WO2024083160A1 - 吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用 - Google Patents

吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用 Download PDF

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WO2024083160A1
WO2024083160A1 PCT/CN2023/125251 CN2023125251W WO2024083160A1 WO 2024083160 A1 WO2024083160 A1 WO 2024083160A1 CN 2023125251 W CN2023125251 W CN 2023125251W WO 2024083160 A1 WO2024083160 A1 WO 2024083160A1
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crystalline form
formula
amorphous
solvent
compound represented
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PCT/CN2023/125251
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English (en)
French (fr)
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林岱宗
黄强
邵文博
孙毅
娄喜余
刘清印
徐学宇
杨方龙
王思勤
金磊
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长春金赛药业有限责任公司
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Publication of WO2024083160A1 publication Critical patent/WO2024083160A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/10—Spiro-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic

Definitions

  • the invention belongs to the field of pharmaceutical compounds, and specifically relates to a crystal form and an amorphous substance of an indoline spirocyclic compound and preparation methods and applications of the two.
  • GH Human growth hormone
  • IGF-1 insulin-like growth factor 1
  • EGF epidermal growth factor
  • Ghrelin is an endogenous growth hormone-releasing peptide containing 28 amino acids and is an endogenous ligand of Growth Hormone Secretagogue Receptor 1a (GHSR 1a). Both in vivo and in vitro experiments have confirmed that ghrelin can significantly promote the secretion of growth hormone. Clinical studies have also found that intravenous injection of ghrelin can strongly stimulate the release of growth hormone in a dose-dependent manner.
  • GH has been found to be promising in the treatment of conditions such as loss of muscle mass, accumulation of adipose tissue, demineralization of bones, and reduced tissue regeneration capacity after injury.
  • GH is synthesized and stored in the pituitary gland, but the release of GH is controlled by hormones from the hypothalamus.
  • Two hormones are known to be involved in the release of GH: growth hormone-releasing hormone (GHRH) and the inhibitory hormone somatostatin (SRIF).
  • GHRH growth hormone-releasing hormone
  • SRIF inhibitory hormone somatostatin
  • GH deficiency involves the release of GH (hypothalamic defect) rather than the synthesis of GH (pituitary defect). Therefore, the use of GHSR agonists to stimulate GH release in the pituitary may become a new therapeutic alternative to recombinant human growth hormone.
  • GHSR has two subtypes, 1a and 1b.
  • Subtype 1a is a functional receptor subtype, and the function of subtype 1b needs further study.
  • GHSR 1a is distributed in the hypothalamus and multiple areas outside the hypothalamus, including the pituitary gland, hypothalamic arch In the periphery, GHSR is also expressed at low levels in the thyroid gland, pancreas, myocardium, etc. Therefore, ghrelin and its receptor GHSR 1a may be involved in the regulation of multiple functions in the body.
  • GHSR agonist activity Studies have found that some clinical peptide or peptidomimetic compounds have shown GHSR agonist activity and have the effect of inducing GH release.
  • the compounds currently entering clinical research include examorelin, tabimorelin, pralmorelin, ibutamoren, tesamorelin, anamorelin and macimorelin, among which the injectable peptide tesamorelin (used to reduce excess abdominal fat in HIV-infected people) 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 adult growth hormone deficiency, but macimorelin also has defects such as poor oral bioavailability and potential risk of cardiac toxicity.
  • GHSR agonists in addition to inducing GH secretion through GHSR 1a activation, also mediate other physiological functions through different receptors of other GHS receptor families or different binding sites on GHSR (such as GHSR 1b, motilin receptor 1a, neurotensin receptor and TRH receptor, etc.). Therefore, the application of GHSR agonists in the field of gastrointestinal indications has been newly developed.
  • GHSR 1b motilin receptor 1a
  • TRH receptor neurotensin receptor and TRH receptor
  • Ghrelin has been shown to have a prokinetic effect on gastrointestinal motility via 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.
  • 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 of 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 ileus; increase in muscle mass and skin thickness, reduction in 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.
  • the solid pharmaceutical form of the above compounds suitable for drug formulation is developed, for example, to improve stability, hygroscopicity and/or efficacy.
  • the present invention provides a crystalline form of a compound represented by formula (1),
  • the crystal form uses Cu-K ⁇ radiation, and X-ray powder diffraction expressed in 2 ⁇ angles has characteristic peaks at 5.43 ⁇ 0.20°, 12.28 ⁇ 0.20°, and 17.91 ⁇ 0.20°.
  • the crystalline form uses Cu-K ⁇ radiation, and the X-ray powder diffraction expressed in 2 ⁇ 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°.
  • the crystalline form uses Cu-K ⁇ radiation
  • the X-ray powder diffraction expressed in 2 ⁇ 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°.
  • the crystalline form has an XRPD pattern substantially as shown in FIG. 9 .
  • the crystalline form is an anhydrate.
  • the weight loss of the crystalline form before 150° C. does not exceed 3 wt %, such as does not exceed 2 wt %.
  • the crystalline form has a TGA spectrum substantially as shown in FIG. 10 .
  • the crystalline form has a sharp endothermic peak at a peak temperature of 130.6 ⁇ 2°C.
  • the crystalline form has a DSC spectrum substantially as shown in FIG. 11 .
  • 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;
  • 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
  • the dissolving is performed under heating conditions.
  • the temperature is lowered to room temperature.
  • the cooling is slow cooling.
  • the method further comprises post-treatment of the solid, such as filtering and/or washing.
  • the present invention also provides an amorphous substance of the compound represented by formula (1), whose XRPD spectrum has no obvious diffraction peak.
  • the amorphous material has an XRPD pattern substantially as shown in FIG. 1 .
  • the present invention also provides a method for preparing the amorphous substance, comprising the following steps: dissolving the compound represented by formula (1) in solvent B, adding the obtained 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), for example, it can be selected from one or more of DMF, DMA, NMP, acetonitrile, THF, DMSO, methyl tert-butyl ether, isopropyl ether, methanol, ethanol, isopropanol, acetone, etc.;
  • 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.
  • the supernatant is added to the solvent C in a dropwise manner.
  • the method further comprises post-treatment of the solid, such as filtering and/or washing.
  • the present invention also provides a pharmaceutical composition containing the above-mentioned crystal form and/or amorphous substance.
  • 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.
  • 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.
  • the pharmaceutical composition further comprises a second active ingredient in addition to the above-mentioned crystalline form and/or amorphous substance, 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.
  • the crystalline form and/or amorphous form and the second active ingredient can be administered separately or together during treatment.
  • the present invention also provides the use of the above-mentioned crystal form, amorphous substance and/or pharmaceutical composition in the preparation of preparations for diagnosing, preventing and/or treating growth hormone deficiency or growth hormone-dependent diseases (or disorders).
  • 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 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, postoperative ileus; 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, as well as wounds and bones of healing.
  • 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, postoperative ileus
  • 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 as well as wounds and bones of healing.
  • the agent may be a GHSR agonist.
  • the present invention also provides a preparation containing the above-mentioned crystal form and/or amorphous substance, or prepared from the above-mentioned pharmaceutical composition.
  • the preparation can be in the form of powder, tablet (e.g., coated tablet, sustained-release or controlled-release tablet), lozenge, capsule (e.g., soft capsule or hard capsule), granule, pill, dispersible powder, suspension, solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.
  • tablet e.g., coated tablet, sustained-release or controlled-release tablet
  • capsule e.g., soft capsule or hard capsule
  • granule, pill dispersible powder
  • suspension solution, emulsion, elixir, syrup, aerosol, cream, ointment, gel, injection, lyophilized powder injection or suppository.
  • the preparation can be administered in any of the following ways: orally, buccal administration, sublingually, by inhalation, by topical application, by intravenous, subcutaneous, acupuncture point or intramuscular injection via parenteral administration, or by rectal administration.
  • 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 crystal form, amorphous substance or the pharmaceutical composition to a patient.
  • the disease has the definitions as indicated above.
  • the present invention provides a crystalline form and an amorphous substance of a compound represented by formula (1), and a preparation method and use thereof.
  • the crystalline form and amorphous substance of the compound represented by formula (1) obtained by the present invention have good stability and solubility, low hygroscopicity, can be stored for a long time, have good repeatability, and are suitable for drug development.
  • 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.
  • the amorphous compound of 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.
  • therapeutically effective amount refers to the 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 diseases as defined below).
  • the therapeutically effective amount may vary depending on 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 disease severity, and the type of disease.
  • the dosage may be determined by the severity of the symptoms and the mode of administration, etc., which can be easily determined by a person of ordinary skill in the art.
  • the specific dosage will vary depending on the following factors: the specific active ingredient selected, the dosage regimen followed, 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.
  • 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.
  • 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.
  • FIG1 is an XRPD pattern of an amorphous compound of formula (1)
  • FIG2 is a DSC spectrum of the amorphous compound of formula (1)
  • FIG3 is a TGA spectrum of the amorphous compound of formula (1)
  • FIG4 is a PLM diagram of the amorphous compound represented by formula (1);
  • FIG5 is a SEM image of the amorphous compound represented by formula (1)
  • FIG6 is a 1 H NMR spectrum of the amorphous compound of formula (1).
  • FIG7 is a DVS spectrum of the amorphous compound represented by formula (1);
  • FIG8 is an XRPD superposition spectrum of the amorphous form of the compound represented by formula (1) before and after DVS testing;
  • FIG9 is an XRPD spectrum of the crystalline form of the compound represented by formula (1);
  • FIG10 is a TGA spectrum of the crystalline form of the compound represented by formula (1);
  • FIG11 is a DSC spectrum of the crystalline form of the compound represented by formula (1);
  • FIG12 is a DVS spectrum of the crystalline form of the compound represented by formula (1);
  • FIG13 is an XRPD superposition spectrum of the crystalline form of the compound represented by formula (1) before and after DVS testing;
  • FIG14 is a 1 H NMR spectrum of the crystalline form of the compound represented by formula (1);
  • FIG15 is an XRPD superposition spectrum of the solid stability of the amorphous compound represented by formula (1);
  • FIG. 16 is an XRPD superposition spectrum of the crystal stability of the compound represented by formula (1).
  • the first step is the preparation of ethyl o-(1-chloroethyl)thiocarbonate (1b)
  • LC-MS Liquid chromatography-mass spectrometry
  • the crystal form of the sample was analyzed by X-ray powder diffractometer.
  • the 2 ⁇ scanning angle of the sample was 3° to 42°, the scanning step was 0.02°, and the scanning time for each step was 0.2s.
  • the light tube voltage and current were 40kV and 40mA respectively.
  • the samples were analyzed using TA Instruments TGA Discovery 550.
  • the samples were placed in a tared aluminum pan, the system automatically weighed the samples, and then the samples were heated from room temperature to the specified temperature at a rate of 10°C/min under nitrogen.
  • the samples were analyzed using TA Instruments Discovery DSC 25.
  • the weighed samples were placed in a sample tray, and the sample temperature was raised from 25°C to the specified temperature at a rate of 10°C/min under the protection of nitrogen (50 ml/min).
  • test sample size was approximately 20 to 30 mg.
  • the temperature of the test chamber was controlled at 25 ⁇ 1 °C, and the relative humidity increased from 0% to 90% and then decreased to 0% at a rate of 10%/h.
  • the quality data was recorded every 20 seconds.
  • the sample was analyzed using Phenom pure+.
  • the sample was gold-sprayed and then placed in the instrument for testing. Different magnifications were adjusted to obtain the sample crystal habits.
  • the samples were analyzed using a polarizing microscope, and the morphology and microstructure of the crystals were obtained by adjusting different magnifications.
  • the samples were analyzed using a Varian Inova 500 MHz NMR analyzer.
  • Chromatographic column C18 column
  • UV detector 210nm
  • Injection volume 5 ⁇ L.
  • 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.
  • the XRPD pattern was collected on a PANalytical X-ray powder diffraction analyzer, and the scanning parameters were as follows:
  • TGA Thermogravimetric analysis
  • DSC differential scanning calorimetry
  • TGA and DSC images were collected on a TA Discovery TGA 5500 thermogravimetric analyzer and a TA Discovery DSC 2500 differential scanning calorimeter, respectively.
  • the test parameters are as follows:
  • the DVS curves were collected on the DVS Intrinsic by SMS (Surface Measurement Systems). The relative humidity at 25°C was calibrated based on the deliquescent points of LiCl, Mg(NO 3 ) 2 and KCl.
  • the DVS test parameters are as follows:
  • Liquid 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.
  • the solid sample prepared by the above method was subjected to XRPD test (as shown in Figure 9), and the result showed that it was in crystalline form.
  • the TGA curve ( Figure 10) shows that the crystal has a weight loss of 1.1% when heated to 150°C
  • the DSC curve ( Figure 11) shows that an endothermic peak is observed at 130.6°C (peak temperature). Based on the smaller TGA weight loss of the crystal form and the absence of DSC signal before 100°C, it is speculated that the crystal form is an anhydrous crystal form.
  • 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 crystal form of the sample was analyzed by X-ray powder diffractometer.
  • a proper amount of the sample was placed on the sample tray and flattened with a spoon or glass sheet to ensure that the surface was smooth and flat. The results are shown in Table 2 and Figure 9.
  • 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 aluminum foil bag is heat-sealed.
  • a bag of desiccant is placed between the aluminum foil bag and the second inner bag and placed in a cardboard barrel.
  • Test Example 1 Determination of the activity of the compounds of the present invention on human GHSR
  • the 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.
  • FBS (Corning, Cat#35-076-CV);
  • Penicillin/Streptomycin (Invitrogen, Cat#15140).
  • Bonine Serum Albumin (Sgima, Cat#B2064-100G).
  • 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.
  • the human GHSR/CHO stable cells were inoculated in a 384-well plate. After overnight, the cell plate was removed, the culture medium was discarded, 20 ⁇ L of buffer was slowly added to each well, and then 20 ⁇ L of 2X Fluo-4Direct TM No-wash Loading Buffer was added to each well. The cell plate was placed in a 37°C 5% CO 2 incubator for 50 min, and the cell plate was removed and placed at room temperature for 10 min. 30uL of buffer was added to each well of the compound plate; another buffer plate was prepared and 30 ⁇ L of buffer was added to each well. For the test of compound agonism: Use the FLIPR instrument and run the software. Transfer 10 ⁇ L of buffer to the cell plate and read the fluorescence signal value.
  • the EC 50 value of the compound can be calculated by the software using the fluorescence values corresponding to different concentrations.
  • the agonist activity of compound 1e on human GHSR was determined by the above experiment, and the measured EC 50 value was 14.6 nM, indicating that compound 1e has good agonist activity on human GHSR. Therefore, it is confirmed that the compound of formula (1) has good agonist activity on human GHSR.
  • the transport buffer in the study was HBSS containing 10.0 mM HEPSS, pH 7.40 ⁇ 0.05.
  • the test compounds were tested at 2.00 ⁇ M in both directions, and the final concentration of DMSO was required to be less than 1%.
  • the cell plates were incubated in a CO 2 incubator at 37 ⁇ 1 °C, 5% CO 2 and saturated humidity for 2 hours. All samples were mixed with acetonitrile containing internal standards and centrifuged at 3200xg for 10 minutes. For the test compounds, 100 ⁇ L of the supernatant was diluted with 100 ⁇ L of ultrapure water for LC-MS/MS analysis.
  • the starting solution, donor solution and receiving solution were analyzed by LC-MS/MS method using the peak area ratio of analyte/internal standard.
  • concentrations of test compounds and control compounds (Digoxin as a model validation compound and ibutamoren as a positive control) in the transporter solution were quantified.
  • the lucifer yellow exclusion assay was applied to determine the integrity of the Caco-2 cell monolayer.
  • 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.
  • the six animals were fasted for the first time two days before administration. After fasting for at least 12 hours, they were fed uniformly. The second fasting was carried out one day before administration. The animals were fasted for at least 12 hours and were fed again 4 hours after administration. Each fasting time was no more than 20 hours. The animals were allowed to drink water freely during the period. Within two days before administration, the same person in charge conducted adaptation training on the animals by touching and grabbing them, and the adaptation was carried out at least once a day.
  • the animals were divided into two groups according to their body weight. There were three animals in each group.
  • the second group of animals was given ibutamoren solution (water, 1 mg/mL) by a single oral administration.
  • the administration volume was 3 mL/kg.
  • the animals were weighed before administration and the administration volume was calculated based on the body weight.
  • 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, the animals were briefly anesthetized with isoflurane, and whole blood samples (approximately 0.23mL per group) were collected by jugular vein puncture. 50 ⁇ L of whole blood sample was quantitatively taken and added to an EP tube containing 50 ⁇ L precooled 1mM PMSF methanol solution, vortexed for ⁇ 3s, and 250 ⁇ L of precipitant containing internal standard was immediately added, vortexed for ⁇ 5s, and centrifuged for 15min to obtain the supernatant for LC-MS/MS analysis.

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Abstract

提供一种式(1)所示的吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用。所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在5.43±0.20°、12.28±0.20°、17.91±0.20°处具有特征峰；所述无定形物的XRPD图谱没有明显衍射峰；提供的晶型及无定形物纯度高，稳定性好，有利于成药。

Description

吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用
本申请要求以下两项在先申请的优先权：2022年10月21日向中国国家知识产权局提交的专利申请号为202211297102.5，发明名称为“吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用”和2023年10月9日向中国国家知识产权局提交的专利申请号为202311300559.1，发明名称为“吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用”的在先申请的优先权。所述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于药物化合物领域，具体涉及一种吲哚啉螺环类化合物的晶型、无定形物及二者的制备方法和应用。
背景技术
人体的生长激素(Growth Hormone,GH)是由脑垂体前叶分泌的一种肽类激素，由191个氨基酸组成，通过诱导类胰岛素生长因子1(IGF-1)或表皮生长因子(EGF)的合成，直接或间接作用于外围器官，主要生理功能包括促进机体的线性生长、促进肌肉和皮肤的细胞增殖，并在外伤后组织的再生中起重要作用。
Ghrelin是一种内源性的含有28个氨基酸的生长激素释放肽，为生长激素促分泌素受体1a型(Growth Hormone Secretagogue Receptor 1a,GHSR 1a)的内源性配体。体内和体外实验均已证实，Ghrelin能显著促进生长激素的分泌。临床研究也发现，静脉注射Ghrelin能够强烈的刺激生长激素释放，并且呈剂量依赖性。
GH的释放被认为可以治疗以生长激素分泌缺陷为特征的生理或病理生理病症，以及治疗被生长激素的合成代谢效应改善的病症。临床发现GH有望治疗肌肉质量损失、脂肪组织的聚集、骨骼的脱矿质、受伤后组织再生能力降低等病症。
GH在垂体腺合成并储存，但GH的释放受到下丘脑激素的控制。目前已知两种激素参与到GH的释放过程：生长激素释放激素(GHRH)和抑制性激素促生长激素抑制素(SRIF)。多数情况下，GH缺陷涉及GH的释放(下丘脑缺陷)而不是GH的合成(垂体缺陷)。因此，采用GHSR激动剂刺激垂体中的GH释放，可能成为替代重组人生长激素的新的疗法。
GHSR具有1a、1b两种亚型，1a亚型是功能性受体亚型，1b亚型的功能有待进一步研究。在中枢神经系统内，GHSR 1a分布在下丘脑及下丘脑以外的多个区域，包括垂体，下丘脑弓 状核，腹内侧核等地方。在外周，GHSR在甲状腺，胰腺，心肌等地方也有低表达。因此Ghrelin及其受体GHSR 1a可能参与体内多种功能的调节。
研究发现，一些肽类或拟肽类的临床化合物表现出了GHSR激动活性，具有诱导GH释放的作用。目前进入临床研究的化合物包括examorelin，tabimorelin，pralmorelin，ibutamoren，tesamorelin，anamorelin和macimorelin，其中，注射多肽tesamorelin(用于减少HIV感染者腹部脂肪过量)和小分子拟肽类macimorelin已被FDA批准上市。Macimorelin是被批准用于成人生长激素缺乏症诊断的唯一可以口服的药物，但macimorelin也存在着口服生物利用度差，潜在心脏毒性的风险等缺陷。
在相关研究中还发现，GHSR激动剂除通过GHSR 1a活化介导诱导GH分泌外，还通过其他GHS受体家族的不同受体，或者GHSR上不同的结合位点(如GHSR 1b、胃肠胃动素受体1a、神经降压素受体和TRH受体等)介导产生其他的生理功能。因此，新开发了GHSR激动剂在胃肠道适应症领域的应用，目前该适应症还没有药物上市，其中，ulimorelin和relamorelin已经进入了临床III期的研究。
Ghrelin已被证明可通过迷走神经和骨盆神经对胃肠蠕动产生促动力作用，但Ghrelin的半衰期较短，阻碍了其成药性，需要开发具有增强药代动力学的GHSR激动剂来改善动物和人类的胃肠功能受损。
为了克服上述技术问题，本申请人自主研发获得了具有全新分子结构的化合物，其结构式为：化学名称为：(4R,11R)-7,7-二甲基-4-(1-(甲基磺酰基)螺[吲哚啉-3,4'-哌啶]-1'-羰基)-6,9-二氧代-1-苯基-2,10-二氧杂-5,8-二氮杂十二烷-11-基异丁酸酯，相关内容记载在专利申请PCT/CN2022/088656中。药效学试验显示，该化合物具有良好的临床应用前景，可用于制备诊断、预防和/或治疗生长激素依赖性疾病或病症；优选的，所述疾病或病症与生长激素缺乏或生长激素依赖有关，如生长激素缺乏患者的诊断，生长激素缺乏的儿童生长缓慢、身材矮小，以及治疗可被生长激素的生理作用改善的其他疾病，包括但不限于：能量平衡和食物摄入调节；脂肪形成、肥胖的治疗与体重减少；恶病质的治疗；胃肠动力改善，胃轻瘫和糖尿病胃轻瘫、术后肠梗阻的治疗；老年病人人群中肌肉质量和皮肤厚度的增加、脂肪物质的减少和骨密度的轻微增加；烧伤、AIDS和癌症情况的治疗，以及伤口和骨骼的愈合。
同时，研发上述化合物适于成药的药物固体形式，例如使稳定性、吸湿性和/或药效等得 到改善的固体形式，从而在制药及用药阶段取得良好效果，成为亟待解决的技术问题。
发明内容
专利申请PCT/CN2022/088656中所涉及的所有内容均以引证的方式添加到本发明中。
为了改善上述技术问题，本发明提供了一种式(1)所示化合物的晶型，
所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在5.43±0.20°、12.28±0.20°、17.91±0.20°处具有特征峰。
根据本发明的实施方案，所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在5.43±0.20°、12.28±0.20°、17.91±0.20°、18.54±0.20°、18.87±0.20°、20.54±0.20°、21.69±0.20°处具有特征峰。
根据本发明的实施方案，所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在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°、21.86±0.20°处具有特征峰。
根据本发明的实施方案，所述晶型具有基本如图9所示的XRPD图谱。
根据本发明的实施方案，所述晶型为无水物。
根据本发明的实施方案，所述晶型在150℃之前失重量不超过3wt％，例如不超过2wt％。
根据本发明的实施方案，所述晶型具有基本如图10所示的TGA图谱。
根据本发明的实施方案，所述晶型在峰值温度为130.6±2℃处具有一个尖锐的吸热峰。
根据本发明的实施方案，所述晶型具有基本如图11所示的DSC图谱。
本发明还提供上述晶型的制备方法，包括如下步骤：将式(1)所示的化合物溶解于溶剂A中，降温析出固体，得到所述晶型；
所述溶剂A可以选自甲醇、乙醇、异丙醇、叔丁醇、正丁醇、丙酮、四氢呋喃、甲基四氢呋喃、甲酸乙酯、乙酸乙酯、乙酸异丙酯、正己烷、正庚烷、环己烷、甲基叔丁醚、甲苯、二氯甲烷、氯仿、DMSO、水、乙腈、异丙醚等中的一种或两种以上，例如选自乙醇/水、DMSO/水、丙酮/水、乙醇/正己烷、乙醇/环己烷、乙腈/正庚烷、乙醇/正庚烷、异丙醇/正庚烷、甲醇/正庚烷、甲醇/正己烷、丙酮/正庚烷的混合溶剂。
根据本发明的实施方案，所述溶解在加热条件下进行。
根据本发明的实施方案，所述降温为降至室温。
根据本发明的实施方案，所述降温为缓慢降温。
根据本发明的实施方案，所述方法还包括对固体的后处理，例如过滤和/或洗涤。
本发明还提供式(1)所示化合物的无定形物，其XRPD图谱没有明显衍射峰。
根据本发明的实施方案，所述无定形物具有基本如图1所示的XRPD图谱。
本发明还提供上述无定形物的制备方法，包括如下步骤：将式(1)所示的化合物溶解于溶剂B中，将所得溶清液加至溶剂C中，搅拌析出固体，得到所述无定形物；
所述溶剂B为式(1)所示化合物的良溶剂，例如可以选自DMF、DMA、NMP、乙腈、THF、DMSO、甲基叔丁醚、异丙醚、甲醇、乙醇、异丙醇、丙酮等中的一种或两种以上；
所述溶剂C为式(1)所示化合物的不良溶剂，例如选自水、正庚烷、正己烷、环己烷中的一种或两种以上。
根据本发明的实施方案，所述溶清液以滴加方式加至溶剂C中。
根据本发明的实施方案，所述方法还包括对固体的后处理，例如过滤和/或洗涤。
本发明还提供一种药物组合物，含有上述晶型和/或无定形物。
根据本发明的实施方案，所述药物组合物还含有药学上可接受的辅料，例如包括但不限于赋形剂、填充剂、润滑剂、粘合剂、崩解剂、无机盐、溶剂、溶解助剂、悬浮剂、等渗剂、缓冲液、防腐剂、抗氧剂、着色剂、起泡剂和调味剂等中的一种或两种以上。
根据本发明的实施方案，所述药物组合物还包括除上述晶型和/或无定形物的第二活性成分，例如所述第二活性成分为与生长发育相关的药物，例如所述第二活性成分为GHSR激动剂或生长激素。
在一些实施方案中，所述晶型和/或无定形物与第二活性成分在治疗时可以分开施用，或者共同施用。
本发明还提供上述晶型、无定形物和/或药物组合物在制备诊断、预防和/或治疗生长激素缺乏或生长激素依赖性疾病(或病症)制剂中的应用。
根据本发明的实施方案，所述疾病例如为生长激素缺乏患者的诊断，生长激素缺乏的儿童生长缓慢、身材矮小，以及治疗可被生长激素的生理作用改善的其他疾病，包括但不限于：能量平衡和食物摄入调节；脂肪形成、肥胖的治疗与体重减少；恶病质的治疗；胃肠动力改善，胃轻瘫和糖尿病胃轻瘫、术后肠梗阻的治疗；老年病人人群中肌肉质量和皮肤厚度的增加、脂肪物质的减少和骨密度的轻微增加；烧伤、AIDS和癌症情况的治疗，以及伤口和骨骼 的愈合。
在一些实施方案中，所述制剂可以为GHSR激动剂。
本发明还提供一种制剂，含有上述晶型和/或无定形物，或由上述药物组合物制备得到。
根据本发明的实施方案，所述制剂可以为散剂、片剂(例如包衣片剂、缓释或控释片剂)、锭剂、胶囊剂(例如软胶囊或硬胶囊)、颗粒剂、丸剂、可分散粉末、混悬剂、溶液剂、乳剂、酏剂、糖浆剂、气雾剂、霜剂、软膏剂、凝胶、注射剂、冻干粉针剂或栓剂等剂型。
根据本发明的实施方案，所述制剂可以以下述任一种方式施用：口服、口腔给药、舌下、吸入、局部涂敷，经胃肠外给药的静脉内、皮下、穴位或肌内注射，直肠给药。
本发明还提供一种诊断、预防和/或治疗生长激素缺乏或生长激素依赖性疾病(或病症)的方法，包括将治疗有效量的所述的晶型、无定形物或所述的药物组合物施用于患者。
根据本发明的实施方案，所述疾病具有如上文所示的限定。
本发明的有益效果
本发明提供了如式(1)所示的化合物的晶型、无定形物及其制备方法和用途。本发明获得的式(1)所示化合物的晶型及无定形物具备良好的稳定性和溶解性，引湿性低，能够长期储存，可重复性好，适合药物开发。
式(1)所示化合物的晶体纯度高，在光照、高温、高湿的条件下晶型稳定性良好，引湿性低，能够长期储存，有利于药物开发，其制备工艺稳定、重复性好，能够适应于工业化生产。
本发明制备获得的式(1)所示化合物的无定形物纯度高，在大多数溶剂中溶解度好，在光照、高温、高湿条件下具有较好的物理和化学稳定性，适合用于药物开发。
术语定义与说明
除非另有说明，本申请说明书和权利要求书中记载的术语定义，包括其作为实例的定义、示例性的定义、优选的定义、实施例中具体化合物的定义等，可以彼此之间任意组合和结合。这样的组合和结合应当属于本申请说明书记载的范围内。
术语“治疗有效量”是指足以实现预期应用(包括但不限于如下定义的疾病治疗)的本发明所述晶型、无定形物、第二活性成分的量。治疗有效量可以取决于以下因素而改变：预期应用(体外或者体内)，或者所治疗的受试者和疾病病症如受试者的重量和年龄、疾病病 症的严重性和给药方式等，其可以由本领域普通技术人员容易地确定。具体剂量将取决于以下因素而改变：所选择的特定活性成分、所依据的给药方案、是否与其它化合物组合给药、给药的时间安排、所给药的组织和所承载的物理递送系统。
术语“患者”是指包括哺乳动物在内的任何动物，优选小鼠、大鼠、其它啮齿类动物、兔、狗、猫、猪、牛、羊、马或灵长类动物，最优选人。
术语“130.6±2℃”代表128.6～132.6℃，例如为128.6℃、129.0℃、130.0℃、131.0℃、132.0℃、132.6℃或前述任意两点之间的值。
附图说明
图1为式(1)所示化合物无定形物的XRPD图谱；
图2为式(1)所示化合物无定形物的DSC图谱；
图3为式(1)所示化合物无定形物的TGA图谱；
图4为式(1)所示化合物无定形物的PLM图；
图5为式(1)所示化合物无定形物的SEM图；
图6为式(1)所示化合物无定形物的1H NMR图谱；
图7为式(1)所示化合物无定形物的DVS图谱；
图8为式(1)所示化合物无定形物DVS测试前后的XRPD叠加图谱；
图9为式(1)所示化合物晶型的XRPD图谱；
图10为式(1)所示化合物晶型的TGA图谱；
图11为式(1)所示化合物晶型的DSC图谱；
图12为式(1)所示化合物晶型的DVS图谱；
图13为式(1)所示化合物晶型DVS测试前后的XRPD叠加图谱；
图14为式(1)所示化合物晶型的1H NMR图谱；
图15为式(1)所示化合物无定形物固体稳定性XRPD叠加图谱；
图16为式(1)所示化合物晶型稳定性XRPD叠加图谱。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
制备例式(1)所示化合物的制备
(4R,11R)-7,7-二甲基-4-(1-(甲基磺酰基)螺[吲哚啉-3,4'-哌啶]-1'-羰基)-6,9-二氧代-1-苯基-2,10-二氧杂-5,8-二氮杂十二烷-11-基异丁酸酯
第一步邻-(1-氯乙基)硫代碳酸乙酯(1b)的制备
将化合物1-氯乙基氯甲酸酯1a(3g,0.02mol)溶于二氯甲烷(10mL)，接着加入四丁基溴化铵(TBAB)(0.34g)，将乙硫醇钠(1.76g；0.02mol)溶入水(10mL)后滴加入反应液，反应液在25℃搅拌16小时。反应液分层，有机层水洗(20mL)，无水硫酸钠干燥，浓缩得到化合物1b(2.0g，收率：56％)为黄色油状物。
1H 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).
第二步1-{[(乙硫基)羰基]氧基}乙基异丁酸酯(1c)的制备
将化合物1b(700mg；4.15mmol)溶于异丁酸(2.2g,25mmol)，再加入N,N-二异丙基乙胺(1.6g,12.5mmol)，反应液在55℃搅拌48小时。反应液加入水(20mL)淬灭，乙酸乙酯萃取(20mL)，饱和碳酸氢钠洗(3x30mL)，饱和食盐水洗(2x20mL)，浓缩得到的残留物1c(850mg,淡黄色油状物)。
1HNMR(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).
第三步1-((氯羰基)氧基异丁酸乙酯(1d)的制备
将磺酰氯(147mg,1.09mmol)在0–5℃慢慢滴加到化合物1c(200mg,0.91mmol)，反应液在25℃搅拌45分钟。浓缩反应液得到的残留物1d可直接用于下一步。
第四步(4R)-7,7-二甲基-4-(1-(甲基磺酰基)螺[吲哚啉-3,4'-哌啶]-1'-羰基)-6,9-二氧代-1-苯基-2,10-二氧杂-5,8-二氮杂十二烷-11-基异丁酸酯(1e)的制备
将化合物1d(60mg,0.11mmol)和伊布莫伦溶于二氯甲烷(3mL)，然后将氢氧化钠(22mg,0.22mmol)溶入5mL水后慢慢滴加入反应液，反应液在25℃搅拌2小时。有机层浓缩得到的残留物用制备色谱(乙腈/水)纯化后得到化合物1e(58mg,白色固体)，收率：72％。
MS m/z(ESI):687.0[M+1]+.
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).
第五步
将上述制备得到的化合物1e通过手性拆分的方式获得式(1)化合物，分离条件：色谱柱:Daicel CHIRALPAK IC_3,3.0*150mm,3μm，流动相:A/B:CO2/MeOH＝60/40，流速:1.5mL/min，柱温:37℃。
tR＝1.582min
MS m/z(ESI):687.0[M+1]+.
1H NMR(300MHz,dmso)δ7.80–7.49(m,2H),7.42–7.15(m,8H),7.08–6.85(m,2H),6.67–6.56(m,1H),4.97(d,J＝7.2Hz,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).
液质联用色谱LC-MS的测定用Agilent 1200 Infinity Series质谱仪。
实施例1无定形物
1.分析方法
1.1 X-射线粉末衍射(XRPD)
利用X-粉末衍射仪对样品进行晶型分析。样品的2θ扫描角度为3°到42°，扫描步长为0.02°，每步的扫描时间为0.2s。光管电压和电流分别为40kV、40mA。制样时将适量样品放到载样盘上，用勺子或玻璃片等工具压平，确保其表面光滑平整。
1.2热重分析(TGA)
采用TA Instruments TGA Discovery 550对样品进行分析。将样品放入去掉皮重的铝盘中，系统自动称重，然后在氮气的保护下将样品以10℃/min的速率从室温升高到指定温度。
1.3差示扫描量热法(DSC)
采用TA Instruments Discovery DSC 25对样品进行分析。将称量过的样品放入载样盘中，在氮气(50ml/min)的保护下将样品以10℃/min的速率从25℃升高到指定温度。
1.4动态水分吸附分析(DVS)
利用Intrinsic DVS(System Measurement System UK)对样品进行分析。测试样品量约为20～30mg。测试室的温度控制在25±1℃之间，相对湿度以10％/h的速率从0％升至90％再降至0％，每20s记录一次质量数据。
1.5扫描电镜(SEM)
利用Phenom pure+对样品进行分析。对样品进行喷金处理后放入仪器中进行测试。调整不同的放大倍数来获取样品晶习。
1.6偏振光显微镜(PLM)
利用偏光显微镜对样品进行分析，通过调节不同的放大倍数，得到晶体的形貌和微观结构。
1.7核磁分析(1H NMR)
采用Varian Inova 500MHz核磁分析仪对样品进行分析。
1.8液相方法(HPLC)
色谱条件：
色谱柱：C18柱；
运行时间：60min；
UV检测器：210nm；
流速：1.0mL/min；
进样体积：5μL。
2.制备方法
称取10g的式(1)所示化合物原料于100ml玻璃瓶中，室温下加入适量的DMSO至完全溶清，将溶清液滴加至100ml水中，室温搅拌析出固体后抽滤得固体。
3.结构性质表征
对所得固体进行检测，XRPD结果(图1)显示所得固体为式(1)所示化合物的无定形物，其1H NMR图谱如图6所示。DSC曲线(图2)显示所述无定形物的玻璃化转变温度在52.69℃左右，TGA曲线(图3)显示所述无定形物在100℃前有大约0.248％的失重。PLM(图4)和SEM(图5)显示所述无定形物为块状非晶体。DVS(图7)结果显示无定形物在80％湿度条件下略有引湿性，DVS测试前后固体的形态未改变(图8)。
4.溶解度测试
称量约10mg的无定形物于8mL的玻璃瓶中，室温下逐步加入溶剂。溶剂每次加入量为 5μL直到固体全部溶解为止，若加入8mL还未溶解，则停止加入溶剂。具体的实验结果如表1所示。
表1.式(1)所示化合物无定形物的溶解度测试结果(室温)
由上表可知，式(1)所示化合物无定形物在水、正庚烷、正己烷和环己烷中溶解度比较差，在其他溶剂中均有较高的溶解度。
实施例2晶型
1.分析方法
1.1 X射线粉末衍射(XRPD)
XRPD图在PANalytical X射线粉末衍射分析仪上采集，扫描参数如下所示：

1.2热重分析(TGA)和差示扫描量热(DSC)
TGA和DSC图分别在TA Discovery TGA 5500热重分析仪和TA Discovery DSC 2500差示扫描量热仪上采集，测试参数如下：
1.3动态水分吸附(DVS)
DVS曲线在通过SMS(Surface Measurement Systems)的DVS Intrinsic上采集。25℃时的相对湿度根据LiCl、Mg(NO3)2和KCl的潮解点进行校准。DVS测试参数如下所示：

1.4液态核磁(Solution NMR)
液态核磁谱图在Bruker 400M核磁共振仪(江苏集萃光电检测中心有限公司)上采集，DMSO-d6作为核磁测试溶剂。
2.制备方法
取约15mg的式(1)所示化合物原料固体于玻璃瓶中，向玻璃瓶中加入适量的丙酮和水的混合溶剂室温下进行悬浮搅拌，后抽滤得到固体。
3.结构性质表征
对上述方法制备得到的固体样品进行XRPD测试(如图9所示)，结果显示为晶体形式。TGA曲线(图10)显示所述晶体加热至150℃有1.1％的失重，DSC曲线(图11)显示在130.6℃(峰值温度)处观测到一个吸热峰。基于该晶型较小的TGA失重和在100℃前无DSC信号，推测该晶型为无水晶型。此外，对该晶型进行了25℃恒温条件下的DVS测试，以评估其引湿性。DVS结果如图12所示，该晶型在25℃/80％RH条件下的水分吸附为0.10％，表明该晶型几乎无引湿性。XRPD结果(图13)显示，该晶型在DVS测试前后未发生晶型变化。
3.1 X-射线粉末衍射(XRPD)
利用X-粉末衍射仪对样品进行晶型分析。制样时将适量样品放到载样盘上，用勺子或玻璃片等工具压平，确保其表面光滑平整。结果如表2和图9所示。
表2.式(1)所示化合物晶型的XRPD衍射峰数据


4.溶解度测试
称量约5mg的晶型样品于8mL的玻璃瓶中，室温下逐步加入溶剂。溶剂每次加入量为10μL直到固体全部溶解为止，若加入5mL还未溶解，则停止加入溶剂。具体的实验结果如表3所示。
表3.式(1)所示化合物晶型的溶解度测试结果(室温)
如表3所示，该晶型在水、正庚烷、甲基叔丁基醚中溶解度比较差，在其他溶剂中均有较高的溶解度。
实施例3稳定性测试一
称取一定量的晶体和无定形原料于液相小瓶中，共制备5份。分别放入80℃(敞口)、25℃/60％RH(敞口)和40℃/75％RH(敞口)以及光照稳定性箱(闭口，5000±500Lx)中，利用液相测定80℃一天，光照10天的稳定性以及25℃,60％RH和40℃,75％RH 1周的化学稳定性，并且测定固体的XRPD。具体结果如表4所示。结果显示，式(1)所示化合物的无定形物和晶体形式在大多数条件下具有较好的物理和化学稳定性，但是在光照条件下，晶体形式的稳定性要明显优于无定形物。
表4.式(1)所示化合物无定形物和晶型的稳定性测试结果

加速稳定性试验：
称取晶体样品，每份4g，在温度40±2℃，相对湿度75±5％的恒温恒湿箱中进行观察。分别在0、1、3个月取样分析，利用液相测定样品的稳定性，利用XRPD测定晶型。具体结果如表5所示。结果显示，式(1)所示化合物的晶型在加速条件下依旧具有良好的稳定性。
表5.式(1)所示化合物晶型的加速稳定性测试结果
实施例4稳定性测试二
包装要求：稳定性样品需模拟市售包装，内两层为药用聚乙烯平底内袋，第一层内袋扎带封口，第二层内袋热封口，外一层铝箔袋热封口，铝箔袋与第二层内袋之间放一包干燥剂，置于纸板桶中。
1.1加速稳定性试验
取式(1)所示化合物晶型，按上述包装要求进行包装，常规样品分装5份，每份10g，在温度40±2℃，相对湿度75±5％的恒温恒湿箱中进行观察。分别在0、1、2、3、6月取样分析。检测项目、检测方法及技术要求详见表6，检测结果与0月比较。
1.2长期稳定性试验
取式(1)所示化合物晶型，按包装要求所述包装，常规样品分装10份，每份10g；在温度30±2℃，相对湿度65±5％的恒温恒湿箱中进行观察。分别在0、3、6月取样分析。检测项目、检测方法及技术要求详见表7，检测结果与0月比较。
表6：加速试验(40±2℃/75±5％RH)

注：0个月的数据由放行结果获得；ND代表未检出。
表7：长期试验(30±2℃/65±5％RH)

备注：0个月的数据由放行结果获得；ND代表未检出。
加速稳定性试验结果显示，6个月与0个月比较，检测结果无显著变化，且未出现大于0.10％的新杂质峰，表明式(1)所示化合物晶型按照以上现有包装方式在加速条件下6个月稳定。
长期稳定性试验结果显示，6个月与0个月比较，检测结果无显著变化，且未出现大于0.10％的新杂质峰，表明式(1)所示化合物晶型按照以上现有包装方式在长期条件下6个月稳定。
实施例4
按照实施例2方法进行扩大化生产，能够制备得到10kg规格的固体样品，经XRPD测试结果显示为图9所示的晶体形式。
实施例5生物学活性测试
测试例1、本发明化合物对人源GHSR活性的测定
该方法用来测定本发明中的化合物对人源GHSR/CHO稳转株细胞中所表达的人源GHSR蛋白活性的激动作用。
一、试验材料及仪器
1、培养基
F12(Gibco,Cat#11765-047)；
FBS(Corning,Cat#35-076-CV)；
Geneticin(Invitrogen,Cat#10131)；
Penicillin/Streptomycin(Invitrogen,Cat#15140)。
2、试剂
Fluo-4Direct(Invitrogen,Cat#F10471)；
HBSS(Gibco,Cat#14025076)；
HEPES(Gibco,Cat#15630080)；
Bonine Serum Albumin(Sgima,Cat#B2064-100G)。
3、仪器耗材
384 well Poly-D-Lysine protein coating plate(Greiner,Cat#781946)；
FLIPR(Molecular Devices)；
Vi-cell XR Cell Viability Analyzer(Beckman Coulter)；
Incubator(Thermo)。
二、实验步骤
化合物梯度配制：将Ghrelin和本发明化合物5倍稀释配制成10个浓度梯度，然后转移到化合物板中，每孔900nL。
配置缓冲液：HBSS(1X):HEPES(1M)＝49:1，加入0.5％BSA。
将含人源GHSR/CHO稳转株细胞接种于384孔板中，过夜后取出细胞板，弃掉培养基，每孔缓慢加入20μL缓冲液，然后每孔各加入20μL 2X Fluo-4DirectTM No-wash Loading Buffer。细胞板放入37℃5％CO2培养箱中孵育50min，取出细胞板室温放置10min。在化合物板中每孔加入30uL缓冲液；另外准备一块缓冲液板，每孔加入30μL缓冲液。对于化合物激动作用的测试：使用FLIPR仪器，运行软件。转移10μL缓冲液于细胞板，读取荧光信号值。转移10uL化合物于细胞板，读取荧光信号值。使用FLIPR程序计算从第91个信号点到第230个信号点的最大值-最小值。化合物的EC50值可采用不同浓度对应的荧光值，经软件计算得到。
三、实验结果：
化合物1e对人源GHSR激动活性通过以上的实验进行测定，测得的EC50值为14.6nM，表明化合物1e对人源GHSR有较好的激动活性。由此确认，式(1)化合物对人源GHSR有较好的激动活性。
测试例2、Caco-2细胞转运实验
研究中的转运缓冲液为含有10.0mM的HEPSS的HBSS，pH 7.40±0.05。待测化合物以2.00μM双向测试，DMSO的终浓度要求至小于1％。将细胞板在37±1℃的CO2培养箱中，在5％湿度的CO2和饱和湿度下静置温育2小时。将所有样品与含有内标的乙腈混合后，以3200xg离心10分钟。测试化合物，将100μL上清液用100μL超纯水稀释，用于LC-MS/MS分析。使用分析物/内标物的峰面积比，通过LC-MS/MS方法对起始溶液，供体溶液和接收 器溶液中测试化合物和对照化合物(Digoxin为模型验证化合物，ibutamoren为阳性对照)的浓度进行定量。转运测定后，应用萤光素黄排斥测定来确定Caco-2细胞单层完整性。
表8实施例化合物1e Caco-2细胞转运实验
以上数据显示，模型构建成功，化合物1e的细胞通透性显著优于参比化合物ibutamoren。
测试例3、化合物1e大鼠体内代谢实验对比
化合物1e采用前药设计，通过大鼠体内代谢实验检验活性成分ibutamoren的量，进而评估候选物与阳性对照ibutamoren的优劣势。
实验操作：
6只动物在给药前两天进行第一次禁食，禁食至少12小时后，统一给予饲料；在给药前一天进行第二次禁食，禁食至少12小时，给药4小时后恢复供食。每次禁食时间不超过20小时。期间自由饮水。给药前两天内由相同负责人通过触摸和抓取动物，对动物进行适应性训练，适应最少1次/天。
第一次给药前，根据动物体重将动物分为2组。每组三只，第1组动物通过单次灌胃给予化合物5的药液(配置方法：中链甘油三酯/聚乙二醇1000维生素E琥珀酸酯/乙醇/丙二醇/水＝6/2/1/1/90，1mg/mL)；第2组动物通过单次灌胃给予ibutamoren药液(水，1mg/mL)。给药体积均为3mL/kg。在给药前称量动物体重，根据体重计算给药体积。
样品采集时间：给药前(约-0.25h)及给药后0.083、0.25、0.5、1、1.5、2、3、5、7、10h；在每个规定的时间点使用异氟烷短暂麻醉动物，通过颈静脉穿刺采集全血样品(每组约0.23mL)，定量取50μL全血样品，加入到装有50μL预冷1mM的PMSF甲醇溶液的EP管中，涡旋～3s，立即加入250μL含内标的沉淀剂，涡旋～5s，离心15min取上清，用于LC-MS/MS分析。
表9大鼠体内药物代谢实验数据
以上数据显示，本发明化合物1e在大鼠灌胃给药的药物代谢实验中，Cmax和AUC明显优于阳性对照ibutamoren，具有更好的成药性。由此确认，式(1)化合物具有更好的成药性。
通过上述实验表明，本发明制备所示化合物的晶型及无定形物纯度高，在高温、高湿的条件下稳定性良好，有利于药物发挥作用，对于工艺优化可满足生产工艺稳定、可重复可控，能够适应于工业化生产。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种式(1)所示化合物的晶型，其中，所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在5.43±0.20°、12.28±0.20°、17.91±0.20°处具有特征峰；
  2. 根据权利要求1所述的晶型，其中，所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在5.43±0.20°、12.28±0.20°、17.91±0.20°、18.54±0.20°、18.87±0.20°、20.54±0.20°、21.69±0.20°处具有特征峰。
  3. 根据权利要求1或2所述的晶型，其中，所述晶型使用Cu-Kα辐射，以2θ角度表示的X-射线粉末衍射在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°、21.86±0.20°处具有特征峰。
  4. 根据权利要求1-3任一项所述的晶型，其中，所述晶型具有基本如图9所示的XRPD图谱；优选地，所述晶型为无水物。
  5. 权利要求1-4任一项所述晶型的制备方法，其中，所述制备方法包括如下步骤：将式(1)所示的化合物溶解于溶剂A中，降温析出固体，得到所述晶型；
    所述溶剂A选自甲醇、乙醇、异丙醇、叔丁醇、正丁醇、丙酮、四氢呋喃、甲基四氢呋喃、甲酸乙酯、乙酸乙酯、乙酸异丙酯、正己烷、正庚烷、环己烷、甲基叔丁醚、甲苯、二氯甲烷、氯仿、DMSO、水、乙腈、异丙醚中的一种或两种以上。
  6. 式(1)所示化合物的无定形物，其中，所述无定形物的XRPD图谱没有明显衍射峰；
    优选地，所述无定形物具有基本如图1所示的XRPD图谱。
  7. 权利要求6所述的无定形物的制备方法，其中，所述制备方法包括如下步骤：将式(1) 所示的化合物溶解于溶剂B中，将所得溶清液加至溶剂C中，搅拌析出固体，得到所述无定形物；
    所述溶剂B为式(1)所示化合物的良溶剂，所述溶剂C为式(1)所示化合物的不良溶剂。
  8. 一种药物组合物，其中，所述药物组合物含有权利要求1-4任一项所述的晶型和/或权利要求6所述的无定形物；
    优选地，所述药物组合物还含有药学上可接受的辅料；
    优选地，所述药物组合物还包括除上述晶型和/或无定形物的第二活性成分，例如所述第二活性成分为与生长发育相关的药物；
    优选地，所述药物组合物为制剂，例如为GHSR激动剂。
  9. 权利要求1-4任一项所述的晶型、权利要求6所述的无定形物和/或权利要求8所述的药物组合物在制备诊断、预防和/或治疗生长激素缺乏或生长激素依赖性疾病(或病症)制剂中的应用。
  10. 一种诊断、预防和/或治疗生长激素缺乏或生长激素依赖性疾病(或病症)的方法，包括将治疗有效量的权利要求1-4任一项所述的晶型、权利要求6所述无定形物或权利要求8所述的药物组合物施用于患者。
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CN115010644A (zh) * 2022-06-09 2022-09-06 五邑大学 一种2,3-二取代吲哚啉类化合物的制备方法
WO2022228318A1 (zh) * 2021-04-25 2022-11-03 长春金赛药业有限责任公司 含吲哚啉螺环类衍生物、其制备方法及其在医药上的应用

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CN1092767A (zh) * 1992-12-11 1994-09-28 麦克公司 促进生长激素释放的螺哌啶和同系物
CN1901908A (zh) * 2003-11-04 2007-01-24 伊利舍医药品公司 治疗性化合物及其用途
CN102906093A (zh) * 2010-04-02 2013-01-30 欧洲筛选有限公司 新型nk-3受体选择性拮抗剂化合物、药物组合物以及在nk-3受体介导的疾病中的使用方法
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