WO2024083164A1 - Solvate of indoline spiro compound, and crystal form thereof, preparation method therefor and use thereof - Google Patents

Solvate of indoline spiro compound, and crystal form thereof, preparation method therefor and use thereof Download PDF

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Publication number
WO2024083164A1
WO2024083164A1 PCT/CN2023/125267 CN2023125267W WO2024083164A1 WO 2024083164 A1 WO2024083164 A1 WO 2024083164A1 CN 2023125267 W CN2023125267 W CN 2023125267W WO 2024083164 A1 WO2024083164 A1 WO 2024083164A1
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formula
compound represented
anisole
solvate
crystal form
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PCT/CN2023/125267
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French (fr)
Chinese (zh)
Inventor
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 WO2024083164A1 publication Critical patent/WO2024083164A1/en

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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

  • Patent application number 202211296034.0 filed with the State Intellectual Property Office of China on October 21, 2022, with the invention name “solvate of indoline spirocyclic compound and its crystal form, preparation method and application”
  • patent application number 202311300572.7 filed with the State Intellectual Property Office of China on October 9, 2023, with the invention name “solvate of indoline spirocyclic compound and its crystal form, preparation method and application”.
  • the full text of the prior application is incorporated into this application by reference.
  • the invention belongs to the field of pharmaceutical compounds, and specifically relates to a solvate of an indoline spirocyclic compound and its crystal form, a preparation method and application.
  • 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 multiple areas of the hypothalamus and outside the hypothalamus, including the pituitary gland, arcuate nucleus of the hypothalamus, ventromedial nucleus, etc.
  • GHSR is also lowly expressed 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 compound has the structural formula:
  • 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.
  • 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.
  • solid pharmaceutical 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 medication stages, has become a technical problem that needs to be solved urgently.
  • the present invention provides an anisole solvate of a compound represented by formula (1),
  • the molar ratio of anisole to the compound represented by formula (1) in the solvate is (1-1.1):1, preferably 1:1.
  • the solvate has a structure shown in the following formula (II):
  • the present invention also provides a crystalline form of anisole solvate of the compound represented by formula (1), wherein the crystalline form uses Cu-K ⁇ radiation and X-ray powder diffraction expressed in 2 ⁇ angles has a characteristic peak at 7.07 ⁇ 0.20°.
  • the crystalline form uses Cu-K ⁇ radiation, and X-ray powder diffraction expressed in 2 ⁇ angles has characteristic peaks at 7.07 ⁇ 0.20°, 15.73 ⁇ 0.20°, and 17.83 ⁇ 0.20°.
  • the crystalline form uses Cu-K ⁇ radiation
  • the X-ray powder diffraction expressed in 2 ⁇ 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°.
  • the crystalline form has an XRPD pattern substantially as shown in FIG. 1 .
  • the crystalline form has a TGA spectrum substantially as shown in FIG. 2 .
  • the crystal form has a sharp endothermic peak at a peak temperature of 82.4 ⁇ 2°C.
  • the crystalline form has a DSC spectrum substantially as shown in FIG. 3 .
  • 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 the anisole solvate crystalline form by dissolution crystallization, gas-liquid diffusion, gas-solid permeation, etc. Prepared by method.
  • 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
  • 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-h
  • the dissolving is performed at room temperature or under heating conditions.
  • the standing time is more than 2 days, such as standing for 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
  • the method further comprises post-treatment of the solid, such as filtering and/or washing.
  • the present invention also provides a pharmaceutical composition comprising anisole solvate of the compound represented by formula (1) and/or its crystal form.
  • the pharmaceutical composition further contains pharmaceutically acceptable excipients, such as but not limited to excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents and regulators.
  • pharmaceutically acceptable excipients such as but not limited to excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents and regulators.
  • excipients such as but not limited to excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents and regulators.
  • flavoring agents such as but not limited
  • 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.
  • the solvate and/or its crystalline form and the second active ingredient may be administered separately or together during treatment.
  • the present invention also provides the use of the above-mentioned solvate, crystal 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 disorders).
  • the disease 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, 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, and healing of wounds and bones.
  • 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
  • the agent may be a GHSR agonist.
  • the present invention also provides the use of the crystal form in purifying the compound represented by formula (1).
  • the present invention also provides a preparation containing the above-mentioned solvate and/or crystal form, 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 solvate, crystal form or pharmaceutical composition to a patient.
  • the disease has the definitions as indicated above.
  • the present invention provides an anisole solvate of a compound represented by formula (1), a crystal form thereof, a preparation method and a use thereof.
  • the crystal form of the compound represented by formula (1) obtained by the present invention has good solubility, low hygroscopicity and good repeatability, and is suitable for drug development. At the same time, it can also be used for impurity removal and purification of the compound represented by formula (1).
  • therapeutically effective amount refers to the amount of the crystalline form, amorphous substance, and second active ingredient of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases 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 the disease condition to be 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 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 based on, whether it is administered in combination with other compounds, the time arrangement 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.
  • FIG1 is an XRPD spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
  • FIG2 is a TGA spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
  • FIG3 is a DSC spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
  • FIG4 is a 1 H NMR spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
  • FIG5 is a PLM diagram of the anisole solvate crystalline form of the compound represented by formula (1);
  • FIG6 is a DVS diagram of the anisole solvate crystalline form of the compound represented by formula (1).
  • 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:
  • 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.
  • 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 samples were analyzed using a polarizing microscope, and the morphology and microstructure of the crystals were obtained by adjusting different magnifications.
  • Chromatographic column C18 column
  • UV detector 210nm
  • Injection volume 5 ⁇ L.
  • LC-MS Liquid chromatography-mass spectrometry
  • the TGA results show that the sample loses 4.8% of its weight when heated to 90°C, and loses 12.9% of its weight when further heated to 200°C.
  • the DSC results Figure 3) show that an endothermic peak is observed at 82.4°C (peak temperature).
  • the 1 H NMR results show that the molar ratio of the residual anisole in the sample to the compound represented by formula (1) is about 1.1:1 ( ⁇ 14.2%, wt%), and the large weight loss of TGA (17.7%) is speculated to be mainly derived from the desorption of anisole. Combined with the sample characterization results and the preparation conditions, it is speculated that the crystals are anisole solvates of the compound represented by formula (1).
  • the crystal was subjected to PLM and DVS measurements, the PLM measurement result of the crystal is shown in FIG5 , and the DVS measurement result thereof is shown in FIG6 .
  • 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.
  • 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 n-heptane: anisole uniform solvent for 5 to 10 minutes, filter with suction, and dry the filter cake.
  • 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 drop n-heptane into the system, stir for 12 hours after the addition is completed, filter with suction, soak the filter cake with n-heptane: anisole uniform solvent for 5 to 10 minutes, filter with suction, and dry the filter cake.
  • 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-4 Direct 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 CO2 incubator at 37 ⁇ 1 °C, 5% humidity of CO2 and saturated humidity for 2 hours. All samples were mixed with acetonitrile containing internal standard and centrifuged at 3200xg for 10 minutes. Test compounds, 100 ⁇ L of supernatant was diluted with 100 ⁇ L of ultrapure water for LC-MS/MS analysis.
  • test compounds and control compounds (Digoxin as a model validation compound and ibutamoren as a positive control) in the starting solution, donor solution and receiver solution were quantified by LC-MS/MS method using the peak area ratio of analyte/internal standard. After the transport assay, 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

Provided are an anisole solvate of an indoline spiro compound represented by formula (1), and a crystal form thereof, a preparation method therefor and the use thereof. The X-ray powder diffraction pattern of the crystal form comprises characteristic peaks at 7.07±0.20° 2θ, as determined by using Cu-Kα radiation. The provided solvate and the crystal form thereof have high purity and good solubility, are beneficial to drug formation, and can also be used for impurity removal and purification of the compound as represented by formula (1).

Description

吲哚啉螺环类化合物的溶剂化物及其晶型、制备方法和应用Solvate of indoline spirocyclic compound and its crystal form, preparation method and application
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202211296034.0，发明名称为“吲哚啉螺环类化合物的溶剂化物及其晶型、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202311300572.7，发明名称为“吲哚啉螺环类化合物的溶剂化物及其晶型、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。This application claims the priority of the following two prior applications: Patent application number 202211296034.0 filed with the State Intellectual Property Office of China on October 21, 2022, with the invention name “solvate of indoline spirocyclic compound and its crystal form, preparation method and application” and patent application number 202311300572.7 filed with the State Intellectual Property Office of China on October 9, 2023, with the invention name “solvate of indoline spirocyclic compound and its crystal form, preparation method and application”. The full text of the prior application is incorporated into this application by reference.
技术领域Technical Field
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溶剂化物及其晶型、制备方法和应用。The invention belongs to the field of pharmaceutical compounds, and specifically relates to a solvate of an indoline spirocyclic compound and its crystal form, a preparation method and application.
背景技术Background technique
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。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.
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。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。 The release of GH is believed to treat physiological or pathophysiological conditions characterized by defective secretion of growth hormone, as well as conditions that are improved by the anabolic effects of growth hormone. 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。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). In most cases, 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。GHSR has two subtypes, 1a and 1b. Subtype 1a is a functional receptor subtype, and the function of subtype 1b needs further study. In the central nervous system, GHSR 1a is distributed in multiple areas of the hypothalamus and outside the hypothalamus, including the pituitary gland, arcuate nucleus of the hypothalamus, ventromedial nucleus, etc. In the periphery, GHSR is also lowly expressed 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.
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。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.
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。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 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. Currently, there are no drugs on the market for this indication. Among them, ulimorelin and relamorelin have entered Phase III clinical studies.
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。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.
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。In order to overcome the above technical problems, the applicant independently developed a chemical with a completely new molecular structure. The compound has the structural formula: 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 a method for diagnosing, preventing and/or treating growth hormone-dependent diseases or conditions; 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 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.
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。At the same time, developing solid pharmaceutical 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 medication stages, has become a technical problem that needs to be solved urgently.
发明内容Summary of the invention
专利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。All contents involved in patent PCT/CN2022/088656 are added to the present invention by reference.
为了改善上述技术问题，本发明提供了一种式(1)所示化合物的苯甲醚溶剂化物，
In order to improve the above technical problems, the present invention provides an anisole solvate of a compound represented by formula (1),
根据本发明的实施方案，所述溶剂化物中苯甲醚与式(1)所示化合物的摩尔比为(1～1.1)：1，优选1：1。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.
根据本发明的实施方案，所述溶剂化物具有如下式(II)所示结构：
According to an embodiment of the present invention, the solvate has a structure shown in the following formula (II):
根据本发明的实施方案，本发明还提供了一种式(1)所示化合物的苯甲醚溶剂化物晶型，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7.07±0.20°处具有特征峰。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 uses Cu-Kα radiation and X-ray powder diffraction expressed in 2θ angles has a characteristic peak at 7.07±0.20°.
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7.07±0.20°、15.73±0.20°、17.83±0.20°处具有特征峰。According to an embodiment of the present invention, the crystalline form uses Cu-Kα radiation, and X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 7.07±0.20°, 15.73±0.20°, and 17.83±0.20°.
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°、26.59±0.20°处具有特征峰。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°.
根据本发明的实施方案，所述晶型具有基本如图1所示的XRPD图谱。According to an embodiment of the present invention, the crystalline form has an XRPD pattern substantially as shown in FIG. 1 .
根据本发明的实施方案，所述晶型具有基本如图2所示的TGA图谱。According to an embodiment of the present invention, the crystalline form has a TGA spectrum substantially as shown in FIG. 2 .
根据本发明的实施方案，所述晶型在峰值温度为82.4±2℃处具有一个尖锐的吸热峰。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.
根据本发明的实施方案，所述晶型具有基本如图3所示的DSC图谱。According to an embodiment of the present invention, the crystalline form has a DSC spectrum substantially as shown in FIG. 3 .
本发明还提供所述式(I)所示的化合物的苯甲醚溶剂化物晶型的制备方法，包括以式(I)所示的化合物为原料，通过溶析结晶、气液扩散、气固渗透等方 法制备得到。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 the anisole solvate crystalline form by dissolution crystallization, gas-liquid diffusion, gas-solid permeation, etc. Prepared by method.
本发明还提供上述晶型的制备方法，包括如下步骤：(1)将式(1)所示的化合物溶解于苯甲醚，然后加入反溶剂，搅拌即得；和/或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
(2)将式(1)所示化合物溶解于苯甲醚中，容器盖盖后扎眼，将所述容器置于含反溶剂的更大体积的容器内，盖盖密封，在室温下静置1天以上，即得；和/或(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; and/or
(3)取一定量式(1)所示化合物置于较小容器中，另取较大容器并向其中加入苯甲醚，将较小容器敞口置于较大容器中，密封后室温下静置1天以上，即得。(3) Place a certain amount of the compound represented by formula (1) in a smaller container, take another larger container and add anisole therein, place the smaller container open in the larger container, seal it and let it stand at room temperature for more than 1 day to obtain the product.
根据本发明的实施方案，所述反溶剂包括甲醇、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。优选地，所述反溶剂选自水、正己烷、正庚烷、环己烷中的一种或两种以上。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.
根据本发明的实施方案，所述溶解在室温或加热条件下进行。According to an embodiment of the present invention, the dissolving is performed at room temperature or under heating conditions.
根据本发明的实施方案，所述静置时间为2天以上，如静置2、3、4、5、6、7、8、9或10天。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.
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。According to an embodiment of the present invention, the method further comprises post-treatment of the solid, such as filtering and/or washing.
本发明还提供一种药物组合物，含有式(1)所示化合物的苯甲醚溶剂化物和/或其晶型。The present invention also provides a pharmaceutical composition comprising anisole solvate of the compound represented by formula (1) and/or its crystal form.
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。According to an embodiment of the present invention, the pharmaceutical composition further contains pharmaceutically acceptable excipients, such as but not limited to excipients, fillers, lubricants, binders, disintegrants, inorganic salts, solvents, dissolution aids, suspending agents, isotonic agents, buffers, preservatives, antioxidants, colorants, foaming agents and regulators. One or more of flavoring agents, etc.
根据本发明的实施方案，所述药物组合物还包括除上述溶剂化物和/或其晶型的第二活性成分，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。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.
在一些实施方案中，所述溶剂化物和/或其晶型与第二活性成分在治疗时可以分开施用，或者共同施用。In some embodiments, the solvate and/or its crystalline form and the second active ingredient may be administered separately or together during treatment.
本发明还提供上述溶剂化物、晶型和/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。The present invention also provides the use of the above-mentioned solvate, crystal 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 disorders).
根据本发明的实施方案，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。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 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, 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, and healing of wounds and bones.
在一些实施方案中，所述制剂可以为GHSR激动剂。In some embodiments, the agent may be a GHSR agonist.
本发明还提供所述的晶型在纯化式(1)所示化合物中的应用。The present invention also provides the use of the crystal form in purifying the compound represented by formula (1).
本发明还提供一种制剂，含有上述溶剂化物和/或晶型，或由上述药物组合物制备得到。The present invention also provides a preparation containing the above-mentioned solvate and/or crystal form, or prepared from the above-mentioned pharmaceutical composition.
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。According to an embodiment of the present invention, 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.
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。 According to an embodiment of the present invention, 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.
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溶剂化物、晶型或所述的药物组合物施用于患者。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.
根据本发明的实施方案，所述疾病具有如上文所示的限定。According to an embodiment of the present invention, the disease has the definitions as indicated above.
本发明的有益效果Beneficial Effects of the Invention
本发明提供了如式(1)所示的化合物的苯甲醚溶剂化物及其晶型、制备方法和用途。本发明获得的式(1)所示化合物的晶型具备良好的溶解性，引湿性低、可重复性好，适合药物开发，同时，亦可以用于式(1)所示化合物的除杂纯化。
The present invention provides an anisole solvate of a compound represented by formula (1), a crystal form thereof, a preparation method and a use thereof. The crystal form of the compound represented by formula (1) obtained by the present invention has good solubility, low hygroscopicity and good repeatability, and is suitable for drug development. At the same time, it can also be used for impurity removal and purification of the compound represented by formula (1).
术语定义与说明Definition and explanation of terms
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。Unless otherwise specified, the definitions of terms recorded in the specification and claims of this application, including their definitions as examples, exemplary definitions, preferred definitions, definitions of specific compounds in the examples, etc., may be arbitrarily combined and coupled with each other. Such combinations and couplings shall fall within the scope of the description of this application.
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。 The term "therapeutically effective amount" refers to the amount of the crystalline form, amorphous substance, and second active ingredient of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases 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 the disease condition to be 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 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 based on, whether it is administered in combination with other compounds, the time arrangement of administration, the tissue to which it is administered, and the physical delivery system carried.
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。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.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1为式(1)所示化合物苯甲醚溶剂化物晶型的XRPD图谱；FIG1 is an XRPD spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
图2为式(1)所示化合物苯甲醚溶剂化物晶型的TGA图谱；FIG2 is a TGA spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
图3为式(1)所示化合物苯甲醚溶剂化物晶型的DSC图谱；FIG3 is a DSC spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
图4为式(1)所示化合物苯甲醚溶剂化物晶型的1H NMR图谱；FIG4 is a 1 H NMR spectrum of the anisole solvate crystalline form of the compound represented by formula (1);
图5为式(1)所示化合物苯甲醚溶剂化物晶型的PLM图；FIG5 is a PLM diagram of the anisole solvate crystalline form of the compound represented by formula (1);
图6为式(1)所示化合物苯甲醚溶剂化物晶型的DVS图。FIG6 is a DVS diagram of the anisole solvate crystalline form of the compound represented by formula (1).
具体实施方式Detailed ways
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。The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions 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 included in the scope that the present invention is intended to protect.
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。Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.
分析方法Analytical method
1.X射线粉末衍射(XRPD)1. X-ray powder diffraction (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：

The XRPD pattern was collected on a PANalytical X-ray powder diffraction analyzer, and the scanning parameters were as follows:

2.热重分析(TGA)和差示扫描量热(DSC)2. Thermogravimetric analysis (TGA) and differential scanning calorimetry (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：
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:
3.液态核磁(SolutionNMR)3. 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。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.
4.动态水分吸附分析(DVS)4. Dynamic moisture sorption analysis (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。The samples were analyzed using Intrinsic DVS (System Measurement System UK). The 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.
5.偏振光显微镜(PLM)5. Polarized Light Microscopy (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。 The samples were analyzed using a polarizing microscope, and the morphology and microstructure of the crystals were obtained by adjusting different magnifications.
6.液相方法(HPLC)6. Liquid phase method (HPLC)
色谱条件：Chromatographic conditions:
色谱柱：C18柱；Chromatographic column: C18 column;
运行时间：60min；Running time: 60min;
UV检测器：210nm；UV detector: 210nm;
流速：1.0mL/min；Flow rate: 1.0 mL/min;
进样体积：5μL。Injection volume: 5 μL.
制备例 式(1)所示化合物的制备Preparation Example Preparation of the compound represented by formula (1)
(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
(4R,11R)-7,7-Dimethyl-4-(1-(methylsulfonyl)spiro[indoline-3,4'-piperidinyl]-1'-carbonyl)-6,9-dioxo-1-phenyl-2,10-dioxa-5,8-diazadodec-11-yl isobutyrate
第一步 邻-(1-氯乙基)硫代碳酸乙酯(1b)的制备Step 1 Preparation of ethyl o-(1-chloroethyl)thiocarbonate (1b)
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。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 then added dropwise to the reaction solution, which was stirred at 25° C. for 16 hours. The reaction solution was separated into layers, the organic layer was washed with water (20 mL), dried over anhydrous sodium sulfate, and concentrated to obtain compound 1b (2.0 g, yield: 56%) as a yellow oil.
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). 1 H NMR (400 MHz, CDCl 3 ) δ 6.60 (q, J = 5.8 Hz, 1H), 2.97-2.86 (m, 2H), 1.81 (d, J = 5.8 Hz, 3H), 1.34 (t, J = 7.4 Hz, 3H).
第二步1-{[(乙硫基)羰基]氧基}乙基异丁酸酯(1c)的制备Step 2 Preparation of 1-{[(ethylthio)carbonyl]oxy}ethyl isobutyrate (1c)
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)。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, and the reaction solution was stirred at 55°C for 48 hours. The reaction solution was quenched by adding water (20 mL), extracted with ethyl acetate (20 mL), washed with saturated sodium bicarbonate (3x30 mL), washed with saturated brine (2x20 mL), and concentrated to obtain the residue 1c (850 mg, light yellow oil).
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). 1 H NMR (400 MHz, CDCl 3 ): δ 6.94 (q, J = 5.4 Hz, 1H), 2.92-2.83 (m, 2H), 2.59 (dt, J = 4.3, 2.4 Hz, 1H), 1.50 (d, J = 5.5 Hz, 3H), 1.32 (td, J = 7.3, 3.6 Hz, 3H), 1.20 (d, J = 7.0 Hz, 6H).
第三步1-((氯羰基)氧基异丁酸乙酯(1d)的制备Step 3 Preparation of ethyl 1-((chlorocarbonyl)oxyisobutyrate (1d)
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。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 solution was stirred at 25°C for 45 minutes. The reaction solution was concentrated to obtain the residue 1d which was used directly in the next step.
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-diazadodecane-11-yl isobutyrate (1e)
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％。Compound 1d (60 mg, 0.11 mmol) and Ibumoren were dissolved in dichloromethane (3 mL), and then sodium hydroxide (22 mg, 0.22 mmol) was dissolved in 5 mL of water and slowly added dropwise to the reaction solution, which was stirred at 25°C for 2 hours. The organic layer was concentrated and the residue was purified by preparative chromatography (acetonitrile/water) to obtain compound 1e (58 mg, white solid), with a yield of 72%.
MS m/z(ESI):687.0[M+1]+.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). 1 H NMR (400 MHz, 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＝6.4 Hz, 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the fifth step
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℃。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: CO 2 /MeOH=60/40, flow rate: 1.5 mL/min, column temperature: 37°C.
tR＝1.582mint R =1.582min
MS m/z(ESI):687.0[M+1]+.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). 1 H NMR (300 MHz, 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=7.2 Hz, 1H), 4.44 (dd, J=30.4, 12.1 Hz, 3H), 3.90 (d, J=6.8 Hz, 3H), 3.72–3.45 (m, 2H), 3.15 (s, 1H), 3.04 (d, J=2.2 Hz, 3H), 2.80 (s, 1H), 1.66 (s, 4H), 1.45–1.28 (m, 9H), 1.06 (d, J=6.9 Hz, 6H).
液质联用色谱LC-MS的测定用Agilent 1200Infinity Series质谱仪。Liquid chromatography-mass spectrometry (LC-MS) was performed using an Agilent 1200 Infinity Series mass spectrometer.
实施例1制备方法Example 1 Preparation method
(1)溶析结晶(1) Dissolution crystallization
取一定量的式(1)所示化合物于4mL玻璃瓶中，加入0.3mL苯甲醚使其溶解，然后快速加入反溶剂，在磁力作用下搅拌5小时后过滤，所得固体在50℃下烘干过夜。实验所用溶剂及结果如表1所示。所得白色固体经鉴定，均为式(1)所示化合物苯甲醚溶剂化物的晶体。A certain amount of the compound represented by formula (1) was placed in a 4 mL glass bottle, 0.3 mL of anisole was added to dissolve it, and then the anti-solvent was quickly added. After stirring for 5 hours under magnetic force, the mixture was filtered and the obtained solid was dried at 50° C. overnight. The solvents used in the experiment and the results are shown in Table 1. The obtained white solids were identified as crystals of anisole solvate of the compound represented by formula (1).
表1.溶析实验条件及结果
Table 1. Dissolution experimental conditions and results
(2)气液扩散(2) Gas-Liquid Diffusion
在2mL小瓶中，将一定量的式(1)所示化合物溶解在0.2mL苯甲醚中，盖盖后扎眼，将小瓶置于含反溶剂的30mL大瓶中，盖盖密封，在室温下放7天，所用溶剂及结果如表2所示。析出固体经鉴定，均为式(1)所示化合物苯甲醚溶剂化物的晶体。 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, and the vial was placed in a 30 mL large bottle containing an anti-solvent, capped and sealed, and left 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).
表2.气液扩散实验条件及结果
Table 2. Gas-liquid diffusion experimental conditions and results
(3)气固渗透(3) Gas-solid permeability
称量约15mg式(1)所示化合物于3mL小瓶中，另取20mL小瓶并向其中加入苯甲醚，将3mL小瓶敞口置于20mL小瓶中，密封后室温下静置5天后收集固体并进行XRPD测试，为式(1)所示化合物苯甲醚溶剂化物的晶体。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 open 3 mL vial was placed in a 20 mL vial, and the mixture was sealed and 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).
实施例2结构表征Example 2 Structural Characterization
(1)X-射线粉末衍射(XRPD)结果显示上述三种方法所得固体均为同一晶型，该晶型的具体表征如表3和图1所示。(1) X-ray powder diffraction (XRPD) results show that the solids obtained by the above three methods are all of the same crystal form, and the specific characterization of the crystal form is shown in Table 3 and Figure 1.
表3.式(1)所示化合物苯甲醚溶剂化物晶型的XRPD衍射峰数据

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

TGA(图2)结果显示样品加热至90℃有4.8％的失重，继续加热至200℃有12.9％的失重。DSC(图3)结果显示样品在82.4℃(峰值温度)处观测到一个吸热峰。1H NMR结果(图4)显示，样品中残留的苯甲醚与式(1)所示化合物的摩尔比约为1.1:1(～14.2％，wt％)，TGA较大的失重(17.7％)推测主要来源于苯甲醚的脱附。结合样品表征结果和制备的条件，推测所述晶体为式(1)所示化合物苯甲醚溶剂合物。The TGA results (Figure 2) show that the sample loses 4.8% of its weight when heated to 90°C, and loses 12.9% of its weight when further heated to 200°C. The DSC results (Figure 3) show that an endothermic peak is observed at 82.4°C (peak temperature). The 1 H NMR results (Figure 4) show that the molar ratio of the residual anisole in the sample to the compound represented by formula (1) is about 1.1:1 (~14.2%, wt%), and the large weight loss of TGA (17.7%) is speculated to be mainly derived from the desorption of anisole. Combined with the sample characterization results and the preparation conditions, it is speculated that the crystals are anisole solvates of the compound represented by formula (1).
进一步地，对所述晶体进行了PLM和DVS的测定，所述晶体的PLM测定结果如图5所示，其DVS的测定结果如图6所示。Furthermore, the crystal was subjected to PLM and DVS measurements, the PLM measurement result of the crystal is shown in FIG5 , and the DVS measurement result thereof is shown in FIG6 .
实施例3溶解度测试Example 3 Solubility Test
称量约5mg的式(1)所示化合物苯甲醚溶剂化物晶体于4mL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4mL还未溶解，则停止加入溶剂。具体的实验结果如下所示。About 5 mg of anisole solvate crystals of the compound represented by formula (1) were weighed into a 4 mL glass bottle, and the solvent was gradually added at room temperature. The amount of solvent added each time was 5 μL until the solid was completely dissolved. If the solid was not dissolved after adding 4 mL, the addition of solvent was stopped. The specific experimental results are shown below.
表4.式(1)所示化合物苯甲醚溶剂化物晶型在不同溶剂中的溶解度(室温)

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

由上表可知，式(1)所示化合物苯甲醚溶剂化物晶体除在水、正庚烷和环己烷中溶解度较差外，在其他常见溶剂中均有较高的溶解度。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.
实施例4重结晶纯化除杂试验Example 4 Recrystallization purification and impurity removal test
工艺1：向容器内加入一定量实施例1制备获得的式(1)所示化合物的苯甲醚溶剂化物晶体，添加适量苯甲醚，调节体系温度40℃，保温搅拌25～35min，目视溶清后，体系以10℃/h的速度缓慢降温至30℃，保温搅拌1h，以5℃/h缓慢降温至20℃，向体系中缓慢滴加正庚烷，滴加完毕搅拌12h，抽滤，滤饼使用正庚烷：苯甲醚均匀溶剂泡洗5～10min，抽滤，滤饼干燥。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 n-heptane: anisole uniform solvent for 5 to 10 minutes, filter with suction, and dry the filter cake.
工艺2：向容器内加入一定量实施例1制备获得的式(1)所示化合物的苯甲醚溶剂化物晶体，添加适量苯甲醚，调节体系温度40℃，保温搅拌25～35min，目视溶清后，体系以10℃/h的速度缓慢降温至30℃，添加式(1)所示的化合物的苯甲醚溶剂化物晶种，养晶0.5～1h，以5℃/h缓慢降温至20℃，向体系中缓慢滴加正庚烷，滴加完毕搅拌12h，抽滤，滤饼使用正庚烷：苯甲醚均匀溶剂泡洗5～10min，抽滤，滤饼干燥。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 drop n-heptane into the system, stir for 12 hours after the addition is completed, filter with suction, soak the filter cake with n-heptane: anisole uniform solvent for 5 to 10 minutes, filter with suction, and dry the filter cake.
工艺1和工艺2获得的固体进行XRPD测试，均为如图1所示的式(1)所示化合物苯甲醚溶剂化物晶型。对所得晶体进行纯度检测，结果如表5所示：The solids obtained by process 1 and process 2 were subjected to XRPD test, 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:
表5.式(1)所示化合物苯甲醚溶剂化物晶型的纯化除杂结果
Table 5. Purification and impurity removal results of the anisole solvate crystalline form of the compound represented by formula (1)
由此可见，在其他操作相同的条件下，加入晶种进行重结晶操作，得到的产品纯度更高，单杂更小，重结晶一次，产品纯度为99.86％，最大单杂0.10％，达到合格标准。It can be seen that under the same 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.
实施例5生物学活性测试Example 5 Biological Activity Test
测试例1、本发明化合物对人源GHSR活性的测定Test Example 1: Determination of the activity of the compounds of the present invention on human GHSR
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。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.
一、试验材料及仪器1. Test materials and instruments
1、培养基1. Culture medium
F12(Gibco,Cat#11765-047)；F12 (Gibco, Cat#11765-047);
FBS(Corning,Cat#35-076-CV)；FBS (Corning, Cat#35-076-CV);
Geneticin(Invitrogen,Cat#10131)；Geneticin (Invitrogen, Cat#10131);
Penicillin/Streptomycin(Invitrogen,Cat#15140)。Penicillin/Streptomycin (Invitrogen, Cat#15140).
2、试剂2. Reagents
Fluo-4Direct(Invitrogen,Cat#F10471)；Fluo-4Direct (Invitrogen, Cat# F10471);
HBSS(Gibco,Cat#14025076)；HBSS (Gibco, Cat#14025076);
HEPES(Gibco,Cat#15630080)；HEPES (Gibco, Cat#15630080);
Bonine Serum Albumin(Sgima,Cat#B2064-100G)。Bonine Serum Albumin (Sgima, Cat#B2064-100G).
3、仪器耗材3. Instrument consumables
384well Poly-D-Lysine protein coating plate(Greiner,Cat#781946)；384well Poly-D-Lysine protein coating plate(Greiner,Cat#781946);
FLIPR(Molecular Devices)；FLIPR (Molecular Devices);
Vi-cell XR Cell Viability Analyzer(Beckman Coulter)；Vi-cell XR Cell Viability Analyzer (Beckman Coulter);
Incubator(Thermo)。Incubator (Thermo).
二、实验步骤2. Experimental steps
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。 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.
配置缓冲液：HBSS(1X):HEPES(1M)＝49:1，加入0.5％BSA。Preparation buffer: HBSS (1X): HEPES (1M) = 49:1, add 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-4 DirectTMNo-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。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 μL of buffer was slowly added to each well, and then 20 μL of 2X Fluo-4 Direct 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 30μL of buffer was added 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. The EC 50 value of the compound can be calculated by the software using the fluorescence values corresponding to different concentrations.
三、实验结果：3. Experimental results:
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。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.
测试例2、Caco-2细胞转运实验Test Example 2: Caco-2 Cell Transport Experiment
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。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 μM in both directions, and the final concentration of DMSO was required to be less than 1%. The cell plates were incubated in a CO2 incubator at 37 ± 1 °C, 5% humidity of CO2 and saturated humidity for 2 hours. All samples were mixed with acetonitrile containing internal standard and centrifuged at 3200xg for 10 minutes. Test compounds, 100 μL of supernatant was diluted with 100 μL of ultrapure water 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 solution, donor solution and receiver solution were quantified by LC-MS/MS method using the peak area ratio of analyte/internal standard. After the transport assay, the lucifer yellow exclusion assay was applied to determine the integrity of the Caco-2 cell monolayer.
表6实施例化合物1e Caco-2细胞转运实验

Table 6 Example Compound 1e Caco-2 cell transport experiment

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。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.
测试例3、化合物1e大鼠体内代谢实验对比Test Example 3: Comparison of compound 1e metabolism in rats
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。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.
实验操作：Experimental operation:
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。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.
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。Before the first administration, the animals were divided into two groups according to their body weight. There were three animals in each group. The first group of animals was given a single oral administration of compound 5 solution (preparation method: medium chain triglycerides/polyethylene glycol 1000 vitamin E succinate/ethanol/propylene glycol/water = 6/2/1/1/90, 1 mg/mL); 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.
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。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. 50μL of whole blood sample was quantitatively taken and added to an EP tube containing 50μL precooled 1mM PMSF methanol solution, vortexed for ~3s, and 250μL of precipitant containing internal standard was immediately added, vortexed for ~5s, and centrifuged for 15min to obtain the supernatant for LC-MS/MS analysis.
表7大鼠体内药物代谢实验数据

Table 7 Drug metabolism experimental data in rats

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。The above data show that in the drug metabolism experiment of rats by oral administration, the Cmax and AUC of the compound 1e of the present invention are significantly better than those of the positive control ibutamoren, and have better drugability. Therefore, it is confirmed that the compound of formula (1) has better drugability.
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。 The above is an explanation of the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (12)

  1. 一种式(1)所示化合物的苯甲醚溶剂化物，
    An anisole solvate of a compound represented by formula (1),
  2. 根据权利要求1所述的溶剂化物，其中，所述苯甲醚与式(1)所示化合物的摩尔比为(1～1.1)：1；优选地，所述苯甲醚与式(1)所示化合物的摩尔比为1：1。The solvate according to claim 1, wherein the molar ratio of anisole to the compound represented by formula (1) is (1-1.1):1; preferably, the molar ratio of anisole to the compound represented by formula (1) is 1:1.
  3. 一种式(1)所示化合物的苯甲醚溶剂化物晶型，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7.07±0.20°处具有特征峰，
    A crystalline form of anisole solvate of a 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 angle using Cu-Kα radiation,
  4. 根据权利要求3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7.07±0.20°、15.73±0.20°、17.83±0.20°处具有特征峰；The crystal form according to claim 3, wherein the crystal form uses Cu-Kα radiation, and X-ray powder diffraction expressed in 2θ angles has characteristic peaks at 7.07±0.20°, 15.73±0.20°, and 17.83±0.20°;
    优选地，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°、26.59±0.20°处具有特征峰。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. 根据权利要求3或4所述的晶型，其中，所述晶型具有基本如图1所示的XRPD图谱。The crystalline form according to claim 3 or 4, wherein the crystalline form has an XRPD pattern substantially as shown in Figure 1.
  6. 根据权利要求3-5任一项所述的晶型，其中，所述式(1)所示化合物的苯甲醚溶剂化物具有如下式(II)所示结构：
    The crystalline 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. 权利要求3-6任一项所述晶型的制备方法，其特征在于，所述制备方法包括以式(I)所示的化合物为原料，通过溶析结晶、气液扩散、气固渗透等方法制备得到；The method for preparing the crystalline form according to any one of claims 3 to 6, characterized in that the method comprises 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;
    优选地，所述制备方法包括如下步骤：Preferably, the preparation method comprises the following steps:
    (1)将式(1)所示的化合物溶解于苯甲醚，然后加入反溶剂，搅拌即得；和/或(1) dissolving the compound represented by formula (1) in anisole, then adding an anti-solvent and stirring; and/or
    (2)将式(1)所示化合物溶解于苯甲醚中，容器盖盖后扎眼，将所述容器置于含反溶剂的更大体积的容器内，盖盖密封，在室温下静置1天以上，即得；和/或(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; and/or
    (3)取一定量式(1)所示化合物置于较小容器中，另取较大容器并向其中加入苯甲醚，将较小容器敞口置于较大容器中，密封后室温下静置1天以上，即得。(3) Place a certain amount of the compound represented by formula (1) in a smaller container, take another larger container and add anisole therein, place the smaller container open in the larger container, seal it and let it stand at room temperature for more than 1 day to obtain the product.
  8. 根据权利要求7所述的制备方法，其特征在于，所述反溶剂包括甲醇、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；The preparation method according to claim 7, characterized in that the anti-solvent comprises 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;
    优选地，所述反溶剂选自水、正己烷、正庚烷、环己烷中的一种或两种以上。Preferably, the anti-solvent is selected from one or more of water, n-hexane, n-heptane and cyclohexane.
  9. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1或2所述的式(1)所示化合物的苯甲醚溶剂化物或权利要求3-6任一项所述的晶型。 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. 根据权利要求9所述的药物组合物，其特征在于，所述药物组合物还含有药学上可接受的辅料；The pharmaceutical composition according to claim 9, characterized in that the pharmaceutical composition further contains a pharmaceutically acceptable excipient;
    优选地，所述药物组合物还包括除上述溶剂化物和/或晶型的第二活性成分，例如所述第二活性成分为与生长发育相关的药物；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;
    优选地，所述药物组合物为制剂，例如为GHSR激动剂。Preferably, the pharmaceutical composition is a preparation, for example, a GHSR agonist.
  11. 权利要求1或2所述的式(1)所示化合物的苯甲醚溶剂化物、权利要求3-6任一项所述的晶型和/或权利要求9-10任一项所述的药物组合物在制备诊断、预防和/或治疗生长激素依赖性疾病(或病症)制剂中的应用；优选地，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。Use of an anisole solvate of the compound of formula (1) according to claim 1 or 2, the crystal form according to any one of claims 3-6 and/or the pharmaceutical composition according to any one of claims 9-10 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 diagnosis of growth hormone-deficient patients, slow growth and short stature of children with growth hormone deficiency, and 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, and healing of wounds and bones.
  12. 权利要求3-6任一项所述的晶型在纯化式(1)所示化合物中的应用。 Use of the crystal form described in any one of claims 3 to 6 in purifying the compound represented by formula (1).
PCT/CN2023/125267 2022-10-21 2023-10-18 Solvate of indoline spiro compound, and crystal form thereof, preparation method therefor and use thereof WO2024083164A1 (en)

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CN1901908A (en) * 2003-11-04 2007-01-24 伊利舍医药品公司 Therapeutic compounds and uses thereof
CN102906093A (en) * 2010-04-02 2013-01-30 欧洲筛选有限公司 Novel nk-3 receptor selective antagonist compounds, pharmaceutical composition and methods for use in nk-3 receptors mediated disorders
WO2016146712A1 (en) * 2015-03-16 2016-09-22 Euroscreen Sa Nk-3 receptor antagonists for therapeutic or cosmetic treatment of excess body fat
CN106008529A (en) * 2016-08-08 2016-10-12 上海工程技术大学 Ibrutinib solvate and preparation method thereof
CN107226816A (en) * 2017-05-27 2017-10-03 重庆文理学院 A kind of preparation method of indoline spiro-compound
CN115010644A (en) * 2022-06-09 2022-09-06 五邑大学 Preparation method of 2, 3-disubstituted indoline compound
WO2022228318A1 (en) * 2021-04-25 2022-11-03 长春金赛药业有限责任公司 Indoline-containing spiro derivative, preparation method therefor and application thereof in medicine

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1092767A (en) * 1992-12-11 1994-09-28 麦克公司 Promote spiroperidol and homologue that tethelin discharges
CN1901908A (en) * 2003-11-04 2007-01-24 伊利舍医药品公司 Therapeutic compounds and uses thereof
CN102906093A (en) * 2010-04-02 2013-01-30 欧洲筛选有限公司 Novel nk-3 receptor selective antagonist compounds, pharmaceutical composition and methods for use in nk-3 receptors mediated disorders
WO2016146712A1 (en) * 2015-03-16 2016-09-22 Euroscreen Sa Nk-3 receptor antagonists for therapeutic or cosmetic treatment of excess body fat
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CN115010644A (en) * 2022-06-09 2022-09-06 五邑大学 Preparation method of 2, 3-disubstituted indoline compound

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