CN111484489A

CN111484489A - Amorphous B-RAF kinase dimer inhibitors

Info

Publication number: CN111484489A
Application number: CN201910077266.9A
Authority: CN
Inventors: 张国良; 周昌友; 孙熀彬
Original assignee: Baiji Shenzhou Beijing Biotechnology Co ltd
Current assignee: Baiji Shenzhou Beijing Biotechnology Co ltd
Priority date: 2019-01-25
Filing date: 2019-01-25
Publication date: 2020-08-04
Anticipated expiration: 2039-01-25
Also published as: CN116715662A; CN111484489B

Abstract

The present invention relates to a stable amorphous form of the B-RAF kinase dimer inhibitor 1- ((1S,1aS,6bS) -5- ((7-oxo-5, 6,7, 8-tetrahydro-1, 8-naphthyridin-4-yl) oxy) -1a, 6B-dihydro-1H-cyclopropa [ B ] benzofuran-1-yl) -3- (2,4, 5-trifluorophenyl) urea (hereinafter sometimes referred to aS compound 1), processes for the preparation of said amorphous compound and therapeutic uses of said amorphous compound.

Description

Amorphous B-RAF kinase dimer inhibitors

Technical Field

Background

Second generation B-RAF inhibitors are disclosed in PCT patent application WO 2014/206343a1, including 1- ((1S,1aS,6bS) -5- ((7-oxo-5, 6,7, 8-tetrahydro-1, 8-naphthyridin-4-yl) oxy) -1a, 6B-dihydro-1H-cyclopropa [ B ] benzofuran-1-yl) -3- (2,4, 5-trifluorophenyl) urea. The structure of compound 1 is shown below:

as a second generation B-RAF inhibitor, the compound 1 has strong inhibition effect on RAF family of serine/threonine kinase, especially BRAF/CRAF dimer. The compound has targeted therapeutic effect on cancers with mutation (including B-RAF mutation and K-RAS/N-RAS mutation) in the MAK channel, and is a molecular targeted therapeutic agent. Compound 1 is improved over first generation B-RAF inhibitors such as vemurafenib (vemurafenib) and dabrafenib (dabrafenib).

The crystalline form of compound 1, as an organically synthesized free base, has low solubility in water and thus low oral bioavailability.

Amorphous form is one form of substance polymorphism, and is an amorphous state. Various physicochemical properties and clinical pharmacodynamic characteristics of amorphous drugs are often different from those of common crystalline drugs. Therefore, in the research of polymorphism of solid drugs, the deep study of amorphous substances is also significant. In general, amorphous forms have a high dispersibility and high energy state compared to crystalline forms, and thus have higher solubility and bioavailability compared to crystalline forms, amorphous forms. However, the amorphous form has a very limited application in the pharmaceutical industry due to its poor stability.

The inventors of the present invention have found a pure amorphous form of compound 1 which has a relatively long stability unlike the general amorphous form and a relatively high bioavailability compared to the crystalline form.

Disclosure of Invention

The present invention provides a pure amorphous form of compound 1, which does not undergo crystalline transformation during a 14 day test period, i.e. still does not show any diffraction peaks during a 14 day test period; and the amorphous form has a relatively high bioavailability compared to the crystalline form. This indicates that the amorphous form of the invention has potential use in the manufacture of a medicament.

In a first aspect, the present invention provides a pure amorphous form of compound 1, characterized in that said pure amorphous form has an X-ray powder diffraction pattern without diffraction peaks, as shown in figure 7.

Preferably, the pure amorphous form of the invention has the structure shown in FIG. 11¹H-NMR spectrum.

Preferably, the glass transition temperature of the pure amorphous form of the present invention is between about 135 to 143℃, more preferably about 138.3C.

Preferably, the pure amorphous form of the present invention has a particle size D of between about 60 to about 80 μm₉₀D between about 2 and about 6 μm₅₀D between about 1 and about 2 μm₁₀(ii) a More preferably D₉₀About 69.9 μm, D₅₀About 3.5 μm, and D₁₀Is about 1.4 μm.

In a second aspect, the present invention provides a process for preparing a pure amorphous form of compound 1, said process comprising: a solution of the crystalline form of compound 1 in a polar solvent is spray dried to give a powdered material.

Preferably, the polar solvent includes ethers, carboxylic acid esters, nitriles, ketones, amides, sulfones, sulfoxides, or halogenated hydrocarbons. More preferably, the polar solvent includes, but is not limited to, acetic acid, acetone, acetonitrile, benzene, chloroform, carbon tetrachloride, dichloromethane, dimethyl sulfoxide, 1, 4-dioxane, ethanol, ethyl acetate, butanol, t-butanol, N-dimethylacetamide, N-dimethylformamide, formamide, formic acid, heptane, hexane, isopropanol, methanol, methyl ethyl ketone, l-methyl-2-pyrrolidone, mesitylene, nitromethane, polyethylene glycol, propanol, 2-acetone, pyridine, tetrahydrofuran, toluene, xylene, mixtures thereof, and the like. Preferably, the polar solvent is a mixture of halogenated hydrocarbons/amides, for example a mixture of DCM/MeOH.

Preferably, the spray drying is performed by a spray dryer. Preferably, the inlet temperature of the spray dryer is set at about 50 to 70 ℃ and the outlet temperature of the spray dryer is set at about 25 to 45 ℃; more preferably, the inlet temperature of the spray dryer is set to about 60 ℃ and the outlet temperature of the spray dryer is set to about 35 ℃.

Preferably, the crystalline form of compound 1 is crystalline form a. In one embodiment, the crystalline form a is characterized by an X-ray powder diffraction pattern comprising at least three, four, five, or six diffraction peaks having independently selected 2 Θ ° values from the group consisting of: 4.7 +/-0.2, 9.4 +/-0.2, 13.6 +/-0.2, 14.0 +/-0.2, 14.9 +/-0.2 and 15.6 +/-0.2 degrees. Preferably, the crystalline form a is characterized by an X-ray powder diffraction pattern comprising at least three, four, five or six diffraction peaks having independently selected 2 Θ ° values from the group consisting of: 4.7 +/-0.2, 9.4 +/-0.2, 13.6 +/-0.2, 14.0 +/-0.2, 14.9 +/-0.2, 15.6 +/-0.2, 21.2 +/-0.2, 24.3 +/-0.2, 24.7 +/-0.2, 25.1 +/-0.2 and 29.1 +/-0.2. More preferably, the crystalline form a is characterized by an X-ray powder diffraction pattern comprising diffraction peaks having values independently selected from the group consisting of the following 2 Θ ° values: 4.7 +/-0.2, 9.4 +/-0.2, 10.2 +/-0.2, 13.6 +/-0.2, 14.0 +/-0.2, 14.9 +/-0.2, 15.6 +/-0.2, 17.2 +/-0.2, 17.4 +/-0.2, 18.7 +/-0.2, 20.0 +/-0.2, 20.4 +/-0.2, 21.2 +/-0.2, 22.3 +/-0.2, 24.3 +/-0.2, 24.7 +/-0.2, 25.1 +/-0.2, 25.5 +/-0.2, 26.8 +/-0.2, 27.4 +/-0.2, 27.8 +/-0.2, 28.6 +/-0.2, 29.1 +/-0.2, 30.2 +/-0.2, 31.8 +/-0.2, 32.0 +/-0.2, 33.1 +/-0.2, 34.1 +/-0.2 and 34.6 +/-0.2. In a preferred embodiment, the crystalline form a is characterized by an X-ray powder diffraction pattern substantially as shown in figure 1.

Preferably, the crystalline form of compound 1 is single crystal a.

In one embodiment, the single crystal form a has unit cell parameters

In one embodiment, the spatial population of single crystal form a is monoclinic (crystal system) P2₁。

In a preferred embodiment, the crystalline form a is prepared by a commercial scale process.

In a third aspect, the present application discloses a method of treating or preventing a disease or disorder responsive to inhibition of Raf kinase in a subject, comprising administering to the subject a therapeutically effective amount of compound 1, wherein compound 1 is in the pure amorphous form disclosed herein.

In one embodiment, the disease or disorder is a cancer selected from the group consisting of: brain cancer, lung cancer, kidney cancer, bone cancer, liver cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer, melanoma, skin cancer, adrenal cancer, cervical cancer, lymphoma, or thyroid tumor and complications thereof.

In another embodiment, the disease is BRAF (V600E or non V600E) or NRAS or KRAS mutant cancer selected from the group consisting of: brain cancer, lung cancer, kidney cancer, bone cancer, liver cancer, bladder cancer, breast cancer, head and neck cancer, ovarian cancer, melanoma, skin cancer, adrenal cancer, cervical cancer, lymphoma, or thyroid tumor and complications thereof.

In another embodiment, compound 1 is administered at a dose of 1-200 mg/day and at a frequency of one to three times per day.

In another embodiment, compound 1 is administered at a dose of 2.5-100 mg/day and at a frequency of one to three times per day.

In another embodiment, compound 1 is administered at a dose of 5-50 mg/day and at a frequency of once daily.

In one embodiment, the subject is a rat, dog, or human.

In a fourth aspect, the present application discloses a pharmaceutical composition comprising a therapeutically effective amount of compound 1, said compound 1 being in the pure amorphous form disclosed herein. Wherein the active compound, Compound 1, may comprise 1-99% by weight, preferably 1-50% by weight, more preferably 1-30% by weight, or most preferably 1-20% by weight of the pharmaceutical composition.

The pharmaceutical composition may be administered as follows: orally in the form of, for example, capsules, tablets, pills, powders, sustained-release injections (such as sterile solutions, suspensions or emulsions); by topical treatment forms, such as pastes, creams or ointments; or by suppositories such as suppositories. The pharmaceutical composition may be in unit dosage form suitable for precision dosage application.

Suitable pharmaceutical carriers include water, various organic solvents, and various inert diluents or fillers. The pharmaceutical composition may contain various additives such as flavors, binders and excipients, if necessary. For oral administration, tablets and capsules may contain various excipients, such as citric acid; various disintegrants such as starch, alginic acid and some silicates; and various binders such as sucrose, gelatin and acacia. Additionally, lubricants comprising magnesium stearate and talc fillers are commonly used in the manufacture of tablets. The same type of solid components can also be used to formulate soft and hard gelatin capsules. When aqueous suspensions are required for oral administration, the active compounds may be combined with various sweetening or flavoring agents, coloring agents or dye combinations. If desired, various emulsifiers may be used or suspensions may be produced; diluents such as water, ethanol, propylene glycol, glycerol, or combinations thereof may be used.

The above pharmaceutical composition is preferably administered orally.

The above pharmaceutical composition is preferably in the form of a capsule or tablet.

Drawings

Figure 1 shows an X-ray diffraction pattern (crystallization from isopropanol/water) for one crystalline form of compound 1 (form a).

Figure 2 shows an X-ray diffraction pattern of another crystalline form (form a) of compound 1.

Figure 3 shows the absolute structure of a single crystal (form a) of compound 1 (single crystal obtained by crystallization from ethyl acetate/heptane).

Figure 4 shows the crystal packing of single crystals (form a) of compound 1.

Figure 5 illustrates hydrogen bonding of single crystals (form a) of compound 1.

Figure 6 shows the theoretical XRPD pattern of single crystals (form a) of compound 1 calculated using the mercure software.

Figure 7 shows an X-ray diffraction pattern of a pure amorphous form of compound 1 (form B).

FIG. 8 shows a crystalline form of Compound 1 (form A)¹H-NMR spectrum.

FIG. 9 shows a crystalline form of Compound 1 (form A)^l3C-NMR spectrum.

Figure 10 shows DVS hygroscopicity (i.e. moisture absorption) of crystalline form a of the compound.

FIG. 11Showing the pure amorphous form (form B) of Compound 1¹H-NMR spectrum.

Figure 12 shows the overlap of pure amorphous form of compound 1 (form B).

Detailed Description

Definitions and general terms

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are described herein.

"crystalline form" or "crystalline form" refers to a solid having a highly regular chemical structure, including, but not limited to, single or multicomponent crystals, and/or polymorphs, solvates, hydrates, clathrates, co-crystals, salts, solvates of salts, hydrates of salts of compounds. Crystalline forms of the substance can be obtained by a variety of methods known in the art. These methods include, but are not limited to, melt crystallization, melt cooling, solvent crystallization, crystallization in a defined space, e.g., in a nanopore or capillary, crystallization on a surface or template, e.g., on a polymer, crystallization in the presence of additives such as co-crystallizing anti-molecules, desolventization, dehydration, fast evaporation, fast cooling, slow cooling, vapor diffusion, sublimation, reactive crystallization, anti-solvent addition, milling, and solvent drop milling, among others.

"amorphous" or "amorphous form" refers to a substance formed when particles (molecules, atoms, ions) of the substance are aperiodically arranged in three-dimensional space, and is characterized by a diffuse, non-peaked, X-ray powder diffraction pattern. Amorphous is a particular physical form of solid material, with locally ordered structural features suggesting a myriad of connections to crystalline materials. Amorphous forms of the substance can be obtained by a variety of methods known in the art. These methods include, but are not limited to, quenching, anti-solvent flocculation, ball milling, spray drying, freeze drying, wet granulation, and solid dispersion techniques, among others. The "pure amorphous form" as referred to herein means an amorphous form consisting only of compound 1, without any other excipients.

The "polar solvent" refers to a solvent containing a polar group such as a hydroxyl group or a carbonyl group, and has a high polarity and a high dielectric constant. Polar solvents suitable for the purposes of the present invention include, but are not limited to, acetic acid, acetone, acetonitrile, benzene, chloroform, carbon tetrachloride, methylene chloride, dimethyl sulfoxide, 1, 4-dioxane, ethanol, ethyl acetate, butanol, tert-butanol, N-dimethylacetamide, N-dimethylformamide, formamide, formic acid, heptane, hexane, isopropanol, methanol, methyl ethyl ketone, l-methyl-2-pyrrolidone, mesitylene, nitromethane, polyethylene glycol, propanol, 2-acetone, pyridine, tetrahydrofuran, toluene, xylene, mixtures of two, three or more thereof, and the like.

Crystalline or amorphous forms can be identified by a variety of techniques, such as X-ray powder diffraction (XRPD), infrared absorption spectroscopy (IR), melting point methods, Differential Scanning Calorimetry (DSC), thermogravimetric analysis (TGA), nuclear magnetic resonance methods, raman spectroscopy, X-ray single crystal diffraction, dissolution calorimetry, Scanning Electron Microscopy (SEM), quantitative analysis, solubility, and dissolution rate, among others.

Information such as change, crystallinity, crystal structure state and the like of the crystal form can be detected by X-ray powder diffraction (XRPD), and the method is a common means for identifying the crystal form. The peak positions of the XRPD patterns depend primarily on the structure of the crystalline form, being relatively insensitive to experimental details, while their relative peak heights depend on a number of factors related to sample preparation and instrument geometry. Accordingly, in some embodiments, the crystalline form of the present invention is characterized by an XRPD pattern having certain peak positions, substantially as shown in the XRPD patterns provided in the figures of the present invention. Meanwhile, the 2 θ measurement of the XRPD pattern may have experimental error, and the 2 θ measurement of the XRPD pattern may be slightly different between different instruments and different samples, so the 2 θ value cannot be regarded as absolute. The diffraction peaks have a tolerance of ± 0.2 ° according to the conditions of the instrument used in the test according to the invention.

Differential Scanning Calorimetry (DSC) is performed by programming, heating or cooling continuously, to measure the temperature of a sample and an inert reference (usually α -Al)₂O₃) The energy difference between them varies with temperature. The melting peak height of the DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Thus, in some embodiments, the crystalline form or amorphous form of the present invention is characterized by a DSC profile with characteristic peak positions substantially as shown in the DSC profiles provided in the figures of the present invention. Meanwhile, the DSC profile may have experimental errors, and the peak position and peak value of the DSC profile may slightly differ between different instruments and different samples, so the peak position or peak value of the DSC endothermic peak cannot be regarded as absolute. According to the conditions of the instrument used in the test of the invention, the melting peak has a tolerance of + -3 ℃.

Glass transition refers to the transition of an amorphous substance between a high elastic state and a glassy state, and is the inherent property of the substance; the transition temperature corresponds to the glass transition temperature (Tg), which is an important physical property of an amorphous substance. Glass transition is a phenomenon associated with molecular motion. Therefore, the glass transition temperature (Tg) depends mainly on the structure of the substance, and is relatively insensitive to experimental details and the like. In some embodiments, the amorphous glass transition temperature (Tg) of the present invention is determined by Differential Scanning Calorimetry (DSC) and is characterized by having a glass transition temperature of 138.3 ℃. The glass transition temperature has a tolerance of + -3 deg.C depending on the condition of the instrument used in the test of the present invention.

Differential Scanning Calorimetry (DSC) can also be used for detecting and analyzing whether the crystal form has crystal transformation or crystal mixing phenomenon.

Solids of the same chemical composition often form isomeric, or referred to as metamorphosis, isomers of different crystal structures under different thermodynamic conditions, and this phenomenon is called polymorphism or homomultiphase phenomenon. When the temperature and pressure conditions are changed, the variants are transformed into each other, and the phenomenon is called crystal transformation. Due to the crystal form transformation, the mechanical, electrical, magnetic and other properties of the crystal can be changed greatly. When the temperature of the crystal form transformation is in a measurable range, the transformation process can be observed on a Differential Scanning Calorimetry (DSC) chart, and the DSC chart is characterized in that the DSC chart has an exothermic peak reflecting the transformation process and simultaneously has two or more endothermic peaks which are respectively characteristic endothermic peaks of different crystal forms before and after transformation.

Thermogravimetric analysis (TGA) is a technique for measuring the change in mass of a substance with temperature by program control, and is suitable for examining the loss of a solvent in a crystal or the sublimation and decomposition of a sample, and it can be presumed that the crystal contains crystal water or a crystal solvent. The change in mass shown by the TGA profile depends on many factors such as sample preparation and instrumentation; the mass change of the TGA detection varies slightly from instrument to instrument and from sample to sample. There is a tolerance of + -0.1% for mass variations depending on the condition of the instrument used in the test of the invention.

In the context of the present invention, the 2 θ values in the X-ray powder diffraction pattern are all in degrees (°).

The term "substantially as shown" means that at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 99% of the peaks in the X-ray powder diffraction pattern or DSC diagram are shown in the figure.

When referring to a spectrogram or/and data appearing in a graph, "peak" refers to a feature that one skilled in the art would recognize as not being attributable to background noise.

"relative intensity" refers to the ratio of the intensity of the first strong peak to the intensity of the other peaks when the intensity of the first strong peak is 100% of all the diffraction peaks in an X-ray powder diffraction pattern (XRPD).

As used herein, unless otherwise indicated, the term "about" means that the amount (e.g., temperature, pH, volume, etc.) can vary within ± 10%, preferably within ± 5%.

In one embodiment, the crystalline forms of the present invention are synthesized according to scheme 1 below. Notably, the methods disclosed herein are particularly useful for the manufacture of compound 1 or its crystalline form a in high quality, reproducible in high yield, on a commercial scale.

Scheme 1

The following synthetic methods, specific examples and efficacy tests further describe the invention, but they should not be construed to limit or restrict the scope of the invention in any way.

Examples

The following embodiments are intended to be exemplary. Although efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), some experimental errors and deviations should be accounted for. Unless otherwise indicated, temperatures are in degrees celsius. Reagents were purchased from commercial suppliers such as Sigma-Aldrich, Alfa Aesar or TCI and used without further purification unless otherwise indicated.

Unless otherwise stated, the following reactions were carried out under a positive pressure of nitrogen or argon or in an anhydrous solvent with a drying tube; the reaction flask was fitted with a rubber septum for introducing substrate and reagents via syringe; the glassware was oven dried and/or heat dried.

Unless otherwise indicated, column chromatographic purification was performed on a Biotage system with silica gel column (manufacturer: DyaxClaration) or on silica SepPak columns (Waters), or on a Teledyne Isco Combiflash purification system using pre-packed silica gel columns.

Recording on a Varian instrument operating at 400MHz¹H NMR spectrum and¹³c NMR spectrum.

Using a Bruker APEX-II CCD diffractometer (Cu K α radiation,

) X-ray intensity data from the colorless plate crystals were measured at 173(2) K. The polarized light microscope photograph was captured at room temperature.

In the following examples, the following abbreviations may be used:

AcOH acetic acid

ACN acetonitrile

API active pharmaceutical ingredient

Aq aqueous or water solution

Brine saturated sodium chloride aqueous solution

Bn benzyl group

BnBr benzyl bromide

CH₂Cl₂Methylene dichloride

DMA N, N-dimethylacetamide

DMF N, N-dimethylformamide

Dppf 1, 1' -bis (diphenylphosphino) ferrocene

DBU 1, 8-diazabicyclo [5.4.0] undec-7-ene

DCM dichloromethane

DIEA or DIPEA N, N-diisopropylethylamine

DMAP 4-N, N-dimethylaminopyridine

DMF N, N-dimethylformamide

DMSO dimethyl sulfoxide

EtOAc ethyl acetate

EtOH ethanol

Et₂O or ether ethyl ether

g

H or hr

HATU O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate

HCl hydrochloric acid

HP L C high performance liquid chromatography

HPMCAS acetic acid succinic acid hydroxypropyl methyl cellulose

IPA or i-PrOH 2-propanol or isopropanol

mg of

m L ml

Mmol millimole

MeCN acetonitrile

MeOH methanol

Min minute

Ms or MS Mass Spectrometry

Na₂SO₄Sodium sulfate

PE Petroleum Ether

PPA polyphosphoric acid

Rt Retention time

Rt or Rt Room temperature

TBAF tetrabutylammonium fluoride

TBSCl tert-butyldimethylsilyl chloride

TEA Triethanolamine

TFA trifluoroacetic acid

THF tetrahydrofuran

T L C thin layer chromatography

TMSCl trimethylchlorosilane

Mu L microliter

XRPD X-ray powder diffraction

Example 1

Preparation of crystalline form a of compound 1

Step 1: synthesis of INTQ-1

1, 4-dioxane (1.5 volumes) was added to a 4-neck round bottom flask of 2L, the flask was evacuated and flushed with nitrogen three times, then Pd (OAc)₂(2 wt%, 0.50kg) and XantPhos (9 wt%, 2.25kg) were added to the flask, and the flask was evacuated and flushed with nitrogen three times. The mixture is stirred at room temperature for 0.5 to 1 hour under a nitrogen atmosphere. NaOH (12.25kg,1.6 equivalents), H₂O (1 vol, 25L) and 1, 4-dioxane (8 vol, 200L) were added to a 20L reactor the mixture was stirred until clear, then SM3(26.75kg,1.2 equivalents) was added to the mixture, the catalyst solution was transferred to a nitrogen atmosphereThe reactor was then charged with SM1(25.00kg,1.0 eq) dropwise to the reactor, the system was heated to 65 ± 5 ℃ and held at 65 ± 5 ℃ for at least 5 hours HP L C was used to monitor the reaction until the content of SM1 did not exceed 1.0%, the reaction mixture was cooled to 30 ± 5 ℃, then filtered, the filter cake was washed with 1, 4-dioxane (1.0 vol.) H was added₂O (4 vol) was added to the filtrate and concentrated to 5 vol. Then H is introduced₂O (2 vol) was added to the residue and concentrated to 5 vol. The residue was cooled to room temperature and filtered. The filter cake is treated with H₂O (2 vol) wash. The filter cake was then slurried with IPA (2 volumes) at 25 + -5 deg.C for 3 hours. The mixture was filtered and the filter cake was washed with IPA (0.5 vol). The solid was dried in an oven under reduced pressure.

And 2, step 3: synthesis of INTQ-3

Adding THF (25 volumes) and INTQ-1(16.00kg,1.0 equivalent) to a reactor, stirring the mixture and cooling to-80 to-70 ℃ then adding n-Bu L i (n-hexane solution, 2.5M,51.20kg,2.5 equivalents) dropwise to the mixture at-80 to-70 ℃ after reacting for 1-2 hours at-80 to-70 ℃, monitoring the reaction by T L C, then adding a solution of DMF (9.92kg,1.8 equivalents) in THF (1.4 volumes) dropwise to the reaction system at-80 to-70 ℃ after reacting for 1-2 hours at-80 to-70 ℃, monitoring the reaction by T L C, adding a solution of AcOH in THF (1.4 volumes) at-80 to-70 ℃ dropwise to the mixture to adjust the pH to 6-7, then adding TEA (8 equivalents) to-70 ℃ after reacting for 1-10 hours at-80 to-70 ℃, monitoring the solution of triphenylphosphine in acetic acid ester (1.4 volumes) dropwise to the reaction mixture at-80 ℃ and then adding a solution of triphenylphosphine (10.05. H) dropwise to the reaction mixture at-80 ℃ and stirring to-70 ℃ after reacting for 1.10 hours, adding a solution of triphenylphosphine (1.19. C) dropwise to the mixture at-80 ℃ and stirring₂O (10.5 vol) and citric acid (32.00kg,2.1 eq.) were added to another reactor, the mixture was stirred to dissolve and cool to 0-5 deg.C, the temperature was cooled to-20 deg.C and the solution was transferred to the above-mentioned 4-necked round bottom flask of 3L, the mixture was then stirred at 20 deg.C or less for 1 hour, confirming the pH was between 4 and 7, the organic layer was separatedAnd washed with 25% NaCl (17 vol). The organic phase was then concentrated to 5 volumes and EtOAc (17 volumes) was added to the mixture and concentrated to 5 volumes. EtOAc (17 vol) was added to the mixture and concentrated to 5 vol. This solution was used directly in the next step.

And 4, step 4: synthesis of INTQ-4

Adding INTQ-3 in EtOAc solution to a reactor, stirring the solution and cooling to-5 ℃, adding HCl to the mixture at-5 ℃ for 2 hours, then heating the mixture to 20-30 ℃, after 5 hours of reaction, monitoring the reaction using HP L C every 2 hours until the content of INTQ-3 is less than 0.5%, concentrating the reaction mixture to 10 volumes and cooling to 0-5 ℃, stirring the residue at 0-5 ℃ for 1 hour, filtering the mixture, adding filter cake to H₂O (15 vol). The mixture is stirred for 2 hours at 20-30 ℃. Filtering the mixture, filtering the filter cake with H₂O (3 vol) wash. The filtrate was then transferred to another reactor and Na was added₂CO₃Adding the mixture to adjust the pH value to 8-9. The mixture is then filtered and the filter cake is washed with H₂O (4 vol) wash. After drying in a vacuum oven, 20.73kg (yield: 69.0%, purity: 95.0%) of INTQ-4 was obtained.

And 5: synthesis of INTQ-5

INTQ-4(10.40kg,1.0 eq), Pd/C (15% wt,1.25kg) and THF (11 vol) were added to a reactor the mixture was stirred and heated to 30-35 ℃. hydrogen was added to a pressure of 10atm, after 15 hours of reaction the reaction was monitored every 2 hours using HP L C until the INTQ-4 content was less than 0.5%. the reaction mixture was cooled to 20-30 ℃ and filtered through celite (0.2wt), the filter cake was washed with THF (2 vol), the filtrate was concentrated to 3 vol and EtOH (6 vol) was added to the mixture, the solution was concentrated to 3 vol and EtOH (6 vol) was added to the mixture, the mixture was concentrated to 3 vol and used directly in the next step.

Step 6: synthesis of BGB-INTQ-6

A solution of INTQ-5 (from the previous step) in EtOH (3 vol), EtOH (7 vol) and Et₃N (22% wt,2.29kg) was added to the reactor, the solution was heated to 70-80 ℃ and after 15 hours of reaction, the reaction was monitored every 2 hours with HP L C until the level of INTQ-5 was less than 1.0%, the reaction mixture was cooled to 30-40 ℃ and concentrated to 5 volumes, the mixture was cooled to-5-0 ℃ and stirred for 2 hours, the mixture was filtered, the filter cake was washed with EtOH (1 volume) and dried in an oven at 45 + -5 ℃ to give 7.58kg (yield: 87.1%, purity: 99.5%) of INTQ-6.

And 7: synthesis of INTQ-7

Potassium hydroxide (49.9Kg,1.7 equiv.) is added to a solution of 4-methoxyphenol (65Kg,1.0 equiv.) in DMSO (65L, 1 vol.) the system is heated to 120 ℃. bromoacetaldehyde diethyl acetal (123.8Kg,1.2 equiv.) is added dropwise while maintaining the temperature at 120-140 ℃. the reaction mixture is cooled to 20-40 ℃ after monitoring the completion of the reaction by HP L C. N-heptane (2 vol) and water (2 vol.) are added to the reaction mixture, the mixture is filtered through celite (0.2 wt.) and the filter cake is washed with N-heptane (0.5 vol.) the filtrate is left to stand for at least 30 minutes.

And 8: synthesis of INTQ-8

Amberlyst-15(3.8Kg,0.1wt) was added to toluene (760L, 20 volumes) in N₂Heating the system to 110 ℃ under protection, adding dropwise a solution of INTQ-7(38 Kg/batch, 3 batches, 1.0 eq) in toluene while maintaining the temperature at 105-110 ℃ after 1 hour of reaction, concentrating the reaction system to 17 volumes at constant pressure of 105-110 ℃, adding toluene (3 volumes) to the system, monitoring the reaction completion by HP L C, cooling the reaction mixture to 20-40 ℃ after completion, filtering the mixture through celite (0.1wt), washing the filter cake with toluene (0.5 volume), washing the filtrate with 2n aoh aqueous solution (2 volume), washing the organic layer twice with 20% aqueous NaCl solution (2 volume), concentrating the organic layer to 2 volume, distilling the crude product below 110 ℃ to give INTQ-8 as an off-white solid (43Kg, yield 61.2%, purity ≥ 98.0%).

And step 9: synthesis of INTQ-9

Adding 1-dodecanethiol (147.0Kg,3.5 equivalents) to a solution of INTQ-8(43Kg,1.0 equivalents) in NMP (260L, 6 volumes), heating the system to 75 ± 5 ℃, adding sodium ethoxide (69.0Kg,3.5 equivalents) in portions while maintaining the temperature below 120 ℃, heating the reaction mixture to 130 ± 5 ℃, sampling the mixture for HP L C per hour until the content of PH-BEI-BGB-3289-INTQ-8 is ≤ 3.0% after 16 hours of reaction at 130 ± 5 ℃, cooling the reaction mixture to 60 ± 5 ℃, then adding 8 volumes of water to the mixture, cooling the reaction mixture to 25 ± 5 ℃, then adding 3 volumes of petroleum ether to the mixture, stirring the mixture for at least 30 minutes and standing for at least 30 minutes, separating the organic phase, adjusting the aqueous phase to PH 1-2 with 6N HCl, extracting the aqueous phase with 5 volumes of ethyl acetate, respectively, adjusting the aqueous phase to 4 volumes of ethyl acetate, and then adjusting the aqueous phase to 30 minutes with at least 6N HCl, and then adjusting the volumes of the aqueous phase with N of the mixtureThe pH value is 1-2. The aqueous phase was extracted with 5 volumes of ethyl acetate. The organic phases of ethyl acetate were combined and concentrated to 3 volumes at a pressure below 50 ℃. To the residue was added 5 volumes of n-heptane and the mixture was adjusted to a PH of 9-10 with 5% NaOH. The mixture was stirred for at least 30 minutes and allowed to stand for at least 30 minutes, and separated. The aqueous phase was adjusted to pH 1-2 with 6N HCl. The aqueous phase was extracted with 5 volumes and 3 volumes of ethyl acetate, respectively. The ethyl acetate organic phases were then combined with 6 vol 10% H₂O₂And concentrated HCl (0.15wt) washes. Then using 6 vol 5% H₂O₂And the organic phase was washed with concentrated HCl (0.15 wt). With 4 vol 5% Na₂SO₃The organic layer was washed. The organic layer was washed three times with 3 volumes of brine. The organic layer was concentrated to 3 volumes. Dichloromethane (5 volumes) was added and concentration continued until no significant fractions (fraction) were present. The crude product of INTQ-9 was used directly in the next step.

Step 10: synthesis of INTQ-10

Adding Et₃N (48.2Kg,2.0 equiv.) is added to a solution of INTQ-9(32Kg,1.0 equiv.) in dichloromethane (10 volumes) at less than 40 ℃. The mixture was cooled to-5. + -. 5 ℃. TMSCl (1.3 equivalents) in dichloromethane (1 volume) was added dropwise while maintaining the temperature at-5 ± 5 ℃. The mixture was sampled hourly for gas chromatography until the level of INTQ-9 was 2.0% or less after 1 hour of reaction at-5. + -. 5 ℃. The mixture was concentrated to 3 volumes at a pressure below 40 ℃. To the residue was added 15 volumes of n-hexane, and the mixture was stirred for at least 30 minutes. The mixture was filtered and the filtrate was concentrated to no significant fractions at a pressure below 40 ℃. The crude product was distilled below 120 ℃ to give INTQ-10 as a pale yellow oil (40Kg, 81.4% yield ≥ 97.5% purity).

Step 11: synthesis of INTQ-11

INTQ-10(20 Kg/batch, 2 batches, 1.0 equiv.) in dichloromethane (5 volumes) was slurried with CuI (0.1wt) at 25 + -5 deg.C for 2-3 hours. In N₂Copper (I) trifluoromethanesulfonate (2:1 complex with toluene, 0.11% wt) and (S, S) -2, 2-bis (4-phenyl-2-oxazolin-2-yl) propane (0.15% wt) were stirred in dichloromethane (4 vol) at 20-30 ℃ for 2-3 hours under an atmosphere. A solution of INTQ-10 in dichloromethane was added through a small pore filter and a solution of ethyl diazoacetate (2.0 equiv.) in dichloromethane (10 volumes) was added slowly dropwise over 15-25 hours at 20-30 ℃. The mixture is stirred at 20-30 ℃ for 30-60 minutes and washed three times with 4 volumes of 0.05N disodium ethylenediaminetetraacetate dihydrate at 20-30 ℃. The organic portion was washed twice with 3 volumes of 25% aqueous NaCl. The organic portion was concentrated under vacuum below 35 ℃ until the system did not exceed 3 volumes. The crude product of INTQ-11 was used directly in the next step.

Step 12 and step 13: synthesis of INTQ-13

Step 12: the crude product of INTQ-11 was dissolved in methanol (3 vol), 38% HCl/EtOH (0.1 vol) was added to the mixture and stirred at 20-30 ℃ for 2-3 hours. Et was added dropwise to the mixture₃N to adjust pH 7. The mixture was concentrated under pressure to 2 volumes. Ethyl acetate (2 vol) was added and concentration continued under pressure to 2 vol. N-heptane (2 vol) was added and concentration continued under pressure to 2 vol. Dichloromethane (2 volumes) was added to completely dissolve the material. The residue was purified by silica gel chromatography (eluting with EtOAc: PE ═ 1:5, total about 100 volumes) to give INTQ-12 as a yellow solid.

Step 13: INTQ-12 was added to EtOAc (1.5 vol) and n-heptane (20 vol) and the mixture was heated to 75-85 ℃ until clear. Stirring the clear solution at 75-85 ℃ for 1 hour, and then gradually cooling to 15-20 ℃. The mixture was filtered and washed with n-heptane (2 vol) to give the product. The wet product was dried at 55 ± 5 ℃ for at least 16 hours to give INTQ-13 as a pale yellow to off-white solid.

Steps 14 and 15: synthesis of INTQ-15

Step 14. add INTQ-13(16Kg,1.0 eq) and INTQ-6(12.7Kg,1.05 eq) to DMF (5 vol.) the system was heated to 55 ± 5 ℃ and cesium carbonate (29.6Kg,1.25 eq) was added, the reaction mixture was heated to 110 ± 5 ℃ and the mixture was sampled for HP L C per hour until the content of INTQ-13 was ≤ 0.5% after 2 hours of reaction at 110 ± 5 ℃, the reaction mixture was cooled to 30 ± 5 ℃, then adjusted to pH 6 with acetic acid (5wt) at 30 ± 5 ℃, water (30 vol) was added to the mixture at 25 ± 5 ℃, the mixture was stirred for 1-2 hours and filtered to give the wet product, the wet product was reslurried with water (5 vol.) the filter cake was used directly in the next step.

Step 15. add wet INTQ-14 to a mixture of 1N NaOH (10 vol) and THF (20 vol.) the system is stirred at 25 ± 5 ℃, sample for HP L C every hour until the content of INTQ-14 is ≤ 0.5% after 4 hours of reaction at 25 ± 5 ℃, adjust the system to pH 4-5 at 25 ± 5 ℃ with 4N HCl and stir for 1 hour, concentrate the system to 8 vol at a pressure below 50 ℃, then filter to obtain wet product, reslurry the wet product with THF (10 vol.), stir the mixture for 1-2 hours and filter to obtain wet product, dry the wet product at 55 ± 5 ℃ for at least 30 hours to obtain INTQ-15 as a light brown to off-white solid.

Steps 16 and 17 and 18: synthesis of INTQ-18

Vacuumizing the reactor to less than or equal to-0.08 MPa, and then filling inert nitrogen. 1, 4-dioxane (10.0 vol), INTQ-15(3.6Kg,1.0 eq) was added to the reactor. The mixture is concentrated below 50 ℃ to 6.0-6.5 volumes and the mixture is sampled to obtain the water content. Adding Et₃N (1.1 eq) was added to the reactor. Heating the mixture to 30 + -5 deg.C, and mixing the DPPA(1.1 equiv.) was added dropwise to the reactor after 2 hours of reaction at 30. + -. 5 ℃ the mixture was sampled for HP L C analysis until the level of INTQ-15 was 1.0% or less and a solution of INTQ-16 was obtained.

Vacuumizing the reactor to less than or equal to-0.08 MPa, and then filling inert nitrogen. Mixing t-BuOH (20.0 vol), (Boc)₂O (0.5 eq) and DMAP (0.02 eq) are added to the reactor, the mixture is heated to 85 ± 5 ℃, stirred for 2-3 hours, and the water content of the mixture is sampled KF ≤ 0.01%. a solution of INTQ-16 is added dropwise at 85 ± 5 ℃ to the reactor of the t-BuOH system described above (for at least 3 hours), after reaction at 85 ± 5 ℃ for 2 hours, the mixture is sampled for HP L C analysis until the content of INTQ-16 is ≤ 1.0%, then the mixture is cooled to less than 50 ℃ and concentrated to 3.0-4.0 volumes below 50 ℃.

DCM (10.0 vol × 2) was added to the residue and the mixture was concentrated to 3.0-4.0 vol below 50 ℃, DCM (10.0 vol) was added to the residue, then 1 wt% aqueous NaOH (20.0 vol) was added to the reactor and stirred at 25 ± 5 ℃ for at least 1 hour, the mixture was filtered through celite, then separated, the organic phase was washed with water (5.0 vol) and separated, the organic phase was further washed with 25 wt% brine (5.0 vol), separated by filtration over a pad of silica gel to remove some of the impurities, the organic phase was concentrated to 6.0-7.0 vol below 40 ℃, DCM was added to 7.0 vol, then the mixture was cooled to not more than 15 ℃, and hydrochloric acid (1.2 vol) was added dropwise to the reactor at a temperature no higher than 15 ℃, after reaction for 3 hours at 15 ± 5 ℃, the mixture was sampled for HP L C analysis until the content of INTQ-17 ≦ 4.0 ≦ 25.0.

INTQ-16:¹H NMR(400MHz,DMSO-d6)10.48(s,1H),7.95(d,J＝5.6Hz,1H),7.34(d,J＝2.4Hz,1H),7.05(d,J＝8.4Hz,1H),7.00(dd,J＝8.8,2.4Hz,1H),6.25(d,J＝5.6Hz,1H),5.42(d,J＝5.2Hz,1H),3.56(dd,J＝5.2,2.8Hz,1H),2.92(t,J＝7.6Hz,2H),2.54(d,J＝8.0Hz,2H),1.51(d,J＝3.2Hz,1H).MS:M/e 364(M+1)⁺.

INTQ-17:¹H NMR(400MHz,DMSO-d6)10.49(s,1H),7.94(d,J＝6.0Hz,1H),7.34(s,1H),7.18(s,1H),6.96–6.83(m,2H),6.22(d,J＝5.6Hz,1H),4.86(d,J＝5.6Hz,1H),2.92(t,J＝7.6Hz,2H),2.86(d,J＝4.8Hz,1H),2.54(t,J＝7.6Hz,2H),2.12(s,1H),1.39(s,9H).MS:M/e 410(M+1)⁺.

And (3) pH adjustment process: a4 wt% NaOH aqueous solution was added dropwise to the reactor to adjust the pH to 2.7-3.1. If the pH is higher>3.1, adding hydrochloric acid (0.2 volume), and then adding a 4 wt% NaOH aqueous solution into the reactor drop by drop to adjust the pH value to 2.7-3.1 (precision pH paper, range 2.7-4.7); the mixture was separated and the emulsion phase was collected as the aqueous phase. The mixture was filtered through celite and the resulting aqueous phase was washed once with DCM (2.0 volumes). DCM (6.0 vol) and EtOH (5.0 vol) were added to the remaining aqueous phase in the reactor. Mixing 10.0 wt% of Na₂CO₃The solution is added dropwise to the reaction to adjust the pH to 8-9 at 25 ± 5 ℃. the mixture is stirred for 10-15 minutes and left to stand for 10-15 minutes, the mixture is separated, the aqueous phase is extracted 2 times with DCM (4.0 vol.) the organic phases are combined, washed with water (2.0 vol.), separated, the organic phases are washed once with 25 wt% brine (5.0 vol.), the organic phases are concentrated to 3.0-4.0 vol.% below 45 ℃, then n-heptane (4.0 vol.) is added to the residue, the mixture is concentrated to 3.0-4.0 vol.% below 45 ℃. the residue is cooled to 25 ± 5 ℃, then centrifuged, the solid is washed with n-heptane (2.0 vol.) the filter cake is transferred to a vacuum oven, dried for 4 hours at 45 ± 5 ℃, the mixture is sampled for drying (24 ± 5 ℃), the mixture is taken for drying until the product is lost in weight loss in bags (24-862.3.2 kg) and the DPE is reported to be packed in a double bag with 2kg product purity.

INTQ-18:¹H NMR(400MHz,DMSO-d6)10.81(s,1H),8.87(s,3H),8.05(d,J＝6.0Hz,1H),7.33(t,J＝1.2Hz,1H),7.07–6.95(m,2H),6.34(d,J＝6.0Hz,1H),5.24(d,J＝6.0Hz,1H),3.32(dd,J＝6.0,2.0Hz,1H),2.97(t,J＝7.6Hz,2H),2.59(t,J＝7.6Hz,2H),2.46(s,1H).MS:M/e 310(M+1)⁺.

Step 19: synthesis of INTQ-19

Vacuumizing the reactor to less than or equal to-0.08 MPa, and then filling inert nitrogen. THF (6.0 vol), H₂O (3.0 vol.), 2,4, 5-trifluoroaniline (1.0 eq.), NaHCO₃(1.2 equiv.) into the reactor, the mixture was cooled to 0 deg.C, phenyl chloroformate was added slowly at 0 + -5 deg.C, the mixture was stirred for at least 2 hours, the mixture was sampled for L CMS until 2,4, 5-trifluoroaniline was < 0.2%, EA (15.0 vol.) was then added with H₂O (5.0 vol) washing of the organic phase followed by 2 washes with 5 wt% aqueous HCl (5.0 vol), 2 washes with saturated NaCl (5.0 vol) 2 washes the organic phase was concentrated to 10.0 vol below 45 deg.C N-heptane (10.0 vol) was added to the residue the mixture was concentrated to 10.0 vol, then N-heptane (10.0 vol) was added to the residue the mixture was concentrated to 10.0 vol and centrifuged, the solids were washed with N-heptane (2.0 vol.) the filter cake was sampled for L CMS analysis with a standard of INTQ-19>99% then the filter cake was transferred to a vacuum oven and dried at 35 + -5 deg.C (oven temperature) for 10 hours before sampling L OD until L OD.2% or less, purity of INTQ-19 was reported, the product was packaged in double L DPE plastic bags and stored at 2-30 deg.C.

INTQ-19:¹H NMR(400MHz,DMSO-d6)10.20(s,1H),7.82(dt,J＝12.0,8.0Hz,1H),7.66(td,J＝10.8,7.6Hz,1H),7.44(t,J＝7.6Hz,2H),7.33–7.20(m,3H).

Step 20: synthesis of crystalline form of Compound 1 (form A)

The reactor was evacuated to ≦ 0.08MPa and then purged with inert nitrogen, DMSO (9.0 vol.), INTQ-18(1.63Kg,1.0 equivalent) and N-methylmorpholine (NMM,1.0 equivalent) were added to the reactor, the mixture was stirred at 20 + -5 ℃ for at least 0.5 hour, INTQ-19(1.27Kg,0.9 equivalent) was added to the reactor at 20 + -5 ℃, after 3 hours of reaction at 20 + -5 ℃, the mixture was sampled for HP L C analysis until the level of INTQ-19 was ≦ 0.3%, after completion of the reaction, the mixture of Compound 1 was added dropwise through a microfilter to a 0.5% hydrochloric acid solution, which was also slowly filtered through a microfilter (30.0 vol) at 20 + -5 ℃, the mixture was stirred for at least 4 hours and the filter cake was centrifuged to wash with (5.0 vol. × 2).

Slurry operation DMSO (9.0 vol) and 0.5% hydrochloric acid are added to the reactor through a micron filter (30.0 vol) and the filter cake is added to the reactor and the mixture is stirred at 20 + -5 ℃ for at least 4 hours and then centrifuged, the filter cake is washed with purified water (5.0 vol ×), the filter cake is sampled for HP L C analysis, the standard for compound 1 is 98.0%. if compound 1 is < 98.0%, the "slurry operation" is repeated, purified water (40.0 vol) and the filter cake are added to the reactor and the mixture is stirred at 20 + -5 ℃ for at least 4 hours and then centrifuged, the filter cake is washed with purified water (5.0 vol ×) and then the filter cake is vacuum dried at 45 + -5 ℃ for at least 8 hours until L OD is 3.0%. if the solvent residue does not meet the standard, the residual solvent is removed by slurrying, the purified water (40.0 vol) and the product are added to the reactor and the mixture is stirred at 20 + -5 ℃ for at least 4 hours and then the mixture is stirred at room temperature for at least 4 hours and the residue is removed by centrifugation through a double slurry operation, the filter cake is centrifuged, the filter cake is collected with purified water (40.0.0%) and the residue is collected in a sample is taken after the sample is dried, the filter cake is added to obtain a sample, the residue is added to obtain a mixture, the residue is added to obtain a mixture is stirred.

Compound 1:¹H NMR(400MHz,DMSO-d6)10.47(s,1H),8.54(s,1H),8.23–8.07(m,1H),7.96(d,J＝5.6Hz,1H),7.65–7.51(m,1H),7.23(s,1H),7.01(d,J＝2.0Hz,1H),6.96–6.87(m,2H),6.25(d,J＝5.6Hz,1H),4.98(d,J＝6.0Hz,1H),2.97(dd,J＝5.6,1.6Hz,1H),2.93(t,J＝7.6Hz,2H),2.54(t,J＝7.6Hz,2H),2.26(s,1H).

the amorphous or crystalline properties of the resulting powder prepared in example 1 were evaluated by X-ray powder diffraction pattern techniques. As evidenced by the crystalline peaks in the XRPD curve in FIG. 1The resulting powder prepared in example 1 was identified as crystalline (sometimes referred to throughout as "form a"). The obtained powder is also prepared by¹H-NMR spectrum and¹³the C-NMR spectrum was as shown in FIGS. 8 and 9, respectively.

The X-ray powder diffraction pattern of the crystalline form of compound 1 (form a) has the following characteristic peak diffraction angles (where the "pitch" is shown as the "d value" in figure 1):

table 1: x-ray powder diffractogram of crystalline form of Compound 1 (form A)

Long term stability of form A

Long-term stability studies of form a show that no significant change in chemical purity occurs when stored for up to 12 months at 25 ℃/60% RH (total impurities: T0 ═ 1.0%, T12 ═ 1.0%) and for up to 6 months at 40 ℃/75% RH (total impurities: T0 ═ 1.0%, T12 ═ 1.0%). Furthermore, no change in optical purity was observed when stored at 25 ℃/60% RH for up to 12 months and at 40 ℃/75% RH for up to 6 months. XRPD data of the test samples showed that form a was stable at 40 ℃/75% RH for 6 months, and form a was also stable at 25 ℃/60% RH for 6 months, but became crystalline form (sometimes referred to as "form a") at 12 months.

XRPD of form a is shown in figure 2. The X-ray powder diffraction pattern of another crystalline form of compound 1 (form a) has the following characteristic peak diffraction angles (where the "pitch" is shown as the "d value" in figure 2):

table 2: x-ray powder diffraction Pattern of Another crystalline form (form A) of Compound 1

Stability studies indicate that form a is chemically stable and can be stored for more than 12 months without significant decomposition.

Form a was also evaluated for hygroscopicity by Dynamic Vapor Sorption (DVS), as shown in fig. 10. Figure 10 shows that form a is moderately hygroscopic with a 4.96% weight gain at 80% RH.

As discussed in the preclinical studies below, oral absorption of form a in rats was relatively poor, with 21% bioavailability, due in part to the low solubility of form a (found to be less than 0.1 μ g/m L in water).

Form a has limited use in direct pharmaceutical formulation due to the low bioavailability observed in preclinical studies. However, form a is a good candidate for purification of APIs and is used as a starting material for the preparation of amorphous solid dispersions. Although a change in crystalline form (i.e. from form a to form a) was observed at 12 months under the above conditions, long term storage (i.e. 12 months) had no effect on the subsequent amorphous MBP preparation, since compound 1 was anyway dissolved in DMA before coprecipitation occurred.

The physicochemical properties of form a prepared in example 1 are summarized in table 3.

Table 3: major physicochemical Properties of form A

Example 2

Single crystals of Compound 1 (form A)

First, 1.8mg of compound 1 was weighed into a 3M L glass vial, 0.5M L EtOAc solvent was added, after vortexing and ultrasonic shaking to accelerate dissolution, the suspension was filtered through a PTFE filter membrane (0.45 μ M), the filtrate was transferred to a clean 4M L shell vial (44.6mm X14.65 mm), then the shell vial was sealed with a PE-Plug with a pinhole thereon and placed in a fume hood to evaporate slowly at ambient temperature and humidity, after six days, a plate crystal sample was obtained.

The structure of the plate-like crystals was determined using a set of diffraction data collected from single crystals grown in EtOAc by slow cooling and was designated as single crystals of compound 1 or form a. The crystal data and structural refinements of form a are listed in figures 3-6.

Table 4: single crystal data and structural finishing of form a

As shown in figure 3, the asymmetric unit of the single structure consists of two separate molecules of compound 1 and two molecules of EtOAc solvent, indicating that the crystal is an EtOAc solvate of compound 1. Single crystal structure determination confirmed that when compound 1 molecule is taken as an example, the absolute configuration of compound 1 is { C15(S), C16(S), C17(S) }. The unit cell of the single crystal consisted of four molecules of compound 1 and four molecules of EtOAc solvent, as shown in figure 4. The potential classical H bonds in the single crystal structure are shown in figure 5. The results of theoretical XRPD patterns calculated using the mercure software for the single crystal form of compound 1 (i.e., form a) are shown in fig. 6.

Example 3

Preparation of pure amorphous form of Compound 1 (form B)

A solution of crystalline form a of compound 1 in DCM/MeOH (2:1) was spray dried to give a white powder spray dried under the conditions that a solution of crystalline form a of compound 1 (2.0g) in 100m L mixed solvent (DCM/MeOH-2: 1 vol) was spray dried through a spray dryer (BUCHI-290& BUCHI-295), the product powder was dried for 16 hours at 50 ℃ by a lamp to obtain 1.06g of powder, the operating parameters of the spray dryer (BUCHI-290& BUCHI-295) were as follows: inlet temperature: 60 ℃; outlet temperature: 35 ℃, aspirator: 100%, infrared pump%: 15%, nozzle cleaner: 2.

The structure of the resulting powder was characterized using a powder X-ray diffraction pattern method, and it was confirmed that the structure was in an amorphous form, for example, the powder X-ray diffraction pattern of FIG. 7 had no diffraction peak. In the present specification, the amorphous form of compound 1 is referred to as the pure amorphous form of compound 1 or form B. Of form B¹The H-NMR spectrum is shown in FIG. 11. The glass transition temperature of form B was determined to be 138.3 ℃. The sample of form B was a white powder with a particle size of D90 ═ 69.9 μm, D50 ═ 3.5 μm, and D10 ═ 1.4 μm. XRPD data of the test samples indicated that form B was stable at 40 ℃/75% RH over 14 days. For example, the XRPD data of fig. 12 confirms that the test sample still does not show any diffraction peaks at 14 days.

Example 14

Form A, B was compared in pharmacokinetics in rats.

1. Drugs and agents:

form a powder with a particle size D90 ═ 62.4 μm after micronization. The content (purity) of the substance is not less than 98.0%.

Form B powder with a particle size after micronization of 69.9 μm D90, 3.5 μm D10, and 1.4 μm D50. The content (purity) of the substance is not less than 98.0%.

2. Experimental animals:

male and female rats were used in this study.

3. The preparation of the medicine comprises the following steps:

the appropriate amount of each material was weighed and dispersed in 0.5% sodium carboxymethyl cellulose. For each material, a suspension was prepared with the desired concentration of compound 1. All doses and concentrations of compound 1 were calculated with the free base in this study.

4. Administration and sample collection:

dosing solutions were prepared fresh prior to dosing. Record actual body weight and notes accordinglyActual volume of the shots rats were fasted overnight and allowed food intake 4 hours after dosing each suspension was orally administered to rats at a dose of 0.5 to 5mg/kg blood samples (-1.0 m L) were collected by the cephalic plexus at various times before and up to 36 hours after dosing, whole blood was processed by centrifugation, plasma samples were collected and stored in a refrigerator before analysis, plasma samples were processed by protein precipitation, the concentration of compound 1 in plasma samples was determined using a validated liquid chromatography-tandem mass spectrometry (L C-MS/MS) method plasma concentration-time data were analyzed using a non-compartmental model using Pharsight WinNonlin_maxAnd the area under the concentration-time curve is shown in table 7.

Table 7: oral PK profiles for forms a and B of compound 1 versus intravenous PK profile for form a in rats

The pure amorphous form of compound 1 (form B) also exhibits higher C than the crystalline form (form a)_max(ng/mL)、AUC_0-inf(ng.h/m L) and F (%). the oral bioavailability of the pure amorphous form of compound 1 (form B) reached approximately 50% of intravenous injection, whereas the oral bioavailability of crystalline form a was only about 20% of intravenous injection, indicating that the pure amorphous form of the present invention greatly enhances oral bioavailability relative to the crystalline form.

The foregoing examples and embodiments are illustrative, but not limiting, of the invention defined by the claims. Various variations and combinations of the features described above may be used without departing from the invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All references cited are incorporated by reference herein in their entirety.

Claims

A pure amorphous form of 1- ((1S,1aS,6bS) -5- ((7-oxo-5, 6,7, 8-tetrahydro-1, 8-naphthyridin-4-yl) oxy) -1a,6 b-dihydro-1H-cyclopropa [ b ] benzofuran-1-yl) -3- (2,4, 5-trifluorophenyl) urea characterized by an X-ray powder diffraction pattern without diffraction peaks aS shown in FIG. 7.
2. The pure amorphous form according to claim 1, characterized in that said pure amorphous form has the structure shown in figure 11¹H-NMR spectrum.
3. The pure amorphous form according to claim 1, characterized in that the glass transition temperature of said pure amorphous form is between about 135 and 143 ℃, more preferably about 138.3 ℃.
4. The pure amorphous form according to claim 1, characterized in that the particle size of said pure amorphous form is D between about 60 to about 80 μm₉₀D between about 2 and about 6 μm₅₀D between about 1 and about 2 μm₁₀(ii) a More preferably D₉₀About 69.9 μm, D₅₀About 3.5 μm, and D₁₀Is about 1.4 μm.
5. A process for preparing the pure amorphous form according to any one of claims 1 to 4, comprising: a solution of a crystalline form of 1- ((1S,1aS,6bS) -5- ((7-oxo-5, 6,7, 8-tetrahydro-1, 8-naphthyridin-4-yl) oxy) -1a,6 b-dihydro-1H-cyclopropa [ b ] benzofuran-1-yl) -3- (2,4, 5-trifluorophenyl) urea in a polar solvent is spray dried to give a powdery substance.
6. The method according to claim 5, characterized in that the polar solvent comprises ethers, carboxylic acid esters, nitriles, ketones, amides, sulfones, sulfoxides or halogenated hydrocarbons.
7. The process according to claim 6, characterized in that the polar solvent is selected from the group consisting of acetic acid, acetone, acetonitrile, benzene, chloroform, carbon tetrachloride, dichloromethane, dimethyl sulfoxide, 1, 4-dioxane, ethanol, ethyl acetate, butanol, tert-butanol, N-dimethylacetamide, N-dimethylformamide, formamide, formic acid, heptane, hexane, isopropanol, methanol, methyl ethyl ketone, l-methyl-2-pyrrolidone, mesitylene, nitromethane, polyethylene glycol, propanol, 2-acetone, pyridine, tetrahydrofuran, toluene, xylene, and mixtures thereof.
8. The method of claim 5, characterized in that the inlet temperature of the spray dryer is set to about 50 to 70 ℃ and the outlet temperature of the spray dryer is set to about 25 to 45 ℃; more preferably, the inlet temperature of the spray dryer is set to about 60 ℃ and the outlet temperature of the spray dryer is set to about 35 ℃.
9. The method of claim 5, characterized in that the crystalline form of the compound is crystalline form A.
10. The process of claim 9, characterized in that said crystalline form a comprises an X-ray powder diffraction pattern having at least three, four, five, or six diffraction peaks independently selected from the following group 2 Θ ° values: 4.7 +/-0.2, 9.4 +/-0.2, 13.6 +/-0.2, 14.0 +/-0.2, 14.9 +/-0.2 and 15.6 +/-0.2 degrees; preferably, the crystalline form a comprises an X-ray powder diffraction pattern having at least three, four, five or six diffraction peaks independently selected from the following group of 2 Θ ° values: 4.7 +/-0.2, 9.4 +/-0.2, 13.6 +/-0.2, 14.0 +/-0.2, 14.9 +/-0.2, 15.6 +/-0.2, 21.2 +/-0.2, 24.3 +/-0.2, 24.7 +/-0.2, 25.1 +/-0.2 and 29.1 +/-0.2 degrees; more preferably, the crystalline form a comprises an X-ray powder diffraction pattern having diffraction peaks independently selected from the group consisting of the following 2 Θ ° values: 4.7 +/-0.2, 9.4 +/-0.2, 10.2 +/-0.2, 13.6 +/-0.2, 14.0 +/-0.2, 14.9 +/-0.2, 15.6 +/-0.2, 17.2 +/-0.2, 17.4 +/-0.2, 18.7 +/-0.2, 20.0 +/-0.2, 20.4 +/-0.2, 21.2 +/-0.2, 22.3 +/-0.2, 24.3 +/-0.2, 24.7 +/-0.2, 25.1 +/-0.2, 25.5 +/-0.2, 26.8 +/-0.2, 27.4 +/-0.2, 27.8 +/-0.2, 28.6 +/-0.2, 29.1 +/-0.2, 30.2 +/-0.2, 31.8 +/-0.2, 32.0 +/-0.2, 33.1 +/-0.2, 34.1 +/-0.2 and 34.6 +/-0.2.
11. The method of claim 10, characterized in that the crystalline form a has an X-ray powder diffraction pattern substantially as shown in figure 1.