Crystalline forms

Abstract

The present specification relates to crystalline forms of (3S)-2-[(5-amino-6-fluoro-1H-pyrrolo [3, 2-b] pyridin-2-yl) methyl]-1 '-butyl-2-alkynyl-6-fluoro-spiro [isoindoline-3, 3'-pyrrolidine]-1, 2 '-dione, and compositions and uses thereof.

Description

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

[0002] This specification relates to crystalline forms of (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, compositions thereof, and uses thereof. BACKGROUND

[0003] Protein arginine methyltransferase 5 (PRMT5) is a member of the PRMT family of arginine methyltransferases that catalyze the addition of a methyl group to the guanidinium motif of arginine residues using S-adenosyl-L-methionine (SAM) as the methyl donor. PRMT5 is a type II arginine methyltransferase that symmetrically dimethylates the guanidinium group of arginine residues, thereby converting the guanidinium NH2 group of arginine to an NMe2 group. PRMT5 methylates a number of different substrates, including histones and non-histone proteins, and in doing so modulates processes such as RNA splicing, cell proliferation, and DNA repair. Notably, PRMT5 is overexpressed in many cancer types and has been identified as a candidate for therapeutic intervention by developing small molecules that inhibit PRMT5 methyltransferase activity (see, e.g., 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 about 15% of cancers. Loss of the 9p21 chromosomal locus results in co-deletion of a number of additional genes, including the gene encoding methylthioadenosine phosphorylase (MTAP). MTAP is a metabolic enzyme involved in methionine salvage, and loss of MTAP results in increased concentrations of the MTAP substrate methylthioadenosine (MTA) in CDKN2A / MTAP deleted cancer cells. MTA itself acts as a weak PRMT5 inhibitor, and accumulation of MTA in CDKN2A / MTAP deleted cancer cell lines correspondingly results in partial inhibition of PRMT5 activity. Impaired PRMT5 activity makes CDKN2A / MTAP deleted cancer cells susceptible to further targeting of PRMT5, for example using short hairpin RNA (shRNA). A "collateral vulnerability" in cancer has been identified, in which CDKN2A / MTAP deleted tumors can be selectively targeted by PRMT5 inhibition (see Marjon et al. (2016) Cell Reports 15, 574-587; Mavrakis et al. (2016) Science 11; 351(6278): 1208-13; 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 a greater inhibitory effect on PRMT5 in environments where there is a relatively high concentration of MTA, such as CDKN2A / MTAP-deleted tumor cells, and do not exert a greater inhibitory effect on PRMT5 in healthy tissues. Thus, “MTA synergistic” PRMT5 inhibitors should have a high therapeutic index (and low off-target toxicity) as their anti-proliferative activity will selectively be exhibited in targeted CDKN2A / MTAP-deleted tumor cells. Several “MTA-synergistic” PRMT5 inhibitors have entered clinical trials, such as MRTX-1719 (NCT05245500); TNG-908 (NCT05275478); TNG-462 (NCT05732831); AMG-193 (NCT05094336, NCT05094336); and AZD3470 (NCT06130553; NCT06137144). However, to date, no PRMT5 inhibitor has been approved for therapeutic use, let alone a “MTA-synergistic” PRMT5 inhibitor. PCT / EP2022 / 075248 (published as International Publication Brochure WO2023 / 036974) discloses PRMT5 inhibitors, in particular MTA-synergistic PRMT5 inhibitors, including the compound (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 (referred to herein as Compound (I)):

[0006]

[0007] and its activity as a PRMT5 enzyme inhibitor in assays in the presence (IC 50 0.006 µM) and absence (IC 50 0.027 µM) of MTA, its activity in HCT116 wild-type cells (IC 50 0.2 µM) and HCT116 MTAP knockout cells (IC 50 0.0059 µM), and its anti-cell proliferative activity in HCT116 wild-type cells (IC 50 6.1 µM) and HCT116 MTAP knockout cells (IC 50 0.24 µM). Thus, Compound (I) is a PRMT5 inhibitor, and in particular a MTA-synergistic PRMT5 inhibitor. In order to further investigate the therapeutic potential of Compound (I), there is a need for solid forms of the compound having suitable properties for drug development.

[0008] In the formulation of a 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 a pharmaceutical formulation comprising the active compound and suitable excipients. The chemical and physical stability of the active compound are important factors in determining the suitability of a solid form for development of a pharmaceutical formulation. The active compound and the formulation containing the active compound should be capable of being effectively stored for a considerable period of time without exhibiting any significant changes in the physicochemical properties (e.g. chemical composition, density, hygroscopicity and solubility) of the active compound.

[0009] There remains a need to provide solid forms of Compound (I) that have suitable physical and chemical stability and other pharmaceutical relevant properties suitable for pharmaceutical development. SUMMARY

[0010] In one aspect, the present specification provides a crystalline form which is (3S)-2-[(5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]-l'-but-2-ynyl-6-fluoro- spiro[isoindoline-3,3'-pyrrolidine]-l,2'-dione:

[0011]

[0012] a co-crystal with adipic acid in a 2: 1 ratio (referred to herein as "Form A of Compound (I)").

[0013] The skilled person will appreciate that, although the specific tautomer of Compound (I) is shown above, Compound (I) can exist in a plurality of tautomeric forms. The present specification encompasses all such tautomeric forms.

[0014] The present specification also provides a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient.

[0015] The present specification also provides Form A of Compound (I) for use as a medicament, for example for use in the prevention or treatment of a disorder mediated by PRMT5.

[0016] The present specification also provides the use of Form A of Compound (I) in the manufacture of a medicament, for example for use in the prevention or treatment of a disorder mediated by PRMT5.

[0017] The present specification also provides a method of prevention or treatment, for example a method of prevention or treatment of a disorder mediated by PRMT5, comprising administration of Form A of Compound (I).

[0018] PCT / EP2022 / 075248 (published as International Publication Brochure WO2023 / 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 drug development. For example, at ambient conditions, Reference Form 1 exists as a hemihydrate that can be reversibly converted to an anhydrous form or monohydrate upon changes in temperature and / or relative humidity. The reversible (dehydrating) hydration behavior of Reference Form 1 can lead to unpredictable changes in physical properties due to interconversion between discrete forms, affecting storage and manufacturing, and can also generate variability in analytical assays, which can complicate its development and use as a pharmaceutically active substance.

[0019] Form A of Compound (I) as described herein has a unique crystal structure (as determined by XRPD) and surprisingly exhibits a combination of attractive properties in the context of drug development. For example, Form A of Compound (I) exhibits high thermal stability (melting point ~ 217.6 °C, as determined by DSC); is unsolvated (as determined by DSC and TGA); exhibits low hygroscopicity (as determined by DVS); appears to be physically stable (as determined by XRPD analysis following water slurry stability experiments); and appears to have improved solubility in a range of biorelevant media compared to Reference Form 1 (as determined by experiments in intestinal and gastric fluid models).

[0020] Compound (I) can exist in other solid forms having alternative counterions such as acetic acid, 1,5-naphthalene disulfonic 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-mesitalic acid, gallic acid, 3,5-dihydroxybenzoic acid, or 2,4-dihydroxybenzoic acid. These can exist in crystalline forms that are: i) co-crystals of Compound (I) with a counterion; ii) salts of Compound (I); or mixtures thereof. However, Form A of Compound (I) is a crystalline form that appears to be particularly suitable for drug development. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1: X-ray powder diffraction pattern of Form A of Compound (I), which is (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 in physical form with adipic acid in a 2:1 ratio.

[0022] Figure 2 : Differential scanning calorimetry and thermogravimetric analysis overlaid thermogram of Reference Form 1.

[0023] Figure 3 : Differential scanning calorimetry and thermogravimetric analysis overlaid thermogram of Form A of Compound (I).

[0024] Figure 4 : Dynamic vapor sorption isotherm plot of Reference Form 1.

[0025] Figure 5 : Dynamic vapor sorption isotherm plot of Form A of Compound (I). DETAILED DESCRIPTION

[0026] X-ray powder diffraction analysis can be performed according to standard methods, examples of which can be found in, for example, Kitaigorodsky, A.I. (1973), Molecular Crystals and Molecules, Academic Press, New York; Bunn, C.W. (1948), Chemical Crystallography, Clarendon Press, London; or Klug, H.P. and Alexander, L.E. (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.

[0027] It is known in the art that X-ray powder diffraction patterns can be obtained with one or more measurement errors depending on the measurement conditions such as the equipment used, sample preparation or machine. In particular, it is generally known that the intensities in X-ray powder diffraction patterns can fluctuate depending on the measurement conditions and sample preparation. For example, the skilled person in the field of X-ray powder diffraction will recognize that the relative intensities of the peaks can vary depending on the orientation of the sample under test and the type and settings of the instrument used. The skilled person will also recognize that the position of the reflections can be influenced by the exact height at which the sample is placed in the diffractometer and the zero calibration of the diffractometer. The surface flatness of the sample can also have a small influence. The skilled person will also recognize that the relative intensities of the peaks can be influenced by, for example, crystallites larger than about 30 microns in size and non- unitary aspect ratios that can affect the analysis of the sample. Furthermore, it will be understood that the intensities can fluctuate depending on the experimental conditions and sample preparation such as preferred orientation of the particles in the sample. The use of an automatic or fixed divergence slit will also affect the relative intensity calculations. The skilled person can account for such influences when comparing diffraction patterns. Therefore, the skilled person will understand that the diffraction pattern data presented herein should not be interpreted as absolute and that any crystalline form providing a powder diffraction pattern substantially the same as those disclosed herein falls within the scope of the present specification (for further information see Jenkins, R & Snyder, R. L. ‘Introduction to X-Ray Powder Diffractometry’ John Wiley & Sons, 1996).

[0028] In general, the measurement error in the diffraction angle in the X-ray powder diffraction pattern is about 5% or less, in particular plus or minus 0.2° 2Θ, and when considering Figure 1 the X-ray powder diffraction patterns in Tables 2 and 3 and when reading Table 2 and Table 3, this degree of measurement error should be taken into account. Furthermore, it will be understood that the intensities can fluctuate depending on the experimental conditions and sample preparation (preferred orientation). The definition of relative intensities is described in Table 1 :

[0029] Table 1. Definition of relative intensity .

[0030] Relative intensity % Definition 25-100 Very strong 10-25 Strong 3-10 Medium <3 Weak

[0031] The X-ray powder diffraction pattern was obtained directly from a sample of Form A of Compound (I) prepared as described herein. From a visual analysis of the pattern (Figure 1) Figure 1 ), it is apparent that Form A of Compound (I) is highly crystalline. The ten most prominent peaks are shown in Table 2:

[0032] Table 2. Ten most prominent peaks in the X-ray powder diffraction pattern of Form A .

[0033] 2-theta, ° Relative intensity 8.9 Very strong 14.5 Strong 16.9 Strong 17.4 Strong 19.1 Very strong 19.7 Very strong 21.2 Strong 22.9 Very strong 23.7 Strong 25.8 Strong

[0034] As used herein, the term "about," when used in reference to any given value, means within ±5% of that value.

[0035] In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least one specific peak at (±0.2) 8.9 degrees 2-theta when measured using CuK alpha radiation. In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least one specific peak at about 8.9 degrees 2-theta when measured using CuK alpha radiation.

[0036] In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least one specific peak at (±0.2) 19.1 degrees 2-theta when measured using CuK alpha radiation. In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least one specific peak at about 19.1 degrees 2-theta when measured using CuK alpha radiation.

[0037] In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least two specific peaks at (±0.2) 8.9 degrees and 19.1 degrees 2-theta when measured using CuK alpha radiation. In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least two specific peaks at about 8.9 degrees and 19.1 degrees 2-theta when measured using CuK alpha radiation.

[0038] In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least one specific peak at (±0.2) 8.9 degrees, 19.1 degrees, 19.7 degrees, and 22.9 degrees 2-theta when measured using CuK alpha radiation. In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising at least one specific peak at about 8.9 degrees, 19.1 degrees, 19.7 degrees, and 22.9 degrees 2-theta when measured using CuK alpha radiation.

[0039] In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising at least two specific peaks selected from (± 0.2) 8.9, 19.1, 19.7, and 22.9 degrees 2-theta, when measured using CuK alpha radiation. In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising at least two specific peaks selected from about 8.9, 19.1, 19.7, and 22.9 degrees 2-theta, when measured using CuK alpha radiation.

[0040] In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising at least three specific peaks selected from (± 0.2) 8.9, 19.1, 19.7, and 22.9 degrees 2-theta, when measured using CuK alpha radiation. In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising at least three specific peaks selected from about 8.9, 19.1, 19.7, and 22.9 degrees 2-theta, when measured using CuK alpha radiation.

[0041] In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising specific peaks at (± 0.2) 8.9, 19.1, 19.7, and 22.9 degrees 2-theta, when measured using CuK alpha radiation. In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising specific peaks at about 8.9, 19.1, 19.7, and 22.9 degrees 2-theta, when measured using CuK alpha radiation.

[0042] In one embodiment, the present specification provides Form A of Compound (I) having an X- ray powder diffraction pattern comprising specific peaks at (± 0.2) 8.9, 19.1, 19.7 and 22.9 degrees 2-theta when measured using CuK alpha radiation, and Form A of Compound (I) having an X-ray powder diffraction pattern comprising one, two, three, four, five or six 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 CuK alpha radiation. In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising specific peaks at about 8.9, 19.1, 19.7 and 22.9 degrees 2-theta when measured using CuK alpha radiation, and Form A of Compound (I) having an X-ray powder diffraction pattern comprising one, two, three, four, five or six specific peaks selected from about 14.5, 16.9, 17.4, 21.2, 23.7 and 25.8 degrees 2-theta when measured using CuK alpha radiation.

[0043] In one embodiment, the present specification provides Form A of Compound (I) having an X- ray powder diffraction pattern comprising 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 when measured using CuK alpha radiation. In one embodiment, the present specification provides Form A of Compound (I) having an X-ray powder diffraction pattern comprising 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 when measured using CuK alpha radiation.

[0044] In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising 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 CuKalphai radiation. In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising specific peaks at 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, when measured using CuKalphai radiation.

[0045] The complete list of XRPD pattern peaks for Form A of Compound (I) is shown in Table 3. In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising specific peaks substantially as set out in Table 3, when measured using CuKalphai radiation.

[0046] In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising 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 CuKalphai radiation, and Form A of Compound (I), which has an X-ray powder diffraction pattern comprising one, two or three specific peaks selected from (± 0.2) 27.0, 28.1, and 29.2 degrees 2-theta, when measured using CuKalphai radiation. In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern comprising specific peaks at 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, when measured using CuKalphai radiation, and Form A of Compound (I), which has an X-ray powder diffraction pattern comprising one, two or three specific peaks selected from about 27.0, 28.1, and 29.2 degrees 2-theta, when measured using CuKalphai radiation.

[0047] In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern substantially as set out in Table 3, when measured using CuKalphai radiation. Figure 1 In one embodiment, the present Specification provides Form A of Compound (I), which has an X-ray powder diffraction pattern substantially as set out in Table 3, when measured using CuKalphai radiation.

[0048] In the description of the present application, where a crystalline form is described as having an "X-ray powder diffraction pattern comprising at least one specific peak at (±0.2)... " or an "X-ray powder diffraction pattern comprising at least one specific peak at about... ", the XRPD of the crystalline form can comprise one or more of the listed 2-theta values, for example one or more of the 2-theta values, 2 or more of the 2-theta values, or 3 or more of the listed 2-theta values. Similar descriptions referring to different numbers of peaks (such as "... comprising at least two specific peaks... " and the like) will be interpreted in the same way.

[0049] Form A of Compound (I) has also been characterized by differential scanning calorimetry (DSC). The skilled person will appreciate that the values or ranges of values observed in the DSC thermogram of a particular compound will show variation between batches of different purities. Thus, although for one compound the range can be small, for other compounds the range can be quite large. Typically, the measurement error in the diffraction angle in a DSC thermal event is about ± 5 °C, and when considering the DSC data included herein, such as for Figure 3 , this degree of measurement error should be taken into account.

[0050] Form A of Compound (I) shows a melting, when heated in a differential scanning calorimeter (as described in the Examples section), with an onset temperature of about 216.6 °C and a peak temperature of about 217.7 °C, as shown in Figure 3 .

[0051] In one embodiment, the present specification provides Form A of Compound (I) having a DSC thermogram with an onset melting temperature of 216.6 °C ± 5 °C and a peak temperature of 217.7 °C ± 5 °C. In one embodiment, the present specification provides Form A of Compound (I) having a DSC thermogram with an onset melting temperature of about 216.6 °C and a peak temperature of about 217.7 °C. In one embodiment, the present specification provides Form A of Compound (I) having a DSC thermogram substantially as shown in Figure 3 .

[0052] Thermogravimetric analysis (TGA), in combination with DSC as described above, also demonstrates that Form A of Compound (I) is unsolvated, as opposed to Reference Form 1 (which, as described above, has variable and reversible (dehydrated) hydration), which can provide advantages during storage, manufacture and drug development.

[0053] In one embodiment, the present specification provides Form A of Compound (I) having a DSC thermogram substantially as shown inFigure 3 DSC and TGA overlay thermograms substantially as shown in

[0054] In one embodiment, the present specification provides a crystalline form which is (3S)-2-[(5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]-l'-but-2-ynyl-6-fluoro- spiro[isoindoline-3,3'-pyrrolidin]-l,2'-dione:

[0055]

[0056] a co-crystal with adipic acid in a 2: 1 ratio (Form A of Compound (I)) having at least one of:

[0057] a) an X-ray powder diffraction pattern comprising specific peaks at (±0.2) 8.9 degrees, 19.1 degrees, 19.7 degrees and 22.9 degrees 2-theta when measured using Cu Ka radiation;

[0058] b) an X-ray powder diffraction pattern comprising specific peaks at (±0.2) 8.9 degrees, 14.5 degrees, 16.9 degrees,

[0059] 17.4 degrees, 19.1 degrees, 19.7 degrees, 21.2 degrees, 22.9 degrees, 23.7 degrees and 25.8 degrees 2-theta when measured using Cu Ka radiation;

[0060] c) an X-ray powder diffraction pattern substantially as shown in Figure 1

[0061] d) a DSC thermogram with an onset melting temperature of 216.6 °C ± 5 °C and a peak temperature of 217.7 °C ± 5 °C;

[0062] e) a DSC thermogram and optionally a TGA thermogram substantially as shown in Figure 3

[0063] When the present specification refers 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%.

[0064] ​​It is expected that this crystalline form is substantially free of other crystalline forms of compound (I) (i.e. one or more crystalline forms other than Form A of compound (I)). Thus, in one embodiment, the crystalline form comprises less than 20%, 15%, 10%, 5%, 3% or 1% by weight of other crystalline forms of compound (I). In one embodiment, the crystalline form comprises more than 80%, 85%, 90%, 95%, 97% or 99% by weight of Form A of compound (I).

[0065] Based on the current experimental data, Form A of compound (I) is believed to be a co-crystal of (3S)-2-[(5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]-l'-but-2-ynyl-6-fluoro- spiro[isoindoline-3,3'-pyrrolidine]-l,2'-dione and adipic acid in a 2:1 ratio, i.e. (3S)-2-[(5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]-l'-but-2-ynyl-6-fluoro- spiro[isoindoline-3,3'-pyrrolidine]-l,2'-dione: adipic acid with a 2:1 stoichiometric ratio. Form A of compound (I) can also be referred to as compound (I): hemi-adipic acid co-crystal.

[0066] Without wishing to be bound by theory, co-crystal formation arises in cases where an acid or base "coformer" is solid at room temperature and there is no or only partial proton transfer between the free compound and such an acid or base coformer. Thus, a co-crystal of the coformer and the free compound is produced rather than a salt. The definition that the coformer acid or base is solid at room temperature is intended to distinguish the co-crystal from a solvate. It is recognised that proton transfer is in fact a continuum and can vary with temperature and thus the point at which the co-crystal is better described as a salt can be somewhat subjective. However, as noted above, based on all current experimental evidence, Form A of compound (I) is believed to be a co-crystal.

[0067] In one embodiment, there is provided Form A of compound (I) obtainable by any of the methods disclosed herein. In one embodiment, there is provided Form A of compound (I) obtainable by the method of Example 1, i.e. Method A. In one embodiment, there is provided Form A of compound (I) obtainable by the method of Example 1, i.e. Method B. In one embodiment, there is provided Form A of compound (I) obtainable by the method of Example 1, i.e. Method C.

[0068] Form A of compound (I) can be further characterised by additional techniques well known to those skilled in the art, such as single crystal X-ray diffraction (e.g. for assessing proton positions, bond lengths or bond angles), solid state NMR, Raman spectroscopy, infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, and the like. 1H-NMR (for assessing e.g. C or N chemical shifts) or spectroscopic techniques (for measuring e.g. O-H or N-H signals and IR peak shifts resulting from hydrogen bonding).

[0069] Form A of Compound (I) can be prepared as described in the Examples herein. Crystallization of the desired Form A of Compound (I) can be assisted by seeding with 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 seeding is particularly advantageous in large scale manufacturing.

[0070] Medical use

[0071] As described above, Compound (I) is an inhibitor of PRMT5, and in particular, Compound (I) is an MTA-cooperative PRMT5 inhibitor. Accordingly, Form A of Compound (I) is expected to be useful as a medicament, such as for preventing or treating a disorder mediated by PRMT5, i.e. a disorder in which inhibition of PRMT5 provides a prophylactic or therapeutic effect.

[0072] As used herein, the term “prevention” is intended to have its normal meaning and includes primary prevention, which prevents the development of a disease or condition, and secondary prevention, in which the disease or condition has already developed and the subject is temporarily or permanently protected against the exacerbation or worsening of the disease or condition or the development of new symptoms associated with the disease or condition. The terms “preventive”, “preventing”, “prevention” are used synonymously with “prophylaxis”.

[0073] As used herein, “treatment” is intended to have its normal meaning of managing a disease or condition so as to completely or partially alleviate one, some or all of its symptoms in a subject, or so as to correct or compensate for an underlying pathology. The terms “treatment” and “treating” are used synonymously with “therapy”.

[0074] A subject will typically be a subject in need of prevention or treatment in accordance with the present specification. In one embodiment, the subject is a human.

[0075] In one embodiment, the present specification provides Form A of Compound (I) for use as a medicament.

[0076] In one embodiment, the present specification provides Form A of Compound (I) for use in the prevention or treatment of a condition mediated by PRMT5 (e.g. a cancer). In one embodiment, the present specification provides Form A of Compound (I) for use in the prevention of a condition mediated by PRMT5 (e.g. a cancer). In one embodiment, the present specification provides Form A of Compound (I) for use in the treatment of a condition mediated by PRMT5 (e.g. a cancer).

[0077] In one embodiment, the present specification provides the use of Form A of Compound (I) in the manufacture of a medicament.

[0078] In one embodiment, the present specification provides the use of Form A of Compound (I) in the manufacture of a medicament for the prevention or treatment of a condition mediated by PRMT5 (e.g. a cancer). In one embodiment, the present specification provides the use of Form A of Compound (I) in the manufacture of a medicament for the prevention of a condition mediated by PRMT5 (e.g. a cancer). In one embodiment, the present specification provides the use of Form A of Compound (I) in the manufacture of a medicament for the treatment of a condition mediated by PRMT5 (e.g. a cancer).

[0079] In one embodiment, the present specification provides a method of preventing or treating a condition mediated by PRMT5 (e.g. a cancer), comprising administering Form A of Compound (I). In one embodiment, the present specification provides a method of preventing a condition mediated by PRMT5 (e.g. a cancer), comprising administering Form A of Compound (I). In one embodiment, the present specification provides a method of treating a condition mediated by PRMT5 (e.g. a cancer).

[0080] In one embodiment, the condition mediated by PRMT5 is a cancer. In one embodiment, the cancer is a MTAP-deleted cancer, i.e. a cancer in which the MTAP gene has been deleted. In one embodiment, the cancer is a CDKN2A-deleted and MTAP-deleted cancer, i.e. a cancer in which the CDKN2A and MTAP genes are deleted.

[0081] In one embodiment, the cancer is selected from gastric cancer, pancreatic cancer, colorectal cancer, uterine cancer, cholangiocarcinoma, gastric 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’s lymphoma), rhabdomyosarcoma and squamous cell carcinoma of the skin.

[0082] 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’s lymphoma).

[0083] 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.

[0084] Combination therapy

[0085] Form A of Compound (I) can be administered in combination with other compounds useful in the treatment of the above-mentioned conditions. In one embodiment, there is provided a combination therapy comprising Form A of Compound (I) and a second active ingredient.

[0086] Form A of Compound (I) and the second active ingredient can be administered simultaneously, sequentially or in admixture for the treatment of one or more of the conditions listed above. Such combinations can be employed in combination with one or more other active ingredients.

[0087] Pharmaceutical composition

[0088] For use in prophylaxis or therapy, Form A of Compound (I) will generally be administered as a pharmaceutical composition. Thus, in one embodiment, the present specification provides a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient.

[0089] In one embodiment, the present specification provides a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient for use as a medicament.

[0090] In one embodiment, the present specification provides a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient for use in the prevention or treatment of a disorder mediated by PRMT5, such as a disorder (e.g. a cancer) disclosed herein. In one embodiment, the present specification provides a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient for use in the prevention of a disorder mediated by PRMT5, such as a disorder (e.g. a cancer) disclosed herein. In one embodiment, the present specification provides a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient for use in the treatment of a disorder mediated by PRMT5, such as a disorder (e.g. a cancer) disclosed herein.

[0091] In one embodiment, the present specification provides the use of a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient in the manufacture of a medicament.

[0092] In one embodiment, the present specification provides the use of a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient in the manufacture of a medicament for preventing or treating a disorder mediated by PRMT5, such as a disorder disclosed herein (e.g., a cancer). In one embodiment, the present specification provides the use of a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient in the manufacture of a medicament for preventing a disorder mediated by PRMT5, such as a disorder disclosed herein (e.g., a cancer). In one embodiment, the present specification provides the use of a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient in the manufacture of a medicament for treating a disorder mediated by PRMT5, such as a disorder disclosed herein (e.g., a cancer).

[0093] In one embodiment, the present specification provides a method of preventing or treating a disorder mediated by PRMT5, such as a disorder disclosed herein (e.g., a cancer), comprising administering a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient. In one embodiment, the present specification provides a method of preventing a disorder mediated by PRMT5, such as a disorder disclosed herein (e.g., a cancer), comprising administering a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient. In one embodiment, the present specification provides a method of treating a disorder mediated by PRMT5, such as a disorder disclosed herein (e.g., a cancer), comprising administering a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient.

[0094] Administration

[0095] Form A of Compound (I) will generally be administered in the form of a pharmaceutical composition via oral, parenteral, intravenous, intramuscular, subcutaneous, or other injectable means, buccal, rectal, vaginal, transdermal, and / or nasal routes and / or via inhalation, which comprises Form A of Compound (I) in a pharmaceutically acceptable dosage form. These compositions can be administered in different dosages depending on the disorder and patient to be treated and the route of administration. In one embodiment, Form A of Compound (I) or a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient is administered orally.

[0096] Dosage forms suitable for oral use constitute one aspect of the present specification. In one embodiment, the present specification provides a solid oral dosage form comprising Form A of Compound (I) or a pharmaceutical composition comprising Form A of Compound (I) and at least one pharmaceutically acceptable excipient. In one embodiment, the solid oral dosage form is a tablet.

[0097] The compositions of the present specification can be obtained by conventional procedures using conventional pharmaceutical excipients well known in the art. Thus, compositions intended to be used for oral use can contain, for example, one or more coloring, sweetening, flavoring and / or preservative agents.

[0098] Suitable pharmaceutically-acceptable excipients for tablet formulations include, for example, inert diluents; granulating and disintegrating agents; binding agents; and lubricating agents. Tablet formulations can be uncoated or coated. Coated tablets can be prepared by either a wet- or dry-coating process.

[0099] Further information on formulations can be found in Chapter 25.2 of Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.

[0100] The amount of active ingredient (i.e. Form A of Compound (I)) combined with one or more excipients to produce a single dose will necessarily vary depending on the host treated and the particular mode of administration.

[0101] In prophylactic or therapeutic treatment of humans, a suitable daily dose of Form A of Compound (I) is from about 0.0001 mg / kg body weight to 100 mg / kg body weight.

[0102] The reader is referred to Chapter 25.3 of Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990 for further information on routes of administration and dosage regimes.

[0103] The present specification can be further illustrated by the following non-limiting examples.

[0104] Examples

[0105] In general:

[0106] (i) unless otherwise stated, operations were carried out at ambient temperature, i.e. in the range 17-25 °C and under an atmosphere of an inert gas, such as nitrogen;

[0107] (ii) evaporation was carried out under vacuum by rotary evaporation or using a Genevac apparatus or Biotage v10 evaporator and work-up procedures were carried out after removal of residual solids by filtration;

[0108] (iii) in an automated Teledyne Isco Rf or Teledyne Isco The above using pre-packed RediSep Rf gold TM Silica column (20 pm - 40 pm, spherical particles), Grace Resolv TM Column ( Silica) or Silicycle column (40 pm - 63 pm) for flash chromatography purification;

[0109] (iv) preparative reverse phase HPLC on an Agilent 1290 Infinity II preparative system equipped with SQ MS detector (multi-mode ESI / APCI source) using Waters CSH C18 OBD column (5 micron silica, 30 mm diameter, 100 mm length, flow rate of 50 mL / min) using a polar decreasing mixture of water (containing 0.1-0.3% ammonia solution) or water (containing 0.1% formic acid) and acetonitrile as eluent. Preparative SFC purification on a Sepiatec P100 SFC system or a Waters Prep 100 SFC system equipped with a QDa MS detector using chromatographic conditions as detailed in the respective experimental data;

[0110] (v) the yield, if present, is not necessarily the maximum obtainable;

[0111] (vi) generally, NMR chemical shift values are measured on the delta scale [proton magnetic resonance spectra were determined using a Bruker Avance 400 (400 MHz) instrument]; measurements were made at ambient temperature unless otherwise stated; the following abbreviations were used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of doublet of doublets;

[0112] dt, doublet of triplets; bs, broad signal;

[0113] (vii) generally, compounds were also characterised by mass spectrometry following liquid chromatography (LCMS or UPLC);

[0114] Analytical UPLC was performed on CSH C18 reversed phase silica using a Waters Acquity UPLC CSH C18 column with dimensions 2.1 mm x 50 mm and particle size 1.7 microns) using a mixture of decreasing polarity as eluent for gradient analysis, for example using a mixture of water containing 0.1 % formic acid or 0.1 % ammonia as solvent A and acetonitrile as solvent B for gradient analysis. A typical 2 minute analytical UPLC method would employ a solvent gradient from a 97:3 mixture of solvent A and solvent B to a 3:97 mixture of solvent A and solvent B over 1.3 min at approximately 1 mL / min. Unless otherwise stated, the molecular ion reported corresponds to [M+H]+; for molecules with multiple isotopic patterns (Br, CI etc) the value reported is the one obtained for the lowest isotopic mass;

[0115] (viii) evaluate the purity of intermediates by thin layer chromatography, mass spectrometry, HPLC (high performance liquid chromatography) and / or NMR analysis.

[0116] Preparation of Reference Form 1

[0117] The synthesis of Compound (I) is described in PCT / EP2022 / 075248 (published as International Publication Brochure WO2023 / 036974), the contents of which are incorporated by reference for the purpose of describing Compound (I) and its synthesis. The compounds described hereinafter are named using Chemdraw version 20.0.2.51 and the synthetic methods described herein are given by way of illustration only and are non-limiting.

[0118] Intermediate AQ: (6-chloro-5-fluoropyridin-3-yl)carbamic acid tert-butyl ester

[0119]

[0120] To 5-bromo-2-chloro-3-fluoropyridine (100 g, 475.22 mmol) in dioxane (1 L) was added tert-butyl carbamate (61.20 g, 522.74 mmol) and cesium carbonate (310.00 g, 950.43 mmol). 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 to 85 °C under nitrogen for 16 hours and then cooled to room temperature. The solids were filtered off and washed with excess dioxane. The solvent was removed in vacuo to give the crude title compound as a dark orange gum (179 g, 153%) which solidified upon standing. The crude gum was used directly 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.

[0121] Intermediate AR: 6-chloro-5-fluoropyridin-3-amine

[0122]

[0123] To a solution of tert-butyl (6-chloro-5-fluoropyridin-3-yl)carbamate (12.4 g, 54.7 mmol) in 1,4-dioxane (20 mL) was added 4M HCI in 1,4-dioxane (137 mL, 547.3 mmol) in one portion at 20 °C. The resulting suspension was stirred at 20 °C for 3 days. The reaction mixture was diluted with water (250 mL) and EtOAc (100 mL). The organic phase was separated and extracted with 2M HCI (3 x 100 mL) until no more product remained in the organic phase. The combined aqueous phases were stirred and cooled to 0 °C in an ice bath. The reaction mixture was basified to pH 14 with 50% NaOH solution. The reaction mixture was then extracted with EtOAc (2 x 250 mL), the combined organics were washed with saturated brine (50 mL), dried over MgS04, filtered and the solvent was removed in vacuo to give the title compound as a brown solid (12.4 g, 77%). Used directly in the next step without further purification. 1 H NMR (400 MHz, CDCl3, 27 °C) 3.88 (s, 2H), 6.80 (dd, J = 9.6, 2.5 Hz, 1H), 7.68 (d, J = 2.5 Hz, 1H).

[0124] Intermediate AS: 2-bromo-6-chloro-5-fluoropyridin-3-amine

[0125]

[0126] 6-chloro-5-fluoropyridin-3-amine (56.8 g, 379.3 mmol) 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 allowed to warm 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 off and washed with water (400 mL). The solid was dried under vacuum to give the title compound as a brown solid (76 g, 89%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 6.01 (2H, s), 7.12 (1H, d); m / z MH + 225.

[0127] Intermediate AT: 5-chloro-6-fluoro-lH-pyrrolo[3,2-b]pyridine-2-carboxylic acid

[0128]

[0129] Pd-acetate (3.35 g, 14.93 mmol), triphenylphosphine (3.92 g, 14.93 mmol), 2-bromo-6-chloro-5-fluoropyridin-3-amine (18.0 g, 74.65 mmol) and pyruvic acid (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 was heated at 100 °C under nitrogen for 2.5 hours. The reaction mixture was cooled to room temperature and filtered to remove unwanted solids. The filtrate was diluted with 2M NaOH (200 mL) and MTBE (200 mL) was added. The reaction mixture was then stirred vigorously and separated. The organic phase was washed with 2M NaOH (100 mL). The combined basic aqueous phases were carefully acidified with concentrated HC1 (aq) 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 under vacuum and dried to give the title compound as a beige solid (14.60 g, 91%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 7.16 (1H, dd), 7.84 (1H, dd), 12.36 (1H, s), 13.46 (1H, s); m / z MH + 214.

[0130] Intermediate AU: methyl 5-chloro-6-fluoro-lH-pyrrolo[3,2-b]pyridine-2-carboxylate

[0131]

[0132] 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 in vacuo. 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 give the title compound as a brown solid (14.20 g, 107%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 3.91 (3H, s), 7.24 (1H, dd), 7.88 (1H, dd), 12.56 (1H, s); m / z MH + 229.

[0133] Intermediate AV: methyl 5-chloro-6-fluoro-l-((2-(trimethylsilyl)ethoxy)methyl)-lH- pyrrolo[3,2-b]pyridine-2-carboxylate Intermediate AW: (5-chloro-6-fluoro-l-((2-(trimethylsilyl)ethoxy)methyl)-lH- pyrrolo[3,2-b]pyridin-2-yl)methanol

[0134]

[0135] Potassium bis(trimethylsilyl)amide (1 M in THF) (101 mL, 100.9 mmol) was added dropwise 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) at 5 °C over 15 min under nitrogen. The reaction mixture was stirred at 5 °C for 30 min. Potassium bis(trimethylsilyl)amide (1 M in THF) (15.52 mL, 15.52 mmol) was added and the reaction mixture was stirred at 5 °C for a further 30 min. (2-(Chloromethoxy)ethyl)trimethylsilane (1.88 mL, 10.09 mmol) was added and stirred at 5 °C for a further 15 min. The reaction mixture was quenched with saturated NH4Cl (400 mL) and extracted with EtOAc (400 mL). The aqueous phase was re-extracted with EtOAc (250 mL). The combined organic phases were dried over MgSO4, filtered and the solvent removed in vacuo. The crude material was suspended in heptane (450 mL) and stirred for 5 min. The unwanted solids were filtered off and washed with heptane (50 mL). The solvent was removed in vacuo to give the title compound (32.50 g, 117%) as a brown gum which solidified on standing. The gum was used directly in the next step without further purification. 1 H NMR (400 MHz, DMSO-d6, 30 °C) - 0.13 (9H, s), 0.70 - 0.80 (2H, m), 3.38 - 3.51 (2H, m), 3.89 (3H, s), 5.95 (2H, s), 7.41 (1H, d), 8.40 - 8.54 (1H, m); m / z MH + 359.

[0136] Intermediate AX: 5-chloro-2-(chloromethyl)-6-fluoro-l-((2-(trimethylsilyl)ethoxy)methyl)- lH-pyrrolo[3,2-b]pyridine Intermediate AE: racemic-l-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-l- carboxylate methyl ester

[0137]

[0138] Diisobutylaluminium hydride (1 M in toluene) (170 mL, 170.42 mmol) was added dropwise 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) at 5 °C over 15 minutes. The reaction mixture was stirred at room temperature for 30 minutes. The reaction mixture was carefully poured into 2 M 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 x 200 mL). The combined organics were dried over MgS04, filtered and the solvent was removed in vacuo. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 40% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a light orange oil (17.60 g, 68%) which solidified on standing. 1 HNMR (400 MHz, DMSO-d6, 30 °C) - 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 MH + 331.

[0139] Intermediate AF: (S)-l-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-l- carboxylate methyl ester Intermediate BQ: (S)-l-allyl-2-((5-chloro-6-fluoro-l-((2-(trimethylsilyl)ethoxy)methyl)- lH-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-3-oxoisoindoline-l-carboxylate methyl ester

[0140]

[0141] 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]pyridin-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. Saturated NaHC03(500 mL) was then added slowly. Once gas evolution had 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 a phase separation paper and the solvent was removed in vacuo to give the title compound as a brown crystalline solid (19.2 g, 91%). 1H NMR (400 MHz, DMSO-d6, 27°C) - 0.10 (9H, s), 0.75 - 0.9 (2H, m), 3.43 - 3.53 (2H, m), 5.07 (2H, s), 5.66 (2H, s), 6.7 - 6.9 (1H, m), 8.32 (1H, dd); m / z MH + 349.

[0142] Intermediate AA: Methyl 2-(2-bromo-4-fluorophenyl)acetate

[0143]

[0144] Thionyl chloride (31.3 mL, 429.1 mmol) was carefully added dropwise to 2-(2-bromo-4-fluorophenyl)acetic acid (CAS Number 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 removed in vacuo. The residue was partitioned between EtOAc (250 mL) and saturated NaHC03(200 mL). The organic phase was washed with water (100 mL), brine (100 mL), passed through phase separation paper and the solvent removed in vacuo to give the title compound as a colourless oil (105 g, 99%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 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 + .

[0145] Intermediate AB: Methyl 5-fluoro-2-(2-methoxy-2-oxoethyl)benzoate

[0146]

[0147] 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(ll) (complex 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 to 7 bar with carbon monoxide. The pressure vessel was heated to 100 °C and stirred for 2 hours. The reaction mixture was allowed to cool, vented and filtered to remove the catalyst. The solvent was removed in vacuo and the residue dissolved in EtOAc (250 mL), washed with water (2 x 200 mL) and brine (100 mL). The organic phase was passed through a phase separation paper and the solvent removed in vacuo. The crude product was purified by flash silica chromatography eluting with a gradient of 0% to 50% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a pale yellow oil (38.40 g, 93%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 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.

[0148] Intermediate AC: rac-Methyl 2-(1-bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoate

[0149]

[0150] Methyl 5-fluoro-2-(2-methoxy-2-oxoethyl)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 at reflux for 72 hours. The reaction mixture was cooled and washed with water (2 x 250 mL), brine (100 mL), passed through a phase separation paper and the solvent removed in vacuo. The crude product was purified by flash silica chromatography eluting with a gradient of 0% to 40% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a colourless oil (50.50 g, 80%). 1HNMR (400 MHz, DMSO-d6, 30 °C) 3.71 (3H, s), 3.86 (3H, s), 6.51 (1H, s), 7.56 (1H, td), 7.66 (1H, dd), 7.81 (1H, dd); m / z MH + .

[0151] Intermediate AD: rac-Methyl 5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1- carboxylate

[0152]

[0153] Methyl 5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1 -carboxylate (Intermediate AD, 45.3 g, 142 mmol) was placed in a flask with MeCN (300 mL) and sodium bicarbonate (23.9 g, 285 mmol) was added. As the reaction mixture was brought to 80 °C, rac-2-(1 -bromo-2-methoxy-2-oxoethyl)-5-fluorobenzoic acid methyl ester (43.5 g, 142 mmol) dissolved in MeCN (100 mL) was slowly added via a dropping funnel. The reaction mixture was stirred at 80 °C for 3 hours. The reaction mixture was allowed to cool, most of the MeCN was removed in vacuo and the residue was partitioned between EtOAc (400 mL) and water (400 mL). The aqueous phase was re-extracted with EtOAc (100 mL), the organics were combined and washed with brine (50 mL). The organic phase was passed through a phase separation paper and the solvent was removed in vacuo. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 50% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a pale yellow oil (45.3 g, 96%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 3.69 (3H, s), 3.73 (3H, s), 4.31 (1H, d), 5.04 (1H, d), 5.18 (1H, s), 6.87 - 6.94 (2H, m), 7.17 - 7.24 (2H, m), 7.50 (1H, ddd), 7.57 (1H, dd), 7.62 (1H, dd); m / z MH + 330.

[0154] Intermediate BR: (S)-l-allyl-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-l-((2- (trimethylsilyl)ethoxy)methyl)-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-3- oxoisoindoline-l-carboxylate methyl ester Intermediate BS: (S)-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-l-((2- (trimethylsilyl)ethoxy)methyl)-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-3- oxo-l-(2-oxoethyl)isoindoline-l-carboxylate methyl ester methyl carboxylate

[0155]

[0156] A solution of rac-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-l- carboxylic acid methyl ester (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'-((lR,2R)-cyclohexane-l,2-diyl)bis(2-(diphenylphosphoryl)benzamide) (2.52 g, 3.64 mmol) in THF (400 mL) was stirred at 5 °C under nitrogen. 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 in vacuo. The reaction mixture was partitioned between EtOAc (400 mL) and water (400 mL) and the organic phase was passed through a phase separation paper. The solvent was removed in vacuo to give an orange oil. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 50% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a cream coloured solid (25.8 g, 96%). 1 H NMR (400 MHz, DMSO-d6, 30 °C) 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 MH + 370.

[0157] (S)-2-((l'-(but-2-yn-l-yl)-5-fluoro-2',3-dioxospiro[isoindoline-l,3'-pyrrolidine]-2- yl)methyl)-6-fluoro-l-((2-(trimethylsilyl)ethoxy)methyl)-lH-pyrrolo[3,2-b]pyridin-5- yl)carbamic acid tert-butyl ester

[0158]

[0159] rac-5-Fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-l-carboxylic acid methyl ester (25.8 g, 69.7 mmol) was purified by SFC chromatography (Column: Phenomenex CI, 30 mm x 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 fractions were evaporated to dryness to give the title compound as a white solid (15.1 g, 56%). 1H NMR (400 MHz, DMSO-d6, 30 °C) 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 MH + 370. (Bioactivity of the bioactive compounds prepared based on the use of this enantiomer of the intermediate (compared to those prepared using the other enantiomer), and X-ray structural evidence that the stereochemical assignment of this intermediate is preferred and more active than the R enantiomer).

[0160] Intermediate AI: (S)-methyl 1-allyl-5-fluoro-3-oxoisoindoline-1-carboxylate

[0161]

[0162] (S)-methyl 1-allyl-5-fluoro-2-(4-methoxybenzyl)-3-oxoisoindoline-1-carboxylate (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 in vacuo 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 a phase separation paper and the solvent was removed in vacuo. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 50% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a crystalline cream solid (12.5 g, 93%). 1 H NMR (400 MHz, DMSO-d6, 27 °C) 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 MH + 250.

[0163] (S)-2-((5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl)-l'-(but-2-yn-l- yl)-5-fluorospiro[isoindoline-l,3'-pyrrolidine]-2',3-dione (Reference Form 1) ​

[0164]

[0165] (S)-1 -allyl-5-fluoro-3-oxoisoindoline-1 -carboxylate (11.80 g, 47.34 mmol) and 5-chloro-2-(chloromethyl)-6-fluoro-1 -((2-(trimethylsilyl)ethoxy)methyl)-1 H- 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 EtOAc (300 mL). The aqueous phase was re-extracted with EtOAc (200 mL). The organic phases were combined, washed with water (3 x 200 mL), brine (200 mL), passed through a phase separation paper and the solvent was removed in vacuo. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 100% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound (22.2 g, 83%) as a yellow gum which slowly set / crystallised to give a yellow solid. 1 H NMR (400 MHz, DMSO-c / 6, 27 °C) - 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, dd), 4.94 - 5.10 (2H, m), 5.26 (1H, d), 5.59 (1H, d), 5.68 (1H, d), 6.76 (1H, s), 7.49 - 7.56 (1H, m), 7.58 - 7.68 (2H, m), 8.25 (1H, dd); m / z MH + 562.

[0166] ​ ​ ​

[0167]

[0168] (S)-1-allyl-2-((5-chloro-6-fluoro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H- pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-3-oxoisoindoline-1-carboxylic acid methyl ester (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 bubbled through the reaction mixture for 10 minutes and then the reaction mixture was refluxed for 3 hours. The reaction mixture was cooled, diluted with water (400 mL) and extracted with EtOAc (2 x 300 mL). The combined organic phases were washed with saturated brine (200 mL), passed through a phase separation paper and the solvent was removed in vacuo. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 50% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a light yellow foam (11.68 g, 68%). 1 HNMR (400 MHz, DMSO-d6, 27 °C) - 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 MH + 643.

[0169] ​ ​ ​

[0170]

[0171] To a solution of (S)-1 -allyl-2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-1 - ((2-(trimethylsilyl)ethoxy)methyl)-1 H-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-3- oxoisoindoline-1 -carboxylate (11.50 g, 17.89 mmol) in 1,4-dioxane (240 mL) and water (60 mL) was added 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). The reaction mixture was stirred at room temperature for 18 h. The reaction mixture was partitioned between DCM (200 mL) and water (100 mL). The aqueous phase was re-extracted with DCM (100 mL) and the organic phases were combined, passed through a phase separation paper and the solvent was removed in vacuo. The crude product was purified by flash silica gel chromatography eluting with a gradient of 0% to 50% EtOAc in heptane. The pure fractions were evaporated to dryness to give the title compound as a beige foam (8.70 g, 75%). 1 H NMR (400 MHz, DMSO-d6, 27 °C) - 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 (1 H, d), 5.06 (1 H, d), 5.52 (1 H, d), 5.61 (1 H, d), 6.57 (1 H, s), 7.48 - 7.57 (1 H, m), 7.64 (1 H, dd), 7.68 (1 H, dd), 8.00 (1 H, d), 9.16 (1 H, s), 9.25 (1 H, s); m / z MH + 645.

[0172] ​ ​

[0173] ​

[0174]

[0175] (S)-methyl 2-((5-((tert-butoxycarbonyl)amino)-6-fluoro-l-((2- (trimethylsilyl)ethoxy)methyl)-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl)-5-fluoro-3- oxo-l-(2-oxoethyl)isoindoline-l-carboxylate (9.50 g, 14.73 mmol) and but-2-yn- 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 NaHC03(100 mL), water (100 mL), and brine (100 mL). The organic phase was passed through a phase separation paper and the solvent was removed in vacuo to give the title compound. The crude compound was used in the next reaction without further purification, assuming 100% yield. m / z MH + 666.

[0176] ​ ​

[0177]

[0178] (S)-tert-butyl 2-((l'-(but-2-yn-l-yl)-5-fluoro-2',3-dioxospiro[isoindoline-l,3'- pyrrolidin]-2-yl)methyl)-6-fluoro-l-((2-(trimethylsilyl)ethoxy)methyl)-lH- pyrrolo[3,2-b]pyridin-5-yl)carbamate (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. The 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 a mixture of water (containing 1 volume% of NH4OH (28-30% in H20)) and MeCN with decreasing polarity as eluent (30-60% gradient). Fractions containing the desired compound were combined, the MeCN was removed in vacuo and the resulting solid was filtered off and dried to give the title compound as a creamy crystalline solid (3.74 g, 58%). 1H NMR (400 MHz, DMSO-d6, 27 °C) 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 MH + 436.

[0179] Example 1 : (3S)-2-[(5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]-l'- but-2-ynyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-l,2'-dione: hemoxalic acid co- crystal ("Form A" of Compound (I)) Preparation of Form A of Compound (I) Method A: single solvent co-crystallization

[0180] Method B: anti-solvent / cooling co-crystallization (without seeding)

[0181] 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 with heating, then the two solutions were combined and stirred with a magnetic stir bar at ambient temperature for 8 days with the cap on the vial slightly loosened to allow for slow evaporation. The resulting material was filtered, the solid recovered and dried under a stream of air overnight to give Form A of Compound (I) in approximately 53% yield.

[0182] Method C: cooling / anti-solvent co-crystallization (with seeding)

[0183] Reference Form 1 (501.51 mg, 1.0942 mmol, 95 mass%) was charged into an 8 mL vial, charged with adipic acid (166.13 mg, 1.1368 mmol, 100 mass%) and dimethyl sulfoxide (3.54 g, 45.3 mmol, 100 mass%) and the contents stirred using a dual 3-pitch blade impeller at 600 rpm, 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 / minute and the temperature held for 20 minutes. Water (1.4 mL, 78 mmol, 100 mass%) was charged to the vial at 80 °C at a rate of 0.012 mL / minute over 2 hours and the temperature held for a further 30 minutes. The contents of the vessel were then cooled to 20 °C at a rate of 0.125 °C / minute over 8 hours and the temperature held for 80 hours. The contents of the vial were further cooled to 5 °C over 15 minutes and the temperature held for 3 hours before heating to 20 °C and charging the vial with water (0.75 mL, 42 mmol, 100 mass%) and crystallisation occurred during this period. The contents of the vial were then discharged into a filter (30 mm diameter filter paper) under ambient conditions and deliquored under vacuum. A pre-mix of dimethyl sulfoxide (0.525 mL, 7.4 mmol, 100 mass%) and water (0.225 mL, 12.5 mmol, 100 mass%) was charged into the vessel for a wash under ambient conditions. The contents of the vial were agitated at 20 °C before being discharged into a filter under ambient conditions and deliquored under vacuum. This step was repeated for a second wash before 2-propanol (1.00 mL, 13.1 mmol, 100 mass%) was charged into the filter and the contents deliquored under vacuum. This step was repeated for a final wash after which the contents of the filter were discharged into a drying tray and dried under vacuum in an oven at 45-50 °C for 24 hours to give Form A of Compound (I) in approximately 92% yield.

[0184] Example 2: XRPD analysis of Form A polymorph of Compound (I)

[0185] Reference Form 1 (8.01 g, 18.2 mmol, 99 mass%) was charged to a 100 mL vessel, hexanedioic acid (2.57 g, 17.6 mmol, 100 mass%) and dimethyl sulfoxide (54.88 g, 702.4 mmol, 100 mass%) were charged and the contents were agitated using a 4-pitch vane impeller at 400 rpm, heated to 25 °C and held at temperature for 30 minutes. The contents of the vessel were then heated to 80 °C at a rate of 5 °C / minute and the temperature was held for 20 minutes. Water (21.6 mL, 1200 mmol, 100 mass%) was charged to the vessel at 80 °C at a rate of 0.1125 mL / minute over 2 hours and the temperature was held for a further 5 minutes. The contents of the vessel were then cooled to 65 °C at a rate of 0.5 °C / minute over 30 minutes and the temperature was held for 30 minutes. Seed crystals (prepared according to Method B; 8.7 mg, 0.017 mmol, 100 mass%) were then charged to the vessel at 65 °C and the temperature was held for 3 hours. The contents of the vessel were then cooled to 20 °C at a rate of 0.125 °C / minute over 6 hours and the temperature was held for a further 3 hours. Antisolvent, water (12 mL, 666.119 mmol, 100 mass%) was charged to the vessel at 20 °C over 6 hours and the temperature was held for a further 6 hours. The contents of the vessel were then discharged into a filter (63 mm diameter) under ambient conditions and deliquored under vacuum to give a 8 mm filter cake height. Pre-mixed dimethyl sulfoxide (7.2 mL, 100 mmol, 100 mass%) and water (4.8 mL, 270 mmol, 100 mass%) were charged to the vessel under ambient conditions for a wash. The contents of the vessel were agitated at 20 °C and then discharged into the filter under ambient conditions and deliquored under vacuum. This step was repeated for a second wash and then 2-propanol (16 mL, 209 mmol, 100 mass%) was charged to the filter and the contents were deliquored under vacuum. This step was repeated for a final wash and then the contents of the filter were discharged into a drying tray and dried under vacuum in an oven at 45-50 °C for 17 hours to give Form A of Compound (I) in approximately 85% yield.

[0186] Table 3. Complete list of peaks in the X-ray powder diffraction pattern of Form A in the range 3°-30° 2Θ

[0187] Rigaku SmartLab (wavelength of X-rays is 1.5418 A) equipped with a D / tex Ultra 250 detector and CBO-E optics Powder X-ray diffraction patterns were recorded using a 2-theta scan axis and in one-dimension scan on a Bruker D4 Endeavor diffractometer (Ni-filtered Cu Ka radiation, 40 kV, 50 mA). During the measurement, the sample was rotated at 30 rotations per minute. The sample was scanned from 3°-40° 2-theta with a step width and scan speed of 0.01° / min and 0.1° / min, respectively. The powder sample was filled in a long glass capillary with an outer diameter of 0.9 mm.

[0188] The 2-theta values (in degrees) and relative intensities of the peaks derived from the XRPD trace for Form A are shown in Table 3 below:

[0189] 2Θ, ° .

[0190] Relative intensity 2Θ, ° Relative intensity Very strong 8.9 Medium 20.5 Medium 11.8 Medium 20.6 Medium 12.2 Strong 21.2 Strong 14.5 Weak 22.2 Weak 15.4 Very strong 22.9 Weak 15.5 Medium 23.4 Medium 15.8 Strong 23.7 Weak 16.1 Weak 24.7 Strong 16.9 Medium 25.1 Strong 17.4 Strong 25.8 Medium 17.6 Weak 26.4 Weak 17.8 Medium 26.6 Weak 17.9 Strong 27.0 Weak 17.9 Medium 27.3 Medium 18.2 Weak 27.9 Weak 18.6 Strong 28.1 Very strong 19.1 Medium 28.6 Medium 19.4 Weak 28.8 Very strong 19.7 Strong 29.2 Results

[0191] Relative intensities refer to the integrated peak size normalized to the size of the strongest peak, where the categories are as defined in Table 1.

[0192] Figure 1

[0193] Example 3: Properties of Reference Form 1 and Form A of Compound (I) The powder X-ray diffraction pattern shown in Figure 1 indicates that Form A of Compound (I) is highly crystalline.

[0194] Differential scanning calorimetry (DSC)

[0195] Thermogravimetric analysis (TGA)

[0196] Thermal events were analyzed by standard mode differential scanning calorimetry on a TA Discovery DSC instrument. Approximately 1.5 mg - 2.0 mg of material contained in a standard hermetic aluminum pan was measured at a constant heating rate of 10 °C / minute over a temperature range of 25 °C to 300 °C. Nitrogen was used as the purge gas at a flow rate of 50 mL / minute.

[0197] Dynamic vapor sorption (DVS)

[0198] Weight loss was analyzed by standard thermogravimetric analysis on a TA Discovery TGA instrument. Approximately 3 mg - 5 mg of material placed on a 100 μL platinum pan was heated from the instrument ambient temperature to 300 °C at a constant heating rate of 10 °C / minute. Nitrogen was used as the purge gas at a flow rate of 25 mL / minute.

[0199] Results

[0200] Approximately 5-10 mg of material was weighed into an aluminium sample pan and subjected to the following relative humidity profile using a Surface Measurement Systems DVS Resolution instrument: 40-90-0-90-0% relative humidity, relative humidity step of 10% and a dm / dt threshold of 0.002% and a maximum step time limit of six hours. The temperature was maintained at approximately 25 °C at all times.

[0201] Figure 2

[0202] The DSC and TGA experimental results for Reference Form 1 and Form A of Compound (I) are shown in Figures Figure 3 and Figure 4 respectively. The DVS experimental results for Reference Form 1 and Form A of Compound (I) are shown in Figures Figure 5 and Figure 2 respectively.

[0203] Thermal analysis of Reference Example 1 by DSC showed a broad endothermic peak indicative of desolvation followed by a single melting endothermic peak (melting peak: endotherm, onset temperature 164.08 °C, peak temperature 168.52 °C, enthalpy 44.746 J / g) Figure 3 ). A single melting endothermic peak was observed for Form A of Compound (I) (melting peak: endotherm, onset temperature 216.63 °C, peak temperature 217.70 °C, enthalpy 122.48 J / g) Figure 2 ).

[0204] TGA of Reference Form 1 showed a weight loss of 1.617% to 100 °C due to desolvation, indicating an initial hydrated form Figure 3 ). TGA of Form A of Compound (I) showed no significant weight loss prior to the melting point, indicating an anhydrous form Figure 4 ).

[0205] DVS analysis showed 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 revealed that Reference Form 1 has a moisture uptake of 3.37% at 80% relative humidity Figure 5 ).

[0206] In contrast, Form A of Compound (I) has a very low moisture uptake comprising a moisture uptake of 0.50% at 80% relative humidity Example 4: Solubility of Reference Form 1 and Form A of Compound (I) ).

[0207] Thus, the results show that Form A of compound (I) exhibits high thermal stability, is unsolvated and has low hygroscopicity.

[0208] Table 4. Solubility of Form A and Reference Form 1 of Compound (I) in biorelevant media

[0209] The results of preliminary solubility tests at 37°C over 24 hours for reference Form (I) and Form A of compound (I) are shown in Table 4.

[0210] Results .

[0211]

[0212] * unsaturated sample; SGF = simulated gastric fluid; FaSSIF = fasted state simulated intestinal fluid; FeSSIF = fed state simulated intestinal fluid

[0213] Example 5: Physical stability of Form A of Compound (I)

[0214] The results show that Form A of compound (I) is more soluble than reference Form 1 in a variety of biorelevant media (Table 4). The solubility test in SGF is inconclusive as both samples were unsaturated.

[0215] Conclusion

[0216] The physical stability of Form A of compound (I) was assessed by the water slurry method. Approximately 15 mg of Form A of compound (I) was weighed into a vial and 250 μΐ^ of water was added. This was stirred at ambient temperature for 10 days using a magnetic stir bar, then filtered using a 0.45 μιη centrifugal filter (13,000 rpm, 5 minutes) and analysed by XRPD, which showed no change in form in the Form A material of compound (I), indicating that Form A of compound (I) appears to be physically stable.

[0217] ​

[0218] The results of the above experiments show that Form A of compound (I) is a discrete crystalline form which is more thermally stable than reference Form 1. Form A of compound (I) is anhydrous, unlike reference Form 1 which shows variable (dehydrated) hydration due to its sensitivity to temperature and / or humidity. Form A of compound (I) is less hygroscopic than reference Form 1, and preliminary solubility data show that Form A of compound (I) is more soluble than reference Form 1 in a variety of biorelevant media. Form A of compound (I) also appears to be physically stable.

Claims

1. A crystalline form of (3S)-2-[(5-amino-6-fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]- 1 '-but-2-ynyl-6-fluoro-spiro[isoindoline-3,3'-pyrrolidine]-l,2'-dione: Form A of Compound (I) is a co-crystal with adipic acid in a 2: 1 ratio.

2. The crystalline form of claim 1, wherein the crystalline form has an X-ray powder diffraction pattern comprising specific peaks at (± 0.2) 8.9, 19.1, 19.7 and 22.9 degrees 2-theta when measured using Cu Ka radiation.

3. The crystalline form of claim 1 or 2, wherein the crystalline form has an X-ray powder diffraction pattern comprising 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 Cu Ka radiation.

4. The crystalline form of any one of claims 1 to 3, wherein the crystalline form has an X-ray powder diffraction substantially as shown in Figure 1 when measured using Cu Ka radiation.

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

6. The crystalline form of claim 1, wherein the crystalline form is (3S)-2-[(5-amino-6- fluoro-lH-pyrrolo[3,2-b]pyridin-2-yl)methyl]-l'-but-2-ynyl-6-fluoro-spiro[isoindoline-3,3'- pyrrolidine]-l,2'-dione: Form A of Compound (I) co-crystal with adipic acid in a 2: 1 ratio, having at least one of: a) an X-ray powder diffraction pattern comprising specific peaks at (± 0.2) 8.9, 19.1, 19.7 and 22.9 degrees 2-theta when measured using Cu Ka radiation; b) an X-ray powder diffraction pattern comprising 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 Cu Ka radiation; c) an X-ray powder diffraction pattern substantially as shown in Figure 1; d) a DSC thermogram with an onset melting temperature of 216.6 °C ± 5 °C and a peak temperature of 217.7 °C ± 5 °C; e) a DSC thermogram and optionally a TGA thermogram substantially as shown in Figure 3.

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

8. The crystalline form of any one of claims 1 to 6 or the pharmaceutical composition of claim 7 for use as a medicament, for example for the prevention or treatment of a disorder mediated by PRMT5. ​ ​ ​ ​ 9. Use of a crystalline form according to any one of claims 1 to 6 or a pharmaceutical composition according to claim 7 in the manufacture of a medicament, for example for the prevention or treatment of a disorder mediated by PRMT5.

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

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

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

13. A crystalline form for use, a pharmaceutical composition for use, a use or a method according to claim 11 or 12, wherein the cancer is selected from gastric cancer, pancreatic cancer, colorectal cancer, uterine cancer, cholangiocarcinoma, gastric 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’s lymphoma), rhabdomyosarcoma and squamous cell carcinoma of the skin.

Images