Prodrugs of NLRP3 modulators

Abstract

The present invention relates to compounds or a pharmaceutically acceptable salt or stereoisomer thereof of formula (I) wherein R1, R2, R2a, R3, X1 have the meaning as indicated in the claims and description. The invention further relates to pharmaceutical compositions comprising said compounds, their use as medicament and in a method for treating and / or preventing of one or more diseases, disorders or conditions associated with NLRP3.

Description

Evotec International GmbH EVO75139PC26 November 2025Prodrugs of NLRP3 ModulatorsBACKGROUND OF THE INVENTION

[0001] NLRP3 is a member of the nucleotide-binding domain (NBD), leucine-rich repeat (LRR)-containing (NLR) protein family that mediates innate inflammatory processes after infection or tissue injury. Upon activation it forms an inflammasome complex with ASC and caspase-1 that leads to IL-ip and IL-18 release and pyroptotic cell death.

[0002] Pathological functions of NLRP3 -mediated inflammation have been described in inherited diseases like cryopyrin-associated periodic syndromes (CAPS), as well as chronic inflammatory (e.g. rheumatoid arthritis), metabolic (e.g. diabetes) or neurological diseases (e.g. Alzheimer’s disease, Parkinson’s disease, multiple sclerosis).

[0003] NLRP3 is an intracellular sensor molecule that is activated in a “priming” and an “activation” step. The priming step includes the stimulation of pattern recognition receptors (PRRs) like Toll-like receptors and the activation of the nuclear factor-KB (NFKB) pathway that increases the expression of NLRP3, caspase- 1, Gasdermin D and pro-inflammatory cytokines. The NLRP3 activation step leads to formation of the active inflammasome that can be triggered by bacterial, viral and fungal infections (PAMPs; pathogen-associated molecule pattern; e.g., nigericin, DNA, RNA), sterile inflammation mediated by endogenous molecules (DAMPs; damage-associated molecule pattern; e.g. ATP, cholesterol crystals, a-synuclein, amyloid-P) and exposure to environmental irritants (Swanson et al., Nature Review Immunology, 2019, Vol 19., 477-489).

[0004] PAMPs and DAMPs cause cellular stress that is sensed by NLRP3. Many of them activate NLRP3 by causing a decrease in cytosolic potassium ions that induces conformational changes in the inactive NLRP3 protein leading to the formation of an NLRP3 complex that recruits ASC (adaptor protein apoptosis-associated speck-like protein containing a CARD). ASC binds pro-caspase-1 which is activated within the multiprotein inflammasome complex (also called ASC speck). Active caspase-1 cleaves the proinflammatory cytokines IL-ip and IL-18, as well as GSDMD (Gasdermin D) which form pores within the membrane, allowing the release of mature IL-ip and IL-18, and can trigger pyroptotic cell death. During pyroptosis, the release of intracellular contents, including NLRP3 inflammasomes, high-mobility group protein Bl (HMGB1), leukotrienes and prostaglandin, amplifies the inflammatory response andEvotec International GmbH EVO75139PC26 November 2025 contributes to inflammatory pathology (Mangan et al., Nature Reviews Drug Discovery, 2018, Vol. 17, 588-606; Swanson et al., Nature Review Immunology, 2019, Vol 19., 477-489).

[0005] Pathological consequences of NLRP3 activation are observed in patients with inherited, autosomal dominant mutations in NLRP3 that promote NLRP3 inflammasome activation. Patients with these gain-of-function mutations develop rare systemic auto- inflammatory syndromes called cryopyrin-associated periodic syndromes (CAPS), characterized by an inflammation-related phenotype with periodic fevers, sterile urticaria, and joint inflammation. CAPS incorporate three overlapping disease entities: familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem autoinflammatory syndrome (NOMID).

[0006] Pre-clinical studies with NLRP3 genetic deletions or small molecule inhibitors associates NLRP3 -mediated inflammation with plenty of peripheral and central diseases. That includes chronic inflammatory diseases, including gout, rheumatoid arthritis, and inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, metabolic diseases like atherosclerosis, diabetes, metabolic syndrome, obesity, and liver steatosis, nonalcoholic steatohepatitis (NASH) and liver fibrosis, and neurological diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), multiple sclerosis (MS), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Prion disease, Traumatic Brain Injury (TBI) and stroke, as well as asthma and allergic airway inflammation, hypertension, myocardial infarction, hyperinflammation following influenza and / or severe acute respiratory syndromecoronavirus 2 (SARS CoV-2), Graft-versus-host disease, silicosis, myelodysplastic syndrome, contact hypersensitivity and joint inflammation triggered by chikungunya virus (Mangan et al., Nature Reviews Drug Discovery, 2018, Vol. 17, 588-606; Holbrook et al., Frontiers in Pharmacology, 2021, Vol. 12, Article 643254; Coll et al., Trends in Pharmacological Science, 2022, Vol. 43, 653-668; Thornton et al., Journal of Pharmacology and Experimental Therapeutics, March 2024, 388(3) 813-826).

[0007] Several small molecules have been reported that directly or indirectly inhibit NLRP3. They have either weak potencies in the micromolar range and / or off target effects (Coll et al., Trends in Pharmacological Science, 2022, Vol. 43, 653-668). MCC950 is used in many pre-clinical studies as a tool compound with good potency and selectivity over NLRC4 and NLRP1. However, high dosing in clinical studies resulted in liver toxicity (Mangan et al., Nature Reviews Drug Discovery, 2018, Vol. 17, 588-606).Evotec International GmbH EVO75139PC26 November 2025

[0008] The pro-inflammatory cytokine IL-ip is a potent NLRP3 -dependent effector and therefore a major target for limiting NLRP3 driven pathology. Biologies like anakinra, canakinumab and rilonacept inhibit the IL1 axis, are approved for therapeutic use in CAPS and tested in clinical trial for rheumatoid arthritis and gout. However, biologies require administration by injection, they cause inflammation at the injection site and have poor brain permeability (Mangan et al., Nature Reviews Drug Discovery, 2018, Vol. 17, 588-606).

[0009] There is a need for orally available and preferably brain-permeable NLRP3 inhibitors with improved potency and selectivity.

[0010] However, in order to enhance pharmaceutical properties of NLRP3 inhibitors prodrugs can be used that can be transformed in the human body to said inhibitors, typically by enzymatic cleavage.SUMMARY OF THE INVENTION

[0011] The present application relates generally to compounds suitable as prodrugs for modulating the activity of NLRP3 after their conversion into NLRP3 active (or more active) compounds. The prodrugs can be e.g. enzymatically converted into NLRP3 modulators, in particular in vivo (such as in intestinal cells), and thus are absorbed into the circulatory system to achieve the purpose of treating NLRP3 related diseases and disorders. The compounds of the present invention can be used in compositions and methods for treating a disease, disorder, or condition which include but are not limited to (i) diseases associated with NLRP3 -mediated inflammation, including but not limited to cryopyrin-associated periodic syndromes (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem autoinflammatory syndrome (NOMID); (ii) chronic inflammatory diseases (e.g., gout, rheumatoid arthritis, inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis), (iii) metabolic diseases (e.g., atherosclerosis, diabetes, metabolic syndrome, obesity, liver steatosis, nonalcoholic steatohepatitis (NASH), and liver fibrosis); (iv) neurological diseases (e.g., Alzheimer’s disease (AD) and Parkinson’s disease (PD), multiple sclerosis (MS), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Prion disease, Traumatic Brain Injury (TBI) and stroke); (v) diseases associated with inherited, autosomal dominant mutations in NLRP3 that promote NLRP3 inflammasome; (vi) asthma and allergic airway inflammation; (vii) hypertension; (viii) myocardial infarction; (ix) hyperinflammation following influenza and / or severe acute respiratory syndrome-coronavirusEvotec International GmbH EVO75139PC26 November 20252 (SARS CoV-2); (x) Graft-versus-host disease; (xi) silicosis; (xii) myelodysplastic syndrome; (xiii) contact hypersensitivity and joint inflammation triggered by chikungunya virus.

[0012] In one aspect, the disclosure encompasses a compound of formula (I)or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, whereinR1is CH3, CF3, CHF2, Cl, OCH3, OCF3or OCHF2;R2, R2aare independently selected from the group consisting of H and R2b;Each R2bis independently selected from the group consisting of-CH2OC(O)OCi-4 alkyl;R3is H or CH3;X1is C(R4) or N;R4is H, Ci-4 alkyl, C3-s cycloalkyl, unsubstituted saturated 4- to 6- membered heterocyclyl, OCH3, CN, Cl or F, wherein Ci-4 alkyl and C3-s cycloalkyl are unsubstituted or substituted with one or more F.

[0013] In another aspect for the compound of formula (I), X1is C(R4).

[0014] In another aspect for the compound of formula (I), X1is CH.Evotec International GmbH EVO75139PC26 November 2025

[0015] In another aspect for the compound of formula (I), R1is CF3.

[0016] In another aspect for the compound of formula (I), R3is H.

[0017] In another aspect for the compound of formula (I), R3is CH3.

[0018] In another aspect for the compound of formula (I), R2bis -CH2OC(O)OCH(CH3)2.

[0019] In another aspect for the compound of formula (I), R2bis -CH2OC(O)OCH2CH3.

[0020] In another aspect for the compound of formula (I), R2, R2aare different.

[0021] In another aspect for the compound of formula (I), one of R2, R2ais H and the other is R2b, in particular one of R2, R2ais H and the other is CH2OC(O)OCH(CH3)2.

[0022] In another aspect for the compound of formula (I), R2, R2aare the same.

[0023] In another aspect for the compound of formula (I), R2, R2aare H.

[0024] In another aspect for the compound of formula (I), R2, R2aare R2b.

[0025] In another aspect of the disclosure for the compounds described herein, the compound is: ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5- (trifluoromethyl)phenoxy}methoxy)phosphonic acid or ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5- (trifluoromethyl)phenoxy}methoxy)({[(propan-2- yloxy)carbonyl]oxy}methoxy)phosphinic acid.

[0026] The invention provides a compound of formula (I). The invention also provides a pharmaceutical composition comprising any of the compounds described herein. The invention also provides the use of a compound of formula (I) as medicament and (for use) in methods ofEvotec International GmbH EVO75139PC26 November 2025 treating and / or preventing of one or more diseases, disorders or conditions associated with NLRP3.

[0027] The compounds of formula (I) are suitable prodrugs for modulating the activity of NLRP3 after their conversion into NLRP3 active (or more active) compounds (parent compounds). In particular, the compounds of formula (I) can be cleaved enzymatically, such as by alkaline phosphatase, into parent compounds according to the following equation:Prodrug Parent Compound

[0028] In yet another aspect, described is a method of treating and / or preventing one or more diseases and / or disorders associated with NLRP3 with a compound described herein, or a compound or composition described herein (for use) in a method of treating and / or preventing one or more diseases, disorders or conditions associated with NLRP3.

[0029] Both the foregoing summary and detailed description are exemplary and explanatory. They are intended to provide further details of the invention, but are not to be construed as limiting. Other objects, advantages, and novel features will be readily apparent to those skilled in the art from the following detailed description of the invention.DETAILED DESCRIPTION OF THE INVENTION

[0030] An object of the present invention is to provide a new class of compounds as prodrugs for modulators of NLRP3, which may be effective in the treatment of NLRP3 related diseases and disorders. The prodrugs can be e.g. enzymatically converted into NLRP3 modulators, in particular in vivo (such as in intestinal cells), and thus are absorbed into the circulatory system to achieve the purpose of treating NLRP3 related diseases and disorders.Evotec International GmbH EVO75139PC26 November 2025The prodrug may show improved solubility compared to the parent compound, thus providing for higher exposure of the parent compound, in particular for toxicology studies.

[0031] Pathological functions of NLRP3 -mediated inflammation have been described in inherited diseases like CAPS, as well as chronic inflammatory (e.g., rheumatoid arthritis), metabolic (e.g., diabetes) or neurological diseases (e.g., Alzheimer’s disease, Parkinson’s disease, multiple sclerosis). Also, as noted above, NLRP3 -mediated inflammation is associated with peripheral and central diseases. These include chronic inflammatory diseases, including gout, rheumatoid arthritis, and inflammatory bowel disease (IBD) including Crohn’s disease and ulcerative colitis, metabolic diseases like atherosclerosis, diabetes, metabolic syndrome, obesity, and liver steatosis, nonalcoholic steatohepatitis (NASH) and liver fibrosis, and neurological diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), multiple sclerosis (MS), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Prion disease, Traumatic Brain Injury (TBI) and stroke, as well as asthma and allergic airway inflammation, hypertension, myocardial infarction, hyperinflammation following influenza and / or severe acute respiratory syndrome-coronavirus 2 (SARS CoV-2), Graft-versus-host disease, silicosis, myelodysplastic syndrome, contact hypersensitivity and joint inflammation triggered by chikungunya virus.

[0032] The present invention provides compounds of the present invention in free or pharmaceutically acceptable salt form or in the form of solvates, hydrates, tautomers or stereoisomers. These can be used in the treatment of diseases or disorders mentioned herein. The same applies to a pharmaceutical composition of the present invention. Accordingly, one aspect of the present invention is a pharmaceutically acceptable salt of a compound of the present invention. Another aspect of the present invention is a stereoisomer of a compound of the present invention. Another aspect of the present invention is a solvate of a compound of the present invention. Another aspect of the present invention is a hydrate of a compound of the present invention. Another aspect of the present invention is a tautomer of a compound of the present invention.

[0033] Accordingly, the present invention provides a compound of formula (I):Evotec International GmbH EVO75139PC26 November 2025(I), or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, whereinR1is CH3, CF3, CHF2, Cl, OCH3, OCF3or OCHF2;R2, R2aare independently selected from the group consisting of H and R2b;Each R2bis independently selected from the group consisting of -CH2OC(O)OCI-4 alkyl;R3is H or CH3;X1is C(R4) or N;R4is H, Ci-4 alkyl, C3-s cycloalkyl, unsubstituted saturated 4- to 6- membered heterocyclyl, OCH3, CN, Cl or F, wherein Ci-4 alkyl and C3-s cycloalkyl are unsubstituted or substituted with one or more F.

[0034] In case a variable or substituent can be selected from a group of different variants and such variable or substituent occurs more than once, then the respective variants can be the same or different.

[0035] Surprisingly, the disclosed example compounds according to the present invention have favourable solubility, stability, in particular as shown in models of the gastrointestinalEvotec International GmbH EVO75139PC26 November 2025 tract, and rapidly convert into the parent compounds in intestinal cells to help to achieve beneficial therapeutic efficacy whilst limiting unintended liabilities.

[0036] Compounds according to the disclosure were tested as detailed in the experimental section below.I. Definitions

[0037] Within the meaning of the present invention the terms are used as follows:

[0038] Technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art, unless otherwise defined. Any suitable materials and / or methodologies known to those of ordinary skill in the art can be utilized in carrying out the methods described herein.

[0039] As used in the description of the invention and the appended claims, the singular forms “a”, “an” and “the” are used interchangeably and intended to include the plural forms as well and fall within each meaning, unless the context clearly indicates otherwise. Also, as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0040] As used herein, “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0041] The term “optionally substituted” means unsubstituted or substituted. Generally - but not limited to-, “one or more substituents” means one, two or three, preferably one or two substituents and more preferably one substituent. Generally these substituents can be the same or different. The term “one or more substituents” also means by way of example one, two, three, four or five, preferably by way of example one, two, three or four.

[0042] “Alkyl” means a straight-chain or branched hydrocarbon chain. Each hydrogen of an alkyl carbon may be replaced by a substituent as further specified.

[0043] “Alkenyl” means a straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon double bond. Each hydrogen of an alkenyl carbon may be replaced by a substituent as further specified.Evotec International GmbH EVO75139PC26 November 2025

[0044] “Alkynyl” means a straight-chain or branched hydrocarbon chain that contains at least one carbon-carbon triple bond. Each hydrogen of an alkynyl carbon may be replaced by a substituent as further specified.

[0045] Ci-4 alkyl” means an alkyl chain having 1 - 4 carbon atoms, e.g. if present at the end of a molecule: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, or e.g. -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-, when two moieties of a molecule are linked by the alkyl group. Each hydrogen of a C1-4 alkyl carbon may be replaced by a substituent as further specified. The term “Ci-3alkyl” is defined accordingly.

[0046] C1-6 alkyl” means an alkyl chain having 1 - 6 carbon atoms, e.g. if present at the end of a molecule: C1-4 alkyl, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, or e.g. -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, - CH(C2HS)-, -C(CH3)2-, when two moieties of a molecule are linked by the alkyl group. Each hydrogen of a C1-6 alkyl carbon may be replaced by a substituent as further specified.

[0047] C2-6 alkenyl” means an alkenyl chain having 2 to 6 carbon atoms, e.g. if present at the end of a molecule: -CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CH-CH2-CH3, - CH=CH-CH=CH2, or e.g. -CH=CH-, when two moieties of a molecule are linked by the alkenyl group. Each hydrogen of a C2-6 alkenyl carbon may be replaced by a substituent as further specified.

[0048] C2-6 alkynyl” means an alkynyl chain having 2 to 6 carbon atoms, e.g. if present at the end of a molecule: -C =CH, -CH2-C =CH, -CH2-CH2-C =CH, -CH2-C =C-CH3, or e.g. -OC- when two moieties of a molecule are linked by the alkynyl group. Each hydrogen of a C2-6 alkynyl carbon may be replaced by a substituent as further specified.

[0049] C3-7 cycloalkyl” or “C3-7 cycloalkyl ring” means a cyclic alkyl chain having 3 - 7 carbon atoms, e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl. Preferably, cycloalkyl refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. Each hydrogen of a cycloalkyl carbon may be replaced by a substituent as further specified herein. The term “C3-s cycloalkyl” or “C3-s cycloalkyl ring” is defined accordingly. The term “C4-6 cycloalkyl” or “C4-6 cycloalkyl ring” is defined accordingly.

[0050] C5 cycloalkylene” refers to a bivalent cycloalkyl with five carbon atoms, i.e. a bivalent cyclopentyl ring.Evotec International GmbH EVO75139PC26 November 2025

[0051] Cs cycloalkenylene” refers to a bivalent cycloalkenylene, i.e. a bivalent cyclopentene or cyclopentadiene.

[0052] C4-12 bicycloalkyl” or “C4-12 bicycloalkyl ring” means a bicyclic fused, bridged or spiro alkyl chain having 4 to 12 carbon atoms, e.g. hexahydroindane, octahydropentalen, bicycle[2.2.1]heptane or spiro(3.2)hexane. Each hydrogen of a bicycloalkyl carbon may be replaced by a substituent as further specified herein.

[0053] “Halogen” means fluoro, chloro, bromo or iodo. It is generally preferred that halogen is fluoro or chloro.

[0054] “4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means a ring with 4, 5, 6 or 7 ring atoms that may contain up to the maximum number of double bonds (aromatic or non-aromatic ring which is fully, partially or un-saturated) wherein at least one ring atom up to 4 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-) and wherein the ring is linked to the rest of the molecule via a carbon or nitrogen atom. Examples for a 4 to 7 membered heterocycle are azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine or homopiperazine. The term “5 to 6 membered heterocyclyl” or “5 to 6 membered heterocycle” is defined accordingly and and includes 5 to 6 membered aromatic heterocyclyl or heterocycle. The term “5 membered heterocyclyl” or “5 membered heterocycle” is defined accordingly and includes 5 membered aromatic heterocyclyl or heterocycle. The term “4 to 6 membered heterocyclyl” or “4 to 6 membered heterocycle” is defined accordingly.

[0055] The term “nitrogen ring atom containing 5-membered heterocyclene” refers to a bivalent 5-membered heterocycle, wherein at least one of the five ring atoms is a nitrogen atom and wherein the ring is linked to the rest of the molecule via a carbon or nitrogen atom.

[0056] Saturated 4 to 7 membered heterocyclyl” or “saturated 4 to 7 membered heterocycle” means fully saturated “4 to 7 membered heterocyclyl” or “4 to 7 memberedEvotec International GmbH EVO75139PC26 November 2025 heterocycle”. “Saturated 4 to 6 membered heterocyclyl” or “saturated 4 to 6 membered heterocycle” means fully saturated “4 to 6 membered heterocyclyl” or “4 to 6 membered heterocycle”.

[0057] “4 to 7 membered at least partly saturated heterocyclyl” or “4 to 7 membered at least partly saturated heterocycle” means an at least partly saturated “4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle”.

[0058] “5 to 6 membered aromatic heterocyclyl” or “5 to 6 membered aromatic heterocycle” means a heterocycle derived from cyclopentadienyl or benzene, where at least one carbon atom is replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-). Examples for such heterocycles are furan, thiophene, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, thiadiazole, triazole, tetrazole, pyridine, pyrimidine, pyridazine, pyrazine, triazine.

[0059] “5 membered aromatic heterocyclyl” or “5 membered aromatic heterocycle” means a heterocycle derived from cyclopentadienyl, where at least one carbon atom is replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-). Examples for such heterocycles are furan, thiophene, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, thiadiazole, triazole, tetrazole.

[0060] "7 to 12 membered heterobicyclyl" or "7 to 12 membered heterobicycle" means a heterocyclic system of two rings with 7 to 12 ring atoms, where at least one ring atom is shared by both rings and that may contain up to the maximum number of double bonds (aromatic or non-aromatic ring which is fully, partially or un-saturated) wherein at least one ring atom up to 6 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-) and wherein the ring is linked to the rest of the molecule via a carbon or nitrogen atom. Examples for a 7 to 12 membered heterobicycle are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine or pteridine. The term 7 to 12 membered heterobicycle also includes spiro structures of two rings like 6-oxa-2-azaspiro[3,4]octane, 2- oxa-6-azaspiro[3.3]heptan-6-yl or 2,6-diazaspiro[3.3]heptan-6-yl or bridged heterocycles likeEvotec International GmbH EVO75139PC26 November 20258-aza-bicyclo[3.2.1]octane or 2,5-diazabicyclo[2.2.2]octan-2-yl or 3,8-diazabicyclo[3.2.1] octane.

[0061] Saturated 7 to 12 membered heterobicyclyl” or “saturated 7 to 12 membered heterobicycle” means fully saturated “7 to 12 membered heterobicyclyl” or “7 to 12 membered heterobicycle”.

[0062] “7 to 12 membered at least partly saturated heterobicyclyl” or “7 to 12 membered at least partly saturated heterobicycle” means an at least partly saturated “7 to 12 membered heterobicyclyl” or “7 to 12 membered heterobicycle”.

[0063] “9 to 11 membered aromatic heterobicyclyl” or “9 to 11 membered aromatic heterobicycle” means a heterocyclic system of two rings, wherein at least one ring is aromatic and wherein the heterocyclic ring system has 9 to 11 ring atoms, where two ring atoms are shared by both rings and that may contain up to the maximum number of double bonds (fully or partially aromatic) wherein at least one ring atom up to 6 ring atoms are replaced by a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-) and wherein the ring is linked to the rest of the molecule via a carbon or nitrogen atom. Examples for an 9 to 11 membered aromatic heterobicycle are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, dihydroquinoline, tetrahydroquinoline, isoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine or pteridine. The terms “9 to 10 membered aromatic heterobicyclyl” or “9 to 10 membered aromatic heterobicycle” are defined accordingly.II. Exemplary Compounds of the Disclosure

[0064] Exemplary compounds of formula (I) are those compounds in which one or more of the residues contained therein have the meanings given above or below, with all combinations of preferred substituent definitions being a subject of the present invention. With respect to all preferred compounds of the formula (I) the present invention also includes all tautomeric and stereoisomeric forms and mixtures thereof in all ratios, and their pharmaceutically acceptable salts.

[0065] In exemplary embodiments of the present invention, the substituents mentioned below independently have the following meaning. Hence, one or more of these substituents can have the meanings given below.Evotec International GmbH EVO75139PC26 November 2025

[0066] In an aspect for the compound of formula (I), X1is C(R4).

[0067] In another aspect for the compound of formula (I), X1is CH.

[0068] In another aspect for the compound of formula (I), R1is CF3.

[0069] In another aspect for the compound of formula (I), R3is H.

[0070] In another aspect for the compound of formula (I), R3is CH3.

[0071] In another aspect for the compound of formula (I), R2bis -CH2OC(O)OCH2CH3.

[0072] In another aspect for the compound of formula (I), R2bis -CH2OC(O)OCH(CH3)2.

[0073] In another aspect for the compound of formula (I), R2, R2aare different.

[0074] In another aspect for the compound of formula (I), one of R2, R2ais H and the other is R2b, in particular one of R2, R2ais H and the other is CH2OC(O)OCH(CH3)2.

[0075] In another aspect for the compound of formula (I), R2, R2aare the same.

[0076] In another aspect for the compound of formula (I), R2, R2aare H.

[0077] In another aspect for the compound of formula (I), R2, R2aare R2b.

[0078] Compounds of the present invention in which some or all of the above-mentioned groups have the preferred or more preferred meanings are also an object of the present invention.

[0079] Exemplary specific compounds of the present invention are selected from the group consisting of:({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5-(trifluoromethyl)phenoxy}methoxy)phosphonic acidEvotec International GmbH EVO75139PC26 November 2025 and({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5-(trifluoromethyl)phenoxy}methoxy)({[(propan-2- yloxy)carbonyl]oxy}methoxy)phosphinic acid.

[0080] Where tautomerism, like e.g., keto-enol tautomerism, of compounds of formula (I) may occur, the individual forms, like e.g., the keto and enol form, are comprised separately and together as mixtures in any ratio. Same applies to stereoisomers, like e.g. enantiomers, cis / trans isomers, conformers and the like.

[0081] Especially, when enantiomeric or diastereomeric forms are given in a compound according to formula (I), each pure form separately and any mixture of at least two of the pure forms in any ratio is comprised by formula (I) and is a subject of the present invention.

[0082] Isotopic labeled compounds of formula (I) are also within the scope of the present invention. Methods for isotope labeling are known in the art. Preferred isotopes are those of the elements H, C, N, O and S. Thus, compounds of the present invention are especially also encompassed, which have one or more hydrogens in the form of2H / deuterium D. Solvates and hydrates of compounds of formula (I) are also within the scope of the present invention.

[0083] If desired, isomers can be separated by methods well known in the art, e.g., by liquid chromatography. Same applies for enantiomers by using e.g., chiral stationary phases. Additionally, enantiomers may be isolated by converting them into diastereomers, i.e., coupling with an enantiomerically pure auxiliary compound, subsequent separation of the resulting diastereomers and cleavage of the auxiliary residue. Alternatively, any enantiomer of a compound of formula (I) may be obtained from stereoselective synthesis using optically pure starting materials, reagents and / or catalysts.

[0084] In case the compounds according to formula (I) contain one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts. Thus, the compounds of the formula (I) which comprise acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine,Evotec International GmbH EVO75139PC26 November 2025 triethanolamine or amino acids. Compounds of the formula (I) which contain one or more basic groups, i.e., groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. If the compounds of the formula (I) simultaneously contain acidic and basic groups in the molecule, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts according to the formula (I) can be obtained by customary methods which are known to the person skilled in the art like, for example by contacting these with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the compounds of the formula (I) which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.III. Pharmaceutical Compositions

[0085] As shown below in the Examples, compounds of the present invention are suitable for modulating NLRP3.

[0086] Accordingly, an aspect of the present invention is a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof of the present invention for use as a medicament as mentioned above. The same applies to a pharmaceutical composition of the present invention.

[0087] A further aspect of the present invention is a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof or a pharmaceutical composition of the present invention for use in a method of treating and / or preventing one or more disorders or diseases mentioned herein.

[0088] A further aspect of the present invention is the use of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof or aEvotec International GmbH EVO75139PC26 November 2025 pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment or prophylaxis of one or more disorders or diseases associated with NLRP3.

[0089] Yet another aspect of the present invention is a pharmaceutical composition comprising at least one compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof of the present invention together with a pharmaceutically acceptable carrier, optionally in combination with one or more other bioactive compounds or pharmaceutical compositions. In one aspect, the one or more bioactive compounds are modulators of NLRP3 other than compounds of the present invention.

[0090] "Pharmaceutical composition" means one or more active ingredients, and one or more inert ingredients that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing a compound of the present invention and a pharmaceutically acceptable carrier.

[0091] A pharmaceutical composition of the present invention may comprise one or more additional compounds as active ingredients like a mixture of compounds of formula (I) in the composition or other modulators of NLRP3.

[0092] The active ingredients may be comprised in one or more different pharmaceutical compositions (combination of pharmaceutical compositions).

[0093] The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids, including inorganic bases or acids and organic bases or acids.

[0094] Starting materials for the synthesis of preferred embodiments of the invention may be purchased from commercially available sources such as Array, Sigma Aldrich, Acros, Fisher, Fluka, ABCR.

[0095] In general, several methods are applicable to prepare compounds of the present invention. In some cases, various strategies can be combined. Sequential or convergent routes may be used. Exemplary synthetic routes are described below.IV. Methods of TreatmentEvotec International GmbH EVO75139PC26 November 2025

[0096] The present invention provides a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof, or a pharmaceutical composition of the present invention, to be used in the treatment or prevention of one or more diseases or disorders associated with NLRP3.

[0097] The therapeutic method described may be applied to mammals such as dogs, cats, cows, horses, rabbits, monkeys and humans. Preferably, the mammalian patient is a human patient.

[0098] Yet another aspect of the present invention is a method for treating, controlling, delaying and / or preventing in a mammalian patient in need of the treatment of one or more diseases, disorders or conditions associated with NLRP3, wherein the method comprises administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof or a pharmaceutical composition of the present invention.

[0099] Yet another aspect of the present invention is a method for treating, controlling, delaying and / or preventing in a mammalian patient in need of the treatment of one or more diseases, disorders or conditions mentioned herein, wherein the method comprises administering to the patient a therapeutically effective amount of a compound or a pharmaceutically acceptable salt, solvate, hydrate, tautomer or stereoisomer thereof or a pharmaceutical composition of the present invention.

[0100] Diseases or disorders amenable to treatment using the compounds and compositions of the invention include but are not limited to: (i) diseases associated with NLRP3 -mediated inflammation, including but not limited to cryopyrin-associated periodic syndromes (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem autoinflammatory syndrome (NOMID); (ii) chronic inflammatory diseases (e.g., gout, rheumatoid arthritis, inflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis), (iii) metabolic diseases (e.g., atherosclerosis, diabetes, metabolic syndrome, obesity, liver steatosis, nonalcoholic steatohepatitis (NASH), and liver fibrosis); (iv) neurological diseases (e.g., Alzheimer’s disease (AD) and Parkinson’s disease (PD), multiple sclerosis (MS), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Prion disease, Traumatic Brain Injury (TBI) and stroke); (v) diseases associated with inherited, autosomal dominant mutations in NLRP3 that promote NLRP3 inflammasome; (vi) asthma andEvotec International GmbH EVO75139PC26 November 2025 allergic airway inflammation; (vii) hypertension; (viii) myocardial infarction; (ix) hyperinflammation following influenza and / or severe acute respiratory syndrome-coronavirus 2 (SARS CoV-2); (x) Graft-versus-host disease; (xi) silicosis; (xii) myelodysplastic syndrome; (xiii) contact hypersensitivity and joint inflammation triggered by chikungunya virus.

[0101] Any suitable route of administration may be employed for providing a mammal, especially a human, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. Preferably compounds of formula (I) are administered orally.

[0102] The effective dosage of active ingredient employed may vary depending on the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosage may be ascertained readily by a person skilled in the art.

[0103] DESCRIPTION OF DRAWINGS

[0104] Figure 1 shows the calibration curve between 1 pg / ml and 1000 pg / ml - UPLC-UV peak area at 290 nm vs concentration of INTERMEDIATE 6.

[0105] Figure 2 shows the calibration curve between Ipg / ml and 1000 pg / ml - UPLC-UV peak area at 290 nm vs concentration of EXAMPLE 1.

[0106] Figure 3 shows the conversion of phosphate prodrug, EXAMPLE 1 into the drug, INTERMEDIATE 6, in Caco-2 Cells at pH 7.4.

[0107] Figure 4 shows INTERMEDIATE 6 rat plasma concentration-time profiles following PO administration of EXAMPLE 1 (A) and INTERMEDIATE 6 (B).

[0108] Figure 5 shows a comparison of INTERMEDIATE 6 AUC following PO administration of EXAMPLE 1 and INTERMEDIATE 6.

[0109] EXAMPLESI. Chemical synthesisExperimental procedures:The following Abbreviations and Acronyms are used:Evotec International GmbH EVO75139PC 26 November 2025ACN acetonitrileALP Alkaline phosphataseAMU atomic mass unitAUC area under the concentration-time curveA>B Apical to Basolateral directionB>A Basolateral to Apical directionBippyPhos 5-(di- / c 7-butylphosphino)- l 3', 5 '-triphenyl- 1'H-[l,4']bipyrazoleBQL Below the limit of quantificationBrine saturated solution of NaCl in waterBU4N(HSO4) T etrabutylammonium hydrogensulfateCaco-2 Human colon carcinoma cell lineCDC13deuterated chloroform cHex cyclohexaneCmax maximum concentrationCyJohnPhos 2-(dicyclohexylphosphino)biphenyl or dicyclohexyl-(2- phenylphenyl)phosphaneCS2CO3 cesium carbonateDCM dichloromethaneDIPEA N -ethyl -N -i sopropy 1 -propan-2 -amineDMAP 4-(Dimethylamino)pyridineDMEM Dulbecco's modified Eagle's mediumDMF N,N-dimethylformamideDMSO dimethylsulfoxideDMSO-t / e deuterated dimethylsulfoxideEDTA ethylenediaminetetraacetic acidER Efflux RatioESI+positive ionisation modeESI’ negative ionisation modeE zO di ethyl etherEtOAc ethyl acetateEtOH ethanolEvotec International GmbH EVO75139PC26 November 2025FBS Fetal Bovine Serum g gram(s)GLP Good laboratory practice h hour(s)HBSSH Hank’s Balanced Salt Solution + 12.5 mM HEPESHCOOH formic acidHI Heat InactivatedHPbCD hydroxypropyl-P-cyclodextrinHPLC high-performance liquid chromatographyIS Internal StandardLC Liquid chromatographyLC / MS / MS liquid chromatography with tandem mass spectrometryLiOH lithium hydroxideLY Lucifer Yellow m multipletMeOH methanolMeTHF 2-Methyltetrahydrofuran mg milligram(s) MgSO4magnesium sulphateMHz megahertz min minutes mg / kg milligram(s) per kilogram mL millilitre (s) mM millimolar MS Mass spectrometryMTBE tert-butyl methyl etherMW Molecular weightN2nitrogen atmosphereNa2CO3sodium carbonateNaHCCh sodium bicarbonateNa2SO4sodium sulfateNCA non-compartmental analysisEvotec International GmbH EVO75139PC26 November 2025NH4CI ammonium chlorideNLRP3 nucleotide-binding domain (NBD), leucine-rich repeat (LRR)- containing (NLR) protein 3NMR Nuclear Magnetic ResonanceOH hydroxyPd2(dba)s tris(dibenzylideneacetone)dipalladium(0)Pd(dppf)C12 [1, 1 ‘-Bis(diphenylphosphino)ferrocene]dichloropalladium(II)P-gp P-glycoproteinPK pharmacokineticsPO oralQC quality controlRFU relative fluorescence units r.t. room temperatureRT retention timeSD Standard deviationSGF Simulated Gastric Fluid pH 1.2SIF Simulated Intestinal Fluid pH 6.8TEA triethylamine tmax time of maximum concentrationUPLC Ultra Performance Liquid ChromatographyUPLC-UV-MS Ultra Performance Liquid Chromatography coupled withUltraViolet and Mass spectrometry detectionUSP United States PharmacopeiaVIT E TPGS D-tocopheryl polyethylene glycol succinate w / v weight per volume w / w weight per weight pM micromolarAnalytical LCMS conditions were as follows:

[0110] UPLC / MS retention times were estimated to be affected by an experimental error of +0.5 min. LCMS may be recorded under the following conditions: diode array DAD chromatographic traces, mass chromatograms and mass spectra may be taken onEvotec International GmbH EVO75139PC26 November 2025UPLC / PDA / MS Acquity™ system coupled with Micromass ZQ™ or Waters SQD single quadrupole mass spectrometer operated in positive and / or negative electron spray (ES) ionization mode and / or Fractionlynx system used in analytical mode coupled with ZQ™ single quadrupole operated in positive and / or negative ES ionisation mode. Quality Control methods used operated under low pH conditions.System 1 (SI): Low pH conditions

[0111] Column: Acquity CSH C18 2.1x50mm 1.7pm, the column temperature was 40 °C; mobile phase solvent A was milliQ water+0.1% HCOOH, mobile phase solvent B ACN+0.1% HCOOH. The flow rate was 0.9 mL / min.

[0112] The gradient table was t= 0 min 97% A 3% B, t= 1.4 min 0.1% A 99.9% B, t= 1.9 min 0.1% A 99.9% B and t= 2 min 97% A 3% B. The UV detection range was 210-350 nm and ES+ZES- range was 100 to 1000 AMU. Data were integrated and reported using Waters MassLynx and OpenLynx software.System 2 (S2): ACIDIC IPC METHOD

[0113] Analytical (MET / uPLC / 1704) UHPLC-MS were performed in reverse phase system using a Waters UPLC™BEH™ C18 column (2.1 mm x 50 mm, 1.7 pm; temperature: 40 °C), with an injection volume of 1 pL at a flow rate of 0.9 mL / min and a gradient of 5 - 100% B over 1.10 min, then 100% B for 0.25 min, where A = 0.1% formic acid in water, and B = 0.1% formic acid in ACN. A second gradient of 100 - 5% B was then applied over 0.05 min and held for 0.10 min. UV spectra were recorded at 215, 254 and 280 nm. Mass spectra were obtained using a Waters QDA detector; ionization mode: electrospray positive or negative. Data were integrated and reported using Waters MassLynx and OpenLynx software.System 3 (S3): ACIDIC FINAL METHOD

[0114] Analytical (MET / uPLC / ABlOl) UHPLC-MS were performed in reverse phase using a Phenom enex Kinetex-XB Cl 8 column (2.1 mm x 100 mm, 1.7 pm; temperature: 40 °C), with an injection volume of 1 pL at a flow rate of 0.6 mL / min and a gradient of 5 - 100% B over 5.30 min, then 100% B for 0.50 min, where A = 0.1% formic acid in water, and B = 0.1% formic acid in ACN. A second gradient of 100 - 5% B was then applied over 0.02 min and held for 1.18 min. UV spectra were recorded at 215, 254 and 280 nm. ELS data wasEvotec International GmbH EVO75139PC26 November 2025 collected on a Waters ELS detector when reported. Mass spectra were obtained using a Waters QDA detector; ionization mode: electrospray positive or negative. Data were integrated and reported using Waters MassLynx and OpenLynx software.

[0115] Purification methods were as follows:

[0116] Purifications by chromatography on silica gel were performed on Biotage Isolera systems using the appropriate Sfar Duo cartridge.

[0117] NMR Conditions

[0118] Unless otherwise stated, 'H-NMR spectra were performed on a Varian MR-400 spectrometer operating at 400 MHz (proton frequency), equipped with: a self-shielded Z- gradient coil 5 mm IH / nX broadband probe head for reverse detection, deuterium digital lock channel unit, quadrature digital detection unit with transmitter offset frequency shift, or on AgilentVNMRS-500, or on a Bruker Avance 400 spectrometers, or on a Agilent Inova 600 operating at 600MHz equipped with 5mm PFG PENTA Probe spectrometers. Chemical shifts are reported as 6 values in ppm relative to trimethylsilane (TMS) as an internal standard. Coupling constants (J values) are given in hertz (Hz) and multiplicities are reported using the following abbreviation (s= singlet, d= doublet, t= triplet, q= quartet, m= multiplet, dd= doubledoublet, ddd= double-double-doublet, dddd= double-double-double-doublet, dt= double triplet, dq= double quartet, dtd= double-triple douplet, qd= quartet of doublet, quin= quintuplet, td= triple doublet, tt= triple triplet).

[0119] General synthesis:

[0120] Scheme for route 1 :Intermediate 1Intermediate 1: 3-methyl-5-(trifluoromethyl)phenolEvotec International GmbH EVO75139PC26 November 2025Intermediate 1

[0121] To a degassed mixture of l-bromo-3-methyl-5-(trifluoromethyl)benzene (100 g, 418.4 mmol) and LiOH (31.3 g, 1255.1 mmol) in 1,4-dioxane (650 mL) and water (122 mL) was added a degassed mixture of Pd2(dba)s (3.8 g, 4.2 mmol) and BippyPhos (4.1 g, 8.0 mmol) in 1,4-dioxane (50 mL). The reaction was stirred at 90 °C under a N2 atmosphere for 18 h. The reaction mixture was cooled to r.t. and filtered through glass fibre filter paper. The filtrate was concentrated in vacuo, dissolved in EtOAc and washed with 1 M HC1. After phase separation, the organic layer was concentrated in vacuo. The crude product was then stirred in an aqueous 5 M NaOH solution for 15 min. Next, heptane was added, and the biphasic mixture was stirred for 5 min. After phase separation, the basic aqueous layer was cooled to 0 °C and acidified with an aqueous 5 M HC1 solution until pH=4 was achieved. The aqueous layer was then extracted with heptane. The combined organic phases were washed with brine, dried over MgSCU and concentrated in vacuo to yield the title compound (55.0 g, 306.2 mmol, 73% yield) as an orange liquid. 'HNMR (400 MHz, DMSO-t / 6) 8 = 9.96 (s, 1H), 6.93 (qd, J= 1.6, 0.9 Hz, 1H), 6.88 - 6.80 (m, 2H), 2.29 (s, 3H). MS (ESI); / Z: 221 [M +HCOOH- H]’, EST, RT = 0.88 (S2).Scheme for route 2:Intermediate 1 Intermediate 2Intermediate 2: 2-iodo-3-methyl-5-(trifluoromethyl)phenolEvotec International GmbH EVO75139PC26 November 2025Intermediate 2

[0122] 3-methyl-5-(trifluoromethyl)phenol (Intermediate 1, 55 g, 306.2 mmol) was dissolved in toluene (500 mL) and cooled to 0 °C under a N2 atmosphere. NaH (60% dispersion in mineral oil, 24.5 g, 612.3 mmol) was added portion-wise over 45 min. Next, a solution of iodine (77.7 g, 306.2 mmol) in toluene (500 mL) was added dropwise over 8 h at 0 °C. After stirring at r.t., the reaction was quenched to pH=7 with an aqueous 6 M HC1 solution at 0 °C. The mixture was partially concentrated in vacuo, before being extracted with EtOAc. The combined organic layers were washed with brine, dried over MgSCU, and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-100% EtOAc in heptane, followed by 0-20% MeOH in EtOAc) to yield the title compound (90% purity, 74.3 g, 221.3 mmol, 72% yield) as an off-white solid. 'H NMR (400 MHz, DMSO-t / 6) 8 = 10.95 (s, 1H), 7.15 - 7.10 (m, 1H), 6.96 - 6.90 (m, 1H), 2.44 (s, 3H); MS (ESI); / Z: 301 [M-H]’, EST, RT = 1.00 (S3).Scheme for route 3:Intermediate 3: 3-methyl-2-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenolEvotec International GmbH EVO75139PC26 November 2025Intermediate 3

[0123] To a degassed solution of 2-iodo-3-methyl-5-(trifluoromethyl)phenol (Intermediate 2, 20.00 g, 49.7 mmol), TEA (21 mL, 149.0 mmol), pinacol borane (22 mL, 149.0 mmol) and CyJohnPhos (3.48 g, 9.9 mmol) in 1,4-dioxane (20 mL) was added Pd(OAc)2 (1.12 g, 5.0 mmol) and the reaction was heated to 80 °C under a N2 atmosphere for 22 h. The reaction mixture was cooled to r.t. and filtered through glass fibre filter paper. The filter cake was washed with EtOAc and the filtrate was partially concentrated in vacuo. The organic layer was washed with a saturated aqueous solution of NH4CI and water, dried over MgSCU and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-12% EtOAc in heptane) to yield the title compound (8.18 g, 26.5 mmol, 53% yield) as a red oil.JH NMR (500 MHz, DMSO-ifc) 8 = 9.79 (s, 1H), 6.92 (s, 1H), 6.83 (s, 1H), 2.30 (s, 3H), 1.31 (s, 12H).19F NMR (376 MHz, DMSO-t / e) 6 = -61.57.Scheme for route 4:Intermediate 4Intermediate 4: oxan-4-yl 4-methylbenzene-l-sulfonateEvotec International GmbH EVO75139PC26 November 2025Intermediate 4

[0124] To a solution of tetrahydropyran-4-ol (9.35 mL, 97.91 mmol) in DCM (200 mL), pyridine (15.84 mL, 195.83 mmol) was added followed by DMAP (1.2 g, 9.79 mmol), and 4- methylbenzenesulfonyl chloride (18.67 g, 97.91 mmol). The reaction was stirred at reflux for 18 h. Then the reaction was cooled down to r.t. and washed with brine. The organic phase was dried through a phase separator and evaporated in vacuo. The residue was purified by chromatography on silica gel (0-20% EtOAc in cHex) to yield the title compound (16 g, 62.42 mmol, 64% yield) as a white solid. M / Z'. 257.1 [M+H]+, ESI+, RT = 0.95 (SI).

[0125] 1H NMR (400 MHz, DMSO-t / 6) 8 = 7.86 - 7.76 (m, 2H), 7.54 - 7.44 (m, 2H), 4.71(tt, J= 8.6, 4.2 Hz, 1H), 3.72 (dt, J = 11.8, 4.6 Hz, 2H), 3.40 (ddd, J= 11.9, 8.9, 3.0 Hz, 2H), 2.43 (s, 3H), 1.76 (dddd, J= 14.2, 7.0, 3.9, 1.3 Hz, 2H), 1.57 (dtd, J= 13.0, 8.8, 4.1 Hz, 2H).Scheme for route 5:Intermediate 5: 6-chloro-2-(oxan-4-yl)-2H-pyrazolo [3, 4-b] pyridineIntermediate 5

[0126] 6 -chloro-lH-pyrazolo[3,4-b]pyridine (5 g, 32.56 mmol) and oxan-4-yl 4- m ethylbenzene- 1 -sulfonate (Intermediate 4, 11.68 g, 45.58 mmol) were dissolved in DMF (180 mL), then CS2CO3 (21.35 g, 65.12 mmol) was added. The reaction was stirred at 100 °C for 3 h. After this time the reaction was cooled down to r.t. and a saturated solution of NaHCCL wasEvotec International GmbH EVO75139PC26 November 2025 added. The resulting mixture was extracted with EtOAc (3x). The combined organic layers were washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The crude product was purified by chromatography on silica gel (0-50% EtOAc in cHex), then further purified by chromatography on silica gel (0-50% EtOAc in DCM) to yield the title compound (2.05 g, 8.62 mmol, 26% yield) as a yellow solid. 'H NMR (400 MHz, DMSO-t / 6) 8 = 8.60 (s, 1H), 8.28 (d, J= 8.6 Hz, 1H), 7.14 (d, J= 8.7 Hz, 1H), 4.87 - 4.68 (m, 1H), 4.05 - 3.94 (m, 2H), 3.57 - 3.43 (m, 2H), 2.17 - 2.00 (m, 4H). MS (ESI); / Z: 238.1 [M+H]+, ESI+, RT = 0.71 (SI).Scheme for route 6:Intermediate 6: 3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5-(trifluoromethyl)phenol

[0127] In a 250 mL round-bottomed flask with screw cap 6-chloro-2-(oxan-4-yl)-2H- pyrazolo[3,4-b]pyridine (Intermediate 5, 2.05 g, 8.62 mmol), 3-methyl-2-(4,4,5,5-tetramethyl- l,3,2-dioxaborolan-2-yl)-5-(trifluoromethyl)phenol (Intermediate 3, 3.47 g, 10.35 mmol) and K3PO4 (5.57 g, 25.87mmol) were dissolved in a mixture of water (12 mL) and 1,4-dioxane (60 mL). The mixture was degassed for 5 min with N2, then Pd(dppf)C12 complex with DCM (0.71 g, 0.86 mmol) was added. The mixture was degassed for further 5 min, the cap sealed, and the mixture was heated at 100 °C for 5 h. After this time the reaction was cooled down to r.t. and a saturated solution of NH4CI was added. The aqueous layer was extracted with EtOAc (3 x). The combined organic layers were washed with brine, dried over Na2SO4 and evaporated in vacuo. The crude material was purified by chromatography on silica gel (0-100% EtOAc / EtOH 3 / 1 in cHex), then by chromatography on silica gel (0-10% MeOH in DCM). Fractions containing theEvotec International GmbH EVO75139PC26 November 2025 desired product were collected and evaporated in vacuo. The residue was taken up with MeOH and filtered washing with MeOH (3 x). The obtained solid was taken up with ACN and filtered again. The solid was washed with ACN (3x) and dried in vacuo to yield the title compound (1.6 g, 4.24 mmol, 49% yield) as a pale yellow solid. 'H NMR (400 MHz, D SO-cf) 5 = 10.09 (s, 1H), 8.55 (s, 1H), 8.23 (d, J= 8.5 Hz, 1H), 7.11 (dd, J= 16.6, 1.8 Hz, 2H), 7.06 (d, J= 8.5 Hz, 1H), 4.82 (tt, J= 10.6, 4.9 Hz, 1H), 4.10 - 4.00 (m, 2H), 3.56 (td, J= 11.6, 3.0 Hz, 2H), 2.25 - 2.10 (m, 7H); M / Z 378.3 [M+H]+, ESI+, RT = 0.98 (SI).Scheme for route 7:Intermediate 7Intermediate 7: Di-tert-butyl chloromethyl phosphateIntermediate 7

[0128] Bu4N(HSO4) (1.03 g, 3.02 mmol) and Na2COs (5.12 g, 48.33 mmol) were added to a mixture of potassium di-tert-butyl phosphate (3 g, 12.08 mmol) in water (7.5 mL) / MeTHF (15 mL). Further Water (15 mL) was added to the mixture, and it was stirred at 50 °C for 5 minutes. Then the mixture was cooled at r.t., chloromethyl chlorosulfate (1.83 mL, 18.12 mmol) was added dropwise and the resulting mixture was stirred at r.t. for 2 h. The mixture was diluted with water and extracted with EtOAc (3x). Organic phases were dried and evaporated in vacuo to yield the title compound (2.96 g, 11.44 mmol, 95% yield) used as such in the next step.JH NMR (400 MHz, CDCh) 5 = 5.66 (d, J= 14.9 Hz, 2H), 1.54 (d, J= 0.7 Hz, 18H).Scheme for route 8:Evotec International GmbH EVO75139PC26 November 2025Intermediate 8Intermediate 8: di-tert-butyl {3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-ylJ- 5-(trifluoromethyl)phenoxy}methyl phosphate

[0129] In a vial, K2CO3 (549.38 mg, 3.97 mmol), Nal (99 mg, 0.66 mmol), DIPEA (0.11 mL, 0.64 mmol) and freshly prepared di-te / 7-butyl chloromethyl phosphate (Intermediate 7, 1.03 g, 3.97 mmol) were added to a solution of 3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4- b]pyridin-6-yl]-5-(trifluoromethyl)phenol (Intermediate 6, 500 mg, 1.32 mmol) in DMF (13 mL).The mixture was stirred at 55 °C for 20 h. Then it was diluted with water and extracted with EtOAc (3x). Organic phases were dried and evaporated in vacuo. The crude material was purified by chromatography on silica gel (0-90% EtOAc in cHex) to yield the title compound (252 mg, 0.42 mmol, 32% yield). 'H NMR (400 MHz, DMSO-t / e) 8 = 8.57 (s, 1H), 8.25 (d, J=8.5 Hz, 1H), 7.45 (s, 2H), 7.04 (d, J= 8.5 Hz, 1H), 5.55 (d, J= 11.3 Hz, 2H), 4.81 (dt, J= 10.7,5.6 Hz, 1H), 4.04 (d, J = 12.2 Hz, 2H), 3.55 (td, J= 11.4, 3.0 Hz, 2H), 2.20 - 2.08 (m, 7H), 1.33 (s, 18H). M / Z-. 600.3 [M+H]+, ESI+, RT = 1.20 (SI).Evotec International GmbH EVO75139PC26 November 2025Scheme for route 9:Intermediate 8 Example 1Example 1: ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5-(trifluoromethyl)phenoxy}methoxy)phosphonic acidExample 1

[0130] TFA (0.24 mL, 3.18 mmol) was added to a stirred solution of di-tert-butyl {3- methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5-(trifluoromethyl)phenoxy (methyl phosphate (Intermediate 8, 248 mg, 0.40 mmol) in DCM (5 mL). The mixture was stirred at r.t. for 2 h, then volatiles were evaporated in vacuo. The residue was triturated with Et2O (2x) and with MTBE (3x20 vol), then the cake was solubilized with 1 mL of ACN (about 5 vol) and, under stirring, 1.5 mL of MTBE (7.5 vol) was added dropwise until slow formation of a white precipitate occurred. After 10 minutes the solid was filtered and washed with further MTBE to yield the title compound (158 mg, 0.32 mmol, 82% yield).JH NMR (400 MHz, DMSO-tL) 5 = 8.57 (s, 1H), 8.25 (d, J= 8.5 Hz, 1H), 7.45 (d, J= 25.0 Hz, 2H), 7.07 (d, J= 8.5 Hz, 1H), 5.47 (d, J= 11.2 Hz, 2H), 4.80 (td, J= 10.5, 4.9 Hz, 1H), 4.04 (d, J= 11.4 Hz, 2H), 3.57 - 3.52 (m, 2H), 2.19 - 2.08 (m, 7H) (2 OH signals not reported in the multiplet list due to water exchange). M / Z: 488.4 [M+H]+, ESI+, RT = 0.89 (SI).Scheme for route 10:Evotec International GmbH EVO75139PC26 November 2025Example 2: ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5-(trifluoromethyl)phenoxy}methoxy)({[(propan-2- yloxy)carbonyl]oxy}methoxy)phosphinic acid

[0131] Chloromethyl isopropyl carbonate (0.02 mL, 0.165 mmol) was added to a mixture of CS2CO3 (65 mg, 0.192 mmol) and ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin- 6-yl]-5-(trifluoromethyl)phenoxy}methoxy)phosphonic acid (Example 1, 20 mg, 0.042 mmol) in DMSO (0.30 mL) at r.t. The reaction was stirred at r.t. for 18 hours, then the mixture was diluted with water and extracted with EtOAc (3x). The organic phases were collected, dried and evaporated in vacuo, the residual material was purified by reverse chromatography on Cl 8 Biotage cartridge (from 15% to 65% of ACN in H2O +0.1% HCOOH) to afford the title compound (8.5 mg, 0.014 mmol, 34% yield). 'H NMR (500 MHz, DMSO-tL ) 6 = 13.22 - 12.00 (m, 1H), 8.60 (s, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.09 (d, J = 8.5 Hz, 1H), 5.45 (d, 7= 11.5 Hz, 2H), 5.33 (d, J= 13.3 Hz, 2H), 4.87 - 4.78 (m, 1H), 4.74 (td, J= 6.2, 12.5 Hz, 1H), 4.10 - 3.51 (m, 4H), 2.22 - 2.06 (m, 7H), 1.18 (d, J = 6.2 Hz, 6H); M / Z: 604.5 [M+H]+, ESI+, RT = 1.13 (SI).II. Thermodynamic solubilityEvotec International GmbH EVO75139PC26 November 2025

[0132] The purpose was to determine the thermodynamic solubility of INTERMEDIATE6 and EXAMPLE 1 compounds in aqueous media vs pH and in biorelevant media.

[0133] Analytical methodUPLC-UV-MS for solubility measurements vs pHSamples were analyzed by UPLC-UV-MS according to the following method:• Column: Waters Acquity BEH C18, 1.7 pm, 2.1 x 50 mm• Flow rate: 0.65 ml / min• UV detection: 290 nm• Column temperature: 40°C ± 2°C• Sample temperature: 23°C ± 2°C• Injection volume: 0.4 pl• Mobile phase: gradient with solutions A and B prepared according to the following Table 1 :Table 1: UPLC gradient method used for solubility measurements• Ionisation mode: ESI+ZESI-• Source temperature: 600 °C• Capillary voltage: 0.8 kV• Cone voltage: +20V / -20 V

[0134] ProtocolThe following protocol has been used to prepare samples for solubility measurements in aqueous media at room temperature and in biorelevant media at 37 °C.• Weigh appropriate amount of selected compound• Addition at room temperature of selected media• 24 h orbital stirring at room temperature or vortex stirring at 37 °C for biorelevant media protected from light.• Separation of the soluble fraction from the non-soluble one by centrifugation (15 min at 18000 rpm) followed by filtration on PTFE 0.45 pm membrane.• Dilution of filtrate (in triplicate) with selected aqueous medium to be in the range of calibration curve.Evotec International GmbH EVO75139PC26 November 2025• Dosage of the diluted and undiluted samples by UPLC-UV-MS.• A calibration curve (chromatographic UV peak area vs concentration) is established in DMSO in the [1 pg / ml - 1000 pg / ml] concentration range.Table 2: Aqueous media of solubility assaysTable 3: Biorelevant media of solubility assaysEvotec International GmbH EVO75139PC26 November 2025

[0135] ResultsThermodynamic solubility results for INTERMEDIATE 6

[0136] A standard calibration curve (chromatographic peak area vs concentration) was established in DMSO between 1 pg / ml and 1000 pg / ml of Intermediate 6.

[0137] Figure 1 shows the calibration curve between 1 pg / ml and 1000 pg / ml - UPLC-UV peak area at 290 nm vs concentration of INTERMEDIATE 6. In more detail, Figure 1 shows the peak area of the Intermediate 6 UV chromatographic peak recorded at 290 nm for Intermediate 6 solution prepared in DMSO at 1 pg / ml, 10 pg / ml, 100 pg / ml, 250 pg / ml, 500 pg / ml, 800 pg / ml and 1000 pg / ml vs Intermediate 6 concentration in DMSO. The Y-axis report peak expressed in AU.min unit. The X-axis reports Intermediate 6 concentration in DMSO expressed in pg / ml. The linear regression establishes the mathematical expression between LC UV peak area of Intermediate 6 and Intermediate 6 concentration in dosed solution. This expression is used to determine amount of Intermediate 6 solubilized in different media.

[0138] After 24 h stirring the sample preparation, the solubility of INTERMEDIATE 6 has been determined in all media by UPLC-UV using the calibration curve established previously.Table 4: Thermodynamic solubility results of INTERMEDIATE 6Evotec International GmbH EVO75139PC26 November 2025Thermodynamic solubility results for EXAMPLE 1

[0139] A standard calibration curve (chromatographic peak area vs concentration) was established in DMSO between 1 pg / ml and 1000 pg / ml of Example 1.

[0140] Figure 2 shows the calibration curve between 1 pg / ml and 1000 pg / ml - UPLC-UV peak area at 290 nm vs concentration of EXAMPLE 1. In more detail, Figure 2 shows the peak area of the Example 1 UV chromatographic peak recorded at 290 nm for Example 1 solution prepared in DMSO at 1 pg / ml, 10 pg / ml, 50 pg / ml, 100 pg / ml, 250 pg / ml, 500 pg / ml, 800 pg / ml and 1000 pg / ml vs Example 1 concentration in DMSO. The Y-axis report peak expressed in AU.min unit. The X-axis reports Example 1 concentration in DMSO expressed in pg / ml. The linear regression establishes the mathematical expression between LC UV peak area of Example 1 and Example 1 concentration in dosed solution. This expression is used to determine amount of Example 1 solubilized in different media.

[0141] After 24 h stirring the sample preparation, the solubility of EXAMPLE 1 has been determined in all media by UPLC-UV using the calibration curve established previously.Table 5: Thermodynamic solubility results of EXAMPLE 1Evotec International GmbH EVO75139PC26 November 2025III. In vitro gastrointestinal stabilityExperimental design:

[0142] This study was designed to investigate the in vitro stability of EXAMPLE 1 in the gastrointestinal tract where degradation and metabolism of a drug can occur.

[0143] The in vitro stability of EXAMPLE 1 and its potential conversion into the drug, INTERMEDIATE 6, was investigated using biorelevant fluids as SGF (Simulated Gastric Fluid) at pH 1.2 and SIF (Simulated Intestinal Fluid) at pH 6.8, following incubation at a concentration of 5 pM at 37 °C for 240 minutes.

[0144] Samples were analyzed by HPLC-MS / MS to measure EXAMPLE 1 disappearance and INTERMEDIATE 6 formation with the compound concentration expressed as area ratio, determined by dividing the analyte peak area by the internal standard peak area.Test system:

[0145] The following buffers were used in this study:• SGF (Simulated Gastric Fluid) was prepared by adding NaCl (2.0 g) and 12 N HC1 (7.0 mL) to Milli Q Water to a final volume of 1 L and mixed. The pH of the solution was checked and adjusted to 1.2 with HC1 or NaOH, if necessary.Evotec International GmbH EVO75139PC26 November 2025• SIF (Simulated Intestinal Fluid) was prepared dissolving KH2PO4 (68 mg) in 500 pL of Milli-Q Water, and adding NaOH 0.2 M (800 pL) and Milli-Q Water to a final volume of 10 mL. The pH of the solution was checked and adjusted to 6.8 with HC1 or NaOH, if necessary.Methods and Procedures:Test and Control Items Stock Solution Preparations

[0146] Stock solutions of EXAMPLE 1 and INTERMEDIATE 6 were prepared at 10 mM, in DMSO as vehicle. Further dilutions, to obtain 500 pM working solutions (WS), were prepared using DMSO.Stability Assay:

[0147] EXAMPLE 1 were added (spiked) to biorelevant simulated fluids (SGF and SIF) in order to obtain a final concentration of 5 pM. SGF and SIF fluids were incubated at 37 °C in a VDRL Stirrer with thermostatic Cupola. At each time point (0, 5, 15, 30, 60, 120 and 240 minutes), 50 pL of spiked incubated fluids were collected. Samples were added to 50 pL of Milli Q water and extracted by protein precipitation with 300 pL of acetonitrile (ACN) containing internal standards (Rolipram for positive ion mode or Diclofenac for negative ion mode), and centrifuged for 10 minutes at 3000 rpm. Supernatants were collected (100 pL), diluted with 18% ACN in Milli Q water (200 pL) and then injected onto the HPLC MS / MS.

[0148] A solution of INTERMEDIATE 6 was prepared at a concentration of 5 pM in biorelevant fluids (SGF and SIF) as known standard solutions to be compared with INTERMEDIATE 6 monitored in samples from the incubation of the EXAMPLE 1.Sample Analysis:

[0149] Samples were analyzed by HPLC-MS / MS to measure EXAMPLE 1 and INTERMEDIATE 6 formed from prodrug conversion, with the compound concentration expressed as area ratio, determined by dividing the analyte peak area by the internal standard peak area. The formation of INTERMEDIATE 6 was monitored in the analysis without carrying out any calibration curve, but assuming that the formation of the drug from the prodrug is in a 1 : 1 ratio and comparing the area ratio between the areas of the incubated samples with that of the known standard solution of the drug at the same incubation concentration of the prodrug.Evotec International GmbH EVO75139PC26 November 2025Data Analysis

[0150] Peak areas for EXAMPLE 1 and INTERMEDIATE 6 were integrated using Analyst or MultiQuant Software from AB Sciex™ and were exported to Morphit (The Edge) designed to calculate in vitro stability parameters.

[0151] The integrated peak areas of EXAMPLE 1 and of INTERMEDIATE 6 formed at each time point were divided by the respective peak areas of the IS. For EXAMPLE 1, the percent of parent remaining was calculated by normalizing the peak area ratio of parent to IS at 0 minutes (as 100 %).

[0152] Ln plots of the peak area ratios for EXAMPLE 1 were used to determine the halflife.

[0153] Half-life values were calculated from the relationship T1 / 2 = 0.693 / k, where k was the slope of the ln% remaining vs. time curve.Results:

[0154] The in vitro gastrointestinal stability of EXAMPLE 1 was investigated in biorelevant fluids as SGF (Simulated Gastric Fluid) at pH 1.2 and SIF (Simulated Intestinal Fluid) at pH 6.8, following incubation at a concentration of 5 pM at 37 °C for 240 minutes.

[0155] A summary of the stability results, expressed as half-life and % remaining values of EXAMPLE 1 and % of INTERMEDIATE 6 formed during the incubation is reported in Table 6.

[0156] Under the experimental conditions used, EXAMPLE 1 resulted to be stable in biorelevant buffers, SGF and SIF, simulating the gastrointestinal tract. EXAMPLE 1 remaining after 240 minutes at 37°C was almost ~ 100% and the % of INTERMEDIATE 6 formed was very small (< 2%).Table 6: In vitro Stability of EXAMPLE 1 tested at 5 pM in biorelevant buffer, SGF at pH 1.2 and SIF at pH 6.8Evotec International GmbH EVO75139PC26 November 2025IV. In vitro investigation of passive permeability and bioconversion of phosphate prodrug, EXAMPLE 1, in Caco-2 Cells at pH 7.4

[0157] The objective of this study was to determine the passive membrane permeability (Papp) of phosphate prodrug, EXAMPLE 1, and the bioconversion of the phosphate prodrug into the drug, INTERMEDIATE 6, by intestinal alkaline phosphatase (ALP) in human colon carcinoma cell line (Caco-2) at pH 7.4.

[0158] The assay was conducted in triplicate using Caco-2 cell monolayers in a bidirectional manner (Apical to Basolateral (A>B) and Basolateral to Apical (B>A)), in the absence and presence of a P-gp inhibitor (GF120918 at 10 pM). The transport buffer was HBSSH at pH 7.4.

[0159] EXAMPLE 1 was tested at the nominal concentration of 1 pM, in triplicate at 60 min. The bioconversion of phosphate prodrug EXAMPLE 1, into the drug, INTERMEDIATE 6, was monitored in each sample. The reference compounds, digoxin (P-gp substrate), was included at a single concentration of 25 pM, while atenolol (low permeability compound) and metoprolol (high permeability compound) were included at a single concentration of 10 pM. Test incubations were run for 60 min.

[0160] Digoxin transport was evaluated in two directions (apical to basolateral [A>B] and basolateral to apical [B>A], whereas atenolol and metoprolol were tested only in the A>B direction. Incubations were conducted in triplicate.

[0161] Samples were analyzed by LC-MS / MS to measure EXAMPLE 1 and INTERMEDIATE 6 formed from EXAMPLE 1 (prodrug) conversion and control item concentration levels, with the compound concentration expressed as area ratio, determined by dividing the analyte peak area by the internal standard peak area.

[0162] Monolayer integrity of Caco-2 cells was evaluated using the paracellular permeability marker LY.Test system:Evotec International GmbH EVO75139PC26 November 2025

[0163] The test system selected for this study was the Caco-2 cell line (Human colon carcinoma cell line) which was obtained from American Type Culture Collection (ATCC) HTB-37.

[0164] Caco-2 cells were kept in culture in a humidified CO2 incubator (5% CO2 / 95% air) at approximately 37 °C, using DMEM (high glucose, GlutaMAX™ Supplemented with pyruvate, 10% FBS HI and Pen / Strep). For the permeability experiment, cells were seeded onto microporous HTS 96-Multiwell Insert plates from Corning plates (CLS3392, 1.0 pm pore polyester membrane) at a density of 5600 cells / well in DMEM medium.

[0165] Cell culture was for 21-28 days at 37 °C- 5% CO2 with medium change twice per week.Methods and Procedures:Test and Control Items Stock Solution Preparations

[0166] Stock solutions of EXAMPLE 1 and INTERMEDIATE 6, control items (digoxin, metoprolol and atenolol) and control inhibitor (GF 120918) were prepared at 10 mM, in DMSO as vehicle.

[0167] Donor working solutions of EXAMPLE 1, with and without 10 pM GF120918, were prepared at a concentration of 1 pM, diluting the stock solutions in transport buffer (HBSSH).

[0168] Donor working solutions of digoxin, atenolol and metoprolol, were prepared at a concentration of 25 pM or 10 pM, diluting the stock solutions in transport buffer (HBSSH).

[0169] Receiver working solutions did contain transport buffer only or transport buffer (HBSSH) with 10 pM GF120918.

[0170] A working solution of INTERMEDIATE 6 was prepared at a concentration of 1 pM, diluting the stock solutions in transport buffer (HBSSH) as known standard solution to be compared with INTERMEDIATE 6 monitored in samples from Caco-2 incubation.

[0171] LY stock solution was prepared at 10 mM in milliQ water. A donor working solution containing LY was also prepared in transport buffer (HBSSH only) at 100 pM.

[0172] All working solutions in Transport Buffer did contain < 1% DMSO and were prepared immediately before the assay.Permeability AssayEvotec International GmbH EVO75139PC26 November 2025

[0173] Caco-2 cells were pre-incubated (37°C, 15 to 30 minutes), on apical (A) and basolateral (B) sides filling both wells with receiver working solutions containing transport buffer. Following pre-incubation, EXAMPLE 1 and control items (digoxin) transport were measured (in triplicate) in two directions: apical to basolateral [A>B] and basolateral to apical [B>A], in the absence and in the presence of 10 pM GF120918. For [A>B] directional transport, 75 pL of donor working solution with EXAMPLE 1 or digoxin were then added to the A (apical) compartment and 235 pL of receiver working solution to the B (basolateral) compartment. For [B>A] directional transport, 235 pL donor working solution with EXAMPLE 1 or digoxin were added to the B compartment and 75 pL receiver working solution to the A compartment. Transport of permeability reference control compounds, atenolol and metoprolol, were measured in one direction ([A>B]) only, also in triplicate.

[0174] The cells were incubated (at 37 °C, with shaking) for 60 min. Samples were removed from donor solutions and transport buffer (blank samples) for t = 0 samples (Co concentration at time 0) and at the end of the incubation period, from the receptor site (basolateral compartment for A>B direction and apical compartment for B>A direction) and from donor side (Cfmai concentration at the end of incubation, i.e. 1 hr). Samples were transferred into 96-well plate.

[0175] Samples were extracted by protein precipitation with acetonitrile containing Rolipram (for positive ion mode) or Diclofenac (for negative ion mode), which served as generic internal standard compounds. Following centrifugation for 10 min at 3000 rpm, supernatants were transferred to a fresh 96-well plate prior to LC-MS / MS analysis using Agilent RapidFire High-Throughput MS system or in a traditional HPLC-MS / MS system.Membrane Integrity

[0176] Additionally, to evaluate the integrity of the cell monolayer, LY permeability was measured in one direction, [A>B], at the end of incubation. Residual solutions in the apical compartment were gently removed and 75 pL of donor working solution in HBSSH pH 7.4 containing LY at 100 pM were then added to the A compartment and 235 pL of receiver working solution to the B compartment. The cells were incubated at 37 °C for 60 min.

[0177] 100 pL from each sample receiver well, 100 pL / well of donor solution containing100 pM LY and 100 pL / well of transport buffer were transferred to 96 well clear bottom blackEvotec International GmbH EVO75139PC26 November 2025 plate. Fluorescence was measured by using a fluorescence plate reader, at Ex = 485 nm (Excitation wavelength), Em = 535 nm (Emission wavelength).Sample Analysis:

[0178] Samples were analyzed by LC-MS / MS to measure EXAMPLE 1 and INTERMEDIATE 6 formed from prodrug conversion and control item concentration levels, with the compound concentration expressed as area ratio, determined by dividing the analyte peak area by the internal standard peak area. The formation of INTERMEDIATE 6 was monitored in the analysis without carrying out any calibration curve, but assuming that the formation of the drug from the prodrug is in a 1 :1 ratio and comparing the area ratio between the areas of the incubated samples with that of the known standard solution of the drug at the same incubation concentration of the prodrug.Data AnalysisPermeability Data

[0179] The apparent permeability (Papp) for EXAMPLE 1 and digoxin was determined in [A>B] and [B>A] directions in the presence or in the absence of GF120918. For metoprolol and atenolol Papp values were measured only in the [A>B] direction.

[0180] In particular, the following parameters were calculated.

[0181] 1. Papp (apparent permeability) values were calculated for direction [A>B] and[B>A], according to the following equation:Where: dQ / dt is the permeability rate (dQ / dt is amount of test / control items within the incubation period, 60 min) normalized by the receiver chamber volume;Amount of test / control items was expressed as area ratio meaning analyte peak area divided by internal standard peak area ratio;Co is the initial concentration of test / control items (internal standard peak area ratios) in the donor compartment;A is the surface area of the filter, which corresponds to the surface area of the cell monolayer.Evotec International GmbH EVO75139PC26 November 2025The Papp value did have the dimension of a rate (nm / sec) and was reported as average Papp (nm / sec) ± standard deviation from three monolayers for [A>B] and for [B>A] directions, where applicable.Permeability in Caco-2 cells in presence of P-gp inhibitor, GF120918, was considered as the passive membrane permeability and classified as:Low: if Papp was < 10 nm / sec or <1 x 10'6cm / sec;Medium: if Papp between 10 and 100 nm / sec or 1 and 10 x 10'6cm / sec;High: if Papp > 100 nm / sec or > 10 x 10'6cm / sec.

[0182] 2. Monolayer efflux ratios (ER) in Caco-2 cells were derived using mean Papp[A>B] and [B>A] direction according to the following equation:' B - APapp^nm! sec)^EffluxRatb =- BPapp(nml sec)?A potential P-gp interaction was classified as follows:Unlikely: if the ER was lower 2;Possible: if the ER was approximately or higher than 2, with no significant reduction (> 50 %) observed in ER in presence of inhibitor;Likely: if ER was higher than 2 and decreased to ~ 1 in presence of inhibitor or at least 50 % reduction was observed in presence of inhibitor.

[0183] 3. Mass balance (MB) or Recovery % for EXAMPLE 1 and control items was calculated from:MB % = [(MD + MR) / M0] x 100Where:MD = amount of EXAMPLE 1 / control items normalized by the donor chamber volume in donor chamber at time = t;MR = amount of EXAMPLE 1 / control items normalized by the receiver chamber volume in receiver chamber at time = t;Evotec International GmbH EVO75139PC26 November 2025MO = amount of EXAMPLE 1 / control items normalized by the donor chamber volume in donor solution at time zero.Amount of test / control items was expressed as area ratio meaning analyte peak area divided by internal standard peak area ratio;Mass Balance values were reported as average MB ± standard deviation from three monolayers, where applicable.

[0184] 4. Mass balance (MB) % for drug INTERMEDIATE 6 formed by the conversion of prodrug EXAMPLE 1 by intestinal ALP was calculated from:MB % = [(MD + MR) / MO] x 100Where:MD = amount of INTERMEDIATE 6 normalized by the donor chamber volume in donor chamber at time = t;MR = amount of INTERMEDIATE 6 normalized by the receiver chamber volume in receiver chamber at time = t;M0 = amount of INTERMEDIATE 6 normalized by the donor chamber volume in known standard solution prepared at the beginning of the experiment at the same concentration of EXAMPLE 1.Amount of INTERMEDIATE 6 was expressed as area ratio meaning analyte peak area divided by internal standard peak area ratio;Mass Balance values were reported as average MB ± standard deviation from three monolayers, where applicable.Membrane Integrity

[0185] To evaluate the cell integrity the percentage of LY rejection was calculated using the following equation:% LY rejection 100 X [1 RFUbasolateral / RFUapical]Where RFU values were subtracted by the background mean values.Evotec International GmbH EVO75139PC26 November 2025

[0186] Integrity of the cell monolayer was considered acceptable as % LY was > 96% in all the wells used for this investigation.Results:

[0187] EXAMPLE 1 transport was measured at 1 pM in the apical to basolateral (A>B) and basolateral to apical (B>A) directions in HBSSH (pH 7.4), in Caco-2 cells. In vitro passive membrane permeability values as Papp (nm / sec) and mass balance results of EXAMPLE 1 are summarized in Table 7. EXAMPLE 1 was not detected in any receiver compartment from any direction (A > B or either B > A), in absence and in presence of GF120918), so no permeability value could be determined.

[0188] In line with expected expression of intestinal ALP on the cell surface of Caco-2 cells (Figure 3), the prodrug EXAMPLE 1 almost disappeared in apical donor compartment (recovery < 10 %), with a corresponding formation of the drug, INTERMEDIATE 6 (recovery 43-48 %) within the one hour incubation on the cells. In the opposite direction (B > A), EXAMPLE 1 remaining in the basolateral donor compartment was almost ~ 100 %. The small amount of INTERMEDIATE 6 detected in any receiver compartment was due to INTERMEDIATE 6 intrinsic permeability through the Caco-2 monolayer.

[0189] These data confirmed the hypothesis that the phosphate prodrug, EXAMPLE 1, can be converted at the intestinal level by ALP to the drug, INTERMEDIATE 6.Table 7: Results of Passive Membrane Permeability of EXAMPLE 1 tested at 1 uM. inHBSSH pH 7.4, in Caco-2 Cell Monolayers

[0190] Figure 3 shows the conversion of phosphate prodrug, EXAMPLE 1 into the drug (parent compound), INTERMEDIATE 6, in Caco-2 Cells at pH 7.4.

[0191] In more detail, Figure 3 reports % of recovery of EXAMPLE 1 and INTERMEDIATE 6 in y-axes measured in each compartments, apical and basolateral from each directions (A>B or B>A) (x-axes), at the end of incubation, considering as 100% theEvotec International GmbH EVO75139PC26 November 2025 concentration of EXAMPLE 1 and INTERMEDIATE 6 at the beginning of incubation. Evidence of very low recovery of EXAMPLE 1 in the donor compartment from the A>B direction (~ 7-10%) with concomitant increase of recovery of INTERMEDIATE 6 (~ 43-48) confirmed the conversion of the phosphate prodrug, EXAMPLE 1, to the drug, INTERMEDIATE 6 at intestinal level. Abbreviations used in Figure 3 are as follows: Rec AB- GF means receiver compartment apical to basolateral direction in the absence of GF120918; Rec AB+GF means receiver compartment apical to basolateral direction in the presence of GF120918; Don AB-GF means donor compartment apical to basolateral direction in the absence of GF120918; Don AB+GF means donor compartment apical to basolateral direction in the presence of GF120918; Rec BA-GF means receiver compartment basolateral to apical direction in the absence of GF120918; Rec BA+GF means receiver compartment basolateral to apical direction in the presence of GF120918; Don BA-GF means donor compartment basolateral to apical direction in the absence of GF120918; Don BA+GF means donor compartment basolateral to apical direction in the presence of GF120918; Co test item means the initial concentration at t=0 in HBSSH buffer so represents 100%.V. Rat PK

[0192] Experimental design:

[0193] The objective of this study was to determine the PK of INTERMEDIATE 6 following PO administration of phosphate prodrug (EXAMPLE 1). Also, the PK of INTERMEDIATE 6 following PO administration of INTERMEDIATE 6 were determined in the rat to compare INTERMEDIATE 6 exposure.In vivo procedure for PO administration of EXAMPLE 1 to rats:

[0194] EXAMPLE 1 was administered via PO gavage in HPbCD / acetate buffer pH 4.6 50 mM (10% / 90%; w / v) with a dosing volume of 5 mL / kg. Plasma samples were collected and stored at -20 °C until analysis. Male Sprague Dawley rats (260-300 g, Janvier Labs, 53941 Saint Berthevin, France) with cannulas implanted in the jugular vein were used in the PK studies of EXAMPLE 1. Rats were fasted overnight prior to dosing and then in ad libitum food regimen. Blood samples (0.1 mL) were collected from the jugular vein in EDTA-containing microtainer tubes (Sarstedt) to obtain plasma following centrifugation. Rats (n=3) received PO doses of 3, 10 and 50 mg / kg EXAMPLE 1 (corresponding to 2.3, 7.7 and 39 mg / kg INTERMEDIATE 6Evotec International GmbH EVO75139PC26 November 2025 as molar equivalents). Serial plasma samples were taken 15, 30, 45, 60, 120, 180, 300, 420, and 1440 min after dosing. The concentration-time data from this study are shown in Table 8 and graphically in Figure 4. The resulting PK parameters are shown in Table 9.In vivo procedure for PO administration of INTERMEDIATE 6 to rats

[0195] INTERMEDIATE 6 was administered via PO gavage to n=3 rats in 0.5 % w / v methylcellulose SM 400, 3% w / v Transcutol HP, 5 % w / v VIT E TPGS in citrate buffer pH 3, 50 mM according to a cross-over design at two dose levels (10 and 50 mg / kg) with a wash-out period of at least one week before each dose. Administration volume was 10 mL / kg. A second group of n=3 rats received the test item formulated in DMSO / (HPbCD / Water 20% / 80%, w / v) 2% / 98% w / w at 3 mg / kg. Plasma samples were collected and stored at -80 °C until analysis. Male Sprague Dawley rats (325-350 g, Charles River Italia, Cal co, Italy) were used in the PK studies of INTERMEDIATE 6. Rats were in ad libitum food regimen. Blood samples (0.2 mL) were collected from the tail vein in K3 EDTA-containing tubes (SARSTEDT, Code 201341, Batch 3071721) to obtain plasma following centrifugation. Serial plasma samples were taken 30, 60, 120, 180, 300, 420, 720 and 1440 min after dosing. The concentration-time data from this study are shown in Table 8 and graphically in Figure 4. The resulting PK parameters are shown in Table 9.Table 8: INTERMEDIATE 6 rat plasma concentration-time data following PO administration of (A) EXAMPLE 1 and (B) INTERMEDIATE 6A)Evotec International GmbH EVO75139PC26 November 2025B)NQ = Not quantifiable

[0196] Figure 4 shows INTERMEDIATE 6 plasma concentration versus time following PO administration of EXAMPLE 1 (A) and INTERMEDIATE 6 (B). Data represents mean ±Evotec International GmbH EVO75139PC26 November 2025 standard deviation. A dose-dependent increase in plasma concentrations of INTERMEDIATE 6 plasma were observed following administration of both INTERMEDIATE 6 and EXAMPLE 1. Plasma concentrations following administration of EXAMPLE 1 showed greater dose proportionality compared to administration of INTERMEDIATE 6.Table 9: INTERMEDIATE 6 pharmacokinetics in rat following PO administration ofEXAMPLE 1 and INTERMEDIATE 6Dose normalised = PK parameter / equivalent INTERMEDIATE 6 doseNo SD provided for INTERMEDIATE 6 following 3 mg / kg PO administration of EXAMPLE 1 due to n=l rat data being deemed an outlier in the studyAUC = AUCo -24hEvotec International GmbH EVO75139PC26 November 2025

[0197] Figure 5 shows a comparison of INTERMEDIATE 6 AUC following PO administration of EXAMPLE 1 and INTERMEDIATE 6. In more detail, Figure 5 shows INTERMEDIATE 6 plasma AUCo-24h (pg.h / mL) versus INTERMEDIATE 6 equivalent dose (mg / kg) or INTERMEDIATE 6 dose (mg / kg) following PO administration of EXAMPLE 1 and INTERMEDIATE 6, respectively. Data represents mean ± standard deviation unless n=2. Filled circles = INTERMEDIATE 6 following PO administration of EXAMPLE 1; filled triangles = INTERMEDIATE 6 following PO administration of INTERMEDIATE 6. A dosedependent increase in INTERMEDIATE 6 plasma AUCo-24h was observed following PO administration of both INTERMEDIATE 6 and EXAMPLE 1. Plasma AUCo-24h following PO administration of EXAMPLE 1 showed greater dose proportionality compared to administration of INTERMEDIATE 6.Quantitation of INTERMEDIATE 6 by LC / MS / MS in rat plasma following administration of EXAMPLE 1

[0198] Plasma samples (20 pL) were mixed with 120 pL of acetonitrile containing tolbutamide as internal standard (IS). The resulting supernatant was separated from the precipitated proteins by centrifugation for 10 minutes. An aliquot (30 pL) was diluted with four volumes of 0.1 % NH4OH in water and INTERMEDIATE 6 was quantified by liquid chromatography with tandem mass spectrometry (LC / MS / MS). An aliquot of 1 pL was injected for analysis.

[0199] The LC / MS / MS system consisted of a Thermo VANQUISH / VH-P10-A pump, a Thermo VANQUISH / VH-A10-A autosampler, and a Thermo VANQUISH / VH-C10-A column oven. The column was a Waters Acquity UPLC BEH C18 (1.7 pm 50 x 2.1 mm), maintained at 40 °C and a flow rate of 800 pL / min. The mobile phase consisted of 0.1 % NH4OH in HPLC water (A) and 0.1 % NH4OH in acetonitrile (B).

[0200] The initial mobile phase composition was 95 % A. 0.1 min after sample injection, the mobile phase was changed to 5 % A / 95% B until 1.2 minutes and held at that composition for an additional 0.3 minute. The mobile phase was then returned to initial conditions and the column re-equilibrated for 1 minute. Total analysis time was 2.5 minutes.

[0201] The UPLC was interfaced to a Thermo TSQ ALTIS mass spectrometer. Ultra-high purity nitrogen was used as the nebulizing and desolvation gas with Sheath gas set to 60, AuxEvotec International GmbH EVO75139PC26 November 2025Gas set to 15 and Sweep gas set to 2. The desolvation temperature was 350 °C and the source temperature was 380 °C. Data acquisition utilized multiple reaction monitoring (MRM). Ions representing the (M+H) + species for INTERMEDIATE 6 and the internal standard were selected in MSI and collisionally dissociated with argon at a pressure of 2x10-3 torr to form specific product ions which were subsequently monitored by MS2. The transitions, voltages and retention times are summarized in Table 10.Table 10: Parameters for MS / MS analysis of INTERMEDIATE 6 and internal standard following administration of EXAMPLE 1

[0202] The plasma standard curve ranged from 2.5 to 5000 ng / mL and was fitted with a linear regression weighted by 1 / x or 1 / x2. Quality control (QC) samples, prepared in blank plasma, at six concentrations within the calibration curve were also analyzed in triplicate. At least 66 % of all QC samples were within 20 % of their nominal value and at least 50 % of QC samples at a given concentration were within 20 % of their nominal value, indicating acceptable assay performance.

[0203] Non-compartmental analysis (NCA) was performed using Phoenix® WinNonlin® version 8.4 (Certara L.P. (Pharsight), St. Louis, MO). Linear up-log down calculation method was used with extravascular dosing option to determine relevant PK parameters.Evotec International GmbH EVO75139PC26 November 2025Quantitation of INTERMEDIATE 6 by LC / MS / MS in rat plasma following administration of INTERMEDIATE 6

[0204] Plasma samples (10 pL) were mixed with acetonitrile (100 pL) containing rolipram as internal standard (IS). The resulting supernatants were separated from the precipitated proteins by centrifugation for 10 minutes. Water (100 pL) was added to the samples, which were then re-centrifuged and 2 pL were injected for quantification by liquid chromatography with tandem mass spectrometry (LC / MS / MS).

[0205] The LC / MS / MS system consisted of a Waters Acquity UPLC, autosampler, and column oven. The column was a Waters Acquity UPLC BEH C18 (1.7 pm 50 x 2.1 mm), maintained at 40 °C and a flow rate of 700 pL / min. The mobile phase consisted of HPLC water containing 0.1 % formic acid (A) and acetonitrile (B). The initial mobile phase composition was 75 % A. After sample injection, the mobile phase was changed to 10 % A / 90 % B over a period of 1.3 minutes and held at that composition for an additional 0.4 minute. The mobile phase was then returned to initial conditions and the column re-equilibrated for 0.5 minute. Total analysis time was 2.5 minute.

[0206] The UPLC was interfaced to an Applied Biosystems / MDS Sciex API-4000 and TurboIonSpray™ as ionisation interface at 600 °C. Ultra high purity nitrogen was used as the curtain gas at 30 psig, while air was used as exhaust gas at 40 psig. Data acquisition utilized multiple reaction monitoring (MRM). Ions representing the (M+H)+ species for INTERMEDIATE 6 and the internal standard were selected in MSI and collisionally dissociated with nitrogen at a pressure of 8 psig to form specific product ions which were subsequently monitored by MS2. The transitions, voltages and retention times are summarized in Table 11.Table 11: Parameters for MS / MS analysis of INTERMEDIATE 6 and internal standard following administration of INTERMEDIATE 6Evotec International GmbH EVO75139PC26 November 2025

[0207] The plasma standard curve ranged from 1 to 1000 ng / mL and was fitted with a linear regression weighted by 1 / x2. Quality control (QC) samples, prepared in blank plasma, at three concentrations within the calibration curve were also analysed in duplicate. At least 2 / 3 of all QC samples and at least 50 % at each concentration level were within ±15 % of their nominal value, indicating acceptable assay performance. Non-compartmental analysis (NCA) was performed using Phoenix® WinNonlin® version 8.0 (Certara L.P. (Pharsight), St. Louis, MO). Linear-log calculation method was used with extravascular dosing option to determine relevant PK parameters.VI. THP1 IL-ip Release Assay (HTRF):

[0208] The purpose of this assay was to evaluate the activity of the parent compound (INTERMEDIATE 6).

[0209] The parent compound was tested in LPS-primed THP1 cells to assess its pharmacological capability to inhibit NLRP3 activation with Nigericin and the release of IL-ip into the supernatant.

[0210] Day 1 : THP1 cells were plated in 384-well Poly L-Lysine coated plates at a density of 18.000 cells per well in differentiation medium containing RPMI 1640 Medium (no Glutamine & Phenol red), 10 % FBS, 2 mM L-Glutamine, 50 pM 2-Mercaptoethanol and 200 ng / ml PMA and incubated for 24 h at 37 °C, 5% CO2.

[0211] Day 2: After 24 h, medium was exchanged to growth medium containing RPMI 1640 Medium (no Glutamine & Phenol red), 10% FBS, 2 mM L-Glutamine, 50 pM 2- Mercaptoethanol and incubated for 24 h at 37 °C, 5 % CO2.

[0212] Day 3: Parent compound was serially diluted in DMSO, spotted into intermediate plates and prediluted with serum-free growth medium (plate 1) or serum-free growth medium containing 50 pM Nigericin (plate 2).Evotec International GmbH EVO75139PC26 November 2025

[0213] In addition to the parent compound testing area, the plates also contained multiples of High control (0.2 % DMSO, 1 pg / ml LPS, 50 pM Nigericin final) and Low control (reference inhibitor MCC950 (sodium salt) at lOx IC50, 1 pg / ml LPS, 50 pM Nigericin final) for assay normalization purposes.

[0214] THP1 cells were washed with serum -free growth medium and 10 pl parent compound from the intermediate plate 1 was added into 40 pl serum-free medium in the assay plate. After 30 minutes incubation at 37 °C, 5 % CO2, LPS in serum-free growth medium was added to a final concentration of 1 pg / ml for priming followed by incubation for 2.5 h at 37°C, 5% CO2.

[0215] After priming, cells were washed with serum -free growth medium containing 50 pM Nigericin and 10 pl parent compound from the intermediate plate 2 as well as 40 pl serum- free medium with Nigericin were added to the cells for activation and incubated for 2 h 37 °C, 5% CO2. Finally, the supernatant was removed and stored at -20°C.

[0216] Day 4: IL-ip in the supernatant was quantified by HTRF (homogenous time resolved fluorescence) analysis using a human IL-ip Kit from Cisbio. Briefly, 8 pl of supernatant as well as 2 pl of pre-mixed anti-ILip-Crypta antibody and anti-ILip-XL antibody were added to a 384 well plate (Greiner BioOne), incubated for 24 h at r.t. and measured with the PHERAstar FSX Reader (BMG LabTech) at excitation 337 nm (donor) and emission 620 / 665 nm (acceptor).

[0217] The HTRF ratio between donor and acceptor signals was calculated and normalized to High and Low controls to calculate IC50 values (nM). NLRP3 inhibitors decrease LPS / Nigericin-induced IL-ip release which is indicated as a reduction in the HTRF ratio.

[0218] THP1 IL-ip release of the tested parent compound is provided in Table 12:Table 12: NLRP3 inhibitory activityEvotec International GmbH EVO75139PC26 November 2025VII. In Vivo LPS Assay:

[0219] A pharmacodynamic model of lipopolysaccharide (LPS)-induced inflammation was employed to assess the test compounds. In this model, male C57BL / 6 mice (aged 8-10 weeks) were used and randomly assigned to experimental groups.Experimental Procedure:

[0220] 1. EXAMPLE 1 Administration: EXAMPLE 1 was administered at time point 0. It was delivered via oral gavage at a predetermined doses, based on preliminary pharmacokinetic and pharmacodynamic data, to ensure optimal bioavailability.

[0221] 2. LPS Challenge: One hour following EXAMPLE 1 administration, animals were challenged with an intraperitoneal injection of LPS (3 mg / kg, Escherichia coli 055:B5).

[0222] 3. Tissue Collection: Two hours post-LPS injection, animals were euthanized, and tissues of interest, including the brain, cerebrospinal fluid (CSF) and blood plasma, were collected for biomarker and exposure analysis of EXAMPLE 1 and INTERMEDIATE 6.

[0223] Mouse CSF samples (typically 1 to 5 pL) were diluted in artificial cerebral spinal fluid (aCSF) supplemented with 1% Tween and protease inhibitor (Roche cat # 11697498001) and measured on the high-sensitivity SMCxPRO platform using an in-house developed semi- quantitative assay with antibodies specific to IL-ip. Briefly, samples were incubated with a capture antibody (abeam cat# ab242452) conjugated to magnetic beads. After repeated washes, bound IL-ip was then measured with a fluorophore-conjugated detection antibody (abeam cat# ab242703). Antibodies were labelled with respective kits from Merck-Millipore following the manufacturer's instructions. Recombinant IL-ip standard protein (R&D cat# 401-ML-010) was used for IL-ip protein back-calculation.

[0224] Cytokines IL-ip, TNF-a, IL-6 and IL- 10 were measured in mouse plasma with the multiplex immunoassay according manufacturer’s instructions (U-PLEX Custom Biomarker Group 1 mouse assays; Meso Scale Diagnostics MSD). Briefly, samples were added to U-PLEXEvotec International GmbH EVO75139PC26 November 2025 plates coated with capture antibodies / linkers against selected cytokines and incubated for 2h at room temperature. After repeated washes, a sulfo-tag detection antibody was added, incubated for 1 hour at room temperature and washed again. Bound cytokines were then immediately measured in MSD Gold Read buffer using an MSD reader. Recombinant cytokine standard proteins were used for back-calculation.

[0225] Blood plasma and brain homogenate were mixed with precipitant solution. EXAMPLE 1 and INTERMEDIATE 6 were measured by liquid chromatography with tandem mass spectrometry (LC-MS / MS) to address concentrations in related matrices for PK analysis.4. Endpoints and Evaluation:

[0226] The pharmacodynamic efficacy of the EXAMPLE 1 was determined by its ability to modulate LPS-induced increases in IL-ip compared to vehicle-treated control animals. EXAMPLE 1 concentration in blood plasma and brain homogenate collected as described above was below quantifiable levels. INTERMEDIATE 6 concentration and IL-1 p concentration are shown in Table 13.Table 13: In Vivo LPS assay for EXAMPLE 1Data analysis was performed by graphpad Prism 10.3.1. Statistical evaluations were done by One-way ANOVA (Dunn Correction).Evotec International GmbH EVO75139PC26 November 2025EQUIVALENTS

[0227] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0228] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.INCORPORATION BY REFERENCE

[0229] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, patent application, or item of information was specifically and individually indicated to be incorporated by reference. To the extent publications, patents, patent applications, and items of information incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

Evotec International GmbH EVO75139PC26 November 2025Claims1. A compound of formula (I)or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR1is CH3, CF3, CHF2, Cl, OCH3, OCF3or OCHF2;R2, R2aare independently selected from the group consisting of H and R2b;Each R2bis independently selected from the group consisting of -CH2OC(O)OCI-4 alkyl;R3is H or CH3;X1is C(R4) or N;R4is H, Ci-4 alkyl, C3-s cycloalkyl, unsubstituted saturated 4- to 6- membered heterocyclyl, OCH3, CN, Cl or F, wherein Ci-4 alkyl and C3-s cycloalkyl are unsubstituted or substituted with one or more F.Evotec International GmbH EVO75139PC26 November 20252. The compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X1is C(R4).

3. The compound of claim 2 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X1is CH.

4. The compound of any one of claims 1 to 3 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R1is CF3.

5. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3is H.

6. The compound of any one of claims 1 to 4 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3is CH3.

7. The compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2bis -CH2OC(O)OCH2CH3.

8. The compound of any one of claims 1 to 6 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2bis -CH2OC(O)OCH(CH3)2.

9. The compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2, R2aare different.

10. The compound of any one of claims 1 to 9 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein one of R2, R2ais H and the other is R2b.

11. The compound of any one of claims 1 to 8 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2, R2aare the same.

12. The compound of claim 11 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2, R2aare H.Evotec International GmbH EVO75139PC26 November 202513. The compound of any one of claims 1 to 9, 11 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R2, R2aare R2b.

14. The compound of claim 1 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the compound is selected from the group consisting of ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5- (trifluoromethyl)phenoxy}methoxy)phosphonic acid and ({3-methyl-2-[2-(oxan-4-yl)-2H-pyrazolo[3,4-b]pyridin-6-yl]-5- (trifluoromethyl)phenoxy}methoxy)({[(propan-2- yloxy)carbonyl]oxy}methoxy)phosphinic acid.

15. A pharmaceutical composition comprising at least one compound or a pharmaceutically acceptable salt or stereoisomer thereof of any one of claims 1 to 14 together with a pharmaceutically acceptable carrier, optionally in combination with one or more other bioactive compounds or pharmaceutical compositions.

16. A compound or a pharmaceutically acceptable salt or stereoisomer thereof of any one of claims 1 to 14 for use as a medicament.

17. A compound or a pharmaceutically acceptable salt or stereoisomer thereof of any one of claims 1 to 14 or a pharmaceutical composition of claim 15 for use in a method of treating and / or preventing of one or more diseases, disorders or conditions associated with NLR.P3.

18. A compound or a pharmaceutically acceptable salt or stereoisomer thereof of any one of claims 1 to 14 or a pharmaceutical composition of claim 15 for use in a method of treating and / or preventing one or more diseases, disorders or conditions associated with NLRP3 -mediated inflammation.Evotec International GmbH EVO75139PC26 November 202519. The compound or composition for use of claim 17 or 18, wherein the one or more diseases, disorders or conditions are selected from the group consisting of cryopyrin- associated periodic syndromes (CAPS), familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), and neonatal-onset multisystem autoinflammatory syndrome (NOMID).

20. The compound or composition for use of any one of claim 17 to 19, wherein the disease, disorder or condition is selected from the group consisting of (a) chronic inflammatory diseases, (b) metabolic diseases, (c) neurological diseases, (d) diseases associated with inherited, autosomal dominant mutations in NLRP3 that promote NLRP3 inflammasome; (e) asthma and allergic airway inflammation; (f) hypertension; (g) myocardial infarction; (h) hyperinflammation following influenza and / or severe acute respiratory syndrome-coronavirus 2 (SARS CoV-2); (i) Graft-versus-host disease; (j) silicosis; (k) myelodysplastic syndrome; and (1) contact hypersensitivity and joint inflammation triggered by chikungunya virus.

21. The compound or composition for use of claim 20, wherein:(a) the chronic inflammatory disease is selected from the group consisting of gout, rheumatoid arthritis, inflammatory bowel disease (IBD), Crohn’s disease and ulcerative colitis;(b) the metabolic disease is selected from the group consisting of atherosclerosis, diabetes, metabolic syndrome, obesity, liver steatosis, nonalcoholic steatohepatitis (NASH), and liver fibrosis; or(c) the neurological disease is selected from the group consisting of Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), Huntington’s disease (HD), Amyotrophic Lateral Sclerosis (ALS), Prion disease, Traumatic Brain Injury (TBI) and stroke.

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