-1,2-ethylenediamine in step (1) of Example 16, while other conditions remained unchanged, to obtain compound A17 (white viscous substance, 108.8 mg, yield about 49.4%).

[0226]

[0227] Referring to the preparation method of compound II-1 in step (3) of Example 16, compound A16 was replaced with an equimolar amount of compound A17, and other conditions remained unchanged to obtain the target compound II-2 (orange-red powder, 33.3 mg, yield 29.7%).

[0228] mp:212.5℃.

[0229] ESI-MS: 561.3672[M+H] + .

[0230] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.74 (s, 1H), 8.04 (t, J = 5.5Hz, 1H), 7.62 (t, J = 5 .6Hz,1H),7.08(d,J=6.8Hz,1H),6.39(s,1H),6.35(d,J=7.0Hz,1H),6.16(dd,J=17. 1,9.8Hz,1H),6.03(dd,J=17.1,2.6Hz,1H),5.53(dd,J=9.8,2.6Hz,1H),3.13-2.79( m,4H),2.09(s,3H),1.38(s,3H),1.21(s,3H),1.08(s,3H),1.04(s,3H),0.53(s,3H).

[0231] Embodiment 18

[0232] Preparation of compound II-3

[0233]

[0234] Substituting N-(tert-butoxycarbonyl)-1,4-butanediamine (304.8 μL, 1.59 mmol) for N-(tert-butoxycarbonyl)-1,2-ethylenediamine in step (1) of Example 16, with other conditions remaining unchanged, yielded compound A18 (white viscous substance, 127.8 mg, yield about 56.6%).

[0235]

[0236] Referring to the preparation method of compound II-1 in step (3) of Example 16, compound A16 was replaced with an equimolar amount of compound A18, and other conditions remained unchanged to obtain the target compound II-3 (orange-red powder, 36.3 mg, yield 31.6%).

[0237] mp:217.0℃.

[0238] ESI-MS: 575.3828[M+H] + .

[0239] 1 H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.73 (s, 1H), 8.02 (t, J = 5.6Hz, 1H), 7.58 (t, J = 5 .5Hz,1H),7.08(d,J=6.7Hz,1H),6.40(s,1H),6.34(d,J=7.0Hz,1H),6.16(dd,J=17. 1,9.9Hz,1H),6.02(dd,J=17.1,2.6Hz,1H),5.52(dd,J=9.9,2.6Hz,1H),3.08-2.82( m,4H),2.09(s,3H),1.38(s,3H),1.21(s,3H),1.07(s,3H),1.04(s,3H),0.53(s,3H).

[0240] Embodiment 19

[0241] Preparation of compound II-4

[0242]

[0243] Substituting N-(tert-butoxycarbonyl)-1,6-hexanediamine (310.9 μL, 1.39 mmol) for N-(tert-butoxycarbonyl)-1,2-ethylenediamine in step (1) of Example 16, with other conditions remaining unchanged, compound A19 (white viscous substance, 178.3 mg, yield about 75.5%) was obtained.

[0244]

[0245] Referring to the preparation method of compound II-1 in step (3) of Example 16, compound A16 was replaced with an equimolar amount of compound A19, and other conditions remained unchanged to obtain the target compound II-4 (orange-red powder, 48.8 mg, yield 40.5%).

[0246] mp:212.1℃.

[0247] ESI-MS: 603.4142 [M+H] + .

[0248] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.74 (s, 1H), 8.03 (t, J = 5.6Hz, 1H), 7.55 (t, J = 6.0Hz, 1H ),7.08(d,J=6.4Hz,1H),6.40(s,1H),6.35(d,J=6.6Hz,1H),6.20(dd,J=17.1,9.9Hz,1H),6. 05(dd,J=17.1,2.5Hz,1H),5.55(dd,J=9.9,2.5Hz,1H),3.07(q,J=6.6Hz,2H),2.90(dq,J=13 .8,6.9Hz,2H),2.09(s,3H),1.38(s,3H),1.21(s,3H),1.07(s,3H),1.04(s,3H),0.53(s,3H).

[0249] Embodiment 20

[0250] Preparation of compound II-5

[0251]

[0252] Substituting tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (293.3 μL, 1.47 mmol) for N-(tert-butyloxycarbonyl)-1,2-ethylenediamine in step (1) of Example 16, while keeping other conditions unchanged, compound A20 (white viscous substance, 156.2 mg, yield about 67.3%) was obtained.

[0253]

[0254] Referring to the preparation method of compound II-1 in step (3) of Example 16, compound A16 was replaced with an equimolar amount of compound A20, and other conditions remained unchanged to obtain the target compound II-5 (orange-red powder, 53 mg, yield 44.9%).

[0255] mp:204.9℃.

[0256] ESI-MS: 591.3771[M+H] + .

[0257] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.70 (s, 1H), 8.09 (t, J = 5.5Hz, 1H), 7.55 (t, J = 5.4Hz, 1H), 7.07(d,J=6.7Hz,1H),6.39(s,1H),6.34(d,J=6.9Hz,1H),6.24(dd,J=17.1,10.0Hz,1H),6.07(d d,J=17.1,2.4Hz,1H),5.57(dd,J=10.0,2.4Hz,1H),3.38(t,J=5.3Hz,4H),3.28-3.22(m,2H),3 .15-3.04(m,2H),2.09(s,3H),1.38(s,3H),1.21(s,3H),1.07(s,3H),1.05(s,3H),0.53(s,3H).

[0258] Embodiment 21

[0259] Preparation of Compound II-6

[0260]

[0261] Replace N-(tert-butyloxycarbonyl)-1,2-ethylenediamine in step (1) of Example 16 with tert-butyl N-[2-[2-(2-aminoethoxy)ethoxy]ethyl]carbamate (293.3 μL, 1.47 mmol) and keep other conditions unchanged to obtain compound A20 (white viscous substance, 186.6 mg, yield about 76.5%).

[0262]

[0263] Referring to the preparation method of compound II-1 in step (3) of Example 16, compound A16 was replaced with an equimolar amount of compound A21, and other conditions remained unchanged to obtain the target compound II-6 (orange-red powder, 71.2 mg, yield 56.2%).

[0264] mp:208.2℃.

[0265] ESI-MS: 635.4045[M+H] + .

[0266] 1H-NMR (300MHz, DMSO-d6, TMS), δppm: 8.71 (s, 1H), 8.14 (t, 1H), 7.59 (t, J = 5.7Hz, 1H), 7.08 (d, J =6.6Hz,1H),6.40(s,1H),6.36(d,J=6.9Hz,1H),6.24(dd,J=17.1,10.0Hz,1H),6.07(dd,J=17. 0,2.3Hz,1H),5.56(dd,J=10.0,2.3Hz,1H),3.42(t,J=7.2Hz,8H),3.26(d,J=5.5Hz,2H),3.02( d,J=3.8Hz,2H),2.09(s,3H),1.38(s,3H),1.21(s,3H),1.07(s,3H),1.04(s,3H),0.54(s,3H).

[0267] Embodiment 22

[0268] The anti-tumor activity of the tripterygium wilfordii acrylamide derivative of the present invention was tested by MTT colorimetry, and tripterygium wilfordii (CEL) and CDDO-Me (Bardoxolone methyl) were selected as positive control drugs. Appropriate amounts of the test compound, CEL and CDDO-Me were weighed, dissolved in DMSO to prepare a mother solution with a concentration of 10 mmol / L, and then diluted with culture medium to a series of solutions of 0.25, 0.5, 1, 2, and 4 μmol / L.

[0269] Instruments: clean bench (SW-CJ-1FD, AIRTECH, Sujing Antai), constant temperature CO2 incubator (3111, Thermo, USA), high pressure sterilizer (YXO.SG41.280, Shanghai Huaxian), centrifuge (SIGMA), multi-biological microscope (IX71, Olympus, Japan), multi-function microplate reader (POLARstar, Omega, USA).

[0270] Reagents: DMEM, 1640 medium (KeyGEN), fetal bovine serum (GIBCO), trypsin (SIGMA), DMSO (SIGMA).

[0271] Cell lines: human breast cancer cell lines MDA-MB-231 and MCF-7, human gastric cancer cell line BGC-823, and human colon cancer cell line HT-29, wherein BGC-823 cells were cultured with RPMI-1640 medium, and MDA-MB-231, MCF-7, and HT-29 cells were cultured with DMEM (high glucose) medium (all purchased from Jiangsu KeyGen Biotech Co., Ltd.).

[0272] Methods: The frozen cell lines were revived and cultured in a constant temperature 37°C, 5% CO2 incubator. The medium was changed once a day. When the cells were in the exponential growth phase and in good condition, they could be plated. 1 mL of 0.25% trypsin digestion solution was added and digested for 1-2 min. The cell state was observed under a microscope. When the adherent cells became round and shrunk, the digestion solution was removed and 1-2 mL of culture medium containing 10% fetal bovine serum was added to make a cell suspension. The cells were counted and 5×10 cells were added to each well. 3 The amount of cell suspension required was calculated based on the number of cells and the total number of wells. The cell suspension was inoculated on a 96-well plate at 100 μL / well, sealed with PBS, and placed in a constant temperature 37°C, 5% CO2 incubator for 24 hours. The old culture medium was discarded and replaced with a series of solutions of the test compound, CEL or CDDO-Me (0.25, 0.5, 1, 2, 4 μmol / L), and a blank control (DMSO was diluted with culture medium to a concentration of 4 μmol / L) was set up. Three replicate wells were set up for each drug concentration and cultured for 48 hours. MTT reagent (5 mg / mL) was added to the 96-well plate at 20 μL / well and continued to incubate for 4 hours. The culture medium in the plate was removed, 100 μL DMSO was added to each well, and the absorbance of each well was detected at a wavelength of 570 nm using a multifunctional microplate reader, and the cell inhibition rate (Inhibition rate) was calculated according to the following formula.

[0273] Cell inhibition rate % = [(blank control OD value - drug administration group OD value) / blank control group OD value] × 100%.

[0274] The average of the three initial screening results is the final inhibition rate, and the IC of the tested drug is calculated 50 The final IC value of the compound was obtained by 3 repeated experiments (graphpad software). 50 value.

[0275] Table 1. Inhibition rate of the test compounds (concentration 0.5 μmol / L) on human breast cancer cell line MDA-MB-231

[0276]

[0277] Table 2. Inhibition of the test compounds on MDA-MB-231, MCF-7, BGC-823 and HT-29 cell lines

[0278]

[0279] Based on Table 1 and Table 2, it can be seen that compared with tripterygium wilfordii, the tripterygium wilfordii acrylamide derivatives of the present invention have significantly enhanced inhibitory effects on MDA-MB-231, MCF-7, BGC-823 and HT-29 cell lines. Among them, compound I-4 has the best activity, which is about 5.3 times higher than that of tripterygium wilfordii, and is expected to become a new anti-tumor candidate, which is worthy of further study.

Claims

1. The structure of the tripterygium wilfordii acrylamide derivative is shown in Formula III: in, A1 and A2 are independently selected from O and NH; L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, n is an integer from 2 to 6, and m is an integer from 1 to 6; R1 and R2 are independently selected from H, R3 and R4 are independently selected from C1-C4 alkyl, or R3 and R4 are connected to each other and together with the oxygen atom to form a 5-7 membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 5-7 membered heterocyclic ring.

2. The tripterygium wilfordii acrylamide derivative according to claim 1, characterized in that: A1 and A2 are independently selected from O; L is selected from -(CH2) n -, n is an integer from 3 to 5; R1 is selected from H, R2 is selected from R3 and R4 are independently selected from C1-C4 alkyl, or R3 and R4 are connected to each other and together with the oxygen atom to form a 5-7 membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 5-7 membered heterocyclic ring; or A1 and A2 are independently selected from NH; L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, n is an integer from 2 to 6, and m is an integer from 1 to 3; R1 and R2 are independently selected from H.

3. The tripterygium wilfordii acrylamide derivative according to claim 2, characterized in that: A1 and A2 are independently selected from O; L is selected from -(CH2) n -, n is an integer from 3 to 5; R1 is selected from H, R2 is selected from R3 and R4 are independently selected from CH3, CH2CH3, CH(CH3)2, or R3 and R4 are connected to each other and together with the oxygen atom to form a 6-membered heterocyclic ring containing 1 oxygen atom, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring; or A1 and A2 are independently selected from NH; L is selected from -(CH2) n -, -(CH2)2O(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, n is an integer from 3 to 6; R1 and R2 are independently selected from H; But not including: A1=A2=O, L is selected from -(CH2)5-, R1 is selected from H, R2 is selected from R3=R4=CH3, R3=R4=CH(CH3)2, or R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring.

4. The tripterygium wilfordii acrylamide derivative according to claim 3, characterized in that: When A1 and A2 are independently selected from O, L is selected from -(CH2) n -, n is 3, R1 is selected from H, R2 is selected from R3, R4 are independently selected from CH3; L is selected from -(CH2) n -, n is an integer of 3 to 5, R1 is selected from H, R2 is selected from R3, R4 are independently selected from CH2CH3; L is selected from -(CH2) n -, n is an integer of 3 to 5, R1 is selected from H, R2 is selected from R3 and R4 are connected to each other and together with the oxygen atom form a 6-membered heterocyclic ring containing 1 oxygen atom; L is selected from -(CH2) n -, n is an integer of 3 to 4, R1 is selected from H, R2 is selected from R3 and R4 are connected to each other and together with the nitrogen atom to form a 6-membered heterocyclic ring; When A1 and A2 are independently selected from NH, L is selected from -(CH2) n -, -(CH2)2O(CH2)2-, -(CH2)2O(CH2)2O(CH2)2-, n is an integer from 4 to 6, and R1 and R2 are independently selected from H.

5. A tripterygium wilfordii acrylamide derivative selected from the group consisting of:

6. A method for preparing the tripterygium wilfordii acrylamide derivative, characterized in that: When A1 and A2 are independently selected from O, L is selected from -(CH2) n -, R1 is selected from H, R2 is selected from The synthetic route is as follows: Wherein, n, R3, and R4 are as described in claim 1; The following steps are involved: Step (1), using anhydrous dichloromethane as a reaction solvent and triethylamine as a base catalyst, maleic anhydride and an amine compound represented by formula IV are reacted at room temperature to obtain intermediate V; Step (2), using N,N-dimethylformamide as the reaction solvent and K2CO3 as the base catalyst, the intermediate V is reacted with the formula The dibromoalkane shown reacts at room temperature to give intermediate VI; Step (3), using N,N-dimethylformamide as a reaction solvent and NaHCO3 as a base catalyst, the intermediate VI reacts with celastrol under heating reflux to obtain a celastrol acrylamide derivative; When A1 and A2 are independently selected from NH, L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, R1, R2 are independently selected from H, the synthesis route is as follows: Wherein, n, m, and L are as described in claim 1; The following steps are involved: Step (1), using anhydrous dichloromethane as the reaction solvent and triethylamine as the base catalyst, the formula The N-(tert-butyloxycarbonyl)diamine compound shown in formula VII reacts with chloroacetamide shown in formula VII at room temperature to obtain intermediate VIII; Step (2), using anhydrous dichloromethane as the reaction solvent, in the presence of trifluoroacetic acid, the intermediate VIII is subjected to acid hydrolysis at room temperature to remove the Boc protecting group to obtain the intermediate IX; Step (3), using N,N-dimethylformamide as the reaction solvent, in the presence of HATU and DIPEA, the intermediate IX undergoes an amidation reaction with the carboxyl group of celastrol at room temperature to obtain a celastrol acrylamide derivative.

7. The method for preparing the tripterygium wilfordii acrylamide derivative according to claim 6, characterized in that: When A1 and A2 are independently selected from O, L is selected from -(CH2) n -, R1 is selected from H, R2 is selected from When, in step (1), the molar ratio of maleic anhydride to amine compound is 1:1.3 to 1:1.5; the molar ratio of maleic anhydride to triethylamine is 1:1.3 to 1:1.5; In step (2), the molar ratio of the intermediate V to the dibromoalkane is 1:3 to 1:4; the molar ratio of the intermediate V to K2CO3 is 1:2 to 1:3; In step (3), the molar ratio of tripterygium wilfordii to intermediate VI is 1:1.5 to 1:2; the molar ratio of tripterygium wilfordii to NaHCO3 is 1:3 to 1:4; When A1 and A2 are independently selected from NH, L is selected from -(CH2) n -、-((CH2)2O) m (CH2)2-, R1 and R2 are independently selected from H, in step (1), the molar ratio of the N-(tert-butoxycarbonyl)diamine compound to chloroacetamide is 1:1.5 to 1:2; the molar ratio of the N-(tert-butoxycarbonyl)diamine compound to triethylamine is 1:2 to 1:3; In step (2), the volume ratio of trifluoroacetic acid to anhydrous dichloromethane is 1:5 to 1:10; In step (3), the molar ratio of tripterygium wilfordii to intermediate IX is 1:4 to 1:5; the molar ratio of tripterygium wilfordii to HATU is 1:1.2 to 1:1.5; and the molar ratio of tripterygium wilfordii to DIPEA is 1:3 to 1:

4.

8. Use of the tripterygium wilfordii acrylamide derivative according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumors are breast cancer, gastric cancer and colon cancer.

10. A pharmaceutical composition, characterized in that: It uses the tripterygium wilfordii acrylamide derivative described in any one of claims 1 to 5 as a main active ingredient and is supplemented with a pharmaceutically acceptable carrier to be prepared into any pharmaceutically acceptable dosage form.

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