Desmuramylpeptide analogue monoester

Abstract

The invention is directed to a monoester compound of Formula (1) which is a desmuramylpeptide (DMP) analogue of muramyl dipeptide (MDP), and also to medical uses of the compound such as treating disorders and diseases (e.g. Crohn's disease) wherein the treatment is affected or facilitated by the compound acting as a potent and selective NOD2 agonist.

Description

[0001] Monoester compounds The present invention relates to a novel desmuramylpeptide (DMP) analogue of muramyl dipeptide (MDP) and medical uses thereof. Nucleotide-binding Oligomerisation Domain (NOD) protein NOD2 is a member of an extended family of inflammatory and immune proteins in animals (NOD families). These proteins combine a central nucleotide-binding domain (NOD) with a C-terminal leucine- rich repeat (LRR) motif and an N-terminal caspase recruitment domain (CARD) or equivalent (Ohto, U. Front Immunol.2022, 13, 953530 ). NOD2 is believed to be mainly^^^^^^^^^ ^^ ^ ^^^^^^^^ ^^^^^^^^^^ ^^^^^ ^^^^^^^^^ ^Ogura, Y et al. J Biol Chem. 2001,276(7), 4812-4818; Segal, A.W. J Intern Med.2019, 286, 373-388).^^^^^^^ ^^^^^^^ ^^ ^^^^^^^^ ^^^^ ^^^^^^^ ^^^^^^^ ^^^^ ^^^^^^^^^^ ^^^^ ^^ ^^^ ^^^^ ^^^^strongly associated with the disease (Jostins, L et al. Nature.2012, 491(7422), 119-124).^^^ ^^^^^^^^^^^ ^^ ^^^^^^^^^ ^^ ^^^^ ^^^^ ^ ^^^^^^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^ ^^^identified by a positional-cloning strategy, based on linkage analysis followed by linkage disequilibrium mapping, of a known susceptibility region on chromosome 16 in 77 multiplex families. Mutations in this gene remain the most strongly associated genetic^^^^ ^^^^^^ ^^^ ^^^^^^^ ^^^^^^^ ^^^^^^^ ^^^^ ^^ ^^^ Nature. 2004, 411, 599-603).^^^^ ^^ ^^^^^^^^^^^^ ^^ ^^^^ ^ ^^^^ ^^ ^^^^^^^ ^^^^^^^ ^^^ ^^^ ^^^^^^^^^^^ ^^ ^ ^^^^^^^^^^^^^^ ^^^^^^ ^^^^^^^^^ ^^^^^^^ ^^ ^^^^^^^^ ^^^^ ^^^^^^^ ^^^^^^^ ^^^^ ^^^^^^^^^^ ^^^^^^^^^^ ^^ ^ ^^^^^^^^^ ^^^^^ ^^^^^^^^^^^^ ^^^^^^^^ ^^ ^^^^^ ^ ^^^^^^ ^^^^^^^^^^^^^n to^^^^^^^ ^^^^^^^ ^^^^^^^ ^^^^^^ ^^ ^^^ Lancet. 2006, 367, 668-678).^^ ^^^ ^^^^ ^^^^^^^^ ^^^^ ^^^^^^^ ^^^^^^^ ^^^^^^^^ ^^ ^^^^^ ^^^^^^^^ ^^^^^^ (Sewell, G.W.et al. Opin Immunol. 2009, 21(5), 506-513). Firstly, a gastrointestinal infection allows faecal bowel contents access to vulnerable tissues within the bowel. Secondly, failure of^^^ ^^^^^ ^^^^^^^^^^^^ ^^^^^^^^ ^^ ^^^^^^ ^^^^^^ ^^ ^^^^^^^ ^^^^^^^ ^^^^^^^^ ^^^^^^^ ^^failure to recruit immune cells to the inflammatory site, resulting, amongst other things, in the clearance of bacteria from the tissues being defective. Thirdly, the retained faecal P90084.WO01 FINAL Application 25.04.2025 products result in the characteristic chronic granulomatous inflammation and adaptive^^^^^^ ^^^^^^^^^ ^^^^^^ ^^^^ ^^ ^^^ ^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^NOD2 is understood to play a role in the secretion of pro-inflammatory molecules such as inflammatory cytokines (Boyle, J.P. et al. Open Biol.2014, 4(12), 140178). In general, NOD proteins are understood to recognise a signal from an invading organism in their LRR domain that induces a polymerisation that triggers a signalling cascade which terminates in the production and release of pro-inflammatory molecules. NOD2 is understood to be activated by muramyl dipeptide (MDP), a component of the cell wall of both Gram negative and Gram positive bacteria. The current theoretical models hypothesise that in its resting state NOD2 is doubled back on itself in an auto-inhibited conformation in the cytoplasm until activated by the binding of MDP to its LRR domain.^^^^ ^^ ^^^^^^^ ^^ ^^^^^^^^ ^^^^^^ ^^^^^^^^^^^^^^^ ^^^^^^^^ ^^^^-oligomerisationleading to production of pro-inflammatory molecules (e.g. pro-inflammatory cytokines) (Maekawa, S. et al. Nat Commun.2016, 7, 711813). It has also been hypothesized that, whilst acute stimulation of the NOD2 receptor leads to secretion of pro-inflammatory mediators from a variety of cell types, chronic stimulation of the NOD2 receptor leads to tolerization of the cells to the effects of subsequent stimulation of both the NOD2 receptor and other pattern recognition receptors. (Hedl, M. et al. PNAS. 2007, 104(49), 119440 and Lessard, A-J. et al. Cell Reports.2017, 20, 1830). This tolerization has been proposed to be a mechanism that could contribute to the restoration of homeostasis in inflamed tissues, and the failure of achieving tolerization may lead to the chronic inflammation^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^^^^^^^^ ^^^^^^^^^ ^^^ ^^^ ^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^ ^^^ ^^^^^^^^^^^^^^^^^ (Cushing,K. et al. JAMA. 2021, 325(1), 69-80). These drugs and biological treatments dampen down the secondary granulomatous and adaptive immune response in patients. Anti-^^^^^ ^^^ ^^ ^^^^^^^ ^^^ ^^ ^^^ ^^^^^^^ ^ ^^^^^^^^^^^^^ ^^^^^^^^^ ^^^^ ^^^^ ^^^ ^^^^^of patients in remission after one year on these treatments (Ding, N.S. Pharmacol Ther. 2016, 43, 30 ^ 51). Immunosuppressant treatment further compromises the underlying innate immune deficit to mucosal damage, thereby increasing the likelihood of further infection and the influx of bowel contents into the tissues, and its impaired clearance. Thus, additional suppression of an already impaired inflammatory response could further

[0002] 2 P90084.WO01 FINAL Application 25.04.2025 impair the clearance of faecal material from the bowel wall, increasing the frequency of^^^^^^^^^ ^^^^^^^^^^^^^ ^^^ ^^^^^^^^^^ ^^^^^^^ ^^^^^^^ ^^^^ ^ ^^^^^^^^ ^^ ^ ^^^^chronic condition (Segal, A.W. J Intern Med.2019, 286, 373-388).^^^^^ ^^ ^^^^^^^^^ ^ ^^^^ ^^^ ^ ^^^^^^^ ^^^^^^^ ^^^^^^^^^ ^^^^ may modulate ^ ^^^^^^^^^innate immunity by acting as a modulator of pro-inflammatory mediator secretion in a subject. The applicants have identified a novel desmuramylpeptide (DMP) compound that is a potent and selective agonist of NOD2. In general, analogues of muramyl dipeptide (MDP) and desmuramylpeptides (DMPs) have at least two stereogenic centres on a dipeptide backbone. Herein, we refer to the stereogenic centre of the glutamic acid residue, analogue or derivative thereof as the ^^^^^^^ stereogenic centre and the stereogenic centre of the other amino acid residue as the ^^^^^^ stereogenic centre (see the dipeptide of Formula (1) below for context). Griffin, M. E. et al. (ACS Chem. Biol. 2023, 18, 1368-1377) describe N -arylpyrazole dipeptides as NOD2 agonists for use in immune checkpoint inhibitor therapy. Gobec, M. et al. (J. Med. Chem. 2018, 61, 2707-2724) and Guzelj, S et al. (ACS Med. Chem. Lett.2022, 13, 8, 1270 ^ 1277) describe desmuramylpeptide compounds as NOD2 agonists with nanomolar potency. Gobec, M. et al. (Eur. J. Med. Chem.2016, 116, 1-12) also describe desmuramylpeptide compounds as NOD2 agonists. Jakopin, Z. et. al. (Int. J. Mol. Sci.2019, 20, 4265) describe an in vitro tool for functional characterization of NOD1 / NOD2 antagonists. Blakskjær, P. et. al. (J. Chem. Soc., Perkin Trans.1, 2001, 1, 910-916) describe a method of synthesising peptides, such as wherein the stereochemistry of the right stereogenic centre is undefined, by C-alkylation or C- allylation using glycyl radical intermediates. P90084.WO01 FINAL Application 25.04.2025US4666890A describe an intermediate compound, ,used in the synthesis of peptides for enhancing protective efficacy in infection. Khan, F-A. et. al. (Eur. J. Org. Chem.2021, 48, 6688-6699) describe desmuramylpeptide which are recognised by NOD2. US4362716A describes dipeptides, wherein the right stereogenic centre is defined (L- alanine) and the left stereogenic centre is defined (D-glutamic acid or derivatives thereof), that are able to stimulate immune reactions. There is no indication of NOD2 activity of these compounds. WO2024 / 224089A1 describes novel analogues of muramyl dipeptide (MDP) and desmuramylpeptides of the following formula: . Summary of the invention An object of the present invention is to provide a compound useful in treating disorders ^^^ ^^^^^^^^ ^^^^^ ^^^^^^^ ^^^^^^^^ ^^^^^^^ ^^^ ^^^^^^^^^ ^^ ^^^^^^^^ ^^ ^^^^^^^^^^^ ^^ ^^^compound acting as a potent and selective NOD2 agonist. In one aspect the present invention, there is provided a compound (a dipeptide) of Formula (1): P90084.WO01 FINAL Application 25.04.2025 or a pharmaceutically acceptable salt thereof. Unless otherwise defined, all the technical and scientific terms used have the same meaning as that usually understood by an ordinary specialist in the field to which the invention belongs. The above compound according to the present invention (i.e. the compound of Formula (1)) may also be referred to as (R)-4-((S)-2-(2-(4-fluorophenyl)-2-methylpropanamido)-3,3-dimethylbutanamido)-5-isopropoxy-5-oxopentanoic acid ^^^ ^^ ^^^^^^^^^.The present invention also relates to a pharmaceutical composition comprising the compound according to the present invention (i.e. the compound of Formula (1)). Preferably, the pharmaceutical composition comprises a pharmaceutically or therapeutically acceptable excipient or carrier. The compound according to the present invention may be a potent and selective agonist of NOD2. By acting as agonist of NOD2, the compound according to the present invention may be capable of modulating proinflammatory mediator secretion in a subject, and may be thus capable of modulating innate immunity in a subject. The compound of Formula (1), which has the same stereochemical configuration as in the natural agonist MDP, may be much more active in the HEK-blue hNOD2 assay than compounds with a stereochemical configuration different to that shown in Formula (1). We believe the absolute configuration of both stereogenic centres of the dipeptide backbone is important, because changing either of them leads to compounds with significantly reduced activity. P90084.WO01 FINAL Application 25.04.2025 A further aspect of the invention is the compound or pharmaceutical composition according to the present invention for use as a medicament for the treatment of a disease^^ ^^^^^^^^^ ^^^^^^^^^^ ^^^ ^^^ ^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^A further aspect of the invention is the compound according to the present invention for use as potent and selective NOD2 agonist. A further aspect of the invention is the compound according to the present invention for use as a medicament capable of modulating innate immunity in a subject. A further aspect of the present invention is the compound according to the present invention for use as a modulator of pro-inflammatory mediator secretion in a subject. The use may be in the treatment of a disease or disorder^^^^^ ^^ ^^^ ^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^^The invention also encompasses a method of treating a disease or disorder, comprising the step of administering the compound according to the present invention or the pharmaceutical composition according to the present invention to a subject in need of the^^^^^ ^^^^^^^^^^ ^^^ ^^^^^^^ ^^ ^^^^^^^^ ^^^^^ ^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^ ^^ ^^^ ^^^^^^^treatment is affected or facilitated by the compound of the invention acting as a potent and selective NOD2 agonist. Comparative compounds The following comparative compound is also disclosed: 4. Ethyl (R)-4-acetamido-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate. The above comparative compound may also be represented by the following structural formula: P90084.WO01 FINAL Application 25.04.2025 . Particular non-limiting examples of the present invention will now be described with reference to the following Figures, in which: Figure 1 is a graph showing dose dependent response of HEK-BlueTMhNOD2 cells to IHXME26 and MDP (control activating ligand) (absorbance measured at 655 nm). Figure 2 is a graph showing dose dependent response of HEK-BlueTMNull2 cells to IHXME26 (absorbance measured at 655 nm). Figure 3 is a graph showing dose dependent response of HEK-BlueTMhNOD1 cells to IHXME26 (absorbance measured at 655 nm). Figures 4A-4F are graphs showing the response of human monocytes to 0.2 µM or 20 µM IHXME26 compared to MDP by inflammatory cytokine secretion (GRO alpha, ENA-78, IL-1 alpha). Figures 5A-5F are graphs showing the response of human monocytes to 0.2 µM or 20 µM (high) IHXME26 compared to MDP by inflammatory cytokine secretion (IL-6, MIP- 1 alpha MMP-1). Figures 6A-6F are graphs showing the response of human monocytes to 0.2 µM or 20 µM (high) IHXME26 compared to MDP by inflammatory cytokine secretion (Eotaxin-2, MCP-1 and VEGF-A). Figure 7 is a graph showing plasma stability of IHXME26 in human, rat, dog, monkey and mini-pig (Cyprotex). P90084.WO01 FINAL Application 25.04.2025 Figure 8 is a graph showing hepatocyte stability of IHXME26 in human, rat, dog, monkey and mini-pig (Cyprotex). Figure 9 shows the results of the EurofinsTMSafetyScreen panel (1 / 4) for IHXME26 at 10 µM. Figure 10 shows the results of the EurofinsTMSafetyScreen panel (2 / 4) for IHXME26 at 10 µM. Figure 11 shows the results of the EurofinsTMSafetyScreen panel (3 / 4) for IHXME26 at 10 µM. Figure 12 shows the results of the EurofinsTMSafetyScreen panel (4 / 4) for IHXME26 at 10 µM. Figure 13 is a graph showing whole blood concentrations of IHXME26 following a 2 mg / kg intravenous dose in rats. Figure 14 is a graph showing whole blood concentrations of IHXME26 following a 10 mg / kg intravenous dose in rats. Figure 15 is a graph showing whole blood concentrations of IHXME26 following a 50 mg / kg oral gavage dose in rats. Figure 16 is a graph showing whole blood concentrations of IHXME26 following a 250 mg / kg oral gavage dose in rats. Figures 17A-17C are graphs showing the tissue concentrations of IHXME26, 6 hours after a 2 mg / kg intravenous dose in rats. Figures 18A-18B are graphs showing tissue concentrations of IHXME26, 1, 4 and 24 hours after a 50 mg / kg oral gavage dose in rats. P90084.WO01 FINAL Application 25.04.2025 Figures 19A-19C are graphs showing tissue concentrations of IHXME2624 hours after a 250 mg / kg oral gavage dose in rats. Figure 20 is a summary plot of whole blood concentrations of IHXME26 measured in PK studies. Figure 21 is a graph showing plasma stability of IHXME26 in human, rat, dog, monkey and mini-pig (Pharmaron). Figure 22 is a graph showing hepatocyte stability of IHXME26 in human, rat, dog, monkey and mini-pig (Pharmaron). Figure 23 is a graph showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on change in bodyweight. Figures 24A-24F are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on clinical chemistry parameters. Figures 25G-25K are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on clinical chemistry parameters. Figures 26L-26P are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on clinical chemistry parameters. Figures 27A-27F are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on haematology parameters. Figures 28G-28K are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on haematology parameters. Figures 29L-29Q are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on haematology parameters.

[0003] 9 P90084.WO01 FINAL Application 25.04.2025 Figures 30R-30T are graphs showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on haematology parameters. Figure 31 is a graph showing the effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on urine albumin concentration. Figures 32A-32F are graphs showing once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on serum cytokine concentrations. Figures 33A-33C is a graph showing the average IHXME26 concentration in rat blood following p.o. dosing at different dose concentrations on study day 0 and day 6. Experimental General Method 1 Compounds of formula (2) depicted below were prepared using the following synthetic procedure. Reference Example 1: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-ethoxy-5-oxopentanoic acid Step 1 To a solution of (R)-2-(((benzyloxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (1 equiv.) in DMF was added K2CO3 (3 equiv.) and iodoethane (2 equiv.). The reaction mixture was stirred at room temperature overnight. The mixture was diluted P90084.WO01 FINAL Application 25.04.2025 with water and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by reverse phase chromatography to afford 5-(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-D-glutamate as a yellow oil. Step 2 To a solution of 5-(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-D-glutamate (1 equiv.) in EtOH was added 10% Pd / C (0.4 equiv.). The reaction mixture was stirred at room temperature under a hydrogen atmosphere overnight. The mixture was filtered and concentrated to afford 5-(tert-butyl) 1-ethyl D-glutamate as a colourless oil, which was used directly in the next step. Step 3 To a solution of ((benzyloxy)carbonyl)-L-valine (1 equiv.) in CH2Cl2was added HATU (1.3 equiv.) and DIPEA (3 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then 5-(tert-butyl) 1-ethyl D-glutamate (1 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by preparative TLC to afford 5-(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-L-valyl-D-glutamate as a white solid. Step 4 To a solution of 5-(tert-butyl) 1-ethyl ((benzyloxy)carbonyl)-L-valyl-D-glutamate (1 equiv.) in EtOH was added 10% Pd / C (0.4 equiv.). The reaction mixture was stirred at room temperature under a hydrogen atmosphere overnight. The mixture was filtered and concentrated to afford 5-(tert-butyl) 1-ethyl L-valyl-D-glutamate as a colourless oil, which was used directly in the next step. Step 5 To a solution of 2-(4-chlorophenyl)-2-methylpropanoic acid (1 equiv.) in CH2Cl2 was added HATU (1.3 equiv.) and DIPEA (3 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then 5-(tert-butyl) 1-ethyl L-valyl-D-glutamate (1 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography

[0004] 11 P90084.WO01 FINAL Application 25.04.2025 to afford 5-(tert-butyl) 1-ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D- glutamate as a white solid. Step 6 To a solution of 5-(tert-butyl) 1-ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl- D-glutamate (1 equiv.) in CH2Cl2was added TFA (2 mL). The reaction mixture was stirred at room temperature for three hours, then the solvent was removed under vacuum to afford (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-ethoxy-5-oxopentanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 12.2 (1H, s), 8.27 (1H, d), 7.45 ^ 7.28 (4H, m), 6.86(1H, d), 4.29 ^ 4.02 (4H, m), 2.27 (2H, t), 2.02 ^ 1.87 (2H, m), 1.84 ^ 1.71 (1H, m), 1.49 (3H, s), 1.46 (3H, s), 1.18 (3H, t), 0.81 (3H, d), 0.73 (3H, d). LCMS m / z = 455.2 [M+H]+General Method 2 Compounds of formula (3) depicted below were prepared using the following synthetic procedure. Reference Example 2: Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D- glutaminate To a solution of (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-ethoxy-5-oxopentanoic acid (1 equiv., prepared as for Example 1) in CH2Cl2was added HATU (1 equiv.) and DIPEA (4 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then NH4Cl (2 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford ethyl (2-(4- chlorophenyl)-2-methylpropanoyl)-L-valyl-D-glutaminate as a white solid. P90084.WO01 FINAL Application 25.04.20251H NMR (400 MHz, DMSO-d6^ ^ 8.26 (1H, d), 7.42 ^ 7.29 (4H, m), 7.25 (1H, s), 6.82(1H, d), 6.77 (1H, s), 4.15 (2H, t), 4.11 ^ 4.03 (2H, m), 2.09 (2H, t), 1.97 ^ 1.84 (2H, m), 1.80 ^ 1.68 (1H ,m), 1.46 (6H, d), 1.17 (3H, t), 0.79 (3H, d), 0.70 (3H, d). LCMS m / z = 454.3 [M+H]+General Method 3 Compounds of formula (4) depicted below were prepared using the following synthetic procedure. Reference Example 3: (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid Step 1 To a solution of 1-(tert-butyl) 5-methyl ((benzyloxy)carbonyl)-D-glutamate (1 equiv.) in EtOH was added Pd / C (0.4 equiv.). The reaction mixture was stirred at room temperature overnight under a hydrogen atmosphere. The solvent was removed under vacuum to afford crude 1-(tert-butyl) 5-methyl D-glutamate which was used without further purification. Step 2 P90084.WO01 FINAL Application 25.04.2025 To a solution of (S)-2-(((benzyloxy)carbonyl)amino)-3,3-dimethylbutanoic acid (1 g, 4.61 mmol) in CH2Cl2 was added HATU (1 equiv.) and the reaction mixture was stirred at room temperature for 30 minutes.1-(tert-butyl) 5-methyl D-glutamate (1 equiv.) and DIPEA (3 equiv.) were added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford 1-(tert-butyl) 5-methyl ((S)-2-(((benzyloxy)carbonyl)amino)- 3,3-dimethylbutanoyl)-D-glutamate. Step 3 To a solution of 1-(tert-butyl) 5-methyl ((S)-2-(((benzyloxy)carbonyl)amino)-3,3- dimethylbutanoyl)-D-glutamate.(1 equiv.) in EtOH was added Pd / C (0.4 equiv.). The reaction mixture was stirred at room temperature overnight under a hydrogen atmosphere. The solvent was removed under vacuum to afford crude 1-(tert-butyl) 5- methyl ((S)-2-amino-3,3-dimethylbutanoyl)-D-glutamate which was used without further purification. Step 4 To a solution of 2-(4-chlorophenyl)-2-methylpropanoic acid (1 equiv.) in DMF was added HATU (1 equiv.) and the reaction mixture stirred at room temperature for 30 minutes.1-(tert-butyl) 5-methyl ((S)-2-amino-3,3-dimethylbutanoyl)-D-glutamate (1 equiv.) and DIPEA (3 equiv.) were added and the reaction was stirred for two hours at room temperature. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine and concentrated. The crude residue was purified by prep-HPLC to afford 1-(tert-butyl) 5-methyl ((S)-2-(2-(4- chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanoyl)-D-glutamate. Step 5 To a solution of 1-(tert-butyl) 5-methyl ((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanoyl)-D-glutamate (1 equiv.) in CH3CN and H2O was added LiOH (2 equiv.) at 0 ^. The reaction mixture was stirred at room temperature for three hours. The reaction solution was purified directly by reverse phase chromatography to afford (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid.

[0005] 14 P90084.WO01 FINAL Application 25.04.20251H NMR (400 MHz, DMSO-d6^ ^ 12.19 (1H, s), 8.39 (1H, d), 7.45 ^ 7.30 (4H, m), 6.38(1H, d), 4.34 (1H, d), 4.12 ^ 3.95 (1H, m), 2.27 (2H, m), 1.99 ^ 1.86 (1H, m), 1.83 ^ 1.68 (1H, m), 1.52 (3H, s), 1.46 (3H, s), 1.40 (9H, s), 0.83 (9H, s). LCMS m / z = 497.4 [M+H]+General Procedure 4 Compounds of formula (5) depicted below were prepared using the following synthetic procedure. Comparative Example 4: Ethyl (R)-4-acetamido-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3-methylbutanamido)butanoate Step 1 To a solution of (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-ethoxy-5-oxopentanoic acid (410 mg, 0.903 mmol) in tert- butanol was added NEt3 (3 equiv.), DPPA (2 equiv.) and (Boc)2O (5 equiv.). The reaction was stirred at 85 °C under a nitrogen atmosphere for 48 hours. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers was dried over Na2SO4, filtered and concentrated. The residue was purified by reverse phase chromatography to afford ethyl (R)-4-((tert-butoxycarbonyl)amino)-2-((S)-2-(2-(4- chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)butanoate. Step 2 P90084.WO01 FINAL Application 25.04.2025 To a solution of ethyl (R)-4-((tert-butoxycarbonyl)amino)-2-((S)-2-(2-(4-chlorophenyl)- 2-methylpropanamido)-3-methylbutanamido)butanoate (1 equiv.) in CH2Cl2 was added a solution of HCl in dioxane (4M, 10 equiv.). The reaction mixture was stirred at room temperature for three hours. The solvent was removed under vacuum to afford ethyl (R)-4-amino-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate hydrochloride salt which was used without purification. Step 3 To a suspension of (R)-4-amino-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate hydrochloride salt (1 equiv.) in CH2Cl2was added NEt3(3 equiv.) and acetyl chloride (2 equiv.) The reaction solution was stirred at room temperature for four hours. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered and concentrated under vacuum. The residue was purified by reverse phase chromatography to afford ethyl (R)-4-acetamido-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)butanoate as a white solid. 1H NMR (400 MHz, DMSO-d6^ ^ ^^^^ ^1H, d), 7.84 (1H, s), 7.34 (4H q),, 6.83 (1H, d),4.18 (2H, dt), 4.11 ^ 4.03 (2H, m), 3.06 (1H, t), 3.01 ^ 2.94 (1H, m), 1.93 ^ 1.80 (2H, m), 1.78 (3H, s), 1.74 ^ 1.65 (1H, m), 1.46 (6H, d), 1.16 (3H, t), 0.79 (3H, d), 0.71 (3H, d). LCMS m / z = 468.2 [M+H]+General Procedure 5 Compounds of formula (6) depicted below were prepared using the following synthetic procedure. P90084.WO01 FINAL Application 25.04.2025 Reference Example 5: (R)-5-ethoxy-4-((S)-2-(3-(4-fluorophenyl)ureido)-3,3- dimethylbutanamido)-5-oxopentanoic acid Step 1 To a solution of 5-(tert-butyl) 1-ethyl ((S)-2-amino-3,3-dimethylbutanoyl)-D-glutamate (1 equiv., prepared as for Example 1) in CH2Cl2 at 0 °C was added DIPEA (4 equiv.) and (4-fluorophenyl)carbamic chloride (1.5 equiv.). The mixture was stirred at room temperature for two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford 5-(tert-butyl) 1-ethyl ((S)-2-(3-(4- fluorophenyl)ureido)-3,3-dimethylbutanoyl)-D-glutamate. Step 2 To a solution of 5-(tert-butyl) 1-ethyl ((S)-2-(3-(4-fluorophenyl)ureido)-3,3- dimethylbutanoyl)-D-glutamate (1 equiv.) in CH2Cl2was added TFA (2 mL). The reaction mixture was stirred at room temperature for three hours, then the solvent was removed under vacuum to afford (R)-5-ethoxy-4-((S)-2-(3-(4-fluorophenyl)ureido)-3,3- dimethylbutanamido)-5-oxopentanoic acid. 1H NMR (400 MHz, DMSO-d6) ^ 8.75 (1H, s), 8.62 (1H, d), 7.44 ^ 7.36 (2H, m), 7.140 7.05 (2H, m), 6.40 (1H, d), 4.29 ^ 4.20 (2H, m), 4.14 ^ 4.03 (2H, m), 2.35 (2H, t), 2.03 ^ 1.93 (1H, m), 1.90 ^ 1.77 (1H, m), 1.18 (3H, t), 0.95 (9H, s). LCMS m / z = 426.2 [M+H]+General Procedure 6 Compounds of formula (7) depicted below were prepared using the following synthetic procedure. Reference Example 6: Ethyl N2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)- 3,3-dimethylbutanoyl)-N5-(methylsulfonyl)-D-glutaminate P90084.WO01 FINAL Application 25.04.2025 To a solution (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-ethoxy-5-oxopentanoic acid (1 equiv., prepared as for Example 1) in CH2Cl2 was added HATU (1 equiv.) and DIPEA (4 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then methanesulfonamide (2 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford ethyl N2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanoyl)-N5- (methylsulfonyl)-D-glutaminate. 1H NMR (400 MHz, DMSO-d6) ^ 8.47 (1H, d), 7.38 (2H, d), 7.33 (2H, d), 6.31 (1H, d), 4.31 (1H, d), 4.17 ^ 4.09 (1H, m), 4.07 (3H, q), 3.16 (3H, s), 2.35 ^ 2.26 (3H, m), 2.01 ^ 1.88 (1H, m), 1.84 ^ 1.71 (1H, m), 1.49 (3H, s), 1.45 (3H, s), 1.17 (3H, t), 0.80 (9H, s). LCMS m / z = 546.3 [M+H]+General Procedure 7 Compounds of formula (8) depicted below were prepared using the following synthetic procedure. Reference Example 7: Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-(methylsulfonamido)pentanoate Step 1 P90084.WO01 FINAL Application 25.04.2025 To a solution (S)-2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid (1 equiv.) in CH2Cl2 was added HATU (1 equiv.) and DIPEA (4 equiv.). The reaction mixture was stirred at room temperature for 30 minutes, then ethyl (R)-2-amino-5- (((benzyloxy)carbonyl)amino)pentanoate 2 equiv.) was added and the reaction was stirred for another two hours. The solvent was removed under vacuum and the residue was purified by reverse phase chromatography to afford ethyl (R)-5- (((benzyloxy)carbonyl)amino)-2-((S)-2-((tert-butoxycarbonyl)amino)-3,3- dimethylbutanamido)pentanoate. Step 2 To a solution of ethyl (R)-5-(((benzyloxy)carbonyl)amino)-2-((S)-2-((tert- butoxycarbonyl)amino)-3,3-dimethylbutanamido)pentanoate (1 equiv.) in CH2Cl2 was added a solution of HCl in dioxane (4 M, 16 equiv.). The reaction mixture was stirred at room temperature for three hours. The solvent was removed under vacuum to afford crude ethyl (R)-2-((S)-2-amino-3,3-dimethylbutanamido)-5- (((benzyloxy)carbonyl)amino)pentanoate hydrochloride salt which was used directly without purification. Step 3 To a solution of 2-(4-chlorophenyl)-2-methylpropanoic acid (1 equiv.) in DMF was added HATU (1 equiv.) and the reaction mixture stirred at room temperature for 30 minutes. ethyl (R)-2-((S)-2-amino-3,3-dimethylbutanamido)-5- (((benzyloxy)carbonyl)amino)pentanoate hydrochloride salt (1 equiv.) and DIPEA (3 equiv.) were added and the reaction was stirred for two hours at room temperature. The mixture was diluted with water and extracted with ethyl acetate. The combined organic layers were washed with brine and concentrated. The crude residue was purified by prep-HPLC to afford ethyl (R)-5-(((benzyloxy)carbonyl)amino)-2-((S)-2-(2-(4- chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanamido)pentanoate. Step 4 To a solution of (R)-5-(((benzyloxy)carbonyl)amino)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)pentanoate (1 equiv.) in EtOH was added Pd / C (0.4 equiv.). The reaction mixture was stirred at room temperature P90084.WO01 FINAL Application 25.04.2025 overnight under a hydrogen atmosphere. The solvent was removed under vacuum to afford ethyl (R)-5-amino-2-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)pentanoate which was used without further purification. Step 5 To a solution of ethyl (R)-5-amino-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)pentanoate (1 equiv.) in CH2Cl2was added methanesulfonyl chloride (1.5 equiv.) and DIPEA (3 equiv.). The reaction mixture was stirred at room temperature overnight. EtOAc and water were added and the organic phase was washed with water, dried over Na2SO4and the solvent was removed under vacuum to afford ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-(methylsulfonamido)pentanoate.1H NMR (400 MHz, DMSO-d6) ^ 8.45 (1H, d) 7.45 ^ 7.31 (4H, m), 6.99 (1H, t), 4.36 (1H, d), 4.18 ^ 4.04 (3H, m), 2.97 ^ 2.91 (2H, m), 2.89 (3H, s), 1.80 ^ 1.70 (1H, m), 1.68 ^ 1.57 (1H, m), 1.47 -1.40 (9H, m), 1.18 (3H, t), 0.82 (9H, s). LCMS m / z = 532.4 [M+H]+Reference Example 8: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-ethoxy-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with (S)- 2-((benzyloxy)carbonyl)-3,3-dimethylbutanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 8.46 (1H, d), 7.43 ^ 7.32 (4H, m), 6.36 (1H, d), 4.34(1H, d), 4.22 ^ 4.14 (1H, m), 4.09 (2H, q), 2.29 (2H, t), 2.01 ^ 1.91 (1H, m), 1.83 ^ 1.72 (1H, m), 1.51 (3H, s), 1.46 (3H, s), 1.18 (3H, t), 0.83 (9H, s). LCMS m / z = 469.2 [M+H]+Reference Example 9: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-methoxy-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with iodomethane and (S)-2-((benzyloxy)carbonyl)-3,3-dimethylbutanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 8.48 (1H, d), 7.44 ^ 7.31 (4H, m), 6.35 (1H, d), 4.34(1H, d), 4.26 ^ 4.18 (1H, m), 2.29 (2H, t), 2.02 ^ 1.91 (1H, m), 1.84 ^ 1.71 (1H, m), 1.51 (3H, s), 1.46 (3H, s), 0.82 (9H, s). P90084.WO01 FINAL Application 25.04.2025 LCMS m / z = 455.2 [M+H]+Reference Example 10: (R)-5-(benzyloxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with benzyl bromide and (S)-2-((benzyloxy)carbonyl)-3,3-dimethylbutanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 8.50 (1H, d), 7.42 ^ 7.30 (9H, m), 5.18 ^ 5.08 (2H, m),4.38 ^ 4.25 (2H, m), 2.29 (2H, t), 2.06 ^ 1.95 (1H, m), 1.87 ^ 1.74 (1H, m), 1.50 (3H, s), 1.44 (3H, s), 0.80 (9H, s). LCMS m / z = 531.2 [M+H]+Reference Example 11: (R)-5-ethoxy-4-((S)-2-(2-(4-fluorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with (S)- 2-((benzyloxy)carbonyl)-3,3-dimethylbutanoic acid and 2-(4-fluorophenyl)-2- methylpropanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 12.11 (1H, s), 8.44 (1H, d), 7.42 ^ 7.29 (2H, m), 7.21^ 7.07 (2H, m), 6.27 (1H, d), 4.32 (1H, d), 4.22 ^ 4.02 (3H, m), 2.27 (2H, t), 2.02 ^ 1.89 (1H, m), 1.84 ^ 1.69 (1H, m), 1.50 (3H, s), 1.46 (3H, s), 1.17 (3H, t), 0.80 (9H, s). LCMS m / z = 453.2 [M+H]+Reference Example 12: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-oxo-5-propoxypentanoic acid The compound was prepared using the procedure as for Reference Example 1 with 1- iodopropane.1H NMR (400 MHz, DMSO-d6^ ^ 12.20 (1H, s), 8.30 (1H, d), 7.43 ^ 7.29 (4H, m), 6.86(1H, d), 4.29 ^ 4.13 (2H, m), 4.09 ^ 3.93 (2H, m), 2.28 (2H, t), 2.04 ^ 1.86 (2H, m), 1.84 ^ 1.72 (1H, m), 1.65 ^ 1.55 (2H, m), 1.49 (3H, s), 1.46 (3H, s), 0.89 (3H, t), 0.81 (3H, d), 0.73 (3H, d). LCMS m / z = 469.3 [M+H]+Reference Example 13: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-isopropoxy-5-oxopentanoic acid

[0006] 21 P90084.WO01 FINAL Application 25.04.2025 The compound was prepared using the procedure as for Reference Example 1 with 2- iodopropane.1H NMR (400 MHz, DMSO-d6^ ^ 12.17 (1H, s), 8.26 (1H, d), 7.44 ^ 7.32 (4H, m), 6.89(1H, d), 4.97 (1H, m), 4.23 ^ 4.12 (2H, m), 2.29 (2H, t), 2.02 ^ 1.87 (2H, m), 1.83 ^ 1.71 (1H, m), 1.50 (3H, s), 1.48 (3H, s), 1.20 (6H, t), 0.83 (3H, d), 0.74 (3H, d). LCMS m / z = 491.2 [M+Na]+Reference Example 14: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3- methylbutanamido)-5-methoxy-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with iodomethane.1H NMR (400 MHz, DMSO-d6^ ^ 12.19 (1H, s), 8.30 (1H, d), 7.44 ^ 7.29 (4H, m), 6.85(1H, d), 4.30 ^ 4.12 (2H, m), 3.64 (3H, s), 2.27 (2H, t), 2.04 ^ 1.87 (2H, m), 1.83 ^ 1.71 (1H, m), 1.49 (3H, s), 1.46 (3H, s), 0.80 (3H, d), 0.73 (3H, d). LCMS m / z = 441.4 [M+H]+Reference Example 15: Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L- phenylalanyl-D-glutaminate The compound was prepared using the procedure as for Reference Example 2 with (R)- 4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-phenylpropanamido)-5-ethoxy- 5-oxopentanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 8.29 (1H, d), 7.33 ^ 7.08 (11H, m), 6.81 (1H, s), 4.67^ 4.59 (1H, m), 4.27 (1H, m), 4.17 ^ 4.07 (2H, m), 2.99 (1H, dd), 2.86 (1H, dd), 2.12 (2H, t), 2.02 ^ 1.91 (1H, m), 1.85 ^ 1.73 (1H, m), 1.35 (3H, s), 1.33 (3H, s), 1.21 (3H, t). LCMS m / z = 502.2 [M+H]+Reference Example 16: (R)-4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)- 3,3-dimethylbutanamido)-5-isopropoxy-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with (S)- 2-((benzyloxy)carbonyl)-3,3-dimethylbutanoic acid and 2-iodopropane.1H NMR (400 MHz, DMSO-d6^ ^ 12.20 (1H, s), 8.39 (1H, d), 7.46 ^ 7.34 (4H, m), 6.26(1H, d), 4.93 ^ 4.83 (1H, m), 4.31 (1H, d), 4.23 ^ 4.12 (1H, m), 2.36 ^ 2.20 (2H, m), P90084.WO01 FINAL Application 25.04.2025 202 ^ 1.90 (1H, m), 1.86 ^ 1.73 (1H, m), 1.52 (3H, s), 1.47 (3H, s), 1.18 (6H, d), 0.84 (9H, s). LCMS m / z = 483.4 [M+H]+Reference Example 17: (R)-5-(tert-butoxy)-4-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 3 with ((benzyloxy)carbonyl)-L-valine.1H NMR (400 MHz, DMSO-d6^ ^ 12.19 (1H, s), 8.19 (1H, d), 7.44 ^ 7.31 (4H, m), 6.89(1H, d), 4.21 ^ 4.07 (2H, m), 2.27 (2H, t), 2.01 ^ 1.86 (2H, m), 1.82 ^ 1.69 (1H, m), 1.51 (3H, s), 1.48 (3H, s), 1.42 (9H, s), 0.83 (3H, d), 0.74 (3H, d). LCMS m / z = 483.2 [M+H]+Reference Example 18: Ethyl ((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)- 3,3-dimethylbutanoyl)-D-glutaminate The compound was prepared using the procedure as for Reference Example 2 with (R)- 4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3,3-dimethylbutanamido)-5- ethoxy-5-oxopentanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 8.46 (1H, d), 7.43 ^ 7.32 (4H, m), 7.27 (1H, s), 6.79(1H, s), 6.34 (1H, d), 4.33 (1H, d), 4.18 ^ 4.03 (3H, m), 2.13 (2H, t), 1.99 ^ 1.87 (1H, m), 1.83 ^ 1.72 (1H, m), 1.50 (3H, s), 1.47 (3H, s), 1.18 (3H, t), 0.82 (9H, s). LCMS m / z = 468.2 [M+H]+Reference Example 19: Ethyl N2-((2-(4-chlorophenyl)-2-methylpropanoyl)-L- valyl)-N5-(2-hydroxyethyl)-D-glutaminate The compound was prepared using the procedure as for Reference Example 2 with 2- aminoethan-1-ol1H NMR (400 MHz, DMSO-d6) ^ 8.28 (1H, d), 7.81 (1H, t), 7.43 ^ 7.31 (4H, m), 6.84 (1H, d), 4.66 (1H, t), 4.22 ^ 4.04 (4H, m), 3.16 ^ 3.06 (2H, m), 2.13 (2H, t), 2.00 ^ 1.87 (2H, m), 1.84 ^ 1.71 (1H, m), 1.49 (2H, s), 1.47 (3H, s), 1.19 (3H, t), 0.81 (3H, d), 0.72 (3H, d). LCMS m / z = 498.2 [M+H]+P90084.WO01 FINAL Application 25.04.2025 Reference Example 20: (R)-5-ethoxy-4-((S)-2-(2-(4-hydroxyphenyl)-2- methylpropanamido)-3-methylbutanamido)-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with 2-(4- hydroxyphenyl)-2-methylpropanoic acid.1H NMR (400 MHz, DMSO-d6) ^ 12.16 (1H, s), 9.30 (1H, s), 8.29 (1H, d), 7.13 (2H, d), 6.72 (2H, d), 6.48 (1H, d), 4.26 ^ 4.04 (4H, m), 2.27 (2H, t), 2.21 ^ 1.84 (2H, m), 1.81 ^ 1.71 (1H, m), 1.43 (6H, s), 1.18 (3H, t), 0.79 (3H, d), 0.69 (3H, d). LCMS m / z = 437.4 [M+H]+Reference Example 21: (R)-5-ethoxy-4-((S)-2-(2-(4-hydroxyphenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-5-oxopentanoic acid The compound was prepared using the procedure as for Reference Example 1 with (S)- 2-((tert-butoxycarbonyl)amino)-3,3-dimethylbutanoic acid and 2-(4-hydroxyphenyl)-2- methylpropanoic acid.1H NMR (400 MHz, DMSO-d6) ^ 9.34 (1H, s), 8.48 (1H, d), 7.15 (2H, d), 6.74 (2H, d), 6.04 (1H, d), 4.29 (1H, d), 4.23 ^ 4.03 (3H, m), 2.29 (2H, t), 2.01 ^ 1.90 (1H, m), 1.84 ^ 1.71 (1H, m), 1.45 (3H, s), 1.42 (3H, s), 1.19 (3H, t), 0.79 (9H, s). LCMS m / z = 451.4 [M+H]+Reference Example 22: Ethyl (2-(4-chlorophenyl)-2-methylpropanoyl)-L-valyl-D- glutaminate The compound was prepared using the procedure as for Reference Example 2 with (R)- 4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)-5-ethoxy-5- oxopentanoic acid.1H NMR (400 MHz, DMSO-d6^ ^ 8.26 (1H, d), 7.42 ^ 7.29 (4H, m), 7.25 (1H, s), 6.82(1H, d), 6.77 (1H, s), 4.15 (2H, t), 4.11 ^ 4.03 (2H, m), 2.09 (2H, t), 1.97 ^ 1.84 (2H, m), 1.80 ^ 1.68 (1H, m), 1.46 (6H, d), 1.17 (3H, t), 0.79 (3H, d), 0.70 (3H, d). LCMS m / z = 454.3 [M+H]+Reference Example 23: Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3-methylbutanamido)-5-hydroxypentanoate The compound was prepared using the procedure as for Reference Example 1 with ethyl (R)-2-((S)-2-amino-3-methylbutanamido)-5-hydroxypentanoate. P90084.WO01 FINAL Application 25.04.20251H NMR (400 MHz, DMSO-d6) ^ 8.19 (1H, d), 7.34 (4H, q), 6.83 (1H, d), 4.46 (1H, t), 4.20 ^ 4.03 (4H, m), 3.37 (2H, d), 1.95 ^ 1.85 (1H ,m), 1.79 ^ 1.68 (1H ,m), 1.61 ^ 1.51 (1H, m), 1.45 (6H, d), 1.43 ^ 1.30 (2H, m), 1.16 (3H, t), 0.78 (3H, d), 0.70 (3H, d). LCMS m / z = 441.3 [M+H]+Reference Example 24: Ethyl N2-((2-(4-chlorophenyl)-2-methylpropanoyl)-L- valyl)-N5,N5-dimethyl-D-glutaminate The compound was prepared using the procedure as for Reference Example 2 with (R)- 4-((S)-2-(2-(4-chlorophenyl)-2-methylpropanamido)-3-methylbutanamido)-5-ethoxy-5- oxopentanoic acid and dimethylamine.1H NMR (400 MHz, DMSO-d6^ ^ 8.24 (1H, d), 7.34 (4H, q), 6.86 (1H, d), 4.26 ^ 4.17(1H, m), 4.17 ^ 4.03 (3H, m), 2.88 (3H, s), 2.80 (3H, s), 2.32 ^ 2.23 (2H, m), 2.00 ^ 1.89 (2H ,m), 1.81 ^ 1.70 (1H, m), 1.46 (6H, d), 1.17 (3H, t), 0.79 (3H, d), 0.71 (3H, d) LCMS m / z = 482.2 [M+H]+Reference Example 25: Ethyl (R)-2-((S)-2-(2-(4-chlorophenyl)-2- methylpropanamido)-3,3-dimethylbutanamido)-4-cyanobutanoate The compound was prepared using the procedure as for Reference Example 1 with ethyl (R)-2-((S)-2-amino-3-methylbutanamido)-4-cyanobutanoate.1H NMR (400 MHz, DMSO-d6^ ^ 8.53 (1H, d), 7.41 ^ 7.30 (4H, m), 6.36 (1H, d), 4.29(1H, d), 4.22 ^ 4.14 (1H, m), 4.09 (2H, q), 2.49 ^ 2.45 (2H, m), 2.08 ^ 1.98 (1H, m), 1.93 ^ 1.81 (1H, m), 1.50 (3H, s), 1.44 (3H, s), 1.17 (3H, t), 0.82 (9H, s). LCMS m / z = 450.3 [M+H]+Example 26: (R)-4-((S)-2-(2-(4-fluorophenyl)-2-methylpropanamido)-3,3- dimethylbutanamido)-5-isopropoxy-5-oxopentanoic acid The compound ^^^^^^^^^ was prepared using the procedure as for Reference Example 1 with 2-iodopropane, (S)-2-((benzyloxy)carbonyl)-3,3-dimethylbutanoic acid and 2-(4- fluorophenyl)-2-methylpropanoic acid.1H NMR (400 MHz, DMSO-d6) ^ 8.41 (1H, d), 7.40 ^ 7.31 (2H, m), 7.19 ^ 7.10 (2H, m), 6.27 (1H, d), 4.93 ^ 4.84 (1H, m), 4.30 (1H, d), 4.15 ^ 4.06 (1H, m), 2.26 (2H, t), P90084.WO01 FINAL Application 25.04.2025 1.98 ^ 1.87 (1H, m), 1.81 ^ 1.68 (1H, m), 1.49 (3H, s), 1.45 (3H, s), 1.15 (3H, t), 0.79 (9H, s). LCMS m / z = 467.2 [M+H]+Reference Example 27: Compound activity in HEK-Blue™ hNOD2 cells All HEK-BlueTMcell lines were purchased from Invitrogen. A dose-reponse curve for each test compound is generated. A dose-response curve is generated on each plate for MDP (control activating ligand) and may be generated for MDP control (a non-activating ligand). Cells treated with the appropriate vehicle (0.05% DMSO) are also included in each plate. Initial cell culture procedure: Cell vials are thawed in a 37°C water bath and cells are transfered to a sterile 15 ml tube containing 15 ml pre-warmed DMEM 10% FBS medium. The tube is centrifuged at 200 x g for 5 minutes. The supernatant is removed and the cells resuspended with 15 ml DMEM 10% FBS medium without selective antibiotics. The vial contents are transfered to a 75 cm3tissue culture flask and the flask placed in and incubator and cultured in a^^^^^^^^^^ ^^^^^^^^^^ ^^ ^^^^ ^^^ ^^ ^^2.Cell maintenance: Cells are maintained and subcultured in growth medium supplemented with 30 µg / ml of blasticidin and 100 µg / ml of Zeocin^. Cells are passaged twice when an 80% confluency is reached. Medium is aspirated and the cells washed with 10 ml PBS. The cells are detached with 5 ml of pre-warmed PBS by pipetting up and down. The PBS cell suspension is diluted 5-fold and cells counted in a haemocytometer.1.5 x 106cells are seeded per 75 cm3tissue culture flask in 15 ml pre-warmed DMEM 4.5 g / L glucose, and 10% FBS. The required selection antibiotics are added. After 2-3 days cells are ready to perform screening experiments. Preparation of compound pre-dilution and master plates:HEK-Blue^ detection medium solution^ ^^^ ^^^^^^^^ ^^^^^^^^^ ^^^ ^^^^^^^^^^^^^instructions and warmed in a 37^C water bath.30 mM DMSO stocks of test compounds P90084.WO01 FINAL Application 25.04.2025 are diluted 1.5-fold in DMSO and 5 ^l added to compound the pre-dilution plate.95 ^l of HEK-Blue detection medium solution is added to all the wells with compound. Test compound solutions from the pre-dilution plate are diluted 50-fold in the master plate. Preparation of test plates: 25 ^l of each test compound and sequential dilutions of the control ligands (MDP and MDP control) are transfered from the master plate to the 384-well test plate. The test plate is incubated at 37^C for at least 1 hour, whilst preparing the cells for loading. Detection of SEAP activity: The 150 cm3tissue culture flasks with HEK-Blue^ cells are removed from the incubator growth medium is aspirated. Cells in each flask are gently rinsed with pre- warmed 10 ml PBS.5 ml pre-warmed PBS is added to the 150 cm3tissue culture flasks and cells are detached by pipetting up and down. The cell suspension is transfered to 50 cm3tube and incubated at 37^C while determining the total number of cells harvested. A cell suspension of 5 x 105cells per ml is prepared by diluting the PBS cell suspension in HEK-Blue^ Detection medium. The cell suspension is transfered into a reservoir and 25 ^l of the cell suspension is added to the relevant wells of the 384-microwell test plate. The plate is incubated the plates at 37 °C in 5% CO2for 16-18 h. SEAP activity is quantified using a spectrophotometer at 655 nm. Results: The reference compounds gave the following EC50^^: Example EC50(nM) Example EC50(nM) 1 1 14 8 2 50 15 13 3 110 16 75 5 15 17 650 6 55 18 8900 7 >10,000 19 >10,000 8 0.1 20 29 9 0.4 21 2 10 0.2 22 50 11 0.1 23 460 12 2 24 810 P90084.WO01 FINAL Application 25.04.2025 13 6 25 3580 The compound of the comparative example gave the following EC50^^^ Comparative EC50 (nM) Example 4 Inactive Example 27(a): IHXME26 activity in HEK-Blue™ hNOD2 cells The same general procedure as Reference Example 27 was used to screen IHXME26. HEK-BlueTMhNOD2 cells stably express human NOD2 gene and an NF-kB inducible SEAP reporter gene. NOD2 recognises MDP structure found in almost all bacteria and therefore acts a positive control. IHXME26 gave the following EC50: Example EC50 (nM) 26 ~0.5 As illustrated in Figure 1, HEK-BlueTMhNOD2 cells treated with increasing concentrations of IHXME26 stimulate secretion of NF-kB inducible SEAP in a dose^^^^^^^^^ ^^^^^^ ^^^^^^^^^ ^^^^^^^^^ ^^^ ^^^ ^^ ^^^^ ^^^ ^^ ^^2.Reference Example 28: Compound activity in HEK-Blue™ Null2 cells A similar general procedure as Reference Example 27 was used to screen compounds in HEK-Blue^ Null2 cells with the exception that Null2 cells were maintained and subcultured in growth medium supplemented with 100 µg / ml of Zeocin^. The reference compounds were typically inactive in the assay. Example 28(a): IHXME26 activity in HEK-Blue™ Null2 cells The same general procedure as Reference Example 28 was used to screen IHXME26. P90084.WO01 FINAL Application 25.04.2025 HEK-BlueTMNull2 cells express the SEAP reporter gene under the control of the IL12- p40 promoter fused to NFkB and AP1 binding sites. This is the parental cell line for the HEK-BlueTMhNOD2 cells. As illustrated in Figure 2, HEK-BlueTMNull2 cells treated with increasing concentrations of IHXME26 show a minimal response of NF-kB inducible SEAP only at high concentrations in comparison to the response with HEK-BlueTMhNOD2 cells. Reference Example 29: Compound activity in HEK-Blue™ hNOD1 cells A similar general procedure as Reference Example 27 was used to screen compounds in HEK-Blue^ hNOD1 cells with the exceptions that C12 iE-DAP was used as the control activing ligand, no non-activating ligand was used and test plates were incubated at room temperature while preparing the cells for loading. The reference compounds were typically inactive in the assay. Example 29(a): IHXME26 activity in HEK-Blue™ hNOD1 cells The same general procedure as Reference Example 29 was used to screen IHXME26. C12-iE-DAP is a NOD1 agonist and acts as a positive control. As illustrated in Figure 3, HEK-BlueTMhNOD1 cells treated with increasing concentrations of IHXME26 had no effect on the secretion of NF-kB inducible SEAP. Reference Example 30: Compound activity in HEK-Blue™ Null1 cells A similar general procedure as Reference Example 28 was used to screen compounds in HEK-Blue^ Null1 cells with the exception that Null1 cells were maintained and subcultured in growth medium supplemented with 100 µg / ml of Zeocin^. The reference compounds were typically inactive in the assay. Reference Example 31: Compound activity in HEK-Blue™ mNOD2 cells P90084.WO01 FINAL Application 25.04.2025 A similar general procedure as Reference Example 28 was used to screen compounds in HEK-Blue^ mNOD2 cells. Results: The reference compounds gave the following EC50^^^ Example EC50 (nM) Example EC50 (nM) 1 79 12 70 2 3100 13 170 3 2100 14 255 5 241 15 1900 8 17 16 5000 9 22 17 >10,000 10 13 20 20 11 1 21 2 22 3100 Reference Example 32: Compound activity in THP-1 cells THP-1 cells are plated with a cell density of, for eample, 8 x 105cells / ml and incubatedwith test compounds at a DMSO concentration of ^ ^^ at 37^C for 20 hours. Plates arethen centrifuged at 100 x g for 10 minutes and 150 µl of supernatant harvested for assessement of compound activity. Compound activity is assessed using, for example, a DuoSet IL-8 / CXCL8 ELISA (R&D systems, DY208) for the determination of secreted IL-8 or a Luminex 65plex detection kit (ThermoFisher Scientific, ProcartaPlex, EPX650-10065-901) for the detection of a range of secreted cytokines and chemokines. Cell viability can addtionally be determined using, for example, a CellTiterGlo^ viability kit (Promega, G7571) with 50 µl CellTiterGlo^ being added to each well and incubated at room temperature for 10 minutes. Luminscence can be read on a FluoStar plate reader. The NOD2 gene in THP-1 cells used in this assay can be modified by, for example, the use of CRISPR to introduce one (heterogenous) or two (homogenous) copies of a singleNOD2 mutations such as L1007fs, G908R or R702W ^^^^ ^^^ ^^^^^ ^^ ^^^^^ ^^^^^^^Disease. P90084.WO01 FINAL Application 25.04.2025 The NOD2 gene in THP-1 cells used in this assay can be modified by, for example, the use of CRISPR to introduce one copy of two different NOD2 mutations (compoundhetereogenous) such as L1007fs, G908R and R702W ^^^^ ^^^ ^^^^^ ^^ ^^^^^ ^^^^^^^Disease. The NOD2 gene in THP-1 cells used in this assay can be modified by, for example, the use of CRISPR to introduce 1 (hetero) or 2 (homo) copies of NOD2 mutations such as R334Wor N670K that are known to cause Blau Syndrome. The compound of the present invention can be tested for activity in these modified THP-1 cells using the general methods described above. Example 33: Compound activity in human monocytes. Human monocytes were isolated from peripheral whole blood from 2 healthy donors via density gradient centrifugation to isolate peripheral blood mononuclear cells (PBMCs) followed by magnetic bead-based negative selection. Cells were incubated withIHXME26 (0.2 µM or 20 µM) for ^^^ ^^ ^^^^ ^^^ ^^ ^^2, and cytokine secretion wasmeasured using multiplex Luminex® bead-based analysis. Initially, 65 cytokines were measured in the supernatants using a Luminex multiplex assay. Of these, 21 cytokines had interpolated values of zero for all samples. A further 5 cytokines were excluded where most of the samples were interpolated as zero and there were no specific treatment effects. Of the remaining 39 cytokines, distinct patterns of clustering were observed by principal component analysis (PCA) and hence a smaller number of 9 representative cytokines that showed key changes with treatment were selected for further investigation. The change in cytokine secretion from primary human monocytes incubated with IHXME26 for 24 hours is shown in Figures 4A-4F, Figures 5A-5F and Figures 6A-6F. The data is plotted as mean and individual wells (n=2 or 4). Values above the top standard were replaced with the top standard; ^ = one replicate was replaced, ^^ = two replicates were replaced. Values below the bottom standard were replaced with half the bottom standard; vv = all replicates were replaced. P90084.WO01 FINAL Application 25.04.2025 The data in Figures 4A-4F, Figures 5A-5F and Figures 6A-6F shows that human^^^^^^^^^ ^^^^^^^^^ ^^^^ ^^^ ^^ ^^ ^^ ^^ ^^^^^^^ ^^^ ^^^ ^^ ^^^^ ^^^ ^^ ^^2secrete a number of cytokines with a secretion profile similar to MDP. The activity of IHXME26 against the EurofinsTMSafetyScreen panel is found in Example 42 and Figures 9-12. Reference Example 34: Compound exposure after oral dosing in mice. C57Bl / 6J female mice were dosed with 100 mg / kg of the reference compounds by oral gavage in PBS or aqueous sodium hydrogencarbonate.9 mice were dosed per compound. Blood:water, liver and caecum samples were collected 4 hours post-dosing and were stored for LC-MS / MS analysis. Tissues were separated from their contents and frozen separately. Results: Concentrations of the reference compounds in various tissues are shown below: Mean Concentration Blood Liver Caecum Caecal Example Contents ng / ml ng / g ng / g ng / g 1 1480 15000 18000 82800 13 663 10200 8320 88200 8 130 BLQ 780 2550 BLQ = Below Limit of Quantification Reference Example 35: Mouse and human hepatocyte stability of the reference compounds The intrinsic clearances (CLint) and half-lives of the reference compounds were measured in either a hepatocyte suspension of cryopreserved male C57BL6 mouse hepatocytes or a mixed hepatocyte suspension of cryopreserved human hepatocytes. Briefly, the compound was incubated with hepatocyte suspensions at 37°C over a time P90084.WO01 FINAL Application 25.04.2025 course and the remaining compound at each time point was assessed by mass spectrometry (UPLC-MS / MS). Mouse and human hepatocyte stablity for IHXME26 is found in Examples 41 and 44. Reference Example 36: Plasma Protein Binding of reference compounds. The extent to which the reference compounds bound to plasma proteins such as albumin and alpha-1 acid glycoprotein within human, rat or mouse plasma was determined by^^^^^ ^^^^^^^^^^^ ^^^^^^^^^ ^^^^^^^^^ ^^^^ ^^^^^^^^^ ^^ ^^^ ^^^ ^ ^^^^^ ^^ ^^^^.Plasma protein binding for IHXME26 is found in Example 44 and plasma stablity for IHXME26 is found in Examples 41 and 44. Reference Example 37: Activity of reference compounds against the HERG channel The reference compounds were tested for inhibition of cardiac potassium (hERG) channels using the QPatch automated patch clamp system (Sophion, Denmark). The compounds were screened at eight concentrations (using 0.5-log unit dilutions) from a top concentration of 30 µM, against a minimum of three separate cells. Each eight-point concentration-response curve was constructed using cumulative single sample additions of each concentration to the same cell. The effect of IHXME26 on the hERG cardiac ion channel, expressed in mammalian HEK-293 cells, was assessed using the electrophysiology platform QPatch HTX. The IC50 was judged to be >10 µM. hERG QPatch results: IHXME26 Concentration0.1 µM 1 µM 10 µM% mean hERG inhibition4.3 4.0 11P90084.WO01 FINAL Application 25.04.2025 Reference Example 38: Activity of reference compounds against a panel of enzymes, ion channels and receptors The reference compounds were tested against the DiscoverX SAFETYscan E / IC50 ELECT - 78 assay panel. The activity of IHXME26 against the EurofinsTMSafetyScreen panel is found in Example 42 and and Figures 9-12. Reference Example 39: Permeability of the reference compounds in Caco 2 cells Caco 2 cells are used as an in vitro model of the human intestinal epithelium and permit assessment of the intestinal permeability of potential drugs. The reference compounds were added to either the apical or basolateral side of a confluent monolayer of Caco 2 cells and permeability was measured by monitoring the appearance of the test compound on the opposite side of the monolayer using LC MS / MS. The efflux ratio (ER) was calculated from the ratio of B A and A B permeabilities. Example 40: Reversible Inhibition of CYPs by the reference compounds. The inhibition of individual CYPs by compounds of was assessed using human liver microsomes in combination with specific probe substrates. Reversible Cytochrome P450 Inhibition IHXME26 was incubated with human liver microsomes and NADPH in the presence of a cytochrome P450 isoform-specific probe substrate. The metabolites were monitored by LC-MS / MS and a decrease in the formation of the metabolite compared to vehicle control was used to calculate an IC50 value. Reversible IC50values: Isoform IC50(µM) CYP2B6 >10 CYP2C8 >10 CYP3A4 (midazolam) >10 CYP3A4 (testosterone) >10 P90084.WO01 FINAL Application 25.04.2025 CYP2C9 >10 CYP1A2 >10 CYP2C19 >10 CYP2D6 >10 Example 41: Multispecies Hepatocyte and Plasma Stability IHXME26 was tested in human, rat, mini-pig, dog and monkey for plasma stability and hepatocyte stability. Plasma Stablity Procedure For each species, the plasma was filtered and adjusted to pH 7.4 at 37 °C. IHXME26 (1 µM, 0.25% DMSO final concentration) was added to afford a final incubation volume of 500 µL. Hepatocyte Stablity Procedure A suspension of cryopreserved hepatocytes (final cell density 0.5 x108viable cells / mL)^^ ^^^^^^^^^^ ^^^^^^^^ ^^^^^^^ ^^^^^^ ^^^^^^ ^^^^^^^^^^^^ ^^^^ ^-(-)-fructose(0.4 g / L) and HEPES (25 mM) was pre-incubated at 37 °C prior to the addition of IHXME26 (1 µM, final DMSO concentration 0.25%). The final incubation volume was 500 µL. Plasma Stablity Results: As illustrated in Figure 7, IHXME26 is stable in human, rat, mini-pig, dog and monkey over 120 minutes (maximum time tested), within the error of the assay (± 20%). The increase in % remaining is due to variability within the assay. Hepatocyte Stablity Results: IHXME26 gave the following results in the species tested for hepatocyte stability: Compound Intrinsic clearance (µL / min / 106cells) Human Rat*Mini-pig Dog Monkey

[0007] 35 P90084.WO01 FINAL Application 25.04.2025 IHXME26 <3.85 12.4 8.68 4.95 12.8 * 40 and 60 minute timepoints outside of assay error range (20%) The low and high intrinsic clearance range for different species is shown below: Clearance Intrinsic clearance (µL / min / 106cells) Category Human Rat*Mini-pig Dog Monkey Low <3.5 <5.1 Not reported <1.9 <5.2 High >19.0 >27.5 Not reported >10.5 >17.9 Source: https: / / www.evotec.com / en / drug-metabolism / hepatocyte-stability. As illustrated in Figure 8, IHXME26 showed moderate intrinsic clearance in all species except human up to 120 minutes. Plasma protein binding, plasma stability and hepatocyte stability for IHXME26 are also found in Example 44. Example 42: Activity of IHXME26 against a panel of enzymes, ion channels and human receptors IHXME26 was tested against the EurofinsTMSafetyScreen87 panel at 10 µM. The binding of a probe to the Ca2+channel (L verapamil site) was increased by ~35-40% at^^ ^^^ ^^^ ^^^^^^^ ^^ ^ ^^^^^ ^^ ^^^ ^^^-selective rat glycine ion channel wasincreased by ~30-35% at 10 µM. There was no other significant binding for any of the other targets at 10 µM. The full profile is shown in Figures 9, 10, 11 and 12. As illustrated in Figures 9, 10, 11 and 12, IHXME26 exhibited a clean off target profile when tested against the EurofinsTMSafetyScreen panel at 10 µM. The activity of the reference compounds against a panel of enzymes, ion channels and receptors is found in Example 38. Example 43: Rat Pharmacokinetic (PK) study

[0008] 36 P90084.WO01 FINAL Application 25.04.2025 The pharmacokinetics of IHXME26 following single intravenous (i.v.) or oral (p.o.) administration were measured. Methodology IHXME26 in PBS / bicarbonate (pH 7) was administered to 4 male and 4 female Han Wistar rats (purchased from Charles River UK) weighing between 150-200 kg for females and 201-225 g for males, intravenously (2 mg / kg and 10 mg / kg). IHXME26 in PBS / bicarbonate (pH 8-8.5) was administered to 8 male and 4 female Wistar rats (purchased from Charles River UK) weighing between 150-200 kg for females and 201-225 g for males, orally (250 mg / kg and 50 mg / kg) by gavage.6 of the male Wistar rats were administered 50 mg / kg orally by gavage. One additional male Wistar Han rat was purchased for the provision of naïve blood and tissue to aid bioanalysis. All dosing was performed using a dose volume of 5 ml / kg. Dosing was staggered (30 minutes) to allow for observation of any acute dose-limiting adverse effects. In Life Blood Sampling^^ ^^ ^^^^^ ^^^^^^^ ^^^^ ^^^^^^^^^ ^^^ ^^^^^ ^^^^ ^^^^^^ ^^ ^^ ^^ ^^^^ ^^^^^-purewater (2 mg / kg i.v. and 250 mg / kg p.o.) or ultra pure water (10 mg / kg i.v. and 50 mg / kgp.o.) ^^ ^^^^ ^ ^^^^^ ^^^^^^ ^^^^^^ ^^ ^^ ^^^Samples for the 10 mg / kg i.v. dosing group were collected at the following time points post dose: 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 24 hr and were stored for LC- MS / MS analysis. Samples for the 2 mg / kg i.v. dosing group were collected at the following time points post dose: 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr and 6 hr and were stored for LC-MS / MS analysis. Samples for the 250 mg / kg and 50 mg / kg p.o. dosing group were collected at the following time points post dose: 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 24 hr and were stored for LC-MS / MS analysis. All samples were stored at -80 °C until required for LC-MS / MS analysis. P90084.WO01 FINAL Application 25.04.2025 For 50 mg / kg p.o. dosed group, 2 male rats were terminated at both 1 and 4 hours post- dosing for collection of terminal tissue samples. The same samples were collected from the remaining 2 male rats 24 hours post-dosing. Terminal Sampling Prior to terminals, urine samples were collected prior to the final bleed and following the final bleed where possible. Where this was not possible, urine was collected directly from the bladder after termination. Following anaesthesia and exsanguination, tissue from the ileum, caecum, proximal colon, distal colon, left lateral liver lobe and front left of the cerebrum as well as caecum and distal colon contents were collected and snap frozen on dry ice. All samples were stored at -80 °C until required for bioanalysis. No abnormal tissue was noted on necropsy and therefore no further tissues were collected. Results The Rat PK profile of IHXME26 following a 2 mg / kg intravenous dose is shown in the Table below: Half-life in blood (t½, min) 15.6 Observed clearance (ml / min / kg) 48.2 C0 (ng / ml) 3,849 AUCall (ng.h / ml) 41,369 AUCINF(ng.h / ml) 41,519 Volume of distribution at steady state (Vss, L / kg) 0.7 The whole blood concentrations of IHXME26 following a 2 mg / kg intravenous dose in rats are shown in Figure 13. Data points represent mean ± SD. BLQ: 10.0 ng / mL. BLQ data points omitted from the above plot. The Rat PK profile of IHXME26 following a 10 mg / kg intravenous dose is shown in the Table below: Half-life in blood (t½, min) 98 Observed clearance (ml / min / kg) 37 C0 (ng / ml) 13,841 P90084.WO01 FINAL Application 25.04.2025 AUCall (ng.h / ml) 264,325 AUCINF (ng.h / ml) 270,798 Volume of distribution at steady state (Vss, L / kg) 2 The whole blood concentrations of IHXME26 following a 10 mg / kg intravenous dose in rats are shown in Figure 14. Data points represent mean ± SD. BLQ: 10.0 ng / mL. BLQ data points omitted from the above plot. The Rat PK profile of IHXME26 following a 50 mg / kg oral gavage dose is shown in the Table below: Half-life in blood (t½, min) 304 Time of maximum concentration (tmax, min) 60 Observed clearance (ml / min / kg) 60 Oral Cmax(ng / ml) 6,558 AUCall (ng.h / ml) 818,055 AUCINF (ng.h / ml) 830,822 Oral Bioavailability (F, AUCINF) 61% The whole blood concentrations of IHXME26 following a 50 mg / kg oral gavage dose in rats are shown in Figure 15. Data points represent mean ± SD. BLQ: 10.0 ng / mL. BLQ data points omitted from the above plot. The Rat PK profile of IHXME26 following a 250 mg / kg oral gavage dose is shown in the Table below: Half-life in blood (t½, min) 209 Time of maximum concentration (tmax, min) 30 Observed clearance (ml / min / kg) 22.4 Oral Cmax (ng / ml) 45,500 AUCall (ng.h / ml) 11,058,110 AUCINF (ng.h / ml) 11,146,913 Oral Bioavailability (F, AUCINF) 215% P90084.WO01 FINAL Application 25.04.2025 The whole blood concentrations of IHXME26 following a 250 mg / kg oral gavage dose in rats are shown in Figure 16. Data points represent mean ± SD. Extrapolated ALQ values have been included in the above plot. The average exposure levels of IHXME26 in tissues, urine and intestinal contents 6 hours following 2 mg / kg intravenous dose are shown in the Table below: ng / g (*ng / ml) nM Ileum 214 459 Caecum 849 1,820 Proximal colon 1,372 2,941 Distal colon 164 352 Brain BLQ BLQ Liver 132 283 Caecum contents 12,914 27,680 Fecal content 10,116 21,683 Urine 349* 748 BLQ = below limit of quantification. BLQ values have been omitted from the calculated mean concentration for Liver (n=1). Figures 17A-17C show the tissue concentrations of IHXME26, 6 hours after a 2 mg / kg iv dose in rats. The average exposure levels of IHXME26 in tissues, and intestinal contents 1,4 and 24 hours following 50 mg / kg oral gavage dose are shown in the Table below: Tissue 1h post dose 4h post dose 24h post dose ng / g nM ng / g nM ng / g nM Ileum 26,587^56,987^ALQ ALQ 1,012 2,169 Caecum 26,728^57,289^45,555^97,643^832 1,782 Proximal colon 6,983 14,967 4,711 10,097 1,630v3,495vDistal colon 2,598 5,568 6,458 13,841 603v1,293vBrain 213 457 170 364 BLQ BLQ Liver 38,895^83,366^33,393^71,574^321 689 Fecal content 6,362 13,636 ALQ ALQ 8,981v19,250vP90084.WO01 FINAL Application 25.04.2025 BLQ = below limit of quantification; ALQ = above limit of quantification.vBLQ values have been omitted from the calculated mean concentration.^ALQ values have been replaced with the maximum in range concentration of 50,000 ng / g prior to calculating the mean concentration. Figures 18A-18B shows the tissue concentrations of IHXME26, 1, 4 and 24 hours after a 50 mg / kg oral gavage dose in rats.Samples with IHXME26 ^^^^^^ ^^^^^ ^^^ ^^^^^ ^^ ^^^^^^^^^^^^^^ ^^^ ^^^^^^^ ^^^^ ^ ^^^symbol. Samples with IHXME26 levels above the limit of quantification are denoted^^^^ ^ ^^^ ^^^^^^ ^^^ ^^^ ^^^^ ^^^^^ ^^^^^^ ^^ ^^^ ^^^^^ ^^^^^ ^^ ^^^^^^^^^^^^^^ ^^^^^^^ng / g). The average exposure levels of IHXME26 in tissues, urine and intestinal contents 24 hours following 250 mg / kg oral gavage dose are shown in the Table below: ng / g (*ng / ml) nM Ileum 14,875 31,883 Caecum 30,608 65,605 Proximal colon 21,670^46,447 Distal colon 6,433 13,788 Brain 749v1,605vLiver 19,883^42,617^Caecum contents ALQ ALQ Fecal content ALQ ALQ Urine 6,233* 13,360 BLQ = below limit of quantification; ALQ = above limit of quantification.vBLQ values have been omitted from the calculated mean concentration.^ALQ values have been replaced with the maximum in range concentration of 50,000 ng / g prior to calculating the mean concentration. Figures 19A-19C show the tissue concentrations of IHXME2624 hours after a 250 mg / kg oral gavage dose in rats. P90084.WO01 FINAL Application 25.04.2025Samples with IHXME26 ^^^^^^ ^^^^^ ^^^ ^^^^^ ^^ ^^^^^^^^^^^^^^ ^^^ ^^^^^^^ ^^^^ ^ ^^^symbol and the data point placed on the upper limit of quantification (50,000 ng / g). Figure 20 shows a summary plot of whole blood concentrations of IHXME26 measured in PK studies. Assessment of tissues from rat PK studies shows moderate levels of IHXME26 are detected in liver tissue up to 6 hours, which is cleared by 24 hours when rats dosed via iv or oral route. There are no detectable levels of IHXME26 in the brain. High concentrations of IHXME26 in gut tissue (ileum, caecum, proximal and distal colon) were observed. The activity of the reference compounds against a panel of enzymes, ion channels and receptors is found in Example 38. Example 44: ADME Studies Plasma Protein Binding The extent to which IHXME26 bound to plasma proteins was determined by rapid equilibrium dialysis. Plasma protein binding (% bound) values were as follows: 60 (rat), 94 (dog), 81 (monkey) and 98 (human). Plasma Stability The stability of IHXME26 was measured in plasma from mixed gender Sprague- Dawley rats, beagle dogs, cynomolgous monkeys or humans. Data was generated from two different suppliers, Pharmaron and Cyprotex. Figure 7 shows the timecourse of % IHXME26 remaining vs time (data generated by Cyprotex). Figure 21 shows the timecourse of % IHXME26 remaining vs time (data generated by Pharmaron). The plasma stablity procedure and results are also found in Example 41. P90084.WO01 FINAL Application 25.04.2025 Hepatocyte Stability The stability of IHXME26 was measured in a suspension of mixed gender cryopreserved CD-1 mouse hepatocytes, Sprague-Dawley rat hepatocytes, beagle dog hepatocytes, cynomolgus monkey hepatocytes, mini-pig or human hepatocytes. Data was generated from two different suppliers, Cyprotex and Pharmaron. The intrinsic clearance values for IHXME26 in hepatocytes are shown in the Table below: Intrinsic clearance (µL / min / 106cells) Supplier Human Rat Mouse Mini-pig Dog Monkey Cyprotex <3.85 12.4 Not tested 8.68 4.95 12.8 Pharmaron <3.85 38.7 140 Not tested 4.62 4.63 Figure 8 shows the timecourse of % IHXME26 remaining vs time (data generated by Cyprotex). Figure 22 shows the timecourse of % IHXME26 remaining vs time (data generated by Pharmaron). The hepatocyte stablity procedure and results are also found in Example 41. Example 45: Toxicology 7-day non-GLP dose range finding study in rats IHXME26 was administered to male and female Han Wistar rats at 50, 250 or 1,000 mg / kg via oral gavage once daily for 7 days. Rats were weighed and scored for clinical observations daily throughout the study, and blood samples were collected from each animal on day 0 and day 6 for toxicokinetic analysis. At the end of the seven-day dosing period a blood sample was collected under terminal anaesthesia for haematology, clinical chemistry, and serum cytokine analysis and the animals were killed prior to tissue collection. The liver, kidney, gastrocnemius muscle, heart, brain, lung, GI tract, stomach and oesophagus, pancreas and mesenteric lymph nodes were collected from all animals for histopathology examination. P90084.WO01 FINAL Application 25.04.2025 All animals administered 50 or 250 mg / kg survived until scheduled necropsy on day 7, except for one male rat in the 250 mg / kg group killed early due to technical issues with dosing. No rats (male or female) administered 1,000 mg / kg IHXME26 survived until scheduled necropsy. No animals treated with 50 mg / kg IHXME26 displayed clinical signs over the duration of the study. In contrast, animals treated with IHXME26 at 250 mg / kg displayed transient to intermittent signs of discomfort / irritation from day 2 of dosing, which resolved within 15 minutes. Animals treated with IHXME26 at the highest dose tested, 1,000 mg / kg, displayed signs of discomfort / irritation from day 1 which persisted for longer than 15 minutes post dose. The clinical signs observed included piloerection, hypoactivity and a hunched posture. One male rat was removed from study on day 2, and two further rats (male and female) were removed from study on day 4 due to displaying weight loss of greater than 15% accompanied with clinical signs. The remaining two rats at 1,000 mg / kg were removed from study on day 6 due to the presence of continuous clinical signs for over 24 hours. The bodyweights of animals treated with 50 mg / kg IHXME26 were generally stable and followed the weight curve of the rats dosed with vehicle. A slight reduction in weight gain compared to vehicle animals was observed in rats dosed with 250 mg / kg IHXME26. Animals (male and female) treated with 1,000 mg / kg decreased in bodyweight throughout the study. The effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on change in bodyweight is shown in Figure 23. Data points represent mean + SEM (n=2- 6). Histologically there were no findings of toxicological significance in animals treated with either 50 or 250 mg / kg IHXME26. Animals (male and female) treated with 1,000 mg / kg IHXME26 showed clear evidence of activation of a systemic acute inflammatory response in their livers with reactive microvascular changes also in the kidney. In addition, there were secondary changes in the muscle and small foci of reactive degenerative changes in the brain. The stomach also displayed degenerative changes in P90084.WO01 FINAL Application 25.04.2025 the glandular mucosa with evidence of increased cornification of the non-glandular stomach, which is consistent with topical irritancy to dosage form. The rat intestines of animals dosed with 1,000 mg / kg IHXME26 also displayed evidence of distention with thickening of the muscularis, both consistent with a bolus effect of dosing form leading to reduced gut transit time. In addition, changes in the lymph node from animals dosed 1,000 mg / kg IHXME26 are also consistent with an effector response on immune activation / selection. Serum cholesterol was significantly lower in animals treated with 250 and 1,000 mg / kg IHXME26 compared to rats treated with vehicle. Rats treated with the highest dose, 1,000 mg / kg IHXME26 showed notable trends for higher total bilirubin and lower serum ALP levels although this did not reach statistical significance compared to rats treated with vehicle. The effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on clinical chemistry parameters are shown in Figures 24A-24F, Figures 25G-25K and Figures 26L-26P. Data points represent individual rats with bars representing mean + SD (n=2-6). Circles represent male rats and triangles represent female rats. Data wereanalysed by one-way ANOVA with all groups compared to vehicle ^^^^^ ^^^^^^^^^multiple comparisons test, ns, * p<0.05, **p<0.01. There were a significantly higher number and percentage of neutrophils observed in the blood from animals treated with 250 mg / kg and 1,000 mg / kg IHXME26 compared animals treated with vehicle. The number and percentage of monocytes in animals treated with the highest dose of 1,000 mg / kg were also significantly higher than rats treated with vehicle. Finally, there was a significantly lower percentage of lymphocytes observed in animals treated with 250 mg / kg and 1,000 mg / kg compared with rats treated with vehicle. The effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on haematology parameters are shown in Figures 27A-25F, Figures 28G-28K, Figures 29L-29Q and Figures 30R-30T. Data points represent individual rats with bars representing mean + SD (n=2-6). Circles represent male rats and triangles represent

[0009] 45 P90084.WO01 FINAL Application 25.04.2025 female rats. Data were analysed by one-way ANOVA with all groups compared to^^^^^^^ ^^^^^ ^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^ ^^^^^ ^^, * p<0.05, *** p<0.001, ****p<0.0001. There were no significant changes in terminal urine albumin levels in rats treated with IHXME26 at all doses compared to animals treated with vehicle. There were also no significant changes in serum cytokines levels including eotaxin, GRO alpha, IL-17A, MCP-3 or RANTES in rats treated with IHXME26 at all doses compared to rats treated with vehicle. Notably, serum MCP-1 was significantly higher in rats treated with 1,000 mg / kg IHXME26 compared to rats treated with vehicle. There was no significant change in MCP-1 levels in rats treated at all other doses of IHXME26. The effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on urine albumin concentration is shown in Figure 31. Data points represent individual rats with lines representing mean (n=1-6). Data were analysed by one-way ANOVA with all^^^^^^ ^^^^^^^^ ^^ ^^^^^^^ ^^^^^ ^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^ ^^^^^ ^^^The effect of once daily dosing of 50, 250, or 1,000 mg / kg IHXME26 over 7 days on serum cytokine concentrations is shown in Figures 32A-32F. Data points represent individual rats with lines representing mean (n=1-6). Data were analysed by one-way^^^^^ ^^^^ ^^^ ^^^^^^ ^^^^^^^^ ^^ ^^^^^^^ ^^^^^ ^^^^^^^^^ ^^^^^^^^ ^^^^^^^^^^^test, * p<0.05. The toxicokinetic properties of IHXME26 in rats following once daily oral gavage dosing are shown in the Table below. Dosage (mg / kg) and sexDay Parameter Unit Male Female 50 250 1000 50 250 1000 ^^^^^^^^^ ^^^^ ^^^^^ ^^^^^ ^^^^^ ^^^^^ ^^^^^ Day 0^^^^^^ ^^^^^^^^ ^^^^ ^^^^^^ ^^^^^^ ^^^^ ^^^^^^ ^^^^^^ ^^^^^^ ^^^^^^^^ ^^^^ ^^^^^^ ^^^^^^ ^^^^ ^^^^^^ ^^^^^^ Day 6 ^^^^ ^^^^^ ^^^^^^ ^^^^^^ ^^ ^^^^^ ^^^^^^ ^^P90084.WO01 FINAL Application 25.04.2025 ^^^^^^^^^^^^^^ ^^^^^^ ^^^^^^^ ^^ ^^^^^ ^^^^^^ ^^ ^^^^^^ ^^^^^^^^ ^^^^^^ ^^^^^^^ ^^ ^^^^^ ^^^^^^^ ^^^^^ ^^ ^^^^^^^^^ ^^ ^^^ ^^^^^^^^ ^^ ^^^^^^^^^^ ^^^^^^^ ^^^^^^^^^ ^^^^^^^^ ^^ ^^^^^^^^^^^^^^^ ^^^^ ^^^^ ^^^^^^ ^^^^ ^^^ ^^^^^^^^^^^The average IHXME26 concentration in rat blood following p.o. dosing at different dose concentrations on study day 0 and day 6 is shown in Figures 33A-33C. Data plotted as mean ± SEM IHXME26 blood concentration of rats dosed with (A) 50 mg / kg, (B) 250 mg / kg, and (C) 1,000 mg / kg. N=3 rats per group. Example 46: Ames reverse mutation assay (microplate fluctuation protocol) IHXME26 was assessed for mutagenic potential in the Ames reverse mutation assay, in the presence and absence of metabolic activation by S9 fraction. An increase in the number of colonies of at least two-fold over baseline and a dose response indicates a positive result. The Ames reverse mutation assay results are below: IHXME26 Test Strain S9 Ames result Concentration ^^^^ ^^^^^ TA98 No Negative^^^^ ^^^^^ TA100 No Negative^^^^ ^^^^^ TA98 Yes Negative^^^^ ^^^^^ TA100 Yes NegativeP90084.WO01 FINAL Application 25.04.2025

Claims

Claims 1. A compound of Formula (1):or a pharmaceutically acceptable salt thereof.

2. A compound according to claim 1 for use as a medicament for the treatment of a disease or disorder.

3. A compound according to claim 1 for use as a potent and selective NOD2 agonist.

4. A compound according to claim 1 for use as a medicament capable of modulating innate immunity in a subject.

5. A compound according to claim 1 for use as a modulator of pro-inflammatory mediator secretion in a subject.6^ ^ ^^^^^^^^ ^^^^^^^^^ ^^ ^^^^^ ^ ^^^ ^^^ ^^ ^^^ ^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^7. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically or therapeutically acceptable excipient or carrier.

8. A method of treating a disease or disorder, comprising the step of administering a compound according to claim 1 or pharmaceutical composition according to claim 7 to a subject in need of the same.

9. A method of modulating innate immunity in a subject, comprising the step of administering a compound according to claim 1 or pharmaceutical composition according to claim 7 to a subject in need of the same. P90084.WO01 FINAL Application 25.04.202510^ ^ ^^^^^^ ^^ ^^^^^^^^ ^^^^^^^ ^^^^^^^^ ^^^^^^^^^^ ^^^ ^^^^ ^^ ^^^^^^^^^^^^^ ^compound according to claim 1 or pharmaceutical composition according to claim 7 to a subject in need of the same.

11. The method according to any of claims 8 to 10 wherein the treatment is affected or facilitated by the compound acting as a potent and selective NOD2 agonist.

12. Use of a compound according to claim 1 in the treatment of a disease or disorder.

13. Use of a compound according to claim 1 in modulating innate immunity in a subject.14^ ^^^ ^^ ^ ^^^^^^^^ ^^^^^^^^^ ^^ ^^^^^ ^ ^^ ^^^ ^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^15. The use according to any of claims 12 to 14 as a potent and selective NOD2 agonist.

16. Use of a compound according to claim 1 in the manufacture of a medicament for the treatment of a disease or disorder.

17. Use of a compound according to claim 1 in the manufacture of a medicament capable of modulating innate immunity in a subject.

18. Use of a compound according to claim 1 in the manufacture of a medicament for the^^^^^^^^^ ^^ ^^^^^^^ ^^^^^^^^19. The use according to any of claims 16 to 18 as a potent and selective NOD2 agonist. P90084.WO01 FINAL Application 25.04.2025

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