Electrophilic warhead derivative based on sinomenine A ring modification as well as preparation method and anti-tumor application thereof

Abstract

The invention belongs to the technical field of medicine and chemical industry, and discloses an electrophilic warhead derivative based on sinomenine A ring modification as well as a preparation method and anti-tumor application thereof. According to the invention, active groups are introduced to the C-1 site and the C-4 site, and the fat solubility is improved, so that relatively high anti-tumor activity is realized; meanwhile, an amide structure is directly connected with a benzene ring, so that the stability of a C-1 site substituent group is greatly improved, and the compound has relatively good inhibition capability on cervical cancer, breast cancer, liver cancer and the like.

Description

Technical Field

[0001] The present invention belongs to the technical fields of medicine and chemical engineering, and relates to a class of electrophilic warhead-containing derivatives based on the modification of the A-ring of sinomenine, a preparation method thereof, and an anti-tumor application. Background Art

[0002] Sinomenine is an alkaloid extracted from Sinomenium acutum, and has pharmacological activities such as anti-inflammatory, immunosuppressive, analgesic, and anti-rheumatic effects. Clinically, it is mainly used to treat rheumatoid arthritis (RA) and autoimmune diseases. In recent years, with the in-depth study of its pharmacological effects, the application potential of sinomenine in the anti-tumor field has gradually emerged, and the structural derivatization and exploration of anti-tumor mechanisms have been concerned by more and more scholars. However, sinomenine in its natural form has problems such as low bioavailability and poor targeting. Therefore, structural derivatization has become an important strategy to optimize its anti-tumor activity.

[0003] The molecular structure of sinomenine contains multiple modifiable functional groups, which provides rich possibilities for its structural modification and derivatization, and also brings difficulties to directed chemical modification. The modification of the A-ring of sinomenine has received main attention in recent years, but most of them focus on C-4 esterification, and functional groups such as covalent warheads are introduced through long fatty chains to improve its anti-tumor activity (Zhu L, Chen C, et al. [J] E. J. Med. Chem, 2024, 268; Gao X, Li H-N, [J] J. Asian. Nat. prod. Res., 2024). However, the modification with too long carbon chains often brings many druggability problems, such as low metabolic stability, abnormal distribution and clearance, and toxicological risks. Due to the special structure of the A-ring of sinomenine, the modification at the C-1 position has great expansion space. In the present invention, the active functional group is introduced based on the C-1 site; and the lipophilicity is improved by the modification at the C-4 position, and good tumor cell inhibition effects can be achieved through the modification with a benzene ring or a short carbon chain. Summary of the Invention

[0004] In order to improve the stability and druggability problems brought by chemical modification, the present invention realizes high anti-tumor activity by introducing active groups at the C-1 and C-4 positions and improving the lipophilicity; at the same time, due to the direct connection of the amide structure to the benzene ring, the stability of the C-1 substituent is greatly improved.

[0005] Another object of the present invention is to propose a synthetic route of the above modification scheme, and to provide the application of the above derivatives in the anti-tumor field.

[0006] The technical solution of the present invention:

[0007] A class of derivatives containing electrophilic warheads based on the modification of the A-ring of sinomenine, in which the structure of the amide-type electrophilic warhead is directly connected to the A-ring derivative of sinomenine and the C-4 position is esterified. The structural formula is as follows:

[0008]

[0009] Among them, R 1 is alkenyl, substituted alkyl or substituted alkenyl;

[0010] The substituent of the substituted alkyl is fluorine, chlorine or bromine;

[0011] The substituent of the substituted alkenyl is fluorine, chlorine, bromine, trifluoromethyl, dimethylaminomethyl or diethylaminomethyl;

[0012] R 2 is alkyl, phenyl or substituted phenyl;

[0013] The substituent of the substituted phenyl is fluorine, chlorine, bromine, iodine, alkyl, methoxy, phenyl, and the number is 1 - 3. The substituents are located at the ortho, meta and para positions of the benzene ring.

[0014] The A-ring derivatives of sinomenine include, but are not limited to, the following structures:

[0015]

[0016]

[0017] The synthesis method of the A-ring derivatives of sinomenine has the following synthetic route:

[0018]

[0019] The specific steps are as follows:

[0020] (1) Using sinomenine as the raw material, in 95% (v / v) ethanol, concentrated hydrochloric acid and a 20% sodium nitrite solution by mass are added. The reaction is carried out at a reaction temperature of 5 - 25 °C and ends within 5 minutes to obtain a crude product. The crude product is slurried in methanol to obtain the intermediate 2 containing nitroso. Among them, the concentration of concentrated hydrochloric acid is 12M, the molar ratio of sinomenine to sodium nitrite is 1:5 - 1:10, and the molar ratio of sodium nitrite to concentrated hydrochloric acid is 1:1 - 1:2;

[0021] (2) Under the condition of a reaction temperature of 5 - 25 °C, the intermediate 2 containing nitroso is reduced with stannous chloride dihydrate in ethanol, and the reaction time is controlled for 20 - 40 minutes to obtain the amino intermediate 3. Among them, the molar ratio of the intermediate 2 containing nitroso to stannous chloride dihydrate is 1:10 - 1:15;

[0022] (3) In dichloromethane, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), triethylamine (TEA), a carboxylic acid and intermediate 3 are subjected to a condensation reaction to obtain intermediate 4; wherein, the reaction temperature is 5 - 25 °C and the reaction time is 2 - 3 h; the molar ratio of intermediate 3, triethylamine, 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate to the carboxylic acid is 1:2:(2 - 3):(3 - 5);

[0023] (4) Intermediate 4 is reacted with an excessive amount of dicyclohexylcarbodiimide (DCC) and a carboxylic acid, and the molar ratio of intermediate 4, the carboxylic acid and dicyclohexylcarbodiimide is 1:(2 - 3):(3 - 5) to obtain a series of sinomenine derivatives A.

[0024] The carboxylic acid in step (3) is but not limited to acrylic acid, 2-chloroacrylic acid, 2-fluoroacrylic acid or 2-trifluoromethylacrylic acid.

[0025] The carboxylic acid in step (4) is but not limited to biphenylacetic acid, 4-chlorophenylacetic acid, 4-methylphenylacetic acid, cyclohexanecarboxylic acid or valeric acid.

[0026] On the other hand, an application of using a derivative containing an electrophilic warhead modified based on the A ring of sinomenine to prepare an anti-tumor drug.

[0027] The types of the tumors include cervical cancer, breast cancer, liver cancer, fibrosarcoma.

[0028] Advantages of the present invention:

[0029] The compound of the present invention covalently binds to a specific target in tumor cells by introducing an active warhead at the 1-position. Compared with non-covalent drugs, due to its special pharmacokinetic properties, the effective concentration of the covalent drug is greatly reduced and the effective action time is greatly increased; at the same time, the lipophilicity is optimized at the 4-position, which is beneficial to drug transmembrane penetration, and the overall structure achieves high anti-tumor activity.

[0030] In terms of compound preparation, such as in reduction reactions and condensation reactions, compared with the previous commonly used methods that use high-boiling solvents such as N,N-dimethylformamide (DMF) and reaction conditions of long-time heating, the method proposed by the present invention mostly uses low-boiling solvents such as dichloromethane, and a high conversion rate can be achieved at room temperature within a short time, and the separation and purification method is simple. Description of the drawings

[0031] Figure 1 is the 1 HNMR spectrum of intermediate 2 in Example 1 of the present application.

[0032] Figure 2 is the 1HNMR spectrum.

[0033] Figure 3 It is the 1 HNMR spectrum of intermediate 4(1) in Example 1 of this application.

[0034] Figure 4 It is the 1 HNMR spectrum of compound A1 in Example 1 of this application.

[0035] Figure 5 It is the 1 HNMR spectrum of intermediate 4(2) in Example 2 of this application.

[0036] Figure 6 It is the 1 HNMR spectrum of compound A2 in Example 2 of this application.

[0037] Figure 7 It is the 1 HNMR spectrum of compound A3 in Example 3 of this application.

[0038] Figure 8 It is the 1 HNMR spectrum of compound A4 in Example 5 of this application. Detailed implementation manners

[0039] The following further illustrates the detailed implementation manners of the present invention in conjunction with the accompanying drawings and technical solutions.

[0040] Example 1

[0041] Preparation of compound A1

[0042] In a 250 mL reaction flask, add 40 mL of 95% (v / v) ethanol solution to 14.7 g of sinomenine hydrochloride, and stir at room temperature to make it evenly dispersed. Add 33 mL of 12 M concentrated hydrochloric acid, stir for 5 min in a cold water bath, slowly dropwise add 22 mL of 20% (mass concentration) NaNO2 solution, complete the dropwise addition within 5 min, continue the reaction for 10 min, and stop stirring. Under an ice-water bath, adjust the pH to 8 with saturated NaOH solution, and a large amount of precipitate will form. Filter by suction and wash the filter cake with absolute ethanol until a colorless filtrate flows out. Evaporate the solvent under reduced pressure for the obtained brownish-black solution, dissolve it with a 1:1 mixed solvent of ethanol and dichloromethane, stir evenly at 40 °C, filter by suction to remove insoluble substances, and alternately rinse with ethanol and dichloromethane to obtain a filtrate without inorganic salts. Evaporate the filtrate to dryness under reduced pressure, add 50 mL of methanol, stir at 60 °C for 1 h, place it indoors and let it naturally cool to room temperature, and then transfer it to an ice-water bath and stir for 30 min. Filter by suction and wash with methanol to obtain intermediate 2 (crude product in khaki color), which can be used for subsequent reaction steps, with a yield of 40%. 11H NMR (400 MHz, DMSO-d6) δ 6.68 (s, 1H), 5.73 (d, J = 2.3 Hz, 1H), 3.75 (d, J = 15.5 Hz, 1H), 3.67 (s, 3H), 3.41 (s, 3H), 3.33 (s, 1H), 3.17 (d, J = 4.6 Hz, 1H), 3.14 (s, 1H), 3.02 (d, J = 20.5 Hz, 1H), 2.88 (s, 1H), 2.53 (s, 1H), 2.29 (s, 3H), 2.08 (t, J = 12.0 Hz, 1H), 1.84–1.66 (m, 2H); ESI-MS (m / z): [M+H] + calcd for C 19 H 23 N2O6 375.16, found 358.98.

[0043] 214.8 mg of compound 2 was added to 8 mL of absolute ethanol, and 2 g of SnCl2·2H2O was slowly added with stirring. The mixture was stirred at room temperature for 30 min under nitrogen protection. The pH value was adjusted to 8 - 9 with 20% NaOH solution by mass fraction, and the insoluble substances were removed by suction filtration. The yellow filtrate was extracted twice with dichloromethane, and the pH (8 - 9) was adjusted again to keep the solution weakly alkaline, and the extraction was continued 2 - 3 times. The organic phases were combined and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure to obtain 154.6 mg of the intermediate 3 as a brown solid, with a yield of 75%. 1 1H NMR (400 MHz, CDCl3): δ 6.18 (s, 1H), 5.45 (d, J = 2.3 Hz, 1H), 4.38 (d, J = 15.7 Hz, 1H), 3.76 (s, 3H), 3.72 (q, J = 7.0 Hz, 2H), 3.47 (s, 3H), 3.29 (t, J = 4.7 Hz, 1H), 2.99 (s, 1H), 2.71 (d, J = 17.6 Hz, 1H), 2.53 (dd, J = 11.9, 2.6 Hz, 1H), 2.44 (s, 3H), 2.40 (d, J = 6.9 Hz, 1H), 2.25 (dd, J = 17.7, 5.8 Hz, 1H), 2.11 (td, 1H), 1.96 (dt, J = 12.3, 2.9 Hz, 1H), 1.86 (td, J = 12.5, 4.6 Hz, 1H); ESI-MS (m / z): [M+H] + calcd for C 19 H 24 N2O4 345.18, found 345.02.

[0044] Under stirring, 311.8 mg of HATU, 56 μL of chloroacetic acid and 1.23 mmol of TEA were added to 5 mL of DCM, and the mixture was stirred at room temperature for 10 min. Subsequently, 0.41 mmol of intermediate 3 was added, and N2 was immediately introduced for protection. The reaction was continued with stirring for 3 h, washed twice with saturated NaHCO3, and dried over anhydrous Na2SO4. After concentrating the organic phase, preparative TLC separation (DCM:MeOH = 10:1) gave 52.4 mg of intermediate 4(2) as a pale yellow solid, with a yield of 43%; 1 1H NMR (400 MHz, CDCl3) δ 7.16 (s, 1H), 5.45 (d, J = 2.2 Hz, 1H), 4.36 (d, J = 15.7 Hz, 1H), 4.22 (s, 2H), 3.81 (s, 3H), 3.49 (s, 3H), 3.31 (t, J = 4.5 Hz, 1H), 3.06 (s, 1H), 2.83 (d, J = 17.9 Hz, 1H), 2.63–2.52 (m, 1H), 2.43 (s, 3H), 2.09 (td, J = 12.2, 3.5 Hz, 1H), 2.02–1.84 (m, 2H); ESI-MS (m / z): [M+H] + calcd for C 21 H 25 ClN2O5 421.16, found 421.03.

[0045] 85 mg of 4-biphenylacetic acid was dissolved in 5 mL of DCM, and 206 mg of DCC was added under stirring. The mixture was stirred at room temperature for 15 min. Then 84.0 mg of intermediate 4(1) and 7.3 mg of DMAP were added. After introducing N2, the reaction was continued with stirring for 3 h, and the reaction progress was monitored by TLC. After the raw materials were completely converted, the insoluble substances were removed by suction filtration, washed once with saturated NaCl, and dried over anhydrous Na2SO4. Preparative TLC separation (DCM:MeOH = 15:1) gave 45.1 mg of off-white solid A1, with a yield of 37%. 11H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.56–7.50 (m, 4H), 7.43 (d, J = 7.9 Hz, 2H), 7.39–7.32 (m, 3H), 7.27 (t, J = 7.3 Hz, 1H), 5.33 (d, J = 2.2 Hz, 1H), 4.16 (s, 2H), 3.89 (q, J = 15.0 Hz, 2H), 3.59 (s, 3H), 3.54 (d, J = 16.2 Hz, 1H), 3.40 (s, 3H), 3.20 (t, J = 4.7 Hz, 1H), 2.91 (s, 1H), 2.73 (d, J = 17.6 Hz, 1H), 2.42 (td, J = 12.0, 5.2 Hz, 2H), 2.34 (s, 3H), 2.23 (d, J = 16.1 Hz, 1H), 2.02 (t, J = 12.3 Hz, 1H), 1.75–1.62 (m, 1H), 1.51–1.38 (m, 1H); HRESI-MS (m / z): [M+H] + calcd for C 35 H 36 ClN2O6 615.2262, found 615.2232.

[0046] Example 2

[0047] Based on Intermediate 3 synthesized in Example 1, Compound A2 was further prepared as follows:

[0048] Under stirring, 354 mg of HATU, 50 μL of 2-acrylic acid and 50 μL of TEA were added to 5 mL of DCM, and the mixture was stirred at room temperature for 10 min. Subsequently, 40.5 mg of Intermediate 3 was added, and N2 protection was immediately charged. The reaction was continued to stir for 3 h, washed twice with saturated NaHCO3, and dried over anhydrous Na2SO4. After concentrating the organic phase, preparative TLC separation (DCM:MeOH = 10:1) was performed to obtain 38.4 mg of Intermediate 4(2) as a pale yellow solid, with a yield of 41%; 11H NMR (500 MHz, CDCl3) δ 7.79 (s, 1H), 7.05 (s, 1H), 6.47–6.32 (m, 2H), 5.75 (dd, J = 9.0, 2.5 Hz, 1H), 5.43 (d, J = 2.1 Hz, 1H), 4.37 (d, J = 15.6 Hz, 1H), 3.77 (s, 3H), 3.54 (s, 1H), 3.45 (s, 3H), 3.34 (s, 1H), 2.99 (d, J = 18.6 Hz, 1H), 2.87 (d, J = 10.2 Hz, 1H), 2.76 (d, J = 18.4 Hz, 1H), 2.56 (s, 3H), 2.50 (d, J = 15.7 Hz, 1H), 2.35 (t, J = 12.3 Hz, 1H), 2.15–2.05 (m, 1H), 2.02 (d, J = 12.2 Hz, 1H); ESI-MS (m / z): [M+H] + calcd for C 22 H 27 N2O5 399.04, found 399.19.

[0049] 44.6 mg of biphenylacetic acid and 72.2 mg of DCC were added to 3 mL of DCM. After stirring at room temperature for 10 min, 28.4 mg of 4(2) and 2.6 mg of DMAP were added. After filling with N2, the reaction was continued with stirring for 2 h. After the raw materials were completely converted, the insoluble substances were removed by suction filtration, washed once with saturated NaCl, and dried over anhydrous Na2SO4. Preparation TLC separation (DCM:MeOH = 10:1) gave 12.2 mg of white solid, yield 29%; 11H NMR (400 MHz, CDCl3) δ 7.60 (td, J = 5.8, 2.7 Hz, 4H), 7.50 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.7 Hz, 2H), 7.37–7.31 (m, 1H), 6.43 (d, J = 16.9 Hz, 1H), 6.35–6.22 (m, 1H), 5.77 (d, J = 10.0 Hz, 1H), 5.39 (d, J = 2.2 Hz, 1H), 3.96 (q, J = 15.0 Hz, 2H), 3.65 (s, 3H), 3.61 (d, J = 16.2 Hz, 1H), 3.44 (s, 3H), 3.23 (t, J = 4.9 Hz, 1H), 2.95 (s, 1H), 2.81 (d, J = 17.9 Hz, 1H), 2.54–2.44 (m, 2H), 2.36 (s, 3H), 2.29 (d, J = 16.2 Hz, 1H), 2.12–2.03 (m, 1H), 1.80–1.70 (m, 1H), 1.58–1.46 (m, 1H). HRESI-MS (m / z): [M+H] + calcd for C 36 H 37 N2O6593.2652, found 593.2647.

[0050] Example 3

[0051] Based on Intermediate 3 synthesized in Example 1, Compound A3 was further prepared as follows:

[0052] 5 mL of DCM was added to 376.4 mg of HATU and 89.1 mg of 2-fluoroacrylic acid, and the mixture was stirred at room temperature for 15 min. Subsequently, 32.4 mg of 3 and 21 μL of TEA were added, and N2 was immediately introduced for protection. The reaction was continued with stirring for 4 h, washed twice with saturated NaHCO3, and dried over anhydrous Na2SO4. After concentrating the organic phase, purification by preparative TLC (DCM:MeOH = 10:1) gave an off-white solid as Intermediate 4(3); 175.4 mg of DCC was added to a DCM solution of 5 mL of 4-biphenylacetic acid (72.2 mg). The mixture was stirred at room temperature for 15 min. Then 71.9 mg of Intermediate 4(3) and 6.2 mg of DMAP were added. After introducing N2, the reaction was continued with stirring for 3 h, and the reaction progress was monitored by TLC. After the raw materials were completely converted, the insoluble matter was removed by suction filtration, washed once with saturated NaCl, and dried over anhydrous Na2SO4. Purification by preparative TLC (DCM:MeOH = 10:1) gave 32.8 mg of an off-white solid Compound A3, with a yield of 32%; 11H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 5.6 Hz, 1H), 7.64–7.55 (m, 5H), 7.53 (s, 1H), 7.50 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.38–7.29 (m, 1H), 5.82 (dd, J = 48.1, 3.4 Hz, 1H), 5.40 (d, J = 2.2 Hz, 1H), 5.28 (dd, J = 15.4, 3.4 Hz, 1H), 3.96 (q, J = 15.0 Hz, 2H), 3.66 (s, 3H), 3.62 (d, J = 16.1 Hz, 1H), 3.47 (s, 3H), 3.27 (t, J = 4.6 Hz, 1H), 2.94 (s, 1H), 2.78 (d, J = 17.7 Hz, 1H), 2.53–2.42 (m, 2H), 2.40 (s, 3H), 2.29 (d, J = 16.1 Hz, 1H), 2.06 (td, J = 12.4, 3.3 Hz, 1H), 1.73 (td, J = 12.2, 4.3 Hz, 1H), 1.56–1.46 (m, 1H); HRESI-MS (m / z): [M+H] + calcd for C 36 H 36 FN2O6 611.2557, found 611.2524.

[0053] Example 4

[0054] Based on Intermediate 3 synthesized in Example 1, Compound A4 was further prepared as follows:

[0055] 513.3 mg of HATU and 143.8 mg of 2-chloroacrylic acid were added to 5 mL of DCM and stirred at room temperature for 15 min. Subsequently, 0.45 mmol of Intermediate 3 and 57 μL of TEA were added, and N2 was immediately charged for protection. The reaction was continued with stirring for 1 h, washed twice with saturated NaHCO3, and dried over anhydrous Na2SO4. After concentrating the organic phase, preparative TLC separation (DCM:MeOH = 10:1) gave orange-yellow Intermediate 4(4); 21.2 mg of 4-biphenylacetic acid was dissolved in 3 mL of DCM, and after stirring to dissolve, 51.6 mg of DCC was added. Stirring was carried out at room temperature for 15 min. Then 22.6 mg of Intermediate 4(4) and 1.83 mg of DMAP were added. After charging N2, the reaction was continued with stirring for 2 h, and the reaction progress was monitored by TLC. After the raw materials were completely converted, the insoluble matter was removed by suction filtration, washed once with saturated NaCl, and dried over anhydrous Na2SO4. Preparative TLC separation (DCM:MeOH = 15:1) gave yellow solid Compound A4, yellow solid, yield 32%; 11H NMR (400 MHz, CDCl3) δ 7.63–7.57 (m, 5H), 7.50 (d, J = 8.3 Hz, 2H), 7.47–7.41 (m, 2H), 7.37–7.32 (m, 1H), 6.73 (d, J = 1.6 Hz, 1H), 5.94 (d, J = 1.5 Hz, 1H), 5.41 (d, J = 2.2 Hz, 1H), 3.96 (q, J = 15.0 Hz, 2H), 3.67 (s, 3H), 3.63 (d, J = 16.1 Hz, 1H), 3.49 (s, 3H), 3.29 (t, J = 4.6 Hz, 1H), 2.96 (s, 1H), 2.80 (d, J = 17.5 Hz, 1H), 2.54–2.43 (m, 2H), 2.42 (s, 3H), 2.29 (d, J = 16.1 Hz, 1H), 2.14–2.05 (m, 1H), 1.74 (td, J = 12.6, 4.5 Hz, 1H), 1.51 (d, J = 12.5 Hz, 1H); HRESI-MS (m / z): [M + H] + calcd for C 36 H 36 ClN2O6 627.2262, found 627.2233.

[0056] Example 5

[0057] Based on the intermediate 4(4) synthesized in Example 4, compound A5 was further prepared as follows:

[0058] Dissolve 32.5 mg of 4-methylphenylacetic acid in 3 mL of DCM. After stirring to dissolve, add 72 mg of DCC. Stir at room temperature for 10 min. Then add 30.6 mg of intermediate 4(4) and 2.3 mg of DMAP. After filling with N2, continue to stir and react for 2 h, and monitor the reaction progress by TLC. After the raw materials are completely converted, filter off the insoluble matter by suction, wash once with saturated NaCl, and dry over anhydrous Na2SO4. Prepare TLC separation (DCM:MeOH = 15:1) to obtain the white solid compound A5 with a yield of 44%

[0059] Example 6

[0060] Based on the intermediate 4(4) synthesized in Example 4, compound A6 was further prepared as follows:

[0061] Dissolve 39.5 mg of 4-chlorophenylacetic acid in 3 mL of DCM. After stirring to dissolve, add 69.2 mg of DCC. Stir at room temperature for 10 min. Then add 28.9 mg of intermediate 4(4) and 2.0 mg of DMAP. After filling with N2, continue stirring and reacting for 2 h, and monitor the reaction progress by TLC. After the raw materials are completely converted, filter off the insoluble substances by suction, wash once with saturated NaCl, and dry with anhydrous Na2SO4. Separate by preparative TLC (DCM:MeOH = 15:1) to obtain off-white solid compound A6 with a yield of 42%

[0062] Example 7

[0063] Based on intermediate 4(4) synthesized in Example 4, further prepare compound A7:

[0064] Dissolve 30.5 mg of cyclohexanecarboxylic acid in 3 mL of DCM. After stirring to dissolve, add 65 mg of DCC. Stir at room temperature for 10 min. Then add 26.2 mg of intermediate 4(4) and 1.8 mg of DMAP. After filling with N2, continue stirring and reacting for 2 h, and monitor the reaction progress by TLC. After the raw materials are completely converted, filter off the insoluble substances by suction, wash once with saturated NaCl, and dry with anhydrous Na2SO4. Separate by preparative TLC (DCM:MeOH = 15:1) to obtain off-white solid compound A7 with a yield of 58%

[0065] Example 8

[0066] Based on intermediate 4(4) synthesized in Example 4, further prepare compound A8:

[0067] Dissolve 28.5 mg of valeric acid in 3 mL of DCM. After stirring to dissolve, add 70.2 mg of DCC. Stir at room temperature for 10 min. Then add 28.5 mg of intermediate 4(4) and 2.2 mg of DMAP. After filling with N2, continue stirring and reacting for 2 h, and monitor the reaction progress by TLC. After the raw materials are completely converted, filter off the insoluble substances by suction, wash once with saturated NaCl, and dry with anhydrous Na2SO4. Separate by preparative TLC (DCM:MeOH = 15:1) to obtain off-white solid compound A8 with a yield of 45% Application Example 1

[0068] The inhibitory activities of some A-ring derivatives in the present invention against tumor cells include a HPV-positive cervical cancer cell Hela, a HPV-positive cervical cancer cell C33A, a Luminal A subtype breast cancer cell MCF-7, a triple-negative breast cancer cell MDA-MB-231, a liver cancer cell HepG2, and a fibrosarcoma cell HT1080.

[0069] Using the MTT method, cells in the exponential growth phase were digested with trypsin and collected. After counting, they were seeded in 96-well plates at a density of 3000 cells / well. After culturing in a cell incubator (saturated humidity, 5% CO2, 37 °C) for 24 h, according to the pre-set drug concentrations, the 10 mmol / L compound stock solution dissolved in DMSO was serially diluted in complete medium (DMEM, containing 10% FBS and 1% PS). After incubation for 24 h, the original culture medium was replaced with DMEM medium containing 10% MTT working solution; after staining for 4 h, the supernatant was aspirated, and the formazan crystals at the bottom were dissolved with DMSO. The absorbance at 490 nm was measured using a microplate reader. According to the calculation method of cell viability: (OD value of control group - OD value of blank group) / (OD value of experimental group - OD value of blank group) × 100%, a logarithmic curve was fitted to calculate the IC 50 value (μM), and the results are shown in Table 1.

[0070] Table 1 IC50 values (μM) of the inhibitory activities of sinomenine derivatives against tumor cell proliferation

[0071]

[0072] The activity evaluation of the synthesized sinomenine A-ring partial derivatives showed that the above compounds all exhibited high anti-tumor activities. Compared with the unesterified products, esterification may promote the permeability of the drug to the cell membrane, which is more conducive to the anti-tumor activity of the drug. Among them, compound A4 had the best inhibitory ability against breast cancer and had important application prospects.

Claims

1. A class of derivatives containing electrophilic warheads based on the modification of the A-ring of sinomenine, characterized in that, The electrophilic warhead-containing derivative has an amide-type electrophilic warhead structure directly connected to the A-ring derivative of sinomenine, and the C-4 position is esterified. The structural formula is as follows: wherein, R 1 is an alkenyl group, a substituted alkyl group or a substituted alkenyl group; The substituent of the substituted alkyl group is fluorine, chlorine or bromine; The substituent of the substituted alkenyl group is fluorine, chlorine, bromine, trifluoromethyl, dimethylaminomethyl or diethylaminomethyl; R 2 is an alkyl group, a phenyl group or a substituted phenyl group; The substituent of the substituted phenyl group is fluorine, chlorine, bromine, iodine, alkyl, methoxy, phenyl, and the number is 1-3. The substituents are located at the ortho, meta and para positions of the benzene ring.

2. The electrophile warhead derivative according to claim 1, characterized in that, The A-ring derivative of sinomenine includes, but is not limited to, the following structures:

3. The preparation method of the electrophilic warhead derivative-containing compound according to claim 1 or 2, characterized in that, The synthesis method of the A-ring derivative of sinomenine has the following synthetic route: The specific steps are as follows: (1) Using sinomenine as the raw material, in 95% ethanol by volume, concentrated hydrochloric acid and a sodium nitrite solution with a mass concentration of 20% are added. The reaction temperature is 5-25°C, and the reaction ends within 5-10 minutes to obtain a crude product. The crude product is slurried in methanol to obtain the nitroso-containing intermediate 2. Among them, the concentration of concentrated hydrochloric acid is 12M, the molar ratio of sinomenine to sodium nitrite is 1:5-1:10, and the molar ratio of sodium nitrite to concentrated hydrochloric acid is 1:1-1:2; (2) Under the condition that the reaction temperature is 5-25°C, intermediate 2 is reduced with stannous chloride dihydrate in ethanol, and the reaction time is controlled to be 20-40 minutes to obtain amino intermediate 3. Among them, the molar ratio of intermediate 2 to stannous chloride dihydrate is 1:10-1:15; (3) In dichloromethane, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, triethylamine, carboxylic acid and intermediate 3 are subjected to a condensation reaction to obtain intermediate 4. Among them, the reaction temperature is 5-25°C, and the reaction time is 2-3 hours; the molar ratio of intermediate 3, triethylamine, HATU and carboxylic acid is 1:2:(2-3):(3-5); (4) Intermediate 4 is reacted with an excessive amount of dicyclohexylcarbodiimide and carboxylic acid, and the molar ratio of intermediate 4, carboxylic acid and DCC is 1:(2-3):(3-5) to obtain A-series sinomenine derivatives.

4. The preparation method according to claim 3, wherein The carboxylic acid in step (3) includes, but is not limited to, acrylic acid, 2-chloroacrylic acid, 2-fluoroacrylic acid or 2-trifluoromethylacrylic acid.

5. The preparation method according to claim 3, characterized in that, The carboxylic acid in step (4) includes, but is not limited to, biphenylacetic acid, 4-chlorophenylacetic acid, 4-methylphenylacetic acid, cyclohexanecarboxylic acid or valeric acid.

6. An application of the electrophilic warhead-containing derivative modified based on the A-ring of sinomenine described in claim 1 or 2 in the preparation of anti-tumor drugs.

7. The application according to claim 6, characterized in that, The types of tumors include cervical cancer, breast cancer, liver cancer, fibrosarcoma.

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