Indoline spiro compound solvate and crystal form, preparation method and application thereof

Abstract

The invention provides an anisole solvate of an indoline spiro compound as shown in a formula (1), and a crystal form, a preparation method and application of the anisole solvate. The crystal form is radiated by Cu-K alpha, and X-ray powder diffraction expressed by a 2 theta angle has a characteristic peak at 7.07 + / -0.20 degrees. The solvate and the crystal form thereof provided by the invention have high purity and good solubility, are beneficial to medicine formation, and can also be used for impurity removal and purification of the compound as shown in the formula (1). # imgabs0 #

Description

Solvates of indoline spirocyclic compounds, their crystal forms, preparation methods and applications

[0001] This application claims priority to the following two prior applications: Patent Application No. 202211296034.0, filed with the State Intellectual Property Office of China on October 21, 2022, entitled “Solvates of Indoline Spirocyclic Compounds, Crystal Forms, Preparation Methods, and Applications thereof”; and Patent Application No. 202311300572.7, filed with the State Intellectual Property Office of China on October 9, 2023, entitled “Solvates of Indoline Spirocyclic Compounds, Crystal Forms, Preparation Methods, and Applications thereof”. The entire contents of the prior applications are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of pharmaceutical compounds, and in particular relates to a solvate of an indoline spirocyclic compound, a crystal form thereof, a preparation method and an application thereof. 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 PCT / CN2022 / 088656 are added to the present invention by reference.

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

[0016] According to an embodiment of the present invention, the molar ratio of anisole to the compound represented by formula (1) in the solvate is (1-1.1):1, preferably 1:1.

[0017] According to an embodiment of the present invention, the solvate has a structure shown in the following formula (II):

[0018] According to an embodiment of the present invention, the present invention also provides a crystalline form of anisole solvate of the compound represented by formula (1), wherein the crystalline form has a characteristic peak at 7.07±0.20° in X-ray powder diffraction expressed in 2θ angles using Cu-Kα radiation.

[0019] According to an embodiment of the present invention, the crystalline form has characteristic peaks at 7.07±0.20°, 15.73±0.20°, and 17.83±0.20° in X-ray powder diffraction expressed in 2θ angle using Cu-Kα radiation.

[0020] 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 7.07±0.20°, 11.35±0.20°, 13.95±0.20°, 14.12±0.20°, 15.73±0.20°, 17.83±0.20°, 19.46±0.20°, 21.26±0.20°, 21.57±0.20°, and 26.59±0.20°.

[0021] According to an embodiment of the present invention, the crystalline form has an XRPD pattern substantially as shown in FIG1 .

[0022] According to an embodiment of the present invention, the crystalline form has a TGA pattern substantially as shown in FIG2 .

[0023] According to an embodiment of the present invention, the crystal form has a sharp endothermic peak at a peak temperature of 82.4±2°C.

[0024] According to an embodiment of the present invention, the crystalline form has a DSC spectrum substantially as shown in FIG3 .

[0025] The present invention also provides a method for preparing the anisole solvate crystalline form of the compound represented by formula (I), comprising using the compound represented by formula (I) as a raw material and preparing it by dissolution crystallization, gas-liquid diffusion, gas-solid permeation and the like.

[0026] The present invention also provides a method for preparing the above-mentioned crystal form, comprising the following steps: (1) dissolving the compound represented by formula (1) in anisole, then adding an anti-solvent and stirring to obtain the crystal form; and / or

[0027] (2) dissolving the compound represented by formula (1) in anisole, closing the container with a lid, placing the container in a larger container containing an anti-solvent, closing the lid, and leaving the container at room temperature for more than 1 day to obtain the product; and / or

[0028] (3) A certain amount of the compound represented by formula (1) is placed in a smaller container, and anisole is added to another larger container. The smaller container is opened and placed in the larger container. After sealing, the mixture is allowed to stand at room temperature for more than 1 day.

[0029] According to an embodiment of the present invention, the anti-solvent includes 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, acetone / n-heptane. Preferably, the anti-solvent is selected from one or more of water, n-hexane, n-heptane, and cyclohexane.

[0030] According to an embodiment of the present invention, the dissolving is performed at room temperature or under heating conditions.

[0031] According to an embodiment of the present invention, the standing time is more than 2 days, such as standing for 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0032] According to an embodiment of the present invention, the method further comprises post-treatment of the solid, such as filtering and / or washing.

[0033] The present invention also provides a pharmaceutical composition comprising anisole solvate of the compound represented by formula (1) and / or its crystal form.

[0034] 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.

[0035] According to an embodiment of the present invention, the pharmaceutical composition further comprises a second active ingredient in addition to the above-mentioned solvate and / or its crystalline 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.

[0036] In some embodiments, the solvate and / or its crystalline form and the second active ingredient can be administered separately or co-administered during treatment.

[0037] The present invention also provides the use of the above-mentioned solvate, crystal form and / or pharmaceutical composition in the preparation of a preparation for diagnosing, preventing and / or treating growth hormone deficiency or growth hormone-dependent diseases (or conditions).

[0038] According to an embodiment of the present invention, the disease (or condition) is related to growth hormone deficiency or growth hormone dependence, such as the diagnosis of growth hormone-deficient patients, 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, 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.

[0039] In some embodiments, the agent may be a GHSR agonist.

[0040] The present invention also provides the use of the crystal form in purifying the compound represented by formula (1).

[0041] The present invention also provides a preparation containing the above-mentioned solvate and / or crystal form, or prepared from the above-mentioned pharmaceutical composition.

[0042] 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.

[0043] 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.

[0044] 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 solvate, crystal form or pharmaceutical composition to a patient.

[0045] According to an embodiment of the present invention, the disease has the definitions shown above.

[0046] Beneficial effects of the present invention

[0047] The present invention provides an anisole solvate of a compound represented by formula (1), its crystal form, preparation method, and use. The crystal form of the compound represented by formula (1) obtained by the present invention has good solubility, low hygroscopicity, and good reproducibility, making it suitable for drug development. Furthermore, it can also be used for impurity removal and purification of the compound represented by formula (1).

[0048] Definitions and Explanations of Terms

[0049] 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.

[0050] 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.

[0051] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIG1 is an XRPD pattern of the anisole solvate crystalline form of the compound represented by formula (1);

[0053] FIG2 is a TGA spectrum of the anisole solvate crystalline form of the compound represented by formula (1);

[0054] FIG3 is a DSC spectrum of the anisole solvate crystalline form of the compound represented by formula (1);

[0055] Figure 4 is a crystalline form of anisole solvate of the compound represented by formula (1) 1 H NMR spectrum;

[0056] FIG5 is a PLM diagram of the anisole solvate crystalline form of the compound represented by formula (1);

[0057] FIG6 is a DVS diagram of the anisole solvate crystalline form of the compound represented by formula (1). DETAILED DESCRIPTION

[0058] 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.

[0059] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0060] Analytical methods

[0061] 1. X-ray powder diffraction (XRPD)

[0062] The XRPD pattern was collected on a PANalytical X-ray powder diffraction analyzer with the following scanning parameters:

[0063] 2. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)

[0064] 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:

[0065] 3. Solution NMR

[0066] 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.

[0067] 4. Dynamic Water Sorption Analysis (DVS)

[0068] 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.

[0069] 5. Polarized Light Microscopy (PLM)

[0070] The samples were analyzed using a polarizing microscope, and the morphology and microstructure of the crystals were obtained by adjusting different magnifications.

[0071] 6. Liquid phase method (HPLC)

[0072] Chromatographic conditions:

[0073] Chromatographic column: C18 column;

[0074] Run time: 60 minutes;

[0075] UV detector: 210nm;

[0076] Flow rate: 1.0 mL / min;

[0077] Injection volume: 5 μL.

[0078] Preparation Example Preparation of the compound represented by formula (1)

[0079] (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

[0080] The first step is the preparation of o-(1-chloroethyl) ethyl thiocarbonate (1b)

[0081] 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.

[0082] 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).

[0083] Step 2 Preparation of 1-{[(ethylthio)carbonyl]oxy}ethyl isobutyrate (1c)

[0084] 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).

[0085] 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,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 Preparation method

[0099] (1) Dissolution crystallization

[0100] A certain amount of the compound represented by formula (1) was placed in a 4 mL glass vial, and 0.3 mL of anisole was added to dissolve it. The antisolvent was then quickly added. The mixture was stirred under magnetic force for 5 hours and then filtered. The resulting solid was dried at 50°C overnight. The solvents used in the experiment and the results are shown in Table 1. The resulting white solid was identified as a crystal of the anisole solvate of the compound represented by formula (1).

[0101] Table 1. Dissolution experimental conditions and results

[0102] (2) Gas-liquid diffusion

[0103] In a 2 mL vial, a certain amount of the compound represented by formula (1) was dissolved in 0.2 mL of anisole. The vial was capped and pierced. The vial was placed in a 30 mL large vial containing an antisolvent, capped and sealed, and stored at room temperature for 7 days. The solvents used and the results are shown in Table 2. The precipitated solids were identified as crystals of anisole solvate of the compound represented by formula (1).

[0104] Table 2. Gas-liquid diffusion experimental conditions and results

[0105] (3) Gas-solid permeability

[0106] About 15 mg of the compound represented by formula (1) was weighed into a 3 mL vial, and anisole was added to another 20 mL vial. The 3 mL vial was opened and placed in a 20 mL vial. After sealing, the mixture was allowed to stand at room temperature for 5 days. The solid was collected and tested by XRPD, which was a crystal of anisole solvate of the compound represented by formula (1).

[0107] Example 2 Structural Characterization

[0108] (1) X-ray powder diffraction (XRPD) results showed that the solids obtained by the above three methods were all of the same crystal form, and the specific characterization of the crystal form is shown in Table 3 and Figure 1.

[0109] Table 3. XRPD diffraction peak data of the anisole solvate crystalline form of the compound represented by formula (1)

[0110] TGA (Figure 2) results show that the sample loses 4.8% weight when heated to 90°C and 12.9% weight when heated to 200°C. DSC (Figure 3) results show that an endothermic peak is observed at 82.4°C (peak temperature). 1H NMR results (Figure 4) show that the molar ratio of residual anisole to the compound represented by Formula (1) in the sample is approximately 1.1:1 (~14.2% by weight). The significant weight loss (17.7%) observed by TGA is presumably due primarily to the desorption of anisole. Based on the sample characterization results and preparation conditions, the crystals are presumed to be anisole solvate of the compound represented by Formula (1).

[0111] Furthermore, the PLM and DVS measurements were performed on the crystal. The PLM measurement results of the crystal are shown in FIG5 , and the DVS measurement results are shown in FIG6 .

[0112] Example 3 Solubility Test

[0113] Approximately 5 mg of anisole solvate crystals of the compound represented by formula (1) were weighed into a 4 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 the solid still did not dissolve after adding 4 mL, the solvent addition was stopped. The specific experimental results are shown below.

[0114] Table 4. Solubility of anisole solvate crystalline form of the compound represented by formula (1) in different solvents (room temperature)

[0115] As can be seen from the above table, the anisole solvate crystals of the compound represented by formula (1) have relatively poor solubility in water, n-heptane and cyclohexane, but have relatively high solubility in other common solvents.

[0116] Example 4 Recrystallization purification and impurity removal test

[0117] Process 1: Add a certain amount of anisole solvate crystals of the compound represented by formula (1) prepared in Example 1 into a container, add an appropriate amount of anisole, adjust the system temperature to 40°C, keep warm and stir for 25 to 35 minutes, after visual dissolution, slowly cool the system to 30°C at a rate of 10°C / h, keep warm and stir for 1 hour, slowly cool to 20°C at 5°C / h, slowly add n-heptane dropwise to the system, stir for 12 hours after the addition is completed, filter with suction, and wash the filter cake with a uniform solvent of n-heptane: anisole for 5 to 10 minutes, filter with suction, and dry the filter cake.

[0118] Process 2: Add a certain amount of anisole solvate crystals of the compound represented by formula (1) prepared in Example 1 into a container, add an appropriate amount of anisole, adjust the system temperature to 40°C, keep warm and stir for 25 to 35 minutes, after visual dissolution, slowly cool the system to 30°C at a rate of 10°C / h, add anisole solvate seed crystals of the compound represented by formula (1), grow the crystals for 0.5 to 1 hour, slowly cool to 20°C at 5°C / h, slowly add n-heptane dropwise to the system, stir for 12 hours after the addition is completed, filter with suction, soak the filter cake with a uniform solvent of n-heptane: anisole for 5 to 10 minutes, filter with suction, and dry the filter cake.

[0119] The solids obtained by process 1 and process 2 were subjected to XRPD testing, and both were anisole solvate crystals of the compound represented by formula (1) as shown in Figure 1. The purity of the obtained crystals was tested, and the results are shown in Table 5:

[0120] Table 5. Purification and impurity removal results of the anisole solvate crystalline form of the compound represented by formula (1)

[0121] It can be seen that under the same other operating conditions, adding seed crystals for recrystallization operation can obtain a product with higher purity and smaller single impurities. After one recrystallization, the product purity is 99.86% and the maximum single impurity is 0.10%, which meets the qualified standards.

[0122] Example 5 Biological Activity Test

[0123] Test Example 1: Determination of the activity of the compounds of the present invention on human GHSR

[0124] 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.

[0125] 1. Test materials and instruments

[0126] 1. Culture medium

[0127] F12 (Gibco, Cat#11765-047);

[0128] FBS (Corning, Cat#35-076-CV);

[0129] Geneticin (Invitrogen, Cat#10131);

[0130] Penicillin / Streptomycin (Invitrogen, Cat#15140).

[0131] 2. Reagents

[0132] Fluo-4Direct(Invitrogen,Cat#F10471);

[0133] HBSS (Gibco, Cat#14025076);

[0134] HEPES (Gibco, Cat#15630080);

[0135] Bonine Serum Albumin (Sgima, Cat#B2064-100G).

[0136] 3. Instrument consumables

[0137] 384well Poly-D-Lysine protein coating plate (Greiner, Cat#781946);

[0138] FLIPR (Molecular Devices);

[0139] Vi-cell XR Cell Viability Analyzer (Beckman Coulter);

[0140] Incubator (Thermo).

[0141] 2. Experimental steps

[0142] 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.

[0143] Preparation buffer: HBSS (1X): HEPES (1M) = 49:1, add 0.5% BSA.

[0144] Stable human GHSR / CHO cells were seeded 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-4 Direct TMNo-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.

[0145] 3. Experimental results:

[0146] 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.

[0147] Test Example 2: Caco-2 cell transport experiment

[0148] 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.

[0149] Table 6 Caco-2 cell transport assay of Example Compound 1e

[0150] 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.

[0151] Test Example 3: Comparison of Compound 1e Metabolism in Rats

[0152] 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.

[0153] Experimental operation:

[0154] 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.

[0155] 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.

[0156] 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.

[0157] Table 7 Drug metabolism experimental data in rats

[0158] The above data show that in the drug metabolism experiment of rats administered orally, the Cmax and AUC of compound 1e of the present invention are significantly better than those of the positive control ibutamoren, thus confirming that the compound of formula (1) has better drugability.

[0159] 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. An anisole solvate of a compound represented by formula (1), 2. The solvate according to claim 1, wherein The molar ratio of the anisole to the compound represented by formula (1) is (1-1.1):1; preferably, the molar ratio of the anisole to the compound represented by formula (1) is 1:

1.

3. A crystalline form of anisole solvate of a compound represented by formula (1), wherein: The crystal form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles has a characteristic peak at 7.07±0.20°.

4. The crystal form according to claim 3, wherein The crystal form has characteristic peaks at 7.07±0.20°, 15.73±0.20°, and 17.83±0.20° in X-ray powder diffraction expressed in 2θ angle using Cu-Kα radiation; Preferably, the crystalline form uses Cu-Kα radiation, and the X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 7.07±0.20°, 11.35±0.20°, 13.95±0.20°, 14.12±0.20°, 15.73±0.20°, 17.83±0.20°, 19.46±0.20°, 21.26±0.20°, 21.57±0.20°, and 26.59±0.20°.

5. The crystal form according to claim 3 or 4, wherein The crystalline form has an XRPD pattern substantially as shown in FIG1 .

6. The crystal form according to any one of claims 3 to 5, wherein The anisole solvate of the compound represented by formula (1) has a structure represented by the following formula (II):

7. The method for preparing the crystal form according to any one of claims 3 to 6, characterized in that: The preparation method comprises using the compound represented by formula (I) as a raw material and preparing the compound by dissolution crystallization, gas-liquid diffusion, gas-solid permeation and the like; Preferably, the preparation method comprises the following steps: (1) dissolving the compound represented by formula (1) in anisole, then adding an anti-solvent and stirring; and / or (2) dissolving the compound represented by formula (1) in anisole, closing the container with a lid, placing the container in a larger container containing an anti-solvent, closing the lid, and leaving the container at room temperature for more than 1 day to obtain the product; and / or (3) A certain amount of the compound represented by formula (1) is placed in a smaller container, and anisole is added to another larger container. The smaller container is opened and placed in the larger container. After sealing, the mixture is allowed to stand at room temperature for more than 1 day.

8. The preparation method according to claim 7, characterized in that The anti-solvent includes 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, acetone / n-heptane; Preferably, the anti-solvent is selected from one or more of water, n-hexane, n-heptane and cyclohexane.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the anisole solvate of the compound represented by formula (1) according to claim 1 or 2 or the crystal form according to any one of claims 3 to 6.

10. The pharmaceutical composition according to claim 9, characterized in that The pharmaceutical composition further contains pharmaceutically acceptable excipients; Preferably, the pharmaceutical composition further comprises a second active ingredient in addition to the above-mentioned solvate and / or crystal 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.

11. Use of an anisole solvate of the compound of formula (1) according to claim 1 or 2, the crystalline form according to any one of claims 3 to 6, and / or the pharmaceutical composition according to any one of claims 9 to 10 in the preparation of a preparation for diagnosing, preventing and / or treating a growth hormone-dependent disease (or condition); preferably, the disease or condition is related to growth hormone deficiency or growth hormone dependence, such as the diagnosis of growth hormone-deficient patients, 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: regulation of energy balance and food intake; treatment of adipogenesis, obesity and weight loss; treatment of cachexia; improvement of gastrointestinal motility, treatment of gastroparesis and diabetic gastroparesis, and postoperative ileus; increase in muscle mass and skin thickness, reduction in fat mass and slight increase in bone density in elderly patients; treatment of burns, AIDS and cancer conditions, and wound and bone healing.

12. Use of the crystal form according to any one of claims 3 to 6 in purifying the compound represented by formula (1).

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