CN109574871B - Acetaminoazobenzene derivative and preparation and application thereof - Google Patents

Acetaminoazobenzene derivative and preparation and application thereof Download PDF

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CN109574871B
CN109574871B CN201811403987.6A CN201811403987A CN109574871B CN 109574871 B CN109574871 B CN 109574871B CN 201811403987 A CN201811403987 A CN 201811403987A CN 109574871 B CN109574871 B CN 109574871B
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朱明彦
东多凌
葛志华
杨卓进
梁婧诗
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Shanghai Jiaotong University
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
    • C07C245/02—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides
    • C07C245/06—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings
    • C07C245/08—Azo compounds, i.e. compounds having the free valencies of —N=N— groups attached to different atoms, e.g. diazohydroxides with nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings with the two nitrogen atoms of azo groups bound to carbon atoms of six-membered aromatic rings, e.g. azobenzene
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Abstract

The invention relates to an acetamido azobenzene derivative, a preparation method and an application thereof, wherein the derivative has a general formula
Figure DDA0001876901480000011
And
Figure DDA0001876901480000012
the structure of (1). The acetamido azobenzene derivative has the characteristics of generating structural change and activity change under the illumination condition, presents the structure in the structural formula I in a dark environment, and presents the structure in the structural formula II after being illuminated by ultraviolet light; structure II has K-Ras (G12C) protein inhibitory activity, and structure I is less active. The compounds can be used as selective drugs for treating tumors related to K-Ras gene mutation, such as rectal cancer, lung cancer, prostate cancer and the like.

Description

Acetaminoazobenzene derivative and preparation and application thereof
Technical Field
The invention relates to the technical field of medicinal chemistry, in particular to an acetamido azobenzene derivative and preparation and application thereof.
Background
Mutations in the Ras gene are associated with many cancers, and the cancer-associated Ras genes are essentially single-base mutations, with 99% of mutations occurring in glycine at positions 12 and 13 and glutamate at position 61 (Nature reviews cancer, 2011,11,761 774). The distribution of Ras mutations is not consistent among cancers, K-Ras is the gene with the highest mutation frequency, reaching 86%, and mutations in K-Ras are most common in colorectal cancer, lung cancer (mainly non-small cell lung cancer), and pancreatic cancer.
K-Ras protein has been considered "druggable" for many years. New technologies in drug discovery have facilitated new therapeutic approaches to the RAS (McCormick, 2015). At present, the research on inhibitors of Ras protein is mainly focused on the following aspects: prevent the formation of Ras-GTP, inhibit the action of Ras-effector protein, reduce the localization of Ras and inhibit the activity of GTP enzyme.
The difficulty in Ras protein inhibitor studies is that the affinity between Ras and GDP (guanosine diphosphate) and GTP (guanosine triphosphate) is strong (reaching the picomolar level) (Nature reviews Molecular cell biology,2012,13,39-51.) and therefore competitive inhibitors acting directly on the GTP/GDP binding pocket hardly attenuate the binding of Ras protein to GTP. The Shokat group reported an inhibitor with electrophilic groups (such as vinylsulfonyl, acryloyl) and the eutectic results showed that an allosteric binding pocket not previously found could lead to a change in the structure of Switch I and Switch II in Ras, which could attenuate the binding of K-Ras (G12C) and GTP (Nature,2013,503(7477): 548). Janes also reports a class of compounds (Cell,2018,172,578-589) on the basis of Janes, has better effect of inhibiting K-Ras protein, and obtains better results in vivo experiments of mice. These inhibitors, although already selective for the mutant K-Ras (G12C) protein and not for the wild-type K-Ras protein, do not feature control of K-Ras protein inhibition under different conditions.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an acetamido-azobenzene derivative, and a preparation method and an application thereof.
The purpose of the invention can be realized by the following technical scheme: an acetamidoazobenzene derivative, the structural formula of which is shown as formula I or formula II:
Figure BDA0001876901460000021
the structure has a hydrophobic benzene ring structure, is combined with a protein allosteric pocket, has an electrophilic group, can be covalently combined with cysteine at the 12 th position of Ras mutein and inhibit protein activation, and can further control the inhibitory activity through illumination after being covalently combined with protein, thereby realizing selectivity aiming at specific tissues or regions.
Preferably, the substituent R on the ring A in the formula1One selected from H, halogen, hydroxyl, nitryl, C1-C10 or alkoxy; substituent R on ring A2One selected from H or halogen; substituent R on ring A3One selected from H or halogen; substituent R on ring A4One selected from H, halogen or C1-C10; n is an integer of 1-3, and X is selected from halogen, alkenyl or maleimide; the C1-C10 are alkyl with 1-10 carbon atoms, and include straight-chain alkyl, branched-chain alkyl or cyclic alkyl, such as methyl, ethyl, isopropyl and cyclopropyl.
More preferably, R1Is halogen, R2Is H, R3Is halogen, R4Is H, n is 1 and X is halogen.
Most preferably, R1Is chlorine, R2Is H, R3Is chlorine, R4Is H, n is 1 and X is chlorine.
Preferably, the derivative comprises one or a mixture of an isotopic compound, a racemate, an optically active isomer, a polymorphism and a pharmaceutically acceptable salt of the substance shown in the formula I or the formula II.
The preparation method of the acetamidoazobenzene derivative is characterized by comprising the following steps:
(1) respectively reacting potassium hydrogen persulfate complex salt with
Figure BDA0001876901460000022
Mixing, stirring and reacting to obtain
Figure BDA0001876901460000031
(2) Subjecting the product obtained in step (1)
Figure BDA0001876901460000032
Dissolving in glacial acetic acid, adding
Figure BDA0001876901460000033
Stirring until the reaction is finished, and filtering to obtain
Figure BDA0001876901460000034
Or, the product obtained in step (1)
Figure BDA0001876901460000035
Dissolving in glacial acetic acid, adding
Figure BDA0001876901460000036
Stirring until the reaction is finished, and filtering to obtain
Figure BDA0001876901460000037
(3) Will be provided with
Figure BDA0001876901460000038
Dissolving in methanol, adding hydrochloric acid, heating under reflux, adjusting pH with 3M sodium hydroxide water solution, adding saturated sodium bicarbonate solution dropwise until precipitate is generated, separating, and drying to obtain
Figure BDA0001876901460000039
(4) Will be provided with
Figure BDA00018769014600000310
Dissolved in dichloromethane and then added
Figure BDA00018769014600000311
Stirring until the reaction is finished, and purifying by column chromatographyTo obtain the product
Figure BDA0001876901460000041
Figure BDA0001876901460000042
Preferably, the substituent R on the A ring present in the reaction step1One selected from H, halogen, hydroxyl, nitryl, C1-C10 or alkoxy; substituent R on ring A2One selected from H or halogen; substituent R on ring A3One selected from H or halogen; substituent R on ring A4One selected from H, halogen or C1-C10; n is an integer of 1-3, and X is selected from halogen, alkenyl or maleimide; the C1-C10 is an alkyl group having 1-10 carbon atoms.
Preferably, the oxone complex salt in step (1) is reacted with
Figure BDA0001876901460000043
Figure BDA0001876901460000044
In a molar ratio of 1: (1.2-2), the stirring speed is 500-1000 rpm, the reaction temperature is 20-30 ℃, and the reaction time is 2-12 hours.
Preferably, in the step (2),
Figure BDA0001876901460000045
and
Figure BDA0001876901460000046
the molar ratio of (1.5-3): 1, the
Figure BDA0001876901460000047
And
Figure BDA0001876901460000048
the molar ratio of (1.5-3): 1, the stirring rate is 500-1000 rpm, and the reaction is carried outThe temperature is 20-30 ℃, and the reaction time is 12-15 hours.
Preferably, in the step (3), the volume ratio of the methanol to the hydrochloric acid is 1: (0.5 to 2) of,
Figure BDA0001876901460000051
the molar ratio of HCl in hydrochloric acid is 1: (50-100), the temperature of the reflux reaction is 80-90 ℃, the time is 10-12 hours, the pH value of the reaction system is adjusted to 8-9 by 3M sodium hydroxide aqueous solution, and the drying temperature is 55-65 ℃.
Preferably, in the step (4),
Figure BDA0001876901460000052
in a molar ratio of 1: (2-3), the reaction temperature is 20-30 ℃, the reaction time is 2-12 hours, and silica gel column chromatography is adopted for purification.
The application of the acetamidoazobenzene derivative is used for preparing a medicament for treating tumors related to K-Ras gene mutation, wherein the tumors related to K-Ras gene mutation comprise one of rectal cancer, lung cancer or prostate cancer.
Compared with the prior art, the beneficial effects of the invention are embodied in the following aspects:
(1) the derivative has the characteristics of generating structural change and activity change under the illumination condition, and shows the structure in the structural formula I in the dark environment, and shows the structure in the structural formula II after being illuminated by ultraviolet light; structure II has K-Ras (G12C) protein inhibitory activity, structure I is less active;
(2) can be used as a selective drug for treating tumors related to K-Ras gene mutation, such as rectal cancer, lung cancer, prostate cancer and the like.
(3) The preparation method is simple and convenient, and is easy to separate and purify.
Drawings
FIG. 1 is the results of the experiment for nucleotide exchange of cis-trans structure derivatives for K-Ras (G12C) protein, prepared in example 2;
FIG. 2 shows the results of the tumor cell inhibitory activity of the trans-structure derivative prepared in example 1;
FIG. 3 shows the results of the tumor cell inhibitory activity of the cis-structure derivative prepared in example 1;
FIG. 4 shows the results of the tumor cell inhibitory activity of the cis-structure derivative prepared in example 2;
FIG. 5 shows the results of the tumor cell inhibitory activity of the trans-structure derivative prepared in example 2;
Detailed Description
The following is a detailed description of the embodiments of the present invention, which is implemented on the premise of the technical solution of the present invention, and a detailed implementation manner and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.
Example 1
(E) The preparation scheme of (E) -2-chloro-N- (4- ((4-chlorophenyl) azo) phenyl) acetamide is as follows:
Figure BDA0001876901460000061
reaction reagents and conditions: (a) k2CO3,H2O,10min；(b)AcOH,DCM,r.t.overnight(c)1) HCl,MeOH,reflux,9h；2)NaOH；(d)chloroacetyl chloride,dry DCM,DIPEA,r.t., overnight.
Specifically, the method comprises the following steps:
(1) intermediate 1[ N- (4-nitrosophenyl) acetamide ]
Figure BDA0001876901460000062
Dissolving potassium hydrogen persulfate composite salt in 50ml of water, adding potassium carbonate to adjust the pH value to be weakly acidic, then quickly adding the potassium hydrogen persulfate composite salt into the 4-aminoacetanilide dissolved in the water, quickly generating gray-green foam and gray-green precipitate in the solution, and stopping the reaction for 10 minutes after the gray-green foam disappears and the precipitate settles. Filtering to obtain a gray-green precipitate, dissolving with hot ethanol while the precipitate is hot, filtering to remove impurities to obtain a green solution, and spin-drying ethanol to obtain the target product, namely an intermediate 1 (green solid). Directly putting into the next reaction.
(2) Intermediate 2[ (E) -N- (4- ((4-chlorophenyl) azo) phenyl) acetamide ]
Figure BDA0001876901460000063
Dissolving the nitroso compound obtained in the previous step in glacial acetic acid, and adding parachloroaniline into the system. Stir at room temperature overnight. After the reaction was completed, a cake-like solid was separated, filtered and washed with water, the solid was dissolved in dichloromethane, and the organic phase was washed with a saturated sodium bicarbonate solution and a saturated brine, dried over anhydrous sodium sulfate, and concentrated to give intermediate 2 (yellow solid, 1.2428g, 50.02%).1H NMR(400MHz,CDCl3)δ2.229 (s，3H),7.473(d，J＝2.2Hz，2H),7.677(d，J＝2.1Hz,2H)，7.845(d，J＝2.2Hz， 2H),7.91(d，J＝2.2Hz，2H)ppm。
(3) And intermediate 3[ (E) -4- ((4-chlorophenyl) azo) aniline ]
Figure BDA0001876901460000071
To methanol was added reactant 2 and 3M hydrochloric acid, and the mixture was refluxed for 9 hours. After the reaction was completed, 3M sodium hydroxide solution was added dropwise to adjust pH 8 to 9, and then saturated sodium bicarbonate solution was slowly added until a yellow precipitate was generated. The yellow precipitate obtained upon isolation was dried below 60 ℃ to give intermediate 3 (yellow solid, 134.3mg, 52.9%)1H NMR(400MHz,CDCl3)δ6.741(d，J＝2.2Hz，2H),7.441(d，J＝2.2Hz，2H),7.795 (dd，J1＝2.2Hz,J2＝2.2Hz，4H)ppm。
(4) And a compound 4[ (E) -2-chloro-N- (4- ((4-chlorophenyl) azo) phenyl) acetamide ]
Figure BDA0001876901460000072
The compound 3 obtained in the previous reaction step was dissolved in redistilled dichloromethane, and chloroacetyl chloride was added to the reaction system and stirred at room temperature overnight. After completion of the reaction, the reaction mixture was eluted with dichloromethane and petroleum ether at a ratio of 2:1, and column chromatography was performed to obtain the objective compound 4 (yellow solid, 150.06mg, 44.92%).1H NMR(400 MHz,CDCl3)δ4.238(s，2H),7.483(d，J＝2.2Hz，2H)，7.739(d，J＝2.2Hz，1H),7.860 (d，J＝2.2Hz，2H)，7.948(d，J＝2.2Hz，2H)ppm。[M-H]–C14H10Cl2N3O, calculated value of Exact mass of target compound: 306.0201, observed value: 306.0204.
example 2
(E) The preparation scheme of (E) -2-chloro-N- (4- ((2, 4-dichlorophenyl) azo) phenyl) acetamide is as follows:
Figure BDA0001876901460000081
reaction reagents and conditions: (a) k2CO3,H2O,30min；(b)AcOH,DCM,r.t.overnight(c)1) HCl,MeOH,reflux,9h；2)NaOH；(d)chloroacetyl chloride,dry DCM,DIPEA,r.t., overnight.
Specifically, the method comprises the following steps:
(1) intermediate 5[2, 4-dichloro-1-nitrosobenzene ]
Figure BDA0001876901460000082
Dissolving 2, 4-dichloroaniline in dichloromethane, dissolving potassium hydrogen persulfate complex salt in water, mixing under nitrogen protection, and stirring at room temperature overnight. After the reaction was completed, the organic phase was washed with 1M hydrochloric acid (10mL) and saturated brine. Finally, the organic phase is dried over anhydrous sodium sulfate, the product intermediate 5 is concentrated and directly taken to the next reaction step.
(2) Intermediate 6[ (E) -N- (4- ((2, 4-dichlorophenyl) azo) phenyl) acetamide ]
Figure BDA0001876901460000083
Dissolving the nitroso compound obtained in the previous step in glacial acetic acid, and adding 4-aminoacetanilide into the system. Stir at room temperature overnight. After the reaction, the cake-like solid was separated, filtered and the solid was washed. The solid was dissolved in dichloromethane, washed with saturated sodium bicarbonate solution and saturated brine, the organic phase was dried over anhydrous sodium sulfate and concentrated to give the product intermediate 6 (yellow solid, 2.6532g, 43.2%).1H NMR(400MHz,CDCl3) δ2.238(s，3H),7.308(d，J＝0.6Hz，1H),7.569(d，J＝0.6Hz,1H)，7.677(d，J＝2.1 Hz，2H),7.679(s，1H),7.962(d，J＝2.2Hz，2H)ppm。
(3) And an intermediate 7[ (E) -4- ((2, 4-dichlorophenyl) azo) aniline ]
Figure BDA0001876901460000091
To methanol was added reactant 6 and 3M hydrochloric acid, and the mixture was heated under reflux for 9 hours. After the reaction, 3M sodium hydroxide solution is added dropwise to adjust the pH of the reaction solution to 8-9, and then saturated sodium bicarbonate solution is added until yellow precipitate is generated. The isolated yellow precipitate was dried below 60 ℃ to give intermediate 7 (yellow solid, 112.3mg, 64.92%).1H NMR(400MHz,CDCl3)δ6.738(d，J＝1.9Hz，2H),7.269(s，1H)，7.531(d，J ＝2.2Hz，1H),7.641(d，J＝2.2Hz，1H)，7.845(d，J＝2.1Hz，2H)ppm。
(4) And a compound 8[ (E) -2-chloro-N- (4- ((2, 4-dichlorophenyl) azo) phenyl) acetamide ]
Figure BDA0001876901460000092
Dissolving the compound obtained in the previous step in redistilled dichloromethane, then dripping chloroacetyl chloride into the reaction system, and stirring at room temperature overnight. After the reaction was completed, the reaction mixture was purified by silica gel chromatography and purified in dichloromethane to petroleum ether ═ 2:1 to yield the title compound 8 (yellow solid, 152.4mg, 57.5%).1H NMR(400 MHz,DMSO-d6)δ4.316(s,2H),7.562(dd,J1＝2.2Hz,J2＝2.2Hz，1H),7.683(d,J＝ 2.1Hz,1H),7.841(d,J＝2.1Hz,2H),7.895(d,J＝0.6Hz,1H),7.941(d,J＝2.1Hz, 2H),10.719(s,1H)ppm；HRMS(ESI):[M-H]–C14H10Cl3N3O, calculated value of Exact mass of target compound: 340.9889, observed value: 340.9886.
example 3
Figure BDA0001876901460000093
(E) Preparation of (E) -2-chloro-N- (4- ((3, 4-dichloro) azo) phenyl) acetamide (9)
A synthesis similar to that of example 2 was used, except that:
(a) the raw material amine in the step 1 adopts 3, 4-dichloroaniline
The final test results were as follows: 1H NMR (400MHz, DMSO-d6): Δ 10.71(s,1H),8.02(s,1H), 7.92(d, J ═ 7.6Hz,2H),7.85(s,1H),7.84(s,1H),7.82(d, J ═ 9.2Hz,2H),4.32(s,2H) ppm.HRMS (ESI): M + H]–C14H11Cl3N3O, calculated value of Exact mass of target compound: 341.9959, observed value: 341.9967.
example 4
Figure BDA0001876901460000101
(E) Preparation of (E) -2-chloro-N- (4- ((2,4, 5-trichlorophenyl) azo) phenyl) acetamide (10)
A synthesis similar to that of example 2 was used, except that:
(a) the raw material amine in the step 1 adopts 2,4, 5-trichloroaniline
The final test results were as follows: 1H NMR (600MHz, DMSO) δ 10.75(s,1H),8.11(s,1H), 7.95(d, J ═ 8.8Hz,2H),7.85(d, J ═ 8.8Hz,2H),7.80(s,1H),4.33(s,2H) ppm hrms (esi):[M+H]–C14H10Cl4N3O, calculated value of Exact mass of target compound: 375.9578, observed value: 375.9568.
example 5
Figure BDA0001876901460000102
(E) Preparation of (E) -2-chloro-N- (4- ((3-bromo-4-chlorophenyl) azo) phenyl) acetamide (11)
A synthesis similar to that of example 2 was used, except that:
(a) the amine raw material in the step 1 adopts 3-bromo-4-chloroaniline
The final test results were as follows: 1H NMR (600MHz, DMSO) δ 10.72(s,1H),8.17(s,1H), 7.95(d, J ═ 7.6Hz,2H),7.91(d, J ═ 8.5Hz,1H), 7.88-7.86 (M,1H),7.85(d, J ═ 8.4Hz, 2H),4.33(s,2H) ppm hrms (ESI): M + H: [ M + H ] M]–C14H11BrCl2N3O, calculated value of Exact mass of target compound: 385.9462, observed value: 385.9456.
example 6
Figure BDA0001876901460000103
(E) Preparation of (E) -2-chloro-N- (4- ((4-chloro-3-cyclopropylphenyl) azo) phenyl) acetamide (12)
A synthesis similar to that of example 2 was used, except that:
(a) the amine raw material in the step 1 adopts 3-cyclopropyl-4-chloroaniline
The final test results were as follows: 1H NMR (600MHz, CDCl3) δ 8.42(s,1H),7.96(dd, J ═ 11.7,5.3Hz,2H),7.76(d, J ═ 8.7Hz,2H),7.67(dt, J ═ 14.2,7.1Hz,1H),7.53(d, J ═ 1.7Hz,1H),7.50(d, J ═ 8.4Hz,1H), 2.37-2.22 (M,1H), 1.16-1.04 (M,2H), 0.90-0.79 (M,2H) ppm hrms (ESI): M + H: [ M + H ], (M,2H) ]]–C17H16Cl2N3O, calculated value of Exact mass of target compound: 348.0670, observed value: 348.0663.
third, biological activity test
(1) Incubation of Compounds with K-Ras (G12C) protein
For example 1 and example 2, K-Ras (FL, G12C) protein was prepared as a 50. mu.M solution of 150. mu.L protein in incubation buffer, to which solutions of cis-and trans-structured compounds in DMSO were added, respectively, to a final concentration of 500. mu.M (the amount of DMSO was 2% (v/v)), and incubated at 4 ℃ for 24 h. 80 μ L of the protein solution was put into a 1.5mL EP tube, and 20 μ L of 5 XSDS was added. Heating at 90 deg.C for 5min, running SDS-PAGE gel. The solution was stained with Coomassie brilliant blue and then decolorized (decolorized solution: 30% absolute ethanol, 10% glacial acetic acid, 60% distilled water). Finding out a protein band according to a protein Maker, cutting gel, carrying out Trypsin cracking, carrying out Zip-desalination, and then feeding a sample to carry out a protein mass spectrum experiment.
Figure BDA0001876901460000111
Figure BDA0001876901460000121
(2) Nucleotide exchange assay
(a) Binding of Compounds to proteins
The protein was added to a buffer solution (20mM HEPES, pH7.5,150mM NaCl,1mM EDTA) to a final concentration of 20. mu.M, and the compound stock solution (20mM) was placed under an ultraviolet lamp at a wavelength of 400nm for 4min and then added to the system in the dark to a final concentration of 400. mu.M in a final volume of 300. mu.L, and incubated at 4 ℃ for 24 h.
(b) Binding of proteins to mant-dGDP
After incubation with compound was complete, 40. mu.L of EDTA buffer (20mM HEPES, pH7.5,150mM NaCl,15mM EDTA, 1mM DTT) was added to the protein solution to give a final EDTA concentration of 2.5 mM; at the same time, 14.4. mu.L of mant-dGDP (initial concentration 10mM) was added to the protein solution, at this time, mant-dGDP concentration was 400. mu.M, and incubated at room temperature for 1 h. Then 2. mu.L MgCl was added2Final concentration 10mM, and incubation at 4 ℃ for 1h, finally removing unbound mant-dGDP with NAP-5 column and measuring protein concentration by Bradford method.
(c) Titration
Using protein reaction buffer (20mM HEPES, pH7.5,150mM NaCl,1mM MgCl)21mM DTT) the protein was diluted to 1.5. mu.M. To a black-backed plate, 10 μ L of protein solution was added per well for 16 parallel samples, two for each parallel sample concentration. After the completion of the dropwise addition, the solution was shaken slightly to allow the protein solution suspended on the walls of the wells to flow into the bottoms of the wells, and the fluorescence value of the protein in the wells was measured with a microplate reader (excitation wavelength: 360 nm; emission wavelength: 440 nm). After the measurement, 5. mu.L of GDP or GTP solution (dissolved in EDTA buffer: 20mM HEPES, pH7.5,150mM NaCl,15mM EDTA, 1mM DTT) at various concentrations was added to the protein solution sample by a shotgun. The GDP/GTP concentrations were: 3000. mu.M, 1200. mu.M, 480.0. mu.M, 192.0. mu.M, 76.80. mu.M, 30.72. mu.M, 12.29. mu.M, 4.915. mu.M, 1.966. mu.M, 0.786. mu.M, 0.314. mu.M, 0.126. mu.M, 0.050. mu.M, 0.020. mu.M, 0.008. mu.M, 0.003. mu.M. When in use, the composition is melted fully and shaken to be mixed uniformly.
The experiment comprises an ultraviolet irradiation group and a non-irradiation group, wherein the ultraviolet irradiation group firstly irradiates for 4min by an ultraviolet lamp with the wavelength of 400nm, then nucleotide (GDP and GTP solution) is added, the distance between a 384-hole plate and a light source is about 5cm, and the experiment is incubated for 2h on a shaking table at room temperature in a dark place. The non-irradiation group does not need illumination, and the nucleotide is directly added dropwise and incubated for 2h at room temperature in a dark place. After the incubation, the fluorescence intensity (excitation wavelength: 360 nm; emission wavelength: 440nm) was measured with a microplate reader. Comparison of fluorescence before and after addition of GDP or GTP solution and calculation of IC with GraphPad Prism 5 software50The value of (c). The fluorescence values before and after addition of GDP or GTP solution are calculated by the formula:
Fn＝Flun is after/Flun is before，Rn＝(Fn-Fmin)/(Fmax-Fmin)
(Flun is after、Flun is beforeBoth are the average of fluorescence values of two multiple wells, resulting in FnI.e. normalized values. According to RnFor concentration (logC)n) Sigmoidal curve was made and nucleotide IC50 was calculated).
The experimental results of nucleotide exchange after cis-trans incubation of the compound prepared in example 2 are shown in FIG. 1, wherein 8-trans represents the experimental results of trans structure, and 8-cis represents the experimental results of trans structure completely changed into cis structure after being irradiated by 400nm ultraviolet light. As can be seen from the figure, after incubation of the photoswitch compound 8 with K-Ras (G12C), part of the trans compound 8 covalently binds to the cysteine at position 12 of the K-Ras mutein into the allosteric pocket. After ultraviolet irradiation, trans-compound 8 and cis-compound 8 are allosterized, and the protein structure is changed accordingly. The experiment shows that the GTP IC50 value is increased, namely the affinity of K-Ras (G12C) and GTP is weakened.
(3) Cell experiments
1. Cell proliferation inhibition assay
(1) And (5) culturing the cells. The tumor cell lines were cultured in DMEM complete medium (high-glucose DMEM medium with the addition of 10% fetal bovine serum, 100units/mL penicillin, 100mg/mL streptomycin). H358 was cultured in DMEM complete medium (high-glucose DMEM medium with 10% fetal bovine serum added). Cells in CO2The cells were cultured in a cell incubator at 37 ℃. Cells were passaged more than three times after recovery, up to 80% full and in good condition for activity testing.
(2) And (5) carrying out specific operation. The proliferation inhibitory activity of the compounds on cells was tested by the tetrazolium (MTT) method. Briefly, cells were seeded in 96-well plates and incubated for 72h with different concentrations of compound in the dark. Then 20. mu.L of MTT (5mg/mL) was added to each well and incubated for 4 h. The supernatant was aspirated, 150. mu.L DMSO was added to each well and shaken for 20 min. The Optical Density (OD) of each well at 550nm was read by a microplate reader (Thermo Varioskan Flash). Each compound was provided with three replicates at each concentration.
(3) The inhibition rate of proliferation of the cell line by the drug was calculated as follows: cell proliferation inhibition rate (OD)Negative control test-ODTest of)/(ODNegative control-ODBlank space) X 100%. Plotting different concentrations of the same sample on the inhibition rate of cell proliferation to obtain a dose response curve, analyzing by using software GraphPad Prism 5, and obtaining half inhibition concentration IC of the sample50。
Figure BDA0001876901460000141
4 kinds of the aboveHalf inhibitory concentration IC of sample50The results are shown in FIGS. 2 to 5. As can be seen from the figure, the compound has good inhibition effect on Ras mutated tumor cells, obvious inhibition effect on cis-structure, weaker inhibition effect on trans-structure, and embodies selectivity and controllability.

Claims (8)

1. The application of the acetamido azobenzene derivative is characterized in that the structural formula of the derivative is shown as a formula I or a formula II:
Figure FDA0003357218440000011
wherein formula I is trans structure, formula II is cis structure;
substituent R on ring A in the structural formula1One selected from H, halogen, hydroxyl, nitryl, C1-C10 or alkoxy; substituent R on ring A2One selected from H or halogen; substituent R on ring A3One selected from H or halogen; substituent R on ring A4One selected from H, halogen or C1-C10; wherein n is an integer of 1-3, and X is halogen; the C1-C10 is alkyl with 1-10 carbon atoms;
the ring A and the ring B are both benzene rings;
the derivative is used for preparing a medicine for treating K-Ras gene mutation-related tumors, shows a structure in a structural formula I in a dark environment, shows a structure in a structural formula II after ultraviolet illumination, and controls the inhibitory activity through illumination after being covalently bound with cysteine at position 12 of Ras mutein, so that the selectivity of the derivative on specific tissues or regions is realized;
the tumor related to the K-Ras gene mutation comprises one of rectal cancer, lung cancer or prostate cancer.
2. The use of an acetamidoazobenzene derivative according to claim 1, wherein said derivative comprises one or a mixture of pharmaceutically acceptable salts of the substance of formula I or formula II.
3. The use of an acetamidoazobenzene derivative according to claim 1, wherein the derivative is prepared by a process comprising the steps of:
(1) respectively reacting potassium hydrogen persulfate complex salt with
Figure FDA0003357218440000012
Mixing, stirring and reacting to obtain
Figure FDA0003357218440000013
(2) Subjecting the product obtained in step (1)
Figure FDA0003357218440000021
Dissolving in glacial acetic acid, adding
Figure FDA0003357218440000022
Stirring until the reaction is finished, and filtering to obtain
Figure FDA0003357218440000023
Or, the product obtained in step (1)
Figure FDA0003357218440000024
Dissolving in glacial acetic acid, adding
Figure FDA0003357218440000025
Stirring until the reaction is finished, and filtering to obtain
Figure FDA0003357218440000026
(3) Will be provided with
Figure FDA0003357218440000027
Dissolving in methanol, adding hydrochloric acid,heating and refluxing, dropwise adding 3M sodium hydroxide aqueous solution after the reaction is finished to adjust the pH, dropwise adding saturated sodium bicarbonate solution until precipitation is generated, separating and drying to obtain the sodium bicarbonate
Figure FDA0003357218440000028
(4) Will be provided with
Figure FDA0003357218440000029
Dissolved in dichloromethane and then added
Figure FDA00033572184400000210
Stirring until the reaction is finished, and purifying by column chromatography to obtain the product
Figure FDA00033572184400000211
Figure FDA0003357218440000031
4. Use of an acetamidoazobenzene derivative according to claim 3, wherein the substituent R on the A ring appearing in the reaction step1One selected from H, halogen, hydroxyl, nitryl, C1-C10 or alkoxy; substituent R on ring A2One selected from H or halogen; substituent R on ring A3One selected from H or halogen; substituent R on ring A4One selected from H, halogen or C1-C10; n is an integer of 1-3, and X is selected from halogen; the C1-C10 is alkyl with 1-10 carbon atoms.
5. The use of an acetamidoazobenzene derivative as claimed in claim 3, wherein said oxone complex salt is used in step (1) with
Figure FDA0003357218440000032
In a molar ratio of 1: (1.2-2) stirring at a speed of 500-1000 rpm, the reaction temperature is 20-30 ℃, and the reaction time is 2-12 hours.
6. The use of an acetamidoazobenzene derivative according to claim 3, wherein, in step (2),
Figure FDA0003357218440000033
and
Figure FDA0003357218440000034
the molar ratio of (1.5-3): 1, the
Figure FDA0003357218440000035
And
Figure FDA0003357218440000036
the molar ratio of (1.5-3) to (1), the stirring speed is 500-1000 rpm, the reaction temperature is 20-30 ℃, and the reaction time is 12-15 hours.
7. The use of an acetamidoazobenzene derivative according to claim 3, wherein in step (3), the volume ratio of methanol to hydrochloric acid is 1: (0.5 to 2) of,
Figure FDA0003357218440000037
the molar ratio of HCl in hydrochloric acid is 1: (50-100), the temperature of the reflux reaction is 80-90 ℃, the time is 10-12 hours, the pH value of the reaction system is adjusted to 8-9 by 3M sodium hydroxide aqueous solution, and the drying temperature is 55-65 ℃.
8. The use of an acetamidoazobenzene derivative according to claim 3, wherein, in step (4),
Figure FDA0003357218440000041
in a molar ratio of 1: (2-3) the reaction temperature is 20-30 ℃, and the reaction temperature isThe time is 2-12 hours, and silica gel column chromatography is adopted for purification.
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