WO2023011547A1 - Crystal form of compound inhibiting function of adamts-5 and/or adamts-4, and preparation method therefor and use thereof - Google Patents

Abstract

The present disclosure relates to a crystal form of a compound inhibiting the function of ADAMTS-5 and/or ADAMTS-4, and preparation method therefor and the use thereof. Specifically, the present disclosure relates to a crystal of and a preparation method for compound A of formula I and the use thereof in the treatment of diseases involving cartilage degradation or disruption of cartilage homeostasis, such as osteoarthritis and/or rheumatoid arthritis.

Description

Crystal form of compound inhibiting ADAMTS-5 and/or ADAMTS-4 function and its preparation method and application

This application claims the priority of Chinese patent application 2021108844766 with a filing date of August 3, 2021. This application cites the full text of the above-mentioned Chinese patent application.

technical field

The present disclosure relates to a crystal form of a compound that inhibits the function of ADAMTS-5 and/or ADAMTS-4, in particular to the crystal form A of the compound of formula I, its preparation method and application.

Background technique

Cartilage is a highly specialized connective tissue in joints. Its main function is to provide load-bearing and compressive capacity to the joints. Chondrocytes are the cellular components of articular cartilage, accounting for only about 5% of the tissue volume. The main component of cartilage is the extracellular matrix, including proteoglycans and collagen. Under physiological conditions, cartilage homeostasis is maintained by a balance between the production (anabolism) and degradation (catabolism) of aggrecan and collagen. However, in diseases such as osteoarthritis, the balance is shifted towards catabolism.

Osteoarthritis is the most common chronic joint disease and a leading cause of pain and disability. It can occur in the hips, knees, spine, hands, and other joints. Osteoarthritis currently affects an estimated 250 million people worldwide, and the prevalence is steadily increasing. Pain and loss of function are accompanied by an increased risk of other diseases such as diabetes, cancer or cardiovascular disease. Osteoarthritis is a general joint disease in which structural changes include degeneration of articular cartilage, synovitis, and changes in subchondral bone and other periarticular tissues. The pathogenesis of osteoarthritis is still not very clear, involving mechanical damage, inflammation, aging, metabolism and other factors. Osteoarthritis is not a passive degenerative disease, but an active dynamic change caused by an imbalance between joint tissue repair and destruction. Currently, the drug treatments available for osteoarthritis are limited to symptomatic relief of pain and inflammation. Drugs that stop or slow disease progression are not currently available.

Progressive loss of articular cartilage is currently considered an early sign of osteoarthritis. Aggrecan may have a protective effect against collagen loss. These studies suggest that aggrecan has a key role in osteoarthritis and other joint diseases. Aggrecan is a proteoglycan whose core protein has covalently linked sulfated glycosaminoglycan (GAG) chains. Its core protein has three globular domains, the N-terminal G1 and G2 domains, and the C-terminal G3 domain. The extensive region between the G2 and G3 domains is heavily modified by the GAGs keratan sulfate (KS) and chondroitin sulfate (CS). Depending on the amino acid sequence, the CS domain is further divided into two subdomains, CS1 and CS2. GAG chains provide a high anionic charge to proteoglycans. Multiple aggrecan monomers associate with hyaluronic acid (HA) through the G1 domain, which is stabilized by connexins, forming large supramolecular aggregates. Aggregates of large aggrecan absorb water and provide elasticity to cartilage. Proper function of cartilage requires high concentrations of aggrecan, a high degree of sulfation, and the ability to form large aggregates.

Proteolytic enzymes can cleave the extended structure of aggrecan, resulting in impairment of the normal function of cartilage. ADAMTS (disintegrins and metalloproteases with thrombospondin motifs) are a family of zinc ion-dependent metalloproteases. ADAMTS-4 and -5, also known as "aggrecanases", degrade aggrecan at several specific locations in the IGD and CS2 domains, (Glasson et al., Nature. 2005, 434:644–648 ; Stanton et al., Nature.2005,434:648–652) showed that ADAMTS-5 deficiency prevented proteoglycan loss and cartilage damage in a surgically induced mouse model of osteoarthritis, suggesting that ADAMTS-5 and Driving cartilage loss is associated with osteoarthritic disease severity. However, some studies in human cartilage graft cultures have shown that not only ADAMTS-5 but also ADAMTS-4 is important for human osteoarthritis (Verma et al., Journal of Cellular Biochemistry. 2011, 112:3507-3514). These studies strongly suggest that inhibition of the enzymatic function of ADAMTS-5 and ADAMT-4 may play a protective role in osteoarthritis.

In conclusion, the role of ADAMTS-5 and/or ADAMTS-4 in cartilage degradation has been recognized. Therefore, compounds capable of inhibiting ADAMTS-5 and/or ADAMTS-4 may have therapeutic value in the treatment of arthritis.

PCT/US2021/016364 provides a new compound (structure shown in formula I) for inhibiting the function of ADAMTS-5 and/or ADAMTS-4, which has better pharmaceutical activity.

The crystal structure of the active ingredient of a drug often affects the chemical stability of the drug. Different crystallization conditions and storage conditions may lead to changes in the crystal structure of the compound, sometimes accompanied by the production of other crystal forms. Generally speaking, amorphous pharmaceutical products have no regular crystal structure and often have other defects, such as poor product stability, fine crystallization, difficult filtration, easy agglomeration, and poor fluidity. Therefore, we need to conduct in-depth research to find crystal forms with high purity and good chemical stability.

Contents of the invention

The present disclosure provides a crystal form A of the compound represented by formula I, which has good stability and can be better applied clinically.

The crystal form A of the compound represented by formula I provided by the present disclosure has characteristic peaks at 2θ angles of 11.2, 13.8, 16.6, 21.0, and 27.6 in its X-ray powder diffraction pattern.

The X-ray powder diffraction pattern of the crystal form A of the compound represented by formula II provided by the present disclosure has characteristic peaks at 2θ angles of 8.4, 11.2, 13.8, 14.9, 16.6, 17.5, 18.5, 21.0, 22.9, and 27.6.

The crystal form A of the compound represented by formula II provided by the present disclosure has characteristic peaks at 2θ angles of 8.4, 11.2, 13.8, 14.9, 15.4, 16.6, 17.5, 18.5, 21.0, 22.9, and 27.6 in the X-ray powder diffraction spectrum .

The crystal form A of the compound represented by formula I provided by the present disclosure has an X-ray powder diffraction pattern as shown in FIG. 2 .

The present disclosure provides the crystal form A of the compound represented by formula I, wherein the error range of the 2θ angle is ±0.2.

The present disclosure further provides a method for preparing crystal form A of the compound represented by formula I, the method comprising: mixing the compound represented by formula I with an appropriate amount of solvent, the solvent is an organic solvent, preferably the solvent is tetrahydrofuran, ethanol, Isopropyl Alcohol, Acetone, Ethyl Acetate.

Through X-ray powder diffraction pattern (XRPD) and differential scanning calorimetry (DSC), the structure determination and crystal form research of the crystal form obtained in the present disclosure are carried out.

The crystallization method of the crystal form in the present disclosure is conventional, such as volatilization crystallization, cooling crystallization or room temperature crystallization.

The starting material used in the preparation method of the disclosed crystal form can be any form of the compound represented by formula I, and the specific form includes but not limited to: amorphous, any crystal form, hydrate, solvate, etc.

The present disclosure further provides a pharmaceutical composition, comprising the crystal form A of the compound represented by formula I, and one or more pharmaceutically acceptable carriers or excipients.

The present disclosure further provides a pharmaceutical composition, which is prepared by the crystal form A of the compound represented by formula I, and one or more pharmaceutically acceptable carriers or excipients.

The present disclosure further provides a preparation method of a pharmaceutical composition, comprising the step of mixing the crystal form A of the compound represented by formula I with one or more pharmaceutically acceptable carriers or excipients.

The present disclosure further provides the use of the aforementioned crystal form A of the compound represented by formula I, or the combination prepared by the aforementioned method, in the preparation of a medicament for inhibiting ADAMTS-5 and/or ADAMTS-4.

The present disclosure further provides the aforementioned crystal form A of the compound represented by formula I, or the combination prepared by the aforementioned method in the preparation of a medicament for preventing or treating inflammatory disorders or diseases involving cartilage degradation and/or cartilage homeostasis disruption the use of.

The present disclosure further provides the use of the crystal form A of the compound represented by the aforementioned formula I, or the combination prepared by the aforementioned method in the preparation of medicines for the prevention or treatment of arthritis, the arthritis is preferably rheumatoid arthritis, Psoriatic arthritis, osteoarthritis, and hypertrophic arthritis.

The present disclosure further provides the use of the crystal form A of the compound represented by the aforementioned formula I, or the combination prepared by the aforementioned method in the preparation of a drug for treating diseases or disorders related to ADAMTS-5 and/or ADAMTS-4 activity , wherein the disease or condition associated with ADAMTS-5 and/or ADAMTS-4 activity is arthritis, preferably rheumatoid arthritis, psoriatic arthritis, osteoarthritis or hypertrophic arthritis.

In the specification and claims of the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. However, for a better understanding of the present disclosure, definitions and explanations of some related terms are provided below. In addition, when the definitions and explanations of terms provided in this application are inconsistent with the meanings commonly understood by those skilled in the art, the definitions and explanations of terms provided in this application shall prevail.

Unless otherwise stated, when a position is specifically designated as deuterium (D), the position is understood to have an abundance of deuterium (i.e., at least 10 % deuterium incorporation). Exemplary compounds having a natural abundance greater than deuterium can be at least 1000 times more abundant deuterium, at least 2000 times more abundant deuterium, at least 3000 times more abundant deuterium, at least 4000 times more abundant deuterium, at least 5000 times more abundant deuterium, at least 6000 times more abundant deuterium, or more abundant deuterium.

The "X-ray powder diffraction pattern or XRPD" described in the present disclosure means that according to the Bragg formula 2d sinθ=nλ (wherein, λ is the wavelength of X-rays, and the order n of diffraction is any positive integer, generally the first-order diffraction peak, n=1), when the X-ray is incident on an atomic surface with d lattice plane spacing of the crystal or part of the crystal sample at the grazing angle θ (the complementary angle of the incident angle, also known as the Bragg angle), the Can satisfy the Bragg equation, thus measured this group of X-ray powder diffraction patterns.

An "X-ray powder diffraction pattern or XRPD" as used in this disclosure is a pattern obtained by using Cu-Kα radiation in an X-ray powder diffractometer.

"Differential scanning calorimetry or DSC" in this disclosure refers to the measurement of the temperature difference and heat flow difference between the sample and the reference during the heating or constant temperature of the sample to characterize all the physical changes and chemical changes related to thermal effects. change to obtain the phase transition information of the sample.

"Thermogravimetric analysis or TGA" as used in this disclosure refers to the continuous measurement of the mass change of a sample with temperature or time at a programmed temperature.

The “2θ or 2θ angle” in this disclosure refers to the diffraction angle, θ is the Bragg angle, the unit is ° or degree, and the error range of 2θ is ±0.3 or ±0.2 or ±0.1.

The "interplanar spacing or interplanar spacing (d value)" in the present disclosure refers to that the spatial lattice selects 3 non-parallel unit vectors a, b, and c that connect two adjacent lattice points. The matrix is divided into juxtaposed parallelepiped units called interplanar spacing. The spatial lattice is divided according to the determined parallelepiped unit connection lines to obtain a set of linear grids, which are called spatial lattices or lattices. Lattice and lattice reflect the periodicity of the crystal structure with geometric points and lines respectively. Different crystal planes have different interplanar spacing (that is, the distance between two adjacent parallel crystal planes); the unit is

or Angstroms.

Description of drawings

Fig. 1 is the XRPD spectrum of formula I compound A crystal form in embodiment 2-2;

Fig. 2 is the DSC spectrum of the crystal form of compound A of formula I in Example 2-2.

Detailed ways

The following will explain the present disclosure in more detail in conjunction with the embodiments, and the embodiments of the present disclosure are only used to illustrate the technical solutions of the present disclosure, and do not limit the essence and scope of the present disclosure. If the specific conditions of the experimental method are not described in the examples of the present disclosure, it is generally carried out according to the conventional conditions or recommended conditions of the raw materials and product manufacturers. Reagents whose specific sources were not indicated were commercially available routine reagents.

The structures of the compounds were identified by nuclear magnetic resonance (NMR) and/or mass spectroscopy (MS). NMR was determined by Bruker AVANCE II (or III)-400MHz. The solvents were deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ) and deuterated methanol (CD ₃ OD), with tetramethylsilane (TMS) as internal standard. NMR chemical shifts (δ) are given in units of 10 ^-6 (ppm).

LC/MS (ESI) was analyzed and determined on the following instruments: Shimadzu LCMS2020 equipped with Sunfire C18 (5μm 50×4.6mm) chromatographic column, equipped with ACQUITY

Waters UPLC-QDa with BEH (2.1*50mm 1.7μm) chromatographic column, Agilent Agilent6120 equipped with Xbridge C18 (5μm 50×4.6mm) chromatographic column.

HPLC was analyzed and determined on the following instruments: Agilent 1200DAD equipped with Sunfire C18 (5μm 150×4.6mm) column and Shimadzu UFLC equipped with Xbridge C18 (5μm 150×4.6mm) column.

Chiral HPLC was analyzed and determined on Waters-UPC2 instrument.

Known materials used in this disclosure are prepared by conventional synthesis methods in the art, or purchased from companies such as Aldrich Chemical, Fisher Scientific or Combi-Blocks.

Unless otherwise stated in the examples, the reactions can be carried out under nitrogen atmosphere.

Unless otherwise stated in the examples, the reaction temperature in the reaction refers to room temperature, and the temperature range is 20°C to 30°C.

The progress of the reaction in the examples is monitored by LC-MS or thin-layer chromatography (TLC), and the developer system includes: A: dichloromethane and methanol, B: hexane and ethyl acetate. The volume ratio of the solvent is adjusted according to the polarity of the compound. The elution systems of column chromatography, thin-layer chromatography and CombiFlash rapid preparation instrument for purifying compounds include: A: dichloromethane and methanol, B: hexane and ethyl acetate. The volume ratio of the solvent is adjusted according to the polarity of the compound, and sometimes it can be adjusted by adding a small amount of basic reagent such as ammonia water or acidic reagent such as acetic acid.

Prep-HPLC analysis using Shimadzu (LC-20AD, SPD20A) Preparative HPLC (Phenomenex Gemini-NX 5μm C18 21.2×100mm column), Waters 2767 equipped with Sunfire Pre C18 (10μm 19×250mm) column and equipped with Xbridge Pre C18 (10μm Waters 2767-QDa on a 19 x 250mm) column.

Waters-SFC80 equipped with Daciel AD/OD/OJ/IC/IA/ID (10μm 20×250mm) column was used for Pre-SFC analysis.

CombiFlash runs on systems from Teledyne ISCO or Agela Technologies.

The abbreviation of the reagent used in the embodiment is as follows:

AIBN is 2,2'-azobis(2-methylpropionitrile),

EDCI is N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride,

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

LDA is lithium diisopropylamide,

NBS is N-bromosuccinimide,

Pd(dppf)Cl is [1,1'- _bis (diphenylphosphino)ferrocene]dichloropalladium(II),

DCE is 1,2-dichloroethane,

DCM is dichloromethane,

DMF is N,N-dimethylformamide,

MeCN or ACN is acetonitrile,

THF is tetrahydrofuran,

HEPES is 4-Hydroxyethylpiperazineethanesulfonic acid,

CHAPS is the inner salt of 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid,

FBS is fetal bovine serum.

The test conditions of the instruments used in the test:

1. Differential Scanning Calorimeter (DSC)

Instrument model: Mettler Toledo DSC 3+

Purge gas: nitrogen, 50mL/min

Heating rate: 10.0°C/min

Temperature range: 30-300°C

2. X-ray Powder Diffraction (XRPD)

Instrument model: BRUKER D8 DISCOVER

Rays: monochromatic Cu-Kα rays

(Cu-Kα1 wavelength is

Cu-Kα2 wavelength is

Cu-Kα wavelength takes the weighted average of Kα1 and Kα2

)

Scanning method: Coupledθ/2θ, scanning mode: step, scanning time: 45s, scanning steps: 3, 2θ start: 10, 2θ end: 48°, each step increment: 19°;

Voltage: 40KV, Current: 40mA

3. X-ray Powder Diffraction (XRPD)

Instrument model: BRUKER D8 Advance

Rays: monochromatic Cu-Kα rays

(Cu-Kα1 wavelength is

Cu-Kα2 wavelength is

Cu-Kα wavelength takes the weighted average of Kα1 and Kα2

)

Scanning method: Coupledθ/2θ, scanning mode: Continuous PSD fast, scanning rate: 0.1s/step, scanning range: 3-50°, scanning steps: 2286, each step increment: 0.02°

Voltage: 40KV, Current: 40mA

4. Thermogravimetric Analysis (TGA)

Instrument model: METTLER TOLEDO TGA 2

Purge gas: nitrogen, 50mL/min

Heating rate: 10.0°C/min

Temperature range: 30-350°C

5. Dynamic Vapor Sorption (DVS)

Instrument model: DVS Intrinsic

Carrier gas: nitrogen, 200sccm

Temperature: 25°C

Humidity level: Cycle 1 from 50%RH-90%RH-95%RH-90%RH-0%RH in 10%RH steps; then from 0%RH-90%RH- in 10%RH steps 95%RH-90%RH-50%RH, cycle 2;

Termination parameters: dM/dT=0.002%/min, Max time=360min, Min time=5min.

Embodiment 1: the preparation of formula I compound

The synthetic route of formula I compound is as follows:

first step:

LDA (15.28g, 142.66mmol, 71.43mL) was dissolved in 50mL of tetrahydrofuran, cooled to -78°C, and Int-1-1 solution (10g, 118.88mmol) dissolved in 10mL of THF was added dropwise. The resulting liquid was warmed to 20°C and stirred for 30 minutes. The reaction mixture was cooled to -78°C again, and tert-butyl 2-bromoacetate dissolved in 10 mL THF was added slowly. The reaction was stirred overnight at room temperature. After the reaction was completed, the reaction solution was quenched by adding saturated ammonium chloride (50mL, aq.), extracted with ethyl acetate (50mL×3), the organic phase was washed with 100mL concentrated brine, dried over sodium sulfate, and concentrated to obtain the crude compound Int -1-2 (22g, 110.97mmol, yield 93.34%).

¹ H NMR (400MHz, CDCl ₃ ): δ2.83(t,2H),2.50(t,2H),1.97-1.92(m,1H),1.45(s,9H),1.06-1.01(m,2H) ,0.91-0.86(m,2H).

Step two:

Seal the mixture of compound Int-1-2 (8.2g, 41.36mmol), ammonium carbonate (33.78g, 351.56mmol), sodium cyanide (5.07g, 103.40mmol), 50mL ethanol and 50mL water, and heat to 80°C , heated for 18 hours. The reaction mixture was cooled, poured into a mixture of 100 mL ethyl acetate and 100 mL water, the layers were separated, and the aqueous layer was extracted with ethyl acetate (100 mL×3). The organic solutions were combined, washed with concentrated brine, dried over sodium sulfate, and concentrated. The resulting residue was purified by silica gel chromatography (ethyl acetate/n-hexane=1/2) to obtain compound Int-1-3 (5.7 g, 21.24 mmol, yield 51.36%).

¹ H NMR(400MHz,DMSO):δ10.61(s,1H),7.66(s,1H),2.29-2.08(m,2H),1.93-1.88(m,2H),1.29(s,9H), 1.09-1.02(m,1H),0.47-0.26(m,3H),0.11-0.04(m,1H).

The third and fourth steps:

Compound Int-1-3 (7.2 g, 26.83 mmol) was dissolved in hydrochloric acid/dioxane solution (4M, 50 mL), stirred at room temperature for 4 hours and concentrated. The resulting solid was triturated with 30 mL of acetonitrile for 1 hour and filtered to give a pure racemic mass as a white solid. With supercritical fluid chromatography SFC (using CHIRALPAK AD-H 10 μm 2.5*25cm chiral column; Flow rate/monitoring: 70g/min; Monitor wavelength: 214nm; Mobile phase A: supercritical carbon dioxide; Mobile phase B: methanol) As a solid, compound Int-1 (2 g, 9.42 mmol, yield 35.12%) was obtained.

¹ H NMR(400MHz,DMSO):δ12.20(s,1H),10.63(s,1H),7.71(s,1H),2.32-2.09(m,2H),1.99-1.87(m,2H), 1.11-1.03(m,1H),0.48-0.27(m,3H),0.12-0.05(m,1H).

Chiral HPLC: 98.04%ee, Rt: 2.918min.

LCMS: MS m/z (ESI): 213.1 [M+1] ⁺ .

the fifth step:

Compound 40a (1 g, 4.87 mmol) was dissolved in 20 mL of DMF, and NBS (870 mg, 4.89 mmol) was added. The mixture was stirred at room temperature for 2 hours, the resulting mixture was poured into 20 mL of ice water, and the mixture was extracted with ethyl acetate (20 mL×2). The combined organic phases were washed with 20 mL of water, 20 mL of concentrated brine, dried over sodium sulfate, and filtered. The filtrate was concentrated to give crude product 40b (1 g, 3.52 mmol, yield 72.22%).

Step six:

Compound 40b (1 g, 3.52 mmol) was dissolved in 10 mL of methanol, and sulfuric acid (18M, 0.7 mL) was added dropwise. The mixed liquid was stirred overnight at 75°C, cooled to room temperature, poured into 20 mL of ice water, the mixed liquid was extracted with 50 mL of ethyl acetate, the organic component was dried over sodium sulfate, and filtered. The filtrate was concentrated to give crude product 40c (1 g, 3.36 mmol, yield 95.29%).

¹ H NMR (400MHz, DMSO-d ₆ ): δ7.57(s, 1H), 7.21(s, 1H), 6.11(brs, 2H), 3.85(s, 3H).

Step seven:

A solution of compound 40c (5.45g, 18.29mmol) and potassium ethylene trifluoroborate (2.45g, 18.29mmol) was dissolved in 50mL of dioxane and 10mL of water, and Pd(dppf)Cl ₂ (1.34g, 1.83mmol) was added and Potassium carbonate (6.35 g, 45.71 mmol). The resulting mixture was evacuated and refilled 3 times with _N2 . The resulting mixture was stirred at 80°C for 16 hours. The mixture was diluted with 100 mL of ethyl acetate, and the combined organic phases were washed with 100 mL of concentrated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography to obtain compound 41b (3.56 g, 14.52 mmol, yield 79.40%).

LCMS: MS m/z (ESI): 246.1 [M+H] ⁺ .

Step eight:

Compound 41b (3.56 g, 14.52 mmol) was dissolved in 20 mL of methanol, and palladium on carbon (1.55 g, 1.45 mmol, 285.48 uL, 10% purity) was added. The resulting mixture was evacuated and gassed with hydrogen. The resulting mixture was stirred at room temperature for 16 hours, liquid chromatography showed the reaction was complete. The mixture was filtered, the block was washed with methanol, and the filtrate was concentrated under reduced pressure to obtain compound 41c (3.45 g, 13.96 mmol, yield 96.12%).

LCMS: MS m/z (ESI): 248.1 [M+H] ⁺ .

Step Nine:

Compound 41c (3.36 g, 13.59 mmol) was dissolved in 34 mL of acetone, and 3.36 mL of hydrochloric acid was added. The resulting mixture was stirred at room temperature for 20 minutes. The mixture was cooled to 0 °C and sodium nitrite (1.88 g, 27.18 mmol) dissolved in 5 mL of water was added. Copper chloride (1.48 g, 14.95 mmol) was added in small amounts at 0 °C. The resulting mixture was stirred at room temperature for 1 hour. The mixture was poured into 1M hydrochloric acid (60 mL), and the aqueous phase was extracted with ethyl acetate (100 mL×3). The combined organic phases were washed with 100 mL of concentrated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluted with n-ethane/ethyl acetate=50/1) to obtain compound 41d (2.23 g, 8.36 mmol, yield 61.53%).

¹ H NMR (400MHz, DMSO-d ₆ ): δ7.99(s, 1H), 7.87(s, 1H), 3.88(s, 3H), 2.92(q, 2H), 1.17(t, 3H).

Step ten:

Compound 41d (2.23 g, 8.36 mmol) was dissolved in 35 mL of carbon tetrachloride, and AIBN (412.00 mg, 2.51 mmol) and NBS (1.64 g, 9.20 mmol) were added. The resulting mixture was stirred at 80°C for 16 hours. Filter the mixture. The solid was washed with DCM, and the filtrate was concentrated in vacuo to give crude compound 41e (2.5 g, 7.24 mmol, 86.51% yield).

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.17(s, 1H), 8.04(s, 1H), 6.08(q, 1H), 3.92(s, 3H), 2.05(d, 3H).

Eleventh step:

Compound 41e (2.5 g, 7.24 mmol) was dissolved in 10 mL of methanol, and NH ₃ /MeOH (7M, 30 mL) was added. The resulting mixture was stirred at room temperature for 16 hours. The mixture was purified by prep-HPLC to obtain compound 41f (1.18 g, 4.73 mmol, yield 65.34%).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ9.11 (brs, 1H), 8.20 (s, 1H), 7.91 (s, 1H), 4.71 (q, 1H), 1.42 (d, 3H).

¹⁹ F NMR (376.5 MHz, DMSO-d ₆ ): δ-60.99.

LCMS: MS m/z (ESI): 250.0 [M+H] ⁺ .

Step twelve:

small scale

Compound 41f (50 mg, 0.2 mmol) was dissolved in THF (2 mL), and borane-d3-THF multiple solution (6 mmol, 6 mL) was added. The reaction solution was stirred at 60°C for 18 hours. 2 mL of methanol was added dropwise, followed by hydrochloric acid (6M, 2 mL). The reaction solution was stirred at 80°C for 2 hours. Next, 5M sodium hydroxide was added to adjust the pH of the mixture to 7, and the liquid was dried and concentrated. The residue was purified by silica gel chromatography (DCM:MeOH=20:1) to obtain compound 71b (39 mg, 0.147 mmol, yield 70%).

massive

Compound 41f (800 mg, 3.20 mmol) was dissolved in 10 mL THF, and BD ₃ (1M in THF, 64 ml, 64 mmol) was added. After the addition, the reaction was stirred at 60°C (in a closed tube) for 10 hours. The reaction was quenched with 10 mL of methanol and hydrochloric acid (6M, 20 mL) was added. It was then stirred at 80° C. for 8 hours. 2N sodium hydroxide was added to adjust the pH value to 7, extracted with ethyl acetate, the combined organic phases were washed with 50 mL concentrated brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel, eluting with 5% methanol in DMC, to afford compound 71b, which was used in the next step.

LCMS: MS m/z (ESI): 238.1 [M+H] ⁺ .

Step Thirteen:

small scale

Compound 71b (20 mg, 0.084 mmol) was dissolved in 2 mL of DMF, triethylamine (40 mg, 0.28 mmol), compound Int-1 (23 mg, 0.108 mmol) and HATU (57 mg, 0.13 mmol) were added. The reaction was stirred at room temperature for 18 hours. 3 mL of water was added, and the mixture was extracted with ethyl acetate (20 mL×3). The combined organic layers were washed with brine, dried over sodium sulfate, and concentrated. The residue was purified by prep-HPLC to obtain compound 71 (24 mg, 0.055 mol, yield 51%).

massive

Compound Int-1 (680mg, 3.2mmol) was dissolved in 10ml DMF, EDCI (920mg, 4.8mmol) and HATU (1.83g, 4.8mmol) were added. After stirring for 10 minutes, compound 71b collected from the previous step was added. The reaction solution was stirred at room temperature for 3 hours. LCMS showed the reaction was complete. Direct purification by reverse phase liquid chromatography afforded compound 71 (1.10 g, 79.6% yield after two steps).

¹ H NMR (400MHz, CD ₃ OD,): 7.76(s, 1H), 7.63-7.60(m, 1H), 5.57-5.53(m, 1H), 2.59-2.40(m, 2H), 2.28-2.19( m, 2H), 1.56-1.50(m, 3H), 1.28-1.21(m, 1H), 0.62-0.58(m, 1H), 0.49-0.41(m, 3H).

LCMS: MS m/z (ESI): 432 [M+H] ⁺ .

Step Fourteen:

Compound 71 (1.10 g) was separated by supercritical fluid chromatography to obtain two isomers (325 mg and 415 mg, respectively).

Enantiomer formula I compound (71-1) (shorter retention time):

¹ H NMR (500MHz,DMSO-d ₆ )δ10.63(s,1H),7.89(d,1H),7.75(t,2H),5.30–5.16(m,1H),4.23(d,1H), 2.37–2.27(m,2H),1.99(dq,2H),1.53(s,1H),1.43(dd,3H),1.11(td,1H),0.49–0.30(m,3H),0.11(dt, 1H).

LCMS: MS m/z (ESI): 432.3 [M+H] ⁺ .

ChirHPLC (1% DEA in ethanol/hexane 60/40, 1.0 mL/min, 35°C, CHIRALPAK IG, 150*4.6mm, 5μm): Rt: 4.594min, de: 100%.

Enantiomer compound 71-2 (longer retention time):

¹ H NMR (500MHz,DMSO-d ₆ )δ10.53(s,1H),7.89(d,1H),7.80–7.66(m,2H),5.30–5.12(m,1H),4.23(d,1H ),2.44–2.36(m,1H),2.31–2.20(m,1H),2.05–1.95(m,2H),1.44(dd,3H),1.11(td,1H),0.50–0.29(m,3H ), 0.16–0.08(m,1H).

LCMS: MS m/z (ESI): 432.3 [M+H] ⁺ .

ChirHPLC (1% DEA in ethanol/hexane 60/40, 1.0 mL/min, 35°C, CHIRALPAK IG, 150*4.6mm, 5μm): Rt: 10.931min, de: 100%.

biological test

The present disclosure will be further described with reference to the following test cases, but these test cases should not be considered as limiting the scope of the present disclosure.

Test Example 1: In Vitro Fluorescence Determination of ADAMTS-4 or ADAMTS-5 Activity

FRET (Fluorescence Resonance Energy Transfer) peptides are split into two separate fragments by recombinant ADAMTS-4 or ADAMTS-5 proteins, resulting in an increase in the quantified fluorescent signal. The polypeptide is 5-FAM-TEGEARGSVILLK(5-TAMRA)K-NH ₂ , customized from ANASPEC. ADAMTS-4 recombinant protein (Cat. No. 4307-AD) and ADAMTS-5 recombinant protein (Cat. No. 2198-AD) were purchased from R&D Systems.

Prepare assay buffer containing 50 mM HEPES pH 7.5, 100 mM NaCl, 5 mM CaCl, 0.1% CHAPS, and 5% glycerol. Dispense 2.5 μL of compound-containing assay buffer into a 384-well plate and add 2.5 μL of ADAMTS-4 or ADAMTS-5 protein (final concentration in the reaction is 10 nM). Compounds and proteins were pre-incubated for 15 min at room temperature. Then, 5 μL of substrate was added to each well. The final substrate concentrations were 15 μM and 8 μM for ADAMTS-4 and ADAMTS-5, respectively. After 3 hours of incubation at 37°C, the fluorescence signal (excitation, 490nm; emission, 520nm) in each well was measured on a TECAN plate reader.

data analysis

The data was entered into the GraphPad Prism software, and the IC ₅₀ value was calculated using the "log(inhibitor)vs.response--Variable slope(four parameters)" function (see Table 1)

Table 1 _IC50 values of exemplary compounds in FRET-peptide enzymatic assays

Conclusion: The compound of formula I of the present disclosure has significant inhibitory effect on the enzymatic activity of ADAMTS-4 and ADAMTS-5.

In vitro ELISA test (enzyme-linked immunosorbent assay) of test example 2.ADAMTS-5 activity

In this assay, the enzymatic activity of recombinant ADAMTS-5 protein (Cat. No. 2198-AD, R&D Systems) is determined using the protein substrate, aggrecan IGD protein. Aggrecan IGD protein is a polypeptide linking human proteoglycan globular domains 1 and 2 (T331-G458) with a histidine tag at the C-terminus (cat. no. 30411000, BIOTEZ), produced by expression in Escherichia coli . The enzymatic product ARGSVIL-peptide was detected using BioTEZ's ELISA kit (Catalog No. 30510111).

Assay buffer was prepared containing 50 mM HEPES pH 7.5, 100 mM NaCl, ₅ mM CaCl2, 0.1% CHAPS and 5% glycerol. Recombinant ADAMTS-5 protein was diluted to 0.3 nM in assay buffer. Transfer 10 µL of buffer and 10 µL of compound solution to each well of a 96-well plate and incubate at room temperature for 15 min. Dilute aprotecan-IGD to 100 nM with assay buffer and add 20 μL to each well. Plates were incubated at 37°C for 45 minutes. After incubation, the newly produced epitope ARGSVIL-peptide was measured using the aggrecanase activity ELISA assay kit following the manufacturer's instructions. Then, add 100 μL of stop solution and read the absorbance of each well at 450 nM on a TECAN plate reader using 620 nM as a reference.

data analysis:

Use the Sigmoidal 4PL function in GraphPad Prism software to obtain the standard curve determined by ELISA, and calculate the corresponding peptide concentration based on the standard curve. _IC50 values were calculated using the "log(inhibitor) vs. response--Variable slope(four parameters)" function (see Table 2).

Table 2 illustrates the _IC50 values of the compounds in the glycan-IGD enzymatic activity assay.

serial number	ADAMTS-5 (IC 50 , nM)
71	29
Compound of formula I	13

Conclusion: the compound of formula I has significant inhibitory effect on the enzymatic activity of ADAMTS-5

Test Example 3. Mouse Explant Test

In this test, fresh mouse femoral head cartilage is treated with IL-1α protein (Sigma-Aldrich, catalog number I2778) in culture medium to induce cartilage catabolism. GAGs attached to cleaved proteoglycan fragments and GAGs attached to intact proteoglycans were then measured by the dimethylmethylene blue dye in the Glycosaminoglycan Detection Kit (Chondrex, Cat# 6022).

Femoral head cartilage samples were isolated from mice (25 days old, male, C57BL/6, from Charles River Lab) and placed in 2.0 ml refill medium (cell culture medium, 10% FBS, 4 mM glutamine, penicillin-strep Mycin, 20mM HEPES). Add 200 µL of FBS-free medium to each well of the 48-well plate and transfer a piece of cartilage to one well of the well plate. The medium was then aspirated and the compound of formula I and IL-la protein were added to the plate in a total volume of 400 μl of fresh medium without FBS. The final concentration of IL-la was 1 ng/mL. The plate was incubated at 37°C for 72 hours in a humidified incubator with 5% carbon dioxide supply.

The supernatant was transferred to a 1.5 mL tube and kept at -20°C. Transfer each cartilage sample to an additional 1.5 mL tube containing 400 µL of freshly prepared papain solution. Papain solution contained 125 μg/mL papain (Sigma-Aldrich, catalog number P3125), 0.1 M sodium acetate (Sigma-Aldrich, catalog number S7899), pH 5.5 and 5 mM EDTA and 5 mM L-cysteine hydrochloride ( Sigma-Aldrich, catalog number C7880). Cartilage samples were kept shaking in a 60 °C water bath for 24 hours.

The lysate was vortexed for 10 seconds and spun at 10,000 rpm for 2 minutes. Dilute the supernatant and lysate samples with phosphate buffer and mix with 100 µL of the dye from the Glycosaminoglycan Detection Kit. The optical density from each well was measured with a TECAN plate reader set to a wavelength of 525 nm.

data analysis:

Concentrations of GAGs in supernatants and lysates were determined based on standard curves using the dose range of chondroitin sulfate provided in the kit.

The proportion of GAG release was calculated as follows:

The tested compound effects are expressed as percent inhibition calculated using the following formula:

Inhibition data for compounds of formula I are presented in Table 3 at concentrations of 2 μM and 20 μM.

The IC _value of table 3 formula I compound in the mouse explant assay

serial number	Inhibition % at 2 μM concentration	% inhibition at 20 μM concentration
Compound of formula I	48	89

The foregoing embodiments and examples are for illustration only, and are not intended to limit the scope of the present disclosure. Various changes and modifications to the disclosed embodiments will become apparent to those skilled in the art from the present disclosure, and such changes and modifications can be made without departing from the spirit and scope of the present disclosure. All cited documents are hereby incorporated by reference in their entirety, without admission that they are prior art.

Example 2-1: Preparation of Formula I Compound A Crystal Form

The compound of formula I (10 mg, 23.16 μmol) was dissolved in 1 mL THF, dissolved, filtered to remove insoluble matter, and allowed to stand overnight. Crystals were precipitated, filtered, and the filter cake was collected and dried in vacuo to obtain Form A of the compound of formula I (2 mg, yield: 20.0%).

Embodiment 2-2: Preparation of Formula I Compound A Crystal Form

The compound of formula I (876mg, 2.03mmol) was dissolved in 10mL of absolute ethanol, the undissolved liquid was stirred at room temperature, the reaction solution was filtered, the filter cake was collected, and dried in vacuum to obtain the crystal form A of the compound of formula I (578mg, yield: 66.0%).

Its characteristic peak positions are shown in Table 4, and the X-ray powder diffraction spectrum is shown in Figure 1.

The DSC spectrum is shown in Figure 2, and the endothermic peak is at 280.16°C.

Table 4 XRD characteristic peak positions of crystal form A

Embodiment 2-3: Preparation of Formula I Compound A Crystal Form

The compound represented by formula I (1.5g, 3.47mmol) was dissolved in 15mL of absolute ethanol, the undissolved liquid was stirred at room temperature, the reaction solution was filtered, the filter cake was collected, and vacuum-dried to obtain the product (1188mg, yield: 79.2%) . According to X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.

Embodiment 2-4: Preparation of formula I compound A crystal form

Dissolve the compound represented by formula I (10.8g, 25.01mmol) in 60mL of isopropanol, stir the undissolved liquid, stir at 50°C, cool down to room temperature, continue stirring, filter the reaction solution, collect the filter cake, and the above wet product Dissolve in 60mL of isopropanol, stir to clear the undissolved solution, stir at 50°C, cool to room temperature, continue stirring overnight, filter the reaction solution, collect the filter cake, and dry in vacuo to obtain the product (6.4g, yield: 59.3%). According to X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.

Embodiment 2-5: Preparation of Formula I Compound A Crystal Form

Dissolve the compound A crystal form (Example 4) (30mg, 69.5μmol) shown in formula I in 0.5mL acetone, stir the undissolved liquid, stir at room temperature, filter the reaction solution, collect the filter cake, and obtain the product (12mg, yield : 40.0%). According to X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.

Embodiment 2-6: Preparation of formula I compound A crystal form

Dissolve the crystal form of Compound A (Example 4) (30 mg, 69.5 μmol) shown in formula I in 0.5 mL of ethyl acetate, stir at room temperature, filter the reaction solution, collect the filter cake, and dry in vacuo to obtain the product ( 17 mg, yield: 56.7%). According to X-ray powder diffraction detection, the product is the crystal form A of the compound of formula I.

Embodiment 3, A crystal form influence factor experiment

Put the crystal form of compound A shown in formula I (prepared in Example 2-3) open and flat, and examine the conditions of heating (40°C, 60°C), ultraviolet light (4500Lux), high humidity (RH 75%, RH 90 %) of the stability of the sample under the conditions, the sampling investigation period is 30 days. The experimental results are shown in Table 5.

Table 5 Formula I Compound A Crystal Form Influencing Factors Experimental Results

Experimental results:

The compound has good physical and chemical stability under light, high temperature, and high humidity conditions. Humidity conditions will affect the sample. It is recommended that the sample be stored in a dry and sealed condition.

Embodiment 4, long-term accelerated stability experiment of crystal form A

The crystal form of compound A of formula I (prepared in Example 2-3) was subjected to a 3-month long-term accelerated stability investigation.

The experimental results are shown in Table 6.

Table 6 Long-term accelerated stability test results of compound A crystal form of formula I:

Experimental results:

Under the condition of avoiding light and sealing (inner packaging is sealed bag, external packaging with aluminum foil), the compound is stored for 3 months under long-term (25°C, 60% RH) and accelerated (40°C, 75% RH) conditions, and the compound is physically and chemically stable Good sex.

Claims (15)

1. A crystal form of a compound shown in formula I,

   

   Its X-ray powder diffraction pattern has characteristic peaks at 2θ angles of 11.2, 13.8, 16.6, 21.0, and 27.6.
2. According to the A crystal form of the compound shown in formula I according to claim 1, its X-ray powder diffraction spectrum has characteristics at 2θ angles of 8.4, 11.2, 13.8, 14.9, 16.6, 17.5, 18.5, 21.0, 22.9, 27.6 peak.
3. According to the A crystal form of the compound shown in formula I according to claim 2, its X-ray powder diffraction pattern is 8.4, 11.2, 13.8, 14.9, 15.4, 16.6, 17.5, 18.5, 21.0, 22.9, 27.6 at the 2θ angle have characteristic peaks.
4. According to claim 1, the crystal form A of the compound represented by formula I has an X-ray powder diffraction pattern as shown in FIG. 1 .
5. The crystal form A of the compound represented by formula I according to any one of claims 1-4, wherein the error range of the 2θ angle is ±0.2.
6. A method for preparing the A crystal form of the compound shown in formula I according to any one of claims 1-5, the method comprising: mixing the compound shown in formula I with an appropriate amount of solvent, the solvent being an organic solvent , the solvent is preferably tetrahydrofuran, ethanol, isopropanol, acetone, ethyl acetate.
7. A pharmaceutical composition, comprising the crystal form A of the compound represented by formula I according to any one of claims 1-5, and one or more pharmaceutically acceptable carriers or excipients.
8. A pharmaceutical composition, which is prepared by the crystal form A of the compound represented by formula I according to any one of claims 1-5, and one or more pharmaceutically acceptable carriers or excipients.
9. A preparation method of a pharmaceutical composition, comprising mixing the A crystal form of the compound shown in formula I according to any one of claims 1-5 with one or more pharmaceutically acceptable carriers or excipients step.
10. The A crystal form of the compound shown in formula I according to any one of claims 1-5, or the pharmaceutical composition according to any one of claims 7-8 is used for inhibiting ADAMTS-5 and/or ADAMTS in the preparation -4 uses of the drug.
11. The A crystal form of the compound shown in formula I according to any one of claims 1-5, or the pharmaceutical composition according to any one of claims 7-8 in the preparation for prevention or treatment involving cartilage degradation and/or or use in medicine for inflammatory conditions or diseases in which cartilage homeostasis is disrupted.
12. The A crystal form of the compound shown in formula I according to any one of claims 1-5, or the pharmaceutical composition according to any one of claims 7-8 in the preparation of medicines for preventing or treating arthritis the use of.
13. The use according to claim 12, the arthritis is selected from rheumatoid arthritis, psoriatic arthritis, osteoarthritis and hypertrophic arthritis.
14. According to the A crystal form of the compound shown in formula I according to any one of claims 1-5, or according to any one of claims 7-8, the pharmaceutical composition is prepared for treatment with ADAMTS-5 and/or Use in medicine for a disease or condition associated with ADAMTS-4 activity.
15. Use according to claim 14, wherein the disease or condition associated with ADAMTS-5 and/or ADAMTS-4 activity is arthritis, preferably rheumatoid arthritis, psoriatic arthritis, osteoarthritis or hypertrophic joint inflammation.

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