CN116670128A

CN116670128A - Crystal form of morpholine-substituted benzopyrimidine compound and preparation method thereof

Info

Publication number: CN116670128A
Application number: CN202180087676.7A
Authority: CN
Inventors: 陈新海; 陈兆国; 于衍新; 胡伯羽; 夏尚华; 王永鑫
Original assignee: Medshine Discovery Inc
Current assignee: Medshine Discovery Inc
Priority date: 2020-12-28
Filing date: 2021-12-28
Publication date: 2023-08-29
Also published as: WO2022143671A1

Abstract

A crystal form of morpholine-substituted benzopyrimidine compound and its preparing process are disclosed.

Description

Crystal form of morpholine-substituted benzopyrimidine compound and preparation method thereof

The application claims the following priority

CN202011596979.5, filing date: 12 months and 28 days 2020.

Technical Field

The application relates to a crystal form of morpholine-substituted benzopyrimidine compounds and a preparation method thereof.

Background

DNA Double Strand Breaks (DSBs), which are a serious DNA damage, cause loss of genetic material, gene recombination, and thus cancer or cell death. Organisms evolved DNA Damage Response (DDR) mechanisms for detection of damage, signaling, and repair of damage to maintain gene stability and cellular activity. DNA double strand break repair consists essentially of two types: homologous end joining (HR) repair and non-homologous end joining (NHEJ) repair. DDR early injury factors such as MRN detect recognition injury sites, recruit members of the phosphoinositide kinase family (ATM, ATR, DNA-PK), phosphorylate H2AX promote formation of gamma H2AX, recruit relevant signal proteins (such as 53BP1, chk2, BRCA1 and NBS 1) and the like to conduct injury signals, enable cells to enter a cell cycle arrest state and recruit relevant repair proteins to repair injury DNA.

DNA-PK is an important member of DNA damage repair, mainly directed to non-homologous end double strand breaks. Consists of six core factors: when KU70, KU80, DNA-PKcs, CRCC4, ligase IV and Artemis are used for repairing DNA double-stranded injury, KU molecules are specifically connected to double-stranded injury parts through a preformed channel, respectively identify and bind with the tail ends of the DNA chains, then slide to two ends along the DNA chains in an ATP dependent manner for a certain distance to form KU-DNA complexes, attract the DNA-PKcs to the injury parts, bind with the KU-DNA complexes and activate kinase activity, and further phosphorylate a series of proteins involved in repair and injury signal transduction, so as to complete the repair.

At present, the induction of DNA damage by means of radiotherapy and chemotherapy (topoisomerase II, bleomycin, doxorubicin, etoposide) and the like is one of the main means for controlling the growth of tumors. However, research shows that DNA-PK in tumor tissues after chemoradiotherapy is highly expressed, and tumor cells damaged by chemoradiotherapy are continuously repaired, so that the method becomes one of main reasons for chemoradiotherapy resistance.

The application aims to discover a DNA-PK small molecule inhibitor, which can inhibit the activity of DNA-PK by being combined with a chemoradiotherapy medicament, thereby greatly reducing the DNA repair of tumors and inducing cells to enter an apoptosis program. Can overcome the drug resistance problem of chemoradiotherapy to a great extent, and enhance the inhibition effect on various tumors such as small cell lung cancer, head and neck cancer, colorectal cancer, pancreatic cancer and the like. The compound has good activity, shows excellent effect and action, and has wide prospect.

Disclosure of Invention

The application provides a compound shown as a formula (I),

wherein n is selected from 0 to 2, preferably 0 to 1, more preferably 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4 or 1.5, most preferably 0.5.

The application provides a crystal form A of a compound shown in a formula (I), which is characterized in that an X-ray powder diffraction pattern of the compound has characteristic diffraction peaks at the following 2 theta angles: 11.22 + -0.20 deg., 24.28 + -0.20 deg., 27.07 + -0.20 deg..

In some aspects of the application, the X-ray powder diffraction pattern of form a has characteristic diffraction peaks at the following 2θ angles: 11.22+ -0.20 °, 13.59+ -0.20 °, 15.73+ -0.20 °, 17.25+ -0.20 °,21.68 + -0.20 °, 24.28+ -0.20 °, 26.27+ -0.20 °, 27.07+ -0.20 °.

In some aspects of the application, the X-ray powder diffraction pattern of form a has characteristic diffraction peaks at the following 2θ angles: 5.26.+ -. 0.20 °, 11.22.+ -. 0.20 °, 13.59.+ -. 0.20 °, 15.73.+ -. 0.20 °, 17.25.+ -. 0.20 °, 18.55.+ -. 0.20 °, 21.68.+ -. 0.20 °, 23.47.+ -. 0.20 °, 24.28.+ -. 0.20 °, 25.02.+ -. 0.20 °, 26.27.+ -. 0.20 °, 27.07.+ -. 0.20 °.

In some aspects of the application, the X-ray powder diffraction pattern of form a has characteristic diffraction peaks at the following 2θ angles: 11.22 + -0.20 deg., 24.28 + -0.20 deg., and/or 27.07 + -0.20 deg., and/or 13.59 + -0.20 deg., and/or 15.73 + -0.20 deg., and/or 17.25 + -0.20 deg., and/or 21.68 + -0.20 deg., and/or 26.27 + -0.20 deg., and/or 5.26 + -0.20 deg., and/or 18.55 + -0.20 deg., and/or 23.47 + -0.20 deg., and/or 25.02 + -0.20 deg., and/or 7.60 + -0.20 deg., and/or 8.24±0.20°, and/or 10.55±0.20°, and/or 14.97±0.20°, and/or 16.59±0.20°, and/or 19.71±0.20°, and/or 23.80±0.20°, and/or 27.43±0.20°, and/or 28.37±0.20°, and/or 29.21±0.20°, and/or 30.90 ±0.20°, and/or 34.44±0.20°, and/or 36.68±0.20°, and/or 37.53±0.20°.

In some aspects of the application, the X-ray powder diffraction pattern of form a has characteristic diffraction peaks at the following 2θ angles: 5.26 °,7.60 °,8.24 °,10.55 °,11.22 °,13.59 °,14.97 °,15.73 °,16.59 °,17.25 °,18.55 °,19.71 °,21.68 °,23.47 °,23.80 °,24.28 °,25.02 °,26.27 °,27.07 °,27.43 °,28.37 °,29.21 °,30.90 °,34.44 °,36.68 °,37.53 °.

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

In some aspects of the application, XRPD pattern analytical data for form a above are shown in table 1:

TABLE 1 XRPD resolution data for form A of Compound (I)

In some embodiments of the application, the form A has a differential scanning calorimetry trace with a onset of an endothermic peak at 144.2+ -3.0deg.C.

In some embodiments of the present application, the DSC profile of the form a is shown in figure 2.

In some aspects of the application, the thermogravimetric analysis of form a above loses 2.19% weight at 170.0±3.0 ℃.

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

The application provides a B crystal form of a compound shown in a formula (I), which is characterized in that an X-ray powder diffraction pattern of the B crystal form has characteristic diffraction peaks at the following 2 theta angles: 4.55 + -0.20 deg., 11.42 + -0.20 deg., 16.30 + -0.20 deg..

In some aspects of the application, the X-ray powder diffraction pattern of form B has characteristic diffraction peaks at the following 2θ angles: 4.55+ -0.20 °, 9.09+ -0.20 °, 11.42+ -0.20 °, 15.24+ -0.20 °, 16.30+ -0.20 °, 18.65+ -0.20 °, 23.94+ -0.20 °, 25.18+ -0.20 °.

In some aspects of the application, the X-ray powder diffraction pattern of form B has characteristic diffraction peaks at the following 2θ angles: 4.55+ -0.20 °, 9.09+ -0.20 °, 11.42+ -0.20 °, 12.68+ -0.20 °, 15.24+ -0.20 °, 16.30+ -0.20 °, 18.65+ -0.20 °, 22.97+ -0.20 °, 23.94+ -0.20 °, 25.18+ -0.20 °.

In some aspects of the application, the X-ray powder diffraction pattern of form a has characteristic diffraction peaks at the following 2θ angles: 4.55±0.20°,11.42±0.20°, and/or 16.30±0.20°, and/or 9.09±0.20°, and/or 15.24±0.20°, and/or 18.65±0.20°, and/or 23.94±0.20°, and/or 25.18±0.20°, and/or 12.68±0.20°, and/or 17.83±0.20°, and/or 22.97±0.20°, and/or 27.14±0.20°.

In some aspects of the application, the above-described form B has an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2θ angles: 4.55 °,9.09 °,11.42 °,12.68 °,15.24 °,16.30 °,17.83 °,18.65 °,22.97 °,23.94 °,25.18 °,27.14 °.

In some embodiments of the application, form B has an XRPD pattern substantially as shown in figure 4.

In some aspects of the application, XRPD pattern analytical data for form B above is shown in table 2:

TABLE 2 XRPD resolution data for form B of Compound (I)

In some embodiments of the application, the form B has a differential scanning calorimetry trace with a onset of an endothermic peak at 128.2+ -3.0deg.C and a peak of an endothermic peak at 198.2+ -3.0deg.C.

In some embodiments of the application, the DSC spectrum of the B-form is shown in FIG. 5.

In some embodiments of the application, the thermogravimetric analysis of form B above loses 2.35% weight at 170.0±3.0 ℃.

In some embodiments of the application, the TGA profile of form B is shown in figure 6.

The application provides a C crystal form of a compound shown in a formula (II), which is characterized in that an X-ray powder diffraction pattern of the C crystal form has characteristic diffraction peaks at the following 2 theta angles: 14.76+ -0.20 °, 18.03+ -0.20 °,23.19 + -0.20 °;

in some aspects of the application, the X-ray powder diffraction pattern of the above-described form C has characteristic diffraction peaks at the following 2θ angles: 10.44+ -0.20 °, 12.22+ -0.20 °, 14.76+ -0.20 °, 18.03+ -0.20 °, 19.09+ -0.20 °, 20.73+ -0.20 °,23.19 + -0.20 °, 28.66+ -0.20 °.

In some aspects of the application, the X-ray powder diffraction pattern of the above-described form C has characteristic diffraction peaks at the following 2θ angles: 10.44+ -0.20 °, 12.22+ -0.20 °, 14.76+ -0.20 °, 18.03+ -0.20 °, 19.09+ -0.20 °, 20.73+ -0.20 °,23.19 + -0.20 °,25.67 + -0.20 °, 27.43+ -0.20 °, 28.66+ -0.20 °.

In some aspects of the application, the X-ray powder diffraction pattern of form a has characteristic diffraction peaks at the following 2θ angles: 14.76±0.20°,18.03±0.20°, and/or 23.19 ±0.20°, and/or 10.44±0.20°, and/or 12.22±0.20°, and/or 19.09±0.20°, and/or 20.73±0.20°, and/or 28.66±0.20°, and/or 25.67 ±0.20°, and/or 27.43±0.20°, and/or 7.08±0.20°, and/or 8.71±0.20°, and/or 13.20±0.20°, and/or 23.37 ±0.20°, and/or 24.24±0.20°.

In some aspects of the application, the X-ray powder diffraction pattern of the above-described form C has characteristic diffraction peaks at the following 2θ angles: 7.08 °,8.71 °,10.44 °,12.22 °,13.20 °,14.76 °,18.03 °,19.09 °,20.73 °,23.19 °,23.37 °,24.24 °,25.67 °,27.43 °,28.66 °.

In some embodiments of the application, form C has an XRPD pattern substantially as shown in figure 7.

In some aspects of the application, XRPD pattern analytical data for the above form C are shown in table 3:

TABLE 3 XRPD resolution data for form C of Compound (II)

In some embodiments of the application, the form C has a differential scanning calorimetry trace with a onset of an endothermic peak at 197.8+ -3.0deg.C.

In some embodiments of the application, the above-mentioned form C has a DSC profile as shown in figure 8.

The application also provides the following experimental method:

experimental example: pharmacokinetic evaluation

The experimental method comprises the following steps:

the tested compound is mixed with 10% DMSO/50% PEG400/40% water, vortexed and sonicated to prepare a near clear solution of 0.2mg/mL or 0.4mg/mL, and the solution is filtered through a microfiltration membrane for later use. 18 to 20 grams of Balb/c female mice were selected and given by intravenous injection of a candidate compound solution at a dose of 1 or 2mg/kg. The tested compound is mixed with 10% DMSO/50% PEG400/40% water, vortexed and sonicated to prepare a near clear solution of 0.2mg/mL or 1mg/mL, and the solution is filtered through a microfiltration membrane for later use. 18 to 20 grams of Balb/c female mice were selected and administered orally with a solution of the candidate compound at a dose of 2 or 10mg/kg. Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and drug substitution parameters were calculated by Phoenix WinNonlin software (Pharsight, USA).

Definition and description

The following terms and phrases used herein are intended to have the following meanings unless otherwise indicated. A particular phrase or terminology, unless otherwise specifically defined, should not be construed as being ambiguous or otherwise clear, but rather should be construed in a generic sense. When trade names are presented herein, it is intended to refer to their corresponding commercial products or active ingredients thereof.

Intermediate compounds of the present application 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 combining them with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present application.

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

The compounds of the present application may be structured by conventional methods well known to those skilled in the art, and if the present application relates to the absolute configuration of a compound, the absolute configuration may be confirmed by conventional means in the art. For example, single crystal X-ray diffraction (SXRD), the grown single crystal is collected from diffraction intensity data using a Bruker D8vent diffractometer, and the light source is cukα radiation, scanning:after scanning and collecting the relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by a direct method (Shellxs 97).

The present application will be specifically described by the following examples, which are not meant to limit the present application in any way.

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

The solvent used in the present application is commercially available. The application adopts the following abbreviations: DCM represents dichloromethane; DMF represents N, N-dimethylformamide; DMSO represents dimethylsulfoxide; etOH stands for ethanol; meOH represents methanol; TFA represents trifluoroacetic acid; ATP represents adenosine triphosphate; HEPES stands for 4-hydroxyethyl piperazine ethane sulfonic acid; mgCl ₂ Represents magnesium dichloride; pd (PPh) ₃ ) ₂ Cl ₂ Representing bis triphenylphosphine palladium dichloride.

Technical effects

The compound has good crystal form stability and is easy to prepare; the compound crystal form has excellent DNA-PK kinase inhibition activity.

X-ray powder diffraction (X-ray powder diffractometer, XRPD) of the application

Instrument model: x-ray diffractometer of PANalytical (Pa NACES) company

The testing method comprises the following steps: approximately 10mg of sample was used for XRPD detection.

The detailed XRPD parameters are as follows:

a radiation source: cu, kα%Kα2/kα1 intensity ratio: 0.5)

Light pipe voltage: 45kV, light pipe current: 40mA

Divergence slit: fixing 1/8deg

First cable slot: 0.04rad, a second cable pull slit: 0.04rad

Receiving slits: no anti-scatter slit: 7.5mm

Measuring time: 5min

Scanning angle range: 4-40deg

Step width angle: 0.0263deg

Step size: 46.665 seconds

Sample disk rotational speed: 15rpm

Differential scanning calorimeter (Differential Scanning Calorimeter, DSC)

Instrument model: TA Discovery DSC 2500 differential scanning calorimeter

The testing method comprises the following steps: samples (about 1-5 mg) were taken and placed in DSC aluminum trays for testing at 50mL/min N ₂ Under the condition, the sample is heated from 25 ℃ to the temperature before the sample is decomposed at the temperature rising rate of 10 ℃/min.

Thermogravimetric analysis according to the application (Thermal Gravimetric Analyzer, TGA)

Instrument model: TA Discovery TGA 5500 thermogravimetric analyzer

The testing method comprises the following steps: samples (about 1-5 mg) were taken and tested in TGA aluminum trays at 10mL/min N ₂ Under the condition, the sample was heated from room temperature to 350 ℃ at a heating rate of 10 ℃/min.

Dynamic moisture adsorption (Dynamic Vapor Sorption, DVS) of the application

Instrument model: SMS Intrinsic dynamic moisture adsorption instrument

Test conditions: samples (10-20 mg) were taken and placed in DVS sample trays for testing.

The detailed DVS parameters are as follows:

temperature: 25 DEG C

Balance: dm/dt=0.002%/min (shortest: 10min, longest: 180 min)

RH (%) test procedure: 10% (0% RH-90% RH,90% RH-0% RH)

5％(90％RH-95％RH，95％RH-90％RH)

RH (%) test step range: 0% RH-95% RH-0% RH

The hygroscopicity evaluation was classified as follows:

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

Note that: Δw% represents the hygroscopic weight gain of the test article at 25±1 ℃ and 80±2% rh.

Drawings

FIG. 1 is an XRPD pattern for Cu-K alpha radiation of form A of compound of formula (I);

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

FIG. 3 is a TGA spectrum of form A of compound of formula (I);

FIG. 4 is an XRPD pattern for Cu-K alpha radiation for form B of compound of formula (I);

FIG. 5 is a DSC of form B of compound of formula (I);

FIG. 6 is a TGA spectrum of form B of compound of formula (I);

FIG. 7 is an XRPD pattern for Cu-K alpha radiation for form C of compound of formula (II);

FIG. 8 is a DSC of form C of compound (II);

FIG. 9 is a DVS spectrum of compound A crystalline form of formula (I).

Detailed Description

For a better understanding of the present application, reference will now be made to the following examples, which are not intended to limit the scope of the present application.

Example 1: preparation of crystalline form A of Compound of formula (I)

First step

17.5L of toluene was added to the reactor, stirring was started, and the rotation speed was 186rpm. Cesium carbonate (4.31 kg,13.2 mol), compound 1 (2.5 kg,9.2 mol), compound 2 (1.66 kg,9.17 mol) and 4, 5-bis (diphenylphosphorus) -9, 9-dimethylxanthene (101.60 g,0.18 mol) were then added, respectively. After 3 times of nitrogen substitution, palladium acetate (19.81 g,0.09 mol) was added thereto, and after 3 times of nitrogen substitution, the temperature was raised to 80 to 90℃and the reaction was continued for 60 hours. After the reaction was completed and the temperature of the reaction vessel was lowered to 25℃with stirring, the mixture was filtered, and the cake was washed 3 times with 3L of xylene, and the filtrate was collected. The above filtrate containing intermediate 3 was slowly charged into a reaction vessel, cesium carbonate (4.31 kg,13.22 mol), compound 4 (858 g,9.86 mol), 4, 5-bis (diphenylphosphorus) -9, 9-dimethylxanthene (101.60 g,0.18 mol) were added, respectively. After 3 times of nitrogen substitution, palladium acetate (19.81 g,0.09 mol) was added, and after 3 times of nitrogen substitution, the temperature was raised to 115 to 125℃for 16 hours. After the reaction was completed and the temperature of the reaction vessel was lowered to 25℃with stirring, the mixture was filtered, and the cake was washed 3 times with 3L of xylene, and the filtrate was collected. 25L of the filtrate of Compound 5 containing 3.31kg of theory was directly fed to the next step.

Second step

The 25L xylene solution containing compound 5 was added to a 50L reactor, stirring was started, and the rotation speed was 192rpm. Slowly dripping 880mL of hydrochloric acid, controlling the reaction temperature between 20 ℃ and 40 ℃ and reacting for 12 to 16 hours. After the reaction was complete, it was filtered and the filter cake was washed once with 3L of ethyl acetate. And (5) collecting a filter cake. The filter cake was diluted with 10L of ethyl acetate and transferred to a 50L knockout with stirring turned on. Then, 803g of a 10L aqueous solution in which sodium hydroxide was completely dissolved was added to the dispenser and stirred for 10 minutes. The organic phase was collected by liquid extraction, the aqueous phase was extracted 2 times with 10L of ethyl acetate, and the organic phases were combined and concentrated under reduced pressure. The solid was collected. The solid was slurried with 10L of methyl tert-butyl ether at 25℃with stirring for 16 hours. Filtering and collecting filter cakes. The filtrate was further concentrated under reduced pressure to 0.5L. Filtering and collecting filter cakes. And combining the two filter cakes, and airing at room temperature to obtain the compound 6.

MS-ESI calculated [ M+H ]] ⁺ 215, measured 215.

¹ H NMR(400MHz,CDCl ₃ )δ:6.17-6.12(m,1H),6.07-6.01(m,1H),3.89-3.82(m,4H),3.78(br s,2H),2.99-3.09(m,4H)。

Third step

12.5L of methylene chloride was added to the reaction vessel, stirring was started, and the rotation speed was 176rpm. Then compound 6 (1.45 kg,6.78 mol) and sodium bicarbonate (700 g,6.6 mol) were added sequentially. Iodine chloride (720 g,6.77 mol) was dissolved in dichloromethane 2L, transferred to a dropping funnel and embedded in a reaction kettle. Starting the dropping funnel to start dropping, keeping the dropping speed at 1L/30min, keeping the temperature range at 20-35 ℃, and then keeping the temperature and stirring for 1 hour. After the completion of the reaction, 10L of the prepared aqueous solution containing 970g of sodium sulfite was added to the reaction vessel and stirred for 30min. The mixture in the reaction kettle is transferred to a 50L knockout for stirring, standing and liquid-separating extraction. The organic phases were collected, the aqueous phase was extracted 3 times with 5L of dichloromethane, the organic phases were combined and transferred to a 50L rotary evaporator for concentration under reduced pressure. The solid was collected and then dried in a vacuum oven to give compound 7.

MS-ESI calculated [ M+H ]] ⁺ 341, measured 341.

¹ H NMR(400MHz,CDCl ₃ )δ:6.32-6.09(m,1H),3.78(br s,2H),3.97-3.80(m,4H),3.19-2.99(m,4H)。

Fourth step

25.5 liters of triethylamine was added to the dried 50L reactor at 20℃under nitrogen protection, stirring was started at a speed of 100-150rpm. Then, compound 7 (5120 g,15.1 mol), compound 8 (1373 g,14.0 mol), cuprous iodide (44.1 g,0.23 mol), and 80.6 g Pd (PPh) were added sequentially ₃ ) ₂ Cl ₂ . Heating to 55-65 deg.c and reaction for 16 hr. The reaction was complete. The reaction solution was filtered, the filter cake was rinsed with 6.5 liters of petroleum ether, and the filtrate was dried by spin to obtain a crude product. The crude product was dispersed with 13l of ethyl acetate and 6.5 l of water. The organic phase is separated and washed with 6.5 l of water. The organic phase was dried by spinning. The crude product obtained was purified by column chromatography to give compound 9.

MS-ESI calculated [ M+H ]] ⁺ 311 found 311.

Fifth step

At 20℃16.5 litres of tetrahydrofuran was added to the 50L reactor and stirring was started at 100-150rpm. Compound 9 (3306 g,10.7 mol) was then added and the internal temperature was cooled to 0-10 ℃.4L of concentrated hydrochloric acid is slowly dripped into the kettle at the internal temperature of 0-40 ℃. Immediately after the completion of the dropping, LCMS detection, the content of compound 9 was less than 2%. T-butyl nitrite (1028 g,9.97 mol) was added dropwise to the autoclave at an internal temperature of 10-15 ℃. Immediately after the completion of the drop LCMS, the reaction was complete. The reaction solution was slowly added to a 27 kg mixture of ice and water of 4.1 kg sodium carbonate, and solids appeared. Filtering, transferring the filter cake to a kettle, adding 30 liters of water, stirring at room temperature for 1 hour (salt washing), filtering, transferring the filter cake to the kettle, adding 25 liters of tert-butyl methyl ether, stirring at room temperature for 0.3 hour, and filtering. The filter cake was again transferred to the kettle, 15 liters of t-butyl methyl ether was added, stirred at room temperature for 0.3 hour, and filtered. Drying in a vacuum oven (< 0.09MPa,50 ℃ C., 15 hours) to obtain the compound 10.

MS-ESI calculated [ M+H ]] ⁺ 286 Measured values 286, 288.

Sixth step

13L of tetrahydrofuran is added into a 30L reaction kettle, nitrogen is replaced for 10 minutes, weak nitrogen flow is kept, stirring is started, the rotating speed is 150-200rpm, methoxy [ cyclooctadiene ] iridium dimer (99.75 g,150.25 mmol), 4-di-tert-butyl-2, 2-bipyridine (80.89 g,300.5 mmol), compound 11 (2602.1 g,15.03mol,98% purity) and bis-pinacolato borate (3112.5 g,12.02mol,98% purity) are sequentially added, heating is started, stirring is carried out for 260 hours at the temperature of 70-75 ℃ in the kettle, cooling is started, cooling is carried out to 25-35 ℃, the reaction liquid is reduced in pressure to remove the solvent, 13L of [ petroleum ether/ethyl acetate=10:1 ] mixed solvent is added for beating, filtering is carried out, the filter cake is put into a vacuum oven, and the organic solvent is distilled off under reduced pressure (0.07 MPa,50 ℃) to obtain 12.

¹ H NMR(400MHz,DMSO-d ₆ )δ:7.84(d,J＝6.4Hz,1H),7.48(d,J＝8.8Hz,1H),4.11(s,2H),1.17(s,12H)。

Seventh step

24L of toluene and 5L of water were added to a 50L reaction vessel, stirring was started, and the rotation speed was 200rpm. Then potassium hydrogencarbonate (1.083 kg,10.83 mol), compound 10 (2.81 kg,9.86 mol), compound 12 (1.90 kg,6.42 mol) were added sequentially. The rotation speed of the stirring paddle of the reaction kettle was adjusted to 50rpm, a polytetrafluoroethylene tube was used to introduce nitrogen flow below the liquid level in the reaction kettle, and bubbling was performed for 10 minutes using a weak nitrogen flow. 75.77g of dichloro bis (triphenylphosphine) palladium (II) is added, the kettle cover is covered, and the temperature is raised to 80-95 ℃ for reaction for 14 hours. After LCMS and HPLC analysis of 10% or less of compound, the reaction kettle temperature was reduced to 50 ℃ with stirring. 11L of water was added to the reaction vessel and stirred at 50℃for 1 hour. Then, the temperature of the reaction kettle is reduced to 25 ℃, and the filtration is carried out to collect filter cakes. The filter cake was washed 1 time with 3L of water. The filter cake was again added to a 50L reactor and 10L methanol was added. And (5) covering a kettle cover, heating to 50 ℃, stirring and pulping for 3 hours. The temperature was then reduced to 25 ℃. Filtering and collecting filter cakes. The filter cake was washed 2 times with 6L of methanol. The solid was collected and then dried in a vacuum oven to give compound 13.

MS-ESI calculated [ M+H ]] ⁺ 419, found 419.

¹ H NMR(400MHz,CDCl3)δ:9.10(s,1H),7.55(d,J＝7.20Hz,1H),7.35(d,J＝8.80Hz,1H)，7.20(dd,J＝6.4,12.8Hz,1H)，3.98-3.88(m,6H),3.45-3.38(m,4H)。

Eighth step

15L of tetrahydrofuran was added to the 50L reactor, stirring was started, and the rotation speed was 189rpm. Compound 13 (2.0 kg,4.78 mol), compound 14 (719 g,4.79 mol) were added sequentially, the cooling circulation bath was started, and after adjusting the internal temperature to 20 ℃ the addition of potassium tert-butoxide 1.15kg was started in 3 portions. Feed frequency: 0.383kg/10min. The kettle cover is covered, the cooling circulation bath is closed, and the reaction liquid is reacted for 1 hour at room temperature. After LCMS and HPLC analysis of compound 13.ltoreq.1%, the reaction was poured into 150L of water containing 15kg of citric acid monohydrate. After stirring evenly, filtering, collecting a filter cake, placing the filter cake in a vacuum drying oven for drying, purifying the obtained crude product by using silica gel column chromatography (eluent: ethyl acetate/dichloromethane: 20% -35%), concentrating, and pulping the obtained solid by using 2 (kg/L) times of volume of dichloromethane to obtain a compound 15.

MS-ESI calculated [ M+H ]] ⁺ 531, found 531.

¹ H NMR(400MHz,CDCl3)δ:9.15(d,J＝36.4Hz,1H),7.72(br t,J＝6.0Hz,1H),7.65-7.61(m,1H),7.59(s,1H),7.37(d,J＝8.8Hz,1H),7.15-7.25(m,1H),5.98(s,1H),3.97-3.91(m,4H),3.45-3.38(m,4H)。

Ninth step

A3L three-neck round bottom flask with a magnetic stirrer is fixed on the magnetic stirrer, an internal temperature thermometer and an oxygen bag connected with a tee joint are fixed, and air tightness is checked. To the reaction flask was added 1.6L of acetonitrile and stirring was started. Compound 15 (199.8 g,0.38 mol) and potassium carbonate (124.8 g,0.91 mol) were added. The air in the reaction flask was replaced with oxygen to place the reaction in an oxygen atmosphere. The reaction was carried out at 20.+ -. 2 ℃ for 51.5 hours. The reaction was completed and stopped. 9 batches were added in parallel, the reaction solution (20L) was combined, slowly added to water (100L) with stirring, and a large amount of solids was precipitated. The solid was collected by filtration and dried in vacuo (< 0.01mpa,50 ℃ for 17 hours) to give the crude product. Methanol (0.9L) and t-butyl methyl ether (2.7L) were added to the reaction vessel, and the crude product was added thereto and stirred at 20℃for 2.5 hours. The filter cake was collected by filtration and dried in vacuo (< 0.01mpa,50 ℃ for 19 hours) to give compound 16.

MS-ESI calculated [ M+H ]] ⁺ 520 Measured values 520, 522.

¹ H NMR(400MHz,CDCl ₃ )δ:9.16(s,1H),8.23(d,J＝8.0Hz,1H),7.77(d,J＝8.0Hz,1H),7.69(d,J＝8.0Hz,1H),7.38(d,J＝12.0Hz,1H),7.24-7.19(m,1H),4.00-3.90(m,4H),3.48-3.38(m,4H)。

Tenth step

Dichloromethane (18.0L) and methanol (9.0L) were added to a 50L reaction vessel, stirring was started, air in the system was replaced with nitrogen, and a nitrogen flow was maintained, ensuring that the reaction solution was placed under a nitrogen atmosphere. To the reaction vessel were added compound 16 (1350.3 g,2.6 mol) and potassium carbonate (674.5 g,4.89 mol). The reaction solution was stirred at 22.+ -. 2 ℃ for 36.5 hours. After completion of the reaction, citric acid (1000 g) was added to the reaction mixture to adjust the pH to 6. Vacuum concentration<0.02MPa,25 ℃) to remove most of the methylene dichloride. To the concentrated solution was added water (90.0L), followed by sodium bicarbonate (500 g), stirred for 0.5 hours to adjust the pH of the solution to 8, filtered, and the cake was collected. Adding the filter cake into water (10.0L) again, stirring for 1 hr, filtering, collecting solid, and vacuum drying<0.02MPa,50 ℃ for 17 hours) to give compound 17.MS-ESI calculated [ M+H ]] ⁺ 516 Actual values 516, 518.

¹ H NMR(400MHz,CDCl ₃ )δ:9.19(s,1H),8.22(d,J＝9.2Hz,1H),7.64(d,J＝7.2Hz,1H),7.37(d,J＝9.2Hz,1H),7.25-7.22(m,1H),7.19(d,J＝9.2Hz,1H),4.27(s,3H),3.97-3.95(m,4H),3.45-3.43(m,4H)。

Eleventh step

Dichloromethane (9.4L) and water (3.2L) were added to a 50L reaction vessel and stirring was started. To the reaction vessel were added compound 17 (1250.9 g,2.42 mol), sodium formate (950.9 g,14.2 mol), cetyltrimethylammonium bromide (85.1 g,0.23 mol) and ruthenium (74.4 g,0.12 mol) chloride as a catalyst (S, S) -N- (p-toluenesulfonic acid) -1, 2-diphenylethylenediamine (p-isopropylbenzene). And nitrogen is replaced and a weak nitrogen flow is maintained, so that the reaction system is ensured to be in a nitrogen atmosphere. The reaction mixture was stirred at 40℃for 1 hour. The reaction was completed, the reaction was stopped, and the internal temperature of the reaction solution was lowered to 23 ℃. Dichloromethane (5.0L) and water (5.0L) were added to the reaction solution. The mixture was stirred, left to stand and separated to collect an organic phase (14.5L). 100-200 mesh silica gel (2.5 kg) was added to the organic phase, and the mixture was concentrated under reduced pressure (< 0.03MPa,50 ℃ C.) to prepare a sand. A silica gel column (6.0 kg of silica gel) was installed, and the organic phase (305L) was collected by rapid elution with methylene chloride/ethyl acetate (4/1 to 1/1). The organic phase was concentrated under reduced pressure (< 0.03MPa,50 ℃ C.), and the resulting solid was further dried in a vacuum oven (< 0.01MPa,50 ℃ C., 13 hours) to give crude product (970.3 g). Methanol (15.0L) was added to a 50L reactor, stirring was started, crude (968 g) was added, heated to 64 ℃, stirred for 2 hours with heat preservation, reduced temperature to 25 ℃, filtered, the filter cake was collected and dried in vacuo (< 0.01mpa,55 ℃,20 hours). The resulting crude product (832 g) was added to a 50L reaction vessel containing methylene chloride (17L) and stirring was started. The reaction mixture was stirred at 18℃for 17 hours after addition of metal-free silica gel iMoLbox-LMat-EH01x (75.3 g). Filtering, concentrating the filtrate under reduced pressure (0.02 MPa,45 ℃) and adding methanol (12.5L) to the solid, heating and stirring (65 ℃ for 19.5 hours), cooling to 25 ℃, filtering, and repeating the step once for the obtained crude product. The solid was dried in vacuo (< 0.01mpa,65 ℃ for 5 days). The crude product (760 g) obtained after drying was added to a 50L reaction vessel containing pure water (15.0L), stirring was started, and stirring was performed at a constant temperature (54 ℃ C., 63.5 hours). Cooling to 25deg.C, filtering, and drying (60deg.C, below 0.01 MPa) to obtain compound A crystal form of formula (I).

MS-ESI calculated [ M+H ]] ⁺ 518 Measured values 518, 520.

¹ H NMR(400MHz,CDCl ₃ )δ:9.03(d,J＝64.0Hz,1H),7.59-7.55(m,1H),7.38(dd,J＝8.0,16.0Hz,1H),7.29-7.27(d,J＝8.0Hz,1H),7.21-7.06(m,1H),6.98(dd,J＝8.0,16.0Hz,1H),6.42(s,1H),5.14(s,1H),4.12(d,J＝16.0Hz,3H),3.92(s,4H),3.39(s,4H)。

Example 2: preparation of crystalline form B of Compound of formula (I)

About 5mg of form A of compound of formula (I) and 5mg of form C of compound of formula (II) are weighed into an HPLC vial, 0.5mL of a presaturated acetone solution is added, and after stirring at room temperature for about 3 days, form B of compound of formula (I) is obtained.

Example 3: preparation of crystalline form C of Compound of formula (II)

About 500mg of the form A of the compound of the formula (I) is weighed into a 20mL vial, 2.0mL of acetonitrile is added, and the mixture is stirred at room temperature for about 2 days to obtain the form C of the compound of the formula (II).

Example 4: competition experiment of A Crystal form and C Crystal form

Under the condition of room temperature (25+/-3 ℃), etOH/H with different water activities is configured ₂ O and Acetone/H ₂ O solvent system (a w =0 to 1), about 5mg of each of the compound a form of the formula (I) and the compound C form of the formula (II) was added to 0.5mL of saturated solution to form a suspension, and the suspension was magnetically stirred at room temperature for 1000 rpm for 2 days, and then sampled and tested for XRPD.

Solvent system (volume ratio, v/v)	Crystal form
Acetone	B crystal form
Acetone/H ₂ O(94/6，aw＝0.2)	Crystal form A
Acetone/H ₂ O(86/14，aw＝0.4)	Crystal form A
Acetone/H ₂ O(73/27，aw＝0.6)	Crystal form A
Acetone/H ₂ O(50/50，aw＝0.8)	Crystal form A
H ₂ O	Crystal form A
EtOH	Crystal form A
EtOH/H ₂ O(93/7，aw＝0.2)	Crystal form A
EtOH/H ₂ O(83/17，aw＝0.4)	Crystal form A
EtOH/H ₂ O(68/32，aw＝0.6)	Crystal form A
EtOH/H ₂ O(55/45，aw＝0.8)	Crystal form A

Conclusion: in the room temperature water activity range of 0-1, the compound A crystal form of the formula (I) is more stable than the compound C crystal form of the formula (II).

Example 5: solid stability test of Compound A Crystal form of formula (I)

The crystal form A of the compound of the formula (I) is respectively placed for 6 days and 11 days under the conditions of high temperature (60 ℃, closed mouth) and high humidity (92.5 percent RH, sealing film is wrapped and 5 small holes are pricked), and the total illumination of the visible light reaches 1.2E+06Lux hrs and the total illumination of the ultraviolet light reaches 200W hrs/m according to ICH conditions ² ) The sample is placed under visible light and ultraviolet light in a closed way (the sample of the shading control group is placed at the same time andwrapped with tinfoil paper), 1,2 months under 60 ℃/75% RH (wrapping and punching 5 holes with sealing film), 1,2, 3 months under 40 ℃/75% RH (wrapping and punching 5 holes with sealing film). The chemical purity (HPLC area purity), relative content (0 day samples stored at-20 ℃) and crystal form of the samples were tested separately at each sampling point to determine the physical/chemical stability of the samples.

Table 4: results of solid stability test of Compound A Crystal form of formula (I)

Note that: relative chromatographic purity = stability sample purity/starting sample purity x 100%; the initial sample mass/initial sample peak area versus the stable sample peak area/stable sample mass relative to the content variation of the initial sample content; peaks with peak areas less than 0.05% were not integrated.

Conclusion: the compound A crystal form of the formula (I) is stable under high-temperature high-humidity illumination and acceleration conditions.

Example 6: hygroscopicity study of the crystalline form A of Compound of formula (I)

Experimental materials:

SMS Intrinsic dynamic moisture adsorption instrument

The experimental method comprises the following steps:

and placing 10-20 mg of the compound A in a DVS sample tray for testing.

Experimental results:

the DVS spectrum of compound a crystalline form of formula (I) is shown in fig. 9, Δw=1.263%.

Conclusion of experiment:

the moisture absorption weight gain of the compound A crystal form of the formula (I) at 25 ℃ and 80% RH is 1.263%, and the compound A crystal form is slightly hygroscopic.

Biological test data

Experimental example 1: in vitro evaluation of DNA-PK kinase inhibitory Activity

The experiment was tested at Eurofins Pharma Discovery Service, reaction Biology corp (RBC.)

Experimental materials and methods:

human DNA-PK; mg/ATP; GST-cMyc-p53; EDTA; ser15 antibody; ATP 10. Mu.M; biotinylated phosphatidylinositol-3, 4, 5-triphosphate; the GST tagged GRP1PH domain; streptavidin allophycocyanin; europium-labeled GST monoclonal antibody.

Experimental method (Eurofins Pharma Discovery Service):

DNA-PK (h) was incubated in assay buffer containing 50nM GST-cMyc-p53, test compound and Mg/ATP (at the desired concentration). The reaction was initiated by addition of Mg/ATP mixture. After incubation for 30 minutes at room temperature, the reaction was stopped by adding a stop solution containing EDTA. Finally, detection buffer (containing a labeled anti-GST monoclonal antibody and a europium-labeled anti-Ser 15 phosphate antibody against phosphorylated p 53) was added. Plates were then read in time resolved fluorescence mode and a uniform time resolved fluorescence (HTRF) signal was determined according to the formula htrf=10000× (Em 665nm/Em620 nm).

Experimental results:

TABLE 5DNA-PK kinase Activity test results

Test article	DNA-PK kinase inhibitory Activity IC 50 (nM)
Crystal form A of the compound of formula (I)	2.0

Conclusion: the compound of formula (I) form A has significant and even unexpected DNA-PK kinase inhibitory activity.

Claims

A compound of the formula (I),

wherein n is selected from 0 to 1, preferably 0.5.
A crystalline form a of a compound of formula (I) characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2Θ angles: 11.22+ -0.20 °, 24.28+ -0.20 °, 27.07+ -0.20 °;
form a of claim 2, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 11.22+ -0.20 °, 13.59+ -0.20 °, 15.73+ -0.20 °, 17.25+ -0.20 °,21.68 + -0.20 °, 24.28+ -0.20 °, 26.27+ -0.20 °, 27.07+ -0.20 °.
A form a of claim 3, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 5.26.+ -. 0.20 °, 11.22.+ -. 0.20 °, 13.59.+ -. 0.20 °, 15.73.+ -. 0.20 °, 17.25.+ -. 0.20 °, 18.55.+ -. 0.20 °, 21.68.+ -. 0.20 °, 23.47.+ -. 0.20 °, 24.28.+ -. 0.20 °, 25.02.+ -. 0.20 °, 26.27.+ -. 0.20 °, 27.07.+ -. 0.20 °.
Form a of claim 4, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 5.26 °,7.60 °,8.24 °,10.55 °,11.22 °,13.59 °,14.97 °,15.73 °,16.59 °,17.25 °,18.55 °,19.71 °,21.68 °,23.47 °,23.80 °,24.28 °,25.02 °,26.27 °,27.07 °,27.43 °,28.37 °,29.21 °,30.90 °,34.44 °,36.68 °,37.53 °.
Form a of claim 5 having an XRPD pattern substantially as shown in figure 1.
Form a according to any one of claims 2 to 6, having a differential scanning calorimetry curve at 144.2±3.0 ℃ with a onset of an endothermic peak.
Form a of claim 7 having a DSC profile as shown in figure 2.
Form a of any one of claims 2 to 6 having a thermogravimetric analysis curve with a weight loss of up to 2.19% at 170.0±3.0 ℃.
Form a of claim 9 having a TGA profile as shown in figure 3.
A crystalline form B of a compound of formula (I), characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2Θ angles: 4.55+ -0.20 °, 11.42+ -0.20 °, 16.30+ -0.20 °;
form B of claim 11, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 4.55+ -0.20 °, 9.09+ -0.20 °, 11.42+ -0.20 °, 15.24+ -0.20 °, 16.30+ -0.20 °, 18.65+ -0.20 °, 23.94+ -0.20 °, 25.18+ -0.20 °.
Form B of claim 12, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 4.55+ -0.20 °, 9.09+ -0.20 °, 11.42+ -0.20 °, 12.68+ -0.20 °, 15.24+ -0.20 °, 16.30+ -0.20 °, 18.65+ -0.20 °, 22.97+ -0.20 °, 23.94+ -0.20 °, 25.18+ -0.20 °.
Form B of claim 13, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 4.55 °,9.09 °,11.42 °,12.68 °,15.24 °,16.30 °,17.83 °,18.65 °,22.97 °,23.94 °,25.18 °,27.14 °.
Form B of claim 14 having an XRPD pattern substantially as shown in figure 4.
Form B of any one of claims 11-15, having a differential scanning calorimetry curve with a onset of an endotherm at 128.2 ± 3.0 ℃ and a peak of an endotherm at 198.2 ± 3.0 ℃.
Form B of claim 16, having a DSC profile as shown in figure 5.
Form B of any one of claims 11-15, having a thermogravimetric analysis curve with a weight loss of up to 2.35% at 170.0±3.0 ℃.
Form B of claim 18 having a TGA profile as shown in figure 6.
A crystalline form C of a compound of formula (II), characterized by an X-ray powder diffraction pattern having characteristic diffraction peaks at the following 2Θ angles: 14.76+ -0.20 °, 18.03+ -0.20 °,23.19 + -0.20 °;
form C of claim 20, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 10.44+ -0.20 °, 12.22+ -0.20 °, 14.76+ -0.20 °, 18.03+ -0.20 °, 19.09+ -0.20 °, 20.73+ -0.20 °,23.19 + -0.20 °, 28.66+ -0.20 °.
Form C of claim 21, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 10.44+ -0.20 °, 12.22+ -0.20 °, 14.76+ -0.20 °, 18.03+ -0.20 °, 19.09+ -0.20 °, 20.73+ -0.20 °,23.19 + -0.20 °,25.67 + -0.20 °, 27.43+ -0.20 °, 28.66+ -0.20 °.
Form C of claim 22, having an X-ray powder diffraction pattern with characteristic diffraction peaks at the following 2Θ angles: 7.08 °,8.71 °,10.44 °,12.22 °,13.20 °,14.76 °,18.03 °,19.09 °,20.73 °,23.19 °,23.37 °,24.24 °,25.67 °,27.43 °,28.66 °.
Form C of claim 23, having an XRPD pattern substantially as shown in figure 7.
Form C of any one of claims 20-24, having a differential scanning calorimetry curve at 197.8 ± 3.0 ℃ with a onset of an endothermic peak.
Form C of claim 25, having a DSC profile as shown in figure 8.