Crystal form and amorphous substance of indoline spiro compound and preparation method and application of crystal form and amorphous substance of indoline spiro compound

Abstract

# imgabs0 # provides a crystal form and an amorphous substance of an indoline spiro compound as shown in a formula (1), and preparation methods and applications of the crystal form and the amorphous substance of the indoline spiro compound as well as the crystal form and the amorphous substance of the indoline spiro compound. The crystal form is radiated by Cu-K alpha, and X-ray powder diffraction expressed by a 2 theta angle has characteristic peaks at 5.43 + / -0.20 degrees, 12.28 + / -0.20 degrees and 17.91 + / -0.20 degrees; the XRPD pattern of the amorphous substance has no obvious diffraction peak; the crystal form and the amorphous substance provided by the invention have high purity and good stability, and are beneficial to medicine formation.

Description

Crystal form and amorphous form of indoline spirocyclic compound and preparation methods and applications thereof

[0001] This application claims priority to the following two prior applications: Patent application No. 202211297102.5, filed with the State Intellectual Property Office of China on October 21, 2022, entitled “Crystalline Spirocyclic Compounds, Amorphous Forms, and Preparation Methods and Applications of the Same”; and Patent application No. 202311300559.1, filed with the State Intellectual Property Office of China on October 9, 2023, entitled “Crystalline Spirocyclic Compounds, Amorphous Forms, and Preparation Methods and Applications of the Same”. The entire text of each of the prior applications is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of pharmaceutical compounds, and in particular relates to a crystalline form and an amorphous form of an indoline spirocyclic compound, and preparation methods and applications of the crystalline form and amorphous form of the indoline spirocyclic compound. Background Art

[0003] Human growth hormone (GH) is a peptide hormone secreted by the anterior pituitary gland. It is composed 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 the linear growth of the body, promoting muscle and skin cell proliferation, and playing an important role in the regeneration of tissues after trauma.

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

[0005] The release of GH is believed to treat physiological or pathophysiological conditions characterized by a deficiency in growth hormone secretion, as well as conditions that are ameliorated by the anabolic effects of growth hormone. GH has been shown to be promising in treating conditions such as loss of muscle mass, accumulation of adipose tissue, bone demineralization, and reduced tissue regeneration after injury.

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

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

[0008] Studies have found that some clinical peptide or peptidomimetic compounds exhibit GHSR agonist activity and have the ability to induce GH release. Compounds currently in clinical development include examorelin, tabimorelin, pralmorelin, ibutamoren, tesamorelin, anamorelin, and macimorelin. Among these, the injectable peptide tesamorelin (used to reduce excess abdominal fat in HIV-infected individuals) and the small molecule peptidomimetic macimorelin have been approved for marketing by the FDA. Macimorelin is the only oral drug approved for the diagnosis of growth hormone deficiency in adults, but macimorelin also has drawbacks such as poor oral bioavailability and potential risk of cardiotoxicity.

[0009] Related studies have also found that GHSR agonists, in addition to inducing GH secretion through GHSR 1a activation, also mediate other physiological functions through different receptors in the GHS receptor family or different binding sites on the GHSR (such as GHSR 1b, motilin receptor 1a, neurotensin receptor, and TRH receptor). Therefore, the application of GHSR agonists in the field of gastrointestinal indications has been newly developed. Currently, there are no drugs available for this indication. Among them, ulimorelin and relamorelin have entered Phase III clinical trials.

[0010] Ghrelin has been shown to have a prokinetic effect on gastrointestinal motility through the vagus nerve and pelvic nerve, but the short half-life of ghrelin hinders its drugability. It is necessary to develop GHSR agonists with enhanced pharmacokinetics to improve impaired gastrointestinal function in animals and humans.

[0011] In order to overcome the above technical problems, the applicant independently developed a compound with a completely new molecular structure, the structural formula of which is: The chemical name is: (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-diazadodec-11-yl isobutyrate, and the relevant content is recorded in patent application PCT / CN2022 / 088656. Pharmacodynamic tests show that the compound has good clinical application prospects and can be used to prepare drugs for the diagnosis, prevention and / or treatment of growth hormone-dependent diseases or conditions; preferably, the diseases or conditions are related to growth hormone deficiency or growth hormone dependence, such as the diagnosis of patients with growth hormone deficiency, slow growth and short stature in children with growth hormone deficiency, and the treatment of other diseases that can be improved by the physiological effects of growth hormone, including but not limited to: energy balance and food intake regulation; treatment of fat formation, obesity and weight loss; treatment of cachexia; improvement of gastrointestinal motility, treatment of gastroparesis and diabetic gastroparesis, and postoperative intestinal obstruction; increase in muscle mass and skin thickness, reduction in fat material and slight increase in bone density in elderly patients; treatment of burns, AIDS and cancer conditions, and wound and bone healing.

[0012] At the same time, developing pharmaceutical solid forms of the above compounds suitable for drug formulation, such as solid forms with improved stability, hygroscopicity and / or efficacy, so as to achieve good results in the pharmaceutical preparation and use stages, has become a technical problem that needs to be solved urgently.

[0013] Summary of the Invention

[0014] All contents involved in patent application PCT / CN2022 / 088656 are added to the present invention by reference.

[0015] In order to improve the above technical problems, the present invention provides a crystalline form of a compound represented by formula (1),

[0016] The crystal form has characteristic peaks at 5.43±0.20°, 12.28±0.20°, and 17.91±0.20° in X-ray powder diffraction expressed in 2θ angles using Cu-Kα radiation.

[0017] According to an embodiment of the present invention, the crystalline form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles 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°.

[0018] According to an embodiment of the present invention, the crystalline form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles is 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° having characteristic peaks.

[0019] According to an embodiment of the present invention, the crystalline form has an XRPD pattern substantially as shown in FIG. 9 .

[0020] According to an embodiment of the present invention, the crystalline form is an anhydrate.

[0021] According to an embodiment of the present invention, the weight loss of the crystalline form before 150° C. is no more than 3 wt %, for example, no more than 2 wt %.

[0022] According to an embodiment of the present invention, the crystalline form has a TGA pattern substantially as shown in FIG10 .

[0023] According to an embodiment of the present invention, the crystal form has a sharp endothermic peak at a peak temperature of 130.6±2°C.

[0024] According to an embodiment of the present invention, the crystalline form has a DSC spectrum substantially as shown in FIG11 .

[0025] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: dissolving the compound represented by formula (1) in solvent A, cooling to precipitate a solid, and obtaining the crystal form;

[0026] The solvent A can be selected from one or more of methanol, ethanol, isopropanol, tert-butanol, n-butanol, acetone, tetrahydrofuran, methyltetrahydrofuran, ethyl formate, ethyl acetate, isopropyl acetate, n-hexane, n-heptane, cyclohexane, methyl tert-butyl ether, toluene, dichloromethane, chloroform, DMSO, water, acetonitrile, isopropyl ether, etc., for example, a mixed solvent selected from ethanol / water, DMSO / water, acetone / water, ethanol / n-hexane, ethanol / cyclohexane, acetonitrile / n-heptane, ethanol / n-heptane, isopropanol / n-heptane, methanol / n-heptane, methanol / n-hexane, and acetone / n-heptane.

[0027] According to an embodiment of the present invention, the dissolving is performed under heating conditions.

[0028] According to an embodiment of the present invention, the temperature is lowered to room temperature.

[0029] According to an embodiment of the present invention, the cooling is slow cooling.

[0030] According to an embodiment of the present invention, the method further comprises post-treatment of the solid, such as filtering and / or washing.

[0031] The present invention also provides an amorphous compound of the compound represented by formula (1), whose XRPD spectrum has no obvious diffraction peak.

[0032] According to an embodiment of the present invention, the amorphous material has an XRPD pattern substantially as shown in FIG1 .

[0033] The present invention also provides a method for preparing the above-mentioned amorphous material, comprising the following steps: dissolving the compound represented by formula (1) in solvent B, adding the resulting clear solution to solvent C, stirring to precipitate a solid, and obtaining the amorphous material;

[0034] The solvent B is a good solvent for the compound represented by formula (1), for example, one or more selected from DMF, DMA, NMP, acetonitrile, THF, DMSO, methyl tert-butyl ether, isopropyl ether, methanol, ethanol, isopropanol, acetone, etc.;

[0035] The solvent C is a poor solvent for the compound represented by formula (1), for example, one or more selected from water, n-heptane, n-hexane, and cyclohexane.

[0036] According to an embodiment of the present invention, the supernatant is added to the solvent C in a dropwise manner.

[0037] According to an embodiment of the present invention, the method further comprises post-treatment of the solid, such as filtering and / or washing.

[0038] The present invention also provides a pharmaceutical composition containing the above-mentioned crystal form and / or amorphous form.

[0039] According to an embodiment of the present invention, the pharmaceutical composition further contains pharmaceutically acceptable excipients, such as, but not limited to, one or more of excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents and flavoring agents.

[0040] According to an embodiment of the present invention, the pharmaceutical composition further comprises a second active ingredient in addition to the above-mentioned crystalline form and / or amorphous form, for example, the second active ingredient is a drug related to growth and development, for example, the second active ingredient is a GHSR agonist or growth hormone.

[0041] In some embodiments, the crystalline form and / or amorphous form and the second active ingredient can be administered separately or co-administered during treatment.

[0042] The present invention also provides the use of the above-mentioned crystal form, amorphous form and / or pharmaceutical composition in the preparation of preparations for diagnosing, preventing and / or treating growth hormone deficiency or growth hormone-dependent diseases (or conditions).

[0043] According to an embodiment of the present invention, the disease is, for example, the diagnosis of patients with growth hormone deficiency, slow growth and short stature in children with growth hormone deficiency, and other diseases that can be improved by the physiological effects of growth hormone, including but not limited to: energy balance and food intake regulation; treatment of adipogenesis, obesity and weight loss; treatment of cachexia; improvement of gastrointestinal motility, treatment of gastroparesis and diabetic gastroparesis, and postoperative intestinal obstruction; increase in muscle mass and skin thickness, reduction of fat material and slight increase in bone density in the elderly patient population; treatment of burns, AIDS and cancer conditions, and healing of wounds and bones.

[0044] In some embodiments, the agent may be a GHSR agonist.

[0045] The present invention also provides a preparation containing the above-mentioned crystal form and / or amorphous form, or prepared from the above-mentioned pharmaceutical composition.

[0046] According to an embodiment of the present invention, the preparation can be in the form of powder, tablet (such as coated tablet, sustained-release or controlled-release tablet), lozenge, capsule (such as soft capsule or hard capsule), granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.

[0047] According to an embodiment of the present invention, the preparation can be administered in any of the following ways: orally, buccal administration, sublingually, inhalation, topical application, parenteral administration, intravenous, subcutaneous, acupuncture point or intramuscular injection, and rectal administration.

[0048] The present invention also provides a method for diagnosing, preventing and / or treating growth hormone deficiency or growth hormone-dependent diseases (or conditions), comprising administering a therapeutically effective amount of the crystalline form, amorphous form or pharmaceutical composition to a patient.

[0049] According to an embodiment of the present invention, the disease has the definitions shown above.

[0050] Beneficial effects of the present invention

[0051] The present invention provides a crystalline form and an amorphous form of a compound represented by formula (1), as well as methods for preparing and using the same. The crystalline form and amorphous form of the compound represented by formula (1) obtained by the present invention have good stability and solubility, low hygroscopicity, long-term storage capability, good reproducibility, and are suitable for drug development.

[0052] The compound represented by formula (1) has high crystal purity, good crystal stability under conditions of light, high temperature and high humidity, low hygroscopicity, and can be stored for a long time, which is beneficial to drug development. Its preparation process is stable and reproducible, and can be adapted to industrial production.

[0053] The amorphous compound of the formula (1) prepared by the present invention has high purity, good solubility in most solvents, good physical and chemical stability under light, high temperature and high humidity conditions, and is suitable for drug development.

[0054] Definitions and Explanations of Terms

[0055] Unless otherwise indicated, the definitions of terms in this specification and claims, including definitions used as examples, exemplary definitions, preferred definitions, and definitions of specific compounds in the Examples, may be arbitrarily combined and coupled with each other. Such combinations and couplings shall fall within the scope of this specification.

[0056] The term "therapeutically effective amount" refers to an amount of the crystalline form, amorphous substance, or second active ingredient of the present invention sufficient to achieve the intended application (including but not limited to the treatment of a disease as defined below). The therapeutically effective amount may vary depending on the following factors: the intended application (in vitro or in vivo), or the subject and disease condition being treated, such as the weight and age of the subject, the severity of the disease condition, and the mode of administration, which can be easily determined by one of ordinary skill in the art. The specific dosage will vary depending on the following factors: the specific active ingredient selected, the dosage regimen used, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.

[0057] The term "patient" refers to any animal including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses or primates, and most preferably humans.

[0058] The term "130.6±2°C" represents 128.6-132.6°C, for example, 128.6°C, 129.0°C, 130.0°C, 131.0°C, 132.0°C, 132.6°C or a value between any two of the foregoing points. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is the XRPD pattern of the amorphous form of the compound represented by formula (1);

[0060] Figure 2 is a DSC spectrum of the amorphous compound represented by formula (1);

[0061] Figure 3 is the TGA spectrum of the amorphous compound represented by formula (1);

[0062] Figure 4 is a PLM diagram of the amorphous form of the compound represented by formula (1);

[0063] Figure 5 is a SEM image of the amorphous compound represented by formula (1);

[0064] Figure 6 shows the amorphous form of the compound represented by formula (1). 1 H NMR spectrum;

[0065] Figure 7 is a DVS spectrum of the amorphous form of the compound represented by formula (1);

[0066] FIG8 is an XRPD superposition pattern of the amorphous form of the compound represented by formula (1) before and after DVS testing;

[0067] Figure 9 is an XRPD pattern of the crystalline form of the compound represented by formula (1);

[0068] Figure 10 is a TGA spectrum of the crystal form of the compound represented by formula (1);

[0069] Figure 11 is a DSC spectrum of the crystalline form of the compound represented by formula (1);

[0070] Figure 12 is a DVS spectrum of the crystal form of the compound represented by formula (1);

[0071] Figure 13 is an XRPD overlay of the crystal form of the compound represented by formula (1) before and after DVS testing;

[0072] Figure 14 is a crystalline form of the compound represented by formula (1) 1 H NMR spectrum;

[0073] Figure 15 is an overlay of XRPD patterns of the solid stability of the amorphous form of the compound represented by formula (1);

[0074] FIG16 is an XRPD superposition spectrum of the crystal stability of the compound represented by formula (1). DETAILED DESCRIPTION

[0075] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0076] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0077] Preparation Example Preparation of the compound represented by formula (1)

[0078] (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-diazadodec-11-yl isobutyrate

[0079] The first step is the preparation of o-(1-chloroethyl) ethyl thiocarbonate (1b)

[0080] 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) was dissolved in water (10 mL) and added dropwise to the reaction mixture, which 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 afford compound 1b (2.0 g, yield: 56%) as a yellow oil.

[0081] 1 H NMR (400MHz, CDCl3) δ6.60 (q, J = 5.8Hz, 1H), 2.97-2.86 (m, 2H), 1.81 (d, J = 5.8Hz, 3H), 1.34 (t, J = 7.4Hz, 3H).

[0082] Step 2 Preparation of 1-{[(ethylthio)carbonyl]oxy}ethyl isobutyrate (1c)

[0083] Compound 1b (700 mg, 4.15 mmol) was dissolved in isobutyric acid (2.2 g, 25 mmol), and N,N-diisopropylethylamine (1.6 g, 12.5 mmol) was added. The reaction mixture was stirred at 55°C for 48 hours. The reaction mixture was quenched by addition of water (20 mL), extracted with ethyl acetate (20 mL), washed with saturated sodium bicarbonate (3 x 30 mL), and then washed with saturated brine (2 x 20 mL). The residue was concentrated to obtain 1c (850 mg, light yellow oil).

[0084] 1 HNMR (400MHz, CDCl3): δ6.94 (q, J=5.4Hz, 1H), 2.92–2.83 (m, 2H), 2.59 (dt, J=4.3, 2.4Hz,

[0085] 1H), 1.50 (d, J = 5.5Hz, 3H), 1.32 (td, J = 7.3, 3.6Hz, 3H), 1.20 (d, J = 7.0Hz, 6H).

[0086] Step 3 Preparation of ethyl 1-((chlorocarbonyl)oxyisobutyrate (1d)

[0087] 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 minutes. The reaction mixture was concentrated to obtain the residue 1d, which was used directly in the next step.

[0088] Step 4 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-diazadodec-11-yl isobutyrate (1e)

[0089] Compound 1d (60 mg, 0.11 mmol) and ibumolen were dissolved in dichloromethane (3 mL). Sodium hydroxide (22 mg, 0.22 mmol) was dissolved in 5 mL of water and slowly added dropwise to the reaction mixture. The reaction mixture was stirred at 25°C for 2 hours. The organic layer was concentrated to obtain a residue which was purified by preparative chromatography (acetonitrile / water) to afford compound 1e (58 mg, white solid) in a 72% yield.

[0090] MS m / z(ESI):687.0[M+1] + .

[0091] 1HNMR(400MHz,MeOD)δ7.79–7.59(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).

[0092] Step 5

[0093] The compound 1e prepared above was subjected to chiral separation to obtain the compound of formula (1). Separation conditions: chromatographic 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°C.

[0094] t R =1.582min

[0095] MS m / z(ESI):687.0[M+1] + .

[0096] 1 H NMR(300MHz,dmso)δ7.80–7.49(m,2H),7.42–7.15(m,8H),7.08–6.85(m,2 H),6.67–6.56(m,1H),4.97(d,J=7.2Hz,1H),4.44(dd,J=30.4,12.1Hz,3H ),3.90(d,J=6.8Hz,3H),3.72–3.45(m,2H),3.15(s,1H),3.04(d,J=2.2Hz ,3H),2.80(s,1H),1.66(s,4H),1.45–1.28(m,9H),1.06(d,J=6.9Hz,6H).

[0097] Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1200 Infinity Series mass spectrometer.

[0098] Example 1 Amorphous

[0099] 1. Analytical methods

[0100] 1.1 X-ray powder diffraction (XRPD)

[0101] Crystalline analysis of the sample was performed using an X-ray powder diffractometer. The sample was scanned at a 2θ angle of 3° to 42°, with a scan step size of 0.02° and a scan time of 0.2 s per step. The light tube voltage and current were 40 kV and 40 mA, respectively. During sample preparation, an appropriate amount of sample was placed on a sample tray and flattened with a spoon or glass slide to ensure a smooth surface.

[0102] 1.2 Thermogravimetric analysis (TGA)

[0103] The samples were analyzed using a TA Instruments TGA Discovery 550. The samples were placed in a tared aluminum pan, weighed automatically by the system, and then heated from room temperature to the specified temperature at a rate of 10°C / min under nitrogen.

[0104] 1.3 Differential Scanning Calorimetry (DSC)

[0105] The samples were analyzed using a TA Instruments Discovery DSC 25. The weighed samples were placed in a sample tray, and under the protection of nitrogen (50 ml / min), the sample was heated from 25°C to the specified temperature at a rate of 10°C / min.

[0106] 1.4 Dynamic Water Sorption Analysis (DVS)

[0107] Samples were analyzed using an Intrinsic DVS (System Measurement System UK). The test sample size was approximately 20-30 mg. The test chamber temperature was controlled at 25 ± 1°C, and the relative humidity was increased from 0% to 90% and then decreased to 0% at a rate of 10% / h. Mass data were recorded every 20 seconds.

[0108] 1.5 Scanning electron microscopy (SEM)

[0109] The sample was analyzed using the Phenom pure+. After being gold-sprayed, the sample was placed in the instrument for testing. Different magnification factors were used to capture the sample's crystal habit.

[0110] 1.6 Polarized Light Microscopy (PLM)

[0111] The samples were analyzed using a polarizing microscope, and the morphology and microstructure of the crystals were obtained by adjusting different magnifications.

[0112] 1.7 NMR analysis ( 1 H NMR)

[0113] The samples were analyzed using a Varian Inova 500 MHz nuclear magnetic resonance analyzer.

[0114] 1.8 Liquid phase method (HPLC)

[0115] Chromatographic conditions:

[0116] Chromatographic column: C18 column;

[0117] Run time: 60 minutes;

[0118] UV detector: 210nm;

[0119] Flow rate: 1.0 mL / min;

[0120] Injection volume: 5 μL.

[0121] 2. Preparation Method

[0122] Weigh 10 g of the compound represented by formula (1) into a 100 ml glass bottle, add an appropriate amount of DMSO at room temperature until it is completely dissolved, add the solution dropwise into 100 ml of water, stir at room temperature to precipitate solid, and then filter to obtain the solid.

[0123] 3. Characterization of structural properties

[0124] The obtained solid was tested, and the XRPD results (Figure 1) showed that the obtained solid was an amorphous substance of the compound represented by formula (1). 1 The H NMR spectrum is shown in Figure 6. The DSC curve (Figure 2) shows that the glass transition temperature of the amorphous material is approximately 52.69°C, and the TGA curve (Figure 3) shows that the amorphous material has a weight loss of approximately 0.248% before 100°C. PLM (Figure 4) and SEM (Figure 5) show that the amorphous material is a bulky non-crystalline solid. DVS (Figure 7) results show that the amorphous material is slightly hygroscopic at 80% humidity, and the morphology of the solid remains unchanged before and after DVS testing (Figure 8).

[0125] 4. Solubility test

[0126] Approximately 10 mg of amorphous material was weighed into an 8 mL glass vial. Solvent was gradually added at room temperature. 5 μL of solvent was added at a time until the solid was completely dissolved. If dissolution was not achieved after adding 8 mL, the solvent addition was stopped. The specific experimental results are shown in Table 1.

[0127] Table 1. Solubility test results of amorphous compound represented by formula (1) (room temperature)

[0128] As can be seen from the above table, the amorphous compound represented by formula (1) has relatively poor solubility in water, n-heptane, n-hexane and cyclohexane, but has relatively high solubility in other solvents.

[0129] Example 2 Crystal form

[0130] 1. Analytical methods

[0131] 1.1 X-ray powder diffraction (XRPD)

[0132] The XRPD patterns were collected on a PANalytical X-ray powder diffraction analyzer with the following scanning parameters:

[0133] 1.2 Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)

[0134] TGA and DSC patterns were collected on a TA Discovery TGA 5500 thermogravimetric analyzer and a TA Discovery DSC 2500 differential scanning calorimeter, respectively. The test parameters were as follows:

[0135] 1.3 Dynamic Water Sorption (DVS)

[0136] DVS curves were acquired on a DVS Intrinsic instrument from SMS (Surface Measurement Systems). Relative humidity at 25°C was calibrated based on the deliquescent points of LiCl, Mg(NO3)2, and KCl. DVS test parameters are as follows:

[0137] 1.4 Solution NMR

[0138] Liquid-state NMR spectra were collected on a Bruker 400M NMR instrument (Jiangsu Jicui Optoelectronics Testing Center Co., Ltd.), and DMSO-d6 was used as the NMR test solvent.

[0139] 2. Preparation Method

[0140] About 15 mg of the raw material solid of the compound represented by formula (1) was placed in a glass bottle, and an appropriate amount of a mixed solvent of acetone and water was added to the glass bottle for suspension and stirring at room temperature, and then filtered to obtain a solid.

[0141] 3. Characterization of structural properties

[0142] The solid sample prepared by the above method was subjected to XRPD testing (as shown in Figure 9), and the results showed that it was in crystalline form. The TGA curve (Figure 10) showed that the crystal had a weight loss of 1.1% when heated to 150°C, and the DSC curve (Figure 11) showed that an endothermic peak was observed at 130.6°C (peak temperature). Based on the smaller TGA weight loss of this crystal form and the absence of a DSC signal before 100°C, it is speculated that this crystal form is an anhydrous crystal form. In addition, the crystal form was subjected to a DVS test at a constant temperature of 25°C to evaluate its hygroscopicity. The DVS results are shown in Figure 12. The moisture adsorption of this crystal form under 25°C / 80%RH conditions was 0.10%, indicating that this crystal form has almost no hygroscopicity. The XRPD results (Figure 13) show that this crystal form did not undergo any crystalline change before and after the DVS test.

[0143] 3.1 X-ray powder diffraction (XRPD)

[0144] The crystal form of the sample was analyzed using an X-ray powder diffractometer. To prepare the sample, place an appropriate amount onto a sample tray and flatten it with a spoon or glass slide to ensure a smooth surface. The results are shown in Table 2 and Figure 9.

[0145] Table 2. XRPD diffraction peak data of the crystal form of the compound represented by formula (1)

[0146] 4. Solubility test

[0147] Approximately 5 mg of the crystalline form sample was weighed into an 8 mL glass vial and solvent was gradually added at room temperature. Solvent was added in 10 μL increments until the solid was completely dissolved. If dissolution was not achieved after adding 5 mL, the solvent addition was stopped. Specific experimental results are shown in Table 3.

[0148] Table 3. Solubility test results of the crystal form of the compound represented by formula (1) (room temperature)

[0149] As shown in Table 3, the solubility of this crystal form in water, n-heptane, and methyl tert-butyl ether is relatively poor, but it has high solubility in other solvents.

[0150] Example 3 Stability Test 1

[0151] A certain amount of crystal and amorphous raw materials were weighed into liquid phase vials, and a total of 5 portions were prepared. They were placed in 80°C (open), 25°C / 60% RH (open), and 40°C / 75% RH (open) and in a light stability chamber (closed, 5000±500Lx). The stability at 80°C for one day and light for 10 days, as well as the chemical stability at 25°C, 60% RH and 40°C, 75% RH for one week, were determined by liquid phase chromatography, and the XRPD of the solid was measured. The specific results are shown in Table 4. The results show that the amorphous and crystalline forms of the compound represented by formula (1) have good physical and chemical stability under most conditions, but under light conditions, the stability of the crystalline form is significantly better than that of the amorphous form.

[0152] Table 4. Stability test results of amorphous and crystalline forms of the compound represented by formula (1)

[0153] Accelerated stability test:

[0154] Crystal samples, 4 g each, were weighed and observed in a constant temperature and humidity chamber at 40 ± 2°C and 75 ± 5% relative humidity. Samples were collected and analyzed at 0, 1, and 3 months. Liquid chromatography was used to determine the stability of the samples, and XRPD was used to determine the crystal form. The results are shown in Table 5. These results demonstrate that the crystal form of the compound represented by formula (1) maintains good stability under accelerated conditions.

[0155] Table 5. Accelerated stability test results of the crystalline form of the compound represented by formula (1)

[0156] Example 4 Stability Test 2

[0157] Packaging requirements: Stability samples must simulate commercial packaging. The inner two layers are medicinal polyethylene flat-bottom inner bags. The first inner bag is sealed with a tie, the second inner bag is heat-sealed, and the outer layer is heat-sealed with an aluminum foil bag. A bag of desiccant is placed between the aluminum foil bag and the second inner bag. The samples are placed in a cardboard barrel.

[0158] 1.1 Accelerated stability test

[0159] The crystalline form of the compound represented by formula (1) was packaged according to the above packaging requirements. A conventional sample was divided into five portions, each containing 10 g. The samples were observed in a constant temperature and humidity chamber maintained at 40 ± 2°C and a relative humidity of 75 ± 5%. Samples were collected and analyzed at 0, 1, 2, 3, and 6 months. The test items, test methods, and technical requirements are detailed in Table 6. The test results were compared with those at 0 months.

[0160] 1.2 Long-term stability test

[0161] The crystalline form of the compound represented by formula (1) was packaged according to the packaging requirements. A conventional sample was divided into 10 portions, each containing 10 g. The sample was observed in a constant temperature and humidity chamber maintained at 30 ± 2°C and a relative humidity of 65 ± 5%. Samples were collected and analyzed in January, March, and June. The test items, test methods, and technical requirements are detailed in Table 7. The test results were compared with those in January.

[0162] Table 6: Accelerated test (40±2℃ / 75±5%RH)

[0163] Note: The data for month 0 was obtained from the release results; ND stands for not detected.

[0164] Table 7: Long-term test (30±2℃ / 65±5%RH)

[0165] Note: The data for month 0 was obtained from the release results; ND stands for not detected.

[0166] The results of the accelerated stability test showed that there was no significant change in the test results after 6 months compared with 0 months, and no new impurity peak greater than 0.10% appeared, indicating that the crystalline form of the compound represented by formula (1) was stable under accelerated conditions for 6 months according to the above existing packaging method.

[0167] The results of the long-term stability test showed that there was no significant change in the test results after 6 months compared with 0 months, and no new impurity peak greater than 0.10% appeared, indicating that the crystal form of the compound represented by formula (1) was stable under long-term conditions for 6 months according to the above existing packaging method.

[0168] Example 4

[0169] By scaling up production according to the method of Example 2, a 10 kg solid sample can be prepared, and the XRPD test results show the crystal form shown in FIG9 .

[0170] Example 5 Biological Activity Test

[0171] Test Example 1: Determination of the activity of the compounds of the present invention on human GHSR

[0172] This method is used to determine the agonistic effect of the compounds of the present invention on the activity of human GHSR protein expressed in human GHSR / CHO stable transfected cells.

[0173] 1. Test materials and instruments

[0174] 1. Culture medium

[0175] F12 (Gibco, Cat#11765-047);

[0176] FBS (Corning, Cat#35-076-CV);

[0177] Geneticin (Invitrogen, Cat#10131);

[0178] Penicillin / Streptomycin (Invitrogen, Cat#15140).

[0179] 2. Reagents

[0180] Fluo-4Direct(Invitrogen,Cat#F10471);

[0181] HBSS (Gibco, Cat#14025076);

[0182] HEPES (Gibco, Cat#15630080);

[0183] Bonine Serum Albumin (Sgima, Cat#B2064-100G).

[0184] 3. Instrument consumables

[0185] 384 well Poly-D-Lysine protein coating plate (Greiner, Cat#781946);

[0186] FLIPR (Molecular Devices);

[0187] Vi-cell XR Cell Viability Analyzer (Beckman Coulter);

[0188] Incubator (Thermo).

[0189] 2. Experimental steps

[0190] Compound gradient preparation: Ghrelin and the compound of the present invention were diluted 5-fold to prepare 10 concentration gradients, and then transferred to the compound plate, with 900 nL per well.

[0191] Preparation buffer: HBSS (1X): HEPES (1M) = 49:1, add 0.5% BSA.

[0192] Stable human GHSR / CHO cells were inoculated into 384-well plates. After overnight, the cell plates were removed and the culture medium was discarded. 20 μL of buffer was slowly added to each well, followed by 20 μL of 2X Fluo-4Direct TM No-wash Loading Buffer. Place the cell plate in a 37°C 5% CO2 incubator and incubate for 50 minutes. Take out the cell plate and place 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. For the test of compound agonism: use the FLIPR instrument and run the software. Transfer 10μL of buffer to the cell plate and read the fluorescence signal value. Transfer 10uL 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 of the compound 50 The value can be calculated by software using the fluorescence values ​​corresponding to different concentrations.

[0193] 3. Experimental results:

[0194] The agonist activity of compound 1e on human GHSR was determined by the above experiments, and the EC 50 The value was 14.6 nM, indicating that compound 1e has good agonist activity against human GHSR. Therefore, it was confirmed that the compound of formula (1) has good agonist activity against human GHSR.

[0195] Test Example 2: Caco-2 cell transport experiment

[0196] The transport buffer used in this study was HBSS containing 10.0 mM HEPSS, pH 7.40 ± 0.05. Test compounds were tested at 2.00 μM in both directions, with a final DMSO concentration of less than 1%. Cell plates were incubated for 2 hours in a CO2 incubator at 37 ± 1°C, 5% CO2 humidity, and saturated humidity. All samples were mixed with acetonitrile containing an internal standard and centrifuged at 3200 × g for 10 minutes. For test compounds, 100 μL of the supernatant was diluted with 100 μL of ultrapure water and used for LC-MS / MS analysis. The concentrations of test compounds and control compounds (Digoxin as a model validation compound and ibutamoren as a positive control) in the starting, donor, and receiver solutions were quantified by LC-MS / MS using the analyte / internal standard peak area ratio. Following the transport assay, the integrity of the Caco-2 cell monolayer was determined using a luciferin yellow exclusion assay.

[0197] Table 8 Caco-2 cell transport assay of Example Compound 1e

[0198] The above data show that the model was successfully constructed and the cell permeability of compound 1e was significantly better than that of the reference compound ibutamoren.

[0199] Test Example 3: Comparison of Compound 1e Metabolism in Rats

[0200] Compound 1e was designed as a prodrug, and the amount of the active ingredient ibutamoren was tested through in vivo metabolism experiments in rats, thereby evaluating the advantages and disadvantages of the candidate and the positive control ibutamoren.

[0201] Experimental operation:

[0202] Two days before dosing, all six animals were fasted for at least 12 hours before being fed. A second fast was conducted one day before dosing, with at least 12 hours of fasting followed, with food resumed 4 hours after dosing. Each fasting period should not exceed 20 hours. Water was freely available during this period. Within two days before dosing, the same person in charge conducted acclimatization training on the animals by touching and handling them at least once daily.

[0203] Before the first dose, animals were divided into two groups based on their body weight. Three animals were assigned to each group. Group 1 received a single oral gavage of compound 5 (preparation: medium-chain triglycerides / polyethylene glycol 1000 vitamin E succinate / ethanol / propylene glycol / water = 6 / 2 / 1 / 1 / 90, 1 mg / mL); Group 2 received a single oral gavage of ibutamoren (water, 1 mg / mL). The dosing volume was 3 mL / kg. Animals were weighed before dosing, and the dosing volume was calculated based on body weight.

[0204] Sample collection time: before administration (approximately -0.25h) and 0.083, 0.25, 0.5, 1, 1.5, 2, 3, 5, 7, and 10h after administration; at each specified time point, animals were briefly anesthetized with isoflurane, and whole blood samples (approximately 0.23mL per group) were collected by jugular vein puncture. 50μL of whole blood sample was quantitatively taken and added to an EP tube containing 50μL of pre-cooled 1mM PMSF methanol solution. The tube was vortexed for ~3s, and 250μL of precipitant containing internal standard was immediately added. The tube was vortexed for ~5s and centrifuged for 15min to obtain the supernatant for LC-MS / MS analysis.

[0205] Table 9 Drug metabolism experimental data in rats

[0206] The above data show that in the drug metabolism experiment of compound 1e of the present invention administered orally to rats, C max The AUC and AUC were significantly better than those of the positive control ibutamoren, indicating better drugability. Therefore, it was confirmed that the compound of formula (1) had better drugability.

[0207] The above experiments show that the crystalline form and amorphous form of the compound prepared by the present invention have high purity and good stability under high temperature and high humidity conditions, which is conducive to the drug's efficacy. The process optimization can meet the requirements of stable, repeatable and controllable production processes and can be adapted to industrial production.

[0208] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A crystalline form of a compound represented by formula (1), wherein The crystal form has characteristic peaks at 5.43±0.20°, 12.28±0.20°, and 17.91±0.20° in X-ray powder diffraction expressed in 2θ angle using Cu-Kα radiation; 2. The crystal form according to claim 1, wherein The crystalline form uses Cu-Kα radiation, and X-ray powder diffraction expressed in 2θ angles 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°.

3. The crystal form according to claim 1 or 2, wherein The crystalline form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles 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°.

4. The crystal form according to any one of claims 1 to 3, wherein The crystalline form has an XRPD pattern substantially as shown in FIG9 ; preferably, the crystalline form is an anhydrate.

5. The method for preparing the crystal form according to any one of claims 1 to 4, wherein: The preparation method comprises the following steps: dissolving the compound represented by formula (1) in solvent A, cooling to precipitate solid, and obtaining the crystal form; The solvent A is selected from one or more of methanol, ethanol, isopropanol, tert-butanol, n-butanol, acetone, tetrahydrofuran, methyltetrahydrofuran, ethyl formate, ethyl acetate, isopropyl acetate, n-hexane, n-heptane, cyclohexane, methyl tert-butyl ether, toluene, dichloromethane, chloroform, DMSO, water, acetonitrile, and isopropyl ether.

6. An amorphous form of a compound represented by formula (1), wherein The XRPD pattern of the amorphous material has no obvious diffraction peaks; Preferably, the amorphous form has an XRPD pattern substantially as shown in FIG1 .

7. The method for preparing the amorphous material according to claim 6, wherein: The preparation method comprises the following steps: dissolving the compound represented by formula (1) in solvent B, adding the resulting clear solution to solvent C, stirring to precipitate a solid, and obtaining the amorphous substance; 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, wherein The pharmaceutical composition contains the crystalline form according to any one of claims 1 to 4 and / or the amorphous form according to claim 6; Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition further comprises a second active ingredient in addition to the above-mentioned crystalline form and / or amorphous form, for example, the second active ingredient is a drug related to growth and development; Preferably, the pharmaceutical composition is a preparation, for example, a GHSR agonist.

9. Use of the crystalline form according to any one of claims 1 to 4, the amorphous form according to claim 6 and / or the pharmaceutical composition according to claim 8 in the preparation of a preparation for diagnosing, preventing and / or treating growth hormone deficiency or growth hormone-dependent diseases (or conditions).

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

Images