Crystalline forms and amorphous substances of indoline spirocyclic compounds, and methods for producing and using both

Abstract

The present invention discloses a crystalline form and an amorphous substance of an indoline spirocyclic compound represented by formula (1), as well as methods for preparing and using both. The crystalline form exhibits characteristic peaks at 5.43±0.20°, 12.28±0.20°, and 17.91±0.20° in X-ray powder diffraction (2θ) using Cu-Kα radiation, while the XRPD spectrum of the amorphous substance exhibits no significant diffraction peaks. The crystalline form and amorphous substance provided by the present invention have high purity and excellent stability, making them advantageous for drug discovery. [Case 1] JPEG2025534100000021.jpg30169

Description

Detailed Description of the Invention

[0001] This application claims priority from two prior applications: Patent Application No. 202211297102.5, filed with the State Intellectual Property Administration of China on October 21, 2022, entitled "Crystalline Forms of Indoline Spirocyclic Compounds, Amorphous Materials, and Methods for Preparing and Using Both," and Patent Application No. 202311300559.1, filed with the State Intellectual Property Administration of China on October 9, 2023, entitled "Crystalline Forms of Indoline Spirocyclic Compounds, Amorphous Materials, and Methods for Preparing and Using Both," both of which are incorporated herein by reference in their entireties.

[0002] [Technical Field] The present invention is in the field of pharmaceutical compounds, and specifically relates to crystalline forms of indoline spirocyclic compounds, amorphous substances, and methods for preparing and using both.

[0003] [Background technology] Human growth hormone (GH) is a peptide hormone secreted by the anterior lobe of the pituitary gland. It consists of 191 amino acids and acts directly or indirectly on peripheral organs by inducing the synthesis of insulin-like growth factor 1 (IGF-1) or epidermal growth factor (EGF). Its main physiological functions include promoting body height growth, promoting cell proliferation in muscles and skin, and playing an important role in tissue regeneration after trauma.

[0004] Ghrelin is an endogenous growth hormone-releasing peptide containing 28 amino acids and is an endogenous ligand for the growth hormone secretagogue receptor type 1a (GHSR 1a). Both in vivo and in vitro experiments have demonstrated that ghrelin significantly stimulates growth hormone secretion. Clinical studies have also shown that intravenous administration of ghrelin can potently stimulate growth hormone release in a dose-dependent manner.

[0005] The release of GH is believed to be capable of treating physiological or pathophysiological disorders characterized by defective growth hormone secretion and disorders that are ameliorated by the anabolic effects of growth hormone. Clinical studies have shown that GH shows promise in treating disorders such as loss of muscle mass, accumulation of adipose tissue, bone demineralization, and a reduced ability of tissue to regenerate after injury.

[0006] GH is synthesized and stored in the pituitary gland, and its release is controlled by hypothalamic hormones. As is known, two hormones are involved in the GH release process: growth hormone-releasing hormone (GHRH) and somatotropin-release inhibitory factor (SRIF). In most cases, GH deficiency is related to GH release (hypothalamic defect) rather than GH synthesis (pituitary defect). Therefore, stimulating GH release in the pituitary gland using GHSR agonists may be a novel therapeutic alternative to recombinant human growth hormone.

[0007] GHSR has two subtypes, 1a and 1b. The 1a subtype is a functional receptor subtype, while the function of the 1b subtype requires further study. In the central nervous system, GHSR 1a is distributed in multiple regions of the hypothalamus and outside the hypothalamus, including the pituitary gland, hypothalamic arcuate nucleus, and ventromedial nucleus. In the periphery, GHSR is also expressed at low levels in the thyroid gland, pancreas, and muscle. Therefore, ghrelin and its receptor, GHSR 1a, may be involved in regulating various functions in the body.

[0008] Research has revealed that several peptide or peptidomimetic clinical compounds exhibit GHSR agonist activity, which can induce GH release. Compounds currently undergoing clinical studies include examorelin, tabimorelin, pralmorelin, ibutamoren, tesamorelin, anamorelin, and macimorelin. The polypeptide tesamorelin (for reducing excess abdominal fat in HIV-infected individuals) and the small molecule peptidomimetic macimorelin have been approved by the FDA and are commercially available. Macimorelin is the only orally administered drug approved for the diagnosis of adult growth hormone deficiency, but it also has drawbacks, including low oral bioavailability and potential cardiac toxicity.

[0009] Related research results have found that GHSR agonists, in addition to inducing GH secretion through the activation of GHSR 1a, also induce other physiological functions through other different receptors in the GHS receptor family or different binding sites in GHSR (GHSR 1b, gastrointestinal motilin receptor 1a, neurotensin receptor, TRH receptor, etc.). Therefore, the application of GHSR agonists in the field of gastrointestinal indications is newly developed, but there are currently no drugs on the market for this indication, and ulimorelin and relamorelin have progressed to clinical phase III research.

[0010] Ghrelin has been shown to promote gastrointestinal peristalsis via the vagus and pelvic nerves. However, the short half-life of Ghrelin hampers its potential for drug discovery. Therefore, it is necessary to develop GHSR agonists with enhanced pharmacokinetics to improve gastrointestinal dysfunction in animals and humans.

[0011] In order to overcome the above technical problems, the present applicant has obtained a compound having a novel molecular structure through its own research and development, and the structural formula is: [ka] The compound is named (4R,11R)-7,7-dimethyl-4-(1-(methylsulfonyl)spiro[indoline-3,4'-piperidine]-1'-carbonyl)-6,9-dioxo-1-phenyl-2,10-dioxa-5,8-diazadodecan-11-yl isobutyrate, and the relevant content is described in patent application PCT / CN2022 / 088656. As shown by the pharmacodynamic test, the compound has a good prospect for clinical application and can be used to manufacture a medicament for the diagnosis, prevention and / or treatment of growth hormone-dependent diseases or disorders, preferably the diseases or disorders are related to growth hormone deficiency or growth hormone dependence, such as the diagnosis of growth hormone-deficient patients, the slow growth and short stature of growth hormone-deficient children, and other diseases that can be improved by the physiological actions of growth hormone, including but not limited to the regulation of energy balance and food intake, lipogenesis, treatment of obesity and weight loss, treatment of cachexia, improvement of gastrointestinal motility, gastroparesis and diabetic gastroparesis, treatment of postoperative ileus, increase in muscle mass and skinfold thickness in patient populations with age-related diseases, reduction in lipids and slight increase in bone mineral density, treatment of burns, AIDS and cancer conditions, and wound and bone healing.

[0012] At the same time, it has become an urgent technical challenge to develop solid drug forms suitable for pharmaceutical use of the above compounds, such as solid forms with improved stability, hygroscopicity and / or efficacy, thereby achieving good results in the pharmaceutical production and administration stages.

[0013] Summary of the Invention The entire contents of patent application PCT / CN2022 / 088656 are incorporated herein by reference.

[0014] In order to solve the above technical problems, the present invention provides a crystalline form of the compound represented by formula (1): [ka]

[0015] The crystalline form has characteristic peaks at 5.43±0.20°, 12.28±0.20° and 17.91±0.20° in X-ray powder diffraction using Cu-Kα radiation and expressed in 2θ angles.

[0016] According to an embodiment of the present invention, the crystalline form has characteristic peaks at 5.43±0.20°, 12.28±0.20°, 17.91±0.20°, 18.54±0.20°, 18.87±0.20°, 20.54±0.20°, and 21.69±0.20° in X-ray powder diffraction using Cu-Kα radiation and expressed in degrees 2θ.

[0017] According to an embodiment of the present invention, the crystalline form has characteristic peaks at 5.43±0.20°, 7.96±0.20°, 9.72±0.20°, 12.28±0.20°, 13.16±0.20°, 17.69±0.20°, 17.91±0.20°, 18.16±0.20°, 18.54±0.20°, 18.87±0.20°, 19.51±0.20°, 20.31±0.20°, 20.54±0.20°, 21.69±0.20°, and 21.86±0.20° in X-ray powder diffraction using Cu-Kα radiation and expressed in degrees 2θ.

[0018] According to an embodiment of the present invention, the crystalline form has an XRPD spectrum substantially as shown in FIG.

[0019] According to an embodiment of the invention, the crystalline form is anhydrous.

[0020] According to an embodiment of the invention, the crystalline form has a weight loss of 3 wt% or less, such as 2 wt% or less, before reaching 150°C.

[0021] According to an embodiment of the present invention, the crystalline form has a TGA spectrum substantially as shown in FIG.

[0022] According to an embodiment of the present invention, the crystalline form has a sharp endothermic peak at a peak temperature of 130.6±2°C.

[0023] According to an embodiment of the present invention, the crystalline form has a DSC spectrum substantially as shown in FIG.

[0024] The present invention further provides a method for preparing the above crystalline form, comprising the steps of dissolving a compound of formula (1) in solvent A, and cooling to precipitate a solid, thereby obtaining the crystalline form;

[0025] The solvent A may be selected from the group consisting of methanol, ethanol, isopropanol, tert-butanol, n-butanol, acetone, tetrahydrofuran, methyltetrahydrofuran, ethyl formate, ethyl acetate, isopropyl acetate, n-hexane, n-dioxane, cyclohexane, methyl tert-butyl ether, toluene, dichloromethane, chloroform, DMSO, water, acetonitrile, isopropyl ether, etc., for example, mixed solvents such as ethanol / water, DMSO / water, acetone / water, ethanol / n-hexane, ethanol / cyclohexane, acetonitrile / n-dioxane, ethanol / n-dioxane, isopropanol / n-dioxane, methanol / n-dioxane, methanol / n-hexane, acetone / n-dioxane, and mixed solvents consisting of combinations thereof.

[0026] According to an embodiment of the present invention, the dissolution process is carried out under heating conditions.

[0027] According to an embodiment of the present invention, the cooling process is down to room temperature.

[0028] According to an embodiment of the present invention, the cooling process is a slow cooling.

[0029] According to an embodiment of the invention, the method further comprises post-treatment of the solids, such as for example filtration and / or washing.

[0030] The present invention further provides an amorphous material of the compound of formula (1), the XRPD spectrum of which does not have any significant diffraction peaks.

[0031] According to an embodiment of the present invention, the amorphous material has an XRPD spectrum substantially as shown in FIG.

[0032] The present invention further provides a method for producing the amorphous substance, comprising the steps of dissolving a compound represented by formula (1) in solvent B, adding the obtained solution to solvent C, and stirring to precipitate a solid to obtain the amorphous substance,

[0033] The solvent B is a good solvent for the compound represented by formula (1), and may be selected from one or more of, for example, DMF, DMA, NMP, acetonitrile, THF, DMSO, methyl tert-butyl ether, isopropyl ether, methanol, ethanol, isopropanol, acetone, etc.;

[0034] The solvent C is a poor solvent represented by formula (1), and is selected from, for example, one or more of water, n-dioxane, n-hexane, and cyclohexane.

[0035] According to an embodiment of the present invention, the dissolving solution is added to the solvent C in a dropwise manner.

[0036] According to an embodiment of the invention, the method further comprises post-treatment of the solids, such as for example filtration and / or washing.

[0037] The present invention further provides pharmaceutical compositions containing the crystalline forms and / or amorphous materials.

[0038] According to an embodiment of the present invention, the pharmaceutical composition further comprises pharmaceutically acceptable auxiliary materials, including, but not limited to, one or more of excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, solubilizers, suspending agents, isotonicity agents, buffers, preservatives, antioxidants, colorants, effervescent agents, and flavoring agents.

[0039] According to an embodiment of the present invention, the pharmaceutical composition further comprises a second active ingredient other than the crystalline and / or amorphous material, for example, the second active ingredient is a drug related to growth or development, for example, the second active ingredient is a GHSR agonist or a growth hormone.

[0040] In some embodiments, the crystalline form and / or amorphous material may be administered separately from or together with a second active ingredient during treatment.

[0041] The present invention further provides the use of the above crystalline form, amorphous substance and / or pharmaceutical composition in the manufacture of a medicament for diagnosing, preventing and / or treating a growth hormone deficiency or growth hormone dependent disease (or disorder).

[0042] According to embodiments of the present invention, the disorders are, for example, the diagnosis of growth hormone deficiency in patients, slow growth and short stature in growth hormone deficient children, and other disorders that can be ameliorated by the physiological actions of growth hormone, including, but not limited to, regulation of energy balance and food intake, lipogenesis, treatment of obesity and weight loss, treatment of cachexia, improvement of gastrointestinal motility, gastroparesis and diabetic gastroparesis, treatment of post-operative ileus, increase in muscle mass and skinfold thickness in patient populations with age-related disorders, reduction in lipids and slight increases in bone mineral density, treatment of burns, AIDS and cancer conditions, and wound and bone healing.

[0043] In some embodiments, the formulation may be a pharmaceutical formulation, such as a GHSR agonist.

[0044] The present invention further provides formulations containing the crystalline and / or amorphous material or prepared from the pharmaceutical compositions.

[0045] According to embodiments of the present invention, the formulation may be in the form of a powder, tablet (e.g., coated tablet, sustained or controlled release tablet), tablet, capsule (e.g., soft capsule or hard capsule), granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, jelly, injection, lyophilized powder injection, or suppository.

[0046] According to embodiments of the present invention, the formulation may be administered orally, bucally, sublingually, by inhalation, topically, parenterally via intravenous, subcutaneous, acupuncture or intramuscular injection, or rectally.

[0047] The present invention further provides a method for diagnosing, preventing and / or treating a growth hormone deficiency or growth hormone dependent disease (or disorder) comprising administering to a patient a therapeutically effective amount of said crystalline form, amorphous material or said pharmaceutical composition.

[0048] According to an embodiment of the present invention, said disease has the above definition.

[0049] The beneficial effects of the present invention are as follows:

[0050] The present invention provides a crystalline form and amorphous substance of the compound of formula (1), as well as methods for preparing and using the same. The crystalline form and amorphous substance of the compound of formula (1) obtained by the present invention have good stability and solubility, low hygroscopicity, long-term storage potential, and high reproducibility, making them suitable for drug development. [ka]

[0051] The compound of formula (1) has high crystalline purity, excellent crystalline stability under conditions of light irradiation, high temperature, and high humidity, low hygroscopicity, and long-term storage, which is advantageous for drug development. The manufacturing process is stable and highly reproducible, and it can be applied to industrial production.

[0052] The amorphous substance of the compound of formula (1) prepared in the present invention has high purity, high solubility in most solvents, and excellent physical and chemical stability under conditions of light exposure, high temperature, and high humidity, making it suitable for drug development.

[0053] The definitions and explanations of terms are as follows:

[0054] Unless otherwise stated, the definitions of terms in the specification and claims of this application, including their exemplary definitions, exemplary definitions, preferred definitions, definitions of specific compounds in the examples, etc., may be combined and linked in any combination with each other, and such combinations and links should be within the scope described in the present specification.

[0055] The term "therapeutically effective amount" refers to an amount of the crystalline form, amorphous material, or second active ingredient of the present invention sufficient to achieve the intended use (including, but not limited to, the treatment of diseases defined below). A therapeutically effective amount can vary based on factors such as the intended use (in vitro or in vivo), or the subject and disease disorder being treated, e.g., the subject's weight and age, the severity of the disease disorder, and the method of administration, and can be readily determined by one of ordinary skill in the art. Specific doses will vary depending on the particular active ingredient selected, the dosing regimen followed, whether it is administered in combination with other compounds, the schedule of administration, the tissue to which it is administered, and the physical delivery system involved.

[0056] The term "patient" refers to any animal, including a mammal, preferably a mouse, rat, other rodent, rabbit, dog, cat, pig, cow, sheep, horse or primate, most preferably a human.

[0057] The term "130.6±2°C" refers to 128.6 to 132.6°C, for example, 128.6°C, 129.0°C, 130.0°C, 131.0°C, 132.0°C, 132.6°C, or any value between said two points.

[0058] BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an XRPD spectrum of an amorphous material of the compound represented by formula (1). FIG. 2 is a DSC spectrum of the amorphous material of the compound represented by formula (1). FIG. 3 is a TGA spectrum of the amorphous material of the compound represented by formula (1). FIG. 4 is a PLM diagram of the amorphous material of the compound represented by formula (1). FIG. 5 is an SEM image of an amorphous material of the compound represented by formula (1). FIG. 6 shows the amorphous material of the compound represented by formula (1). 1 1H NMR spectrum. FIG. 7 is a DVS spectrum of the amorphous material of the compound represented by formula (1). FIG. 8 shows XRPD overlay spectra of an amorphous material of the compound of formula (1) before and after a DVS test. FIG. 9 is an XRPD spectrum of the crystalline form of the compound represented by formula (1). FIG. 10 is a TGA spectrum of the crystalline form of the compound of formula (1). FIG. 11 is a DSC spectrum of the crystalline form of the compound represented by formula (1). FIG. 12 is a DVS spectrum of the crystalline form of the compound represented by formula (1). FIG. 13 shows XRPD overlay spectra of the crystalline form of the compound of formula (1) before and after the DVS test. FIG. 14 shows the crystalline form of the compound of formula (1). 1 1H NMR spectrum. FIG. 15 is an XRPD overlay spectrum of the stability of the compound represented by formula (1) in an amorphous solid state. FIG. 16 is an XRPD overlay spectrum of the stability of the crystalline form of the compound of formula (1).

[0059] [Mode for Carrying Out the Invention] The following provides a more detailed description of the technical solutions of the present invention through specific examples. It should be understood that the following examples are merely for illustrative purposes and are not intended to limit the scope of protection of the present invention. Any technology realized based on the above content of the present invention is included within the intended scope of protection of the present invention.

[0060] Unless otherwise specified, all raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0061] Preparation Example Preparation of the compound represented by formula (1) (4R,11R)-7,7-dimethyl-4-(1-(methylsulfonyl)spiro[indoline-3,4'-piperidine]-1'-carbonyl)-6,9-dioxo-1-phenyl-2,10-dioxa-5,8-diazadodecan-11-yl isobutyrate [ka]

[0062] Step 1: Preparation of ethyl o-(1-chloroethyl)thiocarbonate (1b)

[0063] Compound 1-chloroethyl chloroformate 1a (3 g, 0.02 mol) was dissolved in dichloromethane (10 mL), followed by the addition of tetrabutylammonium bromide (TBAB) (0.34 g). Sodium ethanethiolate (1.76 g, 0.02 mol) dissolved in water (10 mL) was added dropwise to the reaction mixture, and the reaction mixture was stirred at 25° C. for 16 hours. The reaction mixture was separated, and the organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, and concentrated to give compound 1b (2.0 g, yield: 56%) as a yellow oil.

[0064] 1HNMR (400 MHz, CDCl3) δ 6.60 (q, J = 5.8 Hz, 1H), 2.97-2.86 (m, 2H), 1.81 (d, J = 5.8 Hz, 3H), 1.34 (t, J = 7.4 Hz, 3H).

[0065] Second step: Preparation of 1-{[(ethylthio)carbonyl]oxy}ethyl isobutyrate (1c)

[0066] Compound 1b (700 mg, 4.15 mmol) was dissolved in isobutyric acid (2.2 g, 25 mmol), N,N-diisopropylethylamine (1.6 g, 12.5 mmol) was added, and the reaction was stirred at 55° C. for 48 h. The reaction was quenched with water (20 mL), extracted with ethyl acetate (20 mL), washed with saturated sodium bicarbonate (3 × 30 mL), washed with saturated brine (2 × 20 mL), and concentrated to give residue 1c (850 mg, pale yellow oil).

[0067] 1 HNMR(400MHz, CDCl3):δ 6.94 (q, J=5.4Hz, 1H), 2.92-2.83 (m, 2H), 2.59(dt, J=4.3, 2.4Hz, 1H), 1.50(d, J=5.5Hz, 3H),1.32(td, J=7.3, 3.6Hz, 3H), 1.20(d, J=7.0Hz, 6H).

[0068] Step 3: Preparation of ethyl 1-(chlorocarbonyl)oxyisobutyrate (1d)

[0069] Sulfonyl chloride (147 mg, 1.09 mmol) was slowly added dropwise to compound 1c (200 mg, 0.91 mmol) at 0-5 °C, and the reaction mixture was stirred at 25 °C for 45 min. The reaction mixture was concentrated to give the residue 1d, which could be used directly in the next step.

[0070] Fourth step: Preparation of (4R)-7,7-dimethyl-4-(1-(methylsulfonyl)spiro[indoline-3,4'-piperidine]-1'-carbonyl)-6,9-dioxo-1-phenyl-2,10-dioxa-5,8-diazadodecan-11-yl isobutyrate (1e)

[0071] Compound 1d (60 mg, 0.11 mmol) and ibumolene were dissolved in dichloromethane (3 mL), and then sodium hydroxide (22 mg, 0.22 mmol) was dissolved in 5 mL of water and slowly added dropwise to the reaction solution, followed by stirring at 25° C. for 2 hours. The organic layer was concentrated, and the resulting residue was purified by preparative chromatography (acetonitrile / water) to give compound 1e (58 mg, white solid) in a 72% yield.

[0072] MS m / z (ESI): 687.0 [M+1] + .

[0073] 1 HNMR(400MHz, MeOD) δ 7.79-7.59(m, 1H), 7.42-7.26(m, 6H), 7.25-7.16(m, 1H), 6.99-6.90(m, 1H), 6.81-6.66(m, 1H), 5.18-5.11(m, 1H), 4.59-4.49(m, 2H), 4.18-3.97(m, 1H), 3.99-3.84(m, 2H), 3.81-3.63(m, 2H), 3.26-3.16(m, 2H), 2.96(d,J=6.4Hz, 3H), 2.87-2.8 (m, 1H), 2.52-2.50(m, 1H), 2.06-1.55(m, 4H), 1.51-1.31(m, 9H), 1.11-1.02(m, 6H).

[0074] Fifth step The compound 1e prepared above was subjected to optical resolution to obtain the compound of formula (1). The separation conditions were as follows: Chromatography column: Daicel CHIRALPAK IC_3, 3.0*150mm, 3μm, mobile phase: A / B: CO2 / MeOH=60 / 40, flow rate: 1.5mL / min, column temperature: 37℃.

[0075] t R =1.582min

[0076] MS m / z (ESI): 687.0 [M+1] + .

[0077] 1 HNMR (300 MHz, dmso) δ 7.80 - 7.49 (m, 2H), 7.42 - 7.15 (m, 8H), 7.08 - 6.85 (m, 2H), 6.67 - 6.56 (m, 1H), 4.97 (d, J = 7.2 Hz, 1H), 4.44 (dd, J = 30.4, 12.1 Hz, 3H), 3.90 (d, J = 6.8 Hz, 3H), 3.72 - 3.45 (m, 2H), 3.15 (s, 1H), 3.04 (d, J = 2.2 Hz, 3H), 2.80 (s, 1H), 1.66 (s, 4H), 1.45 - 1.28 (m, 9H), 1.06 (d, J = 6.9 Hz, 6H).

[0078] An Agilent 1200 Infinity Series mass spectrometer was used for liquid phase chromatography (LC-MS) measurements.

[0079] Example 1 Amorphous Material 1.Analysis method 1.1 X-ray powder diffraction (XRPD)

[0080] The crystal form of the sample was analyzed using an X-ray powder diffractometer. The 2θ scanning angle of the sample was 3° to 42°, the scanning step width was 0.02°, and the scanning time for each step was 0.2 s. The voltage and current of the light pipe were 40 kV and 40 mA, respectively. When preparing the sample, an appropriate amount of sample was placed on a sample tray and flattened with a tool such as a spoon or glass sheet to ensure that the surface was smooth and flat.

[0081] 1.2 Thermogravimetric analysis (TGA) Samples were analyzed using a TA Instruments TGA Discovery 550. Samples were placed in a tared aluminum tray and automatically weighed by the system, and then the sample was ramped from room temperature to the specified temperature at a rate of 10°C / min under nitrogen protection.

[0082] 1.3 Differential Scanning Calorimetry (DSC) Samples are analyzed using a TA Instruments Discovery DSC 25. A weighed sample is placed in a sample tray and the sample is ramped from 25°C to the specified temperature at a rate of 10°C / min under nitrogen (50 ml / min) protection.

[0083] 1.4 Dynamic Water Sorption Analysis (DVS) The samples were analyzed using an Intrinsic DVS (System Measurement System UK). The sample weight was approximately 20-30 mg. The temperature of the measurement chamber was controlled at 25±1°C, and the relative humidity was increased from 0% to 90% at a rate of 10% / h and then decreased to 0%, with mass data recorded every 20 seconds.

[0084] 1.5 Scanning Electron Microscope (SEM) The sample was analyzed using Phenom pure+. After gold deposition, the sample was placed in the instrument for testing. The crystal habits of the sample were obtained by adjusting the magnification.

[0085] 1.6 Polarized Light Microscope (PLM) The samples are analyzed using a polarized light microscope and adjusted to different magnifications to obtain the morphology and microstructure of the crystals.

[0086] 1.7 Nuclear magnetic analysis ( 1 HNMR) Samples were analyzed using a Varian Inova 500 MHz nuclear magnetic analyzer.

[0087] 1.8 Chromatography (HPLC) Chromatography conditions: Chromatography column: C18 column; Holding time: 60min; UV detector: 210 nm; Flow rate: 1.0mL / min; Injection volume: 5 μL.

[0088] 2. Manufacturing method 10 g of the compound raw material represented by formula (1) is placed in a 100 ml glass bottle, and an appropriate amount of DMSO is added at room temperature to completely dissolve it. The solution is then added dropwise to 100 ml of water and stirred at room temperature to precipitate a solid, which is then filtered by suction to obtain the solid.

[0089] 3. Characterization of Structural Properties The resulting solid was detected, and the XRPD results (FIG. 1) showed that the resulting solid was an amorphous substance of the compound represented by formula (1). 1 The HNMR spectrum is shown in Figure 6. The DSC curve (Figure 2) indicates that the glass transition temperature of the amorphous material is about 52.69°C, and the TGA curve (Figure 3) indicates that the amorphous material has a weight loss of about 0.248% by 100°C. PLM (Figure 4) and SEM (Figure 5) indicate that the amorphous material is bulk amorphous. The DVS (Figure 7) results indicate that the amorphous material is slightly hygroscopic under 80% humidity conditions, and the morphology of the solid did not change before and after the DVS test (Figure 8).

[0090] 4.Solubility test Approximately 10 mg of amorphous material was weighed into an 8 mL glass bottle and solvent was gradually added at room temperature. Add 5 μL of solvent each time until the solid was completely dissolved. If no dissolution occurred after 8 mL, stop adding solvent. The specific experimental results are shown in Table 1.

[0091] [Table 1]

[0092] As can be seen from the above table, the amorphous substance of the compound represented by formula (1) has low solubility in water, n-dioxane, n-hexane and cyclohexane, but high solubility in all other solvents.

[0093] Example 2 Crystalline Form

[0094] 1.Analysis method 1.1 X-ray powder diffraction (XRPD) XRPD spectra were collected on a PANalytical X-ray powder diffractometer, and the scanning parameters were as follows: [Table 2]

[0095] 1.2 Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC) TGA and DSC spectra were collected on a TA Discovery TGA 5500 thermogravimetric analyzer and a TA Discovery DSC 2500 differential scanning calorimeter, respectively, with the following test parameters: [Table 3]

[0096] 1.3 Dynamic Water Sorption (DVS) DVS curves were collected using an SMS (Surface Measurement Systems) DVS Intrinsic. The relative humidity at 25°C was calibrated based on the deliquescence points of LiCl, Mg(NO3)2, and KCl. The DVS test parameters were as follows: [Table 4]

[0097] 1.4 Solution Nuclear Magnetic Resonance (Solution NMR) Solution nuclear magnetic resonance spectra were collected on a Bruker 400M nuclear magnetic resonance instrument (Jiangsu Jixi Photoelectric Testing Center Co., Ltd.), and DMSO-d6 was used as the solvent for the nuclear magnetic resonance studies.

[0098] 2. Manufacturing method Approximately 15 mg of the raw material solid of the compound represented by formula (1) is placed in a glass bottle, and an appropriate amount of a mixed solvent of acetone and water is added to the glass bottle, followed by suspension and stirring at room temperature, and then suction filtration to obtain a solid.

[0099] 3. Characterization of Structural Properties The solid sample prepared by the above method was subjected to XRPD testing (shown in Figure 9), and the results indicated a crystalline form. The TGA curve (Figure 10) showed that the crystals had a weight loss of 1.1% when heated to 150°C, and the DSC curve (Figure 11) showed an endothermic peak at 130.6°C (peak temperature). Based on the small TGA weight loss and the absence of a DSC signal up to 100°C, the crystals were presumed to be anhydrous. The crystalline form was also subjected to DVS testing at a constant temperature of 25°C to evaluate its hygroscopicity. The DVS results are shown in Figure 12. The water adsorption of the crystalline form at 25°C / 80% RH was 0.10%, indicating that the crystalline form has almost no hygroscopicity. As shown in the XRPD results (Figure 13), the crystalline form did not change in shape before and after the DVS testing.

[0100] 3.1 X-ray powder diffraction (XRPD) The crystal form of the sample was analyzed using an X-ray powder diffractometer. When preparing the sample, an appropriate amount of sample was placed on a sample tray and flattened with a tool such as a spoon or glass sheet to ensure that the surface was smooth and flat. The results are shown in Table 2 and Figure 9.

[0101] [Table 5] JPEG2025534100000011.jpg121169

[0102] 4.Solubility test Approximately 5 mg of crystalline sample was weighed into an 8 mL glass bottle and solvent was gradually added at room temperature. 10 μL of solvent was added each time until the solid was completely dissolved. If the solid was not dissolved after 5 mL, the addition of solvent was stopped. The specific experimental results are shown in Table 3.

[0103] [Table 6]

[0104] As shown in Table 3, the crystalline form has low solubility in water, n-dioxane, and methyl tert-butyl ether, but high solubility in all other solvents.

[0105] Example 3 Stability Test 1 A set amount of crystalline and amorphous raw material was weighed and placed into five chromatography vials. They were then placed at 80°C (open), 25°C / 60% RH (open), and 40°C / 75% RH (open), as well as in a sealed light-stability box (5000±500 Lx). The stability was measured by chromatography at 80°C for one day, 10 days under light, and one week under 25°C, 60% RH, and 40°C, 75% RH. XRPD analysis of the solids was also performed. The specific results are shown in Table 4. The results show that both the amorphous and crystalline forms of the compound of formula (1) exhibit good physical and chemical stability under most conditions, but the stability of the crystalline form is significantly superior to that of the amorphous material under light irradiation.

[0106] [Table 7]

[0107] Accelerated Stability Testing:

[0108] Four grams of crystalline samples were weighed and placed in a constant temperature and humidity chamber at a temperature of 40±2°C and a relative humidity of 75±5%. Samples were taken out and analyzed at 0, 1, and 3 months. Chromatography was used to determine the sample stability, and XRPD was used to determine the crystalline form. The specific results are shown in Table 5. The results demonstrate that the crystalline form of the compound of formula (1) remains stable even under accelerated conditions.

[0109] [Table 8]

[0110] Example 4 Stability Test 2 Packaging requirements: The stability samples must be made to simulate commercially available packaging. The inner two layers are medicinal polyethylene flat-bottom liner bags. The first liner bag is sealed with a cable tie, the second liner bag is heat-sealed, and the outer aluminum foil bag is heat-sealed. A desiccant is placed between the aluminum foil bag and the second liner bag, and the sample is then placed in a cardboard barrel.

[0111] 1.1 Accelerated stability testing The crystalline form of the compound of formula (1) is taken and packaged according to the above packaging requirements. Typically, the sample is divided into five 10g packets and observed in a constant temperature and humidity box at a temperature of 40±2°C and a relative humidity of 75±5%. Samples are taken and analyzed at 0, 1, 2, 3, and 6 months. See Table 6 for the detection items, detection methods, and technical requirements, and the detection results are compared with those at month 0.

[0112] 1.2 Long-term stability testing The crystalline form of the compound of formula (1) is taken and packaged according to the above packaging requirements. Typically, the sample is divided into ten 10g packets and observed in a constant temperature and humidity box at a temperature of 30±2°C and a relative humidity of 65±5%. Samples are taken and analyzed at 0, 3, and 6 months. The detection items, detection methods, and technical requirements are listed in Table 7, and the detection results are compared with those at 0 months.

[0113] [Table 9]

[0114] Note: Data for month 0 is taken from published results, ND indicates not detected.

[0115] [Table 10]

[0116] Note: Data for month 0 is taken from published results; ND indicates not detected.

[0117] The results of the accelerated stability test showed that there was no significant change in the detection results between 6 months and 0 months, and no new impurity peaks exceeding 0.10% were observed, indicating that the crystalline form of the compound of formula (1) is stable under accelerated conditions for 6 months using the conventional packaging method described above.

[0118] The results of the long-term stability test showed that there was no significant change in the detection results between 6 months and 0 months, and no new impurity peaks exceeding 0.10% were observed, indicating that the crystalline form of the compound of formula (1) is stable for 6 months under long-term conditions using the conventional packaging method described above.

[0119] Example 4 By carrying out the method in Example 2 on a larger scale, a 10 kg solid sample was produced, and the results of XRPD testing showed the crystalline form shown in FIG.

[0120] Example 5 Biological Activity Test Test Example 1: Measurement of human GHSR activity of the compounds of the present invention

[0121] This method is used to measure the agonistic effect of the compounds of the present invention on the activity of human GHSR protein expressed in human GHSR / CHO stably transformed cells.

[0122] 1. Test materials and equipment 1. Culture medium F12 (Gibco, Cat#11765-047); FBS (Corning, Cat#35-076-CV), Geneticin (Invitrogen, Cat# 10131), Penicillin / Streptomycin (Invitrogen, Cat# 15140).

[0123] 2. Reagents Fluo-4 Direct (Invitrogen, Cat# F10471); HBSS (Gibco, Cat#14025076), HEPES (Gibco, Cat#15630080), Bonine Serum Albumin (Sgima, Cat#B2064-100G).

[0124] 3.Equipment consumables 384 well Poly-D-Lysine protein coating plate (Greiner, Cat#781946); FLIPR (Molecular Devices), Vi-cell XR Cell Viability Analyzer (Beckman Coulter), Incubator (Thermo).

[0125] 2. Experimental steps Compound gradient formulation: Ghrelin and compounds of the present invention were prepared in 10 concentration gradients at 5-fold dilutions, then transferred to the compound plate at 900 nL / well.

[0126] Preparation of buffer: HBSS (1X):HEPES (1M) = 49:1, add 0.5% BSA.

[0127] Stably transformed cells containing human GHSR / CHO were seeded into a 384-well plate. After overnight incubation, the cell plate was removed, the medium was discarded, and 20 μL of buffer solution was slowly added to each well. Then, 20 μL of 2X Fluo-4 Direct was added to each well. TM Add No-wash Loading Buffer. Place the cell plate in a 37°C, 5% CO2 incubator and incubate for 50 minutes. Remove the cell plate and leave it at room temperature for 10 minutes. Add 30uL of buffer to each well of the compound plate. Prepare another buffer plate and add 30μL of buffer to each well. Test the compound for agonist activity using the FLIPR instrument and run the software. Transfer 10μL of buffer to the cell plate and read the fluorescence signal value. Transfer 10μL of compound to the cell plate and read the fluorescence signal value. Use the FLIPR program to calculate the maximum-minimum value from the 91st signal point to the 230th signal point. EC 50 The value of can be calculated using the fluorescence values ​​corresponding to different concentrations via software.

[0128] 3. Experimental results: The agonist activity of compound 1e against human GHSR was measured by the above experiment, and the obtained EC 50 The value of 14.6 nM indicates that compound 1e has good agonistic activity against human GHSR. This confirms that the compound of formula (1) has good agonistic activity against human GHSR.

[0129] Test Example 2: Caco-2 cell transport experiment The transport buffer used in this study was HBSS containing 10.0 mM HEPSS, pH 7.40 ± 0.05. Bidirectional testing was performed on the target compounds at 2.00 μM, with a final DMSO concentration requirement of less than 1%. The cell plates were incubated for 2 hours in a CO2 incubator at 37 ± 1°C under 5% CO2 and saturated humidity. All samples were mixed with acetonitrile containing an internal standard and then centrifuged at 3200 x g for 10 minutes. Test compounds were analyzed by LC-MS / MS after diluting 100 μL of the supernatant with 100 μL of ultrapure water. Analyte / internal standard peak area ratios were used to quantify the concentrations of test and control compounds (digoxin as a model validation compound and ibutamoren as a positive control) in the starting, donor, and receiver solutions. Following transport measurements, the integrity of the Caco-2 cell monolayer was determined using a luciferin yellow rejection assay.

[0130] [Table 11]

[0131] As shown by the above data, the model construction was successful and the cell permeability of compound 1e is significantly superior to that of the reference compound ibutamoren.

[0132] Test Example 3: Comparison of metabolism of compound 1e in rats

[0133] Compound 1e is designed as a prodrug, and the amount of the active ingredient ibutamoren is measured through a rat in vivo metabolism experiment, thereby evaluating the relative merits of the candidate and the positive control ibutamoren.

[0134] Experimental Procedure:

[0135] The six animals underwent a first fasting period two days before administration, followed by at least 12 hours of fasting before being uniformly fed. The animals were fasted for a second period the day before administration, followed by at least 12 hours of fasting, with feeding resumed four hours after administration. Each fasting period did not exceed 20 hours. Water was allowed ad libitum throughout the period. For the two days prior to administration, the same person trained the animals by touching and handling them at least once a day.

[0136] Before the first administration, the animals were divided into two groups based on their body weight. Each group consisted of three animals. Group 1 received a single intragastric administration of Compound 5 (preparation: medium-chain triglyceride / polyethylene glycol 1000 vitamin E succinate / ethanol / propylene glycol / water = 6 / 2 / 1 / 1 / 90, 1 mg / mL). Group 2 received a single intragastric administration of ibutamoren (water, 1 mg / mL). The administration volume was 3 mL / kg. The animals were weighed before administration, and the administration volume was calculated based on their body weight.

[0137] Sampling times were approximately -0.25 h before administration and 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 5, 7, and 10 h after administration. At each time point, animals were briefly anesthetized with isoflurane, and whole blood samples (approximately 0.23 mL per group) were collected by jugular vein puncture. 50 μL of the whole blood sample was quantified and added to an EP tube containing 50 μL of pre-chilled 1 mM PMSF in methanol, vortexed for ~3 s, immediately added with 250 μL of precipitant containing the internal standard, vortexed for ~5 s, and centrifuged for 15 min. The supernatant was collected and analyzed by LC-MS / MS.

[0138] [Table 12]

[0139] As shown by the above data, compound 1e of the present invention exhibited the following metabolic activity in rats after intragastric administration: max The compound of formula (1) has good drug discovery potential.

[0140] As can be seen from the above experiments, the crystalline and amorphous forms of the compounds prepared in the present invention have high purity and high stability under high temperature and humidity conditions, which is beneficial to the effects of the drugs. In terms of process optimization, the requirements for stable production process and repeatable controllability can be met, making them suitable for industrial production.

[0141] Although the embodiments of the present invention have been described above, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made without departing from the spirit and principles of the present invention should be included within the scope of the claims of the present invention. [Brief explanation of the drawings]

[0142] [Figure 1] 1 is an XRPD spectrum of an amorphous material of the compound represented by formula (1). [Figure 2] 1 is a DSC spectrum of an amorphous material of the compound represented by formula (1). [Figure 3] 1 is a TGA spectrum of an amorphous material of the compound represented by formula (1). [Figure 4] FIG. 1 is a PLM diagram of an amorphous material of the compound represented by formula (1). [Figure 5] FIG. 1 is an SEM image of an amorphous material of the compound represented by formula (1). [Figure 6] 1 is a 1H NMR spectrum of an amorphous material of the compound represented by formula (1). [Figure 7] 1 is a DVS spectrum of an amorphous material of the compound represented by formula (1). [Figure 8]1 shows XRPD overlay spectra of an amorphous material of the compound of formula (1) before and after a DVS test. [Figure 9] 1 is an XRPD spectrum of a crystalline form of the compound of formula (1). [Figure 10] 1 is a TGA spectrum of the crystalline form of the compound of formula (1). [Figure 11] 1 is a DSC spectrum of the crystalline form of the compound represented by formula (1). [Figure 12] 1 is a DVS spectrum of the crystalline form of the compound of formula (1). [Figure 13] 1 shows XRPD overlay spectra of the crystalline form of the compound of formula (1) before and after a DVS test. [Figure 14] 1 is a 1H NMR spectrum of the crystalline form of the compound of formula (1). [Figure 15] 1 is an XRPD overlay spectrum of the compound of formula (1) showing its stability in an amorphous solid state. [Figure 16] 1 is an XRPD overlay spectrum of the stability of the crystalline form of the compound of formula (1).

Claims

1. A crystalline form of the compound represented by formula (1), which has characteristic peaks at 5.43±0.20°, 12.28±0.20°, and 17.91±0.20° in X-ray powder diffraction expressed in degrees 2θ using Cu-Kα radiation. 【Chemical 1】

2. 2. The crystalline form of claim 1, wherein the crystalline form has characteristic peaks at 5.43±0.20°, 12.28±0.20°, 17.91±0.20°, 18.54±0.20°, 18.87±0.20°, 20.54±0.20°, and 21.69±0.20° in X-ray powder diffraction using Cu-Kα radiation and expressed in degrees 2θ.

3. 3. The crystalline form according to claim 1, wherein the crystalline form has characteristic peaks at 5.43±0.20°, 7.96±0.20°, 9.72±0.20°, 12.28±0.20°, 13.16±0.20°, 17.69±0.20°, 17.91±0.20°, 18.16±0.20°, 18.54±0.20°, 18.87±0.20°, 19.51±0.20°, 20.31±0.20°, 20.54±0.20°, 21.69±0.20°, and 21.86±0.20° in X-ray powder diffraction analysis using Cu-Kα radiation and expressed in degrees 2θ.

4. 4. A crystalline form according to any one of claims 1 to 3, having an XRPD spectrum substantially as shown in Figure 9, preferably anhydrous.

5. A method for producing the crystalline form according to any one of claims 1 to 4, comprising: The method comprises the steps of dissolving the compound of formula (1) in solvent A, cooling to precipitate a solid, and obtaining the crystalline form; The method for producing a crystalline form, wherein the solvent A is selected from the group consisting of methanol, ethanol, isopropanol, tert-butanol, n-butanol, acetone, tetrahydrofuran, methyltetrahydrofuran, ethyl formate, ethyl acetate, isopropyl acetate, n-hexane, n-dioxane, cyclohexane, methyl tert-butyl ether, toluene, dichloromethane, chloroform, DMSO, water, acetonitrile, isopropyl ether, and combinations of two or more of these solvents.

6. An amorphous material of the compound of formula (1), the XRPD spectrum of which has no significant diffraction peaks; 【Chemistry 2】 Preferably, the amorphous material has an XRPD spectrum substantially as shown in FIG.

7. 7. A method for producing an amorphous substance according to claim 6, comprising the steps of dissolving a compound represented by formula (1) in solvent B, adding the resulting solution to solvent C, and stirring to precipitate a solid to obtain the amorphous substance, A method for producing an amorphous substance, wherein the solvent B is a good solvent for the compound represented by formula (1), and the solvent C is a poor solvent for the compound represented by formula (1).

8. A pharmaceutical composition comprising the crystalline form of any one of claims 1 to 4 and / or the amorphous substance of claim 6, Preferably, it further contains pharmaceutically acceptable auxiliary materials, Preferably, it further comprises a second active ingredient other than the crystalline and / or amorphous material, such as, for example, a drug related to growth or development, Preferably, the pharmaceutical composition is a formulation of, for example, a GHSR agonist.

9. Use of the crystalline form of any one of claims 1 to 4, the amorphous substance of claim 6 and / or the pharmaceutical composition of claim 8 in the manufacture of a preparation for the diagnosis, prevention and / or treatment of growth hormone deficiency or growth hormone dependent diseases (or disorders).

10. A method for diagnosing, preventing and / or treating a growth hormone deficiency or growth hormone dependent disease (or disorder) comprising administering to a patient a therapeutically effective amount of the crystalline form of any one of claims 1 to 4, the amorphous material of claim 6 or the pharmaceutical composition of claim 8.

Images