CN107955019B - Salt form and crystal form of EGFR inhibitor and preparation method thereof - Google Patents

Abstract

The invention discloses a salt form and a crystal form of an EGFR inhibitor and preparation methods thereof, and also discloses application of the salt form and the crystal form in preparation of a medicament for treating non-small cell lung cancer.

Description

Salt form and crystal form of EGFR inhibitor and preparation method thereof

Technical Field

The invention relates to a salt form and a crystal form of a T790M mutant EGFR inhibitor and preparation methods thereof, and also relates to application of the salt form and the crystal form in medicaments for treating non-small cell lung cancer.

Background

Protein tyrosine kinases are a class of enzymes that catalyze the transfer of a phosphate group located on a protein substrate from ATP or GTP to a tyrosine residue. Receptor tyrosine kinases activate secondary signaling pathways by transmitting signals from the outside of the cell to the inside of the cell causing phosphorylation. A variety of cellular processes are regulated by these signals, including proliferation, carbohydrate utilization, protein synthesis, angiogenesis, cell growth and cell survival. In addition, many diseases or conditions are associated with aberrant, abnormal or deregulated activity of one or more kinases.

The epidermal growth factor receptor belongs to the ErbB family of transmembrane tyrosine kinase receptors, which include EGFR (also known as ErbB or HER1), ErbB2 (HER2 or neu gene), ErbB3 (HER3) and ErbB4(HER 4). All have tyrosine kinase activity except HER 3. The EGFR/ErbB family tyrosine kinase receptors have an indispensable role in cell proliferation, differentiation and apoptosis, and thus are effective targets for preventing tumor growth and metastasis. The first generation of epidermal growth factor receptor tyrosine kinase inhibitors (in EGFR-TKI) including gefitinib (J Med 2004; 350:2129-39) and erlotinib (Lancet Oncol 2011; 12:735-42) have been shown to be effective in advanced NSCLC patients with somatic activating mutations. These mutations are point mutations (L858R) encoding in-frame deletions in the kinase domain encoding epidermal growth factor receptor, e.g., exon 19 of polynucleotide, and a substitution of leucine to arginine at position 858 on exon 21 (Nat Rev Cancer 2007; 7: 169-81). However, patients receiving the first generation of EGFR-TKIs eventually develop secondary tumor growth due to drug resistance. The secondary mutation of threonine at position 790 to methionine (T790M) is the most widely recognized mechanism of resistance. This mutation was detected in tumor cells from 50% to 60% of patients with disease progression (N Engl J Med 2005; 353: 207-8). Second generation EGFR-TKIs, such as Afatinib (Lancet Oncol 2014; 15:213-22) and dacomitinib (Cancer 2014; 120:1145-54), were developed to overcome drug resistance generated by the first generation TKIs. They may irreversibly bind covalently to cysteine at position 797 on EGFR. The covalent mechanism is thought to overcome the increased affinity of ATP by the double mutant. Cysteine-797 is however present in all forms of EGFR. These second generation compounds are therefore active not only against active mutant and second mutant EGFR, but also against wild type EGFR. Inhibition of wild-type EGFR is not considered to contribute to its clinical efficacy, but rather leads to side effects of rash and diarrhea (curr. med. chem.2006,13, 3483-.

Thus, third generation EGFR-TKIs, including AZD9291(Cancer Discov 2014; 4:1046-61), CO-1686(Cancer Res.2013; 19: 2240-. They are highly effective in T790M positive tumors, but they still have some toxicity, like still producing diarrhea, rash, nausea and even hyperglycemia clinical side effects ((J Clin Oncol 2014; 32: abstr 8009; J Clin Oncol 2014; 32: abstr 8010). it is clear that a compound with higher activity and lower toxicity brings greater benefit.

The Aslican AZD9291 is an oral small molecule third generation epidermal growth factor tyrosine kinase inhibitor, and the AZD9291 has stronger specific binding capacity for drug-resistant mutation T790M and sensitive mutation sites (19Del and L858R), so the AZD9291 has good treatment effect on drug-resistant non-small cell lung cancer patients after first-line TKI treatment, but has certain inhibition on EGFR wild type, so the side effects of diarrhea, rash and the like can be generated clinically.

CO1686 is a novel, oral, selective inhibitor for treating EGFR mutant non-small cell lung cancer patients, which can inhibit key activation and T790M drug resistance mutation, which leaves wild type EGFR signal idle and has good tolerance, and the metabolite produced by CO1686 has inhibitory effect on IGF1R and INSR two targets, so that the clinical side effect is shown as hyperglycemia.

Disclosure of Invention

The present invention provides compounds of formula (II).

The present invention also provides a crystalline form a of the compound of formula (II) characterised by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 Θ angles: 4.55 +/-0.2 degrees, 13.84 +/-0.2 degrees, 15.97 +/-0.2 degrees and 18.91 +/-0.2 degrees.

In some embodiments of the present invention, the above form a has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 4.55 +/-0.2 degrees, 10.21 +/-0.2 degrees, 11.35 +/-0.2 degrees, 13.84 +/-0.2 degrees, 15.97 +/-0.2 degrees, 17.22 +/-0.2 degrees, 18.91 +/-0.2 degrees and 22.54 +/-0.2 degrees.

In some embodiments of the present invention, the above form a has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 4.55 +/-0.2 degrees, 9.27 +/-0.2 degrees, 10.21 +/-0.2 degrees, 11.35 +/-0.2 degrees, 13.84 +/-0.2 degrees, 14.45 +/-0.2 degrees, 15.97 +/-0.2 degrees, 16.58 +/-0.2 degrees, 17.22 +/-0.2 degrees, 18.91 +/-0.2 degrees, 22.54 +/-0.2 degrees and 23.67 +/-0.2 degrees.

In some embodiments of the invention, the form a of the above form a has an XRPD pattern as shown in figure 1.

In some embodiments of the invention, the XRPD pattern analysis data for form a above is shown in table 1:

TABLE 1

In some embodiments of the invention, the differential scanning calorimetry curve of form A above has endothermic peaks at 71.51 ℃ ± 3 ℃ and 154.71 ℃ ± 3 ℃.

In some embodiments of the invention, the DSC pattern of form a above is shown in figure 2.

In some embodiments of the invention, the thermogravimetric analysis curve of form a above shows a weight loss of 2.676% at 126.44 ℃ ± 3 ℃ and a weight loss of 2.9411% at 172.01 ℃ ± 3 ℃.

In some embodiments of the invention, the TGA profile of the form a is as shown in figure 3.

The invention also provides a preparation method of the compound A crystal form of the formula (II), which comprises the following steps:

(a) adding a compound shown in the formula (I) into a solvent for dissolving;

(b) slowly adding a mixed solution of methanesulfonic acid and a solvent under stirring;

(c) stirring for 8-16 hours at 20-30 ℃;

(d) centrifuging and drying for 8-16 hours;

wherein the solvent is tetrahydrofuran, and the volume ratio of the methanesulfonic acid to the solvent in the mixed solution is 1: 9.

The invention also provides a crystal form B of the compound of formula (II), which is characterized in that the X-ray powder diffraction pattern thereof has characteristic diffraction peaks at the following 2 theta angles: 9.89 +/-0.2 degrees, 15.46 +/-0.2 degrees, 19.60 +/-0.2 degrees and 21.44 +/-0.2 degrees.

In some embodiments of the invention, the form B has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 5.00 +/-0.2 degrees, 9.89 +/-0.2 degrees, 14.86 +/-0.2 degrees, 15.46 +/-0.2 degrees, 19.60 +/-0.2 degrees, 21.44 +/-0.2 degrees and 24.64 +/-0.2 degrees.

In some embodiments of the invention, the form B has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2 Θ angles: 5.00 +/-0.2 degrees, 9.89 +/-0.2 degrees, 12.00 +/-0.2 degrees, 14.86 +/-0.2 degrees, 15.46 +/-0.2 degrees, 19.60 +/-0.2 degrees, 20.11 +/-0.2 degrees, 21.44 +/-0.2 degrees, 24.05 +/-0.2 degrees and 24.64 +/-0.2 degrees.

In some embodiments of the invention, the XRPD pattern of form B above is as shown in figure 5.

In some embodiments of the invention, the XRPD pattern analysis data for form B above is shown in table 2:

TABLE 2

In some embodiments of the invention, the differential scanning calorimetry curve of form B above has an endothermic peak at 199.07 ℃ ± 3 ℃.

In some embodiments of the invention, the DSC pattern of form B above is shown in figure 6.

In some embodiments of the present invention, the thermogravimetric analysis curve of the form B has a thermogravimetric analysis curve with a weight loss of 0.1283% at 173.87 ℃ ± 3 ℃ and a weight loss of 0.9514% at 212.74 ℃ ± 3 ℃.

In some embodiments of the invention, the TGA profile of form B above is shown in figure 7.

The invention also provides a preparation method of the compound B crystal form shown in the formula (II), which comprises the following steps:

(a) adding the compound of formula (II) to a solvent to form a suspension;

(b) stirring the suspension for 8-16 hours at 35-45 ℃;

(c) centrifuging and drying for 8-16 hours;

wherein the solvent is selected from the group consisting of acetone, isopropanol, ethyl acetate and 2-methyltetrahydrofuran.

The invention also provides application of the compound or the crystal form A or the crystal form B in preparation of a medicine for treating non-small cell lung cancer.

Technical effects

The compound has better PK property and oral absorption rate, stable crystal form, good hygroscopicity and small influence of light and heat.

The compounds of the present invention show excellent activity against sensitive mutant and double mutant EGFR (sensitive and T790M resistant) and have high selectivity against wild type EGFR. They are likely to provide more effective treatment of diseases caused by abnormalities in epidermal growth factor receptor enzymes.

Definitions and explanations

As used herein, the following terms and phrases are intended to have the following meanings unless otherwise indicated. A particular phrase or term should not be considered as ambiguous or unclear without special definition, but rather construed in a generic sense. When a trade name appears herein, it is intended to refer to its corresponding commodity or its active ingredient.

The intermediate compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof well known to those skilled in the art, with preferred embodiments including, but not limited to, the examples of the present invention.

The chemical reactions of the embodiments of the present invention are carried out in a suitable solvent that is compatible with the chemical changes of the present invention and the reagents and materials required therefor. In order to obtain the compounds of the present invention, it is sometimes necessary for a person skilled in the art to modify or select the synthesis steps or reaction schemes based on the existing embodiments.

The present invention will be specifically described below by way of examples, which are not intended to limit the present invention in any way.

All solvents used in the present invention are commercially available and can be used without further purification.

The invention employs the following abbreviations: r.t. represents room temperature; THF represents tetrahydrofuran; NMP stands for N-methylpyrrolidone; MeSO₃H represents methane sulfonic acid; DME represents ethylene glycol dimethyl ether; DCM represents dichloromethane; xphos represents 2-dicyclohexylphosphine-2 ', 4 ', 6 ' -triisopropylbiphenyl; EtOAc for ethyl acetate; MeOH represents methanol; acetone represents acetone; 2-Me-THF represents 2-methyltetrahydrofuran; IPA stands for isopropyl alcohol.

The compound is made by hand or

The software names, and the commercial compounds are under the supplier catalog name.

The powder X-ray diffraction (XRPD) method of the invention

The instrument model is as follows: bruker D8advance X-ray diffractometer

The test method comprises the following steps: about 10-20 mg of sample was used for XRPD detection.

The detailed XRPD parameters are as follows:

light pipe: the concentration of Cu, k alpha,

voltage of light pipe: 40kV, light tube current: 40mA

Divergent slit: 0.60mm

Detector slit: 10.50mm

Anti-scattering slit: 7.10mm

Scanning range: 4-40deg

Step diameter: 0.02deg

Step length: 0.12 second

Sample pan rotation speed: 15rpm

Differential thermal analysis (DSC) method of the present invention

The instrument model is as follows: TA Q2000 differential scanning calorimeter

The test method comprises the following steps: a sample (. about.1 mg) was placed in a DSC aluminum pan for testing at 50mL/min N₂The sample was heated from 30 deg.C (room temperature) to 300 deg.C (or 350 deg.C) at a ramp rate of 10 deg.C/min.

The present invention is a Thermal Gravimetric Analysis (TGA) method

The instrument model is as follows: TA Q5000IR thermogravimetric analyzer

The test method comprises the following steps: a sample (2-5 mg) was placed in a TGA platinum pan for testing at 25mL/min N₂Under the condition, the sample is heated from room temperature to 350 ℃ or the weight loss is 20 percent at the heating rate of 10 ℃/min.

The invention relates to a Dynamic Vapor adsorption analysis (DVS) method

The instrument model is as follows: SMS DVS Advantage dynamic vapor adsorption instrument

And (3) testing conditions are as follows: samples (10-15 mg) were placed in a DVS sample pan for testing.

The detailed DVS parameters are as follows:

temperature: 25 deg.C

Balancing: dm/dt is 0.01%/min (shortest: 10min, longest: 180min)

And (3) drying: drying at 0% RH for 120min

RH (%) test step: 10 percent of

RH (%) test step range: 0 to 90 to 0 percent

The hygroscopicity evaluation was classified as follows:

moisture absorption classification	ΔW％
		Deliquescence	Absorb sufficient water to form liquid
Is very hygroscopic	ΔW％≥15％
		Has moisture absorption	15％>ΔW％≥2％
Slightly hygroscopic	2％>ΔW％≥0.2％
		No or almost no hygroscopicity	ΔW％<0.2％

Note: Δ W% represents the moisture pick-up weight of the test article at 25. + -. 1 ℃ and 80. + -. 2% RH.

Drawings

FIG. 1 is an XRPD spectrum of Cu-Ka radiation of compound A form (II);

FIG. 2 is a DSC of form A of compound (II);

FIG. 3 is a TGA spectrum of the crystalline form of Compound A of (II);

figure 4 is a DVS spectrum of form a of compound (II);

FIG. 5 is a Cu-Ka radiation XRPD spectrum of compound B form (II);

FIG. 6 is a DSC of form B of Compound (II);

FIG. 7 is a TGA spectrum of form B of compound (II);

fig. 8 is a DVS spectrum of compound (II) form B.

Detailed Description

For better understanding of the present invention, the following description is given with reference to specific examples, but the present invention is not limited to the specific embodiments.

Example 1: preparation of Compounds of formula (I)

Step 1:

N₂pyridine (1.74Kg, 22.01mol, 1.10 equivalents) in dichloromethane (20L) was cooled to-20 ℃ under protection, trifluoromethanesulfonic anhydride (5.87Kg,20.81mol,1.04 equivalents) was slowly added dropwise, the reaction system was stirred at-20 ℃ for 0.5 hour, 2-bromoethanol (2.50Kg,20.01mol,1.0 equivalents) was slowly added dropwise, the reaction temperature was maintained below 0 ℃ and stirred for 1.0 hour, TLC (petroleum ether: ethyl acetate: 5:1) indicated completion of the reaction. After the reaction, the reaction system was directly filtered, the filtrate was concentrated, petroleum ether (15L) was added, the precipitated solid was filtered, and the filtrate was concentrated to obtain compound b (4.80Kg, yield 93.33%) as a dark yellow oil.¹HNMR(400MHz,CDCl₃):4.77(t,J＝6.40Hz,2H),3.63(t,J＝6.40Hz,2H).

Step 2:

compound b (8.5Kg, 33.07mol, 1 eq.) and diphenyl sulfide (6.78Kg,36.38mol, 1.10 eq.) were placed in dry toluene (22.0L) and the reaction mixture was heated to 100 ℃ and stirred for 6 hours under nitrogen.¹HNMR showed the reaction was complete. The reaction was cooled to room temperature, and methyl t-butyl ether (20L) was added, the precipitated solid was filtered, and the filter cake was dried in a vacuum oven at 40 ℃ to give compound c (7.4Kg, yield 50.48%) as a brown solid.¹H NMR(400MHz,CDCl₃):8.08-8.15(m,4H),7.69-7.83(m,6H),4.85-4.93(m,2H),3.66-3.72(m,2H).

And step 3:

compound c (7.4Kg, 16.69mol, 1 equivalent) and potassium bicarbonate (2.01Kg,20.03mol, 1.20 equivalents) were placed in tetrahydrofuran (18.2L) and H₂O (9.1L), the reaction mixture was stirred at 15 ℃ for 3 hours.¹HNMR showed the reaction was complete. Addition of H₂O (10L), the aqueous phase was extracted with DCM (10L. times.4), the organic phases were combined and concentrated to give compound d (5.72Kg, yieldRate 94.57%) was a dark yellow oil.¹H NMR(400MHz,CDCl₃):7.87(d,J＝7.60Hz,8H),7.62-7.78(m,12H),7.48(dd,J＝16.00,8.80Hz,2H),6.68(dd,J＝8.80,1.60Hz,2H),6.53(dd,J＝16.00,2.20Hz,2H).

And 4, step 4:

indole-2-carboxylic acid (3Kg, 18.62mol, 1.0 eq) was dissolved in THF (15L), lithium aluminium hydride (900g, 23.7mol, 1.3 eq) was added in portions at 20 ℃, the temperature was programmed to 70 ℃, after the lithium aluminium hydride addition was completed, stirring was carried out at 70 ℃ for 3 hours, TLC (petroleum ether: ethyl acetate 1:1) monitored for completion of the reaction, the temperature was reduced to 20 ℃, water (900mL) was slowly added, NaOH (1N,900mL) was quenched, after complete quenching, suction filtration was carried out under reduced pressure, the filtrate was spin-dried to give a reddish brown oil, which was dissolved in EA (3L), petroleum ether (30L) was added under stirring, and a pale yellow solid precipitated from the solution. Filtration was carried out, the solid was slurried with petroleum ether and ethyl acetate 10:1(15L), suction filtered under reduced pressure, and the solid was dried in a vacuum oven at 50 ℃ to constant weight to give compound r (2.14kg, yield: 74%) as a pale yellow solid.¹H NMR(400MHz,CDCl₃)δ8.36(br s,1H),7.60(d,J＝8.0Hz,1H),7.35(d,J＝8.0Hz,1H),7.20(dt,J＝1.6,8.0Hz,1H),7.15-7.09(m,1H),6.42(d,J＝1.6Hz,1H),4.83(s,2H),1.93(br s,1H).

And 5:

in N₂Under protection, dissolving compound r (2.14Kg,14.04mol, 1.0 equivalent) in DCM (50L), cooling to 0 deg.C, adding KOH (1.97Kg, 35.1mol, 2.5 equivalents), stirring at 0 deg.C for 30min, dissolving diphenyl (vinyl) sulfonium (6.36Kg, 17.54mol, 1.25 equivalents) in DCM (10L), slowly dropwise adding into the reaction system, controlling the reaction temperature at 0-5 deg.C, heating to 30 deg.C, stirring for 5 hr, monitoring reaction completion by TLC (petroleum ether: ethyl acetate ═ 2:1), adding water (30L) to quench the reaction, separating, washing the organic phase with saline (60L), drying, spin-drying to obtain black oily substance, adding DME (1.2L), stirring at-10 deg.C overnight, precipitating yellow solid, filtering, and collecting solidPulping with DME (1.2L) at-10 deg.C, filtering, pulping the filter cake with petroleum ether (800mL) once at room temperature, filtering, and drying the filter cake in a vacuum oven to constant weight to obtain compound e as white solid (1.1Kg, yield: 44.5%).¹HNMR(400MHz,CDCl₃)δ7.63-7.57(m,1H),7.31(dd,J＝0.8,8.0Hz,1H),7.21(dt,J＝0.8,8.0Hz,1H),7.18-7.11(m,1H),6.24(d,J＝0.8Hz,1H),5.01(d,J＝0.8Hz,2H),4.22-4.16(m,2H),4.13-4.07(m,2H)

Step 6:

5-fluoro-2, 4-dichloropyrimidine (2.12kg, 12.67mol, 1.05 eq.) was added to a solution of ethylene glycol dimethyl ether (10L), the solution was cooled to 0-5 ℃ and N was added₂Aluminum trichloride (3.22kg,24.13mol, 2.0 eq.) was added in portions under protection and the reaction was stirred at 30 ℃ for 1 hour. Compound e (3.22kg,24.13mol,1.0 eq) was then slowly added to the reaction mixture, stirred at 20 ℃ for 15 hours, and the completion of the reaction was monitored by TLC (dichloromethane: methanol ═ 6: 1). Slowly adding the reaction solution into water (40L), separating out a large amount of solid, continuously stirring for 0.5h, filtering, adding the filter cake into methanol (16L), pulping at room temperature for 1 h, cooling to room temperature, and filtering. The filter cake was dried to give compound g (3.36kg, 90% yield) as a tan solid.¹HNMR(400MHz,CDCl₃)δ8.43–8.42(d,J＝4.0Hz,1H),7.98-7.96(m，1H),7.40-7.32(m，3H),5.28(s,2H),4.27-4.22(m,4H)

LCMS(ESI)(20-80AB):m/z:304.1[M+1].

And 7:

30L of 1, 4-dioxane was charged into a 50L kettle (20 ℃ C.), and Compound (f) (3.0Kg, 9.88mol, 1.0 equivalent), Compound (g) (1.93Kg, 10.37mol,1.05 equivalent), Palladium acetate (110.91g, 0.494mol, 0.05 equivalent), XPhos (471g, 0.988mol, 0.1 equivalent), Potassium phosphate (4.19Kg, 19.76mol, 2.0 equivalent) were charged into the reaction kettle (20 ℃ C.), N₂The replacement was performed 3 times. The temperature is programmed to 100 +/-5 ℃, and after the sampling HPLC central control detection reaction is finished, the temperature is cooled to the room temperature. The reaction solution was filtered under reduced pressure. Filter cakePulping with 40L purified water at 25 + -5 deg.C for 2 hr. The filter cake was slurried with methanol (30L) for 1 hour and dried under vacuum at 50 ℃ to constant weight. Compound i (4.18kg, 91.6% yield) was obtained as a yellow solid.¹H NMR(400MHz,DMSO-d6)δ8.89(d,J＝8.0Hz,1H)，8.59(d,J＝1.92Hz,1H)，8.50(s,1H)，7.78(dd,J＝7.2,3.58Hz,1H)，7.55(d,J＝8.0Hz,1H)，7.37(d,J＝13.30Hz,1H)，7.17-7.29(m,2H)，5.11(s,2H)，4.21(br dd,J＝12.99,4.83Hz,4H)，4.02(s,3H).LCMS(ESI)(20-80AB):m/z:454.2[M+1].

And 8:

10.4L N-Methylpyrrolidone was added to a 50L kettle (20 ℃ C.), and Compound (h) (4.15Kg, 9.15mol, 1.0 eq.), N, N, N-trimethylethylenediamine (1.12Kg, 10.98mol,1.2 eq.), potassium carbonate (2.53Kg, 18.30mol, 2.0 eq.) were added to the kettle (20 ℃ C.). The temperature is programmed to 100 +/-5 ℃, and after the sampling HPLC central control detection reaction is finished, the temperature is cooled to 25 +/-5 ℃. 10.4L of purified water was added dropwise to the above solution, and a large amount of red solid was precipitated. And (3) carrying out vacuum filtration, washing the filter cake with purified water (20L) until the filtrate is colorless, pulping the filter cake with 20L of methanol at 25 +/-5 ℃ for 1 hour, carrying out vacuum filtration, and carrying out vacuum drying on the filter cake at 50 ℃ until the weight is constant. Compound k (4.26kg, 86.4% yield) was obtained as a red solid.¹H NMR(400MHz,CDCl₃)δ9.06(s,1H)，8.35(d,J＝2.89Hz,1H)，7.91(br,dd,J＝4.96,3.70Hz,1H)，7.54(s,1H)，7.35-7.43(m,1H)，7.27-7.33(m,3H)，6.69(s,1H)，5.29(s,2H)，4.30(t,J＝5.08Hz,2H)，4.22(t,J＝5.14Hz,2H)，4.00(s,3H)，3.28(t,J＝7.15Hz,2H)，2.89(s,3H)，2.57(t,J＝7.15Hz,2H)，2.28(s,6H)，1.69(s,3H).LCMS(ESI)(20-80AB):m/z:536.3[M+1].

And step 9:

NMP (6L) was charged to a 10L autoclave (RT), 0.75kg of compound (L) was charged to the autoclave (RT), N₂Bubbling for 5 min. 75g of wet Pd-C (50%) are added to the reaction mixture, washed with H₂Replace qi for 4 times. The reaction pressure is adjusted to 2.0MPa, and the reaction is heated to 25 ℃ under the stirring condition. Stirring at 25 deg.C for 20 hr (when R1 pressure is less than 1.5MPa, adding H₂To 2.0MPa), central control sampling (HPLC), sampling (sampling method: dissolving 0.1mL of the extract in1mL MeOH). Stopping the reaction, and then reducing the pressure of the high-pressure kettle to normal pressure; the reaction solution was filtered through celite (750g) under reduced pressure; the filter cake was washed 1 time with 6L DCM and the filtrate was adjusted to pH 1 with aq.hcl (1M, 0.4L); DCM (6L x 5) was added to the filtrate for extraction and TLC check (sampling method: 0.1ml of aqueous phase was taken directly; developing solvent: DCM/MeOH 10:1, iodine color development, essentially no NMP was seen). Adding NaOH (5M) into water phase to adjust pH to 5, separating out solid, and stirring for 0.5 h. And (4) carrying out suction filtration under reduced pressure, washing a filter cake with water, and collecting the filter cake. The solid was dried under vacuum at 50 ℃ for 48 hours to give compound i.¹HNMR(400MHz,CD₃OD):δ,8.55(s,1H),8.41(d,J＝3.14Hz,1H),7.83-7.89(m,1H),7.49(dd,J＝7.22,1.19Hz,1H),7.27(ddd,J＝8.82,7.62,1.13Hz,2H),7.16(s,1H),5.14(s,2H),4.18-4.30(m,4H),4.04(s,3H),3.37-3.49(m,4H),2.92(s,6H),2.74(s,3H)LCMS(ESI)(20-80AB):m/z:506.4[M+1].

Step 10:

10L of purified water, 458.76g of NaHCO₃15L of dichloromethane were added to a 50L reactor and stirred at room temperature until the solid was clear. Slowly adding 1.48Kg of compound (J), stirring for 10min, and standing the reaction solution for 10min for layering. The organic dichloromethane layer was washed 2 times with saturated brine (10L x 2). Dried over anhydrous sodium sulfate. The organic layer was spin dried to give 1.26Kg of brown solid. Free amine (1.26Kg,2.49mol,1.0 eq) was dissolved in 12.6L DME and 1.26L purified water and transferred to a 50L autoclave, cooled to-5-0 ℃, chloropropionyl chloride (347.77g, 2.74mol, 1.1 eq) was slowly added dropwise to the above solution, the addition was complete. Stirring was continued for 0.5 h. LCMS 20-80AB 35MIN monitors less than 1% of starting material remaining and the reaction is complete. Sodium hydroxide (398.74g, 9.96mol, 4.0 equiv) was added to the above solution, the temperature was raised to 70 ℃ and stirred for 6-8 hours, and the reaction was completed. The reaction solution was cooled to room temperature. The reaction solution was poured into 65L of purified water to precipitate a large amount of brown solid, which was then filtered under reduced pressure and the filter cake was dried under vacuum for 24 hours to constant weight to obtain the compound of formula (I) as a brown solid (1.16Kg, yield 78%).¹H NMR(400MHz,CDCl₃):δ,10.12(s,1H),9.45(s,1H),8.39(d,J＝3.01Hz,1H),7.88-7.96(m,1H),7.55(s,1H),7.24-7.40(m,4H),6.81(s,1H),6.25-6.45(m,2H),5.68(dd,J＝9.79,2.01Hz,1H),5.31(s,2H),4.14-4.27(m,4H),3.90(s,3H),2.86-2.92(m,2H),2.73(s,3H),2.25-2.32(m,8H)LCMS(ESI)(20-80AB):m/z:560.3[M+1].

Example 2: preparation of the Compound of formula (II)

Adding 8L of acetone into a 50L reaction kettle, adding the compound (1.10Kg,1.97mol, 1.0 equivalent) of the formula (I) into the reaction kettle at room temperature, stirring until the compound is dissolved and clarified, adding thiourea resin (220.44g, 20% by mass ratio) into the reaction solution, stirring overnight at 25 ℃, carrying out suction filtration under reduced pressure, and washing a filter cake with 3L of acetone. The acetone was combined, the solution was transferred to a 50L reactor, warmed to 65 ℃ and methanesulfonic acid (185.34g, 1.93mol, 0.98 eq.) was slowly added dropwise from a constant pressure dropping funnel, and the solution was clear after the addition. After 30min, an off-white solid precipitated from the solution. Stirring for 2 hours under heat preservation, and cooling to 25 ℃. N is a radical of₂Suction filtration under reduced pressure under protection, vacuum drying of the filter cake to constant weight in a vacuum oven gave compound of formula (II) as a brown solid (965g, 73% yield).¹H NMR(400MHz,CD₃OD):δ,8.37(s,1H),8.34(d,J＝3.07Hz,1H),7.87(dd,J＝7.78,3.14Hz,1H),7.44(d,J＝7.91Hz,1H),7.15-7.26(m,2H),6.96(s,1H),6.42-6.55(m,2H),5.86(dd,J＝8.72,3.07Hz,1H),5.05(s,2H),4.13-4.22(m,4H),4.00(s,3H),3.49(t,J＝5.65Hz,2H),3.28(t,J＝5.71Hz,2H),2.87(s,6H),2.72(d,J＝1.51Hz,6H).LCMS(ESI)(20-80AB):m/z:560.3[M+1].

Example 3: preparation of compound A crystal form of formula (II)

120mg of the compound of formula (I) are taken, dissolved in 0.5mL of tetrahydrofuran and then 0.14mL of methanesulfonic acid-tetrahydrofuran solution (V/V,1:9) is slowly added with stirring. Stirring overnight at 25 ℃ and then centrifuging, and placing the residual solid sample in a vacuum drying oven (30 ℃) to dry overnight to obtain the crystal form A of the compound shown in the formula (II).

Example 4: preparation of compound B crystal form of formula (II)

50mg of the compound of formula (II) was weighed into a 4.0mL glass vial, and an appropriate amount of acetone was added to make a suspension. And (3) adding magnetons, placing the suspension sample on a magnetic heating stirrer (at 40 ℃) for testing (keeping out of the sun), stirring at 40 ℃ overnight, centrifuging, and placing the residual solid sample in a vacuum drying oven (at 30 ℃) for drying overnight to obtain the crystal form B of the compound shown in the formula (II).

50mg of the compound of formula (II) was weighed into a 4.0mL glass vial and suspended in an appropriate amount of isopropanol. And (3) adding magnetons, placing the suspension sample on a magnetic heating stirrer (at 40 ℃) for testing (keeping out of the sun), stirring at 40 ℃ overnight, centrifuging, and placing the residual solid sample in a vacuum drying oven (at 30 ℃) for drying overnight to obtain the crystal form B of the compound shown in the formula (II).

50mg of the compound of formula (II) was weighed into a 4.0mL glass vial, and an appropriate amount of ethyl acetate was added to make a suspension. And (3) adding magnetons, placing the suspension sample on a magnetic heating stirrer (at 40 ℃) for testing (keeping out of the sun), stirring at 40 ℃ overnight, centrifuging, and placing the residual solid sample in a vacuum drying oven (at 30 ℃) for drying overnight to obtain the crystal form B of the compound shown in the formula (II).

50mg of the compound of formula (II) was weighed into a 4.0mL glass vial, and an appropriate amount of 2-methyltetrahydrofuran was added to make a suspension. And (3) adding magnetons, placing the suspension sample on a magnetic heating stirrer (at 40 ℃) for testing (keeping out of the sun), stirring at 40 ℃ overnight, centrifuging, and placing the residual solid sample in a vacuum drying oven (at 30 ℃) for drying overnight to obtain the crystal form B of the compound shown in the formula (II).

A compound of formula (II) (700g) and absolute ethanol (5.6L) were added to the reaction vessel at room temperature. Stirring and heating to 85 deg.c until the solid is completely dissolved. N-heptane (3.5L) was slowly added to the kettle and some solid precipitated from the solution. Stirring for 1 hour under heat preservation, and cooling the reaction solution to 25 ℃. N is a radical of₂And (5) carrying out vacuum filtration under the protection, and drying the solid in a vacuum drying oven to constant weight. Adding the solid and 7L of acetone into a reaction kettle, stirring, heating to an internal temperature of 60 ℃, stirring for 12-40 hours, carrying out crystal transformation, and cooling to room temperature after crystal transformation is completed. N is a radical of₂And (3) carrying out vacuum filtration under reduced pressure under protection, and drying a filter cake in a vacuum drying oven to constant weight to obtain the crystal form B (530g, yield 75%) of the compound shown in the formula (II).¹H NMR(400MHz,CD₃OD):δ,8.37(s,1H),8.34(d,J＝3.07Hz,1H),7.87(dd,J＝7.78,3.14Hz,1H),7.44(d,J＝7.91Hz,1H),7.15-7.26(m,2H),6.96(s,1H),6.42-6.55(m,2H),5.86(dd,J＝8.72,3.07Hz,1H),5.05(s,2H),4.13-4.22(m,4H),4.00(s,3H),3.49(t,J＝5.65Hz,2H),3.28(t,J＝5.71Hz,2H),2.87(s,6H),2.72(d,J＝1.51Hz,6H).LCMS(ESI)(20-80AB):m/z:560.3[M+1].

Example 5: study of hygroscopicity of Compound A Crystal form of formula (II)

Experimental materials:

SMS DVS Advantage dynamic vapor adsorption instrument

The experimental method comprises the following steps:

10-15 mg of the compound A crystal form of the formula (II) is placed in a DVS sample tray for testing.

The experimental results are as follows:

the DVS spectrum of the form a of compound of formula (II) is shown in figure 4, with Δ W ═ 7.86%.

And (4) experimental conclusion:

the crystal form A of the compound of the formula (II) has the moisture absorption weight gain of 7.86% at 25 ℃ and 80% RH and is hygroscopic.

Example 6: study of hygroscopicity of Crystal form B of Compound of formula (II)

Experimental materials:

SMS DVS Advantage dynamic vapor adsorption instrument

The experimental method comprises the following steps:

and (3) placing 10-15 mg of the compound B crystal form shown in the formula (II) in a DVS sample tray for testing.

The experimental results are as follows:

the DVS spectrum of the form B of compound of formula (II) is shown in figure 8, with Δ W ═ 0.830%.

And (4) experimental conclusion:

the compound of formula (II) form B has a moisture pick-up of 0.83% at 25 ℃ and 80% RH, and is slightly hygroscopic.

Example 7: solid stability test of Compound B form of formula (II)

According to the guidelines of bulk drug and preparation stability tests (9001 of the four pharmacopoeias 2015 edition), the stability of the crystal form B of the compound of formula (II) under the conditions of high temperature (60 ℃, open), high humidity (92.5% of room temperature/relative humidity, open) and strong light (5000lx, closed) is investigated.

Compound B of formula (II) was weighed as 15mg, placed at the bottom of a glass sample bottle and spread into a thin layer. Sealing the bottle mouth of a sample placed under high-temperature and high-humidity conditions by using aluminum foil paper, and pricking small holes in the aluminum foil paper to ensure that the sample can be fully contacted with ambient air; the samples placed under intense light were sealed with a threaded cap. Samples placed under different conditions were sampled and tested (XRPD) at day 5 and day 10, and the test results were compared with the initial test results at day 0, and the test results are shown in table 3 below:

TABLE 3 solid stability test results for Compound B form of formula (II)

And (4) conclusion: the compound B crystal form of the formula (II) has good stability under the conditions of high temperature, high humidity and strong illumination.

In vivo pharmacodynamic study of form B of compound of formula (II) in the NCI-H1975 xenograft (CDX) model

Experimental materials:

BALB/c nude mice, female, 6-8 weeks, weighing about 18-22 grams, were kept in a special pathogen-free environment in single ventilated cages (10 mice per cage). All cages, bedding and water were sterilized prior to use. All animals were free to obtain a standard certified commercial laboratory diet. There were 100 mice purchased from Weitonglihua, Beijing for study. Each mouse was implanted subcutaneously in tumor tissue (20-30 cubic millimeters) in the right flank for tumor growth. The experiment was started when the mean tumor volume reached about 140-. The experimental method comprises the following steps:

in vivo efficacy was performed by subcutaneous implantation of NCI-H1975 lung cancer patient-derived xenograft (CDX) BALB/c nude mice. Experiment test compounds were administered orally daily for 21 consecutive days. Tumor volume was measured twice a week with a two-dimensional caliper, the volume being measured in cubic millimeters and calculated by the following formula: v ═ 0.5a × b2, where a and b are the major and minor diameters of the tumor, respectively. The anti-tumor efficacy was determined by dividing the mean tumor gain volume of animals treated with the compound by the mean tumor gain volume of untreated animals.

The experimental results are as follows: see table 4.

TABLE 4

And (4) experimental conclusion:

the compound B crystal form (12mg/Kg) of the formula (II) has the equivalent tumor inhibition activity to AZD9291(3mg/Kg) and CO1686(50 mg/Kg).

In vivo study of pharmaceutical efficacy of form B of Compound of formula (II) in A431 xenograft (CDX) model

Experimental materials:

BALB/c nude mice, female, 6-8 weeks, weighing about 18-20 grams, were kept in a special pathogen-free environment in a single ventilated cage (5 mice per cage). All cages, bedding and water were sterilized prior to use. All animals were free to obtain a standard certified commercial laboratory diet. All experimental mice were purchased from BK Laboratory Animal co, shanghai, LTD mice for study. Each mouse was implanted subcutaneously in tumor tissue (20-30 cubic millimeters) in the right flank for tumor growth. The experiment was started when the mean tumor volume reached about 150-

The experimental method comprises the following steps:

in vivo selectivity experiments were performed on subcutaneously implanted human skin squamous cell A431 xenograft (CDX) BALB/c nude mice.

Test compounds were administered orally for 11 consecutive days. Tumor volume was measured twice a week with a two-dimensional caliper, the volume being measured in cubic millimeters and calculated by the following formula: v ═ 0.5a × b2, where a and b are the major and minor diameters of the tumor, respectively. The anti-tumor efficacy was determined by dividing the mean tumor gain volume of animals treated with the compound by the mean tumor gain volume of untreated animals.

The experimental results are as follows: see table 5.

TABLE 5

And (4) experimental conclusion:

the compound B crystal form (12mg/Kg) of the formula (II) has better selectivity than AZD9291(3 mg/Kg).

Claims (8)

1. Form B of a compound of formula (II) characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2 θ angles: 5.00 +/-0.2 degrees, 9.89 +/-0.2 degrees, 10.04 +/-0.2 degrees, 12.00 +/-0.2 degrees, 14.86 +/-0.2 degrees, 15.46 +/-0.2 degrees, 19.60 +/-0.2 degrees, 20.11 +/-0.2 degrees, 21.44 +/-0.2 degrees, 24.05 +/-0.2 degrees, 24.64 +/-0.2 degrees, 24.82 +/-0.2 degrees

2. The form B according to claim 1, having an XRPD pattern as shown in figure 5.

3. Form B according to claim 1 or 2, having a differential scanning calorimetry curve with an endothermic peak at 199.07 ± 3 ℃.

4. The form B according to claim 3, having a DSC profile as shown in figure 6.

5. Form B according to claim 1 or 2 having a thermogravimetric analysis curve with a weight loss of 0.1283 at 173.87 ± 3 ℃ and a weight loss of 0.9514 at 212.74 ± 3 ℃.

6. The crystalline form B according to claim 5 having a TGA profile as shown in figure 7.

7. A process for preparing the crystalline form B of compound of formula (II) according to claims 1 to 6, comprising:

(a) adding the compound of formula (II) to a solvent to form a suspension;

(b) stirring the suspension for 8-16 hours at 35-45 ℃;

(c) centrifuging and drying for 8-16 hours;

wherein the solvent is selected from the group consisting of acetone, isopropanol, ethyl acetate and 2-methyltetrahydrofuran.

8. Use of the form B according to claims 1-6 in the preparation of a medicament for treating non-small cell lung cancer.

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