CN104744350A - Pyridine-substituted diarylurea compound and preparation method and application thereof - Google Patents
Pyridine-substituted diarylurea compound and preparation method and application thereof Download PDFInfo
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- CN104744350A CN104744350A CN201510106888.1A CN201510106888A CN104744350A CN 104744350 A CN104744350 A CN 104744350A CN 201510106888 A CN201510106888 A CN 201510106888A CN 104744350 A CN104744350 A CN 104744350A
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- FUWGAABFLXHHHW-UHFFFAOYSA-N O=C(Nc(cc1)ccc1-c1cccnc1)Nc(cc1Cl)ccc1Cl Chemical compound O=C(Nc(cc1)ccc1-c1cccnc1)Nc(cc1Cl)ccc1Cl FUWGAABFLXHHHW-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/36—Radicals substituted by singly-bound nitrogen atoms
- C07D213/40—Acylated substituent nitrogen atom
Abstract
The invention provides a pyridine-substituted diarylurea compound and a preparation method and application thereof. The structural formula of the compound is shown in the specification, wherein R1 and R2 are any one of trifluoromethyl, trifluoromethoxyl and halogen respectively. The compound is prepared through two organic synthesis reaction steps; and the preparation method has the advantages of simplicity in operation in the reaction process, easily available raw materials, mild reaction conditions, cheap reagents and the like, and is suitable for large-scale production and manufacturing of pharmaceutical enterprises. The compound has high activity in inhibiting kinase VEGFR-2, can inhibit the proliferative activity of tumor cells, can be used for preparing anti-tumor drugs and drugs for inhibiting the activity of kinase VEGFR-2, and has favorable application prospects and high scientific research values.
Description
Technical field
The present invention relates to biomedicine technical field, relate to a kind of antineoplastic compound, be specifically related to di-aryl urea compounds of a kind of pyridine replacement and its preparation method and application.
Background technology
Malignant tumour as one of larger public health problem in the whole world, the greatly health of harm humans, and will first killer of the new millennium mankind be become.Malignant tumour is no longer the serious disease of advanced industrial country, and developing country is faced with larger Disease Spectrum.
Chemotherapy, as one of important means for the treatment of tumour, has had huge development and progress at nearly 30 years, has obtained large quantities of clinical antitumor agents with different mechanism of action.But antitumour drug also exists many untoward reactions, such as alopecia, vomiting, produces bone marrow depression, and produce resistance etc. fast, these all cause chemicals cannot reach the result for the treatment of of expection.The research and development of therefore new antitumor drug are one of the focus and difficulties of current pharmaceutical field.
Summary of the invention
Di-aryl urea compounds that the object of the present invention is to provide a kind of pyridine to replace and its preparation method and application, this compound embodies good anti-tumor activity in vitro, can be applied to the preparation of antitumor drug.
For achieving the above object, the technical solution used in the present invention is:
The di-aryl urea compounds that pyridine replaces, its structural formula is as follows:
Wherein, R
1, R
2be respectively in trifluoromethyl, trifluoromethoxy or halogen any one.
Described halogen is F, Cl or Br.
The preparation method of the di-aryl urea compounds that pyridine replaces, comprises the following steps:
1) para-bromoaniline and the reaction of connection pinacol borate, obtain p-aminophenyl pinacol borate, then p-aminophenyl pinacol borate and 3-bromopyridine obtain 4-(3-pyridine) aniline by Suzuki linked reaction;
Or reacted by Suzuki, 3-pyridine boronic acid is connected with para-bromoaniline, obtain biphenyl compound 4-(3-pyridine) aniline;
2) 4-(3-pyridine) aniline becomes urea with containing disubstituted aniline condensation, obtains the di-aryl urea compounds that pyridine replaces.(the R in the di-aryl urea compounds that pyridine replaces
1, R
2introduce by containing the substituting group on phenyl ring in disubstituted aniline)
Described step 1) concrete steps be: para-bromoaniline is dissolved in 1, in 4-dioxane, add connection pinacol borate, catalyst P d (pddf) Cl and Potassium ethanoate, react under nitrogen protection, room temperature is cooled under nitrogen protection after reaction terminates, obtain p-aminophenyl pinacol borate, then anhydrous sodium carbonate is added, 3-bromopyridine, 1, 4-dioxane and water, react under nitrogen protection, room temperature is cooled to after reaction terminates, suction filtration, with 1, 4-dioxane washing leaching cake, collect filtrate and be spin-dried for, the residue obtained is separated through chromatography column, obtain 4-(3-pyridine) aniline.
Described step 2) concrete steps be: under condition of ice bath, with heavily steaming methylene dichloride, two (trichloromethyl) carbonic ether is dissolved and stirs, then the dichloromethane solution containing disubstituted aniline is dripped, stir after dropwising, continue the dichloromethane solution dripping triethylamine, stir after dropwising, continue the dichloromethane solution dripping 4-(3-pyridine) aniline and triethylamine, dropwise rear stirring reaction, after having reacted, reaction solution is washed, dry, be spin-dried for, the residue chromatography column obtained is separated the di-aryl urea compounds obtaining pyridine and replace.
The di-aryl urea compounds that pyridine replaces suppresses the application in VEGFR-2 kinase activity medicine in preparation.
The di-aryl urea compounds that pyridine replaces is preparing the application in antitumor drug.
The medicine that described antitumor drug is is target spot with VEGFR-2 kinases, comprises the medicine of anti-lung cancer, anti-liver cancer, anti-cancer of the stomach, leukemia, anti-neural cancer, inhibitor against colon carcinoma cells, anti-breast cancer, anti-cervical cancer and anti-carcinoma of the pancreas.
Described antitumor drug is the medicine of inhibition tumor cell proliferation activity.
Described antitumor drug is the medicine suppressing lung cell A549 and liver cancer cell SMCC-7721 proliferation activity.
Relative to prior art, beneficial effect of the present invention is:
The di-aryl urea compounds that pyridine provided by the invention replaces, be a kind of novel compound with anti-tumor activity obtained through chemosynthesis, it has good inhibit activities to VEGFR-2 kinases, and can the proliferation activity of inhibition tumor cell.Scientific research has found that the generation of the generation of blood vessel and tumour, development and migration have substantial connection, suppress the formation of new vessel can the effectively growth of Tumor suppression and migration, many somatomedins all participate in the generation of new vessel, and wherein VEGFR-2 is the strongest known positive regulatory factor.The di-aryl urea compounds that pyridine provided by the invention replaces is by suppressing the kinase whose activity of VEGFR-2, block the signal path of its induction, thus can the hyperplasia of inhibition tumor cell and migration, therefore the di-aryl urea compounds that pyridine provided by the invention replaces can be used for antitumor drug and suppresses the preparation of VEGFR-2 kinase activity medicine, has a good application prospect and scientific research value.
The preparation method of the di-aryl urea compounds that pyridine provided by the invention replaces, target compound can be obtained by the conventional organic synthesis of five steps, there is the advantages such as reaction process is simple to operate, raw material is easy to get, reaction conditions gentle, agents useful for same is cheap, be suitable for the scale operation manufacture of pharmacy corporation.
In addition, the di-aryl urea compounds that pyridine provided by the invention replaces, to its cell-proliferation activity of inhibiting tumour cells comprising Non-small cell lung carcinoma cell (A549) and liver cancer cell (SMCC-7721), the preparation of antitumor drug can be can be applicable to.
Accompanying drawing explanation
Fig. 1 is the synthetic route chart of the di-aryl urea compounds that pyridine provided by the invention replaces;
Wherein compound 1 is para-bromoaniline, and compound 2 is p-aminophenyl pinacol borate, and compound 3 is 3-bromopyridine, and compound 4 is 4-pyridine boronic acid; Compound is 5 is 4-(3-pyridine) aniline, and compound 6 is containing disubstituted aniline, the di-aryl urea compounds that compound 7 replaces for the pyridine with anti-tumor activity.
What mark in figure is specially: a:B
2o
4(CH)
4(CH
3)
8, dioxane, Pd (pddf) Cl
2, KOAc, 100 DEG C; B:dioxane, Pd (pddf) Cl
2, K
2cO
3, 100 DEG C; C:dioxane, Pd (pddf) Cl
2, K
2cO
3, 100 DEG C; D:BTC, Et
3n, DCM.
Embodiment
The invention provides the di-aryl urea compounds that a kind of pyridine replaces, the di-aryl urea compounds that this pyridine replaces has anti-tumor activity, can be applied to the preparation of antitumor drug.
Be described in detail the present invention below in conjunction with drawings and Examples, the explanation of the invention is not limited.
The di-aryl urea compounds that the pyridine with anti-tumor activity provided by the invention replaces, its chemical structural formula is:
Wherein, R
1, R
2be respectively in trifluoromethyl, trifluoromethoxy or halogen any one.
Preparation method and the method for screening active ingredients with the di-aryl urea compounds that anti-swollen active knurl drug candidate pyridine replaces of the present invention is described in detail below in conjunction with the synthetic route shown in Fig. 1 and concrete synthetic example.
Embodiment 1
In the structural formula of this compound, R
1for trifluoromethyl, R
2for bromine, by following steps preparation (see Fig. 1):
1) p-aminophenyl pinacol borate (compound 2) is prepared by para-bromoaniline (compound 1) and the reaction of connection pinacol borate
Para-bromoaniline (compound 1) 1g (5.8mmol) is dissolved in 40mL 1; in 4-dioxane; add connection pinacol borate 1.62g (6.4mmol); catalyst P d (pddf) Cl 20.42g (0.58mmol) and Potassium ethanoate 2.28g (23mmol); under nitrogen protection; in 100 DEG C of reaction 5h; be cooled to room temperature under nitrogen protection and obtain product p-aminophenyl pinacol borate (compound 2), solution is not treated carries out next step operation.
2) p-aminophenyl pinacol borate (compound 2) and 3-bromopyridine (compound 3) prepare 4-(3-pyridine) aniline (compound 5) by Suzuki linked reaction
In above-mentioned solution, add anhydrous sodium carbonate 2.4g (17.4mmol), 3-bromopyridine (compound 3) 0.82g (5.2mmol) and 20mL Isosorbide-5-Nitrae-dioxane, 20mL H2O, spends the night in 100 DEG C of reactions under nitrogen protection.Be cooled to room temperature, suction filtration, 1,4-dioxane washing leaching cake, collect filtrate, be spin-dried for obtain black residue, be separated (eluting solvent is by sherwood oil: ethyl acetate=3:1) through chromatography column and obtain solid 4-(3-pyridine) aniline (compound 5) 0.35g, productive rate about 35%;
Physico-chemical property: mp:114 ~ 116 DEG C, MS (EI) [M]+: m/z=170
Hydrogen spectrum nuclear magnetic resonance data:
1h NMR (400MHz, CDCl
3) δ 8.82 (d, J=1.6Hz, 1H), 8.53 (d, J=4.0Hz, 1H), 7.83 (d, J=7.9Hz, 1H), 7.43 (d, J=8.4Hz, 2H), 7.33 (m, 1H), 6.81 (d, J=8.3Hz, 2H).
3) 4-(3-pyridine) aniline (compound 5) and 3-bromo-5-5-trifluoromethylaniline (compound 6) prepare 1-[the bromo-5-of 3-(trifluoromethyl) phenyl]-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) by condensation reaction
Under condition of ice bath, heavily steam methylene dichloride with 15mL two for 0.14g (0.48mmol) (trichloromethyl) carbonic ether (BTC) is dissolved and stir 5min, slowly drip the dichloromethane solution of the bromo-5-5-trifluoromethylaniline of 0.3g (1.2mmol) 3-(compound 6) again, dropwise and stir 15min, the dichloromethane solution 10mL dripping 0.2mL (1.44mmol) triethylamine is continued in turbid solution, dropwise rear continuation and stir 15min, then in reaction soln, drip the dichloromethane solution 10mL of 4-(3-pyridine) aniline (compound 5) 0.2g (1.2mmol) and 0.2mL (1.2mmol) triethylamine, dropwise rear continuation and stir 20min, reaction solution is used saturated sodium bicarbonate solution successively, saturated common salt water washing, anhydrous sodium sulfate drying, be spin-dried for and obtain residue, be separated with chromatography column and obtain solid 1-[the bromo-5-of 3-(trifluoromethyl) phenyl]-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) 0.38g, productive rate 73%,
Gained compound structure is as follows:
Physico-chemical property: mp:200 ~ 202 DEG C, MS (ESI) [M+H]+: m/z=436
Hydrogen spectrum nuclear magnetic resonance data is:
1h NMR (400MHz, DMSO) δ 9.28 (s, 1H), 9.12 (s, 1H), 8.89 (d, J=2.0Hz, 1H), 8.53 (m, 1H), 8.08 – 8.03 (m, 1H), 7.98 (s, 1H), 7.89 (s, 1H), 7.70 (d, J=8.7Hz, 2H), 7.62 (d, J=8.6Hz, 2H), 7.53 (s, 1H), 7.46 (m, 1H).
Embodiment 2
In the structural formula of this compound, R
1for trifluoromethoxy, R
2for bromine.
Step 1) ~ 2) with step 1 in embodiment 1) ~ 2) identical, namely prepare p-aminophenyl pinacol borate (compound 2) by initial by compound para-bromoaniline (compound 1) and the reaction of connection pinacol borate, then p-aminophenyl pinacol borate and 3-bromopyridine (compound 3) prepare 4-(3-pyridine) aniline (compound 5) by Suzuki linked reaction.
3) 4-(3-pyridine) aniline (compound 5) and 4-bromo-2-trifluoro-methoxyaniline (compound 6) prepare 1-[the bromo-2-of 4-(trifluoromethoxy) phenyl]-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) by condensation reaction, and concrete operation steps is:
Under condition of ice bath, heavily steam methylene dichloride with 15mL two for 0.14g (0.48mmol) (trichloromethyl) carbonic ether (BTC) is dissolved and stir 5min, slowly drip the dichloromethane solution of the bromo-2-trifluoro-methoxyaniline of 0.28g (1.1mmol) 4-(compound 6) again, dropwise and stir 15min, the dichloromethane solution 10mL dripping 0.2mL (1.44mmol) triethylamine is continued in turbid solution, dropwise rear continuation and stir 15min, then in reaction soln, drip the dichloromethane solution 10mL of 4-(3-pyridine) aniline (compound 5) 0.2g (1.2mmol) and 0.2mL (1.2mmol) triethylamine, dropwise rear continuation and stir 20min, reaction solution is used saturated sodium bicarbonate solution successively, saturated common salt water washing, anhydrous sodium sulfate drying, be spin-dried for and obtain residue, be separated with chromatography column and obtain solid 1-[the bromo-2-of 4-(trifluoromethoxy) phenyl]-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) 0.24g, productive rate 44%,
Gained compound structure is as follows:
Physico-chemical property: mp:216 ~ 218 DEG C, MS (ESI) [M+H]+: m/z=452
Nuclear magnetic resonance data is:
1h NMR (400MHz, DMSO) δ 9.47 (s, 1H), 8.89 (d, J=1.8Hz, 1H), 8.64 (s, 1H), 8.54 (d, J=3.4Hz, 1H), 8.28 (d, J=9.0Hz, 1H), 8.06 (d, J=7.9Hz, 1H), 7.71 (d, J=8.6Hz, 2H), 7.65 (s, 1H), 7.61 (d, J=8.4Hz, 3H), 7.47 (m, 1H).
Embodiment 3
In the structural formula of this compound, R
1, R
2be chlorine.
Step 1) ~ 2) with step 1 in embodiment 1) ~ 2) identical, namely prepare p-aminophenyl pinacol borate (compound 2) by initial by compound para-bromoaniline (compound 1) and the reaction of connection pinacol borate, then p-aminophenyl pinacol borate and 3-bromopyridine (compound 3) prepare 4-(3-pyridine) aniline (compound 5) by Suzuki linked reaction.
3) 4-(3-pyridine) aniline (compound 5) and 3,4-dichlorphenamide bulk powder (compound 6) prepares 1-(3 by condensation reaction, 4-dichlorophenyl)-3-[4-(pyridin-3-yl) phenyl] urea (compound 7), concrete operation steps is:
Under condition of ice bath, heavily steam methylene dichloride with 15mL two for 0.21g (0.7mmol) (trichloromethyl) carbonic ether (BTC) is dissolved and stir 5min, slowly drip 0.26g (1.6mmol) 3 again, the dichloromethane solution of 4-dichlorphenamide bulk powder (compound 6), dropwise and stir 15min, the dichloromethane solution 10mL dripping 0.3mL (2.1mmol) triethylamine is continued in turbid solution, dropwise rear continuation and stir 15min, then in reaction soln, drip the dichloromethane solution 10mL of 4-(3-pyridine) aniline (compound 5) 0.3g (1.8mmol) and 0.3mL (2.1mmol) triethylamine, dropwise rear continuation and stir 20min, reaction solution is used saturated sodium bicarbonate solution successively, saturated common salt water washing, anhydrous sodium sulfate drying, be spin-dried for and obtain residue, be separated with chromatography column and obtain solid 1-(3, 4-dichlorophenyl)-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) 0.16g, productive rate 28%,
Gained compound structure is as follows:
Physico-chemical property: mp:228 ~ 330 DEG C, MS (ESI) [M+H]+: m/z=358
Nuclear magnetic resonance data is:
1h NMR (400MHz, DMSO) δ 9.06 (s, 1H), 9.00 (s, 1H),
8.89(d,J＝1.8Hz,1H),8.53(d,J＝3.7Hz,1H),8.05(d,J＝8.0Hz,1H),7.91(d,J＝2.4Hz,1H),7.69(d,J＝8.6Hz,2H),7.60(d,J＝8.6Hz,2H),7.54(d,J＝8.8Hz,1H),7.46(dd,J＝7.9,4.8Hz,1H),7.36(m,1H)。
Embodiment 4
In the structural formula of this compound, R
1, R
2be trifluoromethyl.
Step 1) ~ 2) with step 1 in embodiment 1) ~ 2) identical, namely prepare p-aminophenyl pinacol borate (compound 2) by initial by compound para-bromoaniline (compound 1) and the reaction of connection pinacol borate, then p-aminophenyl pinacol borate and 3-bromopyridine (compound 3) prepare 4-(3-pyridine) aniline (compound 5) by Suzuki linked reaction.
3) 4-(3-pyridine) aniline (compound 5) and 3,5-bis-5-trifluoromethylaniline (compound 6) prepares 1-(3 by condensation reaction, 5-bis-trifluoromethyl)-3-[4-(pyridin-3-yl) phenyl] urea (compound 7), concrete operation steps is:
Under condition of ice bath, heavily steam methylene dichloride with 15mL two for 0.14g (0.14mmol) (trichloromethyl) carbonic ether (BTC) is dissolved and stir 5min, slowly drip 0.25g (1.1mmol) 3 again, the dichloromethane solution of 5-bis-5-trifluoromethylaniline (compound 6), dropwise and stir 15min, the dichloromethane solution 10mL dripping 0.2mL (1.44mmol) triethylamine is continued in turbid solution, dropwise rear continuation and stir 15min, then in reaction soln, drip the dichloromethane solution 10mL of 4-(3-pyridine) aniline (compound 5) 0.2g (1.2mmol) and 0.2mL (1.44mmol) triethylamine, dropwise rear continuation and stir 20min, reaction solution is used saturated sodium bicarbonate solution successively, saturated common salt water washing, anhydrous sodium sulfate drying, be spin-dried for and obtain residue, be separated with chromatography column and obtain solid 1-(3, 5-bis-trifluoromethyl)-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) 0.37g, productive rate 65%,
Gained compound structure is as follows:
Physico-chemical property: mp:214 ~ 215 DEG C, MS (ESI) [M+H]+: m/z=425
Nuclear magnetic resonance data is:
1h NMR (400MHz, DMSO) δ 9.47 (s, 1H), 9.19 (s, 1H), 8.90 (s, 1H), 8.54 (d, J=4.1Hz, 1H), 8.16 (s, 2H), 8.06 (d, J=8.0Hz, 1H), 7.71 (d, J=8.5Hz, 2H), 7.65 (t, J=8.9Hz, 3H), 7.47 (dd, J=7.8,4.7Hz, 1H).
Embodiment 5
In the structural formula of this compound, R
1, R
2be bromine.
1) reacted by Suzuki, 3-pyridine boronic acid (compound 4) is connected with para-bromoaniline (compound 1), obtain biphenyl compound 4-(3-pyridine) aniline (compound 5);
2) 4-(3-pyridine) aniline (compound 5) and 3,5-dibromo aniline (compound 6) prepares 1-(3,5-dibromo phenyl)-3-[4-(pyridin-3-yl) phenyl] urea (compound 7) by condensation reaction
Under condition of ice bath, heavily steam methylene dichloride with 15mL two for 0.14g (0.14mmol) (trichloromethyl) carbonic ether (BTC) is dissolved and stir 5min, slowly drip 1.1mmol 3 again, the dichloromethane solution of 5-bis-bromo-amine (compound 6), dropwise and stir 15min, the dichloromethane solution 10mL dripping 0.2mL (1.44mmol) triethylamine is continued in turbid solution, dropwise rear continuation and stir 15min, then in reaction soln, drip the dichloromethane solution 10mL of 4-(3-pyridine) aniline (compound 5) 0.2g (1.2mmol) and 0.2mL (1.44mmol) triethylamine, dropwise rear continuation and stir 20min, reaction solution is used saturated sodium bicarbonate solution successively, saturated common salt water washing, anhydrous sodium sulfate drying, be spin-dried for and obtain residue, be separated with chromatography column and obtain solid 1-(3, 5-dibromo phenyl)-3-[4-(pyridin-3-yl) phenyl] urea (compound 7), the structure of gained compound 7 is as follows:
The generation of blood vessel and the generation of tumour, development and migration have substantial connection, suppress the formation of new vessel can the growth of effective Tumor suppression.Many somatomedins all participate in the generation of new vessel, and wherein VEGFR-2 is the strongest known positive regulatory factor.By suppressing VEGFR-2 kinase whose activity, block the signal path of its induction, can the hyperplasia of inhibition tumor cell and migration, and then reach the object for the treatment of tumour.
Anti-tumor activity experiment is carried out to the di-aryl urea compounds that pyridine provided by the invention replaces below.
1, the di-aryl urea compounds that pyridine replaces screens the kinase whose inhibit activities of VEGFR-2
Kinases VEGFR-2 and substrate A bltide is purchased from Signal-Chem company, and select the ADP-GlobTM Kinase Assays detection kit of Promega company to detect the Inhibiting enzyme activity of target compound, working method illustrates according to test kit carries out.
By ATP (10mM) buffer (2 ×) (Tris 80mM, MgCl
220mM, BSA 0.2mg/mL, DTT 2mM) dilute buffer (2 ×) solution that 40 times are mixed with ATP (250 μMs); The mixing solutions ATP solution of 250 μMs and Abltide liquor capacity 1:1 being hybridly prepared into ATP (125 μMs)-Abltide (0.5 μ g/ μ l) is for subsequent use; VEGFR-2 kinase solution buffer (1 ×) (Tris 40mM, MgCl
210mM, BSA0.1mg/mL, DTT 1mM) dilute buffer (1 ×) solution for standby that 66 times are mixed with VEGFR-2 (1.5ng/ μ l); Target compound and positive control drug (Sorafinib) buffer (1 ×) are mixed with 6 × 10 respectively
-5mol/L, 6 × 10
-6mol/L, 6 × 10
-7mol/L, 6 × 10
-8mol/L, 6 × 10
-9mol/L, 6 × 10
-10the sample solution of mol/L concentration gradient, on 384 orifice plates, every hole adds the mixing solutions of 2 μ L ATP-Abltide successively, 1 μ L sample solution, 2 μ L enzyme solution; Blank well adds the mixing solutions of 3 μ L damping fluids and 2 μ LATP-Abltide; Control wells adds the mixing solutions of 2 μ L ATP-Abltide, 1 μ L damping fluid, and 2 μ L enzyme solution, finish, hatch 60min at 30 DEG C; Add ADP-Glo reagent 5 μ L, at 25 DEG C, hatch 40min; Add Kinase detection reagent 10 μ L, at 25 DEG C, hatch 30min.Adopt the chemoluminescence module of the multi-functional microplate reader of PerkinElmer to measure the luminous value in every hole, computerized compound is to the inhibiting rate of VEGFR-2 and IC
50.
The di-aryl urea compounds that different pyridines replaces is specifically as shown in table 1 to the result of VEGFR-2 kinase inhibitory activity:
The di-aryl urea compounds that table 1 pyridine replaces is to the kinase whose IC50 of VEGFR-2
Can find out that the di-aryl urea compounds that pyridine provided by the invention replaces has the good kinase whose activity of suppression VEGFR-2 by table 1, part of compounds is suitable with positive control drug BAY 43-9006 to the kinase whose suppression of VEGFR-2, even has some activity to be higher than positive control.Illustrate that the di-aryl urea compounds that pyridine provided by the invention replaces can suppress to apply in the medicine of VEGFR-2 kinase activity in preparation.
2, the antitumor activity screening of the di-aryl urea compounds of pyridine replacement
The di-aryl urea compounds that employing mtt assay checks pyridine to be measured to replace is to the growth inhibitory activity of tumour cell:
The di-aryl urea compounds that pyridine provided by the invention replaces has antitumor action, vitro inhibition proliferation activity is had to tumour cell, at lung carcinoma cell (A549), there is in liver cancer cell (SMCC-7721) clone the proliferation activity of inhibition tumor cell, may be used for the treatment to these cancers; Compared with positive drug BAY 43-9006 (Sorafinib), individual compound shows higher inhibition tumor cell proliferation activity.
To be in the lung carcinoma cell (A549) of logarithmic phase, liver cancer cell (SMCC-7721), the trysinization with 0.25% 3 ~ 5 minutes, being diluted to concentration after piping and druming is evenly 1 × 10
4~ 2 × 10
4the single cell suspension of individual/mL, is parallelly inoculated in 96 well culture plates, and every hole inoculation volume is 180 μ L; In 37 DEG C, 5%CO
2cultivate 24 hours in incubator; With the aqueous solution containing 0.25%DMSO (N, N-dimethyl sulfoxide (DMSO)) for negative control, take BAY 43-9006 as positive control, the di-aryl urea compounds (1 × 10 that the pyridine that testing sample adds 4 different concns replaces
-4mol/L, 2 × 10
-5mol/L, 4 × 10
-6mol/L, 8 × 10
-7mol/L), each concentration establishes 3 multiple holes, continues cultivation 48 hours; Then every hole adds the MTT working fluid 22 μ L of 5mg/mL, mixing, 37 DEG C of incubators are hatched after 4 hours and are taken out, nutrient solution is abandoned in careful suction, every hole adds 150 μ L DMSO, vibration 10min, and euzymelinked immunosorbent assay (ELISA) detector measures 490nm place, each hole ultraviolet absorption value (OD value), then calculate cell inhibitory rate, and obtain IC according to inhibiting rate
50value; The calculation formula of cell inhibitory rate is:
Inhibiting rate %=(positive controls OD value-medication group OD value)/negative control cell OD value × 100%
Detected result shows: compared with negative control group, and the di-aryl urea compounds that pyridine replaces has In-vitro Inhibitory Effect in various degree to above-mentioned 2 kinds of tumour cells, and result is as shown in table 2.
The di-aryl urea compounds that table 2 pyridine replaces is to the IC50 (μM) of different cell strain
The suppression of result display section compound on tumor cell is suitable with positive control drug BAY 43-9006, even has some activity to be higher than positive control.Illustrate that the proliferation inhibiting effect of di-aryl urea compounds to tumour cell (lung cell A549 and liver cancer cell SMCC-7721) that pyridine provided by the invention replaces is obvious, can apply preparing in antitumor drug.
And the structure of the di-aryl urea compounds that the higher pyridine of above-mentioned activity replaces is specifically as shown in table 3:
The structural formula of the di-aryl urea compounds that the pyridine that table 3 activity is higher replaces
Because VEGFR-2 kinases is to the growth of tumour cell, and the di-aryl urea compounds that pyridine provided by the invention replaces is to the kinase whose restraining effect of VEGFR-2, therefore the di-aryl urea compounds that the pyridine with anti-tumor activity provided by the invention replaces can be applied in the antitumor drug being target spot with VEGFR-2 kinases, not only comprise anti-lung cancer and the medicines resistant to liver cancer of above-mentioned citing, in like manner, the medicines such as anti-cancer of the stomach, leukemia, anti-neural cancer, inhibitor against colon carcinoma cells, anti-breast cancer, anti-cervical cancer and anti-carcinoma of the pancreas can also be comprised.
Claims (10)
1. a di-aryl urea compounds for pyridine replacement, it is characterized in that, its structural formula is as follows:
Wherein, R
1, R
2be respectively in trifluoromethyl, trifluoromethoxy or halogen any one.
2. the di-aryl urea compounds of pyridine replacement according to claim 1, is characterized in that: described halogen is F, Cl or Br.
3. the preparation method of the di-aryl urea compounds that the pyridine described in claim 1 or 2 replaces, is characterized in that, comprise the following steps:
1) para-bromoaniline and the reaction of connection pinacol borate, obtain p-aminophenyl pinacol borate, then p-aminophenyl pinacol borate and 3-bromopyridine obtain 4-(3-pyridine) aniline by Suzuki linked reaction;
Or reacted by Suzuki, 3-pyridine boronic acid is connected with para-bromoaniline, obtain biphenyl compound 4-(3-pyridine) aniline;
2) 4-(3-pyridine) aniline becomes urea with containing disubstituted aniline condensation, obtains the di-aryl urea compounds that pyridine replaces.
4. the preparation method of the di-aryl urea compounds of pyridine replacement according to claim 3, it is characterized in that: described step 1) concrete steps be: para-bromoaniline is dissolved in 1, in 4-dioxane, add connection pinacol borate, catalyst P d (pddf) Cl and Potassium ethanoate, react under nitrogen protection, room temperature is cooled under nitrogen protection after reaction terminates, obtain p-aminophenyl pinacol borate, then anhydrous sodium carbonate is added, 3-bromopyridine, 1, 4-dioxane and water, react under nitrogen protection, room temperature is cooled to after reaction terminates, suction filtration, with 1, 4-dioxane washing leaching cake, collect filtrate and be spin-dried for, the residue obtained is separated through chromatography column, obtain 4-(3-pyridine) aniline.
5. the preparation method of the di-aryl urea compounds of pyridine replacement according to claim 3, it is characterized in that: described step 2) concrete steps be: under condition of ice bath, with heavily steaming methylene dichloride, two (trichloromethyl) carbonic ether is dissolved and stirs, then the dichloromethane solution containing disubstituted aniline is dripped, stir after dropwising, continue the dichloromethane solution dripping triethylamine, stir after dropwising, continue the dichloromethane solution dripping 4-(3-pyridine) aniline and triethylamine, dropwise rear stirring reaction, after having reacted, reaction solution is washed, dry, be spin-dried for, the residue chromatography column obtained is separated the di-aryl urea compounds obtaining pyridine and replace.
6. the di-aryl urea compounds that the pyridine described in claim 1 or 2 replaces suppresses the application in VEGFR-2 kinase activity medicine in preparation.
7. the di-aryl urea compounds that the pyridine described in claim 1 or 2 replaces is preparing the application in antitumor drug.
8. apply as claimed in claim 7, it is characterized in that: the medicine that described antitumor drug is is target spot with VEGFR-2 kinases, comprise the medicine of anti-lung cancer, anti-liver cancer, anti-cancer of the stomach, leukemia, anti-neural cancer, inhibitor against colon carcinoma cells, anti-breast cancer, anti-cervical cancer and anti-carcinoma of the pancreas.
9. apply as claimed in claim 7, it is characterized in that: described antitumor drug is the medicine of inhibition tumor cell proliferation activity.
10. apply as claimed in claim 9, it is characterized in that: described antitumor drug is the medicine suppressing lung cell A549 and liver cancer cell SMCC-7721 proliferation activity.
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| WO2018209030A1 (en) * | 2017-05-12 | 2018-11-15 | Rti International | Diarylureas as cb1 allosteric modulators |
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