WO2025234887A1

WO2025234887A1 - Nek7 degraders and methods of use thereof

Info

Publication number: WO2025234887A1
Application number: PCT/PL2024/000026
Authority: WO
Inventors: Sylvain Cottens; Przemyslaw GLAZA; Marta KLEJNOT; Roman PLUTA; Michal Jerzy WALCZAK
Original assignee: Captor Therapeutics SA
Current assignee: Captor Therapeutics SA
Priority date: 2024-05-09
Filing date: 2024-05-09
Publication date: 2025-11-13
Anticipated expiration: 2026-11-09

Abstract

The present invention provides a compound of formula: (I) and pharmaceutically acceptable salts and methods of use thereof.

Description

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 10 (IL-10) and IL-18 from their precursors (pro- IL-10 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-10 and IL-18 from their precursor forms (pro-IL-10 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-KB (nuclear factor kappa-light-chain-enhancer of activated B cells) or cytokines such as TNF or IL-10, 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 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 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: or a pharmaceutically acceptable salt thereof.

In some embodiments, the pharmaceutically acceptable salt is a formic acid salt. In some embodiments, the compound i

In accordance with a second aspect of the invention, there is provided a method of making the compound or pharmaceutically acceptable salt thereof of the first aspect, the method comprising:

(i) separation of stereoisomers of tert-Butyl (2S)-5-amino-2-(5-bromo-3-methyl-l- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-{(R)-5-bromo-3- methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate;

(ii) conversion of tert-butyl (S)-5-amino-2-((/?)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5- to tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)-5-oxopentanoate;

(iii) hydrogenation of tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin- 2-yl)-5-oxopentanoate to provide tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-(piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate;

(iv) separation of stereoisomers of tert-butyl (5)-5-amino-2-((f?)-3-methyl-l-oxo-5-(piperidin- 2-yl)isoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((/?)-3- methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)- 3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate; and

(v) conversion of tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((R)-piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate to (S)-3-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2- yl)piperidine-2, 6-dione or a pharmaceutically acceptable salt thereof.

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 pm, 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 A = 254 nm. In some such embodiments, when subjected to analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), a mobile phase of hexane/AcOEt/EtOH/iPrNHz at 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 R_t: tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rt of 7.974 min; and tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-l-oxoisoindolin- 2-yl)-5-oxopentanoate has a retention time R_t of 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-l-oxoisoindolin-2-yl)-5-oxopentanoate with 2-(tributylstannyl)pyridine and subsequent reaction with XPhos Pd G2, to provide tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5- (pyridin-2-yl)isoindolin-2-yl)-5-oxopentanoate.

In some embodiments of the second aspect, the hydrogenation in step (iii) is carried out using PtO?.

In some embodiments of the second aspect, the separation by chiral HPLC in step (iv) is carried out at 35°C using a Pic Solution 175 instrument, a Knauer 40D Detector, a CHIRALPAK IC column (5 pm, 250 x 30 mm), a flowrate of 90 mL/min, a mobile phase of isocratic 65% CO2 in supercritical state and 35% of 0.1% /PrNH2 in MeOH/ACN (ratio 1/1 v/v), maintaining isobaric conditions of 100 bar, and wherein monitoring is carried out by UV at A = 243 nm. In some such embodiments, when subjected to analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), a mobile phase of 40% modifier (ACN/MeOH/iPrNH2 at a ratio of 50/50/0.3 v/v/v) in SCCO2, and a flow rate of 4 mL/min the stereoisomers separated by chiral HPLC separation have the following retention times R_t: tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate has a retention time R_t of 4.77 min; and tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo- 5-((R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate has a retention time Rt of 5.41 min.

In some embodiments of the second aspect, step (v) comprises reaction of tert-butyl (S)-5-amino-2- ((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate with benzenesulfonic acid at 130"C under microwave irradiation.

In some embodiments of the second aspect, the method further comprises reacting tert-butyl L- glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(l-bromoethyl)benzoate to provide the tert-Butyl (25)-5-amino-2-(5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate of step (i).

In some embodiments of the second aspect, the method further comprises:

(vi) conversion of (S)-3-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)piperidine-

2.6-dione formic acid salt to (S)-3-((R)-3-methyl-l-oxo-5-({R)-piperidin-2-yl)isoindolin-2-yl)piperidine-

2.6-dione.

In accordance with a third aspect of the invention, there is provided a compound of formula: or a pharmaceutically acceptable salt thereof, produced by the method of any of the above embodiments of the second aspect of the invention.

In some embodiments of the third aspect, the pharmaceutically acceptable salt is a formic acid salt.

In some embodiments of the third aspect, the compound is In accordance with a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising the compound of any of the above embodiments of the first and third aspects of the invention.

In accordance with a fifth aspect of the present invention, there is provided the compound of any of the above embodiments of the first and third aspects of the invention, or the pharmaceutical composition of the fourth aspect of the invention, for use in medicine.

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 third aspects of the invention, or the pharmaceutical composition of the fourth 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 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 sixth 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 sixth 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 ( H I DS), Tumour Necrosis Factor (TN F) 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 (CKD), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn's disease.

In accordance with a seventh 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 third aspects of the invention.

In some embodiments of the seventh aspect, the compound is formulated in a pharmaceutical composition.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the crystal structure of ((S)-3-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2- yl)piperidine-2, 6-dione) (Compound A2), 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 pharmaceutically acceptable salt thereof.

In some embodiments, the compound is provided as a pharmaceutically acceptable salt, in some embodiments the compound is provided as a formic acid salt. In other embodiments, the compound is provided as a free base.

In the second aspect of the invention, there is provided a method of making the compound or pharmaceutically acceptable salt as defined above, the method comprising:

(i) separation of stereoisomers of tert-Butyl (2S)-5-amino-2-(5-bromo-3-methyl-l- oxoisoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((S)-5- bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate and tert-butyl (5)-5-amino-2-((R)-5-bromo-3- methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate;

(ii) conversion of tert-butyl (S)-5-amino-2-((R)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5- to tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)-5-oxopentanoate;

(iii) hydrogenation of tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin- 2-yl)-5-oxopentanoate to provide tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-{piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate;

(iv) separation of stereoisomers of tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-(piperidin-

2-yl)isoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (5)-5-amino-2-((R)-3- methyl-l-oxo-5-({S)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-

3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate; and

(v) conversion of tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((R)-piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate to (S)-3-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2- y l)piperid i ne-2, 6-dione or a pharmaceutically acceptable salt thereof.

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 pm, 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 A = 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 pm), a mobile phase of hexane/AcOEt/EtOH/iPrNH2 at a ratio of 50/25/25/0.1 v/v/v/v, and a flow rate of 1 mL/min, monitored by UV at A = 254 nm, the stereoisomers separated by chiral HPLC separation have the following retention times R_t: tert-butyl (5)-5-amino-2-((S)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5- oxopentanoate has a retention time R_t of 7.974 min; and tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time R_t of 9.692 min.

In some embodiments, step (ii) comprises reaction of tert-butyl (S)-5-amino-2-((R)-5-bromo-3- methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate with 2-(tributylstannyl)pyridine and subsequent reaction with XPhos Pd G2, to provide tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-(pyridin-2- yl)isoindolin-2-yl)-5-oxopentanoate.

In some embodiments, the hydrogenation in step (iii) is carried out using PtOj. 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.

In some embodiments, the separation by chiral HPLC in step (iv) is carried out at 35°C using a Pic Solution 175 instrument, a Knauer40D Detector, a CHIRALPAK IC column (5 pm, 250 x 30 mm), a flowrate of 90 mL/min, a mobile phase of isocratic 65% CO2 in supercritical state and 35% of 0.1% /PrNH₂ in MeOH/ACN (ratio 1/1 v/v), maintaining isobaric conditions of 100 bar, and wherein monitoring is carried out by UV at X = 243 nm. In some such embodiments, when subjected to analytical LC performed at 35°C using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), a mobile phase of 40% modifier (ACN/MeOH/iPrNH₂ at a ratio of 50/50/0.3 v/v/v) in SCCO2, and a flow rate of 4 mL/min, monitored by UV at X = 250 nm, the stereoisomers separated by chiral HPLC separation have the following retention times R_t: tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((S)-piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate has a retention time R_t of 4.77 min; and tert-butyl (S)-5-amino-2- ((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate has a retention time R_t of 5.41 min.

In some embodiments, step (v) comprises reaction of tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5- {{R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate with benzenesulfonic acid at 130’C under microwave irradiation. In some embodiments, the method further comprises reacting tert-butyl L-glutaminate hydrochloride with DIPEA and methyl 4-bromo-2-(l-bromoethyl)benzoate to provide the tert-Butyl (2S)-5-amino-2-(5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate of step (i).

In some embodiments, the method further comprises:

(vi) conversion of (S)-3-((R)-3-methyl-l-oxo-5-((/?)-piperidin-2-yl)isoindolin-2-yl)piperidine-

2.6-dione formic acid salt to (S)-3-({/?)-3-methyl-l-oxo-5-((/?)-piperidin-2-yl)isoindolin-2-yl)piperidine-

2.6-dione.

In accordance with a third aspect of the invention, there is provided a compound of formula: or a pharmaceutically acceptable salt thereof, produced by the method as described above.

In some embodiments, the compound is produced by the method as a pharmaceutically acceptable salt. In some embodiments, the compound is produced as a formic acid salt. In other embodiments, the compound is produced as a free base.

In accordance with a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising a compound as described above.

In accordance with a fifth aspect of the present invention, there is provided a compound or pharmaceutical composition as described above, for use in medicine.

In accordance with a sixth 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.

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 ( I LD), 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 (CKD), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn's disease. In accordance with a seventh 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.

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 (6 [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-dg (62.50) 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, ACN, 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

AcOH Acetic acid

DCM Dichloromethane

DIPEA N,N-Diisopropylethylamine

DMSO Dimethyl sulfoxide

Et Ethyl

EtOH Ethanol

FA Formic acid

HPLC High performance liquid chromatography

/PrNH₂ Isopropylamine LCMS Liquid chromatography mass spectrometry

Me Methyl

MeOH Methanol

MHz Megahertz

NMR Nuclear magnetic resonance

Rt Retention time

RT Room temperature

SFC Supercritical fluid chromatography tBu tert-Butyl

TFA Trifluoroacetic acid

XPhos Pd G2 Chloro(2-dicyclohexylphosphino-2',4',6'-triisopropyl-l,r- biphenyl)[2-(2'-amino-l,r-biphenyl)]palladium(ll)

Synthesis of (S)-3-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (Compound A2) and (S)-3-((R)-3-methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)piperidine-2,6- dione (Compound Al)

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-(l-bromoethyl)benzoate (1.15 g, 3.57 mmol, 1 equiv) was then added at 0°C, the cooling bath was removed and the reaction mixture was stirred at RT for 16 h under agon. After completion, the reaction mixture was diluted with AcOEt, washed with water and brine. The organic fraction was dried over Na₂SO₄ and concentrated in vacuo to provide crude product. tert-Butyl (25)-5-amino-2-(5-bromo-3-methyl-l-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-(l-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 pm, 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 A = 254 nm. The pure stereoisomers isolated by the chiral HPLC separation were then subjected to analytical LC. The retention times R_t of the pure stereoisomers in the analytical LC process are indicated below.

Isomer 1: tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate (360 mg, 23% yield) - Intermediate i( 1)

Rt = 7.974 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), 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 A = 254 nm)

LCMS (ESI+) m/z 411.0, 413.0 [M+H]⁺

^XH 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: tert-butyl (S)-5-amino-2-((/?)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate (360 mg, 23% yield) - Intermediate i(2)

Rt = 9.692 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), mobile phase: hexane/AcOEt/EtOH//PrNH₂, 50/25/25/0.1, v/v/v/v, and flow rate: 1 mL/min, operating at ambient temperature and monitored by UV at A = 254 nm)

LCMS (ESI+) m/z 411.0, 413.0 [M+H] *H NMR (400 MHz, DMSO-ds) 6 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-l-oxoisoindolin-2-yl)- 5-oxopentanoate (450 mg, 1.1 mmol, 1 equiv) (Intermediate i(2)) in 1,4-dioxane (5 mL) was added 2- (tributylstannyl)pyridine (1.5 equiv) under argon and the mixture was further bubbled with argon for 20 min. XPhos Pd G2 (0.1 equiv) was then added and the reaction was stirred under argon at 100‘C for 7 h. After completion, the reaction mixture was concentrated in vacuo and tert-butyl (S)-5-amino- 2-((/?)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)-5-oxopentanoate (300 mg, 67% yield) (Intermediate ii) was purified by flash column chromatography (SiCh, AcOEt/DCM, 1/1, v/v).

LCMS (ESI+) m/z 409.9 [M+H]

Step 3: To a stirred solution of tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin- 2-yl)-5-oxopentanoate (580 mg, 1.42 mmol, 1 equiv) (Intermediate ii) in AcOH (10 mL) was added PtOz (290 mg) and the reaction mixture was stirred at RT for 5 h under hydrogen atmosphere (balloon). After completion, the reaction mixture was filtered and the filtrate was evaporated to give crude product as mixture of stereoisomers. The stereoisomers were separated by SFC HPLC described below.

Chiral separation: The SFC HPLC separation was performed on Pic Solution 175 instrument equipped with Knauer40D Detector, using CHIRALPAK IC column (5 pm, 250 x 30 mm) operating at temperature of 35°C, with flowrate 90 mL/min, mobile phase isocratic 65% CO2 in supercritical state and 35% of modifier (0.1% /PrNHj in MeOH/ACN 1/1, v/v), duration time: 20 min; maintaining isobaric conditions of 100 bar and monitoring by UV at X = 243 nm. The pure stereoisomers isolated by the chiral HPLC separation were then subjected to analytical LC. The retention times Rt of the pure stereoisomers in the analytical LC process are indicated below.

Isomer 1: tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate (250 mg, 42% yield) - Intermediate iii(l)

Rt = 4.77 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), mobile phase: 40% modifier (ACN/MeOH/iPrNH2, 50/50/0.3, v/v/v) in SCCO2, and flow rate: 4 mL/min, operating at 35°C and monitored by UV at A = 250 nm)

LCMS (ESI+) m/z 416.7 [M+H]⁺ *H NMR (401 MHz, DMSO-ds) 8 7.60 - 7.55 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.15 (s, 1H), 7.12 (s, 1H), 4.61 (q, J = 6.6 Hz, 1H), 4.45 - 4.39 (m, 1H), 3.63 (dd, J = 10.8, 2.4 Hz, 1H), 3.05 (d, J = 11.7 Hz, 1H), 2.64 (d, J = 11.8 Hz, 1H), 2.15 (dd, J = 8.1, 4.9 Hz, 3H), 1.79 (m, 3H), 1.70 (d, J = 11.8 Hz, 1H), 1.57 (d, J = 10.6 Hz, 1H), 1.49 - 1.41 (m, 2H), 1.36 (s, 9H), 1.14 (d, J = 6.4 Hz, 3H).

Isomer 2: tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate (190 mg, 32% yield) - Intermediate iii(2)

Rt = 5.41 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), mobile phase: 40% modifier (ACN/MeOH/iPrNHj, 50/50/0.3, v/v/v) in scCOz, and flow rate: 4 mL/min, operating at 35°C and monitored by UV at A = 250 nm)

LCMS (ESI+) m/z 416.4 [M+H]⁺

^JH NMR (401 MHz, DMSO-d6) δ 7.60 - 7.55 (m, 2H), 7.48 (d, J = 7.9 Hz, 1H), 7.16 (s, 1H), 7.12 (s, 1H), 4.60 (q, J = 6.7 Hz, 1H), 4.45 - 4.38 (m, 1H), 3.63 (d, J = 10.6 Hz, 1H), 3.05 (d, J = 11.6 Hz, 1H), 2.63 (d, J = 11.4 Hz, 1H), 2.20 - 2.09 (m, 3H), 1.96 (m, 2H), 1.79 (s, 1H), 1.70 (d, J = 11.9 Hz, 1H), 1.56 (d, J = 10.2 Hz, 1H), 1.45 (d, J = 11.6 Hz, 1H), 1.36 (s, 9H), 1.21 (m, 1H), 1.09 (d, J = 6.4 Hz, 3H).

Step 4: tert-Butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate (29 mg, 0.071 mmol, 1 equiv) (Intermediate iii(2)) was dissolved in ACN (2 mL) and benzenesulfonic acid (1.1 equiv) was added. The reaction was carried at 130°C under microwave irradiation. After completion the solvent was evaporated and (S)-3-((R)-3-methyl-l-oxo-5-((R)- piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione formic acid salt (3 mg, 11% yield) (Compound A2) was purified by preparative HPLC.

Preparative HPLC was performed using Waters auto purification instrument. Column name: Luna Omega polar C18 (5 pm, 250 x 21.1 mm), operating at ambient temperature and flowrate of 16 mL/min. Mobile phases: A: 0.1% FA in water, B: ACN. The product was then subjected to analytical LC. The retention time R_t of the product in the analytical LC process is indicated below.

Rt = 9.39 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), mobile phase: hexane/DCM/EtOH/iPrNH?, 20/40/40/0.1, v/v/v/v, and flow rate: 1 mL/min, operating at ambient temperature)

LCMS (ESI+) m/z 342.4 [M+H] ^JH NMR (400 MHz, DMSO-ds) 8 10.95 (s, 1H), 7.64 - 7.58 (m, 2H), 7.51 (d, J = 8.0 Hz, 1H), 4.74 (dd, J = 12.6, 5.2 Hz, 1H), 4.63 (q, J = 6.6 Hz, 1H), 3.79 - 3.71 (m, 1H), 3.10 (d, J = 11.9 Hz, 1H), 2.89 - 2.55 (m, 4H), 1.97 (dd, J = 8.8, 3.9 Hz, 1H), 1.87 - 1.70 (m, 2H), 1.61 (d, J = 9.0 Hz, 1H), 1.46 (d, J = 9.5 Hz, 2H), 1.41 (d, J = 6.7 Hz, 3H).

Step 5: tert-Butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate (42 mg, 0.102 mmol, 1 equiv) (Intermediate iii(l)) was dissolved in ACN (2 mL) and benzenesulfonic acid (1.1 equiv) was added. The reaction was carried at 130°C under microwave irradiation. After completion the solvent was evaporated and (S)-3-((/?)-3-methyl-l-oxo-5-((S)- piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione formic acid salt (3 mg, 7% yield) (Compound Al) was purified by preparative HPLC.

Rt = 8.78 min (analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), mobile phase: hexane/DCM/EtOH/iPrNH₂, 20/40/40/0.1, and flow rate: 1 mL/min, operating at ambient temperature)

LCMS (ESI+) m/z 342.3 [M+H]⁺

^JH NMR (401 MHz, DMSO-d₆) δ 10.94 (s, 1H), 7.63 (s, 1H), 7.60 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 4.73 (dd, J = 12.6, 5.2 Hz, 1H), 4.64 (q, J = 6.6 Hz, 1H), 3.72 (d, J = 10.1 Hz, 1H), 3.09 (d, J = 12.1 Hz, 1H), 2.90 - 2.76 (m, 1H), 2.75 - 2.55 (m, 3H), 2.03 - 1.93 (m, 1H), 1.81 (s, 1H), 1.74 (d, J = 12.0 Hz, 1H), 1.60 (d, J = 9.5 Hz, 1H), 1.43 (dd, J = 18.4, 8.7 Hz, 6H).

Structure determination of (S)-3-((R)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2- yl)piperidine-2, 6-dione (Compound A2)

Co-crystallisation of CRBN_TBD (Cereblon thalidomide-binding domain), IKZF2_ZF2 (Helios protein zinc finger 2) and ((S)-3-((/?)-3-methyl-l-oxo-5-((/?)-piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione) (Compound A2) was performed using sitting drop vapor diffusion method at the room temperature. Crystallization trials were performed with CRBN_TBD at 130 pM and molar excess of IKZF2_ZF2 (1:1.3) and Compound A2 (1:4). Crystals formed within 2-3 days in the shape of thick rods in 0.1 M BIS-TRIS pH 6.5, 25% w/v Polyethylene glycol 3,350 (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 suite¹ (Potterton, 2018). Post data processing at beamline using XDS (Kabsh, 20100), structure was solved using molecular replacement with PHASER² (McCoy, 2007) with PDB ID: 7BQV as a search model. Refinement was carried out using REFMAC³ (MURshudov, 2011), with model building performed and compound topology generated using COOT (Emsley, 2010) and AceDRG⁴ (Long, 2017) respectively. The structure is shown in Figure 1.

Electron density inspection allowed Compound A2 chirality assignment due to visible favorable interaction between CRBN_TBD GLU377 and Compound A2 piperidine.

1. 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" . 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/ . REFMAC: Murshudov, G. N.; Skubak, 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://ioumaL5.iucr.Org/d/L5sues/2011/04/00/ba5152/irideK.htmi . AceDRG: Long, F.; Nicholls, R. A.; Emsley, P.; Graaeulis, 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.nim. nih.gov/pmc/articles/PMC5297914/rf:"^J:text.:'The%20progr3m%20AceDRG%20gene rate5%20accurate,from%20the%20Crystaiiography%200pen%20Database.

Synthesis of 3-(3-methyl-l-oxo-5-(piperldin-2-yl)lsoindolin-2-yl)piperidine-2, 6-dione (racemic mixture)

Step 1: 3-(5-Bromo-3-methyl-l-oxoisoindolin-2-yl)piperidine-2,6-dione was synthesized in 80% yield by the following method: Methyl 4-bromo-2-(l-bromoethyl)benzoate (70mg, 0.217 mmol, 1 equiv) and 3-aminopiperidine-2, 6-dione hydrochloride {1.5 equiv) were suspended in dry ACN. NaOAc (4 equiv) was added and the reaction was stirred at 60-140°C for 5-48 h. The volatiles were removed under reduced pressure, water was added and the solids were agitated for 1-6 h. The product was filtered, washed with water and EtjO, and dried under reduced pressure.

Methyl 4-bromo-2-(l-bromoethyl)benzoate was prepared as described in W0202220342A1.

LCMS (ESI+) m/z 337.2, 339.2 [M+H]⁺

Step 2: 3-(3-Methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione was synthesized using the following method (95% yield): 3-(5-bromo-3-methyl-l-oxoisoindolin-2-yl)piperidine-2, 6-dione (500 mg, 1.48 mmol, 1 equiv), 2-(tributylstannyl)pyridine (1.5 equiv), Pd(PPhs)4 catalyst (0.1 equiv) were purged with argon and dissolved in 1,4-dioxane as solvent. The reaction mixture was stirred at 90-120’C for 5-24 h. The solution was cooled to ambient temperature, filtered through pad of Celite® and concentrated under reduced pressure. The crude product was purified by flash column chromatography and/or preparative HPLC.

LCMS (ESI+) m/z 336.1 [M+H]⁺

*H NMR (500 MHz, DMSO-d₆) δ 10.96 (d, J = 16.6 Hz, 1H), 8.74 (ddd, J = 4.8, 1.9, 0.9 Hz, 1H), 8.37 (dd, J = 4.7, 1.5 Hz, IH), 8.25 (ddd, J = 7.9, 4.2, 1.5 Hz, 1H), 8.12 (ddt, 7 = 8.1, 2.1, 1.1 Hz, 1H), 7.97 (tt, 7 = 7.8, 1.5 Hz, IH), 7.79 (dd, J = 8.0, 6.8 Hz, IH), 7.45 (ddd, J = 7.5, 4.7, 1.0 Hz, IH), 4.92 - 4.75 (m, 2H), 2.92 - 2.59 (m, 3H), 2.11 - 2.01 (m, IH), 1.53 (dd, J = 11.7, 6.7 Hz, 3H). Step 3: 3-(3-Methyl-l-oxo-5-(piperidin-2-yl)isoindolin-2-yl)piperidine-2, 6-dione (formic acid salt) was synthesized in 20% yield by PtOj reduction of 3-(3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2- yl) piperidine-2, 6-dione (47.0 mg, 1 equiv) under hydrogen atmosphere (1-30 bar) at RT for 5-24 h. The solid particles were filtered off on a pad of Celite* and washed with EtOH. The filtrate was concentrated under reduced pressure and the crude product was purified by flash column chromatography and/or preparative HPLC.

LCMS (ESI+) m/z 341.9 [M+H]⁺ ^XH NMR (500 MHz, DMSO-d₆) δ 10.92 (s, 1H), 7.64 - 7.58 (m, 2H), 7.50 (ddd, J = 13.5, 7.8, 1.3 Hz, 1H), 4.73 (dd, J = 12.8, 5.3 Hz, 1H), 4.64 (p, J = 6.5 Hz, 1H), 3.73 (dd, J = 10.6, 2.6 Hz, 1H), 3.10 (d, J = 11.9 Hz, 1H), 2.87 - 2.77 (m, 1H), 2.74 - 2.64 (m, 1H), 2.62 - 2.56 (m, 1H), 1.98 (ddt, J = 10.4, 5.3, 2.7 Hz, 1H), 1.86 - 1.79 (m, 2H), 1.78 - 1.71 (m, 2H), 1.60 (d, J = 9.6 Hz, 1H), 1.51 - 1.35 (m, 6H).

Ternary complex formation assay

The effect of the molecular glue compound of the invention, i.e. Compound A2, and also its stereoisomer Compound Al on the formation of a ternary complex composed of [NEK7]-[Compound]- [CRBN/DDB1] was investigated with two methods: AlphaLISA dose response assay and HTRF ternary complex assay.

AlphaLISA dose response assay:

Two types of protein solution were prepared:

- 200 nM biotinylated NEK7, 40 pg/ml AlphaScreen Streptavidin-coated Donor Beads in HBS (10 mM HEPES, 150 mM NaCL, pH 7.4) buffer with 0.1% Tween-20 and ImM DTT,

- 200 nM 6XHis-CRBN/Strep-DDBl, 40 pg/ml AlphaUSA Anti-6xHis Acceptor beads in HBS buffer with 0.1% Tween-20 and ImM 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. 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 pl 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 ref, 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: Aexc = 680 nm, A_em = 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-l-oxo-5-(piperidin-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 A2 (the compound of the present invention) has much stronger capability to induce the formation of the [NEK7]-[Compound]-[CRBN/DDBl] complex than Compound Al.

HTRF ternary conwLex_assay:

The effect of the molecular glue compounds, i.e. Compound A2, and also its stereoisomer Compound Al on the formation of a ternary complex composed of [NEK7]-[Compound]-[CRBN/DDBl] was investigated.

Mix solution of proteins and reagents was prepared:

- 24 nM NEK7, 52.8 nM 6XHis-CRBN/Strep-DDBl, 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. 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 ref) 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 337 665 620.

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/DDBl] complex for each compound was determined based on obtained dose response curves;

5) Ternary complex response level at 1 pM compound was normalized to that of the racemic mixture as a reference for Compound Al and Compound A2. Normalized values were expressed as a percentage.

Off-target ternary complex formation assay

The effect of the molecular glue compounds of the invention on the formation of a ternary complex composed of [SALL4 ZF2]-[Compound]-[CRBN/DDBl] was investigated by AlphaLISA dose response assay.

Two types of protein solution were prepared:

- 800 nM Strep-tagged SALL4 ZF2, 40 pg/ml AlphaScreen StrepTactin-coated Donor Beads in PBS (10 mM phosphate buffer, 137 mM NaCL, 2.7 mM KCI, pH 7.4) buffer with 0.1% Tween-20 and ImM DTT,

- 200 nM 6XHis-CRBN/Strep-DDBl, 40 pg/ml AlphaLISA Anti-6xHis Acceptor beads in PBS buffer with

0.1% Tween-20 and ImM 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.

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 pl 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 ref, room temperature) and incubated at 25*C for 30 min.

The results were analyzed as follows:

2) average of the maximum measured luminescence for racemic mixture was calculated and used as a an internal positive control;

Table 1: Ternary complex assay results for the compound of the invention i.e. Compound A2, and also its stereoisomer Compound Al in relation to the racemic mixture. As shown in Table 1, Compound A2 surprisingly demonstrated a much greater capability than its stereoisomer Compound Al in inducing formation of the [NEK7]-[Compound]-[CRBN/DDBl] complex. Compound A2 also showed surprisingly lower activity than its stereoisomer Compound Al in formation of the off-target ternary complex [SALL4 ZF2]-[Compound]-[CRBN/DDBl]. Compound A2 therefore shows surprisingly high activity and selectivity in formation of the [NEK7]-[Compound]- [CRBN/DDB1] complex.

Biological Testing Methodology

Ceil culture

Human peripheral blood mononuclear cells (PBMCs)

Human PBMCs were isolated from buffy coats of healthy volunteers. Buffy coats were diluted 1:1 (v/v) with DPBS (Sigma-Aldrich) in falcon tubes. Then, 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 lOOx (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^A6 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.

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 lOOx (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^A6 of U937 cells 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 pg/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 lOOx (Biowest) at 37°C, 5% CO₂ and subcultured every 2-3 days.

Nano-Gio HiBiT Lytic Assay

For Nano-Gio HiBiT Lytic Assay, HEK293 NEK7-HiBiT cells were seeded at the density 2xl0^A3 cells in triplicates (pilot study with three concentrations) or duplicates (12-point dose-response study) in the 40 pL 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% CO₂ for 24 hours. After incubation, 40 pL of Nano-Gio HiBiT Lytic Reagent (prepared according to the manufacturer protocol) were added to 40 pL 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 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 bloting

Cell lysates from Human PBMCs or U937 cells were prepared by direct lysis in 40-50 pl RIPA lysis buffer (50mM Tris-HCI pH 7.4, 150mM NaCI, 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 -80°C. Following thawing, lysates were centrifuged at 4’C, 19 OOOxg 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 NaCI, 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 - 0-Actin (Ih, 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 DCso values (the concentration of the compound leading to 50% protein 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 (cone. 0.1, 1 and 10 pM in the pilot study or series of concentrations in dose-response study starting from 10 pM 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 A2 leads to efficient degradation of NEK7-HiBiT protein, unlike the inactive Compound Al (Table 2). The activity of Compound A2 in the NEK7-HiBiT Lytic assay is nearly 4 times higher than that of the racemic mixture when comparing DCso values (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.

A - Levels of NEK7-HiBiT Protein < 10%

B - Levels of NEK7-HiBiT Protein > 10% and < 30%

C - Levels of NEK7-HiBiT Protein > 30%

NEK7 protein degradation determined by Western blot

NEK7 protein degradation was tested in human U937 monocytic cell line. Table 3 and Figure 2 show the results of NEK7 levels in cells treated with Isomers or DMSO for 24h. It has been demonstrated that Compound A2, unlike Compound Al, leads to strong degradation of NEK7 after 24 hours of treatment with a single dose of 1 pM.

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.

A - Levels of NEK7 Protein < 10%

B - Levels of NEK7 Protein > 10% and < 50%

C - Levels of NEK7 Protein > 50%

The activity of Compound A2 was further confirmed across a wide range of concentrations in a degradation assay performed on human PBMCs. Figure 3 illustrates the dose-dependent effect of NEK7 protein degradation in the tested model after 24 hours of cell incubation with the compound. The Table 4 presents DCso values for Compound A2 compared to the racemic mixture, indicating a higher activity of the compound covered by the present invention.

Table 4: Absolute DCso values (mean of biological replicates) following treatment with the compounds in human PBMCs.

Claims

1. A compound of formula: or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the pharmaceutically acceptable salt is a formic acid salt.

3. The compound of claim 1, which is

4. A method of making the compound of any one of claims 1-3, comprising:

(iii) hydrogenation of tert-butyl (5)-5-amino-2-((/?)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin- 2-yl)-5-oxopentanoate to provide tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-(piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate;

2-yl)isoindolin-2-yl)-5-oxopentanoate by chiral HPLC, to provide tert-butyl (S)-5-amino-2-((/?)-3- methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate and tert-butyl (S)-5-amino-2-((R)-

3-methyl-l-oxo-5-((ff)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate; and

(v) conversion of tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-((R)-piperidin-2- yl)isoindolin-2-yl)-5-oxopentanoate to {S)-3-((/?)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2- yl)piperidine-2, 6-dione or a pharmaceutically acceptable salt thereof.

5. The method of claim 4, 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 pm, 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 X = 254 nm.

6. The method of claim 5 wherein, when subjected to analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), a mobile phase of hexane/AcOEt/EtOH/iPrNH₂ at 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 R_t: tert-butyl (S)-5-amino-2-((S)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time R_t of 7.974 min; tert-butyl (S)-5-amino-2-((/?)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate has a retention time Rt of 9.692 min.

7. The method of any one of claims 4-6, wherein step (ii) comprises reaction of tert-butyl (S)-5- amino-2-((/?)-5-bromo-3-methyl-l-oxoisoindolin-2-yl)-5-oxopentanoate with 2- (tributylstannyl)pyridine and subsequent reaction with XPhos Pd G2, to provide tert-butyl (S)-5- amino-2-((/?)-3-methyl-l-oxo-5-(pyridin-2-yl)isoindolin-2-yl)-5-oxopentanoate.

8. The method of any one of claims 4-7, wherein the hydrogenation in step (iii) is carried out using PtO₂.

9. The method of any one of claims 4-8, wherein the separation by chiral HPLC in step (iv) is carried out at 35°C using a Pic Solution 175 instrument, a Knauer40D Detector, a CHIRALPAK IC column (5 pm, 250 x 30 mm), a flowrate of 90 mL/min, a mobile phase of isocratic 65% CO₂ in supercritical state and 35% of 0.1% /PrNH₂ in MeOH/ACN (ratio 1/1 v/v), maintaining isobaric conditions of 100 bar, and wherein monitoring is carried out by UV at A = 243 nm.

10. The method of claim 9 wherein, when subjected to analytical LC performed using a CHIRALPAK IC column (250 x 4.6 mm, 5 pm), a mobile phase of 40% modifier (ACN/MeOH/iPrNH₂ at a ratio of 50/50/0.3 v/v/v) in scCOz, and a flow rate of 4 mL/min the stereoisomers separated by chiral HPLC separation have the following retention times R_t: tert-butyl (S)-5-amino-2-((/?)-3-methyl-l-oxo-5-((S)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate has a retention time R_t of 4.77 min; tert-butyl (S)-5-amino-2-((R)-3-methyl-l-oxo-5-((/?)-piperidin-2-yl)isoindolin-2-yl)-5- oxopentanoate has a retention time R_t of 5.41 min.

11. The method of any one of claims 4-10, wherein step (v) comprises reaction of tert-butyl (S)- 5-amino-2-((/?)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)-5-oxopentanoate with benzenesulfonic acid at 130°C under microwave irradiation.

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

13. The method of any one of claims 4-12, further comprising:

(vi) conversion of (S)-3-((/?)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)piperidine-

2.6-dione formic acid salt to (S)-3-((/?)-3-methyl-l-oxo-5-((R)-piperidin-2-yl)isoindolin-2-yl)piperidine-

2.6-dione.

14. A compound of formula: or a pharmaceutically acceptable salt thereof, produced by the method of any one of claims 4-13.

15. The compound of claim 14, wherein the pharmaceutically acceptable salt is a formic acid salt.

16. The compound of claim 14, which is

17. A pharmaceutical composition comprising the compound of any one of claims 1-3 or 14-16.

18. The compound of any one of claims 1-3 or 14-16, or the pharmaceutical composition of claim 17 for use in medicine.

19. The compound of any one of claims 1-3 or 14-16or the pharmaceutical composition of claim 17 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.

20. The compound or pharmaceutical composition for use of claim 19, 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.

21. The compound or pharmaceutical composition for use of claim 19 or 20, 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 (CKD), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), ulcerative colitis (UC) or Crohn's disease.

22. A method of degrading NEK7 protein comprising contacting said protein with the compound of any one of claims 1-3 or 14-16.

23. The method of claim 22, wherein the compound is formulated in a pharmaceutical composition.