Nek7 degraders and methods of use thereof

Abstract

The present invention provides a compound of formula (I) and solvates and methods of use thereof.

Description

[0001]NEK7 DEGRADERS AND METHODS OF USE THEREOF FIELD OF THE INVENTION The present invention relates to novel compounds which can act as degraders of NEK7, and methods of use thereof. BACKGROUND Inflammasomes are a group of intracellular complexes located in the cytosol, which are an element of innate immunity, responsible for the detection of either pathogen-associated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs). Inflammasome multiprotein complexes are composed of three parts: a sensor protein, an adaptor, and pro-caspase-1, responsible for the production of pro-inflammatory cytokines - interleukin 1β (IL-1β) and IL-18 from their precursors (pro- IL-1β and pro-IL-18, respectively). Among all the known inflammasomes, the NLRP3 inflammasome plays a central role in innate immunity. NLRP3 inflammasome is composed of NLRP3 as a sensor protein, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) as an adaptor and pro-caspase-1. The interactions among these proteins are closely associated with the formation of NLRP3 inflammasome. NLRP3 has an N-terminal pyrin domain, which interacts with the adaptor protein ASC via interactions between pyrin domains; a central adenosine triphosphatase (ATPase) domain known as NACHT, which comprises an NBD, helical domain 1 (HD1), winged helix domain (WHD) and helical domain 2 (HD2) and a C-terminal LRR domain. ASC also has a caspase recruitment domain, which recruits caspase-1 via interactions between the caspase recruitment domains, to promote caspase dimerization and activation. Caspase 1 causes maturation of pro-inflammatory cytokines - IL-1β and IL-18 from their precursor forms (pro-IL-1β and pro-IL-18 respectively). The formation and activation of the inflammasome requires the synergistic effect of two signals. First, as a result of the initiation signal from TOLL-like receptors (TLR), proinflammatory transcription factors are induced, especially NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) or cytokines such as TNF or IL-1β, which upregulates the inflammasome components as well as NEK7. NEK7 has recently been identified as an important requirement in NLRP3 inflammasome activation via direct interaction with NLRP3. Human NEK7, a member of the family of mammalian NIMA-related 1 251982040 v1 kinases (NEK proteins), consists of a non-conserved and disordered N-terminal regulatory domain as well as a conserved C-terminal catalytic domain - serine / threonine kinase. NEK7 binds directly to the leucine-rich repeat (LRR) domain of NLRP3. The interaction stimulates the assembly and activation of the NLRP3 inflammasome and promotes its oligomerization through the bridging of adjacent subunits of the NLRP3 protein. NLRP3 is associated with the catalytic domain of NEK7, but the catalytic activity of NEK7 was shown to be dispensable for activation of the NLRP3 inflammasome. NEK7 is expressed in a variety of tissues and is essential for cell division and growth, as well as the survival of mammalian cells. Low activity status of NEK7 protein in resting cells is critical to the maintenance of homeostasis. However, once homeostasis is disordered, an aberrant expression of NEK7 occurs, which is closely related to neoplastic progression. Overexpression of NEK7 promotes the production of abnormal cells, including the multinucleated cells and apoptotic cells which are related to inflammation. With the inappropriate release of proinflammatory cytokines, the NLRP3 inflammasome is involved in various inflammatory diseases, such as atherosclerosis, type 2 diabetes, metabolic syndrome, multiple sclerosis, Alzheimer’s disease, gout, rheumatoid arthritis, and inflammatory bowel disease. Mechanism of NLRP3 inflammasome activation by NEK7 strongly indicates promising roles for targeting NEK7 in treating inflammation-related diseases. There are several pathways that are essential for the activation of NLRP3 inflammasome, including ROS signaling, K+efflux, Ca2+signaling, chloride efflux and lysosomal destabilization. Thus, a great number of inhibitors have been widely used to disturb these signaling pathways. Compounds focused on NEK7 may regulate NLRP3 to abolish the inflammation response with improved specificity and potency. Apart from NLRP3 inflammasome activation, NEK7 plays significant role in mitotic entry, cell cycle progression, cell division, mitotic progression. In last years the potential role of NEK7 in the cancer development of various tissues has been demonstrated. Although inhibitors in general can inhibit protein of interest (POI) activity, targeted degradation appears as an attractive therapeutic alternative. Protein degradation is mainly regulated by the ubiquitin–proteasome pathway, in which proteins are tagged for degradation by covalent conjugation of multiples ubiquitin molecules. Manipulation of the ubiquitin–proteasome system to achieve targeted degradation of proteins within cells is possible using chemical tools and drugs. Targeted protein degradation (TPD) rather than inhibition could provide advantages such as reduced drug exposure time required to suppress signaling, it provides more complete and lasting inactivation of 251982040 v1 downstream signaling since cell needs time to express POI in required quantity again. TPD also can overcome intrinsic feedback activation or overexpression of the target protein. Protein degraders may potentially be used as a general way to solve compensatory upregulation of proteins that contributes to illness, adverse effects, and drug resistance. Therefore, there is a great need to provide NEK7 degraders as a key to downregulate inflammasome activation in NLRP3 inflammasome-related diseases as well as in cancer treatment. SUMMARY OF INVENTION In accordance with a first aspect of the invention, there is provided a compound of formula: In accordance with a second aspect of the invention, there is provided a method of making the compound or solvate thereof of the first aspect, the method comprising: (i) separation of stereoisomers of tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate; (ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (iii) hydrogenation of tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate to provide tert-Butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5- (tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate; 251982040 v1 (iv) conversion of tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)-5-oxopentanoate to (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione; (v) separation of stereoisomers of (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione by chiral HPLC, to provide (S)-3-((R)-3-methyl-1-oxo-5-((R)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione and (S)-3-((R)-3-methyl-1-oxo-5-((S)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. In some embodiments of the second aspect, the separation by chiral HPLC in step (i) is carried out at 20-25°C using an Agilent 1200 series instrument, a CHIRALPAK IC column (5 μm, 250 x 20 mm), a mobile phase of hexane / DCM / EtOH at a ratio of 60 / 20 / 20 v / v / v, and a flowrate of 18 mL / min for 30 min, and wherein monitoring is carried out by UV at λ = 254 nm. In some such embodiments, when subjected to analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 μm), a mobile phase of hexane / AcOEt / EtOH / iPrNH2at a ratio of 50 / 25 / 25 / 0.1 v / v / v / v, and a flow rate of 1 mL / min), the stereoisomers separated by chiral HPLC separation have the following retention times Rt: tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rtof 7.974 min; and tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin- 2-yl)-5-oxopentanoate has a retention time Rtof 9.692 min. In some embodiments of the second aspect, step (ii) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate with 2-(3,4-dihydro-2H-pyran-6-yl)- 4,4,5,5-tetramethyl-1,3,2-dioxaborolane and K3PO4, and subsequent reaction with PdCl2(dtbpf) under inert atmosphere at 70°C under microwave irradiation, to provide tert-butyl (S)-5-amino-2- ((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate. In some embodiments of the second aspect, the hydrogenation in step (iii) is carried out using PtO2. In some embodiments of the second aspect, the separation by chiral HPLC in step (v) is carried out at 20-25°C using an Agilent 1200 series instrument, a CHIRALPAK IG column (5 μm, 250 x 21 mm), a mobile phase which is an isocratic mixture of hexane / AcOEt / iPrOH at a ratio of 2 / 1 / 1 v / v / v, a flowrate of 21 mL / min and duration time of 60 min, and wherein monitoring is carried out by UV at λ = 254 nm. In some such embodiments, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of hexane / AcOEt / iPrOH / iPrNH2at a 251982040 v1 ratio of 50 / 25 / 25 / 0.1 v / v / v / v, and a flowrate of 1 mL / min, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro- 2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 38.993 min; and (S)-3- ((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 51.878 min. In some embodiments, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of ACN / water / FA at a ratio of 70 / 30 / 0.1 v / v / v, and a flowrate of 1 mL / min, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 10.53 min; (S)-3-((R)-3-methyl-1- oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 8.34 min. In some embodiments of the second aspect, step (iv) comprises reaction of tert-butyl (2S)-5-amino- 2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate with benzenesulfonic acid at 130°C under microwave irradiation, to provide (3S)-3-((3R)-3-methyl-1-oxo- 5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. In some embodiments of the second aspect, the method further comprises reacting tert-butyl L- glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(1-bromoethyl)benzoate to provide the tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate of step (i). In accordance with a third aspect of the invention, there is provided a method of making the compound or solvate thereof of the first aspect, the method comprising: (i) separation of stereoisomers of tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate; (ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate; (iii) conversion of tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate; 251982040 v1 (iv) conversion of tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (v) separation of stereoisomers of tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3-en-1- yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate by Supercritical Fluid Chromatography (SFC) to give tert-butyl (S)-5-amino-2-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (vi) conversion of tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate to (S)-3-((R)-5-((R)-1-(allyloxy) but-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione and (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (vii) conversion of (S)-3-((R)-5-((R)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione to (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione; and (viii) conversion of (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione to (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione. In some embodiments of the third aspect, step (ii) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate with potassium vinyltrifluoroborate and K2CO3and reaction with PdCl2(dppf) to provide tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5- vinylisoindolin-2-yl)-5-oxopentanoate. In some embodiments of the third aspect, step (iii) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5-oxopentanoate with osmium tetroxide and sodium periodate monohydrate to provide tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin- 2-yl)-5-oxopentanoate. In some embodiments of the third aspect, step (iv) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate with potassium allyltrifluoroborate and boron trifluoride diethyl etherate to provide tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3- en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate. 251982040 v1 In some embodiments of the third aspect, the separation by SFC in step (v) is carried out at 30°C using a CHIRAL ART Cellulose-SC column (5 μm, 150 x 25 mm), a flowrate of 80g / min, a CO2backpressure of 100bar with 50% isopropanol modifier, and wherein monitoring is carried out by UV at λ = 236 nm. In some embodiments of the third aspect, when subjected to analytical SFC performed at 30°C using a CHIRALPAK IC column (150 x 4.6 mm, 5 μm), a mobile phase of 75% CO2 / 25% methanol, a flowrate of 3 g / min, a backpressure of 100bar and monitoring carried out by UV at λ = 254 nm, the stereoisomers separated by SFC have the following retention times Rt: tert-butyl (S)-5-amino-2-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate has a retention time Rtof 3.11 min tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate has a retention time Rtof 3.85 min. In some embodiments of the third aspect, step (vi) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate with sodium hydride and allyl bromide to provide (S)-3-((R)-5-((R)-1-(allyloxy) but-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione and (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione. In some embodiments of the third aspect, step (vii) comprises reaction of (S)-3-((R)-5-((R)-1- (allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione with Grubbs Catalyst 1st Generation to provide (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione. In some embodiments of the third aspect, step (viii) comprises reduction of (S)-3-((R)-5-((R)-3,6- dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione to provide (S)-3-((R)-3- methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. In some embodiments of the third aspect, the method further comprising reacting tert-butyl L- glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(1-bromoethyl)benzoate to provide the tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate of step (i). 251982040 v1 In accordance with a fourth aspect of the invention, there is provided a compound of formula: , or a solvate thereof, produced by the method of any of the above embodiments of the second or third aspects of the invention. In some embodiments of the fourth aspect, the compound In some embodiments of any of the first and fourth aspects, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of ACN / water / FA at a ratio of 70 / 30 / 0.1 v / v / v, and a flowrate of 1 mL / min, the compound has a retention time Rtof 10.53 min. In accordance with a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising the compound of any of the above embodiments of the first and fourth aspects of the invention. In accordance with a sixth aspect of the present invention, there is provided the compound of any of the above embodiments of the first and fourth aspects of the invention, or the pharmaceutical composition of the fifth aspect of the invention, for use in medicine. In accordance with a seventh aspect of the present invention, there is provided the compound of any of the above embodiments of the first and fourth aspects of the invention, or the pharmaceutical composition of the fifth aspect of the invention, for use in a method of treating a disease or condition in a subject in need thereof, wherein the disease or condition is an inflammatory disease or condition, an autoinflammatory disease or conditions, an auto-immune disease or condition, a respiratory disease 251982040 v1 or condition, a cardiovascular disease or condition, a gastro-intestinal disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a skin disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, a wound, a burn, or cancer. In some embodiments of the seventh aspect, the disease or condition is an inflammatory disease or condition, an autoinflammatory disease or conditions, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, or a wound or burn. In some embodiments of the seventh aspect, the disease or condition is cryopyrin-associated periodic syndromes (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), neonatal onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA), hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), Tumour Necrosis Factor (TNF) Receptor-Associated Periodic Syndrome (TRAPS), systemic juvenile idiopathic arthritis, adult-onset Still’s disease (AOSD), relapsing polychondritis, Schnitzler’s syndrome, Sweet’s syndrome, Behcet’s disease, anti-synthetase syndrome, deficiency of interleukin 1 receptor antagonist (DIRA), haploinsufficiency of A20 (HA20), lupus nephritis, pulmonary arterial hypertension, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, asthma, amyotrophic lateral sclerosis, rheumatoid arthritis, gout, Alzheimer’s disease, Parkinson’s disease, Huntington’s diseases, spinal cord injury, atherosclerosis, heart failure, dilated cardiomyopathy (DCM), pericarditis, myocarditis, myocardial infarction, obesity, type II diabetes, nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, chronic kidney disease (CDK), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn’s disease. 251982040 v1 In accordance with an eighth aspect of the present invention, there is provided a method of degrading NEK7 protein comprising contacting said protein with the compound of any of the above embodiments of the first and fourth aspects of the invention. In some embodiments of the eighth aspect, the compound is formulated in a pharmaceutical composition. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the possible crystal structure of (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl- 1-oxoisoindolin-2-yl)piperidine-2,6-dione (intermediate ix(2)), under co-crystallisation with CRBN_TBD (Cereblon thalidomide-binding domain) and IKZF2_ZF2 (Helios protein zinc finger 2). Figure 2 shows NEK7 levels in cells treated with the tested compounds or with DMSO for 24 hours. Figure 3 shows the dose-dependent effect of NEK7 protein degradation after 24 hours of cell incubation with the tested compounds. DETAILED DESCRIPTION OF THE INVENTION In a first aspect of the present invention, there is provided a compound of formula: , or a solvate thereof. In the second aspect of the invention, there is provided a method of making the compound or solvate as defined above, the method comprising: (i) separation of stereoisomers of tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate; (ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; 251982040 v1 (iii) hydrogenation of tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate to provide tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5- (tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate; (iv) conversion of tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)-5-oxopentanoate to (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione; (v) separation of stereoisomers of (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione by chiral HPLC, to provide (S)-3-((R)-3-methyl-1-oxo-5-((R)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione and (S)-3-((R)-3-methyl-1-oxo-5-((S)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. In some embodiments, the separation by chiral HPLC in step (i) is carried out at 20-25°C using an Agilent 1200 series instrument, a CHIRALPAK IC column (5 μm, 250 x 20 mm), a mobile phase of hexane / DCM / EtOH at a ratio of 60 / 20 / 20 v / v / v, and a flowrate of 18 mL / min for 30 min, and wherein monitoring is carried out by UV at λ = 254 nm. In some such embodiments, when subjected to analytical LC performed at 20-25°C using a CHIRALPAK IC column (250 x 4.6 mm, 5 μm), a mobile phase of hexane / AcOEt / EtOH / iPrNH2at a ratio of 50 / 25 / 25 / 0.1 v / v / v / v, and a flow rate of 1 mL / min, monitored by UV at λ = 254 nm, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate has a retention time Rtof 7.974 min; and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rtof 9.692 min. In some embodiments, step (ii) comprises reaction of tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate with 2-(3,4-dihydro-2H-pyran-6-yl)-4,4,5,5- tetramethyl-1,3,2-dioxaborolane and K3PO4, and subsequent reaction with PdCl2(dtbpf) under inert atmosphere at 70°C under microwave irradiation, to provide tert-butyl (S)-5-amino-2-((R)-5-(3,4- dihydro-2H-pyran-6-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate. In some embodiments of the second aspect, the hydrogenation in step (iii) is carried out using PtO2. Other possible hydrogenating agents include: platinum on activated charcoal; palladium on activated charcoal; rhodium on activated charcoal; rhodium on alumina; palladium hydroxide on activated charcoal; and raney nickel. 251982040 v1 In some embodiments, the separation by chiral HPLC in step (v) is carried out at 20-25°C using an Agilent 1200 series instrument, a CHIRALPAK IG column (5 μm, 250 x 21 mm), a mobile phase which is an isocratic mixture of hexane / AcOEt / iPrOH at a ratio of 2 / 1 / 1 v / v / v, a flowrate of 21 mL / min and duration time of 60 min, and wherein monitoring is carried out by UV at λ = 254 nm. In some such embodiments, when subjected to analytical LC performed at 20-25°C using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of hexane / AcOEt / iPrOH / iPrNH2at a ratio of 50 / 25 / 25 / 0.1 v / v / v / v, and a flowrate of 1 mL / min, monitored by UV at λ = 254 nm, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: (S)-3-((R)-3-methyl-1-oxo-5-((R)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 38.993 min; and (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6- dionehas a retention time Rtof 51.878 min. In some embodiments, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of ACN / water / FA at a ratio of 70 / 30 / 0.1 v / v / v, and a flowrate of 1 mL / min, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H- pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 10.53 min; (S)-3-((R)-3- methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione has a retention time Rtof 8.34 min. In some embodiments, step (iv) comprises reaction of tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1- oxo-5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate with benzenesulfonic acid at 130°C under microwave irradiation, to provide (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran- 2-yl)isoindolin-2-yl)piperidine-2,6-dione. In some embodiments, the method further comprises reacting tert-butyl L-glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(1-bromoethyl)benzoate to provide the tert-Butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate of step (i). In the third aspect of the invention, there is provided a method of making the compound or solvate thereof as described above, the method comprising: (i) separation of stereoisomers of tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate; 251982040 v1 (ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate; (iii) conversion of tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate; (iv) conversion of tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (v) separation of stereoisomers of tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3-en-1- yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate by Supercritical Fluid Chromatography (SFC) to give tert-butyl (S)-5-amino-2-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (vi) conversion of tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate to (S)-3-((R)-5-((R)-1-(allyloxy) but-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione and (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (vii) conversion of (S)-3-((R)-5-((R)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione to (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione; and (viii) conversion of (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione to (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione. In some embodiments of the third aspect, step (ii) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate with potassium vinyltrifluoroborate and K2CO3and reaction with PdCl2(dppf) to provide tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5- vinylisoindolin-2-yl)-5-oxopentanoate. In some embodiments of the third aspect, step (iii) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5-oxopentanoate with osmium tetroxide and sodium 251982040 v1 periodate monohydrate to provide tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin- 2-yl)-5-oxopentanoate. In some embodiments of the third aspect, step (iv) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate with potassium allyltrifluoroborate and boron trifluoride diethyl etherate to provide tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3- en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate. In some embodiments of the third aspect, the separation by SFC in step (v) is carried out at 30°C using a CHIRAL ART Cellulose-SC column (5 μm, 150 x 25 mm), a flowrate of 80g / min, a CO2backpressure of 100bar with 50% isopropanol modifier, and wherein monitoring is carried out by UV at λ = 236 nm. In some such embodiments, when subjected to analytical SFC performed at 30°C using a CHIRALPAK IC column (150 x 4.6 mm, 5 μm), a mobile phase of 75% CO2 / 25% methanol, a flowrate of 3 g / min, a backpressure of 100bar and monitoring carried out by UV at λ = 254 nm, the stereoisomers separated by SFC have the following retention times Rt: tert-butyl (S)-5-amino-2-((R)- 5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rtof 3.11 min; and tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rtof 3.85 min. In some embodiments of the third aspect, step (vi) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate with sodium hydride and allyl bromide to provide (S)-3-((R)-5-((R)-1-(allyloxy) but-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione and (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione. In some embodiments of the third aspect, step (vii) comprises reaction of (S)-3-((R)-5-((R)-1- (allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione with Grubbs Catalyst 1st Generation to provide (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione. In some embodiments of the third aspect, step (viii) comprises reduction of (S)-3-((R)-5-((R)-3,6- dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione to provide (S)-3-((R)-3- methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. 251982040 v1 In some embodiments of the third aspect, the method further comprises reacting tert-butyl L- glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(1-bromoethyl)benzoate to provide the tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate of step (i). In accordance with a fourth aspect of the invention, there is provided a compound of formula: , or a solvate thereof, produced by the method as described above. In some embodiments of the first and fourth aspects, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of ACN / water / FA at a ratio of 70 / 30 / 0.1 v / v / v, and a flowrate of 1 mL / min, the compound has a retention time Rtof 10.53 min. In accordance with a fifth aspect of the present invention, there is provided a pharmaceutical composition comprising a compound as described above. In accordance with a sixth aspect of the present invention, there is provided a compound or pharmaceutical composition as described above, for use in medicine. In accordance with a seventh aspect of the present invention, there is provided a compound or pharmaceutical composition as described above, for use in a method of treating a disease or condition in a subject in need thereof, wherein the disease or condition is an inflammatory disease or condition, an autoinflammatory disease or conditions, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a gastro-intestinal disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a skin disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, a wound, a burn, or cancer. 251982040 v1 In some embodiments, the disease or condition is an inflammatory disease or condition, an autoinflammatory disease or conditions, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, or a wound or burn. In some embodiments, the disease or condition is cryopyrin-associated periodic syndromes (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), neonatal onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA), hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), Tumour Necrosis Factor (TNF) Receptor-Associated Periodic Syndrome (TRAPS), systemic juvenile idiopathic arthritis, adult-onset Still’s disease (AOSD), relapsing polychondritis, Schnitzler’s syndrome, Sweet’s syndrome, Behcet’s disease, anti-synthetase syndrome, deficiency of interleukin 1 receptor antagonist (DIRA), haploinsufficiency of A20 (HA20), lupus nephritis, pulmonary arterial hypertension, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, asthma, amyotrophic lateral sclerosis, rheumatoid arthritis, gout, Alzheimer’s disease, Parkinson’s disease, Huntington’s diseases, spinal cord injury, atherosclerosis, heart failure, dilated cardiomyopathy (DCM), pericarditis, myocarditis, myocardial infection, obesity, type II diabetes, nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, chronic kidney disease (CKD), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn’s disease. In accordance with a eighth aspect of the present invention, there is provided a method of degrading NEK7 protein comprising contacting said protein with a compound as described above. In some such embodiments, the compound is formulated in a pharmaceutical composition. 251982040 v1 EXAMPLES Synthesis of compounds The reagents and solvents were used as received from the commercial sources. Proton nuclear magnetic resonance (NMR) spectra were recorded on 400MHz Bruker Avance spectrometers. The spectra are reported in terms of chemical shift (δ [ppm]), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m = multiplet), coupling constant (J [Hz]), and integration. Chemical shifts are reported in ppm relative to dimethyl sulfoxide-d6(δ 2.50) as indicated in NMR spectra data. The samples were prepared by dissolving a dry sample (0.2-2 mg) in an appropriate deuterated solvent (0.7-1 mL). LCMS were collected using Waters SQD2 or API 2000 Mass Spectrometers. All masses reported are the m / z of the protonated parent ions unless otherwise stated. The sample was dissolved in an appropriate solvent (e.g. DMSO, CAN, water) and was injected directly into the column using an automated sample handler. The chemical names were generated using ChemDraw Professional v. 18.2.0.48 from PerkinElmer Informatics, Inc. Abbreviations used in the following examples are presented below in the alphabetical order: ACN Acetonitrile AcOEt Ethyl acetate DCM Dichloromethane DIPEA N,N-Diisopropylethylamine DMSO Dimethyl sulfoxide Et Ethyl EtOH Ethanol FA Formic acid HPLC High performance liquid chromatography LC Liquid chromatography LCMS Liquid chromatography mass spectrometry Me Methyl MHz Megahertz NMR Nuclear magnetic resonance 251982040 v1 PdCl2(dtbpf) [1,1'-Bis(di-tert-butylphosphino)ferrocene]palladium(II) dichloride PdCl2(dppf) [1,1'-Bis(diphenylphosphino)ferrocene]palladium iPrOH Isopropanol iPrNH2Isopropylamine RtRetention time RT Room temperature SFC Supercritical Fluid Chromatography tBu tert-butyl TFA Trifluoroacetic acid THP Tetrahydropyran Synthesis of (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine- 2,6-dione (Compound X1) and (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin- 2-yl)piperidine-2,6-dione (Compound X2) Step 1: To a stirred solution of tert-butyl L-glutaminate hydrochloride (2 g, 8.38 mmol, 2.35 equiv) in dry ACN (20 mL) under argon, cooled in an ice-water bath, was added DIPEA (2.28 mL, 3.67 equiv) and the mixture was stirred for 10 min. Methyl 4-bromo-2-(1-bromoethyl)benzoate (1.15 g, 3.57 mmol, 1 251982040 v1 equiv) was then added at 0°C, the cooling bath was removed and the reaction mixture was stirred at RT for 16 h under argon. After completion, the reaction mixture was diluted with AcOEt, washed with water and brine. The organic fraction was dried over Na2SO4and concentrated in vacuo to provide crude product. tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (mixture of stereoisomers) was purified by flash column chromatography (SiO2, AcOEt / hexanes, 45 / 55, v / v) and forwarded for chiral separation of stereoisomers. Methyl 4-bromo-2-(1-bromoethyl)benzoate was prepared according to the procedure described in WO2022216644A1. Chiral separation The separation of stereoisomers was performed by chiral HPLC, using Agilent 1200 series instrument. Column name: CHIRALPAK IC (5 μm, 250 x 20 mm). Mobile phase: hexane / DCM / EtOH, 60 / 20 / 20, v / v / v. Operating at ambient temperature and flowrate 18 mL / min for 30 min, monitoring by UV at λ = 254 nm. The pure stereoisomers isolated by the chiral HPLC separation were then subjected to analytical LC. The retention times Rtof the pure stereoisomers in the analytical LC process are indicated below. Isomer 1 (Intermediate i(1)): tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate (360 mg, 23% yield) Rt= 7.974 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 μm), mobile phase: hexane / AcOEt / EtOH / iPrNH2, 50 / 25 / 25 / 0.1, v / v / v / v, and flow rate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 254 nm) LCMS (ESI+) m / z 411.0, 413.0 [M+H]+1H NMR (401 MHz, DMSO-d6) δ 7.92 (d, J = 1.7 Hz, 1H), 7.68 (dd, J = 8.1, 1.7 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 7.22 (s, 1H), 4.70 (q, J = 6.6 Hz, 1H), 4.46 (dd, J = 8.9, 5.1 Hz, 1H), 2.30 – 2.02 (m, 4H), 1.49 (d, J = 6.7 Hz, 3H), 1.37 (s, 9H). Isomer 2 (Intermediate i(2)): tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate (360 mg, 23% yield) Rt= 9.692 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 μm), mobile phase: hexane / AcOEt / EtOH / iPrNH2, 50 / 25 / 25 / 0.1, v / v / v / v, and flow rate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 254 nm) LCMS (ESI+) m / z 411.0, 413.0 [M+H]+251982040 v11H NMR (400 MHz, DMSO-d6) δ 7.90 (s, 1H), 7.69 (dd, J = 1.8, 8.0 Hz, 1H), 7.60 (d, J = 8.2 Hz, 1H), 7.20 (s, 1H), 7.14 (s, 1H), 4.65 (q, J = 6.8 Hz, 1H), 4.45 – 4.34 (m, 1H), 2.39 – 2.22 (m, 1H), 2.22 – 2.03 (m, 3H), 1.41 (d, J = 6.8 Hz, 3H), 1.36 (s, 9H). Step 2: To a stirred solution of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)- 5-oxopentanoate (Intermediate i(2)) (300 mg, 0.73 mmol, 1 equiv) in the mixture of 1,4-dioxane / water (9 mL, 10 / 1, v / v) were added K3PO4(2 equiv) and 2-(3,4-dihydro-2H-pyran-6-yl)-4,4,5,5-tetramethyl- 1,3,2-dioxaborolane (1.1 equiv) under argon and the slurry was further bubbled with argon for 20 min. PdCl2(dtbpf) (0.15 equiv) was then added and the reaction was stirred in inert atmosphere at 70°C for 1 h under microwave irradiation. After completion, the reaction mixture was concentrated in vacuo and tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate (Intermediate ii) (250 mg, 82% yield) was purified by flash column chromatography (SiO2, AcOEt / hexane, 1 / 1, v / v). LCMS (ESI+) m / z 415.4 [M+H]+Step 3: To a stirred solution of tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate (Intermediate ii) (250 mg, 0.53 mmol, 1 equiv) in AcOEt (8 mL) was added PtO2(220 mg) and the reaction mixture was stirred at RT for 3 h under hydrogen atmosphere (balloon). After completion, the reaction mixture was filtered and the filtrate was evaporated to give crude product. tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H- pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate (Intermediate iii) (120 mg, 54% yield) was purified by flash column chromatography (SiO2, AcOEt / hexane, 55 / 45, v / v) as a mixture of stereoisomers. LCMS (ESI+) m / z 417.4 [M+H]+Step 4: To a solution of tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)-5-oxopentanoate (Intermediate iii) (110 mg, 0.264 mmol, 1 equiv) in ACN (3 mL) was added benzenesulfonic acid (2 equiv) and the reaction mixture was stirred at 130°C for 1 h under microwave irradiation. After completion the solvent was evaporated and the stereoisomers of the product were separated by chiral HPLC. Chiral separation: The chiral HPLC separation was performed on Agilent 1200 series instrument, using CHIRALPAK IG column (5 μm, 250 x 21 mm) operating at ambient temperature, with flowrate 21 251982040 v1 mL / min, mobile phase isocratic mixture of 2 / 1 / 1 hexane / AcOEt / iPrOH, v / v / v, duration time of 60 min and monitored by UV at λ = 254 nm. The pure stereoisomers isolated by the chiral HPLC separation were then subjected to analytical LC. The retention times Rtof the pure stereoisomers in the analytical LC process are indicated below. Isomer 1 (Compound X1): (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione (18 mg, 20% yield) Rt= 38.993 min (analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), mobile phase: hexane / AcOEt / iPrOH / iPrNH2, 50 / 25 / 25 / 0.1, v / v / v / v; and flowrate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 254 nm) Rt= 10.53 min (analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), mobile phase: ACN / water / FA, 70 / 30 / 0.1, v / v / v, and flowrate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 250 nm) LCMS (ESI-) m / z 341.2 [M+H]-1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.57 (s, 1H), 7.45 (d, J = 7.9 Hz, 1H), 4.74 (dd, J = 12.4, 5.2 Hz, 1H), 4.64 (q, J = 6.7 Hz, 1H), 4.45 (d, J = 11.0 Hz, 1H), 4.05 (d, J = 11.5 Hz, 1H), 3.61 – 3.51 (m, 1H), 2.82 (dd, J = 11.9, 8.3 Hz, 1H), 2.69 – 2.54 (m, 2H), 1.98 (dd, J = 10.7, 5.0 Hz, 1H), 1.86 (s, 2H), 1.65 (dd, J = 11.5, 7.8 Hz, 1H), 1.57 (d, J = 3.5 Hz, 2H), 1.49 – 1.35 (m, 4H). Isomer 2 (Compound X2): (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione (17 mg, 19% yield) Rt= 51.878 min (analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), mobile phase: hexane / AcOEt / iPrOH / iPrNH2, 50 / 25 / 25 / 0.1, v / v / v / v; and flowrate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 254 nm) Rt= 8.34 min (analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), mobile phase: ACN / water / FA, 70 / 30 / 0.1, v / v / v, and flowrate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 250 nm) LCMS (ESI+) m / z 343.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.95 (s, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.57 (s, 1H), 7.46 – 7.42 (m, 1H), 4.74 (dd, J = 12.4, 5.1 Hz, 1H), 4.65 (q, J = 6.6 Hz, 1H), 4.48 – 4.41 (m, 1H), 4.06 (d, J = 11.5 Hz, 1H), 3.62 – 3.49 (m, 1H), 2.82 (t, J = 13.9 Hz, 1H), 2.67 – 2.55 (m, 2H), 2.02 – 1.92 (m, 1H), 1.92 – 1.80 (m, 2H), 1.74 – 1.61 (m, 1H), 1.61 – 1.51 (m, 2H), 1.45 – 1.35 (m, 4H). 251982040 v1 Synthesis of (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine- 2,6-dione (Compound X2) Step 1: To a solution of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate (Intermediate i(2)) (5.6 g, 13.6 mmol, 1 equiv) in 1,4-dioxane (80 mL) and water (15 mL) was added K2CO3(5.65 g, 40.8 mmol, 3 equiv), followed by potassium vinyltrifluoroborate (2.74 g, 20.4 mmol, 1.2 eq) and PdCl2(dppf) (0.60 g, 0.81 mmol, 0.06 eq). The reaction mixture was stirred at 70°C for 12h. After completion, the mixture was filtered through celite bed and the solids were washed with ethyl acetate (100 mL). The filtrate was concentrated under reduced pressure and the crude product was purified by flash column chromatography to give tert-butyl (S)-5-amino-2-((R)-3- methyl-1-oxo-5-vinylisoindolin-2-yl)-5-oxopentanoate (Intermediate iv) (4.8 g, >95% yield). LCMS (ESI+) m / z 359.2 [M+H]+1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.41 (s, 1H), 6.80 (dd, J = 17.5, 10.9 Hz, 1H), 5.87 (d, J = 17.5 Hz, 1H), 5.71 (s, 1H), 5.39 (d, J = 10.9 Hz, 1H), 5.33 (s, 1H), 4.66 – 4.57 (m, 2H), 2.61 – 2.39 (m, 2H), 2.29 (t, J = 7.3 Hz, 2H), 1.49 (d, J = 6.8 Hz, 3H), 1.42 (s, 9H). Step 2: To a solution of tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate (5.5 g, 15.3 mmol, 1.0 equiv) in THF / water (200 mL, 1:1), were added osmium tetroxide (22 mL, 4% in water, 3.4 mmol, 0.22 equiv) and sodium periodate monohydrate (9.9 g, 46.2 mmol, 3 equiv) at 0°C and the reaction mixture was stirred for 1h. After completion, the reaction 251982040 v1 mixture was diluted with water (50 mL) and the product was extracted into ethyl acetate (150 mL). The organic layer was dried over Na2SO4, and concentrated under reduced pressure to give tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (4.70 g, 85% yield) as an off white solid. The product was directly used in the next step without further purification. LCMS (ESI+) m / z 305.2 [M−C4H8+H]+1H NMR (400 MHz, CDCl3) δ 10.13 (s, 1H), 8.03 – 7.96 (m, 2H), 7.95 (s, 1H), 5.57 (s, 1H), 5.27 (s, 1H), 4.71 (q, J = 6.8 Hz, 1H), 4.62 (dd, J = 9.8, 5.6 Hz, 1H), 2.65 – 2.41 (m, 2H), 2.32 (t, J = 7.2 Hz, 2H), 1.54 (d, J = 6.8 Hz, 3H), 1.42 (s, 9H). Step 3: To a solution of tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate (4.70 g, 14.0 mmol, 1.0 equiv) and potassium allyltrifluoroborate (4.85 g, 36.6 mmol, 1.2 equiv) in DCM (70 mL) was added slowly boron trifluoride diethyl etherate (8.5 ml, 65.2 mmol, 5.0 equiv) at −45°C, the reaction mixture was stirred at the same temperature for 1h, and then warmed to 0°C. After completion, the reaction was quenched with NaHCO3solution (50 mL) and the product extracted into DCM (2 x 50 mL). The organic layer was dried over Na2SO4, concentrated under reduced pressure, and the crude product was purified by flash column chromatography to give tert-butyl (2S)- 5-amino-2-((3R)-5-(1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate (3.6 g, 64%) as a light-yellow solid. LCMS (ESI+) m / z 403.2 [M+H]+Chiral separation The separation of diastereoisomers was performed by Supercritical Fluid Chromatography. Column name: CHIRAL ART Cellulose-SC (5 μm, 150 x 25 mm) operating at 30°C, flowrate 80 g / min, CO2backpressure 100 bar with 50% modifier (isopropanol), and monitoring by UV at λ = 236 nm. The pure stereoisomers isolated by the chiral SFC separation were then subjected to analytical SFC. The retention times Rtof the pure stereoisomers in the analytical SFC process are indicated below. Isomer 1 1H NMR (400 MHz, CDCl3) δ 7.78 (d, J = 7.8 Hz, 1H), 7.46 (s, 2H), 7.42 (d, J = 7.9 Hz, 1H), 5.88 – 5.75 (m, 1H), 5.68 (s, 1H), 5.26 (s, 1H), 5.23 – 5.19 (m, 1H), 5.19 – 5.16 (m, 1H), 4.85 (dd, J = 8.2, 4.6 Hz, 1H), 4.68 – 4.55 (m, 2H), 2.62 – 2.39 (m, 4H), 2.28 (t, J = 7.3 Hz, 2H), 1.48 (d, J = 6.8 Hz, 3H), 1.42 (s, 9H). 251982040 v1 tR= 3.11 min (analytical SFC performed using a Chiralpak IC column (150 x 4.6 mm, 5 μm), mobile phase: 75% CO2 / 25% methanol, flowrate: 3 g / min, operating at 30°C, backpressure: 100 bar, and monitored by UV at λ = 254 nm) Isomer 2 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.2 Hz, 1H), 7.49 – 7.39 (m, 2H), 5.90 – 5.75 (m, 2H), 5.64 (s, 1H), 5.30 (s, 1H), 5.23 – 5.19 (m, 1H), 5.19 – 5.15 (m, 1H), 4.85 (dd, J = 8.2, 4.6 Hz, 1H), 4.66 – 4.53 (m, 2H), 2.65 – 2.38 (m, 4H), 2.29 (t, J = 7.2 Hz, 2H), 1.49 (d, J = 6.7 Hz, 3H), 1.42 (s, 9H). tR= 3.85 min (analytical SFC performed using a Chiralpak IC column (150 x 4.6 mm, 5 μm), mobile phase: 75% CO2 / 25% methanol, flowrate: 3 g / min, operating at 30°C, backpressure: 100 bar, and monitored by UV at λ = 254 nm) Step 4: To the solution of tert-butyl (S)-5-amino-2-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate (1.60 g, 3.98 mmol, 1 eq) in dry THF (50 mL) was added slowly sodium hydride (60% oil dispersion, 0.239 g, 9.94 mmol, 2.5 eq) at 0°C. The reaction mixture was stirred for 30 min, a solution of allyl bromide (0.962 g, 7.95 mmol, 2 eq) in THF (10 ml) was added dropwise, and the reaction mixture was stirred at RT for 5h. After completion, the reaction mixture was quenched with a solution of 10% acetic acid, the product was extracted into AcOEt (5 x 100 mL), the organic phase was dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by flash column chromatography and SFC to give (S)-3-((R)-5-((S)-1-(allyloxy)but- 3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (513 mg, 35% yield) and (S)-3-((R)-5- ((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (588 mg, 45% yield). (S)-3-((R)-5-((S)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Intermediate viii(1)) LCMS (ESI+) m / z 369.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 7.65 (d, J = 7.9 Hz, 1H), 7.55 (s, 1H), 7.43 (d, J = 7.9 Hz, 1H), 5.87 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H), 5.75 (ddt, J = 17.1, 10.2, 6.9 Hz, 1H), 5.24 (dq, J = 17.3, 1.8 Hz, 1H), 5.13 (dq, J = 10.4, 1.5 Hz, 1H), 5.07 – 4.95 (m, 2H), 4.75 (dd, J = 12.4, 5.2 Hz, 1H), 4.67 (q, J = 6.6 Hz, 1H), 4.53 (t, J = 6.5 Hz, 1H), 3.90 – 3.77 (m, 2H), 2.90 – 2.75 (m, 1H), 2.71 – 2.64 (m, 1H), 2.60 – 2.53 (m, 2H), 2.47 – 2.36 (m, 1H), 2.06 – 1.92 (m, 1H), 1.41 (d, J = 6.7 Hz, 3H). (S)-3-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Intermediate vii(1)) 251982040 v1 LCMS (ESI+) m / z 329.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 7.64 – 7.55 (m, 2H), 7.43 (d, J = 7.8 Hz, 1H), 5.78 (ddt, J = 17.2, 10.3, 7.0 Hz, 1H), 5.47 (d, J = 4.4 Hz, 1H), 5.07 – 4.92 (m, 2H), 4.73 (dt, J = 10.6, 5.4 Hz, 2H), 4.65 (q, J = 6.6 Hz, 1H), 2.91 – 2.74 (m, 1H), 2.70 – 2.55 (m, 3H), 2.48 – 2.36 (m, 1H), 2.07 – 1.93 (m, 1H), 1.41 (d, J = 6.7 Hz, 3H). Step 5: To a solution of (S)-3-((R)-5-((S)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione (130 mg, 0.353 mmol, 1 equiv) in DCM (10 mL) was added Grubbs Catalyst 1st Generation (21 mg, 0.035 mmol, 0.1 equiv) and the reaction mixture was stirred at RT for 16 h. After completion, the mixture was diluted with DCM (15 mL) and filtered through Celite® pad. The solids were washed with DCM and the filtrates were concentrated under reduced pressure. The crude product was purified by flash column chromatography to give (S)-3-((R)-5-((S)-3,6-dihydro-2H-pyran- 2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione (95 mg, 79%) as an off white solid. LCMS (ESI+) m / z 341.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 7.67 – 7.60 (m, 2H), 7.49 (d, J = 8.6 Hz, 1H), 5.97 – 5.83 (m, 2H), 4.75 (dd, J = 12.6, 5.2 Hz, 1H), 4.70 – 4.61 (m, 2H), 4.35 – 4.26 (m, 2H), 2.93 – 2.76 (m, 1H), 2.72 – 2.56 (m, 2H), 2.42 – 2.30 (m, 1H), 2.25 – 2.11 (m, 1H), 2.04 – 1.92 (m, 1H), 1.42 (d, J = 6.7 Hz, 3H). Step 6: To a solution of (S)-3-((R)-5-((S)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione (60 mg, 0.176 mmol, 1 equiv) in tert-butanol / ACN (6 mL, 1:1) was added 10% platinum on activated carbon (13 mg). The reaction mixture was stirred was stirred at RT for 24h under hydrogen atmosphere (balloon). After completion, the mixture was filtered through Celite® pad, the solids were washed with 10% MeOH / DCM (2x5 ml), and the filtrates were concentrated under reduced pressure. The crude compound was triturated with diethyl ether (10 mL) and dried under reduced pressure to give (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione (45 mg, 75%) as an off white solid. Rt= 8.34 min (analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), mobile phase: ACN / water / FA, 70 / 30 / 0.1, v / v / v, and flowrate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 250 nm) LCMS (ESI+) m / z 343.3 [M+H]+251982040 v11H NMR (400 MHz, DMSO-d6) δ 10.93 (s, 1H), 7.61 (d, J = 7.8 Hz, 1H), 7.59 – 7.54 (m, 1H), 7.43 (dd, J = 7.9, 1.4 Hz, 1H), 4.74 (dd, J = 12.5, 5.2 Hz, 1H), 4.65 (q, J = 6.6 Hz, 1H), 4.44 (dd, J = 11.2, 2.1 Hz, 1H), 4.06 (d, J = 11.4 Hz, 1H), 3.65 – 3.49 (m, 1H), 2.83 (ddd, J = 15.9, 13.7, 5.3 Hz, 1H), 2.71 – 2.55 (m, 2H), 2.04 – 1.93 (m, 1H), 1.92 – 1.81 (m, 2H), 1.74 – 1.62 (m, 1H), 1.61 – 1.52 (m, 2H), 1.49 – 1.35 (m, 4H). Synthesis of (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine- 2,6-dione (Compound X1) Step 1: To the solution of tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate (1.60 g, 3.98 mmol, 1 eq) in dry THF (50 mL) was added slowly sodium hydride (60% oil dispersion, 0.239 g, 9.94 mmol, 2.5 eq) at 0°C. The reaction mixture was stirred for 30 min, a solution of allyl bromide (0.962 g, 7.95 mmol, 2 eq) in THF (10 ml) was added dropwise, and the reaction mixture was stirred at RT for 5h. After completion, the reaction mixture was quenched with a solution of 10% acetic acid, the product was extracted into AcOEt (5 x 100 mL), the organic phase was dried over Na2SO4and concentrated under reduced pressure. The crude product was purified by flash column chromatography and SFC to give (S)-3-((R)-5-((R)-1-(allyloxy) but- 3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (910 mg, 62% yield) and (RS)-3-((R)-5- ((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (196 mg, 15% yield). (S)-3-((R)-5-((R)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Intermediate viii(2)) LCMS (ESI+) m / z 369.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.56 (s, 1H), 7.43 (d, J = 7.8 Hz, 1H), 5.87 (ddt, J = 17.3, 10.5, 5.3 Hz, 1H), 5.74 (ddt, J = 17.2, 10.3, 6.9 Hz, 1H), 5.24 (dq, J = 17.2, 1.8 Hz, 1H), 5.13 (dq, J = 10.5, 1.5 Hz, 1H), 5.09 – 4.91 (m, 2H), 4.75 (dd, J = 12.4, 5.2 Hz, 1H), 4.67 (q, J = 251982040 v1 6.6 Hz, 1H), 4.53 (t, J = 6.5 Hz, 1H), 3.94 – 3.77 (m, 2H), 2.90 – 2.77 (m, 1H), 2.71 – 2.54 (m, 3H), 2.48 – 2.37 (m, 1H), 2.06 – 1.93 (m, 1H), 1.43 (d, J = 6.7 Hz, 3H). (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (Intermediate vii(2)) LCMS (ESI+) m / z 329.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 0.67H), 10.90 (s, 0.33H), 7.66 – 7.53 (m, 2H), 7.51 – 7.41 (m, 1H), 5.77 (ddt, J = 17.3, 10.3, 6.9 Hz, 1H), 5.54 – 5.40 (m, 1H), 5.07 – 4.93 (m, 2H), 4.80 – 4.68 (m, 2.33H), 4.65 (q, J = 6.6 Hz, 0.67H), 2.89 – 2.74 (m, 1H), 2.66 – 2.55 (m, 3H), 2.45 – 2.37 (m, 1H), 2.07 – 1.93 (m, 1H), 1.51 – 1.35 (m, 3H). Step 2: To a solution of (S)-3-((R)-5-((R)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione (130 mg, 0.353 mmol, 1 equiv) in DCM (10 mL) was added Grubbs Catalyst 1st Generation (21 mg, 0.035 mmol, 0.1 equiv) and the reaction mixture was stirred at RT for 16 h. After completion, the mixture was diluted with DCM (15 mL) and filtered through Celite® pad. The solids were washed with DCM and the filtrates were concentrated under reduced pressure. The crude product was purified by flash column chromatography to give (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran- 2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione (85 mg, 75%) as an off white solid. LCMS (ESI+) m / z 341.3 [M+H]+1H NMR (400 MHz, DMSO-d6) δ 10.94 (s, 1H), 7.67 – 7.59 (m, 2H), 7.50 (d, J = 8.6 Hz, 1H), 6.00 – 5.81 (m, 2H), 4.75 (dd, J = 12.5, 5.3 Hz, 1H), 4.71 – 4.61 (m, 2H), 4.38 – 4.22 (m, 2H), 2.90 – 2.75 (m, 1H), 2.72 – 2.54 (m, 2H), 2.40 – 2.28 (m, 1H), 2.26 – 2.11 (m, 1H), 2.05 – 1.92 (m, 1H), 1.42 (d, J = 6.7 Hz, 3H). Step 3: To a solution of (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione (60 mg, 0.176 mmol, 1 equiv) in tert-butanol / ACN (6 mL, 1:1) was added 10% platinum on activated carbon (13 mg). The reaction mixture was stirred was stirred at RT for 24h under hydrogen atmosphere (balloon). After completion, the mixture was filtered through Celite® pad, the solids were washed with 10% MeOH / DCM (2x5 ml), and the filtrates were concentrated under reduced pressure. The crude compound was triturated with diethyl ether (10 mL) and dried under reduced pressure to give (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione (40 mg, 72%) as an off white solid. 251982040 v1 Rt= 10.53 min (analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), mobile phase: ACN / water / FA, 70 / 30 / 0.1, v / v / v, and flowrate: 1 mL / min, operating at ambient temperature and monitored by UV at λ = 250 nm) LCMS (ESI+) m / z 343.3 [M+H]+1H NMR (400 MHz, DMSO) δ 10.93 (s, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.56 (s, 1H), 7.45 (d, J = 7.9 Hz, 1H), 4.74 (dd, J = 12.4, 5.2 Hz, 1H), 4.65 (q, J = 6.7 Hz, 1H), 4.45 (dd, J = 11.0, 2.1 Hz, 1H), 4.05 (d, J = 11.5 Hz, 1H), 3.56 (ddd, J = 14.3, 7.5, 4.4 Hz, 1H), 2.91 – 2.75 (m, 1H), 2.71 – 2.53 (m, 2H), 2.05 – 1.93 (m, 1H), 1.93 – 1.80 (m, 2H), 1.74 – 1.52 (m, 3H), 1.50 – 1.35 (m, 4H). Structure determination of (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione (Intermediate ix(2)) Co-crystallisation of CRBN_TBD (Cereblon thalidomide-binding domain), IKZF2_ZF2 (Helios protein zinc finger 2) and Intermediate ix(2) ((S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione) was performed using sitting drop vapor diffusion method at the room temperature. This intermediate compound was utilized in crystallography to take advantage of the double bond presence in compound modelling and chirality assignment of Compound X1. Crystallization trials were performed with CRBN_TBD at 130 μM and molar excess of IKZF2_ZF2 (1:1.3) and Intermediate ix(2) (1:4). Crystals formed within 2-3 days in the shape of thick rods in 0.15 M DL-Malic acid pH 7.0, 20% w / v Polyethylene glycol 3350 (1:1 ratio). Prior data collection crystals were flash-frozen in liquid nitrogen in the presence of 25% ethylene glycol. X-Ray diffraction data were collected at the Deutsches Elektronen-Synchrotron DESY (Hamburg, Germany). Structure was solved using the CCP4 program suite (Potterton, 2018). Post data processing at beamline using XDS (Kabsh, 20100), structure was solved using molecular replacement with PHASER (McCoy, 2007) with PDB ID: 7BQV as a search model. Refinement was carried out using REFMAC (MURshudov, 2011), with model building performed and compound topology generated using COOT (Emsley, 2010) and AceDRG (Long, 2017) respectively. Electron density inspection revealed hydrogen bonding between Compound ix oxygen from THP ring and CRBN HIS353 and stacking with Helios HIS146. High quality data with 1.4 Å resolution allowed to model in tail part of the ligand and assigning its chirality. The structure is shown in Figure 1. 251982040 v11. CCP4 suite: Potterton, L.; Agirre, J.; Ballard, C.; Cowtan, K.; Dodson, E.; Evans, P. R.; Jenkins, H. T.;Keegan, R.; Krissinel, E.; Stevenson, K.; Lebedev, A.; McNicholas, S. J.; Nicholls, R. A.; Noble, M.; Pannu, N. S.; Roth, C.; Sheldrick, G.; Skubak, P.; Turkenburg, J.; Uski, V.; von Delft, F.; Waterman, D.; Wilson, K.; Winn, M.; Wojdyr, M. CCP4i2: the new graphical user interface to the CCP4 program suite. Acta Crystallogr., Sect. D: Struct. Biol.2018, 74,68−84. “CCP4i2: the new graphical user interface to the CCP4 program suite” https: / / journals.iucr.org / d / issues / 2018 / 02 / 00 / rr5150 / rr5150.pdf2. PHASER: McCoy, A. J.; Grosse-Kunstleve, R. W.; Adams, P. D.; Winn, M. D.; Storoni, L. C.; Read, R.J. Phaser crystallographic software. J. Appl. Crystallogr.2007, 40, 658−674; https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC2483472 / 3. REFMAC: Murshudov, G. N.; Skubák, P.; Lebedev, A. A.; Pannu, N. S.; Steiner, R. A.; Nicholls, R. A.;Winn, M. D.; Long, F.; Vagin, A. A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr., Sect. D: Biol. Crystallogr.2011, 67, 355−367; https: / / journals.iucr.org / d / issues / 2011 / 04 / 00 / ba5152 / index.html4. AceDRG: Long, F.; Nicholls, R. A.; Emsley, P.; Graǽulis, S.; Merkys, A.; Vaitkus, A.; Murshudov, G.N. AceDRG: a stereochemical description generator for ligands. Acta Crystallogr., Sect. D: Struct. Biol.2017, 73, 112−122; https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC5297914 / #:~:text=The%20program%20AceDRG %20generates%20accurate,from%20the%20Crystallography%20Open%20Database.Synthesis of 3-(3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione(racemic mixture) Step 1: In a vial 3-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (200 mg, 0.59 mmol,1 equiv), copper(I) iodide (0.2 equiv), sodium iodide (2 equiv) and N,N’-dimethylethane-1,2-diamine (0.4 equiv) were suspended in dioxane (6 mL) and purged with argon for 5 min. The reaction vial was sealed and the reaction mixture was stirred at 125°C for 48h. After completion 3-(5-iodo-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione (67 mg, 0.17 mmol, 29% yield) was purified by flash column chromatography.LCMS (ESI+) m / z 385.0 [M+H]+251982040 v1 Step 2: In a vial were placed 3-(5-iodo-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione (33 mg, 0.077mmol, 1 equiv), tributyl(tetrahydro-2H-pyran-2-yl)stannane (2 equiv), bis(dibenzylideneacetone)palladium(0) (0.05 equiv), bis(3,5-bis(trifluoromethyl)phenyl)(2',4',6'- triisopropyl-3,6-dimethoxy-[1,1'-biphenyl]-2-yl)phosphane (0.1 equiv), potassium fluoride (2 equiv), copper(I) chloride (2 equiv) and tBuOH (1 mL). The slurry was purged with argon for 10 min, the vial was sealed and the reaction mixture was stirred at 80°C for 18 h. The reaction mixture was filtered, the volatiles were removed under reduced pressure and the crude product was purified by preparative HPLC to give 3-(3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione as white solid (1.8 mg, 6.6% yield). LCMS (ESI+) m / z 343.2 [M+H]+1H NMR (500 MHz, DMSO-d6) δ 10.91 (d, J = 16.5 Hz, 1H), 7.63 – 7.55 (m, 2H), 7.44 (td, J = 8.2, 6.0 Hz, 1H), 4.78 – 4.70 (m, 1H), 4.70 – 4.61 (m, 1H), 4.49 – 4.41 (m, 1H), 4.06 (d, J = 11.5 Hz, 1H), 3.56 (ddd, J = 14.5, 7.8, 4.9 Hz, 1H), 2.79 (dddd, J = 37.7, 18.1, 14.1, 5.8 Hz, 1H), 2.66 – 2.53 (m, 2H), 1.99 (pt, J = 7.7, 3.4 Hz, 1H), 1.93 – 1.81 (m, 2H), 1.66 (dt, J = 15.9, 10.6 Hz, 1H), 1.57 (qt, J = 6.6, 3.7 Hz, 2H), 1.47 – 1.39 (m, 4H). Ternary complex formation assay The effect of the molecular glue compound of the invention, i.e. Compound X1, and also its stereoisomer Compound X2 on the formation of a ternary complex composed of [NEK7]-[Compound]- [CRBN / DDB1] was investigated with two methods: AlphaLISA dose response assay or HTRF ternary complex assay. AlphaLISA dose response assay: Two types of protein solution were prepared: - 200 nM biotinylated NEK7, 40 μg / ml AlphaScreen Streptavidin-coated Donor Beads in HBS (10 mM HEPES, 150 mM NaCL, pH 7.4) buffer with 0.1% Tween-20 and 1mM DTT, - 200 nM 6XHis-CRBN / Strep-DDB1, 40 μg / ml AlphaLISA Anti-6xHis Acceptor beads in HBS buffer with 0.1% Tween-20 and 1mM DTT. The prepared solutions were incubated at room temperature for 30 min and then the solution containing the donor beads was mixed with the solution containing the acceptor beads. 251982040 v1 The tested compounds were dispensed onto a white 384-well AlphaPlate 384 SW. DMSO was backfilled to all wells, resulting in a final DMSO content of 2%. Wells containing only DMSO served as background. Next, 10 μl of solution with donor and acceptor beads was added to the wells. The plate was sealed with transparent film and shaken using a VibroTurbulator for 60 sec at room temperature, level 3. The plate was then spun down shortly (10 s, 1000 rcf, room temperature) and incubated at 25°C for 30 min. The read-out was performed with PerkinElmer Enspire Multimode Plate Reader (method for AlphaLISA 384-well low volume, Filterset: λexc= 680 nm, λem= 615 nm). The results were analyzed as follows: 1) an average of luminescence for background signal was calculated and used as a negative control; 2) average of the maximum measured luminescence for 3-(3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione racemic mixture was calculated and used as an internal positive control; 3) raw luminescence values were normalized against positive and negative controls; 4) the responses normalized to that of the racemic mixture were determined and expressed as a percentage. As illustrated in Table 1, Compound X1 (the compound of the present invention) has much stronger capability to induce the formation of the [NEK7]-[Compound]-[CRBN / DDB1] complex than Compound X2. HTRF ternary complex assay: The effect of the molecular glue compounds, i.e. Compound X1, and also its stereoisomer Compound X2 on the formation of a ternary complex composed of [NEK7]-[Compound]-[CRBN / DDB1] was investigated. Mix solution of proteins and reagents was prepared: - 24 nM NEK7, 52.8 nM 6XHis-CRBN / Strep-DDB1, 3 nM of Streptavidin-Eu cryptate (acceptor) and 6.67 nM of Anti-6XHis-d2 (donor) was prepared in PPI Europium detection buffer (Cisbio) with 1 mM DTT. 251982040 v1 The tested compounds in dose-response were dispensed onto a white 384-well low volume plate (Greiner, 784075). DMSO was backfilled to all wells, resulting in a final DMSO content of 0.5%. Wells containing only DMSO served as background. The plate was sealed with transparent film and shaken using a VibroTurbulator for 60 sec at level 3. The plate was then spun down shortly (10 s, 1000 rcf) and incubated at 25°C for 180 min. The read-out was performed with plate reader (Pherastar, BMG Labtech) in time resolved fluorescence mode. Filterset: TR 337665620. The results were analyzed as follows: 1) an average of fluorescence for background signal was calculated and used as a negative control; 2) raw fluorescence values for tested compounds were normalized against negative controls; 3) saturation curve was fitted to specific binding with Hill slope model; 4) The level of [NEK7]-[Compound]-[CRBN / DDB1] complex for each compound was determined based on obtained dose response curves; 5) Ternary complex response level at 1 μM compound was normalized to that of the racemic mixture as a reference for Compound X1 and Compound X2. Normalized values were expressed as a percentage. As illustrated in Table 1, Compound X1 (the compound of the present invention) has much stronger capability to induce the formation of the [NEK7]-[Compound]-[CRBN / DDB1] complex than Compound X2. 251982040 v1 Table 1: Ternary complex assay results for the compound of the invention in relation to racemic mixture. Compound Normalized AlphaLISA Normalized HTRF NEK7 NEK7 ternary complex ternary complex average average signal at 1 μM signal at 1 μM molecule molecule [%] [%] Racemic 100 100 mixture Compound 491 367 X1 Compound 11 17 X2 Biological Testing Methodology Cell culture Human peripheral blood mononuclear cells (PBMCs) Human PBMCs were isolated from buffy coats from healthy volunteers. Buffy coats were diluted 1:1 (v / v) with DPBS (Sigma-Aldrich) in falcon tubes. After reconstitution, suspension was carefully layered on Histopaque-1077 solution (Sigma-Aldrich) and centrifuged (760 x g, RT, 20 min; Brakes Off). PBMCs were collected and washed with DPBS + 2% FBS (Biowest) + 5mM EDTA (ThermoFisher Scientific) (5x at 200 x g, RT, 10 min; Brakes Off); the last wash step was conducted using DPBS + 2% FBS without EDTA. Subsequently, cells were resuspended in appropriate volume of RPMI 1640 medium (ATCC modification; Gibco) supplemented with 10% of heat-inactivated FBS (Gibco) and 1% of Penicillin- Streptomycin Solution 100x (Biowest). Cell density and viability were assessed in cell counting chamber using trypan blue solution (Sigma-Aldrich). Then, the cells were either used in the experiment or frozen in liquid nitrogen for further analysis. The purity of isolated cells was assessed using flow cytometry. 3.5 x 10^6 of freshly isolated cells or cells thawed from liquid nitrogen were seeded on 6-well plate in 2.5 mL of complete medium and subjected on WB degradation assay. 251982040 v1 U937 human monocytic cell line The U937 cells were cultured in RPMI 1640 medium (ATCC modification; Gibco), supplemented with 10% heat-inactivated FBS and 1% of Penicillin-Streptomycin Solution 100x (Biowest) at 37°C, 5% CO2. The cells were subcultured twice a week when the culture reached optimal confluence. Cell density and viability were assessed in LUNA Automated Cell Counter using trypan blue solution (Sigma- Aldrich). 1.25 x 10^6 of U937 were seeded on 6-well plate in 2.5 mL of complete medium and subjected on WB degradation assay. HEK293 NEK7-HiBiT cell line HEK293 NEK7-HiBiT cells were generated using CRISPR-Cas9 system. HEK293 cells were transformed with pSpCas9-BB-2A-Puro v2.0 plasmid carrying gRNA targeting the N-terminus of NEK7 and ssODN template containing the HiBiT tag sequence with flanking homology sequences. Neon Transfection System (Thermo Fisher Scientific) was used for electroporation. HEK293 NEK7-HiBiT cells were cultured with DMEM Glutamax (Gibco) supplemented with 10% heat inactivated FBS (Gibco). After transfection the culture medium was changed to DMEM Glutamax with 10% heat-inactivated FBS and 1% Penicillin-Streptomycin (Biowest) supplemented with Puromycin (2 μg / ml; Invivogen) for clonal selection. In order to isolate single cell clones for further validation and analysis, limiting dilution cloning in 96-well plates was performed. When the single clones reached confluency, HiBiT Lytic Assay (Promega) was performed in order to identify HiBiT positive clones. The clone selected for further studies was verified and validated using genotyping and HiBiT Blotting (Promega). Selected clone of HEK293 NEK7-HiBiT cells was maintained in the DMEM-Glutamax medium supplemented with 10% of heat-inactivated FBS (Gibco) and 1% of Penicillin-Streptomycin Solution 100x (Biowest) at 37°C, 5% CO2and subcultured every 2-3 days. Nano-Glo HiBiT Lytic Assay For Nano-Glo HiBiT Lytic Assay, HEK293 NEK7-HiBiT cells were seeded at the density 2x10^3 cells in triplicates (pilot study with three concentrations) or duplicates (12-point dose-response study) in the 40 μL of growth medium per single well on 384 well plate (Greiner Bio-One). Compounds or DMSO were added to treatment plates using Echo555 Liquid Handler and incubated at 37°C, 5% CO2for 24 hours. After incubation, 40 μL of Nano-Glo HiBiT Lytic Reagent (prepared according to the manufacturer protocol) were added to 40 μL of the cell culture medium present in each well. The plate content was briefly mixed (460 rpm) on an orbital shaker to ensure cell lysis. The plate was left at RT 251982040 v1 protected from light for another 10 min to stabilize the luminescent signal. The luminescence signal was measured using CLARIOstar Multimode Plate Reader. Focus and gain were adjusted to DMSO treated cells. The results were calculated as the NEK7-HiBiT % relative to the DMSO control of 2 or 3 technical replicates relative to the DMSO control. Degradation assay Human PBMCs or U937 cells were treated for 24 hours with selected compounds at specified concentrations. After that, the cells were harvested from the wells into falcon tubes, centrifuged in DPBS twice, and lysed with RIPA buffer. Western blotting Cell lysates from human PBMCs or U937 cells were prepared by direct lysis in 40-50 μl of RIPA lysis buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 0.1% SDS and 1 mM EDTA) supplemented with protease and phosphatase inhibitors (cOmplete EDTA-free Protease Inhibitor Cocktail, Roche; Halt™ Phosphatase Inhibitor Cocktail, Thermo Scientific). Subsequently, lysates were snap frozen in liquid nitrogen and stored in -20°C. Following thawing, lysates were centrifuged at 4°C, 19000×g for 15 min for supernatants collection. The protein concentration in each sample was determined by BCA method (Pierce BCA Protein Assay Kit, Thermo Fischer Scientific). The absorbance was measured using CLARIOstar Multimode Plate Reader at 562 nm. SDS-PAGE samples were prepared by mixing the lysates with 5xSB and RIPA buffer. Denaturation of the samples was performed by incubation at 95°C for 5 minutes. The protein samples were resolved on 4-20% TGX Stain-Free™ protein gels (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad) using Trans-Blot® Turbo system (Bio-Rad). Membranes were blocked in 5% non- dried milk (NFM) or 5% BSA in TBS-T (10 mM Tris, 150 mM NaCl, 0.1% Tween-20) for 1 h at room temperature (RT). Membranes were incubated with primary antibody for NEK7 (O / N, 4°C), followed by incubation with the appropriate horseradish peroxidase (HRP) conjugated secondary antibody diluted in 5% NFM in TBS-T for 1 h in RT. In parallel, membranes were incubated with antibodies for loading controls – β-Actin (1h, RT) or Vinculin (O / N, 4°C) diluted in 5% NFM or 5% BSA in TBS-T, respectively, Between each antibody incubation, the membranes were washed in TBS-T. Membranes were developed using SuperSignal West Pico PLUS chemiluminescent substrate (ThermoScientific). Membrane images were captured using Chemi Doc Imager. The analysis was performed in Image Lab software. Densitometric values for NEK7 protein were normalized to the loading control in the Excel spreadsheet and calculated as a relative to the cells treated with DMSO control. The absolute DC50values (the concentration of the compound leading to 50% protein 251982040 v1 degradation) were calculated in GraphPad Prism Software using following equation: log(inhibitor) vs. response - Variable slope (four parameters). Results NEK7-HiBiT degradation assay HEK293 NEK7-HiBiT cells were treated with the compounds (concentrations 0.1, 1 and 10 μM in the pilot study or series of concentrations in dose-response study starting from 10 μM with half-log dilutions) or DMSO for 24h. After incubation with compounds NEK7-HiBiT degradation was measured as a luminescence signal using CLARIOstar Multimode Plate reader. Compound X1 leads to robust degradation of NEK7-HiBiT protein, as shown in Table 2. In contrast, Compound X2 is inactive in the NEK7-HiBiT Lytic Assay. The activity of Compound X1 in the NEK7-HiBiT Lytic assay is nearly 4 times higher than that of the racemic mixture when comparing DC50values (the concentration of the compound leading to 50% protein degradation). Table 2: Levels of NEK7-HiBiT protein presented as a % of DMSO control (mean of experimental replicates) following treatment with the compounds. Levels of NEK7- Number of Number of Absolute DC50HiBiT Protein (as experimental experimental Compound ± SD value a % of DMSO replicates replicates (DR (nM) control) (pilot study) study) Racemic mixture 9.27 ± 1.31 A 1 11 Compound X1 2.47 ± 1.19 A - 3 Compound X2 - C - 2 A – Levels of NEK7-HiBiT Protein ≤ 15% B – Levels of NEK7-HiBiT Protein > 15% and ≤ 30% C – Levels of NEK7-HiBiT Protein > 30% 251982040 v1 NEK7 protein degradation determined by Western blot NEK7 protein degradation was investigated in human U937 monocytic cell line. Table 3 and Figure 2 show the results of NEK7 levels in cells treated with Compound X1, Compound X2 or DMSO for 24h. It has been shown that in contrast to Compound X2, Compound X1 efficiently degrades NEK7 protein after 24 hours of treatment with a single dose of 1 μM. Table 3: Levels of NEK7 protein presented as a % of DMSO control (mean of 3 experimental replicates) following treatment with the compounds in U937 monocytic cell line. Compound Levels of NEK7 Protein (as a % of DMSO control) Compound X1 A Compound X2 C A – Levels of NEK7 Protein ≤ 10% B – Levels of NEK7 Protein > 10% and ≤ 50% C – Levels of NEK7 Protein > 50% The activity of Compound X1 was further confirmed in human PBMCs in the degradation assay. Figure 3 shows the dose-dependent effect of NEK7 protein degradation in those cells after 24 hours of incubation with Compound X1. Table 4 presents DC50values for Compound X1 compared to the racemic mixture, indicating a higher activity of the former. Table 4: Absolute DC50values (mean of biological replicates) following treatment with the compounds in human PBMCs. Compound Absolute DC50± SD (nM) Number of experimental replicates Racemic mixture 16.8 ± 14.1 n=4 Compound X1 4.24 ± 0.50 n=3 251982040 v1

Claims

CLAIMS 1. A compound of formula:or a solvate thereof.

2. The compound of claim 1, which is.

3. A method of making the compound of any one of claims 1-2, comprising: (i) separation of stereoisomers of tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate; (ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (iii) hydrogenation of tert-butyl (S)-5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate to provide tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5- (tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate; (iv) conversion of tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)-5-oxopentanoate to (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione; (v) separation of stereoisomers of (3S)-3-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2- yl)isoindolin-2-yl)piperidine-2,6-dione by chiral HPLC, to provide (S)-3-((R)-3-methyl-1-oxo-5-((R)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione and (S)-3-((R)-3-methyl-1-oxo-5-((S)- tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. 251982040 v14. The method of claim 3, wherein the separation by chiral HPLC in step (i) is carried out at 20- 25°C using an Agilent 1200 series instrument, a CHIRALPAK IC column (5 μm, 250 x 20 mm), a mobile phase of hexane / DCM / EtOH at a ratio of 60 / 20 / 20 v / v / v, and a flowrate of 18 mL / min for 30 min, and wherein monitoring is carried out by UV at λ = 254 nm.

5. The method of claim 4 wherein, when subjected to analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 μm), a mobile phase of hexane / AcOEt / EtOH / iPrNH2at a ratio of 50 / 25 / 25 / 0.1 v / v / v / v, and a flow rate of 1 mL / min), the stereoisomers separated by chiral HPLC separation have the following retention times Rt: tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rtof 7.974 min; tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rtof 9.692 min.

6. The method of any one of claims 3-5, wherein step (ii) comprises reaction of tert-butyl (S)-5- amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate with 2-(3,4-dihydro-2H- pyran-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane and K3PO4, and subsequent reaction with PdCl2(dtbpf) under inert atmosphere at 70°C under microwave irradiation, to provide tert-butyl (S)- 5-amino-2-((R)-5-(3,4-dihydro-2H-pyran-6-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate.

7. The method of any one of claims 3-6, wherein the hydrogenation in step (iii) is carried out using PtO2.

8. The method of any one of claims 3-7, wherein the separation by chiral HPLC in step (v) is carried out at 20-25°C using an Agilent 1200 series instrument, a CHIRALPAK IG column (5 μm, 250 x 21 mm), a mobile phase which is an isocratic mixture of hexane / AcOEt / iPrOH at a ratio of 2 / 1 / 1 v / v / v, a flowrate of 21 mL / min and duration time of 60 min, and wherein monitoring is carried out by UV at λ = 254 nm.

9. The method of claim 8 wherein, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of hexane / AcOEt / iPrOH / iPrNH2at a 251982040 v1ratio of 50 / 25 / 25 / 0.1 v / v / v / v, and a flowrate of 1 mL / min, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6- dione has a retention time Rtof 38.993 min; (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6- dione has a retention time Rtof 51.878 min.

10. The method of claim 8 or 9 wherein, when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of ACN / water / FA at a ratio of 70 / 30 / 0.1 v / v / v, and a flowrate of 1 mL / min, the stereoisomers separated by chiral HPLC separation have the following retention times Rt: (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6- dione has a retention time Rtof 10.53 min; (S)-3-((R)-3-methyl-1-oxo-5-((S)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6- dione has a retention time Rtof 8.34 min.

11. The method of any one of claims 3-10, wherein step (iv) comprises reaction of tert-butyl (2S)-5-amino-2-((3R)-3-methyl-1-oxo-5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)-5-oxopentanoate with benzenesulfonic acid at 130°C under microwave irradiation, to provide (3S)-3-((3R)-3-methyl-1- oxo-5-(tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione.

12. The method of any one of claims 3-11, further comprising reacting tert-butyl L-glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(1-bromoethyl)benzoate to provide the tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate of step (i).

13. A method of making the compound of any one of claims 1-2, comprising: (i) separation of stereoisomers of tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate; (ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate; 251982040 v1(iii) conversion of tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5- oxopentanoate to tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate; (iv) conversion of tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate to tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (v) separation of stereoisomers of tert-butyl (2S)-5-amino-2-((3R)-5-(1-hydroxybut-3-en-1- yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate by Supercritical Fluid Chromatography (SFC) to give tert-butyl (S)-5-amino-2-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate and tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate; (vi) conversion of tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)-5-oxopentanoate to (S)-3-((R)-5-((R)-1-(allyloxy) but-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione and (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1- oxoisoindolin-2-yl)piperidine-2,6-dione; (vii) conversion of (S)-3-((R)-5-((R)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2- yl)piperidine-2,6-dione to (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione; (viii) conversion of (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl) piperidine-2,6-dione to (S)-3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2- yl)piperidine-2,6-dione.

14. The method of claim 13, wherein step (ii) comprises reaction of tert-butyl (S)-5-amino-2-((R)- 5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate with potassium vinyltrifluoroborate and K2CO3and reaction with PdCl2(dppf) to provide tert-butyl (S)-5-amino-2-((R)-3-methyl-1-oxo-5- vinylisoindolin-2-yl)-5-oxopentanoate.

15. The method of claim 13 or 14 wherein step (iii) comprises reaction of tert-butyl (S)-5-amino- 2-((R)-3-methyl-1-oxo-5-vinylisoindolin-2-yl)-5-oxopentanoate with osmium tetroxide and sodium periodate monohydrate to provide tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin- 2-yl)-5-oxopentanoate. 251982040 v116. The method of any one of claims 13-15, wherein step (iv) comprises reaction of tert-butyl (S)-5-amino-2-((R)-5-formyl-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate with potassium allyltrifluoroborate and boron trifluoride diethyl etherate to provide tert-butyl (2S)-5-amino-2-((3R)- 5-(1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate.

17. The method of any one of claims 13-16, wherein the separation by SFC in step (v) is carried out at 30°C using a CHIRAL ART Cellulose-SC column (5 μm, 150 x 25 mm), a flowrate of 80g / min, a CO2backpressure of 100bar with 50% isopropanol modifier, and wherein monitoring is carried out by UV at λ = 236 nm.

18. The method of claim 17 wherein, when subjected to analytical SFC performed at 30°C using a CHIRALPAK IC column (150 x 4.6 mm, 5 μm), a mobile phase of 75% CO2 / 25% methanol, a flowrate of 3 g / min, a backpressure of 100bar and monitoring carried out by UV at λ = 254 nm, the stereoisomers separated by SFC have the following retention times Rt: tert-butyl (S)-5-amino-2-((R)-5-((S)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate has a retention time Rtof 3.11 min tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5- oxopentanoate has a retention time Rtof 3.85 min.

19. The method of any one of claims 13-18, wherein step (vi) comprises reaction of tert-butyl (S)-5-amino-2-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate with sodium hydride and allyl bromide to provide (S)-3-((R)-5-((R)-1-(allyloxy) but-3-en-1-yl)-3- methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione and (RS)-3-((R)-5-((R)-1-hydroxybut-3-en-1-yl)-3- methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione.

20. The method of any one of claims 13-19, wherein step (vii) comprises reaction of (S)-3-((R)-5- ((R)-1-(allyloxy)but-3-en-1-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione with Grubbs Catalyst 1st Generation to provide (S)-3-((R)-5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1- oxoisoindolin-2-yl) piperidine-2,6-dione.

21. The method of any one of claims 13-20, wherein step (viii) comprises reduction of (S)-3-((R)- 5-((R)-3,6-dihydro-2H-pyran-2-yl)-3-methyl-1-oxoisoindolin-2-yl)piperidine-2,6-dione to provide (S)- 3-((R)-3-methyl-1-oxo-5-((R)-tetrahydro-2H-pyran-2-yl)isoindolin-2-yl)piperidine-2,6-dione. 251982040 v122. The method of any one of claims 13-21, further comprising reacting tert-butyl L-glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(1-bromoethyl)benzoate to provide the tert-butyl (2S)-5-amino-2-(5-bromo-3-methyl-1-oxoisoindolin-2-yl)-5-oxopentanoate of step (i).

23. A compound of formula:or a solvate thereof, produced by the method of any one of claims 3-22.

24. The compound of claim 23, which is.

25. The compound of any one of claims 1-2 or 23-24, wherein when subjected to analytical LC performed using a CHIRALPAK IG column (250 x 4.6 mm, 5 μm), a mobile phase of ACN / water / FA at a ratio of 70 / 30 / 0.1 v / v / v, and a flowrate of 1 mL / min, the compound has a retention time Rtof 10.53 min.

26. A pharmaceutical composition comprising the compound of any one of claims 1-2 or 23-25.

27. The compound of any one of claims 1-2 or 23-25, or the pharmaceutical composition of claim 26 for use in medicine.

28. The compound of any one of claims 1-2 or 23-25 or the pharmaceutical composition of claim 26 for use in a method of treating a disease or condition in a subject in need thereof, wherein the disease or condition is an inflammatory disease or condition, an autoinflammatory disease or conditions, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a gastro-intestinal disease or condition, a renal 251982040 v1disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a skin disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, a wound, a burn, or cancer.

29. The compound or pharmaceutical composition for use of claim 28, wherein the disease or condition is an inflammatory disease or condition, an autoinflammatory disease or conditions, an auto-immune disease or condition, a respiratory disease or condition, a cardiovascular disease or condition, a renal disease or condition, a disease or condition of the central nervous system (CNS), a disease or condition of the endocrine system, a metabolic disease or condition, a liver disease or condition, an ocular disease or condition, a lymphatic disease or condition, a psychological disease or condition, graft versus host disease or condition, allodynia, pain, a condition associated with diabetes, a condition associated with arthritis, or a wound or burn.

30. The compound or pharmaceutical composition for use of claim 28 or 29, wherein the disease or condition is cryopyrin-associated periodic syndromes (CAPS), Muckle-Wells syndrome (MWS), familial cold autoinflammatory syndrome (FCAS), neonatal onset multisystem inflammatory disease (NOMID), familial Mediterranean fever (FMF), pyogenic arthritis, pyoderma gangrenosum and acne syndrome (PAPA), hyperimmunoglobulinemia D and periodic fever syndrome (HIDS), Tumour Necrosis Factor (TNF) Receptor-Associated Periodic Syndrome (TRAPS), systemic juvenile idiopathic arthritis, adult-onset Still’s disease (AOSD), relapsing polychondritis, Schnitzler’s syndrome, Sweet’s syndrome, Behcet’s disease, anti-synthetase syndrome, deficiency of interleukin 1 receptor antagonist (DIRA), haploinsufficiency of A20 (HA20), lupus nephritis, pulmonary arterial hypertension, interstitial lung disease (ILD), idiopathic pulmonary fibrosis, asthma, amyotrophic lateral sclerosis, rheumatoid arthritis, gout, Alzheimer’s disease, Parkinson’s disease, Huntington’s diseases, spinal cord injury, atherosclerosis, heart failure, dilated cardiomyopathy (DCM), pericarditis, myocarditis, myocardial infarction, obesity, type II diabetes, nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, chronic kidney disease (CDK), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn’s disease.

31. A method of degrading NEK7 protein comprising contacting said protein with the compound of any one of claims 1-2 or 23-25. 44 251982040 v132. The method of claim 31, wherein the compound is formulated in a pharmaceutical composition. 251982040 v1