WO2025162419A1 - Crystal form of bicyclic derivative parp inhibitor, preparation method therefor, and use thereof - Google Patents

Abstract

Provided in the present invention are a compound of formula (I), a crystal form thereof, a preparation method therefor, a pharmaceutical composition thereof, and the use of the crystal form and the pharmaceutical composition in preparation of related drugs.

Description

A crystal form of a bicyclic derivative PARP inhibitor, its preparation method and application Technical Field

The present invention relates to multiple crystal forms of a compound, a preparation method and applications thereof, and particularly to multiple crystal forms of a bicyclic derivative PARP inhibitor, a preparation method and applications thereof, belonging to the technical field of medicinal chemistry.

Background Art

Approximately 5% of breast cancer patients are associated with germline mutations in the BRCA1/2 genes (3% for BRCA1 and 2% for BRCA2). Breast cancers caused by BRCA1 mutations are mostly triple-negative (70%), while BRCA2 mutations are more likely to cause estrogen receptor-positive (ER-positive) breast cancer (70%). The BRCA1/2 genes are tumor suppressor genes that play an important role in DNA damage repair and normal cell growth. Mutations in these genes can inhibit the normal ability to repair DNA damage, causing homologous recombination deficiency (HRD). This is caused by loss of BRCA function or mutations or loss of function in other homologous recombination-related genes, preventing double-strand breaks from being repaired by homologous recombination (HRR), ultimately leading to cancer.

Poly(ADP-ribose) polymerase (PARP) is a DNA repair enzyme that plays a key role in the DNA repair pathway. PARP is activated when DNA is damaged and broken. As a molecular sensor of DNA damage, it has the function of recognizing and binding to the site of DNA breakage, thereby activating and catalyzing the poly(ADP-ribosylation) of the receptor protein and participating in the DNA repair process. PARP plays a key role in the excision and repair of single-stranded DNA bases. In HRD tumor cells, double-stranded DNA cannot be repaired, and PARP inhibitors block single-strand repair, resulting in a "synthetic lethality" effect, leading to tumor cell death.

PARP inhibitors have a "trapping" effect on PARP proteins, causing PARP proteins bound to damaged DNA to be trapped on the DNA and unable to get off. This directly prevents other DNA repair proteins from binding, ultimately leading to cell death. Currently, a number of PARP inhibitors have been successfully developed, such as Olaparib, Rucaparib, and Niraparib. However, adverse reactions limit their ability to be used in combination with chemotherapy drugs. This may be related to the lack of selectivity of marketed PARP inhibitors for the PARP family. These side effects include intestinal toxicity caused by end-ankyropolymerase inhibition and hematotoxicity caused by PARP-2 inhibition. Therefore, it is of great clinical significance to develop highly selective PARP-1 inhibitors to reduce the related toxic side effects of non-selective PARP inhibitors.

Patent WO2023227052 describes a compound of formula (I), which has good PARP inhibitory activity.

It is important to select a crystalline agent form that is stable, reproducible, and possesses physicochemical properties that are favorable for its use as a therapeutic agent.

Summary of the Invention

The present invention provides compounds represented by formula (I), their crystal forms, preparation methods, pharmaceutical compositions, and their use in preparing medicaments for treating PARP-mediated diseases. The various crystal forms provided by the present invention have excellent properties, including high purity, good solubility, stable physical and chemical properties, ease of processing, crystallization, and handling, resistance to high temperatures, high humidity, and strong light, and low hygroscopicity.

The present invention provides a crystalline form of a compound of formula (I):

In certain specific embodiments, the crystalline form is crystalline form I, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 4.63°±0.2°, 9.26°±0.2°, 10.45°±0.2°, 13.95°±0.2° and 16.33°±0.2°.

In certain specific embodiments, the crystalline form is crystalline form I, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 4.63°±0.2°, 9.26°±0.2°, 10.45°±0.2°, 13.95°±0.2°, 15.97°±0.2°, 16.33°±0.2°, 17.14°±0.2°, 20.62°±0.2° and 22.37°±0.2°.

In certain specific embodiments, the crystalline form is Form I, and its X-ray powder diffraction pattern using Cu-Kα radiation is substantially as shown in Figure 2.

In certain specific embodiments, the differential scanning calorimetry (DSC) curve thereof shows that the peak temperature of Form I is 274.95° C., and ΔH=97.746 J/g.

In certain specific embodiments, the differential scanning calorimetry (DSC) curve of Form I of the compound of formula (I) is substantially as shown in FIG3 .

In certain embodiments, the thermogravimetric analysis (TGA) curve of Form I of the compound of formula (I) shows a weight loss of about 0.13% before 100°C and a weight loss of about 0.34% from 100°C to 250°C.

In certain specific embodiments, the thermogravimetric analysis curve of Form I of the compound of formula (I) is substantially as shown in FIG4 .

In certain specific embodiments, the hygroscopicity (DVS) isotherm curve of Form I of the compound of formula (I) shows that the water adsorption is 1.273% at 0 RH%-80% RH, indicating slight hygroscopicity. The isotherm adsorption curve is substantially as shown in FIG5 .

In certain specific embodiments, the crystalline form is crystalline form II, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 7.59°±0.2°, 11.41°±0.2°, 11.79°±0.2°, 14.53°±0.2°, 17.40°±0.2°, 21.54°±0.2°, 24.93°±0.2° and 25.45°±0.2°.

In certain specific embodiments, the crystalline form is crystalline form II, and its X-ray powder diffraction pattern using Cu-Kα radiation has characteristic diffraction peaks at the following 2θ positions: 3.74°±0.2°, 7.59°±0.2°, 11.41°±0.2°, 11.79°±0.2°, 14.53°±0.2°, 17.09°±0.2°, 17.40°±0.2°, 18.15°±0.2°, 19.15°±0.2°, 21.54°±0.2°, 22.16°±0.2°, 22.66°±0.2°, 24.93°±0.2°, 25.45°±0.2° and 26.58°±0.2°.

In certain specific embodiments, Form II has an X-ray powder diffraction pattern substantially as shown in FIG6 using Cu-Kα radiation.

In certain specific embodiments, the differential scanning calorimetry (DSC) curve thereof shows that the peak temperature of Form II is 277.66° C., and ΔH=89.910 J/g.

In certain specific embodiments, the differential scanning calorimetry curve of Form II of the compound of formula (I) is substantially as shown in FIG7 .

In certain embodiments, the thermogravimetric analysis (TGA) curve of Form II of the compound of formula (I) shows a weight loss of about 0.13% before 100°C and a weight loss of about 1.34% from 100°C to 270°C.

In certain specific embodiments, the thermogravimetric analysis curve of Form II of the compound of formula (I) is substantially as shown in FIG8 .

In certain specific embodiments, the hygroscopicity (DVS) isotherm curve of Form II of the compound of formula (I) shows that the water adsorption at 0 RH%-80% RH is 1.148%, which is slightly hygroscopic. The isotherm adsorption curve is substantially as shown in FIG9 .

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned compound or any crystalline form thereof, and a pharmaceutically acceptable carrier and/or excipient, preferably wherein the therapeutically effective amount is 1-1440 mg. The pharmaceutical composition may be in the form of a unit dosage form (a unit dosage form is also referred to as a "dose strength").

The present invention also provides use of the compound, crystal form, or composition of any of the aforementioned embodiments in the preparation of a medicament for treating a PARP-mediated disease. Furthermore, the PARP-mediated disease is selected from breast cancer, uterine cancer, cervical cancer, ovarian cancer, and prostate cancer.

The present invention also provides a method for treating a PARP-mediated disease, comprising administering to a subject a therapeutically effective amount of a compound, crystalline form, or composition thereof according to any of the preceding embodiments, wherein the disease is preferably breast cancer, uterine cancer, cervical cancer, ovarian cancer, or prostate cancer, and wherein the therapeutically effective amount is preferably 1-1440 mg. In some embodiments, the mammal of the present invention includes a human.

As used herein, an "effective amount" or "therapeutically effective amount" refers to administering a sufficient amount of a compound or crystalline form disclosed herein to alleviate, to some extent, one or more symptoms of the disease or condition being treated. In some embodiments, the result is a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic uses is the amount of a composition comprising a compound or crystalline form disclosed herein required to provide a clinically significant reduction in disease symptoms. Examples of therapeutically effective amounts include, but are not limited to, 1-1440 mg, 1-1400 mg, 1-1300 mg, 1-1200 mg, 1-1000 mg, 1-900 mg, 1-800 mg, 1-700 mg, 1-600 mg, 1-500 mg, 1-400 mg, 1-300 mg, 1-250 mg, 1-200 mg, 1-150 mg, 1-125 mg, 1-100 mg, 1-80 mg, 1-60 mg, 1-50 mg, 1-40 mg, 1-25 mg, 1-20 mg, 5-1000 mg, 5-900 mg, 5-800 mg, 5-700 mg, 5-600 mg, 5 -500mg, 5-400mg, 5-300mg, 5-250mg, 5-200mg, 5-150mg, 5-125mg, 5-100mg, 5-90mg, 5-70mg, 5-80mg, 5-60mg, 5-50mg, 5-40mg, 5-30mg, 5-25mg, 5 -20mg, 10-1000mg, 10-900mg, 10-800mg, 10-700mg, 10-600mg, 10-500mg, 10-450mg, 10-400mg, 10-300mg, 10-250mg, 10-200mg, 10-150mg, 10-12 5mg, 10-100mg, 10-90mg, 10-80mg, 10-70mg, 10-60mg, 10-50mg, 10-40mg, 10-30mg, 10-20mg; 20-1000mg, 20-900mg, 20-800mg, 20-700mg, 20-60 0mg, 20-500mg, 20-400mg, 20-350mg, 20-300mg, 20-250mg, 20-200mg, 20-150mg, 20-125mg, 20-100mg, 20-90mg, 20-80mg, 20-70mg, 20-60mg, 20- 50mg, 20-40mg, 20-30mg; 50-1000mg, 50-900mg, 50-800mg, 50-700mg, 50-600mg, 50-500mg, 50-400mg, 50-300mg, 50-250mg, 50-200mg, 50-150mg , 50-125mg, 50-100mg; 100-1000mg, 100-900mg, 100-800mg, 100-700mg, 100-600mg, 100-500mg, 100-400mg, 100-300mg, 100-250mg, 100-200mg;

In some embodiments, the pharmaceutical composition or formulation of the present invention contains the above-mentioned therapeutically effective amount of the compound or crystal form of the present invention;

The present invention relates to a pharmaceutical composition or pharmaceutical preparation comprising a therapeutically effective amount of a compound or crystalline form of the present invention and a carrier and/or excipient. The pharmaceutical composition may be in the form of a unit dosage form (the amount of the active ingredient in a unit dosage form is also referred to as the "dose strength"). In some embodiments, the pharmaceutical composition includes but is not limited to 1-1440 mg, 5-1000 mg, 10-800 mg, 20-600 mg, 25-500 mg, 40-200 mg, 50-100 mg, 1 mg, 1.25 mg, 2.5 mg, 5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg, 90 mg, 95 mg, 100 mg, 110 mg, 120 mg, 125 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 , 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg of a compound or crystalline form of the present invention.

A method for treating a disease in a mammal, comprising administering to a subject a therapeutically effective amount of a compound or crystalline form of the present invention, and a pharmaceutically acceptable carrier and/or excipient, the therapeutically effective amount preferably being 1-1500 mg, wherein the disease is selected from breast cancer, uterine cancer, cervical cancer, ovarian cancer, and prostate cancer.

A method for treating a disease in a mammal, comprising administering a compound or crystalline form of the present invention and a pharmaceutically acceptable carrier and/or excipient to a subject at a daily dose of 1-1440 mg/day, wherein the daily dose can be a single dose or divided doses. In some embodiments, the daily dose includes but is not limited to 10-1440 mg/day, 20-1440 mg/day, 25-1440 mg/day, 50-1440 mg/day, 75-1440 mg/day, 100-1440 mg/day, 200-1440 mg/day, 10-1000 mg/day, 20-1000 mg/day, 25-1000 mg/day, 50-1000 mg/day, 75-1000 mg/day, 100-1000 mg/day, g/day, 200-1000 mg/day, 25-800 mg/day, 50-800 mg/day, 100-800 mg/day, 200-800 mg/day, 25-400 mg/day, 50-400 mg/day, 100-400 mg/day, 200-400 mg/day, in some embodiments, daily doses include but are not limited to 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 25 mg/day, 50 mg/day, 75 mg/day, 100 mg/day, 125 mg/day, 150 mg/day, 200 mg/day, 400 mg/day, 600 mg/day, 800 mg/day, 1000 mg/day, 1200 mg/day, 1400 mg/day, 1440 mg/day.

The present invention relates to a kit, which may include a compound or a crystal form in a single-dose or multi-dose form. The kit contains the compound or crystal form of the present invention, and the amount of the compound or crystal form of the present invention is the same as that in the above-mentioned pharmaceutical composition.

The amounts of the crystalline forms according to the invention in the present invention are in each case calculated as the free base.

"Preparation specifications" refers to the weight of the main drug contained in each vial, tablet or other unit preparation.

The compound or crystalline form of the present invention is present in an amount of about 5% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 10% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 15% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 20% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 25% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 30% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 35% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 40% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 45% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 50% to about 100% by weight of the drug substance; in certain embodiments, it is present in an amount of about 55% to about 100% by weight of the drug substance. In certain embodiments, the drug substance is present at about 60% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 65% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 70% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 75% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 80% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 85% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 90% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 95% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 98% to about 100% by weight of the drug substance; in certain embodiments, the drug substance is present at about 99% to about 100% by weight of the drug substance; in certain embodiments, substantially all of the drug substance is a substantially pure compound or crystal.

The crystalline form of the present invention can be prepared by the following preparation method:

1. Volatilization experiment: Add the compound of formula (I) into a selected single solvent or binary solvent to form a clear sample solution, and evaporate it open at different temperatures until the solvent is dry.

2. Suspension method: Add the compound of formula (I) to a selected single solvent or binary solvent until a suspension is formed. After suspension and stirring at room temperature to 50°C for a certain period of time (e.g., 1 hour to 3 days, or 2 hours to 24 hours, or 2 hours to 12 hours, or 3 hours to 5 hours), the suspension is centrifuged and dried to obtain the product.

3. Dissolution crystallization method: dissolve the compound of formula (I) in a good solvent, add a certain amount of the solution dropwise to a poor solvent or add a poor solvent dropwise to the solution, stir to precipitate a solid, separate and dry to obtain the product.

4. Cooling method: dissolve a certain amount of sample in the corresponding solvent at high temperature, transfer the solution to room temperature for cooling, let it stand or stir for crystallization, separate, and dry to obtain the product.

5. Thermal method experiment: Take a certain amount of sample, place it on a glass slide and place it on a hot table. Heat it to the target temperature at a certain rate (such as 5-20℃/min, or 10-15℃/min), keep it at a constant temperature for a period of time (such as 0.5-5min, or 1-3min, or 1-2min), and then cool it naturally to room temperature to obtain a solid.

6. Vapor Diffusion Assay: Add a suitable amount of a good solvent dropwise to a certain amount of the compound of formula (I) at room temperature until the sample is completely dissolved or a saturated solution of the good solvent is prepared. A certain amount of each solution is taken and the clear solution is placed in a poor solvent atmosphere and allowed to stand at room temperature until solid precipitates. This is followed by separation. Alternatively, the solid compound of formula (I) can be directly placed in a solvent atmosphere and allowed to stand at room temperature for 1 to 7 days to obtain the product.

The good solvent and poor solvent of the present invention are relative. In a pair of solvents, the one with higher solubility is a good solvent, and the one with lower solubility is a poor solvent. In some embodiments, the good solvent is selected from ethylene glycol methyl ether, ethylene glycol dimethyl ether, dioxane, DMF, DMSO, methanol, ethanol, n-propyl alcohol, butyl formate, 4-methyl-2-pentanone, tetrahydrofuran, isopropyl alcohol, ethyl acetate, n-heptane, ether, water, acetonitrile, toluene, chloroform, acetone, butyl formate, MTBE, and cyclohexane with higher solubility, and the poor solvent is selected from the above-mentioned solvent with lower solubility. In some embodiments, the good solvent is selected from ethylene glycol methyl ether, ethylene glycol dimethyl ether, dioxane, DMF, DMSO, methanol, ethanol, n-propyl alcohol, butyl formate, 4-methyl-2-pentanone, tetrahydrofuran, or a mixed solvent thereof. In some embodiments, the poor solvent is selected from isopropyl alcohol, ethyl acetate, n-heptane, diethyl ether, water, acetonitrile, toluene, chloroform, acetone, butyl formate, MTBE, cyclohexane, or a mixed solvent thereof.

In some embodiments, the solvents of the evaporation method are water and acetone;

In some embodiments, the solvent used in the dissolution crystallization method is dichloromethane and n-heptane; in some embodiments, the solvent used in the dissolution crystallization method is dichloromethane and isopropyl ether; in some embodiments, the solvent used in the dissolution crystallization method is ethyl acetate and n-heptane; in some embodiments, the solvent used in the dissolution crystallization method is 4-methyl-2-pentanone and n-heptane; in some embodiments, the solvent used in the dissolution crystallization method is dioxane and n-heptane;

In some embodiments, the solvents used in the cooling method are ethylene glycol dimethyl ether and cyclohexane; in some embodiments, the solvents used in the cooling method are toluene and cyclohexane; in some embodiments, the solvents used in the cooling method are isopropyl acetate and cyclohexane; in some embodiments, the solvents used in the cooling method are dioxane and cyclohexane; in some embodiments, the solvents used in the cooling method are dioxane and water; in some embodiments, the solvents used in the cooling method are DMSO and water.

In some embodiments, the vapor diffusion method employs vapor diffusion in ethyl acetate.

The good solvent and poor solvent described in the present invention are relative. In a pair of solvents, the one with higher solubility is a good solvent, and the one with lower solubility is a poor solvent.

The solvent used in the above preparation method, unless otherwise specified, may be a single solvent or a combination of two or more solvents.

The X-ray powder diffraction, DSC pattern, and TGA pattern disclosed in the present invention, and those substantially the same also fall within the scope of the present invention.

Unless stated otherwise, the terms used in the specification and claims have the following meanings.

" _IC50 " refers to the half-maximal inhibitory concentration, which is the concentration at which half of the maximal inhibitory effect is achieved.

As used herein, "the crystal of the present invention", "the crystal form of the present invention", "the crystal form of the present invention" and the like can be used interchangeably.

The "room temperature" mentioned in the present invention generally refers to 4-30°C, preferably 20±5°C.

The crystalline structure of the present invention can be analyzed using various analytical techniques known to those skilled in the art, including but not limited to, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC) and/or thermogravimetric analysis (TGA), also known as thermogravimetry (TG).

As used herein, "2θ or 2θ angle" refers to the peak position expressed in degrees (°) based on the setup of an X-ray diffraction experiment, and is typically the unit of the abscissa in a diffraction pattern. If the incident beam forms an angle θ with a certain lattice plane and the reflection is diffracted, the experimental setup requires recording the reflected beam in 2θ angles. It should be understood that the specific 2θ value of a specific crystal form mentioned herein is intended to represent the 2θ value (expressed in degrees) measured using the X-ray diffraction experimental conditions described herein, and the error range of the 2θ may be ±0.3, ±0.2, or ±0.1.

It is understood that the numerical values described and protected by the present invention are approximate values. Variations in the numerical values may be due to equipment calibration, equipment errors, crystal purity, crystal size, sample size and other factors.

It is understood that the crystal form of the present invention is not limited to the characteristic spectra that are exactly the same as the characteristic spectra described in the drawings disclosed in the present invention, such as XRD, DSC, TGA, and DVS. Any crystal form having characteristic spectra that are substantially the same or essentially the same as those described in the drawings falls within the scope of the present invention.

It is understood that, as is well known in the art of differential scanning calorimetry (DSC), the melting peak height of a DSC curve depends on many factors related to sample preparation and instrument geometry, while the peak position is relatively insensitive to experimental details. Therefore, in some embodiments, the crystalline compound of the present invention is characterized by a DSC pattern having a characteristic peak position, having substantially the same properties as the DSC pattern provided in the accompanying drawings of the present invention, with a measurement error tolerance of within ±5°C, generally required to be within ±3°C.

"Carrier" refers to a system that does not cause significant irritation to the organism and does not eliminate the biological activity and properties of the administered compound, and can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug and deliver the drug to the target organ. Non-limiting examples include microcapsules and microspheres, nanoparticles, liposomes, etc.

An "excipient" is a substance that is not itself a therapeutic agent but serves as a diluent, adjuvant, binder, and/or vehicle that is added to a pharmaceutical composition to improve its handling or storage properties or to allow or facilitate the formation of a compound or pharmaceutical composition into a unit dosage form for administration. As known to those skilled in the art, pharmaceutical excipients can serve a variety of functions and can be described as wetting agents, buffers, suspending agents, lubricants, emulsifiers, disintegrants, absorbents, preservatives, surfactants, colorants, flavoring agents, and sweeteners. Examples of pharmaceutical excipients include, but are not limited to: (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate, hydroxypropyl methylcellulose, hydroxypropyl cellulose, microcrystalline cellulose, and cross-linked carboxymethylcellulose (e.g., cross-linked sodium carboxymethylcellulose); (4) tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn starch, and maltodextrin. oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer’s solution; (19) ethanol; (20) pH buffer solutions; (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is an X-ray powder diffraction pattern of the amorphous compound represented by formula (I).

Figure 2 is an X-ray powder diffraction pattern of Form I of the compound represented by formula (I).

FIG3 is a differential scanning calorimetry analysis curve of Form I of the compound represented by formula (I).

FIG4 is a thermogravimetric analysis spectrum of Form I of the compound represented by formula (I).

FIG5 is an isothermal adsorption curve of Form I of the compound represented by formula (I).

FIG6 is an X-ray powder diffraction pattern of Form II of the compound represented by formula (I).

FIG7 is a differential scanning calorimetry analysis curve of Form II of the compound represented by formula (I).

FIG8 is a thermogravimetric analysis spectrum of Form II of the compound represented by formula (I).

FIG9 is an isothermal adsorption curve of Form II of the compound represented by formula (I).

DETAILED DESCRIPTION

The structures of the compounds were confirmed by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). NMR shifts (δ) are given in units of ^10-6 (ppm). NMR measurements were performed using a Bruker Avance III 400 and Bruker Avance 300 NMR spectrometer. The solvents used were deuterated dimethyl sulfoxide (DMSO- _d6 ), deuterated chloroform ( _CDCl3 ), and deuterated methanol ( _CD3OD ), with tetramethylsilane (TMS) as the internal standard.

MS was performed using (Agilent 6120B (ESI) and Agilent 6120B (APCI)).

HPLC determination was performed using a LC-20AT (Shimadzu) high pressure liquid chromatograph (Shim-pack GIST C18, 4.6×250 mm (HSS), 5 μm).

Crystal form detection instrument information and detection method parameter table (see Table 1 below):

Table 1. Instrument information and detection method parameters

The known starting materials of the present invention can be synthesized by methods known in the art, or can be purchased from companies such as Titan Technology, Anage Chemical, Shanghai Demer, Chengdu Kelon Chemical, Shaoyuan Chemical Technology, and Bailingwei Technology.

Unless otherwise specified in the examples, the solution refers to an aqueous solution.

Unless otherwise specified in the examples, the room temperature is 20°C to 30°C.

The following describes in detail the implementation process of the present invention and the beneficial effects produced by specific embodiments, which is intended to help readers better understand the essence and characteristics of the present invention and is not intended to limit the scope of implementation of this case.

Example 1: Preparation of compound of formula (I)

Synthesis of compound of formula (I):

Step 1: 3-Bromo-1,1-difluoropropan-2-one (1A)

3-Bromo-1,1-difluoropropane-2-one (5 g, 40.27 mmol) was dissolved in anhydrous tetrahydrofuran, the nitrogen atmosphere was replaced three times, and dibromomethane (14 g, 80.54 mmol) was added. The reaction system was cooled to -78°C, and methyllithium (80.54 mmol) was added dropwise. The reaction was allowed to react at -78°C for 2 h. After completion of the reaction, saturated ammonium chloride solution was added at 0°C to quench the reaction. The mixture was extracted with ethyl acetate (50 ml x 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was then purified by rapid column chromatography (eluent ratio: EA/PE = 0% to 20%) to obtain compound 1A (5.01 g, 72%).

LC-MS (ESI): m/z = 173.2 [M + 1 ^{] +}

Step 2: 6-Bromo-2-(difluoromethyl)imidazo[1,2-a]pyrazine (1B)

Compound 1A (49.42 g, 287.35 mmol) and 2-amino-5-bromopyrazine (10 g, 57.47 mmol) were dissolved in 1,4-dioxane (100 mL) and stirred at 100°C for 1 h. The reaction was monitored by LCMS. The filter cake was collected by filtration and dispersed in 300 mL of 1,4-dioxane. The mixture was stirred at 100°C overnight. After completion of the reaction, the mixture was directly spin-dried and purified by rapid separation (PE/EA = 0-20%) to obtain compound 1B (10 g, 80%).

MS m/z＝248.0[M+1] ⁺

Step 3: tert-Butyl 4-(2-(difluoromethyl)imidazo[1,2-a]pyrazin-6-yl)piperazine-1-carboxylate (1C)

Compound 1B (5 g, 20.2 mmol), tert-butyl piperazine-1-carboxylate (5.6 g, 30 mmol), tris(dibenzylideneacetone)dipalladium (1.92 g, 2 mmol), 2-(di-tert-butylphosphino)biphenyl (0.6 g, 2 mmol), sodium tert-butoxide (5.8 g, 60.6 mmol), and toluene (50 mL) were added to a reaction flask and stirred at 110°C under nitrogen for 2 h. After completion of the reaction, the reaction solution was extracted three times with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by flash column chromatography (EA:PE = 20%) to obtain the target compound 1C (3 g, 42%).

LC-MS (ESI): m/z = 354.2 [M + 1] ⁺

Step 3: 6-(piperazin-1-yl)-2-(difluoromethyl)imidazo[1,2-a]pyrazine (1D)

Compound 1C (330 mg, 0.89 mmol) and trifluoroacetic acid (3 mL) were added to the reaction flask and stirred at 25°C for 1 h. After the reaction, the mixture was concentrated under reduced pressure to obtain crude compound 1D (240 mg, 100%), which was directly used in the next step.

Step 4: 7-((4-(2-(difluoromethyl)imidazo[1,2-a]pyrazin-6-yl)piperazin-1-yl)methyl)-8-fluoro-3-methylquinoxalin-2(1H)-one (Formula I)

7-((4-(2-(difluoromethyl)imidazo[1,2-a]pyrazin-6-yl)piperazin-1-yl)methyl)-8-fluoro-3-methylquinoxalin-2(1H)-one

1E (synthesized according to US20220009901A1) (1.28 g, 4.74 mmol), 1D (1.0 g, 3.95 mmol), N,N-diisopropylethylamine (1.54 g, 11.86 mmol), potassium iodide (61.42 mg, 0.37 mmol), and acetonitrile (3 mL) were added to a reaction flask and stirred at 60°C for 1 h. The reaction was monitored by LCMS. After completion, the reaction system was directly sent to preparative HPLC for separation: 1. Instrument: Waters 2767 Preparative HPLC; Chromatographic column: SunFire@Prep C18 (19 mm × 250 mm). 2. The sample was filtered through a 0.45 μm filter to prepare a sample solution. 3. Preparative Chromatography Conditions: a. Mobile Phase A, B Composition: Mobile Phase A: Acetonitrile; Mobile Phase B: Water (containing 0.1% ammonium acetate); b. Gradient elution, Mobile Phase A content from 10% to 55%; c. Flow rate 12 mL/min. Retention time 7.0 min to yield Compound (I) (1.0 g, 57%).

LCMS m/z＝444.4[M+1] ⁺

¹ H NMR(400MHz,DMSO-d6)δ8.90(s,1H),8.21(s,1H),7.93(d,1H),7.52(d,1H),7.3 0(d,1H),7.16(t,1H),3.70(s,2H),3.31(d,5H),2.63–2.55(m,4H),2.42(s,3H).

Example 2 Preparation of the amorphous compound represented by formula (I)

50 mg of the compound of formula (I) obtained in Example 1 was added to 20 mL of a mixed solvent of acetonitrile and water, dissolved and then freeze-dried. The amorphous form of the compound of formula (I) was characterized by XRD. Its X-ray powder diffraction pattern is shown in Figure 1.

Example 3 Preparation of Crystalline Form I of the Compound Represented by Formula (I)

150 mg of the amorphous form of the compound represented by formula (I) obtained in Example 2 was taken, dichloromethane (3 mL) and methanol (1 ml) were added, the temperature was raised to 50°C to dissolve, and methyl tert-butyl ether (2.0 mL) was slowly added after cooling to room temperature. After the addition, the mixture was stirred at room temperature overnight. The precipitated solid was filtered, and the filter cake was washed with a small amount of methyl tert-butyl ether and then vacuum-dried at 50°C overnight to obtain Form I of the compound represented by formula (I). Form I of the compound represented by formula (I) was characterized by XRD, DSC, TGA and DVS, and its X-ray powder diffraction pattern, differential scanning calorimetry analysis curve, thermogravimetric analysis diagram, and isothermal adsorption curve are shown in Figures 2-5, respectively.

¹ H NMR(400MHz,DMSO-d6)δ8.90(s,1H),8.21(s,1H),7.93(d,1H),7.52(d,1H),7.3 0(d,1H),7.16(t,1H),3.70(s,2H),3.31(d,5H),2.63–2.55(m,4H),2.42(s,3H).

The X-ray powder diffraction pattern (XRD) of the crystalline form I of the compound of formula (I) is shown in Figure 2. The specific peaks are shown in Table 2.

Table 2. XRD specific peaks of Form I

Example 4 Preparation of Crystalline Form II of the Compound Represented by Formula (I)

150 mg of the amorphous compound of formula (I) obtained in Example 2 was added to 4 mL of a mixed solvent of acetonitrile and water, dissolved at 50°C, and hot filtered. The resulting filtrate was cooled at room temperature for crystallization and stirred overnight at room temperature. Filtered, the resulting solid was vacuum-dried at 50°C overnight to obtain Form II of the compound of formula (I). Form II of the compound of formula (I) was characterized by XRD, DSC, TGA, and DVS. Its X-ray powder diffraction pattern, differential scanning calorimetry curve, thermogravimetric analysis diagram, and isothermal adsorption curve are shown in Figures 6-9, respectively.

¹ H NMR(400MHz,DMSO-d6)δ8.90(s,1H),8.21(s,1H),7.93(d,1H),7.52(d,1H),7.3 0(d,1H),7.16(t,1H),3.70(s,2H),3.31(d,5H),2.63–2.55(m,4H),2.42(s,3H).

The X-ray powder diffraction pattern (XRD) of the crystalline form II of the compound of formula (I) is shown in Figure 6. The specific peaks are shown in Table 3.

Table 3. XRD specific peaks of Form II

Example 5 Study on the stability of related crystal forms

1. Crystal stability of the compound of formula (I) crystal form I

The measurement conditions are shown in Table 4, and the experimental results are shown in Table 5.

Table 4. Solid-state stability test of Form I

Table 5. Test results of solid-state stability test of Form I

Conclusion: The crystalline form I of the compound of formula (I) has good solid-state stability.

2. Crystal stability of Form II of the compound of formula (I)

The experimental conditions are shown in Table 6, and the experimental results are shown in Table 7.

Table 6. Solid-state stability test of Form II

Table 7. Test results of solid-state stability test of Form II

Conclusion: The solid-state stability of the crystalline form II of the compound of formula (I) is good.

Example 6 Study on the transformation of the compound of formula (I) into Form I and Form II

10 mg of Form I and 10 mg of Form II samples were taken respectively, mixed evenly, and sampled for XRPD characterization; the mixed sample was added to 0.5 mL of a mixed solvent of dimethyl sulfoxide and water (1:1) to form a suspension, stirred at room temperature for 5 days, centrifuged, and vacuum-dried at 40°C overnight, and sampled for XRPD characterization.

Table 8. Crystal form conversion test results of Form I and Form II

Conclusion: At room temperature, the thermodynamic stability of form I is better than that of form II.

Example 7 DSC test of the crystal form of the compound of formula (I)

A TA Instruments Q200DSC differential scanning calorimeter was used. A 0.5–5 mg sample was accurately weighed and placed in a perforated DSC Tzero pan. The sample was heated to the final temperature at a rate of 10°C/min, with a nitrogen purge rate of 50 mL/min.

The test results of Form 1 and Form 2 of the compound of formula (I) are shown in Figures 3 and 7.

Example 8 TGA test of the crystal form of the compound of formula (I)

The thermogravimetric analyzer (TGA) was a TA Instruments Q500TGA. Samples (1–10 mg) were placed in a pre-equilibrated open aluminum sample pan and automatically weighed within the TGA furnace. The samples were heated to the final temperature at a rate of 10°C/min, with nitrogen purge rates of 60 mL/min at the sample and 40 mL/min at the balance.

The test results of Form 1 and Form 2 of the compound of formula (I) are shown in Figures 4 and 8.

Example 9 PARP enzyme activity test experiment

Compounds were tested for inhibition of PARP1/2 enzyme activity using the FP method. The substrate tracer, Tracer (from ICE), was prepared in reaction buffer (50 mM Tris (pH 8.0), 10 mM MgCl2, 150 mM NaCl, 0.001% Triox-100). PARP1/2 (BPS, Cat#80501/80502) was added to the reaction buffer and gently mixed. The final concentrations of PARP1/2 and Tracer in the reaction mixture were 5/10 nM and 2.5 nM, respectively. The positive reference, Olaparib, started at 1 μM and was diluted 3-fold to 10 concentrations. 0.1 μL of compound in 100% DMSO was delivered to a 384-well plate (Corning 4514) using acoustic liquid delivery technology (Echo 655) and centrifuged at 1000 rpm for 1 minute. 5 μL of PARP enzyme solution was transferred to a 384-well plate and centrifuged at 1000 rpm for 1 minute, followed by incubation at 25°C for 10 minutes. 5 μL of substrate solution was transferred to a 384-well plate, centrifuged at 1000 rpm for 1 minute, and incubated at 25°C for 60 minutes. Finally, the mP signal of FP (ex/em: 485 nm/520 nm) was read using the fluorescence polarization module of the BMG PHERAstar FSX. GraphPad Prism 8 software was then used to perform a four-parameter nonlinear regression fit to the IC50 value.

Experimental results: The _IC50 values of the compound of formula (I) against PARP-1 and PARP-2 in vitro are expressed on a scale of A, B, C, or D, where A represents 0 < IC50 ≤ _{10 nM} , B represents 10 nM < IC50 ≤ 50 _nM , C represents 50 nM < _IC50 ≤ 500 nM, and D represents 500 nM < _IC50 .

Table 9. PARP enzyme activity

Conclusion: The compound of formula (I) of the present invention has a significant selective inhibitory effect on PARP-1 enzyme activity in vitro.

Example 10 Pharmacokinetic test in mice

Experimental animals: Male Balb/c mice, 20-25 g, 12 mice per compound, purchased from Chengdu Dashuo Experimental Animal Co., Ltd.

Experimental Design: On the day of the experiment, Balb/c mice were randomly divided into groups according to body weight. They were fasted but not watered for 12-14 hours before administration and fed 4 hours after administration.

Table 10. Dosing Information

Note: Intravenous administration solvent: 10% DMA + 10% Solutol + 80% Saline; oral administration solvent: 5% DMSO + 30% PEG400 + 65% (20% SBE-CD), Solutol is polyethylene glycol-15-hydroxystearate; Saline is normal saline

Before and after administration, 0.06 mL of blood was collected from the orbital cavity under isoflurane anesthesia. The blood was placed in an EDTAK2 centrifuge tube and centrifuged at 5000 rpm at 4°C for 10 minutes to collect plasma. Blood was collected from both the intravenous and oral gavage groups at 0, 5, 15, 30 minutes, and 1, 2, 4, 6, 8, and 24 hours. Brain tissue was collected at 30 minutes, 2, and 24 hours after administration of compounds 12, 15, and 18, respectively. Brain tissue was collected at 24 hours after administration of compounds 2 and 5. The brain tissue was rinsed with cold saline to remove any residual blood, blotted dry, and homogenized. All samples were stored at -80°C prior to analysis and quantitative analysis was performed using LC-MS/MS.

Table 11. Pharmacokinetic parameters of test compounds in mouse plasma

Conclusion: The compound of formula (I) has excellent pharmacokinetic characteristics in mouse plasma.

Example 11 Stability Study of Crystal Form

Crystal samples were taken and placed independently at 40°C + 75% RH and 30°C + 60% RH for 0 month, 1 month, 2 months, 3 months, 6 months and 9 months, respectively. The purity was tested by HPLC. The results showed that the chemical stability of crystal forms I and II of the compound of formula (I) of the present invention was good.

Example 12 Experimental Study of Influencing Factors

(1) Crystal samples were placed at 40°C, 60°C, and 92.5% RH for 0 and 6 days respectively, and the purity was determined by HPLC.

(2) Under light (4500 Lx), the crystal samples were placed independently at 40°C, 60°C, and 92.5% RH for 6 days, 10 days, and 30 days, and the purity was tested.

The research results show that the crystal forms I and II of the compound of formula (I) of the present invention can maintain good chemical stability under the experimental conditions of the above-mentioned influencing factors.

Claims (8)

A crystalline form of a compound of formula (I):
The crystalline form according to claim 1, wherein the crystalline form is Form I, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 4.63°±0.2°, 9.26°±0.2°, 10.45°±0.2°, 13.95°±0.2°, and 16.33°±0.2°; preferably, it has characteristic diffraction peaks at the following 2θ positions: 4.63°±0.2°, 9.26°±0.2°, 10.45°±0.2°, 13.95°±0.2°, 15.97°±0.2°, 16.33°±0.2°, 17.14°±0.2°, 20.62°±0.2°, and 22.37°±0.2°. The crystalline form according to claim 2, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in Figure 2; preferably, its differential scanning calorimetry analysis curve, thermogravimetric analysis curve, and isothermal adsorption curve are substantially as shown in Figures 3, 4, and 5, respectively. The crystalline form according to claim 1, wherein the crystalline form is crystalline form II, and using Cu-Kα radiation, its X-ray powder diffraction pattern has characteristic diffraction peaks at the following 2θ positions: 7.59°±0.2°, 11.41°±0.2°, 11.79°±0.2°, 14.53°±0.2°, 17.40°±0.2°, 21.54°±0.2°, 24.93°±0.2° and 25.45°±0.2°; preferably, it has characteristic diffraction peaks at the following 2θ positions: 3 .74°±0.2°, 7.59°±0.2°, 11.41°±0.2°, 11.79°±0.2°, 14.53°±0.2°, 17.09°±0.2°, 17.40°±0.2°, 18.15°±0.2°, 19.15°±0.2°, 21.54°±0.2°, 22.16°±0.2°, 22.66°±0.2°, 24.93°±0.2°, 25.45°±0.2° and 26.58°±0.2°. The crystalline form according to claim 4, using Cu-Kα radiation, has an X-ray powder diffraction pattern substantially as shown in Figure 6; preferably, its differential scanning calorimetry analysis curve, thermogravimetric analysis curve, and isothermal adsorption curve are substantially as shown in Figures 7, 8, and 9, respectively. A pharmaceutical composition comprising a therapeutically effective amount of the crystalline form according to any one of claims 1 to 5, and a pharmaceutically acceptable carrier and/or excipient, wherein the therapeutically effective amount is preferably 1-1440 mg. Use of the crystalline form according to any one of claims 1 to 5, or the pharmaceutical composition according to claim 6, in the preparation of a medicament for treating PARP-mediated diseases. A method for treating a PARP-mediated disease, comprising administering to a subject a therapeutically effective amount of the crystalline form of any one of claims 1 to 5, wherein the therapeutically effective amount is preferably 1 to 1440 mg, and the disease is preferably breast cancer, uterine cancer, cervical cancer, ovarian cancer, or prostate cancer.