WO2020011020A1

WO2020011020A1 - Mefatinib composition, related compound, preparation method therefor and use thereof

Info

Publication number: WO2020011020A1
Application number: PCT/CN2019/093051
Authority: WO
Inventors: 吕裕斌; 黄雪惠; 金燕芬; 殷建明; 李邦良
Original assignee: 杭州华东医药集团生物医药有限公司
Priority date: 2018-07-10
Filing date: 2019-06-26
Publication date: 2020-01-16
Also published as: CN108853109B; CN108853109A

Abstract

A mefatinib composition, related compounds, a preparation method therefor and use thereof. The mefatinib composition comprises mefatinib represented by formula (I) and a compound represented by formula (II) having a normalized percentage content of 1.0% or less but not zero. The mefatinib composition can be used to treat or prevent a variety of indications associated with EGFR and HER2 kinase functions, and the incidence of toxic response is small. The compound of formula (II) can be used, as a standard for related materials in mefatinib, for quality control of mefatinib active pharmaceutical ingredients or formulations.

Description

Maihuatinib composition, related compounds, preparation method and application thereof

Technical field

The invention relates to a mehuatinib composition, related compounds, a preparation method and uses thereof.

Background technique

Mefatinib, its chemical name is: 2-butenal amino, N- [4-[(3-chloro-4-fluorophenyl) amino] -7-difluoromethyloxy]- 6-quinazolinyl] -4- (dimethylamino)-(2E) -dimaleate; molecular formula: C ₂₁ H ₁₉ ClF ₃ N ₅ O ₂ · 2C ₄ H ₄ O ₄ ; molecular weight: 698.0 The structural formula is as follows:

Patent document CN102838550A discloses a quinazoline crotonyl compound (Mevatinib free base) represented by the following formula,

The compound proved to be an ideal and highly effective dual non-reversible tyrosine kinase inhibitor, which can competitively bind to ATP by acting on the intracellular part of EGFR, inhibit the activity and phosphorylation of the kinase, and block EGFR tyrosine kinase ATP Binding site to achieve the purpose of specifically inhibiting EGFR. The compound can be used to treat or prevent various indications related to EGFR and HER2 kinase function, including but not limited to breast cancer, ovarian cancer, gastrointestinal cancer, esophageal cancer, lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, epidermal scale Cancer, prostate cancer, glioma, and nasopharyngeal cancer.

The above-mentioned patent document CN102838550A Example 1 discloses a method for synthesizing mehuatinib free base, specifically obtained through the following synthetic route:

CN105859641 uses the mehuatinib free base prepared in CN102838550A to prepare mehuatinib crystals.

In the above route, the total molar yield from compound 2 to mehuatinib free base is only about 37%. In the preparation of Formula 1b, the raw materials and products are insoluble in organic solvents, and are always in a suspension state, which is not conducive to reaction monitoring. The reaction from compound 1 to the free base of mehuatinib is an anhydrous reaction, and the conversion rate is only about 38%. Moreover, Compound 1 cannot be completely converted, the purity of the crude product is less than 70%, there are many impurities, and the product-related substances are complicated. The obtained crude mehuatinib free base is an oil and is difficult to solidify. It must be purified by column chromatography, and the batch yield is low. , Only suitable for the scale of 10g level. Overall, the method has low yield, difficult purification, high cost, and low batch yield. Therefore, the synthetic process and the resulting product are not suitable for industrial applications.

Patent document CN104151359A discloses another synthetic route of mehuatinib free base (that is, the compound of formula A10 in the following synthetic route), specifically as follows:

This synthetic route has the following advantages: In the step of preparing A10E from A9, adding an appropriate solvent after the reaction can precipitate the resulting product, which is easy to handle and has high purity; in the step of preparing A10 from A10E, the reaction is in the organic phase / It is carried out in the water phase. After the reaction is completed, water can be added to precipitate the product, which is easy to handle and has high purity. The crude product content is above 96% and the yield is close to 100%. The total reaction yield from A9 to A10 is greater than 60%. The product obtained by this process route does not need to be purified by column chromatography, and is suitable for large-scale industrial production.

Summary of the invention

As mentioned above, the free mehuatinib product disclosed in CN102838550A and its synthetic process are not suitable for large-scale industrial production. The inventor used the product A10 in the patent document CN104151359A, which is suitable for large-scale industrial production, to prepare its dimaleate, and optimized the process to obtain a high-purity dimaleate. During mass production, product yield and quality are stable. However, the study found that the related substances contained in A10 prepared by the synthetic route of patent document CN104151359A are different from the related substances contained in the product obtained in CN102838550A. In particular, A10 in CN104151359A contains a small amount of cis isomer, so that the dimaleate obtained contains the cis isomer of dimaleate, that is, the compound represented by formula (II). Further research found that, compared to mehuatinib, compounds of formula (II) have a significant inhibitory effect on normal cells, produce strong toxic and side effects, and have a weak inhibitory effect on tumor cells. Therefore, it is of great significance to control the content of the compound of formula (II) in the product obtained by this process. The present inventors completed the present invention by controlling the content of the compound of formula (II) in the product obtained by the synthesis process to a specific range, reducing the toxic and side effects, and bringing the toxic and side effects of the obtained mehuatinib composition into an acceptable range. invention.

To this end, in a first aspect, a mehuatinib composition is provided, characterized in that the composition comprises mehuatinib represented by formula (I) and the normalized percentage content is 1% or less and A compound represented by formula (II) which is zero,

Optionally, the normalized percentage content of the compound represented by formula (II) in the Myhuatinib composition is 0.9% or less, optionally 0.8% or less, optionally 0.5% or less, optionally Ground is 0.3% or less, optionally 0.2% or less.

Optionally, from the perspective of production cost and actual needs, the normalized percentage content of the compound represented by formula (II) in the above-mentioned mehuatinib composition may be 0.01% or more, optionally 0.02% or more, Optionally, it is 0.05% or more.

Optionally, in the mehuatinib composition, the normalized percentage of mehuatinib represented by formula (I) is 98.0% or more, preferably 98.5% or more, and more preferably 99.0% or more It is more preferably 99.3% or more, and still more preferably 99.5% or more.

In a second aspect, a method for preparing the above-mentioned mehuatinib composition is provided, which includes:

(1) Prepare A10 by referring to the preparation method disclosed in patent CN104151359A;

The preparation method of the A10 adopts the following synthetic route,

Each R in the above structural formula is independently a C _1-6 alkyl group, preferably a methyl group or an ethyl group, and more preferably an ethyl group.

In the step of preparing the A9 compound, the A8 compound is reacted with hydrogen in the presence of a catalyst. The catalyst may be a hydrogenation catalyst commonly used in the art, such as Raney nickel, platinum, palladium, rhodium and the like, and Raney nickel catalyst is preferred. The reaction is preferably performed in the presence of ammonium chloride, preferably the mass ratio of the A8 compound to the ammonium chloride is 1-20: 1, more preferably 8-12: 1, and still more preferably 10: 1. Preferably, the reaction solvent is selected from N ′, N′-dimethylformamide (DMF), tetrahydrofuran, a mixed solvent of tetrahydrofuran and methanol; preferably a mixed solvent of tetrahydrofuran and methanol, and more preferably a tetrahydrofuran to methanol volume ratio of 3 A mixed solvent of -8: 1, more preferably a mixed solvent of tetrahydrofuran to methanol in a volume ratio of 5: 1. Preferably, the obtained A9 compound is dried under vacuum to less than 1% moisture.

Preferably, the A8 compound is added to the reaction kettle, and a mixed solvent of tetrahydrofuran and methanol is added to the reaction kettle, and the mixture is dissolved by stirring. After adding Raney nickel and ammonium chloride, the temperature is increased, and hydrogen gas is passed through after nitrogen substitution, and the reaction is maintained while stirring. After the reaction was completed, the mixture was filtered, and the filtrate was washed twice with saturated brine. The aqueous layers were combined and back-extracted with an appropriate amount of tetrahydrofuran. The organic layers were combined and spin-dried, and dried under vacuum to less than 1.0%.

In the preparation step of the A10E compound, a reaction between the A9 compound and dialkylphosphoacetic acid is performed in the presence of N, N'-carbonyldiimidazole (CDI). Preferably, the amounts of N, N'-carbonyldiimidazole (CDI) and dialkylphosphoacetic acid are each 1.5 times equivalent to 3 equivalents of the A9 compound, more preferably 2.05 to 2.22 equivalents each, and more preferably 2 each. Times equivalent. Preferably, the reaction solvent is selected from tetrahydrofuran, toluene, dioxane, DMF, dichloromethane, and the like, and tetrahydrofuran is preferred. The reaction product is preferably slurried with tetrahydrofuran, methyl tert-butyl ether, methyl tert-butyl ether + tetrahydrofuran, DMF + water, dioxane, ethyl acetate, dichloromethane or ethanol. Tetrahydrofuran, methyl tert-butyl ether, methyl tert-butyl ether + tetrahydrofuran, DMF + water are preferably used for slurry washing. The reaction temperature is preferably from ordinary temperature to 50 ° C.

Preferably, CDI is added to the reaction kettle, and then THF is added, stirred and heated. Diethylphosphoacetic acid was dissolved in an appropriate amount of THF, added dropwise to the reaction kettle, and the mixture was stirred for later use.

A9 was dissolved in THF, the temperature was raised, and the above mixture was added dropwise to the THF solution of A9. After the dropwise addition was completed, the reaction was kept warm and stirred. After the reaction was completed, methyl tert-butyl ether was added to the reaction system and stirred. After filtration, the filter cake was washed with an appropriate amount of a mixed slurry of MTBE: THF = 1: 1.2 (w / w), and then with water, and then with an appropriate amount of a mixed slurry of MTBE: THF = 1: 1.2 (w / w). Vacuum dried.

In the preparation step of the A10 compound, dimethylaminoacetaldehyde dialkanol is first converted into N, N-dimethylaminoacetaldehyde, and then the reaction is performed in the presence of a base. Preferably, the base is selected from sodium hydroxide, potassium hydroxide, sodium alkoxide, potassium alkoxide, and sodium tert-butoxide is preferred. The amount of sodium tert-butoxide is preferably 10 times equivalent to 15 equivalents, and more preferably 10 times equivalent. The amount of dimethylaminoacetaldehyde dialkanol used is preferably 2 times to 6 equivalents, and preferably 4 times the equivalent, of the A10E compound. The reaction temperature is preferably -30 to -20 ° C.

Preferably, the concentrated hydrochloric acid is diluted with water, transferred to a reaction kettle, and heated. Under the protection of nitrogen, dimethylaminoacetaldehyde diethanol was slowly added dropwise to the diluted hydrochloric acid, and the reaction was stirred after the addition was completed. Neutralize to neutral with solid sodium carbonate before use, and filter off the inorganic salt for later use.

Add A10E and THF to the reaction kettle, stir, and lower the temperature to -30 ° C to -20 ° C. The sodium tert-butoxide was added to the reactor in batches, the temperature was maintained at -30 ° C to -20 ° C, and the stirring was completed after the addition. At -30 ° C to -20 ° C, an aqueous aldehyde solution is added dropwise to carry out the reaction. After the reaction is completed, transfer to a post-treatment kettle with an appropriate amount of water in advance, stir, filter, and wash the filter cake with purified water to neutrality, then wash with an appropriate amount of n-heptane slurry, and dry under vacuum.

(2) Preparation of dimaleate;

The A10 compound is reacted with a maleate in a solvent to obtain a dimaleate product. The solvent is a mixed solvent of ethyl acetate and water, wherein the mass ratio of water to ethyl acetate is 1-4: 20, and preferably 1:10. 1 to 4 parts by mass of water, preferably 2 parts by mass of water, is used per part by mass of the A10 compound. When the ratio and amount of water are too small, the content of mehuatinib is lower and the compound of formula (II) is higher.

Preferably, ethyl acetate and water are added to the reaction kettle and the temperature is raised. Add A10 to the reaction kettle and stir until completely dissolved. The maleic acid was dissolved in an appropriate amount of ethyl acetate, and the ethyl acetate solution of maleic acid was added dropwise to the reaction kettle to perform the reaction. After the reaction was completed, the temperature was lowered and the crystals were stirred and crystallized. After filtration, the filter cake was washed with a small amount of ethyl acetate slurry and dried under vacuum to obtain the dimaleate (Mevatinib composition).

Optionally, the following (3) recrystallization step is further included.

(3) recrystallization;

The dimaleate product is recrystallized using a solvent, the solvent is a mixed solvent of ethyl acetate and water, wherein the mass ratio of water to ethyl acetate is 1-4: 20, preferably 1:10. 1 to 4 parts by mass of water, preferably 2 parts by mass of water, is used per part by mass of the dimaleate. The recrystallization temperature is preferably 45-65 ° C, and more preferably 50 ° C. It is preferred to add a mehuatinib seed during crystallization.

Preferably, ethyl acetate and water are added to the reaction kettle and the temperature is raised. Bubbling nitrogen, the dimaleate was added to the reaction kettle, stirred until fully dissolved, lowered to room temperature, crystallized, filtered, the filter cake was washed with a small amount of ethyl acetate slurry, and dried under vacuum.

During the salt formation and recrystallization, the crystallization is preferably performed under the protection of nitrogen, which can improve the purity.

In a third aspect, the use of the above-mentioned mehuatinib composition for preparing a medicament for preventing or treating an indication related to EGFR and HER2 kinase function is provided, and the above-mentioned mehuatinib composition is used for preventing or treating the Use of EGFR and HER2 kinase function-related indications.

Optionally, the indications related to EGFR and HER2 kinase functions include breast cancer, ovarian cancer, gastrointestinal cancer, esophageal cancer, lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, epidermal squamous cell carcinoma, prostate cancer, and neuroglia Plasmoma and nasopharyngeal carcinoma;

The pharmaceutical formulation of the drug includes the above-mentioned mehuatinib composition and a pharmaceutically acceptable excipient, which can be administered in a single dose or in multiple doses by any acceptable method of administration of a medicament having similar uses, such as rectum Oral (including buccal or sublingual), intranasal and transdermal routes, or by intra-arterial injection, intravenous, intraperitoneal, intramuscular, subcutaneous, oral, topical, as an inhalant.

The dosage forms of the drugs for preventing or treating indications related to the function of EGFR and HER2 kinase include liquid preparations, gas preparations, solid preparations and semi-solid preparations, and suitable preparations include tablets (including enteric-coated tablets) , Pills, powders, lozenges, capsules, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, ointments, soft and hard gelatin capsules, sterile injectable solutions and sterile Packaging powder form. It may be a formulation in which the active ingredient is released rapidly, continuously, or delayed.

In a fourth aspect, a compound represented by the formula Z-A10 is provided,

In a fifth aspect, a compound represented by formula (II) is provided,

The compound of the formula (II) can be used as a standard (reference substance) for detecting and controlling the content of the compound of the formula (II) in the mehuatinib composition to ensure the quality and safety of the product.

According to a sixth aspect of the present invention, a method for preparing a compound of formula Z-A10 or a compound of formula (II) is provided, wherein the compound of formula Z-A10 has the following synthetic route,

A method for preparing a compound of formula Z-A10 includes: reacting 1-dimethylamine-2-propyne with carbon dioxide (preferably dry ice) in the presence of a base to prepare 4- (dimethylamino) -2-propargyric acid, Then 4- (dimethylamino) -2-propargylic acid was reacted with A9 in the presence of isobutyl chloroformate and N-methylmorpholine to prepare Z-A10-01 compound. Z-A10-01 compound is hydrogenated in the presence of Lindlar catalyst to prepare Z-A10 compound;

The synthetic route of the compound of formula (II) is as follows:

The method for preparing the compound of formula (II) includes: after preparing the compound of formula Z-A10 as described above, salting the compound of formula Z-A10 with maleic acid to obtain the compound of formula (II).

In a seventh aspect, a compound of formula (II) is provided as a standard (reference) for use in detecting a compound of formula (II) or its content in a mehuatinib composition.

According to an eighth aspect, a method for detecting the content of a compound of formula (II) in a mehuatinib composition is provided. The method is characterized by using a standard compound of formula (II) as a reference substance for detection by high performance liquid chromatography. Preferably, the mobile phase of high-performance liquid chromatography is disodium hydrogen phosphate-phosphate buffer solution, methanol, or a mixed solvent of acetonitrile and water, and gradient elution is preferably performed using disodium hydrogen phosphate-phosphate buffer solution and methanol.

Beneficial effects:

The provided mehuatinib composition and the preparation method thereof are suitable for industrial production, and the content of specific related substances, namely the compound of formula (II), is low. The inventors have conducted in-depth research on relevant substances in the mehuatinib composition that can be industrially produced, and the toxic and side effects of the provided mehuatinib composition are significantly reduced, and the acute toxicity test shows that the toxic and side effects are within an acceptable range.

The provided compound of formula (II) can be used as a standard (reference) for detecting and controlling the content of related substances in the mehuatinib composition, and it can guarantee the quality of mehuatinib APIs and preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a ¹ HNMR spectrum of a compound of formula (II).

2 is a HPLC chromatogram of a test solution in Example 4. FIG.

3 is an HPLC chromatogram of a cis-isomer control solution in Example 4. FIG.

4 is an HPLC overlay chromatogram of a cis-isomer control solution and a test solution in Example 4. FIG.

detailed description

Hereinafter, specific embodiments of the present invention will be described in detail. It should be understood that the specific embodiments described herein are only used to describe the present invention by way of example, and are not intended to limit the present invention.

The used mehuatinib free base (A10) was prepared according to the method disclosed in patent document CN104151359A.

Example 1 Preparation of Myhuatinib Composition

1.Salt formation step

479.7 g of ethyl acetate and 812.6 g of purified water were added to the reaction kettle, and the temperature was raised to about 50 ° C. Then 406.3 g of A10 was added to the reaction kettle and stirred to completely dissolve; 203.1 g of maleic acid was added to 1145.5 g. In ethyl acetate, stir to dissolve it all; add the ethyl acetate solution of maleic acid to the reaction kettle at 50 ° C. After the dropwise addition, continue to stir for 1 hour, naturally cool down, stir the liquid to crystallize, and filter. The filter cake was washed with 3.44 Kg of ethyl acetate, and dried under vacuum at 35 ° C to obtain a crude Maihuatinib.

2.Recrystallization step

10.51Kg of ethyl acetate and 1.05Kg of water were added to a 10L reaction kettle, the temperature was raised to 50 ° C, 525.7g of the crude Maihuatinib prepared above was added, and the mixture was stirred until fully dissolved and then cooled to room temperature. Add 3.0 g of mehuatinib seed crystals, and then continue to crystallize after stirring for 4 h. After filtration, the filter cake was washed with 1.60 Kg of ethyl acetate, and dried under vacuum at 35 ° C. overnight to obtain 503.4 g of a mehuatinib composition with a yield of 95.8% and a normalized purity of 99.2% (lot number MET306-111041).

With reference to the above method, two additional batches of mehuatinib composition were prepared with normalized purity of 99.2% (lot numbers MET306-111043, MET306-111061).

Referring to the above salt formation steps, a toxicological batch of mehuatinib composition was prepared separately, with a normalized purity of 98.5% (toxicological batch).

Example 2 Salt formation and crystallization conditions

Referring to the method of salt formation in Example 1, the mass ratio of fixed ethyl acetate to water was about 10: 1, and the input amount and results are shown in the table below.

The results show that the amount of water added has a great effect on the purity of the product and the content of the residual compound of formula (II) in the product. Adding more water will help to improve the purity of the product and reduce the content of the compound of formula (II). However, if the amount of water is too large, the compound of formula (II) will be generated instead. In addition, the amount of water added also has an impact on the yield of the product, but tests have found that stirring overnight can ensure that the yield is above 90%.

Referring to the recrystallization method of Example 1, the amount ratio of crude Maihuatinib, ethyl acetate and water, and the purity of the obtained product are shown in the following table.

The results showed that too much ethyl acetate would lead to a decrease in purity. However, if the amount of ethyl acetate is too small, crystal precipitation is not favorable.

Comparative Example 1 refers to the crystallization process in patent CN105859641 to prepare a mehuatinib composition

In a three-necked flask, 10.22 g of mehuatinib free base was added, 17 ml of ethyl acetate was added, and the solution was turbid by mechanical stirring and heating to 50 ° C. A 0.15 mol / L ethyl acetate solution of maleic acid was added dropwise to Precipitation (approximately 125ml) was added, then 4.1ml of deionized water was added, followed by the addition of about 175ml of a 0.15mol / L maleic acid ethyl acetate solution at a rate of 1ml / min with a syringe, and the temperature was kept at about 50 ° C and the temperature was stirred for 15 After being allowed to cool down to room temperature for hours, it was filtered with suction and dried under vacuum at 35 ° C. for 5 hours to obtain the crystal form K of mehuatinib dimaleate. The purity by HPLC was 98.23%.

Example 3 Synthesis of a compound of formula (II) (cis isomer)

Step 1: Synthesis of 4- (dimethylamino) -2-propargyric acid

(At -78 ° C, n-butyllithium hexane solution (2.5M, 33.4mmol, 13.4ml) was slowly added dropwise to 1-dimethylamine-2-propyne (33.4mmol, 3.6ml, 2.78g) In dry tetrahydrofuran (15ml) solution. The resulting mixture was stirred at -78 ° C for 1 hour. Crushed dry ice (335mmol, 11.72g) was added in one portion and stirred for 15 minutes. The reaction solution was poured into 300ml water and 100ml ethyl acetate was used. After washing three times, the aqueous phase was concentrated under reduced pressure. The resulting residue was dissolved in methanol, and the insoluble salt was removed by filtration. The filtrate was concentrated under reduced pressure to obtain 6.05 g of crude 4- (dimethylamino) -2-propargylic acid containing a large amount of inorganic salt.

MS (ESI) m / z = 128.2 [M + 1].

Step 2: Synthesis of intermediate Z-A10-01

Add 3 g of the crude 4- (dimethylamino) -2-propargylic acid obtained above, 300 ml of tetrahydrofuran, and N-methylmorpholine (31.8 mmol, 3.22 g) to the reaction flask, and add isobutyl chloroformate at -10 ° C. Esters (12.7 mmol, 1.74 g), incubated for 30 minutes. A solution of A9 (8.3 mmol, 2.94 g) in 80 ml of pyridine was added dropwise, and stirring was continued for 1 hour. 200 ml of ice water and 200 ml of a saturated sodium bicarbonate solution were added. Extracted with ethyl acetate (300ml × 3), dried the organic phase over anhydrous sodium sulfate, filtered, and concentrated the filtrate under reduced pressure. The residue was separated and purified by silica gel column chromatography (methanol / ethyl acetate (v / v) = 1: 10 ) To obtain Z-A10-01 (2.4 mmol, 1.12 g), yield: 28.9%.

MS (ESI) m / z = 464.2 [M + 1].

Step 3: Synthesis of intermediate Z-A10

Z-A10-01 (0.2 mmol, 92.8 mg) and 20 ml of methanol were added to the reaction flask. Lindlar catalyst (12 mg) was continuously added. The system was evacuated and replaced with hydrogen 3 times. The reaction was hydrogenated at room temperature for 16 hours. Filtered with celite, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column chromatography (methanol / ethyl acetate (v / v) = 1:10) to obtain Z-A10 (0.13 mmol, 60 mg). Yield: 65%.

MS (ESI) m / z = 466.9 [M + 1].

Step 4: Synthesis of compound of formula (II)

In a three-necked flask, add Z-A10 (0.16 mmol, 75 mg) and 0.2 ml of ethyl acetate, heat to 50 ° C, add 0.5 ml of a 0.1 mol / L ethyl acetate maleate solution, and then add 0.15 ml of deionized water Then add about 0.8ml of 0.1mol / L maleic acid ethyl acetate solution at a speed of 1ml / min with a syringe, keep it stirred at a temperature of about 50 ° C for 2 hours, naturally lower the temperature to room temperature, suction filter and vacuum at 35 ° C Drying for 5 hours gave 101 mg of the compound of formula (II). The ¹ HNMR spectrum and H atom assignment are shown in FIG. 1.

Example 4 Identification of a compound of formula (II) in a mehuatinib composition

Take an appropriate amount of the mehuatinib composition (prepared according to the method of Example 1), add acetonitrile: water (1: 1) to dissolve it to prepare a test solution.

Another appropriate amount of the cis-isomer reference substance was added to the acetonitrile: water (1: 1) solution to prepare a control solution.

Take the test solution and inject it into the liquid chromatograph. The chromatographic conditions are: using octadecylsilane bonded silica as a filler; the detection wavelength is 245nm; the column temperature is 30 ° C; and 5mM disodium hydrogen phosphate-phosphate buffer solution (take 1.78g disodium hydrogen phosphate, add 1000ml purified water) Dissolve, stir and adjust the pH to 7.4 with a phosphoric acid solution after stirring uniformly. Use mobile phase A as methanol; use methanol as mobile phase B, and perform gradient elution according to the following table. Record the chromatogram, see Figure 2.

时间(分钟)Time (minutes)	流动相A(％)Mobile phase A (%)	流动相B(％)Mobile phase B (%)
00	4040	6060
55	2525	7575
1818	22twenty two	7878
24twenty four	55	9595
2828	55	9595

Take the control solution into the liquid chromatograph, perform gradient elution according to the above method, and record the chromatogram, as shown in Figure 3.

Take the control solution and the test solution separately, mix them and inject them into the liquid chromatograph, and perform gradient elution according to the above method. Record the overlay chromatograms of the mehuatinib composition and the reference product, as shown in Figure 4.

The results show that under this chromatographic condition, mehuatinib and its cis isomer can be separated well, and the retention times are about 14.7min and 16.7min, respectively. In the overlay chromatogram, a single superimposed peak appeared at a retention time of about 16.7 minutes, indicating that a component consistent with the cis isomer peak position exists in the Myhuatinib composition. For further confirmation, the component corresponding to the peak position was collected, and concentrated under reduced pressure to perform LCMS detection. The measured molecular ion peak m / z = 466.2, which is consistent with the cis isomer.

Example 5 Application of a compound of formula (II) as a reference

Take appropriate amounts of different batches of mehuatinib composition, accurately weigh them, and add an acetonitrile: water (1: 1) solution to prepare a test solution with a concentration of 0.5 mg / ml.

In addition, take appropriate amounts of Maihuatinib reference substance and cis-isomer reference substance, accurately weigh, add acetonitrile: water (1: 1) solution to prepare a reference solution mixed solution, where the concentration of Maihuatinib and cis-isomer is Both were 5 μg / ml.

Take 10 μl of the reference solution and inject it into the liquid chromatograph. The detection conditions are the same as in Example 4. The peaks of mehuatinib and cis-isomer appeared sequentially.

The test solution and 10 μl were taken and injected into a liquid chromatograph, and the detection conditions were the same as in Example 4. Record the chromatogram. If there is a peak in the chromatogram of the test solution that is consistent with the retention time of the cis-isomer reference, the cis-isomer content is calculated from the peak area according to the normalization and the self-control method.

That is, by using the compound of formula (II) as a reference substance for positioning, the normalized content of the cis isomer and the self-control content of the mehuatinib composition can be determined. In addition, the compound of formula (II) can also be used as a reference substance to calculate the weight content of cis isomers in mehuatinib using an external standard method.

Test example 1, cytostatic test

Cells: NCI-H292 was purchased from the Cell Bank (SIBCB) of Shanghai Academy of Life Sciences, Chinese Academy of Sciences, NCI-H1650 was purchased from ATCC, and A549 was a gift from University of Michigan. The human small intestinal mucosal epithelial cell line was isolated from the small intestine of an aborted fetus. The cells were all cultured at 37 ° C in a 5% CO ₂ incubator. The cell lines tested were in RPMI-1640 medium containing 10% fetal bovine serum.

Test sample: a mehuatinib composition (prepared by the method of Example 1, the normalized content of the compound of Formula II is 0.015%), and a compound of the formula II (prepared by the method of Example 3).

experimental method:

Firstly, the compound of formula II and the mehuatinib composition were dissolved in 100% DMSO, and then diluted to the required concentration with the medium. Five concentration gradients of 0.001, 0.01, 0.1, 1, 10 μM were prepared. 0.1%. A 0.1% DMSO medium solution was used as a negative control, and cells were seeded in a 0.1% DMSO medium solution as a cell control.

WST method single drug test experiment

Dosing (protection from light): In 96-well culture plates, make 3 replicates of each experimental dose, 100 μl / well, 5 dose concentrations each of the compound of formula II and the mehuatinib composition; cell control wells and negative controls The wells were replaced with 0.1% DMSO medium solution.

Seeding cells (protected from light): In addition to the negative control wells, add 100 μl of complete medium cell suspension to each well to be tested, which contains the corresponding number of cells to ensure that the cells of the cell control group are just full when the time required for culture is reached Hole bottom surface. The negative control well was replaced with 100 μl of 0.1% DMSO medium solution.

Incubate in a CO ₂ incubator at 37 ° C for 72 hours. Aspirate the liquid in the well to be measured. Add 100 μl of CCK-8 detection solution to each test well, and incubate in a CO ₂ incubator at 37 ° C for 3-4 hours. The microplate reader A450nm detects the OD value.

The cell survival percentage was calculated using the following formula: (O.D.sample-O.D.blank) / (O.D.Control-O.D.blank) × 100.

IC _{50 was} calculated using a non-linear regression data analysis method using Graphpad Prism software. The results are shown in the table below.

Test Example 2: Acute toxicity study in rats

A maihuatinib composition (a toxicological batch with a normalized content of the compound of formula II of 0.76%) was used to evaluate the possible toxicity of maihuatinib after intragastric and intravenous administration to SD rats.

Fifty SD rats were randomly divided into five groups (five / sex / group). The first group of animals was given orally with 0.5% CMC-Na solution, and the second, third, and fourth groups were given orally with mehuati Nivar 200, 500, 1000 mg / kg, the 5th group was given mehuatinib 100 mg / kg by intravenous injection, and the administration volume was 10 mL / kg. Observe 4 hours after the administration, and then observe it once a day in the morning and afternoon each for a period of 14 days. During the experiment, the animals' body weight and food intake were regularly measured. After the observation period, the animals were euthanized, grossly dissected, and abnormal tissues and organs seen in the grossly dissected were examined under a microscope.

During the experiment, a total of 9 animals in the 1000 mg / kg gavage group were found dead, of which 4 males died on D6 (day 6, the same meaning as D below) and D8, and 5 females died on D3 to D8; A total of three female animals in the 500 mg / kg dose group were found dead at D7 and D9; one male animal in the 100 mg / kg dose group died within 1 min after intravenous administration.

During the test period, no abnormal reaction was observed in the vehicle control group of the first group.

Clinical observation of intragastric administration: In the 200 mg / kg dose group, nasal secretions, soft stools, loose stools, and perianal fouling were observed in 4 male animals during the test period, and eye or nasal secretions were seen in 2 female animals.

In the 500mg / kg dose group, male animals D2 ～ D15 showed nasal secretions, soft stools, perianal fouling, perianal skin redness, loose stools, mental weakness, abnormal movements, arch back, eye secretions, excessive saliva secretion, Coat fluffy, swelling of foreskin, swelling of penis, hair loss, skin ulceration and red urine; female animals D3 ～ D15 show nasal secretions, eye secretions, loose stools, perianal fouling, debilitating, abnormal movement, coat fluffy , Prone, arched back and soft stools, three females were found dead on D7 and D9.

In the 1000 mg / kg dose group, male animals showed nasal secretions, soft stools, loose stools, perianal fouling, fluffy coats, arched back, eye secretions, and thin coats at D3 to D15. There were 4 male animals at D6. D8 and D8 were found dead; females began to show nasal secretions, loose stools, perianal fouling, lack of energy, arched back, fluffy hair, and abnormal movement from D2 to D7. Five females were found dead at D3 to D8.

Intravenous administration: One male animal in the 100 mg / kg dose group died within 1 min after the drug was taken, and the systemic abnormal reaction of the remaining animals was mainly manifested as a state of mental weakness, shortness of breath, and proneness immediately after the drug, and recovered within 60 minutes after the drug; 2 female animals D3 showed nasal secretions and recovered on D4. The local reaction of injection mainly showed tail swelling, tail discoloration and crusting.

During administration, compared with the same-sex vehicle control group during the same period, the weight of male animal D15 in the gavage 200mg / kg group was reduced, and the weight of female animal D4 was reduced, ranging from 8% to 9%; the 500mg / kg gavage group Male animals D8, D11, and D15 lost weight, and female animals D4 and D8 lost weight, ranging from 20% to 34%; gavage at 1000 mg / kg dose group animals D4, weight reduction, ranging from 11% to 16% ; There was no significant difference in the body weight of the intravenous injection of 100 mg / kg.

During the experiment, the food intake of the animals in the 200, 500 and 1000 mg / kg dose groups in the first week (W1) after drug administration was lower than that of the same-sex vehicle control group during the same period. The control group was similar.

The results of pathological examination showed that one single intragastric administration of mehuatinib to SD rats did not show any significant changes and histological changes that were clearly euthanized at the planned time. Pathological examinations of dead animals in the 500 mg / kg dose group and the 1000 mg / kg dose group may indicate that gastrointestinal lesions are related to the test product; abnormalities such as the thymus, spleen, and adrenal gland have not been determined to be relevant to the test product.

A single intravenous injection of mehuatinib was given to SD rats. Except for the injection site and surrounding tissues, no other major changes and histological changes clearly related to the test product were seen. Swelling, discoloration, and crusting in the tail of some animals after injection were considered to be related to the local irritation of the test article.

Mevatinib was administered to SD rats by a single intragastric administration. The main toxic reaction was gastrointestinal reactions. The lethal dose (LD) was 500 mg / kg, and the maximum tolerated dose (MTD) was 200 mg / kg. A single intravenous injection of mehuatinib was administered to SD rats. The main toxic reaction was a local stimulus injection with a lethal dose (LD) of 100 mg / kg.

Claims

A mehuatinib composition, characterized in that the composition comprises mehuatinib represented by formula (I) and formula (II) with a normalized percentage content of 1% or less and not zero. Shown compound,

Optionally, the normalized percentage content of the compound represented by formula (II) in the Myhuatinib composition is 0.9% or less, optionally 0.8% or less, optionally 0.5% or less, optionally Ground is 0.3% or less, optionally 0.2% or less.
The mehuatinib composition according to claim 1, wherein the normalized percentage content of the compound represented by formula (II) in the mehuatinib composition is 0.01% or more, optionally 0.02% or more , Optionally 0.05% or more.
The mehuatinib composition according to claim 1 or 2, wherein the normalized percentage of mehuatinib represented by formula (I) is 98.0% or more, preferably 98.5% or more, and more preferably 99.0 % Or more, more preferably 99.3% or more, and still more preferably 99.5% or more.
The method for preparing a mehuatinib composition according to any one of claims 1-3, comprising:

(1) Preparation of A10, using the following synthetic route,

Each R in the above structural formula is independently a C 1-6 alkyl group, preferably a methyl group or an ethyl group, and more preferably an ethyl group,

In the step of preparing the A9 compound, the A8 compound is reacted with hydrogen in the presence of a catalyst;

In the step of preparing the A10E compound, a reaction between the A9 compound and dialkylphosphoacetic acid is performed in the presence of N, N'-carbonyldiimidazole (CDI);

In the preparation step of the A10 compound, dimethylaminoacetaldehyde dialkanol is first converted into N, N-dimethylaminoacetaldehyde, and then the reaction is performed in the presence of a base;

(2) Preparation of dimaleate;

The A10 compound is reacted with maleate in a solvent to obtain a dimaleate product. The solvent is a mixed solvent of ethyl acetate and water, wherein the mass ratio of water to ethyl acetate is 1-4: 20, preferably 1:10; using 1-4 parts by mass of water, preferably 2 parts by mass of water, per part by mass of the A10 compound;

Optionally, further comprising (3) a recrystallization step;

The dimaleate product is recrystallized using a solvent, the solvent is a mixed solvent of ethyl acetate and water, wherein the mass ratio of water to ethyl acetate is 1-4: 20, preferably 1:10; per mass 1-4 parts by mass of water, preferably 2 parts by mass of water, for parts of dimaleate;

It is preferred to add a mehuatinib seed during crystallization.
The use of the mehuatinib composition according to any one of claims 1 to 3 for the preparation of a medicament for preventing or treating an indication related to the function of EGFR and HER2 kinase; optionally, said EGFR and HER2 Kinase-related indications are selected from breast cancer, ovarian cancer, gastrointestinal cancer, esophageal cancer, lung cancer, head and neck squamous cell carcinoma, pancreatic cancer, epidermal squamous cell carcinoma, prostate cancer, glioma, and nasopharyngeal carcinoma. Or more.
A pharmaceutical preparation for preventing or treating indications related to the function of EGFR and HER2 kinase, comprising a mehuatinib composition according to any one of claims 1-3 and a pharmaceutically acceptable excipient.
A compound represented by formula (II),
A compound represented by formula Z-A10,
A method for preparing a compound of formula Z-A10 or a compound of formula (II), wherein the compound of formula Z-A10 is synthesized as follows,

The preparation method of the compound of formula Z-A10 includes: reacting 1-dimethylamine-2-propyne with carbon dioxide in the presence of a base to prepare 4- (dimethylamino) -2-propargylic acid, and then 4- (Dimethylamino) -2-ynylbutyric acid is reacted with A9 in the presence of isobutyl chloroformate and N-methylmorpholine to prepare a Z-A10-01 compound, and the Z-A10-01 compound is then used in a Lindlar catalyst Preparation of Z-A10 compound by hydrogenation in the presence;

The synthetic route of the compound of formula (II) is as follows:

The method for preparing the compound of formula (II) includes: after the compound of formula Z-A10 is obtained as described above, the compound of formula Z-A10 and maleic acid are salted.
The use of the compound of formula (II) according to claim 7 as a reference substance for detecting the compound of formula (II) or its content in a mehuatinib composition.
A method for detecting the content of a compound of formula (II) in a mehuatinib composition, characterized in that a standard compound of formula (II) is used as a reference substance for detection by high performance liquid chromatography;

Preferably, the mobile phase of high-performance liquid chromatography is disodium hydrogen phosphate-phosphate buffer solution, methanol, or a mixed solvent of acetonitrile and water, and gradient elution is preferably performed using disodium hydrogen phosphate-phosphate buffer solution and methanol.