Vinylpyridine prodrug compounds, and methods of making and using the same

Abstract

The application discloses a kind of vinyl pyridine prodrug compounds and preparation method and application thereof, the compound structure is as shown in formula I, the prodrug compound of the application is activated prodrug by palladium resin catalytic reaction specificity, releases active ingredient under the activation of palladium resin, targets microtubule protein and inhibits the microtubule polymerization of tumor cell, to effectively inhibit the growth of tumor cell, reach high safety, effectively increase treatment window, produce high-efficiency low-toxicity therapeutic effect, while realizing high-efficiency treatment in tumor site, can reduce the toxicity to normal cell, can realize the selectivity and precision of treatment, with potential extensive application prospect.

Description

Technical Field

[0001] This invention relates to a vinylpyridine prodrug compound, and also to the preparation and application of the above compound. Background Technology

[0002] Cancer treatment has always been a focus of medical research; however, traditional chemotherapy methods are often accompanied by widespread side effects, especially toxicity to normal tissues. To improve treatment precision and reduce toxic side effects, targeted prodrug activation technology has received widespread attention in recent years. Bioorthogonal chemical reactions, as a technology that can activate prodrugs under specific conditions in vivo, offer new possibilities for precision medicine. Through this technology, drugs remain in a low-activity state in vivo and are activated only in the specific environment of tumor cells, significantly reducing toxicity to normal tissues.

[0003] Microtubules are a crucial component of the cytoskeleton, participating in numerous biological processes such as cell division, morphology maintenance, and substance transport. The dynamic stability of microtubules is particularly important for tumor cell proliferation; therefore, microtubules and related proteins have become key targets in cancer therapy. Tubulin, the main protein composing microtubules, can polymerize or depolymerize to form microtubule fibers, regulating cell cycle and cell division. Inhibiting microtubule polymerization can prevent normal tumor cell division and proliferation, leading to cell cycle arrest and ultimately cell death. Therefore, targeting tubulin, especially by binding to it to inhibit microtubule polymerization, has become an effective anti-cancer strategy. However, microtubule inhibitors (such as colchicine-like compounds) often exhibit strong toxicity in vivo, not only inhibiting cancer cells but also affecting normal cells, causing systemic side effects. Therefore, developing microtubule inhibitors that can target tumor cells while minimizing toxicity to normal tissues is a significant challenge in cancer treatment research. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a vinylpyridine prodrug compound that has a precise targeting effect on the colchicine binding site of tubulin, and to provide a method for preparing the above compound and its application in the preparation of tubulin inhibitors or antitumor drugs.

[0005] Technical solution: This invention discloses a vinylpyridine prodrug compound with the structure shown in Formula I:

[0006]

[0007] Among them, R1, R2, R4, and R5 are each independently selected from hydrogen, halogen, hydroxyl, mercapto, cyano, nitro, carboxyl, ester, acyl, amide, hydroxymethyl, aldehyde, sulfonyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 alkylamino, C1-C4 imino, C6-C 10 Aryl, C3-C 10 heteroaryl, C4-C8 heterocyclic;

[0008] R3 is selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, C3-C 10 heteroaryl, C4-C8 heterocyclic group.

[0009] Preferably, R1, R2, R4, and R5 are each independently selected from hydrogen, halogen, hydroxyl, acyl, amide, hydroxymethyl, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C4 alkoxy, C3-C6 cycloalkyl, C1-C4 alkylamino, C6-C 10 Aryl, C3-C 10 heteroaryl, C4-C8 heterocyclic;

[0010] R3 is selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, C3-C 10 heteroaryl, C4-C8 heterocyclic group.

[0011] Preferably, R1, R2, R4, and R5 are each independently selected from hydrogen, halogen, C1-C4 alkoxy, C1-C4 alkylamino, and C4-C8 heterocyclic groups;

[0012] R3 is selected from hydrogen, C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C3-C8 cycloalkyl, C6-C 10 Aryl, C3-C 10 Mixed aromatic compounds.

[0013] Preferably, R1 and R2 are selected from hydrogen, fluorine, chlorine, bromine, methoxy, ethoxy, methylamino, ethylamino, N,N-dimethylamino, and tetrahydropyrrolyl.

[0014] R3 is selected from hydrogen, methyl, ethyl, allyl, propyne, 1-butyn-3-yl, 3-methyl-1-butyn-3-yl, 1-buten-3-yl, benzyl, 4-propynoxybenzyl, 3-propynoxybenzyl, 2-propynoxybenzyl, 4-allyloxybenzyl, 3-allyloxybenzyl, 2-allyloxybenzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-cyanobenzyl, and 4-methylbenzyl.

[0015] R4 and R5 are selected from hydrogen, hydroxyl, methoxy, ethoxy, methylamino, and N,N-dimethylamino.

[0016] The compounds also include pharmaceutically acceptable salts.

[0017] The above compounds are selected from any one of compounds 1 to 16:

[0018]

[0019]

[0020]

[0021] The preparation method of the above-mentioned vinylpyridine prodrug compound includes the following steps: using 2,6-dichloroisonicotinic acid as a raw material, adding a condensing agent to condense it with N,O-dimethylhydroxylamine hydrochloride to obtain Weinreb amide, which is then reduced to obtain ketone iii. After protecting the carbonyl group with 2-bromoethanol, the chlorine atom is substituted with different groups to obtain intermediate vi. After deprotection under acidic conditions, intermediate vii is obtained. Intermediate vii is reacted with p-toluenesulfonyl hydrazine to obtain intermediate viiii. Different substituted halogenated phenols are protected with tert-butyldimethylchlorosilane to obtain intermediate x. Intermediate viiii and intermediate x are subjected to the Heck reaction, and after deprotection, the original drug xii is obtained. Finally, the vinylpyridine prodrug compound can be obtained by nucleophilic substitution of the hydroxyl group with different halogenated hydrocarbons. The synthetic route is as follows:

[0022]

[0023] The present invention also discloses a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

[0024] The aforementioned vinylpyridine prodrug compounds or pharmaceutical compositions can be used in the preparation of microtubule inhibitors.

[0025] The aforementioned vinylpyridine prodrug compounds or pharmaceutical compositions can also be used in the preparation of antitumor drugs.

[0026] Invention Principle: The vinylpyridine prodrug compounds of this invention are novel bioorthogonal prodrug molecules. Targeting the colchicine binding site of tubulin, a bioorthogonal strategy is employed to activate the prodrug, thereby targeting tubulin and inhibiting microtubule polymerization in tumor cells, achieving precise targeted therapy for tumors. Molecular docking results and structure-activity relationship studies show that the hydroxyl group in xii of the original drug molecule is the key group for maintaining efficacy. By using different hydrocarbon substitutions, the interaction between the hydroxyl group and the binding pocket can be blocked, resulting in a significant reduction in activity and toxicity. This prodrug molecule exhibits extremely low activity in vivo. Injecting palladium resin into the tumor to catalyze the prodrug molecule enables specific activation of the prodrug within tumor cells, releasing a drug with antitumor activity, thus improving the selectivity and precision of treatment. The compounds of this invention have extremely low toxicity, and the palladium resin catalyst also has good safety, producing effects superior to the original drug after activation. Therefore, this series of prodrug molecules can not only reduce or even avoid toxicity to normal cells but also achieve highly efficient treatment at the tumor site, showing broad application prospects.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: (1) The prodrug compound of the present invention can effectively inhibit tubulin activity and has significant anti-tumor activity, high selectivity for tumor cells, and low toxicity; (2) The preparation method is simple, the reaction conditions are relatively mild, there is almost no high temperature and high pressure reaction, and the operation is convenient. There are no dangerous reactions, no highly toxic reagents, and the reaction is safe and green. The reaction yield is high, the product purification operation is simple, and it is energy-saving and efficient. Attached Figure Description

[0028] Figure 1 The structural formula of the vinylpyridine prodrug compound of this invention is shown below;

[0029] Figure 2 These are the experimental results showing the effect of the compound of this invention on the body weight of mice. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the embodiments. The test materials used in the embodiments can all be purchased through conventional means.

[0031] Example 1

[0032] Compound 1: 2,6-Dimethoxy-4-{1-[4-methoxy-3-(prop-2-ynyloxy)phenyl]vinyl}pyridine

[0033] Step 1: 2,6-Dichloro-N-methoxy-N-methylpyridine-4-carboxamide

[0034]

[0035] 2,6-Dichloroisonicotinic acid (4 g, 20.83 mmol), N,O-dimethylhydroxylamine hydrochloride (2.44 g, 25.00 mmol), EDCI (4.39 g, 22.91 mmol), and HOBt (3.09 g, 22.91 mmol) were dissolved in DCM. Et3N (6.83 mL, 31.25 mmol) was added to the reaction solution, and the mixture was stirred at room temperature for 12 h. After the reaction was complete, the reaction solution was filtered to remove solid impurities, and the filtrate was concentrated under reduced pressure to obtain a pale yellow oily crude product. The crude product was purified by silica gel chromatography (PE:EA = 4:1) to obtain the title compound as a white solid (4.35 g, 88.92%). ESI-MS m / z: 234.67 [M+H] + .

[0036] Step 2: 1-(2,6-Dichloropyridin-4-yl)ethyl-1-one

[0037]

[0038] 2,6-Dichloro-N-methoxy-N-methylpyridine-4-carboxamide (4 g, 17.02 mmol) was dissolved in anhydrous THF and placed in an ice bath. Methylmagnesium bromide (3 M, 5.67 mL, 17.02 mmol) was slowly added to the reaction flask under nitrogen protection. The reaction was monitored by TLC after 1 hour. After completion, the reaction was quenched with saturated ammonium chloride solution, extracted with dichloromethane, washed with saturated brine, and the organic layer was dried over anhydrous Na₂SO₄. The solvent was removed by distillation under reduced pressure, yielding a yellow liquid (2.86 g, 88.45%), requiring no further purification. ESI-MS m / z: 189.81 [M+H] + .

[0039] Step 3: 2,6-Dichloro-4-(2-methyl-1,3-dioxacyclopentan-2-yl)pyridine

[0040]

[0041] 1-(2,6-dichloropyridin-4-yl)ethyl-1-one (2.86 mL, 15.05 mmol) and 2-bromoethanol (3.76 mL, 30.10 mmol) were dissolved in toluene. 1,8-diazabicyclo[5.4.0]undec-7-ene (5.62 mL, 37.63 mmol) was slowly added dropwise to the mixture, and the mixture was stirred at 90 °C for 12 h. After the reaction was complete as detected by TLC, toluene was removed under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (PE:EA = 30:1) to give a white powder solid (3.01 g, 85.44%). ESI-MS m / z: 234.67 [M+H]+ .

[0042] Step 4: 2,6-Dimethoxy-4-(2-methyl-1,3-dioxacyclopent-2-yl)pyridine

[0043]

[0044] In a sealed tube, 2,6-dichloro-4-(2-methyl-1,3-dioxanepent-2-yl)pyridine (3.00 g, 12.82 mmol) was dissolved in 20 mL of MeOH, and sodium methoxide solid (6.92 g, 128.16 mmol) was added. After stirring at 100 °C for 12 h, the reaction mixture was cooled to room temperature, and the reaction was monitored by TLC. After the reaction was complete, methanol was removed under reduced pressure. The reaction mixture was extracted with ethyl acetate, the organic phase was washed with saturated brine, and dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure to give a yellow solid (2.51 g, 86.95%). ESI-MS m / z: 226.21 [M+H] + .

[0045] The crude product can be directly proceeded to the next reaction.

[0046] Step 5: 1-(2,6-Dimethoxypyridin-4-yl)ethyl-1-one

[0047]

[0048] 2,6-Dimethoxy-4-(2-methyl-1,3-dioxacyclopentan-2-yl)pyridine (2.5 g, 11.11 mmol) was dissolved in 30 mL of concentrated hydrochloric acid and reacted at 80 °C for 1.5 h. After the reaction was complete as determined by TCL, the pH of the reaction solution was adjusted to 8 using 2 mol / L NaOH solution. The reaction solution was extracted with EA, the organic phase was washed with saturated brine, and dried over anhydrous Na₂SO₄. The solvent was removed by distillation under reduced pressure to obtain the crude product. The crude product was purified by silica gel chromatography (PE:EA = 40:1) to give a white solid (1.87 g, 92.99%). ESI-MS m / z: 182.25 [M+H] + .

[0049] Step 6: N-{[(1Z)-1-(2,6-dimethoxypyridin-4-yl)ethenyl]amino}-4-methylbenzenesulfonamide

[0050]

[0051] 1-(2,6-dimethoxypyridin-4-yl)ethyl-1-one (1.80 g, 9.93 mmol) was dissolved in anhydrous ethanol, and 4-methylbenzenesulfonyl hydrazine (2.13 g, 11.42 mmol) was slowly added dropwise. The mixture was stirred at 80 °C for 2 hours, and the reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered, and the solid was collected and dried to give the title compound as a white powder (2.84 g, 81.91%). ESI-MS m / z: 350.12 [M+H] + .

[0052] Step 7: [(5-bromo-2-methoxyphenyl)oxy]dimethyl(2-methylpropyl-2-yl)silane

[0053]

[0054] 5-Bromo-2-methoxyphenol (2.30 g, 11.33 mmol) was dissolved in 15 mL of DMF, and imidazole (1.90 g, 28.30 mmol) and TBSCl (3.40 g, 15.30 mmol) were added. The mixture was stirred at room temperature for 4 hours. After the reaction was complete, the reaction was quenched with water. The reaction solution was extracted with ethyl acetate, and the organic phase was washed with saturated brine and dried over anhydrous Na2SO4. The solvent was removed by distillation under reduced pressure to obtain a yellow oily liquid. The crude product was purified by column chromatography using PE / EA (10:1) to obtain a colorless, transparent oily liquid (3.30 g, 91.81%). ESI-MS m / z: 237.35 [M+H] + .

[0055] Step 8: 4-[1-(3-{[dimethyl(2-methylprop-2-yl)silyl]oxy}-4-methoxyphenyl)vinyl]-2,6-dimethoxypyridine

[0056]

[0057] In a reaction flask, N-{[(1Z)-1-(2,6-dimethoxypyridin-4-yl)ethylidene]amino}-4-methylbenzenesulfonamide (1.0 g, 2.86 mmol), N-{[(1Z)-1-(2,6-dimethoxypyridin-4-yl)ethylidene]amino}-4-methylbenzenesulfonamide (908 mg, 2.86 mmol), Xphos (136 mg, 286.20 μmol), Pd(CH3CN)2Cl2 (59 mg, 228.96 μmol), and t-BuOLi (504 mg, 6.30 mmol) were added. The starting materials were dissolved in dioxane under nitrogen protection. The reaction system was stirred at 90 °C for 5 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by column chromatography using PE / EA (8:1) to obtain a light yellow, transparent oily liquid (986 mg, 85.79%). ESI-MS m / z: 402.31 [M+H] + .

[0058] Step 9: 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol

[0059]

[0060] 4-[1-(3-{[dimethyl(2-methylprop-2-yl)methsilyl]oxy}-4-methoxyphenyl)vinyl]-2,6-dimethoxypyridine

[0061] (986 mg, 2.46 mmol) was dissolved in THF, and tetrabutylammonium fluoride (TBAF) (963 mg, 3.68 mmol) was slowly added dropwise to the reaction solution. After stirring for 30 minutes, the reaction was monitored by TLC. After the reaction was complete, the solvent was removed under reduced pressure, and the crude product was purified by column chromatography using PE / EA (6:1) to give a yellow solid (607 mg, 86.04%). ESI-MS m / z: 288.11 [M+H] + .

[0062] Step 10: 2,6-Dimethoxy-4-{1-[4-methoxy-3-(prop-2-ynyloxy)phenyl]vinyl}pyridine

[0063] 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol (100 mg, 348.05 μmol), 3-bromopropyne (124.01 mg, 1.04 mmol), and anhydrous potassium carbonate (144 mg, 1.04 mmol) were dissolved in acetone and the mixture was heated under reflux for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain the product, a pale yellow oily liquid (74 mg, 65.35%). 1 H NMR (300MHz, DMSO-d6) δ7.05(dd,J=7.5,2.0Hz,1H),6.91(d,J=2.0Hz,1H),6.84(d,J=7.5Hz,1H),6.58(s ,2H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.4Hz,1H),4.81(s,2H),3.95(s,6H),3.84(s,3H),2.77(s,1H). 13 C NMR (100MHz, DMSO-d6) δ164.57,150.65,149.18,138.61,135.92,133.20,124. 48,115.02,113.80,113.71,101.89,80.82,78.21,59.21,56.50,55.14.ESI-MS m / z:326.41[M+H] + .

[0064] Example 2

[0065] Compound 2: 2,6-Dimethoxy-4-{1-[4-methoxy-3-(prop-2-enyloxy)phenyl]vinyl}pyridine

[0066] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol (100 mg, 348.05 μmol), 3-bromopropene, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a light yellow oily liquid (79 mg, 69.33%) was obtained. 1H NMR (300MHz, DMSO-d6) δ7.06(dd,J=7.5,2.0Hz,1H),6.95(d,J=2.0Hz,1H),6.83(d,J=7.5Hz,1H),6.58(s,2H),5.99(s,1H),5.73 (d,J=12.4Hz,1H),5.48(d,J=12.4Hz,1H),5.38(d,J=12.4Hz,1H),5.13(d,J=12.4Hz,1H),4.54(s,2H),3.95(s,6H),3.84(s,3H). 13 C NMR(100MHz,DMSO-d6)δ164.57,151.64,150.03,138.60,135.68,133.20,133.1 3,124.52,118.88,115.02,113.19,112.42,101.89,74.88,56.50,55.14.ESI-MS m / z:328.26[M+H] + .

[0067] Example 3

[0068] Compound 3: 4-{1-[3-(benzyloxy)-4-methoxyphenyl]vinyl}-2,6-dimethoxypyridine

[0069] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol (100 mg, 348.05 μmol), benzyl bromide, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a light yellow oily liquid (98 mg, 74.60%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ7.40–7.24(m,5H),7.06(dd,J=7.5,2.0Hz,1H),6.93(d,J=2.0Hz,1H),6.83(d,J=7.5Hz,1 H), 6.58 (s, 2H), 5.73 (d, J = 12.4Hz, 1H), 5.48 (d, J = 12.5Hz, 1H), 5.10 (t, J = 1.0Hz, 2H), 3.95 (s, 6H), 3.84 (s, 3H). 13C NMR(100MHz,DMSO-d6)δ164.57,151.45,150.23,140.03,138.60,135.75,133.20,132.4 7,132.28,132.20,124.52,115.02,113.19,111.91,101.89,81.70,56.50,55.12ESI-MS m / z:378.24[M+H] + .

[0070] Example 4

[0071] Compound 4: 4-{1-[3-(but-3-yn-2-yloxy)-4-methoxyphenyl]vinyl}-2,6-dimethoxypyridine

[0072] 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol (150 mg, 522.08 μmol), 3-butyn-2-ol (43 mg, 622.49 μmol), and triphenylphosphine (274 mg, 1.04 mmol) were dissolved in anhydrous dichloromethane, cooled to 0 °C, and protected with nitrogen. Diisopropyl azodicarbonate (211 mg, 1.04 mmol) was added dropwise, and the reaction was carried out at room temperature for 3 hours. The reaction was quenched with water and extracted with dichloromethane. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain the target product, a colorless oily liquid (101 mg, 57.00%). 1 H NMR(300MHz, DMSO-d6)δ7.05(dd,J=7.5,2.0Hz,1H),6.87–6.81(m,2H),6.58(s,2H),5.73(d,J=12. 4Hz,1H),5.48(d,J=12.4Hz,1H),4.87(s,1H),3.95(s,6H),3.84(s,3H),2.60(s,1H),1.49(s,3H). 13 C NMR(100MHz,DMSO-d6)δ164.57,150.52,149.62,138.61,135.64,133.20,124.45, 115.02,113.36,112.32,101.89,80.65,74.96,74.68,56.50,55.14,21.70.ESI-MS m / z:340.25[M+H] + .

[0073] Example 5

[0074] Compound 5: 2,6-Dimethoxy-4-(1-{4-methoxy-3-[(2-methylbut-3-yn-2-yl)oxy]phenyl}vinyl)pyridine

[0075] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol (100 mg, 348.05 μmol), 3-chloro-3-methylbut-1-yne, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a light yellow oily liquid (59 mg, 47.96%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ7.05(dd,J=7.5,2.0Hz,1H),6.98(d,J=2.0Hz,1H),6.85(d,J=7.5Hz,1H),6.58(s ,2H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.4Hz,1H),3.95(s,6H),3.84(s,3H),2.52(s,1H),1.47(s,6H). 13 C NMR(100MHz,DMSO-d6)δ164.57,148.57,148.46,138.63,135.30,133.20,124.90, 115.02,113.18,112.00,101.89,86.18,80.10,74.43,56.50,55.14,30.19.ESI-MS m / z:354.26[M+H] + .

[0076] Example 6

[0077] Compound 6: 4-{1-[3-(but-3-en-2-yloxy)-4-methoxyphenyl]vinyl}-2,6-dimethoxypyridine

[0078] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol (100 mg, 348.05 μmol), 3-chloro-3-methylbut-1-yne, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a light yellow oily liquid (81 mg, 68.17%) was obtained. 1H NMR (300MHz, DMSO-d6) δ7.05(dd,J=7.5,2.0Hz,1H),6.88(d,J=2.0Hz,1H),6.84(d,J=7.5Hz,1H),6.58(s,2H),5.73(d,J= 12.4Hz,1H),5.58(s,1H),5.51-5.41(m,2H),5.19(d,J=12.4Hz,1H),5.07(s,1H),3.95(s,6H),3.84(s,3H),1.38(s,3H). 13 C NMR(100MHz,DMSO-d6)δ164.57,150.38,150.00,138.60,137.70,135.43,133.20,1 24.52,115.75,115.02,113.06,112.99,101.89,78.06,56.50,55.14,21.87.ESI-MS m / z:342.55[M+H] + .

[0079] Example 7

[0080] Compound 7: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[2-(prop-2-ynyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0081] Step 1: [2-(prop-2-alkynyloxy)phenyl]methanol

[0082]

[0083] 2-Hydroxymethylphenol (300 mg, 2.42 mmol), 3-bromopropyne (862 mg, 7.25 mmol), and anhydrous potassium carbonate (1.34 g, 9.67 mmol) were dissolved in acetone and the mixture was heated under reflux for 12 hours. After the reaction was complete, the solvent was removed under reduced pressure, and the mixture was extracted with ethyl acetate. The combined organic phases were washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated to obtain the crude product. The crude product was purified by column chromatography to obtain the title compound (315 mg, 80.37%). ESI-MS m / z: 163.31 [M+H] + .

[0084] Step 2: 1-(bromomethyl)-2-(prop-2-alkynyloxy)benzene

[0085]

[0086] [2-(propan-2-alkynyloxy)phenyl]methanol (315 mg, 1.94 mmol) was dissolved in dichloromethane and placed in an ice bath. Phosphorus tribromide (365 μL, 3.88 mmol) was added dropwise. After reacting at 0 °C for 1 hour, the reaction was monitored by TLC. After the reaction was complete, the reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, washed with saturated brine, and dried over anhydrous Na₂SO₄. The solvent was removed by distillation under reduced pressure to obtain a yellow oily crude product. The crude product was purified by column chromatography to obtain the product (281 mg, 64.28%). ESI-MS m / z: 224.71 [M+H] + .

[0087] Step 3: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[2-(prop-2-alkynyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0088] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol, 1-(bromomethyl)-2-(prop-2-ynyloxy)benzene, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a white solid (98 mg, 65.26%) was obtained. 1 H NMR(300MHz,DMSO-d6)δ7.35(ddt,J=7.5,2.1,1.0Hz,1H),7.29–7.23(m,1H),7.09–7.01(m,2H),6.92–6.81(m,3H),6.58(s,2 H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.5Hz,1H),5.10(d,J=0.9Hz,2H),4.76(s,2H),3.95(s,6H),3.84(s,3H),2.68(s,1H). 13 CNMR(100MHz,DMSO-d6)δ164.57,162.05,150.58,150.47,138.60,135.75,133.22,133.20,131.44,128.15, 125.57,124.52,116.44,115.02,113.19,112.50,101.89,80.53,78.61,78.28,60.84,56.50,55.14.ESI-MS m / z:432.66[M+H] + .

[0089] Example 8

[0090] Compound 8: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[3-(prop-2-ynyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0091] Using 3-hydroxymethylphenol and 3-bromopropyne as raw materials, and following the procedure in Example 7, a white solid (84 mg, 55.93%) was obtained. 1 H NMR(300MHz,DMSO-d6)δ7.27(dtd,J=5.9,2.0,1.0Hz,1H),7.22(t,J=7.4Hz,1H ),7.06(dd,J=7.5,2.0Hz,1H),6.93(d,J=2.0Hz,1H),6.88–6.81(m,2H),6.70( tt,J=2.0,1.0Hz,1H),6.58(s,2H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.5Hz, 1H), 5.02 (t, J = 1.0Hz, 2H), 4.73 (s, 2H), 3.95 (s, 6H), 3.84 (s, 3H), 2.68 (s, 1H). 13 C NMR(100MHz,DMSO-d6)δ164.57,158.06,150.95,150.42,138.60,136.67,135.75,133.20,131.10,125.98, 124.52,118.22,115.70,115.02,113.19,111.91,101.89,80.74,80.07,78.28,60.45,56.50,55.14.ESI-MS m / z:432.35[M+H] + .

[0092] Example 9

[0093] Compound 9: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[4-(prop-2-alkynyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0094] Using 4-hydroxymethylphenol and 3-bromopropyne as raw materials, and following the procedure in Example 7, a white solid (109 mg, 72.58%) was obtained. 1H NMR (300MHz, DMSO-d6) δ7.29–7.22(m,2H),7.06(dd,J=7.5,2.0Hz,1H),6.93(d,J=2.0Hz,1H),6.86–6.80(m,3H),6.58(s,2H ),5.73(d,J=12.4Hz,1H),5.48(d,J=12.5Hz,1H),5.11(t,J=1.0Hz,2H),4.68(s,2H),3.95(s,6H),3.84(s,3H),2.68(s,1H). 13 CNMR(100MHz,DMSO-d6)δ164.57,161.59,151.45,150.27,138.60,135.75,134.34,133.20,133.08, 124.52,117.02,115.02,113.19,111.91,101.89,81.20,80.74,78.28,59.69,56.50,55.14.ESI-MS m / z:432.61[M+H] + .

[0095] Example 10

[0096] Compound 10: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[2-(prop-2-enyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0097] Using 2-hydroxymethylphenol and 3-bromopropene as raw materials, and following the procedure in Example 7, a white solid (84 mg, 55.67%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ7.36 (ddt, J=7.6, 2.0, 1.0Hz, 1H), 7.23 (td, J=7.4, 2.0Hz, 1H ),7.09–7.01(m,2H),6.90(d,J=2.0Hz,1H),6.83(dd,J=7.4,1.9Hz,2H),6.58(s,2H), 5.99(s,1H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.5Hz,1H),5.38(d,J=12.4Hz,1H), 5.13(d,J=12.5Hz,1H),5.10(d,J=0.9Hz,2H),4.51(s,2H),3.95(s,6H),3.84(s,3H). 13C NMR(100MHz,DMSO-d6)δ164.57,162.04,150.58,150.47,138.60,135.75,133.64,133.20,133.17,130.99,1 27.34,125.03,124.52,118.84,115.02,114.23,113.19,112.50,101.89,78.13,75.89,56.50,55.14.ESI-MS m / z:434.29[M+H] + .

[0098] Example 11

[0099] Compound 11: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[3-(prop-2-enyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0100] Using 3-hydroxymethylphenol and 3-bromopropene as raw materials, and following the procedure in Example 7, a white solid (79 mg, 52.36%) was obtained. 1 H NMR(300MHz, DMSO-d6)δ7.30–7.19(m,2H),7.06(dd,J=7.5,2.0Hz,1H),6.93(d,J=2.0Hz, 1H),6.88(dt,J=7.5,2.1Hz,1H),6.83(d,J=7.5Hz,1H),6.77(tt,J=2.1,0.9Hz,1H),6.58 (s,2H),5.99(s,1H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.5Hz,1H),5.38(d,J=12.4Hz,1 H),5.13(d,J=12.5Hz,1H),5.02(t,J=1.0Hz,2H),4.50(s,2H),3.95(s,6H),3.84(s,3H). 13 C NMR(100MHz,DMSO-d6)δ164.57,158.12,150.95,150.42,138.60,136.14,135.75,133.20,133.16,131.04,1 25.98,124.52,118.84,118.06,116.48,115.02,113.19,111.91,101.89,80.07,75.71,56.50,55.19.ESI-MS m / z:434.35[M+H] + .

[0101] Example 12

[0102] Compound 12: 2,6-Dimethoxy-4-{1-[4-methoxy-3-({[4-(prop-2-enyloxy)phenyl]methyl}oxy)phenyl]vinyl}pyridine

[0103] Using 4-hydroxymethylphenol and 3-bromopropene as raw materials, and following the procedure in Example 7, a white solid (91 mg, 60.31%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ7.27(p,J=1.5Hz,1H),7.25(q,J=1.3Hz,1H),7.06(dd,J=7.5,2.0Hz,1H),6.93(d,J=2.0Hz,1H),6.85–6.83(m,3H),6.58(s,2H ),5.99(s,1H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.5Hz,1H),5.38(dd,J= 12.4,0.0Hz,1H),5.13–5.11(m,3H),4.49(s,2H),3.95(s,6H),3.84(s,3H). 13 C NMR(100MHz,DMSO-d6)δ164.57,162.96,151.45,150.27,138.60,135.75,134.34,133.20,133.16,1 33.06,124.52,118.84,116.63,115.02,113.19,111.91,101.89,81.20,74.53,56.50,55.14.ESI-MS m / z:340.25[M+H] + .

[0104] Example 13

[0105] Compound 13: 2,6-Dimethoxy-4-[1-(4-methoxy-3-{[(4-methoxyphenyl)methyl]oxy}phenyl)vinyl]pyridine

[0106] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol, p-methoxybenzyl bromide, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a white solid (99 mg, 69.81%) was obtained. 1H NMR (300MHz, DMSO-d6) δ7.30–7.27(m,3H),7.06(dd,J=7.5,2.0Hz,1H),6.93(d,J=2.0Hz,1H),6.90–6.86(m,2H),6.83(d,J=7.5H z,1H),6.58(s,2H),5.73(d,J=12.4Hz,1H),5.48(d,J=12.4Hz,1H),5.11(t,J=1.0Hz,3H),3.95(s,6H),3.84(s,3H),3.79(s,3H). 13 C NMR(100MHz,DMSO-d6)δδ164.57,162.15,151.45,150.27,138.60,135.75,134.34,133.20, 132.87,124.52,115.64,115.02,113.19,111.91,101.89,81.20,56.50,56.03,55.1.ESI-MS m / z:408.19[M+H] + .

[0107] Example 14

[0108] Compound 14: 2,6-Dimethoxy-4-[1-(4-methoxy-3-{[(4-methylphenyl)methyl]oxy}phenyl)vinyl]pyridine

[0109] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol, p-methylbenzyl bromide, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a white solid (94 mg, 68.99%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ7.28–7.22(m,2H),7.15–7.04(m,3H),6.93(d,J=2.0Hz,1H),6.83(d,J=7.5Hz,1H),6.58(s ,2H),5.73(d,J=12.5Hz,1H),5.48(d,J=12.5Hz,1H),5.10(t,J=1.0Hz,2H),3.95(s,6H),3.84(s,3H),2.37(s,3H). 13C NMR(100MHz,DMSO-d6)δ164.57,151.45,150.23,141.29,138.89,138.60,135.75,133.20,1 31.29,130.45,124.52,115.02,113.19,111.91,101.89,81.68,56.50,55.14,22.32.ESI-MS m / z:317.27[M+H] + .

[0110] Example 15

[0111] Compound 15: 2,6-Dimethoxy-4-[1-(4-methoxy-3-{[(4-nitrophenyl)methyl]oxy}phenyl)vinyl]pyridine

[0112] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol, p-nitrobenzyl bromide, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a yellow powdery solid (62 mg, 42.17%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ8.12(d,J=1.6Hz,1H),8.10(d,J=1.6Hz,1H),7.61(p,J=1.4Hz,1H),7.59(q,J=1.2Hz,1H),7.06(dd,J=7.5,2.0Hz,1H),6.93( d,J=2.0Hz,1H),6.83(d,J=7.5Hz,1H),6.58(s,2H),5.73(d,J=12.4Hz,1H ),5.48(d,J=12.5Hz,1H),5.11(d,J=0.9Hz,2H),3.95(s,6H),3.84(s,3H). 13 CNMR(100MHz,DMSO-d6)δ164.57,151.45,150.45,149.29,142.24,138.60,135.75,133.2 0,131.03,125.27,124.52,115.02,113.19,111.91,101.89,81.39,56.50,55.14.ESI-MS m / z:423.51[M+H] + .

[0113] Example 16

[0114] Compound 16: 4-[({5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenyl}oxy)methyl]benzene-1-carboxynitrile

[0115] Using 5-[1-(2,6-dimethoxypyridin-4-yl)vinyl]-2-methoxyphenol, 4-(bromomethyl)benzene-1-carboxynitrile, and anhydrous potassium carbonate as raw materials, and following step 10 of Example 1, a white solid (101 mg, 72.11%) was obtained. 1 H NMR (300MHz, DMSO-d6) δ7.60(d,J=1.6Hz,1H),7.59(d,J=1.6Hz,1H),7.41(q,J=1.3Hz,1H),7.40(dt,J=2.0,1.0Hz,1H),7.06(dd,J=7.5,2.0Hz,1H),6. 93(d,J=2.0Hz,1H),6.83(d,J=7.5Hz,1H),6.58(s,2H),5.73(d,J=12.4Hz,1 H), 5.48 (d, J = 12.4Hz, 1H), 5.12 (t, J = 1.0Hz, 2H), 3.95 (s, 6H), 3.84 (s, 3H). 13 CNMR(100MHz,DMSO-d6)δ164.57,151.45,150.27,138.60,137.65,135.75,133.20,133.05,1 31.52,124.52,121.03,115.22,115.02,113.19,111.91,101.89,80.22,56.50,55.14.ESI-MS m / z:403.16[M+H] + .

[0116] Example 17

[0117] Tablets prepared from the compounds of this invention:

[0118] tablet

[0119]

[0120] Take the above formula and prepare it into tablets using conventional methods.

[0121] Example 18

[0122] Antiproliferation experiments were conducted on the compounds of this invention:

[0123] Experimental methods: Cell digestion, counting, and preparation of a solution with a concentration of 3.5 × 10⁻⁶ cells. 4100 μl of cell suspension per well was added to each well of a 96-well plate. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 24 hours. The drug was diluted to the required concentration with complete culture medium, and 100 μL of the corresponding drug-containing culture medium was added to each well. A negative control group was set up. The 96-well plate was incubated at 37°C in a 5% CO2 incubator for 72 hours.

[0124] CCK-8 method:

[0125] 1) The 96-well plate was stained with CCK-8 at λ = 450 nm, and the OD value was measured;

[0126] 2) Add 10 μL CCK-8 to each well and continue incubation in an incubator for 2-3 hours;

[0127] 3) Shake gently for 10 minutes to mix; read the OD value of each well using a λ=450nm microplate reader and calculate the inhibition rate.

[0128]

[0129] Table 1. IC50 values ​​of the antiproliferative activity of the compounds of the present invention against human cancer cell lines and normal human cell lines. 50 (nM)

[0130] Example HCT-8 K562 HepG2 MDA-MB-231 HFL-1 Mother medicine 86 28 39 84 792 1 D C C E F 2 C C D F F 3 A C A B F 4 E D D F D 5 C D C D F 6 B D D E E 7 D A B C E 8 C E B D F 9 A A B A E 10 C A A C C 11 C C D B E 12 A C D F A 13 B B D E F 14 D C D E E 15 B A C A B 16 B B B A A

[0131] Note: Grade A indicates 1μM < IC 50 ≤3μM, Grade B indicates 3μM < IC 50 ≤6μM, Grade C indicates 6μM < IC 50 ≤10μM, Grade D indicates 10μM < IC 50 ≤20μM, Class E indicates 20μM < IC 50 ≤30μM, F grade indicates IC 50 >30μM.

[0132] The results are shown in Table 1. For tumor cell lines, the IC50 of the compounds in this invention... 50 When the concentration was greater than 1 μM, its cytotoxicity was significantly reduced, IC50 50 The lowest level has exceeded 10 times. For normal cell lines, its safety has also been improved to some extent, with IC50... 50 The results generally exceeded 2 times, with most reaching 10 times and a few exceeding 20 times. These results indicate that the small molecule bio-orthogonal prodrug of this invention has fewer toxic side effects, can effectively increase the therapeutic window, and improve safety.

[0133] Example 19

[0134] The growth inhibition experiment of K562 cells after palladium resin catalytic release of the compound of the present invention was carried out:

[0135] Experimental Methods: The compound was prepared as a 10 mM stock solution according to the relevant requirements, and then diluted to the final concentration for subsequent experiments. Different concentrations of the bio-orthogonal prodrug compound and different doses of palladium resin were co-incubated with K562 cells in 96-well plates for 72 h. The inhibition rate was then measured and calculated using the CCK-8 assay.

[0136] Table 2. Experimental results of the growth inhibition of K562 cells by the compounds of the present invention after being catalytically released by palladium resin.

[0137]

[0138]

[0139] As shown in Table 2, some of the bio-orthogonal prodrug compounds of the present invention recovered their tumor cell killing effect after co-incubation with palladium resin. This indicates that these bio-orthogonal prodrug compounds can release active ingredients and effectively inhibit the growth of tumor cells under the activation of palladium resin, thus achieving a highly effective and low-toxicity therapeutic effect.

[0140] Example 20

[0141] In vivo safety evaluation experiments were conducted on the compounds of this invention:

[0142] Experimental Methods: Twenty-one 4-week-old female ICR mice were provided by Shanghai Silex Laboratory Animal Co., Ltd. Animals were randomly divided into three groups of three: a blank control group, a mother drug group, and a prodrug group. The blank control group was injected with DMF:Tween 80:9% saline solution = 10:2:88 (V:V:V). The mother drug group and the prodrug group received intravenous administration of 40 mg / kg, 80 mg / kg, and 160 mg / kg of DMF:Tween 80:9% saline solution = 10:2:88 (V:V:V) at the same dosage. Administration continued for 21 days, and body weight was measured every two days. *P<0.05;

[0143] **P<0.01;***P<0.001 compared with the blank group.

[0144] Depend on Figure 2 It can be seen that some compounds in this invention do not significantly affect the body weight of mice at concentrations above the effective dose. In contrast, the original drug significantly reduces mouse body weight at therapeutic concentrations. Therefore, compared to the original drug, the bioorthogonal prodrug molecule in this invention has better safety in vivo.

[0145] Example 21

[0146] In vivo antitumor experiments were conducted on the compounds of this invention:

[0147] Experimental methods: Twenty-four female ICR mice, aged 4 weeks, were provided by Shanghai Silex Laboratory Animal Co., Ltd. Suspensions of cultured mouse hepatocellular carcinoma cells H22 were collected at a concentration of 1 x 10⁻⁶. 7 The xenograft was administered subcutaneously to the right forelimb axilla of mice at a dose of 0.1 ml per mouse. The diameter of the xenograft was measured using calipers; tumors were considered xenografts when they reached 100 mm in size. 3 Animals were randomly divided into groups of 6 each: a blank control group, a palladium resin control group, a mother drug group, and a prodrug + palladium resin group. The blank control group received an injection of DMF:Tween 80:9% saline solution in a ratio of 10:2:88 (V:V:V). Both the palladium resin control group and the prodrug + palladium resin group received an intratumoral injection of Pd resin (1 mg in 50 μL of PBS) before administration. The blank control group and the palladium resin control group received no administration. The mother drug group and the prodrug + palladium resin group received the drug via tail vein at a dose of 20 mg / kg, with DMF:Tween 80:9% saline solution as the solvent.

[0148] The ratio of drug concentration to drug concentration was 10:2:88 (V:V:V), and the drugs were administered continuously for 21 days. Mice were sacrificed after 21 days of administration, and tumor masses were surgically removed and weighed. The tumor growth inhibition rate (%) was calculated, and the results were analyzed using SPSS 17.0. Statistical analysis between groups was performed using a t-test, and the calculation formula is as follows:

[0149]

[0150] Table 3. In vivo antitumor activity of the compounds of this invention

[0151]

[0152] As shown in Table 3, the prodrug compound of the present invention can produce a higher concentration of the original drug molecule at the same dose, thus having a stronger effect on tumor growth in vivo.

[0153] Therefore, the vinylpyridine prodrug compound of the present invention specifically activates the prodrug through a palladium resin catalytic reaction to target tubulin and inhibit microtubule polymerization in tumor cells. The active ingredient is released upon activation by the palladium resin, effectively inhibiting tumor cell growth. It exhibits high safety and minimal toxicity, effectively increasing the therapeutic window and achieving highly effective and low-toxicity treatment, thus improving the selectivity and precision of treatment. This invention not only reduces toxicity to normal cells but also achieves highly effective and precise treatment at the tumor site, demonstrating broad application potential.

Claims

1. A vinylpyridine prodrug compound, characterized in that, The structure of the compound is shown in Formula I: ； Selected from any one of the following compounds 1 to 16: ； 。 2. The compound according to claim 1, characterized in that, The compounds also include pharmaceutically acceptable salts.

3. A method for preparing the vinylpyridine prodrug compound of claim 1, characterized in that, Includes the following steps: ； Using 2,6-dichloroisonicotinic acid as a raw material, a condensing agent was added to condense it with N,O-dimethylhydroxylamine hydrochloride to obtain Weinreb amide, which was then reduced to ketone iii. After protecting the carbonyl group with 2-bromoethanol, the chlorine atom was substituted with different groups to obtain intermediate vi. After deprotection under acidic conditions, intermediate vii was obtained. Intermediate vii was reacted with p-toluenesulfonyl hydrazine to obtain intermediate viiii. Different substituted halogenated phenols were protected with tert-butyldimethylchlorosilane to obtain intermediate x. Intermediate viiii and intermediate x were subjected to the Heck reaction, and after deprotection, the original drug xii was obtained. Finally, the hydroxyl group was nucleophilically substituted with different halogenated hydrocarbons to obtain vinylpyridine prodrugs as shown in Formula I.

4. A pharmaceutical composition, characterized in that, It comprises the compound of formula I of claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

5. The use of a vinylpyridine prodrug compound of claim 1 or a pharmaceutical composition of claim 4 in the preparation of a microtubule inhibitor.

6. The use of a vinylpyridine prodrug compound of claim 1 or a pharmaceutical composition of claim 4 in the preparation of an antitumor drug.

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