Crystal morphology

Abstract

This specification relates to the crystalline form of (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione, as well as its composition and use.

Description

[Technical Field]

[0001] This specification claims the benefit of priority to European Patent Application No. 23161547.7, filed on 13 March 2023, the contents of which are incorporated herein by reference in their entirety for all purposes.

[0002] (Field of Invention) This specification relates to the crystalline form of (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione, as well as its composition and use. [Background technology]

[0003] Protein arginine methyltransferase 5 (PRMT5) is a member of the PRMT family of arginine methyltransferase enzymes that catalyzes the addition of a methyl group to the guanidine motif of an arginine residue using S-adenosyl-L-methionine (SAM) as a methyl donor. PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates the guanidine group of an arginine residue, thereby converting the guanidine NH2 group of arginine to an NMe2 group. PRMT5 methylates a wide variety of substrates, including histone and non-histone proteins, regulating processes such as RNA splicing, cell proliferation, and DNA repair. Importantly, PRMT5 is overexpressed in numerous cancer types and has been identified as a candidate for therapeutic intervention through the development of small molecules that inhibit PRMT5 methyltransferase activity (see, for example, Kim et al., (2020) Cell Stress 4(8) 199-2151).

[0004] Cyclin-dependent kinase inhibitor 2A (CDKN2A) is a tumor suppressor gene that is homozygously deleted in approximately 15% of cancers. Loss of the 9p21 chromosome locus results in co-deletion of numerous additional genes, including the gene encoding methylthioadenosine phosphorylase (MTAP). MTAP is a metabolic enzyme involved in methionine salvage, and loss of MTAP leads to increased concentrations of the MTAP substrate methylthioadenosine (MTA) in CDKN2A / MTAP-deficient cancer cells. MTA itself acts as a weak PRMT5 inhibitor, and therefore, MTA accumulation in CDKN2A / MTAP-deficient cancer cell lines results in partial inhibition of PRMT5 activity. Reduced PRMT5 activity makes CDKN2A / MTAP-deficient cancer cells more sensitive to further targeting of PRMT5, for example, using short hairpin RNA (shRNA). A “secondary vulnerability” in cancer has been identified, in which CDKN2A / MTAP-deficient tumors can be selectively targeted through PRMT5 inhibition (see Marjon et al., (2016) Cell Reports 15, 574-587, Mavrakis et al., (2016) Science 11;351(6278):1208-13, and Kryukov et al., (2016) Science 11;351(6278):1214-8).

[0005] "MTA-synergistic" PRMT5 inhibitors (i.e., inhibitors that preferentially bind to PRMT5 in the presence of MTA) exert greater inhibitory effects against PRMT5 in environments with relatively high concentrations of MTA, such as CDKN2A / MTAP-deficient tumor cells, but not in healthy tissues. Therefore, "MTA-synergistic" PRMT5 inhibitors should have a high therapeutic index (and low off-target toxicity) because their antiproliferative activity is selectively expressed in targeted CDKN2A / MTAP-deficient tumor cells. Several "MTA synergistic" PRMT5 inhibitors, such as MRTX-1719 (NCT05245500), TNG-908 (NCT05275478), TNG-462 (NCT05732831), AMG-193 (NCT05094336, NCT05094336), and AZD3470 (NCT06130553, NCT06137144), are in clinical trials. However, to date, no PRMT5 inhibitors, let alone "MTA synergistic" PRMT5 inhibitors, have been approved for therapeutic use. International application PCT / EP2022 / 075248 (published as international publication 2023 / 036974) is for compound (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione (referred to herein as compound (I)):

[0006] [ka] PRMT5 inhibitors, including MTA synergistic PRMT5 inhibitors, MTA presence (IC 50 0.006 μM) and absence of MTA (IC 50 Its activity as an inhibitor of the PRMT5 enzyme in a 0.027 μM assay, and its activity in HCT116 wild-type cells (IC) 50 0.2 μM) and HCT116 MTAP knockout cells (IC 50 Its activity at 0.0059 μM, and HCT116 wild-type cells (IC) 506.1 μM) and its activity against cell proliferation in HCT116 MTAP knockout cells (IC 50 0.24 μM) have been disclosed. Thus, compound (I) is a PRMT5 inhibitor, specifically, a MTA synergistic PRMT5 inhibitor. To further study the therapeutic potential of compound (I), it is desirable to have a solid form of the compound with properties suitable for pharmaceutical development.

[0007] In the formulation of the drug substance, it is important that the drug substance (active compound) is in a form that can be conveniently handled and processed. This is important not only from the perspective of obtaining a commercially viable manufacturing process for the drug substance itself, but also from the perspective of the subsequent manufacture of pharmaceutical formulations containing the active compound and suitable excipients. The chemical stability and physical stability of the active compound are important factors in determining the suitability of the solid form for use in the development of pharmaceutical formulations. The active compound and the formulations containing it should be able to be effectively stored for a significant period without showing any significant changes in the physicochemical properties (e.g., chemical composition, density, hygroscopicity, and solubility) of the active compound.

[0008] There is still a need to provide a solid form of compound (I) with appropriate physical and chemical stability and other drug-related properties suitable for pharmaceutical development. SUMMARY OF THE INVENTION

[0009] In one aspect, the present specification provides a (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridin-2-yl)methyl]-1'-but-2-ynyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione:

[0010]

Chemical formula

[0011] Those skilled in the art will understand that while a specific tautomer of compound (I) is shown above, compound (I) can exist in multiple tautomeric forms. This specification encompasses all such tautomeric forms.

[0012] This specification also provides a pharmaceutical composition comprising form A of compound (I) and at least one pharmaceutically acceptable excipient.

[0013] This specification also provides form A of compound (I) for use as a medicament in the prevention or treatment of, for example, disorders mediated by PRMT5.

[0014] This specification also provides the use of form A of compound (I) in the manufacture of a medicament for the prevention or treatment of, for example, disorders mediated by PRMT5.

[0015] This specification also provides a method of prevention or treatment, for example, of preventing or treating a disorder mediated by PRMT5, the method comprising administering form A of compound (I).

[0016] International Application PCT / EP2022 / 075248 (published as International Publication No. WO 2023 / 036974) describes the synthesis of a crystalline form of compound (I) (referred to herein as "Reference Form 1"). However, as discussed in the following Examples, experimental investigation of its properties has revealed that Reference Form 1 exhibits characteristics that can be detrimental to pharmaceutical development. For example, under ambient conditions, Reference Form 1 exists as a hemihydrate that can be reversibly converted to an anhydrous form or a monohydrate upon changes in temperature and / or relative humidity. The reversible (de)hydration behavior of Reference Form 1 results in unpredictable changes in physical properties due to the interconversion between individual forms, which can affect storage and manufacture, and can also cause variability in analytical assays that can complicate its development and use as a pharmaceutical active substance.

[0017] Compound (I) form A described herein has a distinct crystalline structure (as determined by XRPD) and, remarkably, exhibits a combination of features that are attractive in the context of pharmaceutical development. For example, compound (I) form A exhibits high thermal stability (as determined by DSC).

[0018]

number

[0019] Compound (I) may exist in other solid forms with alternative counterions such as acetic acid, 1,5-naphthalenedisulfonic acid, glutaric acid, oxalic acid, propionic acid, camphoric acid, 5-nitroisophthalic acid, 5-chlorosalicylic acid, 3,5-dinitrobenzoic acid, benzoic acid, fumaric acid, sulfuric acid, 2-mesitylenesulfonic acid, gallic acid, 3,5-dihydroxybenzoic acid, or 2,4-dihydroxybenzoic acid. These may exist in crystalline forms, i) as cocrystals of compound (I) and its counterion, ii) as salts of compound (I), or as mixtures thereof. However, form A of compound (I) is a crystalline form that appears particularly suitable for pharmaceutical development. [Brief explanation of the drawing]

[0020] [Figure 1] X-ray powder diffraction pattern of compound (I) form A, which is the physical form of (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione and adipic acid in a 2:1 ratio. [Figure 2] Differential scanning calorimetry and thermogravimetric analysis overlay thermogram of reference form 1. [Figure 3] Differential scanning calorimetry and thermogravimetric overlay thermogram of compound (1) form A. [Figure 4] Dynamic vapor adsorption isotherm plot of reference form 1. [Figure 5] Dynamic vapor adsorption isotherm plot of compound (1) form A. [Modes for carrying out the invention]

[0021] X-ray powder diffraction analysis can be performed according to standard methods, examples of which can be found, for example, in Kitaigorodsky, AI (1973), "Molecular Crystals and Molecules," Academic Press, New York; Bunn, CW (1948), Chemical Crystallography, Clarendon Press, London; or Klug, H.P. & Alexander, LE (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.

[0022] It is known in the art that, depending on the measurement conditions (such as the equipment, sample preparation, or machinery used), an X-ray powder diffraction pattern with one or more measurement errors may be obtained. Specifically, it is generally known that the intensity in an X-ray powder diffraction pattern can vary depending on the measurement conditions and sample preparation. For example, a person skilled in the art of X-ray powder diffraction will recognize that the relative intensity of peaks can vary depending on the orientation of the sample under test and the type and settings of the equipment used. A person skilled in the art will also recognize that the position of reflection can be affected by the precise height at which the sample is positioned in the diffractometer and the zero calibration of the diffractometer. Surface flatness of the sample can also have a small effect. A person skilled in the art will also recognize that the relative intensity of peaks can be affected, for example, by grains larger than about 30 micrometers and non-unitaria aspect ratios, which can affect the analysis of the sample. Furthermore, it should be understood that the intensity can vary depending on experimental conditions and sample preparation, such as the preferred orientation of particles in the sample. The use of automatic or fixed divergent slits also affects the relative intensity calculation. A person skilled in the art can handle such effects when comparing diffraction patterns. Therefore, those skilled in the art will understand that the diffraction pattern data presented herein should not be interpreted as absolute, and that any crystal morphology providing substantially the same force diffraction pattern as disclosed herein falls within the scope of this specification (for further information, see Jenkins, R & Snyder, RL, "Introduction to X-Ray Powder Diffractometry," John Wiley & Sons, 1996).

[0023] Generally, the measurement error of the diffraction angle in X-ray powder diffraction patterns is approximately 5% or less, specifically plus or minus 0.2°²theta. This level of measurement error should be taken into account when considering the X-ray powder diffraction pattern in Figure 1, and when reading Tables 2 and 3. Furthermore, it should be understood that the intensity may vary depending on the experimental conditions and sample preparation (preferred orientation). The definition of relative intensity is shown in Table 1:

[0024] [Table 1]

[0025] X-ray powder diffraction patterns were obtained directly from a sample of compound (I) form A prepared as described herein. Visual analysis of the patterns (Figure 1) clearly shows that compound (I) form A is highly crystalline. The ten most prominent peaks are shown in Table 2:

[0026] [Table 2]

[0027] As used herein, the term "approximately" means within ±5% of any given number.

[0028] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least one specific peak at (±0.2)8.9 degrees 2-theta.

[0029] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least one specific peak at (±0.2)19.1 degrees 2-theta.

[0030] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least two specific peaks at (±0.2)8.9 and 19.1 degrees 2-theta.

[0031] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least one specific peak selected from (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2-theta. In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least one specific peak selected from about 8.9, 19.1, 19.7, and 22.9 degrees 2-theta.

[0032] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least two specific peaks selected from (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2-theta.

[0033] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least three specific peaks selected from (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2-theta.

[0034] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes specific peaks at (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2-theta.

[0035] In one embodiment, the Specified Classification of Compound (I) A has an X-ray powder diffraction pattern that includes specific peaks at (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2-theta when measured using CuKα radiation, and 1, 2, 3, 4, 5, or 6 specific peaks selected from (±0.2)14.5, 16.9, 17.4, 21.2, 23.7, and 25.8 degrees 2-theta when measured using CuKα radiation. In one embodiment, the Specified Provision provides a compound (I) form A having an X-ray powder diffraction pattern that includes specific peaks at approximately 8.9, 19.1, 19.7, and 22.9 degrees 2-theta when measured using CuKα radiation, and 1, 2, 3, 4, 5, or 6 specific peaks selected from approximately 14.5, 16.9, 17.4, 21.2, 23.7, and 25.8 degrees 2-theta when measured using CuKα radiation.

[0036] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least two peaks selected from (±0.2)8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2-theta. In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes at least two peaks selected from about 8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2-theta.

[0037] In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes specific peaks at (±0.2)8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2 theta. In one embodiment, the Specified provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes specific peaks at approximately 8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2 theta.

[0038] Table 3 shows a complete list of XRPD pattern peaks for compound (I) form A. In one embodiment, this specification provides compound (I) form A having an X-ray powder diffraction pattern that, when measured using CuKα radiation, substantially includes specific peaks as shown in Table 3.

[0039] In one embodiment, the Specified Classification of Compound (I) A has an X-ray powder diffraction pattern that includes specific peaks at (±0.2)8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2-theta when measured using CuKα radiation, and one, two, or three specific peaks selected from (±0.2)27.0, 28.1, and 29.2 degrees 2-theta when measured using CuKα radiation. In one embodiment, the Specified Classification of Compound (I) A has an X-ray powder diffraction pattern that includes specific peaks at approximately 8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2-theta when measured using CuKα radiation, as well as one, two, or three specific peaks selected from approximately 27.0, 28.1, and 29.2 degrees 2-theta when measured using CuKα radiation.

[0040] In one embodiment, this specification provides compound (I) form A, which, when measured using CuKα radiation, has substantially the X-ray powder diffraction pattern shown in Figure 1.

[0041] In this specification, when a crystalline form is described as having an "X-ray powder diffraction pattern containing at least one specific peak at (±0.2)..." or an "X-ray powder diffraction pattern containing at least one specific peak at approximately...", the XRPD of the crystalline form may contain one or more of the enumerated 2-theta values, for example, one or more of the enumerated 2-theta values, two or more of the 2-theta values, or three or more of the 2-theta values. Similar descriptions relating to different numbers of peaks (e.g., "...containing at least two specific peaks...") should be interpreted similarly.

[0042] Compound (I) Form A is also characterized by differential scanning calorimetry (DSC). Those skilled in the art will understand that the values ​​or ranges of values ​​observed in the DSC thermogram of a particular compound will vary between batches of different purities. Thus, for some compounds, the range may be small, while for others, the range may be considerably larger. Generally, the measurement error of the diffraction angle in DSC thermal events is about plus or minus 5°C, and the degree of such measurement error should be considered when examining the DSC data included herein, such as in Figure 3.

[0043] When heated with a differential scanning calorimeter (under the conditions described in the Examples section), compound (I) form A exhibits melting with an onset temperature of approximately 216.6°C and a peak temperature of approximately 217.7°C, as illustrated in Figure 3.

[0044] In one embodiment, this specification provides compound (I) form A having a DSC thermogram with an onset melting point at 216.6°C plus or minus 5°C and a peak at 217.7°C plus or minus 5°C. In one embodiment, this specification provides compound (I) form A having a DSC thermogram with an onset melting point at approximately 216.6°C and a peak at approximately 217.7°C. In one embodiment, this specification provides compound (I) form A having a DSC thermogram substantially as shown in Figure 3.

[0045] Thermogravimetric analysis (TGA) (combined with DSC as described above) also demonstrates that compound (I) form A is unsolvated, in contrast to reference form 1 (which has variable and reversible (de)hydration as discussed above), which may offer advantages during storage, manufacturing, and pharmaceutical development.

[0046] In one embodiment, this specification provides compound (I) form A having a DSC and TGA overlay thermogram substantially as shown in Figure 3.

[0047] In one embodiment, this specification refers to (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione:

[0048] [ka] The cocrystal of compound (I) form A and adipic acid in a 2:1 ratio is as follows: a) X-ray powder diffraction pattern including specific peaks at (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2-theta when measured using CuKα radiation. b) X-ray powder diffraction pattern, when measured using CuKα radiation, including specific peaks at (±0.2) 8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2-theta. c) An X-ray powder diffraction pattern substantially as shown in Figure 1, d) A DSC thermogram showing an initial melting point at 216.6°C plus or minus 5°C and a peak at 217.7°C plus or minus 5°C. e) Provides a crystalline morphology having at least one of a DSC thermogram and optionally a TGA thermogram, substantially as shown in Figure 3.

[0049] Where this specification relates to a crystalline form, the degree of crystallinity is greater than about 60%. In one embodiment, the degree of crystallinity is greater than about 80%. In one embodiment, the degree of crystallinity is greater than about 90%. In one embodiment, the degree of crystallinity is greater than about 95%. In one embodiment, the degree of crystallinity is greater than about 98%.

[0050] The crystalline form is preferably substantially free from other crystalline forms of compound (I) (i.e., crystalline forms other than compound (I) form A). Therefore, in one embodiment, the crystalline form contains less than 20% by weight, less than 15% by weight, less than 10% by weight, less than 5% by weight, less than 3% by weight, or less than 1% by weight of other crystalline forms of compound (I). In one embodiment, the crystalline form contains more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 95% by weight, more than 97% by weight, or more than 99% by weight of compound (I) form A.

[0051] Based on the experimental data, compound (I) form A is considered to be a cocrystal of (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione and adipic acid in a 2:1 ratio, i.e., a cocrystal having (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione:adipic acid in a 2:1 stoichiometric ratio. Compound (I) form A can also be referred to as compound (I):hemiadipic acid cocrystal.

[0052] While we do not wish to be constrained by theory, cocrystal formation arises when the acid or base "co-forming agent" is solid at room temperature, and there is little to no proton transfer between the free compound and such acid or base co-forming agent. The result is a cocrystal of the co-forming agent and the free compound, rather than a salt. The definition of an acid or base as a co-forming agent that is solid at room temperature is intended to distinguish the cocrystal from a solvate. It is acknowledged that proton transfer is actually a continuum and can change with temperature, and therefore, the point that cocrystals are better described as salts may be somewhat subjective. However, as stated above, based on all current experimental evidence, compound (I) form A is considered to be a cocrystal.

[0053] In one embodiment, compound (I) form A is provided, which can be obtained by any of the methods disclosed herein. In one embodiment, compound (I) form A is provided, which can be obtained by the method of Example 1, Method A. In one embodiment, compound (I) form A is provided, which can be obtained by the method of Example 1, Method B. In one embodiment, compound (I) form A is provided, which can be obtained by the method of Example 1, Method C.

[0054] Compound (I) Form A is obtained by single-crystal X-ray diffraction (for example, to evaluate proton positions, bond lengths, or bond angles), solid. 1 It may be further characterized by additional techniques well known to those skilled in the art, such as 1H-NMR (for example, to evaluate C or N chemical shifts) or spectroscopic techniques (for example, to measure OH or NH signals and IR peak shifts arising from hydrogen bonds).

[0055] Compound (I) form A can be prepared as described in the examples herein. Crystallization of the desired compound (I) form A can be assisted by seeding crystals of the desired form. Seed crystals can be obtained using one of the methods described in the examples, such as method B. The use of seed crystals is particularly advantageous in larger-scale production.

[0056] medical use As described above, compound (I) is a PRMT5 inhibitor, specifically, a MTA synergistic PRMT5 inhibitor. Therefore, compound (I) form A is expected to be useful as a drug in the prevention or treatment of PRMT5-mediated disorders, i.e., disorders for which inhibition of PRMT5 provides a preventive or therapeutic effect.

[0057] As used herein, “preventive measures” is intended to have its ordinary meaning and includes primary and secondary preventive measures for preventing the onset of a disease or condition, where the disease or condition has already developed and the subject is temporarily or permanently protected from exacerbation or worsening of the disease or condition, or from the onset of new symptoms associated with the disease or condition. The terms “preventive,” “preventing,” and “prevention” are used synonymously with “preventive measures.”

[0058] As used herein, the term “treatment” is intended to have its ordinary meaning of addressing a disease or condition in order to completely or partially alleviate one, some or all of the symptoms of the disease or condition in the subject, or to correct or compensate for an underlying medical condition. The terms “treatment” and “treating” are used synonymously with “therapy.”

[0059] The subjects are typically those who require preventive measures or treatments in accordance with this specification. In one embodiment, the subjects are human.

[0060] In one embodiment, this specification provides compound (I) form A for use as a pharmaceutical agent.

[0061] In one embodiment, this specification provides compound (I) form A for use in the prevention or treatment of PRMT5-mediated disorders (e.g., cancer). In one embodiment, this specification provides compound (I) form A for use in the prevention of PRMT5-mediated disorders (e.g., cancer). In one embodiment, this specification provides compound (I) form A for use in the treatment of PRMT5-mediated disorders (e.g., cancer).

[0062] In one embodiment, this specification provides the use of compound (I) form A in the manufacture of a pharmaceutical product.

[0063] In one embodiment, this specification provides the use of compound (I) form A in the manufacture of a drug for the prevention or treatment of PRMT5-mediated disorders (e.g., cancer). In one embodiment, this specification provides the use of compound (I) form A in the manufacture of a drug for the prevention of PRMT5-mediated disorders (e.g., cancer). In one embodiment, this specification provides the use of compound (I) form A in the manufacture of a drug for the treatment of PRMT5-mediated disorders (e.g., cancer).

[0064] In one embodiment, this specification provides a method for preventing or treating a PRMT5-mediated disorder (e.g., cancer), comprising administering compound (I) form A. In one embodiment, this specification provides a method for preventing a PRMT5-mediated disorder (e.g., cancer), comprising administering compound (I) form A. In one embodiment, this specification provides a method for treating a PRMT5-mediated disorder (e.g., cancer).

[0065] In one embodiment, the disorder mediated by PRMT5 is cancer. In one embodiment, the cancer is MTAP deletion cancer, i.e., cancer in which the MTAP gene is deleted. In one embodiment, the cancer is CDKN2A deletion and MTAP deletion cancer, i.e., cancer in which both the CDKN2A and MTAP genes are deleted.

[0066] In one embodiment, the cancer is selected from gastric cancer, pancreatic cancer, colorectal cancer, uterine cancer, bile duct cancer, abdominal cancer, bladder cancer, cervical cancer, testicular germ cell cancer, lung cancer (e.g., non-small cell lung cancer), multiple myeloma, lymphoma (e.g., diffuse large B-cell lymphoma or Hodgkin lymphoma), rhabdomyosarcoma, and cutaneous squamous cell carcinoma.

[0067] In one embodiment, the cancer is selected from gastric cancer, lung cancer (e.g., non-small cell lung cancer), and lymphoma (e.g., diffuse large B-cell lymphoma or Hodgkin lymphoma).

[0068] In one embodiment, the cancer is non-small cell lung cancer. In one embodiment, the cancer is diffuse large B-cell lymphoma. In one embodiment, the cancer is Hodgkin lymphoma.

[0069] Combination therapy Compound (I) form A may be administered in combination with other compounds used to treat the above-mentioned conditions. In one embodiment, a combination therapy comprising Compound (I) form A and a second active ingredient is provided.

[0070] Compound (I) form A and the second active ingredient may be administered simultaneously, sequentially, or in combination for the treatment of one or more of the conditions listed above. Such combinations may be used in combination with one or more further active ingredients.

[0071] Pharmaceutical composition For use in preventive or therapeutic purposes, compound (I) form A is typically administered as a pharmaceutical composition. Accordingly, in one embodiment, this specification provides a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient.

[0072] In one embodiment, this specification provides a pharmaceutical composition for use as a drug, comprising compound (I) form A and at least one pharmaceutically acceptable excipient.

[0073] In one embodiment, this specification provides a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient for use in a method of preventing or treating a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer). In one embodiment, this specification provides a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient for use in a method of preventing a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer). In one embodiment, this specification provides a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient for use in a method of treating a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer).

[0074] In one embodiment, this specification provides the use of a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient in the manufacture of a drug.

[0075] In one embodiment, this specification provides the use of a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient in the manufacture of a drug for the prevention or treatment of a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer). In one embodiment, this specification provides the use of a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient in the manufacture of a drug for the prevention of a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer). In one embodiment, this specification provides the use of a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient in the manufacture of a drug for the treatment of a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer).

[0076] In one embodiment, this specification provides a method for preventing or treating a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer), comprising administering a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient. In one embodiment, this specification provides a method for preventing a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer), comprising administering a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient. In one embodiment, this specification provides a method for treating a PRMT5-mediated disorder, such as a disorder disclosed herein (e.g., cancer), comprising administering a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient.

[0077] Administration Compound (I) form A is usually administered in the form of a pharmaceutical composition containing compound (I) form A in a pharmaceutically acceptable dosage form, typically by oral, parenteral, intravenous, intramuscular, subcutaneous, or other injectable means, via the oral, rectal, vaginal, transdermal and / or nasal routes, and / or inhalation. Depending on the disorder being treated and the patient, and the route of administration, the composition may be administered in various doses. In one embodiment, compound (I) form A, or a pharmaceutical composition containing compound (I) form A and at least one pharmaceutically acceptable excipient, is administered orally.

[0078] Dosage forms suitable for oral use form one aspect of this specification. In one embodiment, this specification provides a solid oral dosage form comprising compound (I) form A, or a pharmaceutical composition comprising compound (I) form A and at least one pharmaceutically acceptable excipient. In one embodiment, the solid oral dosage form is a tablet.

[0079] The compositions described herein can be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Accordingly, compositions intended for oral use may contain, for example, one or more colorants, sweeteners, flavorings, and / or preservatives.

[0080] Suitable pharmaceutically acceptable excipients for tablet formulations include, for example, inert diluents, granulators and disintegrants, binders, and lubricants. Tablet formulations may be uncoated or may be coated using conventional coating agents and procedures well known in the art.

[0081] For further information regarding the formulation, readers should refer to Chapter 25.2, Volume 5 of "Comprehensive Medicinal Chemistry" (Corwin Hansch; Chairman of Editorial Board), Pergamon Press, 1990.

[0082] The amount of the active ingredient (i.e., compound (I) form A) combined with one or more excipients to produce a single dosage form will inevitably vary depending on the host being treated and the specific route of administration.

[0083] The preferred daily dose of compound (I) form A for prophylactic or therapeutic use in humans is approximately 0.0001 to 100 mg / kg body weight.

[0084] For further information regarding the route of administration and the administration plan, readers should refer to Chapter 25.3, Volume 5 of "Comprehensive Medicinal Chemistry" (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0085] This specification can be further illustrated by the following non-limiting embodiments. [Examples]

[0086] Generally: (i) Unless otherwise specified, the operations were carried out at ambient temperature, i.e., in the range of 17–25°C, and in an atmosphere of an inert gas such as nitrogen. (ii) Evaporation was performed by rotary evaporation or by using a Genevac apparatus or Biotage v10 evaporator in a vacuum, and after removing the residual solid by filtration, the work-up procedure was carried out. (iii) Flash chromatography purification was performed using an automated Teledyne Isco CombiFlash® Rf or Teledyne Isco CombiFlash® Companion® with a pre-packed RediSep Rf Gold® silica column (20-40 μm, spherical particles), a GraceResolv® cartridge (Davisil® silica), or a Silicycle cartridge (40-63 μm). (iv) Preparative reverse-phase HPLC was performed using an Agilent 1290 Infinity II preparative system equipped with an SQ MS detector (Multimode ESI / APCI source) and a Waters CSH C18 OBD column (5 micron silica, 30 mm diameter, 100 mm length, 50 mL / min flow rate), with water (containing 0.1-0.3% aqueous ammonium) or a mixture of water (containing 0.1% formic acid) and acetonitrile that reduced polarity as the eluent. Preparative SFC purification was performed using either a Sepiatec P100 SFC system or a Waters Prep 100 SFC system equipped with a QDa MS detector, using the chromatographic conditions detailed in the corresponding experimental data. (v) The yield, if any, is not necessarily the maximum achievable value. (vi) Generally, NMR chemical shift values ​​were measured on a delta scale [proton magnetic resonance spectra were determined using a Bruker Avance 400 (400 MHz) instrument]. Unless otherwise specified, measurements were performed at ambient temperature. The following abbreviations were used: s, singleline; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doubleline doubleline; ddd, doubleline doubleline doubleline; dt, tripleline doubleline; bs, broad signal. (vii) Generally, compounds were also characterized by mass spectrometry after liquid chromatography (LCMS or ULC). Reverse-phase C18 silica was used at a flow rate of 1 mL / min, and detection was by electrospray mass spectrometry and UV absorbance recording in the wavelength range of 220–320 nm. Analytical ULC was performed with CSH C18 reverse-phase silica using a Waters Acquity UPLC CSH C18 column (dimensions 2.1 × 50 mm and particle size 1.7 microns). Gradient analysis was used with a mixture that reduces polarity as the eluent, e.g., water (containing 0.1% formic acid or 0.1% ammonia) as solvent A and a mixture that reduces polarity of acetonitrile as solvent B. A typical 2-minute analytical ULC method uses a solvent gradient over 1.3 minutes at approximately 1 mL / min, from a 97:3 mixture of solvents A and B to a 3:97 mixture of solvents A and B, respectively. Unless otherwise specified, the reported molecular ions correspond to [M+H]+, and for molecules with multiple isotopic patterns (such as Br and Cl), the reported values ​​are obtained for the lowest isotopic mass unless otherwise specified. (viii) The purity of the intermediate was evaluated by thin-layer chromatography, mass spectrometry, HPLC (high-performance liquid chromatography), and / or NMR analysis.

[0087] Preparation of Reference Form 1 The synthesis of compound (I) is described in international application PCT / EP2022 / 075248 (published as international publication 2023 / 036974), which is incorporated by reference for the purpose of describing compound (I) and its synthesis. The compounds described below are named using Chemdraw version 20.0.2.51, and the synthetic methods described herein are given for illustrative purposes only and are not limiting.

[0088] Intermediate AQ: (6-chloro-5-fluoropyridine-3-yl)carbamate tert-butyl

[0089] [ka]

[0090] To 5-bromo-2-chloro-3-fluoropyridine (100 g, 475.22 mmol) in dioxane (1 L), tert-butyl carbamate (61.20 g, 522.74 mmol) and cesium carbonate (310.00 g, 950.43 mmol) were added. The solution was degassed under vacuum and purged with an inert atmosphere of nitrogen for 5 minutes, followed by the addition of tris(dibenzylideneacetone)dipalladium(0) (13.06 g, 14.26 mmol) and (9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane) (xantphos) (11.00 g, 19.01 mmol). The reaction mixture was heated at 85 °C for 16 hours under nitrogen and then cooled to room temperature. The solid was filtered off and washed with excess dioxane. The solvent was removed in vacuo to give the crude title compound (179 g, 153%) as a dark orange gum, which was allowed to stand to solidify. The crude gum was used in the next step without further purification. 1 H NMR (400 MHz, DMSO-d6, 30 °C) 1.49 (9H, s), 7.98 (1H, dd), 8.29 (1H, d), 9.98 (1H, s); m / z MH + 247.

[0091] Intermediate AR: 6-chloro-5-fluoropyridin-3-amine

[0092]

Chemical formula

[0093] 4M HCl (137 mL, 547.3 mmol) in 1,4-dioxane was added all at once at 20°C to a solution of tert-butyl (6-chloro-5-fluoropyridine-3-yl)carbamate (36 g, 109.5 mmol) in 1,4-dioxane (20 mL). The resulting suspension was stirred at 20°C for 3 days. The reaction mixture was diluted with water (250 mL) and ethyl acetate (100 mL). The organic phase was separated and extracted with 2M HCl (3 × 100 mL) until no product remained in the organic phase. The combined aqueous phase was stirred and cooled to 0°C in an ice bath. The reaction mixture was basicized to pH 14 with 50% NaOH solution. Next, the reaction mixture was extracted with dimethylammonium ammonium (2 × 250 mL), the combined organic matter was washed with saturated brine (50 mL), dried with MgSO4, filtered, and the solvent was removed under vacuum to obtain the title compound (12.4 g, 77%) as a brown solid. This compound was used directly in the next step without further purification. 1 H NMR (400MHz, CDCl3, 27℃) 3.88 (s, 2H), 6.80 (dd, J = 9.6, 2.5Hz, 1H), 7.68 (d, J = 2.5Hz, 1H).

[0094] Intermediate AS: 2-bromo-6-chloro-5-fluoropyridine-3-amine

[0095] [ka]

[0096] 6-chloro-5-fluoropyridine-3-amine (56.8 g, 379.3 mmol) in MeCN (250 mL) was cooled to 5°C, and a solution of NBS (67.50 g, 379.3 mmol) in MeCN (500 mL) was added over 15 minutes. The reaction mixture was warmed to room temperature and stirred for 45 minutes. Water (2 L) was added, and the reaction mixture was stirred for 30 minutes. The resulting solid was filtered and washed with water (400 mL). The solid was dried under vacuum to obtain the title compound (76 g, 89%) as a brown solid. 1 H NMR(400MHz,DMSO-d6,30℃)6.01(2H,s),7.12(1H,d);m / z MH + 225.

[0097] Intermediate AT: 5-chloro-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-carboxylic acid

[0098] [ka]

[0099] Palladium acetate (3.35 g, 14.93 mmol), triphenylphosphine (3.92 g, 14.93 mmol), 2-bromo-6-chloro-5-fluoropyridine-3-amine (18.0 g, 74.65 mmol), and pyruvate (15.57 mL, 224 mmol) were placed in a flask with 1,4-dioxane (88 mL). Triethylamine (45.80 mL, 328.5 mmol) was added, and the reaction mixture was heated under nitrogen at 100 °C for 2.5 hours. The reaction mixture was cooled to room temperature, filtered, and unwanted solids were removed. The filtrate was diluted with 2 M NaOH (200 mL), and MTBE (200 mL) was added. The reaction mixture was then vigorously stirred and separated. The organic phase was washed with 2 M NaOH (100 mL). The combined basic aqueous phase was carefully acidified with concentrated HCl (aqueous solution), and a brown solid precipitated, which was collected by filtration and dried. The dark brown solid was suspended in MeOH (90 mL) and stirred at room temperature for 2 hours. The solid was filtered and dried under vacuum to obtain the title compound (14.60 g, 91%) as a beige solid. 1 H NMR(400MHz,DMSO-d6,30℃)7.16(1H,dd),7.84(1H,dd),12.36(1H,s),13.46(1H,s);m / z MH + 214.

[0100] Intermediate AU:5-chloro-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-carboxylate methyl

[0101] [ka]

[0102] Sulfuric acid (3.08 mL, 57.83 mmol) was carefully added dropwise to 5-chloro-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-carboxylic acid (14.60 g, 57.83 mmol) in MeOH (113 mL) at room temperature. The reaction mixture was stirred under reflux for 18 hours. The reaction mixture was allowed to cool, and the solvent was removed under vacuum. Saturated NaHCO3 (400 mL) was carefully added to the residue, and the resulting precipitate was filtered off, washed with water, and dried under vacuum to obtain the title compound (14.20 g, 107%) as a brown solid. 1 H NMR(400MHz,DMSO-d6,30℃)3.91(3H,s),7.24(1H,dd),7.88(1H,dd),12.56(1H,s);m / z MH + 229.

[0103] Intermediate AV: 5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-carboxylate methyl

[0104] [ka]

[0105] Potassium bis(trimethylsilyl)amide (1M in THF) (101 mL, 100.9 mmol) was added dropwise over 15 minutes under nitrogen at 5°C to a solution of methyl 5-chloro-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-carboxylate (21.74 g, 77.60 mmol) and 2-(chloromethoxy)ethyl)trimethylsilane (18.83 mL, 100.9 mmol) in THF (419 mL). The reaction mixture was stirred at 5°C for 30 minutes. Potassium bis(trimethylsilyl)amide (1M in THF) (15.52 mL, 15.52 mmol) was added, and the reaction mixture was stirred for a further 30 minutes at 5°C. (2-(chloromethoxy)ethyl)trimethylsilane (1.88 mL, 10.09 mmol) was added, and the mixture was stirred for a further 15 minutes at 5°C. The reaction mixture was quenched with saturated NH4Cl (400 mL) and diluted with HCl (400 mL). The aqueous phase was re-extracted with HCl (250 mL). The combined organic phase was dried over MgSO4, filtered, and the solvent was removed under vacuum. The crude material was suspended in heptane (450 mL) and stirred for 5 minutes. Unwanted solids were filtered off and washed with heptane (50 mL). The solvent was removed under vacuum to obtain the title compound (32.50 g, 117%) as a brown gum, which was allowed to stand and solidify. The gum was used in the next step without further purification. 1 m / z MH + 359.

[0106] Intermediate AW: (5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methanol

[0107] [ka]

[0108] Diisobutylaluminum hydride (1M in toluene) (170 mL, 170.42 mmol) was added dropwise over 15 minutes at 5°C to a stirred solution of methyl 5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-carboxylate (27.80 g, 77.47 mmol) in DCM (333 mL). The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was carefully poured into 2M NaOH (500 mL), diluted with DCM (500 mL), and stirred for 1 hour. The organic phase was separated, and the aqueous phase was extracted with DCM (2 × 200 mL). The combined organic matter was dried over MgSO4, filtered, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography and elution gradient of 0-40% siRNA in heptane. The pure fraction was evaporated to dryness to obtain the title compound (17.60 g, 68%) as a pale orange oil, which was then allowed to stand and solidify. 1 H NMR(400MHz,DMSO-d6,30℃)-0.10(9H,s),0.75-0.83(2H,m),3.36-3.56(2H ,m),4.72(2H,d),5.50(1H,t),5.60(2H,s),6.57(1H,d),8.24(1H,dd);m / z M.H. + 331.

[0109] Intermediate AX: 5-chloro-2-(chloromethyl)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine

[0110] [ka]

[0111] Thionyl chloride (13.2 mL, 181.4 mmol) was carefully added dropwise to (5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methanol (20.0 g, 60.45 mmol) in DCM (200 mL) at room temperature. The reaction mixture was stirred at room temperature for 1 hour. Then, saturated NaHCO3 (500 mL) was slowly added. Once gas generation ceased, the phases were separated, and the aqueous phase was re-extracted with DCM (300 mL). The organic phases were combined, washed with brine (200 mL), passed through phase separation filter paper, and the solvent was removed under vacuum to obtain the title compound (19.2 g, 91%) as a brown crystalline solid. 1 m / z MH + 349.

[0112] Intermediate AA: 2-(2-bromo-4-fluorophenyl)methyl acetate

[0113] [ka]

[0114] Thionyl chloride (31.3 mL, 429.1 mmol) was carefully added dropwise to 2-(2-bromo-4-fluorophenyl)acetic acid (CAS No. 61150-59-2) (100 g, 429.1 mmol) in MeOH (400 mL) at room temperature. The reaction mixture was stirred at 60°C for 4 hours, cooled, and the solvent was removed under vacuum. The residue was partitioned between ELISA (250 mL) and saturated NaHCO3 (200 mL). The organic phase was washed with water (100 mL) and brine (100 mL), passed through phase separation filter paper, and the solvent was removed under vacuum to obtain the title compound (105 g, 99%) as a colorless oil. 1H NMR(400MHz,DMSO-d6,30℃)3.64(3H,s),3.83(2H,s),7.25(1H,td),7.48(1H,dd),7.58(1H,dd));m / z MH + Not observed.

[0115] Intermediate AB: 5-Fluoro-2-(2-methoxy-2-oxoethyl)methyl benzoate

[0116] [ka]

[0117] Methyl 2-(2-bromo-4-fluorophenyl)acetate (45.0 g, 182.14 mmol) and triethylamine (27.90 mL, 200.35 mmol) were placed in a steel pressure vessel with MeOH (300 mL). [1,1'-Bis(diphenylphosphino)ferrocene]dichloropalladium(II) (complexed with dichloromethane) (4.46 g, 5.46 mmol) was added, and the vessel was sealed. The vessel was purged with carbon monoxide, and then filled with carbon monoxide up to 7 bar. The pressure vessel was heated to 100°C and stirred for 2 hours. The reaction mixture was cooled, aerated, and filtered to remove the catalyst. The solvent was removed under vacuum, and the residue was dissolved in ELISA (250 mL) and washed with water (2 × 200 mL) and brine (100 mL). The organic phase was passed through phase separation filter paper, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography with an elution gradient of 0–50% siRNA in heptane. The pure fraction was evaporated to dryness to obtain the title compound (38.40 g, 93%) as a pale yellow oil. 1 H NMR(400MHz,DMSO-d6,30℃)3.60(3H,s),3.80(3H,s),3.99(2H,s),7.42-7.49(2H,m),7.66(1H,ddd);m / z MH + 227.

[0118] Intermediate AC:rac-methyl-2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoate

[0119] [ka]

[0120] 5-Fluoro-2-(2-methoxy-2-oxoethyl)methyl benzoate (47.0 g, 207.8 mmol) was dissolved in chloroform (450 mL). 1-Bromopyrrolidine-2,5-dione (55.5 g, 311 mmol) was added, followed by 2,2'-azobis(2-methylpropionitrile) (3.41 g, 20.8 mmol), and the reaction mixture was stirred under reflux for 72 hours. The reaction mixture was cooled, washed with water (2 × 250 mL) and brine (100 mL), passed through phase-separating filter paper, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography and elution gradient of 0-40% ethyl acetate in heptane. The pure fraction was evaporated to dryness to obtain the title compound (50.50 g, 80%) as a colorless oil. 1 m / z MH + Not observed.

[0121] Intermediate AD:rac-methyl5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate

[0122] [ka]

[0123] 4-methoxybenzylamine (23.5 g, 171 mmol) was placed in a flask with MeCN (300 mL), and sodium bicarbonate (23.9 g, 285 mmol) was added. rac-methyl 2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoate (43.5 g, 142 mmol), dissolved in MeCN (100 mL), was slowly added via a dropping funnel while raising the reaction mixture temperature to 80°C. The reaction mixture was stirred at 80°C for 3 hours. The reaction mixture was cooled, most of the MeCN was removed under vacuum, and the residue was partitioned between HCl (400 mL) and water (400 mL). The aqueous phase was re-extracted with HCl (100 mL), the organic matter was combined, and washed with brine (50 mL). The organic phase was passed through phase-separating filter paper, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography with an elution gradient of 0–50% siRNA in heptane. The pure fraction was evaporated to dryness to obtain the title compound (45.3 g, 96%) as a pale yellow oil. 1 H NMR(400MHz,DMSO-d6,30℃)3.69(3H,s),3.73(3H,s),4.31(1H,d),5.04(1H,d),5.18(1H, m / z M.H. + 330.

[0124] Intermediate AE:rac-methyl1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate

[0125] [ka]

[0126] rac-methyl 5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate (24.0 g, 72.9 mmol), allyl acetate (11.8 mL, 109 mmol), tris(dibenzylideneacetone)dipalladium(0) (1.67 g, 1.82 mmol), and N,N'-((1R,2R)-cyclohexane-1,2-diyl)bis(2-(diphenylphosphanail)benzamide) (2.52 g, 3.64 mmol) were stirred in THF (400 mL) under nitrogen at 5°C. Then, 1,1,3,3-tetramethylguanidine (13.7 mL, 109 mmol) was added dropwise. The reaction mixture was stirred at 5°C for 5 minutes. The THF was removed under vacuum. The reaction mixture was partitioned between Depositphotos (400 mL) and water (400 mL), and the organic phase was passed through phase-separating filter paper. The solvent was removed under vacuum to obtain an orange oil. The crude product was purified by flash silica chromatography with an elution gradient of 0–50% Depositphotos in heptane. The pure fraction was evaporated to dryness to obtain the title compound (25.8 g, 96%) as a cream solid. 1 H NMR(400MHz,DMSO-d6,30℃)3.04-3.20(2H,m),3.26(3H,s),3.73(3H,s),4.52(1H,d),4.71(1H,d ),4.74-4.94(3H,m),6.82-6.96(2H,m),7.28-7.39(2H,m),7.45-7.58(2H,m),7.63(1H,dd);m / z M.H. + 370.

[0127] Intermediate AF:(S)-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate methyl

[0128] [ka]

[0129] rac-methyl-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate (approximately 70:30 favorably to the desired (S) enantiomer) (25.8 g, 69.7 mmol) was purified by SFC chromatography (column: Phenomenex C1, 30 × 250 mm, 5 microns, mobile phase: 10% IPA + 0.1% DEA / 90% scCO2, flow rate: 90 mL / min, BPR: 120 bar, column temperature: 40 °C, UV max 210 nm). The pure fraction was evaporated to dryness to obtain the title compound (15.1 g, 56%) as a white solid. 1 H NMR(400MHz,DMSO-d6,30℃)3.04-3.20(2H,m),3.26(3H,s),3.73(3H,s),4.52(1H,d),4.71(1H,d ),4.74-4.94(3H,m),6.82-6.96(2H,m),7.28-7.39(2H,m),7.45-7.58(2H,m),7.63(1H,dd);m / z M.H. + 370. (The presumed stereochemical assignment of this intermediate was made based on the biological activity of the bioactive compound prepared using this enantiomer of the intermediate (compared to those prepared using the other enantiomer), along with X-ray structural evidence that the S enantiomer is preferred and more active than the R enantiomer.)

[0130] Intermediate AI:(S)-1-allyl-5-fluoro-3-oxoisoindoline-1-carboxylate methyl

[0131] [ka]

[0132] (S)-1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate methyl (20.0 g, 54.1 mmol) was placed in a flask with MeCN (200 mL) and water (100 mL). Cerium(IV) ammonium nitrate (74.2 g, 135 mmol) was added, and the reaction mixture was stirred at room temperature for 30 minutes. The MeCN was removed under vacuum, and the reaction mixture was partitioned between DCM (400 mL) and water (250 mL). The aqueous phase was extracted with DCM (200 mL). The organic phases were combined, washed with brine (100 mL), passed through phase separation filter paper, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography with an elution gradient of 0-50% ethyl acetate in heptane. The pure fraction was evaporated to dryness to obtain the title compound (12.5 g, 93%) as a cream crystalline solid. 1 H NMR(400MHz,DMSO-d6,27℃)2.79(1H,dd),2.94(1H,dd),3.68(3H,s),4.93-5.15(2H,m), 5.35-5.57(1H,m),7.37-7.46(1H,m),7.50(1H,ddd),7.63-7.79(1H,m),9.32(1H,s);m / z M.H. + 250.

[0133] Intermediate BQ: (S)-1-allyl-2-((5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-5-fluoro-3-oxoisoindoline-1-carboxylate methyl

[0134] [ka]

[0135] (S)-1-allyl-5-fluoro-3-oxoisoindoline-1-carboxylate methyl (11.80 g, 47.34 mmol) and 5-chloro-2-(chloromethyl)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine (16.9 g, 48.3 mmol) were placed in a flask with dry DMF (60 mL). Cesium carbonate (38.60 g, 118.4 mmol) was added, and the reaction mixture was stirred at 60°C for 2 hours. The reaction mixture was cooled and partitioned between water (300 mL) and toluene (300 mL). The aqueous phase was re-extracted with toluene (200 mL). The organic phases were combined, washed with water (3 × 200 mL) and brine (200 mL), passed through phase separation filter paper, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography with an elution gradient of 0-100% siRNA in heptane. The pure fraction was evaporated to dryness to obtain the title compound (22.2 g, 83%) as a yellow gum, which was slowly solidified / crystallized to obtain a yellow solid. 1 H NMR(400MHz,DMSO-d6,27℃)-0.09(9H,s),0.79-0.88(2H,m),3.03(3H,s),3.16-3.29(2H,m),3.46-3.60(2H,m),4.73(1H,d),4.89(1H m / z M.H. + 562.

[0136] Intermediate BR:(S)-1-allyl-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-5-fluoro-3-oxoisoindoline-1-carboxylate methyl

[0137] [ka]

[0138] (S)-1-allyl-2-((5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-5-fluoro-3-oxoisoindoline-1-carboxylate methyl (15 g, 26.69 mmol), cesium carbonate (21.74 g, 66.72 mmol), BrettPhos Pd G3 (2.42 g, 2.67 mmol), dicyclohexyl (2',4',6'-triisopropyl-3,6-dimethoxy-[1,1'-biphenyl]-2-yl)phosphane (1.43 g, 2.67 mmol), and tert-butyl carbamate (6.25 g, 53.37 mmol) were placed in a flask with degassed 2-methyltetrahydrofuran (150 mL). Nitrogen was passed through the reaction mixture and foamed for 10 minutes, then the reaction mixture was refluxed for 3 hours. The reaction mixture was cooled, diluted with water (400 mL), and extracted with dimethyl phosphate (2 × 300 mL). The combined organic phase was washed with saturated brine (200 mL), passed through phase-separating filter paper, and the solvent was removed under vacuum. The crude product was purified by flash silica chromatography with an elution gradient of 0–50% dimethyl phosphate in heptane. The pure fraction was evaporated to dryness to obtain the title compound (11.68 g, 68%) as a pale yellow foam. 1 H NMR(400MHz,DMSO-d6,27℃)-0.08(9H,s),0.82-0.86(2H,m),1.42(9H,s),2.99(3H,s),3.15-3.30(2H,m),3.52(2H,dtd),4.71(1H,d),4.88(1H, dd),4.94-5.10(2H,m),5.26(1H,d),5.55(1H,d),5.64(1H,d),6.69(1H ,s),7.46-7.56(1H,m),7.57-7.66(2H,m),7.99(1H,d),9.18(1H,s);m / z M.H. + 643.

[0139] Intermediate BS: Methyl(S)-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-5-fluoro-3-oxo-1-(2-oxoethyl)isoindoline-1-carboxylate carboxylate

[0140] [ka]

[0141] To a solution of (S)-1-allyl-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-5-fluoro-3-oxoisoindoline-1-carboxylate methyl (11.50 g, 17.89 mmol) in 1,4-dioxane (240 mL) and water (60 mL), osmium(VIII) oxide (4% in water) (1.14 mL, 0.18 mmol), sodium periodate (9.57 g, 44.73 mmol), and 2,6-dimethylpyridine (4.17 mL, 35.78 mmol) were added. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was then partitioned between DCM (200 mL) and water (100 mL). The aqueous phase was re-extracted with DCM (100 mL), the organic phase was combined, and the mixture was passed through phase-separating filter paper to remove the solvent under vacuum. The crude product was purified by flash silica chromatography with an elution gradient of 0-50% siRNA in heptane. The pure fraction was evaporated to dryness to obtain the title compound (8.70 g, 75%) as a beige foam. 1H NMR(400MHz,DMSO-d6,27℃)-0.07(9H,s),0.82(2H,ddd),1.42(9H,s),3.25(3H,s),3.46-3.54(2H,m),3.67(2H,s),4.92(1H,d),5.06(1 m / z M.H. + 645.

[0142] (S)-(2-((1'-(buta-2-in-1-yl)-5-fluoro-2',3-dioxospiro[isoindoline-1,3'-pyrroridine]-2-yl)methyl)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-5-yl) tert-butyl carbamate

[0143] [ka]

[0144] (S)-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-5-fluoro-3-oxo-1-(2-oxoethyl)isoindoline-1-carboxylate methyl (9.50 g, 14.73 mmol) and buta-2-in-1-amine hydrochloride (2.33 g, 22.10 mmol) were placed in a flask with 1,2-dichloroethane (100 mL). Triethylamine (3.08 mL, 22.10 mmol) was added, and the reaction mixture was stirred at room temperature for 30 minutes. Sodium triacetoxyborohydride (6.25 g, 29.47 mmol) was added, and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with DCM (250 mL) and washed with saturated NaHCO3 (100 mL), water (100 mL), and brine (100 mL). The organic phase was passed through phase-separating filter paper, and the solvent was removed under vacuum to obtain the title compound. The crude compound was used in the next reaction without further purification, assuming a 100% yield. m / z MH + 666.

[0145] (S)-2-((5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl)-1'-(buta-2-in-1-yl)-5-fluorospiro[isoindoline-1,3'-pyrrolidine]-2',3-dione (reference form 1)

[0146] [ka]

[0147] (S)-(2-((1'-(buta-2-in-1-yl)-5-fluoro-2',3-dioxospiro[isoindoline-1,3'-pyrrolidine]-2-yl)methyl)-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[3,2-b]pyridine-5-yl)carbamate tert-butyl (9.81 g, 14.73 mmol) was placed in a flask with 2,2,2-trifluoroacetic acid (22.55 mL, 294.7 mmol), and the solution was stirred at room temperature for 2 hours. 2,2,2-trifluoroacetic acid was removed under vacuum, and the residue was dissolved in MeCN (20 mL). Ammonium hydroxide (28-30% in water) (22.95 mL, 589.4 mmol) was added, and the reaction mixture was stirred at 40°C for 2 hours. The crude product was purified by reverse-phase chromatography (Interchim C18-HP Flash column, 415 g) using water (containing 1 vol% NH4OH (28-30% of H2O)) and a mixture that reduced the polarity of MeCN as the eluent (30-60% gradient). The fractions containing the desired compound were combined, MeCN was removed under vacuum, and the resulting solid was filtered and dried to obtain the title compound (3.74 g, 58%) as a cream-colored crystalline solid. 1 H NMR(400MHz,DMSO-d6,27℃)1.88(3H,t),2.36-2.44(2H,m),3.54(1H,ddd),3.76(1H,dt),4.08(2H,qq), 4.24(1H,d),5.03(1H,d),5.49(2H,s),6.11(1H,d),7.36(1H,dd),7.49-7.64(3H,m),10.69(1H,d);m / z M.H. + 436.

[0148] Example 1: Preparation of (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione:hemiadipic acid cocrystal ("Compound (I) Form A") Method A: Single-solvent cocrystallization Approximately 27.6 mg of adipic acid was dissolved in 500 μL of methanol and heated. Separately, approximately 150 mg of reference form 1 was dissolved in 2 mL of methanol while heating. These two solutions were then combined, the vial cap was slightly loosened, and the mixture was allowed to evaporate slowly at ambient temperature for 8 days while stirring with a magnetic stirring rod. The resulting material was filtered, the solid was collected, and dried overnight under a stream of air to obtain compound (I) form A in approximately 53% yield.

[0149] Method B: Poor solvent / cold cocrystallization (without seed addition) Reference Form 1 (501.51 mg, 1.0942 mmol, 95% by mass) was packed into an 8 mL vial, and adipic acid (166.13 mg, 1.1368 mmol, 100% by mass) and dimethyl sulfoxide (3.54 g, 45.3 mmol, 100% by mass) were added. The contents were stirred at 600 rpm using a double 3-pitch blade impeller, heated to 25°C, and held at this temperature for 5 minutes. The contents of the vial were then heated to 80°C at a rate of 5°C / min and held at this temperature for 20 minutes. Water (1.4 mL, 78 mmol, 100% by mass) was added to the vial at a rate of 0.012 mL / min over 2 hours at 80°C, and the temperature was held for a further 30 minutes. The contents of the bath were then cooled to 20°C at a rate of 0.125°C / min over 8 hours, and the temperature was held at this temperature for 80 hours. The contents of the vial were further cooled to 5°C over 15 minutes, the temperature was maintained for 3 hours, and then heated to 20°C. Water (0.75 mL, 42 mmol, 100% by mass) was then added to the vial, during which crystallization occurred. The contents of the vial were then drained into a filter (30 mm diameter filter paper) under ambient conditions and dehydrated under vacuum. Premixed dimethyl sulfoxide (0.525 mL, 7.4 mmol, 100% by mass) and water (0.225 mL, 12.5 mmol, 100% by mass) were added to the tank under ambient conditions for washing. The contents of the vial were stirred at 20°C, then drained into a filter under ambient conditions and dehydrated under vacuum. This process was repeated for a second wash, and then 2-propanol (1.00 mL, 13.1 mmol, 100% by mass) was added to the filter and the contents were dehydrated under vacuum. This process was repeated for final cleaning, after which the contents of the filter were discharged into a drying dish and dried under vacuum in an oven at 45-50°C for 24 hours to obtain compound (I) form A in approximately 92% yield.

[0150] Method C: Cooling / poor solvent cocrystallization (seed addition) Reference Form 1 (8.01 g, 18.2 mmol, 99% by mass) was packed into a 100 mL tank, and adipic acid (2.57 g, 17.6 mmol, 100% by mass) and dimethyl sulfoxide (54.88 g, 702.4 mmol, 100% by mass) were added. The contents were stirred at 400 rpm using a 4-pitch blade impeller, heated to 25°C, and held at this temperature for 30 minutes. The contents of the tank were then heated to 80°C at a rate of 5°C / min and held at this temperature for 20 minutes. Water (21.6 mL, 1200 mmol, 100% by mass) was added to the tank at a rate of 0.1125 mL / min over 2 hours at 80°C and held at this temperature for a further 5 minutes. The contents of the tank were then cooled to 65°C at a rate of 0.5°C / min over 30 minutes and held at this temperature for 30 minutes. Next, seed (prepared according to Method B, 8.7 mg, 0.017 mmol, 100% by mass) was packed into the tank at 65°C and the temperature was maintained for 3 hours. Then, the contents of the tank were cooled to 20°C at a rate of 0.125°C / min over 6 hours and the temperature was maintained for a further 3 hours. A poor solvent, water (12 mL, 666.119 mmol, 100% by mass) was packed into the tank at 20°C over 6 hours and the temperature was maintained for a further 6 hours. Then, the contents of the tank were drained into a filter (63 mm in diameter) under ambient conditions and dehydrated under vacuum to obtain a cake height of 8 mm. Premixed dimethyl sulfoxide (7.2 mL, 100 mmol, 100% by mass) and water (4.8 mL, 270 mmol, 100% by mass) were packed into the tank under ambient conditions for washing. The contents of the tank were stirred at 20°C and then drained into a filter under ambient conditions and dehydrated under vacuum. This process was repeated for a second wash, and then 2-propanol (16 mL, 209 mmol, 100% by mass) was packed into the filter, and the contents were removed under vacuum. This process was repeated for a final wash, and then the contents of the filter were discharged into a drying dish and dried under vacuum in an oven at 45-50°C for 17 hours to obtain compound (I) form A in approximately 85% yield.

[0151] Example 2: XRPD analysis of compound (I) form A polymorph. Powder X-ray diffraction patterns were recorded using a Rigaku SmartLab equipped with a D / tex Ultra 250 detector and CBO-E optical elements (X-ray wavelength 1.5418 Å, nickel-filtered Cu Kα radiation, 40 kV, 50 mA) with two-theta scanning axes and one-dimensional scanning. During measurement, the sample was rotated at 30 revolutions per minute. The sample was scanned from 3 to 40° two-theta axes using step widths and scanning speeds of 0.01° and 0.1° / min, respectively. The powder sample was packed into a long glass capillary with an outer diameter of 0.9 mm.

[0152] Table 3 below shows the 2-theta values ​​(degrees) and relative intensities of the peaks derived from the XRPD traces characterizing morphology A.

[0153] [Table 3]

[0154] Relative intensity refers to the normalized integrated peak size for the largest peak having the category defined in Table 1.

[0155] result The powder X-ray diffraction pattern shown in Figure 1 demonstrates that compound (I) form A is highly crystalline.

[0156] Example 3: Properties of Reference Form 1 and Compound (I) Form A Differential Scanning Calorimetry (DSC) Thermal phenomena were analyzed by standard-mode differential scanning calorimetry using a TA Discovery DSC instrument. Approximately 1.5–2.0 mg of material contained in a standard sealed aluminum pan was measured over a temperature range of 25–300°C at a constant heating rate of 10°C / min. Nitrogen was used as a purge gas at a flow rate of 50 mL / min.

[0157] Thermogravimetric analysis (TGA) Weight loss was analyzed by standard thermogravimetric analysis using a TA Discovery TGA instrument. Approximately 3–5 mg of material placed on a 100 μL platinum pan was heated from ambient temperature to 300 °C at a constant heating rate of 10 °C / min. Nitrogen was used as a purge gas at a flow rate of 25 mL / min.

[0158] Dynamic vapor solvation (DVS) Approximately 5-10 mg of the material was weighed into an aluminum sample pan and subjected to the following relative humidity profiles using a Surface Measurement Systems DVS Resolution instrument: 40-90-0-90-0% relative humidity with a 10% relative humidity process and a dm / dt threshold of 0.002%, and a maximum process time limit of 6 hours. The temperature was maintained at approximately 25°C throughout.

[0159] result The results of the DSC and TGA experiments for Reference Form 1 and Compound (I) Form A are shown in Figures 2 and 3, respectively. The results of the DVS experiments for Reference Form 1 and Compound (I) Form A are shown in Figures 4 and 5, respectively.

[0160] Thermal analysis of Reference Example 1 by DSC showed a broad endothermic reaction indicating desolvation, followed by a single endothermic reaction during fusion (fusion peak: endothermic, onset 164.08°C, peak 168.52°C, enthalpy 44.746 J / g) (Figure 2). A single endothermic reaction during fusion was observed for compound (I) form A (fusion peak: endothermic, onset 216.63°C, peak 217.70°C, enthalpy 122.48 J / g) (Figure 3).

[0161] TGA of reference form 1 shows a weight loss of 1.617% by 100°C due to desolvation, indicating its initial hydrated form (Figure 2). TGA of compound (I) form A shows no significant weight loss before the melting point, indicating its anhydrous form (Figure 3).

[0162] DVS analysis shows that reference form 1 undergoes reversible hydration from hemihydrate to monohydrate at high relative humidity and reversible dehydration from hemihydrate to anhydrous at low relative humidity (Figure 4). DVS analysis also reveals that reference form 1 has a moisture absorption of 3.37% at 80% relative humidity (Figure 4).

[0163] In contrast, compound (I) form A has very low hygroscopicity, with a moisture absorption of 0.50% at 80% relative humidity (Figure 5).

[0164] Therefore, the results indicate that compound (I) form A exhibits high thermal stability, is not solvated, and has low hygroscopicity.

[0165] Example 4: Solubility of Reference Form 1 and Compound (I) Form A Table 4 shows the results of preliminary solubility tests of reference form (I) and compound (I) form A over 24 hours at 37°C.

[0166] [Table 4] * Unsaturated sample, SGF (simulated gastric fluid) = artificial gastric fluid, FaSSIF (fasted state simulated intestinal fluid) = artificial intestinal fluid in a fasted state, FeSSIF (fed state simulated intestinal fluid) = artificial intestinal fluid in a fed state

[0167] result The results indicate that compound (I) form A is more soluble than reference form 1 in various biologically related media (Table 4). Solubility tests in SGF were inconclusive because both samples were unsaturated.

[0168] Example 5: Physical stability of compound (I) form A The physical stability of compound (I) form A was evaluated by the water slurry method. Approximately 15 mg of compound (I) form A was weighed into a vial and 250 μL of water was added. This was stirred at ambient temperature for 10 days using a magnetic stirring rod, and then filtered using a 0.45 μm centrifuge filter (13,000 rpm for 5 minutes). Analysis by XRPD showed no morphological changes in the compound (I) form A material, indicating that compound (I) form A appears to be physically stable.

[0169] conclusion The results of the above experiments demonstrate that compound (I) form A is a distinct crystalline form that is more thermally stable than reference form 1. Unlike reference form 1, compound (I) form A is anhydrous, exhibiting variable (de)hydration due to its sensitivity to temperature and / or humidity. Compound (I) form A is less hygroscopic than reference form 1, and preliminary solubility data suggest that compound (I) form A is more soluble than reference form 1 in various biomedical media. Compound (I) form A also appears to be physically stable.

Claims

1. (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluorospiro[isoindoline-3,3'-pyrrolidine]-1,2'-dione: 【Chemistry 1】 The crystalline form is a cocrystal of adipic acid in a 2:1 ratio (compound (I) form A).

2. The crystal morphology according to claim 1, wherein the crystal morphology has an X-ray powder diffraction pattern that includes specific peaks at (±0.2)8.9, 19.1, 19.7, and 22.9 degrees 2 theta when measured using CuKα radiation.

3. The crystal morphology according to claim 1 or 2, wherein the crystal morphology has an X-ray powder diffraction pattern that, when measured using CuKα radiation, includes specific peaks at (±0.2) 8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2 theta.

4. The crystal morphology according to any one of claims 1 to 3, wherein the crystal morphology has substantially the same X-ray powder diffraction as shown in Figure 1 when measured using CuKα radiation.

5. The crystal morphology according to any one of claims 1 to 4, wherein the crystal morphology has a DSC thermogram substantially as shown in Figure 3.

6. The aforementioned crystalline form is (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo[3,2-b]pyridine-2-yl)methyl]-1'-buta-2-inyl-6-fluoro-spiro[isoindoline-3,3'-pyrroridine]-1,2'-dione: 【Chemistry 2】 The cocrystal of compound (I) form A and adipic acid in a 2:1 ratio is as follows: a) X-ray powder diffraction pattern, when measured using CuKα radiation, including specific peaks at (±0.2) 8.9, 19.1, 19.7, and 22.9 degrees 2-theta. b) X-ray powder diffraction pattern, when measured using CuKα radiation, including specific peaks at (±0.2) 8.9, 14.5, 16.9, 17.4, 19.1, 19.7, 21.2, 22.9, 23.7, and 25.8 degrees 2-theta. c) An X-ray powder diffraction pattern substantially as shown in Figure 1, d) A DSC thermogram showing an initial melting point at 216.6°C plus or minus 5°C and a peak at 217.7°C plus or minus 5°C. e) The crystal morphology according to claim 1, having at least one of a DSC thermogram and optionally a TGA thermogram, substantially as shown in Figure 3.

7. A pharmaceutical composition comprising the crystalline form described in any one of claims 1 to 6 and at least one pharmaceutically acceptable excipient.

8. For example, a crystalline form according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 for use as a drug in a method of preventing or treating a PRMT5-mediated disorder.

9. For example, the use of the crystalline form according to any one of claims 1 to 6 or the pharmaceutical composition according to claim 7 in the manufacture of a drug for the prevention or treatment of PRMT5-mediated disorders.

10. A method for prevention or treatment, for example, for a PRMT5-mediated disorder, comprising administering the crystalline form described in any one of claims 1 to 6 or the pharmaceutical composition described in claim 7.

11. The crystalline form for use, pharmaceutical composition for use, use, or method according to any one of claims 8 to 10, wherein the disorder is cancer.

12. The crystalline form for use, pharmaceutical composition for use, use, or method according to claim 11, wherein the cancer is MTAP-deficient cancer.

13. The crystalline form for use, pharmaceutical composition for use, use, or method according to claim 11 or 12, wherein the cancer is selected from gastric cancer, pancreatic cancer, colorectal cancer, uterine cancer, bile duct cancer, abdominal cancer, bladder cancer, cervical cancer, testicular germ cell cancer, lung cancer (e.g., non-small cell lung cancer), multiple myeloma, lymphoma (e.g., diffuse large B-cell lymphoma or Hodgkin lymphoma), rhabdomyosarcoma, and cutaneous squamous cell carcinoma.

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