CN109336867A

CN109336867A - Carbazoles STAT3 inhibitor crystal form A and preparation method thereof

Info

Publication number: CN109336867A
Application number: CN201811282931.XA
Authority: CN
Inventors: 郭程杰
Original assignee: Nanjing Advanced Biomaterials And Process Equipment Research Institute Co Ltd
Current assignee: Nanjing Advanced Biomaterials And Process Equipment Research Institute Co Ltd
Priority date: 2018-10-31
Filing date: 2018-10-31
Publication date: 2019-02-15

Abstract

The invention belongs to medicinal chemistry arts, more particularly to compound 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1 with function of tumor, 3, a kind of novel crystal forms, preparation method, the composition comprising the crystal form of 5- triazine -2- amine mesylate, and the purposes of the crystal form or the composition comprising the crystal form in medicine preparation, the A crystal form has good physical and chemical stability, solubility and bioavilability, is suitble to formulation development.

Description

Carbazoles STAT3 inhibitor crystal form A and preparation method thereof

Technical field

The invention belongs to medicinal chemistry arts, and in particular to the compound 4- (9- ethyl -9H- carbazole-with function of tumor 4- yl) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A kind of novel crystal forms, preparation method, the composition comprising the crystal form and the crystal form or the combination comprising the crystal form Purposes of the object in medicine preparation.

Background technique

Cancer is the general designation of a major class malignant tumour, its main feature is that without limitation, without end hyperplasia.Cancer cell makes patient's body Interior nutriment is largely consumed, while releasing a variety of toxin, and human body is made to generate a series of symptoms, cause human body it is thin, Inability, anaemia, loss of appetite, fever and serious organ function are impaired, cause necrotic hemorrhage concurrent infection, patient finally by It is dead in organ failure.

Signal transduction and (the Signal Transducer and Activator of of activating transcription factor -3 Transcription-3, STAT3) be a kind of GAP-associated protein GAP that can be activated by different cytokine receptors, cell because Carrier is served as during son-acceptor interaction, the inherence specificity for keeping signal to transmit in the cell, and pass through induction target The effect effect of biostimulation is expressed in genetic transcription, other than participating in angiogenesis and immune response, also with the increasing of cell It grows, survive, breaking up, the close associations such as anti-apoptotic.STAT3 is in kinds of tumor cells (including blood such as leukaemia, Huppert's disease A variety of entity tumors such as liquid tumour and lung cancer, breast cancer, prostate cancer) in abnormal expression increase, generation with malignant tumour, Develop closely related.

By inhibiting STAT3 activity to be expected to that cancer cell is made apoptosis occur to achieve the purpose that treating cancer, study recently It was found that inhibiting STAT3 signal that can overcome the chemical drug resistance including kinds of tumors such as retinoblastoma, lung cancer, leukaemia Property, successfully research and development are a new antitumor target to STAT3.Therefore, new STAT3 inhibitor is actively found for cancer Treatment have particularly important meaning.

Compound 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene - 4- ketone group) -1,3,5-triazines -2- amine mesylate, there is formula:

The Compound ira vitro cytoactive detection finds that it has good inhibitory activity to prostate gland cancer cell DU-145, IC₅₀Value is 8.4nM, is had a good application prospect.

This field knows that the difference of drug crystal forms will cause the difference of various physicochemical properties, as solubility, dissolution rate, Fusing point, density, hardness, optical and electrical properties, steam pressure etc..These differences can be reflected in thermodynamic stability, as stable type, Metastable type and instability mode；It can also be reflected on physical and chemical stability, such as hygroscopicity, crystal transfer, sample degradation.This A little differences directly affect prescription preparation process, storage method, internal medicine the generation dynamic performance of drug, and then influence the safety of drug Property and validity.

Therefore, 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color is studied Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate polymorphism and select significant and steady on clinical treatment Fixed controllable crystal form tool is of great significance to.

Summary of the invention

To 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- Ketone group) -1,3,5-triazines -2- amine mesylate crystal form research in, the present inventor has found tens kinds of crystal forms altogether, real It tests and shows in obtained numerous crystal forms, wherein A crystal form has good physical and chemical stability, solubility and biological utilisation Degree is suitble to formulation development.

It is an object of the present invention to provide compound 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl acylamino-s Phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate a kind of crystalline substance for being named as A crystal form Type, the crystal form have good physical and chemical stability, solubility and bioavilability.

It is a further object to provide the preparation methods of the A crystal form, and this method is easy to operate, convenient for industrialization Mass production.

It is also another object of the present invention to provide the medicines for containing the A crystal form and at least one pharmaceutically acceptable carrier Use composition.

Fourth object of the present invention is to provide a kind of A crystal form in preparation prevention and/or the drug for the treatment of tumour In application.

For foregoing invention purpose, in a first aspect, the present invention provides 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl Amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form, use Cu- Ka radiation, X-ray powder diffraction 2 θ of the angle of diffraction be 3.7 ± 0.2 °, 4.5 ± 0.2 °, 11.7 ± 0.2 °, 13.6 ± 0.2 °, Characteristic peak is shown at 19.5 ± 0.2 °, 22.9 ± 0.2 ° and 26.0 ± 0.2 °.

This field knows, when with the crystallization of X-ray diffraction measure compound, due to the instrument of measurement or condition of measurement etc. Influence, for measured summit, there are the relative intensities of certain measurement error, especially x-ray diffraction pattern with test The variation of condition and change.For example, the evaluated error of 2 θ values can be about ± 0.2 °, the evaluated error of relative intensity can for ± 20%.Therefore, when determining every kind of crystalline texture, it should take into account this error.It can be understood as any and the application A There is crystal form the crystal form of essentially identical or similar x-ray diffraction pattern to belong within scope of the present application.

Specifically, 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- provided by the invention Methyl -4H- chromene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form, it is radiated using Cu-Ka, X-ray powder Diffraction 2 θ of the angle of diffraction be 3.7 ± 0.2 °, 4.5 ± 0.2 °, 6.0 ± 0.2 °, 10.4 ± 0.2 °, 11.7 ± 0.2 °, 13.6 ± Characteristic peak is shown at 0.2 °, 15.0 ± 0.2 °, 19.5 ± 0.2 °, 22.9 ± 0.2 °, 26.0 ± 0.2 °.

4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl-provided by the invention 4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form have X- diffraction pattern as described in Figure 1.

Second aspect, the present invention provide 4- of the invention (9- ethyl -9H- carbazole -4- base) -6- (4- acrylyl aminobenzene Base)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form preparation method, the side Method includes the following steps:

The compound of formula (1) is made in step a:2- hydroxy acetophenone acetylation；

Step b: the compound of formula (2) is made in the compound cyclization of formula (1)；

Step c: the compound of formula (3) is made in the compound bromo of formula (2)；

Step d: the compound amino of formula (3) replaces the compound that formula (4) are made；

The compound of formula (5) is made in step e:4- nitrobenzene methyl and biuret cyclization；

Step f: the compound of formula (6) is made in the compound chloro of formula (5)；

Step g: the compound of formula (6) reacts the compound that formula (7) are made with the compound of formula (4)；

Step h: the compound and 4- (4,4,5,5- tetramethyl -1,3,2- dioxolane borine) -9- ethyl-of formula (7) The compound of formula (8) is made in 9H- carbazole reaction；

Step i: the compound reduction of formula (8) hydrogenates the compound that formula (9) are made；

Step j: the compound of formula (9) reacts the compound that formula (I) is made with acryloyl chloride；

Step k: the compound of formula (I) is reacted with methanesulfonic acid is made formula (I) compound methanesulfonic acid salt, by gained mesylate It is added in alcohol-water mixed solvent, reflux, 0-5 DEG C of crystallization is to get 4- (9- ethyl -9H- carbazole -4- base) -6- (4- acrylyl ammonia Base phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form, reaction route is as follows:

According to the preparation method, the solvent that formula (I) compound is reacted with methanesulfonic acid in step k is selected from alcohol, ketone, acetic acid second Ester or their mixture；Preferably, the solvent that formula (I) compound is reacted with methanesulfonic acid is selected from methanol, ethyl alcohol, normal propyl alcohol, different Propyl alcohol, acetone, methyl ethyl ketone, methyl iso-butyl ketone (MIBK), ethyl acetate or their mixture；It is further preferred that formula (I) is changed It closes the solvent that object is reacted with methanesulfonic acid and is selected from methanol, ethyl alcohol, acetone, ethyl acetate or their mixture.

According to the preparation method, the molar ratio that formula (I) compound is reacted with methanesulfonic acid in step k is about 1:1-1.1；It is excellent Selection of land, the molar ratio that formula (I) compound is reacted with methanesulfonic acid are about 1:1.

According to the preparation method, alcohol described in step k is selected from methanol or ethyl alcohol；Preferably, the alcohol is ethyl alcohol.

According to the preparation method, the volume ratio of alcohol and water is 2~4:1 in alcohol-water mixed solvent described in step k； Preferably, the volume ratio of alcohol and water is 3:1 in alcohol-water mixed solvent described in step k.

According to the preparation method, alcohol-water mixed solvent usage amount described in step F is every gram of 4- (9- ethyl- 9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine Mesylate uses alcohol-water mixed solvent 8-12mL；Preferably, alcohol-water mixed solvent usage amount described in step F is every Gram 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3, 5- triazine -2- amine mesylate uses alcohol-water mixed solvent 10mL.

The third aspect, the present invention provide pharmaceutical composition, and it includes 4- of the invention (9- ethyl -9H- carbazole -4- base) - 6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form and Pharmaceutically acceptable carrier.By 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl - 4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form and pharmaceutically acceptable carrier be mixed with patent medicine Object preparation, to be suitable for oral or parenteral.Medication includes, but are not limited in intradermal, intramuscular, peritonaeum, is intravenous, Subcutaneously, intranasally and peroral route.The preparation can be applied by any approach, such as by being transfused or injecting, by through upper The approach application that skin or mucocutaneous (such as oral mucosa or rectum etc.) absorb.Administration can be whole body or local.Through The example of mouth application preparation includes solid or liquid dosage form, specifically, including tablet, pill, granula, pulvis, capsule, sugar Slurry, emulsion, suspension etc..The preparation can be prepared by methods known in the art, and include that field of pharmaceutical preparations routinely makes Carrier.

Fourth aspect, the present invention provide 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- Methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form or 4- (9- ethyl -9H- carbazole -4- base) -6- The medicine of (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form Application of the compositions in the drug of preparation prevention and/or treatment tumour, including easily crowd is sent out to tumour or tumor patient is applied With 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl-of the invention of therapeutically effective amount 4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form or 4- (9- ethyl -9H- carbazole -4- base) -6- (4- alkene Propionamido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form medicine group Object is closed, Tumor incidence is effectively reduced, extends tumor patient life, the tumour includes but is not limited to prostate cancer, cream Gland cancer.

4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color of the invention Alkene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form is with good stability, solubility and bioavilability, it fits It shares in pharmaceutical preparation is made.

Detailed description of the invention

Fig. 1 is 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene - 4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form x-ray diffraction pattern.

Specific embodiment

The present invention is explained in more detail with reference to embodiments, the embodiment of the present invention is merely to illustrate technology of the invention Scheme not limits the scope of the invention.

The preparation of 1 2- aminomethyl -4H- chromene -4- keto hydrochloride of embodiment

The preparation of step 1 2- acetoxy acetophenone

2- hydroxy acetophenone (100mmol), chloroacetic chloride (250mmol) and potassium carbonate (500mmol) are added in reaction flask, 300ml acetone is added, back flow reaction 12h, after reaction, water, ethyl acetate extraction, anhydrous sulphur is added in evaporating solvent under reduced pressure Sour sodium is dry, is concentrated to give grease, directly throws in next step.

The preparation of step 2 2- methyl -4H- chromene -4- ketone

2- acetoxy acetophenone (50mmol) is weighed in reaction flask, 100ml DMSO is added and dissolves, at 0-5 DEG C in batches It is added sodium hydrogen (150mmol), finishes, be warmed to room temperature stirring 3h and water, dilute hydrochloric acid tune pH are added into reaction solution after reaction It is worth faintly acid, ethyl acetate extraction, anhydrous sodium sulfate is dry, is concentrated to give grease, and 100ml second is added into gained grease Acid and 5 drop concentrated hydrochloric acids, back flow reaction about 3h, reaction terminate, reaction solution are spin-dried for, and water, ethyl acetate extraction, anhydrous slufuric acid is added Sodium is dry, and column chromatographic purifying obtains title compound.

ES:M/Z 161 [M+H]⁺。

The preparation of step 3 2- bromomethyl -4H- chromene -4- ketone

By step 2 gained 2- methyl -4H- chromene -4- ketone (10mmol), N-bromosuccinimide (NBS, 10mmol) and Benzoyl peroxide (BPO, 0.95mmol) is added in reaction flask, and 20ml carbon tetrachloride is added to dissolve, back flow reaction 12h, reaction knot Shu Hou, reaction solution add water, and ethyl acetate extraction, anhydrous sodium sulfate is dry, and column chromatographic purifying obtains title compound.

ES:M/Z 239 [M+H]⁺。

The preparation of step 4 2- aminomethyl -4H- chromene -4- keto hydrochloride

Step 3 gained 2- bromomethyl -4H- chromene -4- ketone (0.5mmol) is added in reaction flask, it is molten that 5ml DMF is added Solution is added 2ml ammonium hydroxide, 12h is stirred at room temperature, and after reaction, water, ethyl acetate extraction is added in reaction solution, and anhydrous sodium sulfate is done Dry, filtering is spin-dried for, and ethyl acetate 5ml is added, and the ethyl acetate hydrogen chloride solution of saturation is added after stirring and dissolving to supernatant layer nothing Precipitating generates, and filters, dry, obtains title compound.

ES:M/Z 239 [M+H]⁺。

The preparation of chloro- 6- (4- the nitrobenzophenone) -1,3,5- triazine of 2 2,4- of embodiment bis-

The preparation of step 1 4- nitrobenzene methyl

4- nitrobenzoic acid (250mmol) is weighed in reaction flask, the dissolution of 300mL methanol is added, thionyl chloride is added dropwise (375mmol) drips and finishes back flow reaction 12h, and after reaction, decompression is spin-dried for, and saturated sodium bicarbonate solution is added and adjusts pH to 7- 8, ethyl acetate extraction, anhydrous sodium sulfate is dry, is concentrated to give title compound, directly throws in next step.

The preparation of step 2 6- (4- nitrobenzophenone) -1,3,5- triazine -2,4- (1H, 3H)-diketone

Biuret (100mmol) is weighed in reaction flask, the dissolution of 150mL glycol dimethyl ether is added, adds in batches at 0-5 DEG C Enter sodium hydride (83.4mmol), finish, be stirred to react 1h at 50 DEG C, adds 4- nitrobenzene methyl (83.4mmol), add Finish, be warming up to 85 DEG C of reaction 20h and be after reaction poured into water reaction solution, adjusts pH to acidity with concentrated hydrochloric acid, filter, filter Cake drying, obtains title compound.

ES:M/Z 235 [M+H]⁺。

The preparation of step 3 2,4- bis- chloro- 6- (4- nitrobenzophenone) -1,3,5- triazine

6- (4- nitrobenzophenone) -1,3,5-triazines -2,4- (1H, 3H)-diketone (200mmol) is added in reaction flask, is added Entering 200mL phosphorus oxychloride, phosphorus pentachloride (800mmol), reaction solution is poured into water by 105 DEG C of reaction 12h after reaction, and two Chloromethanes extraction, anhydrous sodium sulfate is dry, is concentrated to give title compound.

ES:M/Z 275 [M+H]⁺。

The chloro- 6- of 3 4- of embodiment (4- nitrobenzophenone)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine Preparation

Chloro- 6- (4- the nitrobenzophenone) -1,3,5- triazine (50mmol) of 2 gains 2,4- of embodiment bis- is weighed in reaction flask In, the dissolution of 100mL tetrahydrofuran is added, 1 gains 2- aminomethyl -4H- chromene -4- keto hydrochloride 2- (trifluoro of embodiment is added Methyl)-pyridine -4- amine (55mmol), sodium carbonate (100mmol), back flow reaction 72h, filtering, column chromatographic purifying obtains title compound Object.

ES:M/Z 416 [M+H]⁺。

The preparation of 4 4- of embodiment (4,4,5,5- tetramethyl -1,3,2- dioxolane borine) -9- ethyl -9H- carbazole

The preparation of the chloro- 9- ethyl -9H- carbazole of step 1 4-

4- chlorine carbazole (5.46mmol) is weighed in reaction flask, 20mL THF dissolution is added, is cooled to -10 DEG C, NaH is added (14mmol), finishes, and after stirring 30 minutes, adds bromoethane (6mmol), finishes, reacts at room temperature 3h, and reaction terminates, and water quenching is added to go out, Ethyl acetate extraction, dry, concentration is prepared chromatogram purification and obtains the chloro- 9- ethyl -9H- carbazole of 4-.

ES:M/Z 230 [M+H]⁺。

The preparation of step 2 4- (4,4,5,5- tetramethyl -1,3,2- dioxolane borine) -9- ethyl -9H- carbazole

Weigh the chloro- 9- ethyl -9H- carbazole (1mmol) of step 1 gains 4-, connection pinacol borate (1.1mmol), vinegar Sour potassium (2mmol) and 1,1'- bis(diphenylphosphino) ferrocene dichloropalladium (Pd (dppf) Cl₂, 2mmol) and in reaction flask, add Entering 5mL Isosorbide-5-Nitrae-dioxane, for 24 hours, after reaction, water is added in 100 DEG C of reactions under the conditions of nitrogen protection, and ethyl acetate extracts, Merge organic phase, saturated common salt water washing merges organic phase, dries, filters, and is concentrated, and column chromatographic purifying obtains title compound.

5 4- of embodiment (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine preparation

Step 1 4- (9- ethyl -9H- carbazole -4- base) -6- (4- nitrobenzophenone)-N- (2- methyl -4H- chromene -4- ketone Base) -1,3,5- triazine -2- amine preparation

In 30ml microwave reaction bottle, embodiment 3 gained mixture 4- chloro- 6- (4- nitrobenzophenone)-N- (2- is sequentially added Methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine (10mmol), 4 gains 4- (4,4,5,5- tetramethyl-of embodiment 1,3,2- dioxolane borine) -9- ethyl -9H- carbazole (10mmol), [bis- (diphenylphosphine) ferrocene of 1,1'-] dichloride Palladium dichloromethane complex (0.1mmol), x-phos (0.4mmol), cesium carbonate (100mmol) and 1,4- dioxane/H2O (60ml/10ml), dissolution, argon gas displacement, 105 DEG C of microwave reaction 90min, concentration, column chromatographic purifying obtain title compound.

ES:M/Z 575 [M+H]⁺。

Step 2 4- (9- ethyl -9H- carbazole -4- base) -6- (4- aminophenyl)-N- (2- methyl -4H- chromene -4- ketone Base) -1,3,5- triazine -2- amine preparation

Weigh step 1 gains 4- (9- ethyl -9H- carbazole -4- base) -6- (4- nitrobenzophenone)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5-triazines -2- amine (1mmol), 10%Pd-C (10mg) in reaction flask, be added 15ml methanol, at 1 Normal atmosphere pressure, H₂1h is restored, reaction is stopped, title compound is concentrated in filtering, is directly used in next step.

ES:M/Z 545 [M+H]⁺。

Step 3 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene - 4- ketone group) -1,3,5- triazine -2- amine preparation

Weigh step 2 gains 4- (9- ethyl -9H- carbazole -4- base) -6- (4- aminophenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5-triazines -2- amine (0.1mmol), diisopropylethylamine (0.3mmol) in reaction flask, be added 15ml Anhydrous methylene chloride dissolution, is slowly dropped into methylene chloride (1ml) solution dissolved with allyl acyl chlorides (0.12mmol), 10min Fully reacting, concentration, column chromatographic purifying obtain title compound.

¹H NMR(600MHz,CDCl₃) (δ, ppm): 10.12 (s, 1H), 8.12 (m, 1H), 7.80~7.82 (m, 2H), 7.78~7.79 (m, 1H), 7.67~7.69 (m, 2H), 7.50~7.52 (m, 2H), 7.43~7.45 (m, 2H), 7.31~ 7.32 (m, 2H), 7.07~7.09 (m, 2H), 6.97~6.99 (m, 1H), 6.68~6.70 (m, 1H), 6.02~6.04 (m, 1H), 5.56 (s, 1H), 5.50~5.52 (m, 1H), 4.88 (s, 1H), 4.46~4.51 (m, 3H), 3.06~3.08 (m, 2H), 2.81~2.84 (m, 2H), 2.01~2.03 (brs, 2H), 1.91~1.93 (brs, 1H), 1.30~1.31 (t, 3H)

ES:M/Z 599 [M+H]⁺。

Embodiment 6:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate preparation

5 compound of embodiment (0.5mmol) is weighed in reaction flask, adds ethyl alcohol 10mL, at room temperature after stirring and dissolving, is added dropwise Methanesulfonic acid acetone soln (containing methanesulfonic acid 0.5mmol) 1mL, drop finish, continue to stir 0.5h, evaporating solvent under reduced pressure, room temperature at room temperature Lower vacuum drying obtains title compound.

Embodiment 7:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate preparation

5 compound of embodiment (0.5mmol) is weighed in reaction flask, adds ethyl acetate 10mL, at room temperature after stirring and dissolving, Methanesulfonic acid acetone soln (contain methanesulfonic acid 0.55mmol) 1mL is added dropwise, drop finishes, continue to stir 0.5h at room temperature, evaporating solvent under reduced pressure, Vacuum drying obtains title compound at room temperature.

Embodiment 8:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form preparation

Weigh 5.0g4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) in reaction flask, the alcohol-water that addition 10mL volume ratio is 3:1 mixes -1,3,5-triazines -2- amine mesylate Solution, flow back 10min, cooled to room temperature, and 0-5 DEG C of crystallization 12h obtains 4- (9- ethyl -9H- carbazole -4- base) -6- (4- Allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form.

Embodiment 9:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form preparation

Weigh 1.0g4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) in reaction flask, the methanol-water that addition 12mL volume ratio is 4:1 mixes -1,3,5-triazines -2- amine mesylate Solution, flow back 10min, cooled to room temperature, and 0-5 DEG C of crystallization 12h obtains 4- (9- ethyl -9H- carbazole -4- base) -6- (4- Allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form.

Embodiment 10:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form preparation

Weigh 1.0g4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) in reaction flask, the alcohol-water that addition 8mL volume ratio is 2:1 mixes -1,3,5-triazines -2- amine mesylate Solution, flow back 10min, cooled to room temperature, and 0-5 DEG C of crystallization 12h obtains 4- (9- ethyl -9H- carbazole -4- base) -6- (4- Allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form.

Embodiment 11:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form preparation

Weigh 1.0g4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) in reaction flask, the methanol-water that addition 12mL volume ratio is 3:1 mixes -1,3,5-triazines -2- amine mesylate Solution, flow back 10min, cooled to room temperature, and 0-5 DEG C of crystallization 12h obtains 4- (9- ethyl -9H- carbazole -4- base) -6- (4- Allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form.

4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- of above embodiments 8-11 preparation (2- methyl -4H- chromene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form through measurement there is essentially identical X to penetrate Ray diffraction diagram spectrum, is shown in Fig. 1.

Embodiment 12: biological activity determination

Compound prepares: the compound that the full-automatic microwell plate pretreatment system of POD810 is prepared the embodiment of the present invention 6 It is added in orifice plate, compound initial concentration is 100uM, and each compound does duplicate hole, 2 times of dilutions, 10 points.

The culture of cell: prostate gland cancer cell DU-145, human breast cancer cell MDA-MB-468 are used contain 15% tire respectively The RPMI-1640 culture medium of cow's serum (FBS) is cultivated in 37 degree of incubators, and logarithmic growth phase cell is for testing.

The experiment of MTT cells viability: prostate gland cancer cell DU-145, human breast cancer cell MDA-MB-468 are inoculated with respectively In 96 orifice plates (5-10 × 104cells/well), handle 48h with the compound of embodiment 6, be added in every hole 20 μ l 3- (4, 5- dimethylthiazole -2) -2,5- diphenyltetrazolium bromide bromide (MTT) hatching 4h, 100 μ l DMSO in every hole is then added, sets and shakes Low-speed oscillation 10min on bed, dissolves crystal sufficiently.The extinction in each hole is measured at enzyme-linked immunosorbent assay instrument OD 490nm Value, handles to obtain homologous thread and IC using GraphPad Prism₅₀Value, the results are shown in Table 1.

Table 1

The experimental results showed that 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl - 4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate is to the active MDA-MB-468 of STAT3 with sustained activation Cell, DU-145 cell have good inhibiting effect.

Embodiment 13: water solubility evaluation

By pharmacopeia four water-soluble experiments in 2015, experimental result was shown in Table 2.

Table 2

Compound	It is water-soluble
		8 compound of embodiment	42.81mg/ml

Embodiment 14:4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form stability test

Weigh 3 parts of 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- color Alkene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form 0.5g, it is laid in culture dish respectively, 1 part of opening is placed in light It is placed under the conditions of being 4500Lx ± 500Lx according to illumination 10 days, 1 part of opening is placed under the conditions of being placed in 85% relative humidity of room temperature (RH) 10 days, 1 part of opening was placed 10 days under the conditions of being placed in 95% relative humidity of room temperature (RH), is sampled in the 0th, 5,10 day, and examination is maximum Single miscellaneous, total impurities, crystal form and appearance variations, experimental result are shown in Table 3.

Table 3

Above-mentioned experimental result shows 4- of the invention (9- ethyl -9H- carbazole -4- base) -6- (4- acrylyl aminobenzene Base)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form have good chemical stabilization Property and physical stability.

Claims

1.4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) - The A crystal form of 1,3,5-triazines -2- amine mesylate, is radiated using Cu-Ka, and X-ray powder diffraction is 3.7 in 2 θ of the angle of diffraction It is shown at ± 0.2 °, 4.5 ± 0.2 °, 11.7 ± 0.2 °, 13.6 ± 0.2 °, 19.5 ± 0.2 °, 22.9 ± 0.2 ° and 26.0 ± 0.2 ° Show characteristic peak.

2. A crystal form as described in claim 1, it is characterised in that: radiated using Cu-Ka, X-ray powder diffraction is in diffraction 2 θ of angle be 3.7 ± 0.2 °, 4.5 ± 0.2 °, 6.0 ± 0.2 °, 10.4 ± 0.2 °, 11.7 ± 0.2 °, 13.6 ± 0.2 °, 15.0 ± Characteristic peak is shown at 0.2 °, 19.5 ± 0.2 °, 22.9 ± 0.2 °, 26.0 ± 0.2 °.

3. the preparation method of A crystal form of any of claims 1 or 2, includes the following steps:

Step h: the compound and 4- (4,4,5,5- tetramethyl -1,3,2- dioxolane borine) -9- ethyl -9H- click of formula (7) Azoles reacts the compound that formula (8) are made；

Step k: the compound of formula (I) is reacted with methanesulfonic acid is made formula (I) compound methanesulfonic acid salt, and gained mesylate is added In alcohol-water mixed solvent, reflux, 0-5 DEG C of crystallization is to get 4- (9- ethyl -9H- carbazole -4- base) -6- (4- acrylyl aminobenzene Base)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5-triazines -2- amine mesylate A crystal form, reaction route is as follows:

4. preparation method as claimed in claim 3, it is characterised in that: formula (I) compound reacts molten with methanesulfonic acid in step k Agent is selected from alcohol, ketone, ethyl acetate or their mixture；Preferably, the solvent that formula (I) compound is reacted with methanesulfonic acid is selected from first Alcohol, ethyl alcohol, normal propyl alcohol, isopropanol, acetone, methyl ethyl ketone, methyl iso-butyl ketone (MIBK), ethyl acetate or their mixture；Into Preferably, the solvent that formula (I) compound is reacted with methanesulfonic acid is selected from methanol, ethyl alcohol, acetone, ethyl acetate or theirs is mixed for one step Close object.

5. preparation method as claimed in claim 3, it is characterised in that: what formula (I) compound was reacted with methanesulfonic acid in step k rubs Your ratio is about 1:1-1.1；Preferably, the molar ratio that formula (I) compound is reacted with methanesulfonic acid is about 1:1.

6. preparation method as claimed in claim 3, it is characterised in that: alcohol described in step k is selected from methanol or ethyl alcohol, pure and mild The volume ratio of water is 2~4:1；Preferably, the alcohol is ethyl alcohol, and the volume ratio of alcohol and water is in the alcohol-water mixed solvent 3:1.

7. preparation method as claimed in claim 3, it is characterised in that: alcohol-water mixed solvent usage amount described in step F For every gram of 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) - 1,3,5- triazine -2- amine mesylate uses alcohol-water mixed solvent 8-12mL；Preferably, alcohol described in step F-water mixing The usage amount of solvent is every gram of 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl)-N- (2- methyl -4H- Chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate use alcohol-water mixed solvent 10mL.

8. a kind of pharmaceutical composition includes the described in any item 4- of claim 1-2 (9- ethyl -9H- carbazole -4- base) -6- (4- Allyl amido phenyl)-N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form and pharmacy Upper acceptable carrier.

9. claim 1-2 described in any item 4- (9- ethyl -9H- carbazole -4- base) -6- (4- allyl amido phenyl) - N- (2- methyl -4H- chromene -4- ketone group) -1,3,5- triazine -2- amine mesylate A crystal form or medicine group according to any one of claims 8 Close application of the object in the drug of preparation prevention and/or treatment tumour.