EP4642763A1 - Crystalline forms of nlrp3 inflammasome inhibitors, chemical processes and chemical compounds - Google Patents

Crystalline forms of nlrp3 inflammasome inhibitors, chemical processes and chemical compounds

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Publication number
EP4642763A1
EP4642763A1 EP23841209.2A EP23841209A EP4642763A1 EP 4642763 A1 EP4642763 A1 EP 4642763A1 EP 23841209 A EP23841209 A EP 23841209A EP 4642763 A1 EP4642763 A1 EP 4642763A1
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EP
European Patent Office
Prior art keywords
salt
trifluoromethyl
compound
pyran
tetrahydro
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23841209.2A
Other languages
German (de)
French (fr)
Inventor
Helena Bernadette LEUSER
Staffan Karlsson
Venkata Subhash PITHANI
Ryuichi FUCHIGAMI
Christopher Breen
Mary Grace RUSSELL
Yuanqing Fang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tanabe Pharma Corp
AstraZeneca AB
Original Assignee
Mitsubishi Tanabe Pharma Corp
AstraZeneca AB
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Filing date
Publication date
Application filed by Mitsubishi Tanabe Pharma Corp, AstraZeneca AB filed Critical Mitsubishi Tanabe Pharma Corp
Publication of EP4642763A1 publication Critical patent/EP4642763A1/en
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/30—Phthalazines
    • C07D237/34—Phthalazines with nitrogen atoms directly attached to carbon atoms of the nitrogen-containing ring, e.g. hydrazine radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C65/00—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups
    • C07C65/32—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups
    • C07C65/40—Compounds having carboxyl groups bound to carbon atoms of six—membered aromatic rings and containing any of the groups OH, O—metal, —CHO, keto, ether, groups, groups, or groups containing keto groups containing singly bound oxygen-containing groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/79—Acids; Esters
    • C07D213/80—Acids; Esters in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/30—Phthalazines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D237/00—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings
    • C07D237/26—Heterocyclic compounds containing 1,2-diazine or hydrogenated 1,2-diazine rings condensed with carbocyclic rings or ring systems
    • C07D237/30—Phthalazines
    • C07D237/32—Phthalazines with oxygen atoms directly attached to carbon atoms of the nitrogen-containing ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D309/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings
    • C07D309/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
    • C07D309/08—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only ring hetero atom, not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D309/10—Oxygen atoms
    • C07D309/12—Oxygen atoms only hydrogen atoms and one oxygen atom directly attached to ring carbon atoms, e.g. tetrahydropyranyl ethers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/13—Crystalline forms, e.g. polymorphs

Definitions

  • Described in this specification are crystalline forms of inhibitors of the NLRP3 inflammasome, pharmaceutical compositions comprising such crystalline forms, chemical processes useful for the preparation of inhibitors of the NLRP3 inflammasome, and intermediate compounds useful in such processes.
  • the NLRP3 inflammasome is a multi-protein complex consisting of the NLR family pyrin domain containing 3 (NLRP3) protein, PYD and CARD domain containing (ASC, also known as PYCARD), and caspase 1 (CASP1), and is a stress sensing pathway leading to an inflammatory response (Swanson KV et al. Nat Rev Immunol. 2019 Aug;19(8):477-489). When activated, these three proteins condense into a large multiprotein complex; a speck.
  • the NLRP3 protein consists of three domains, PYD, NACHT and LRR (Sharif H et al. Nature. 2019 Jun;570(7761):338-343).
  • the aminoterminal PYD domain is thought to be important in the binding of NLRP3 to the PYD domain of ASC
  • the NACHT domain has ATPase activity suggested to regulate the oligomerization, potentially through conformational change of the LRR domain
  • the LRR domain is considered to induce autoinhibition by folding onto the NACHT domain.
  • the activity of the NLRP3 protein is further regulated by a multitude of posttranslational modifications including phosphorylations and ubiquitinylations.
  • pyroptosis Downstream effects of an activated NLRP3 inflammasome is further expanded through caspase- 1 mediated cleavage and hence activation of gasdermin D.
  • gasdermin D forms a large pore leading to a regulated form of lytic cell death called pyroptosis (Kovacs SB et al. Trends Cell Biol. 2017 Sep;27(9):673-684).
  • pyroptosis amplifies inflammation through release of cellular contents subsequently leading to the recruitment and influx of additional immune cells.
  • cryopyrin-associated periodic syndromes 1, 2 and 3 where causative genetic lesions in NLRP3 have been identified (Kacar M et al. Rheumatology (Oxford). 2019 Nov l;58(Suppl 6):vi31-vi43).
  • NLRP3 inflammasome activation has been linked to multiple indications (as discussed herein) often with demonstrated presence or activity in the affected tissue, and inhibition of the NLRP3 inflammasome will therefore resolve unfavorable inflammation.
  • the NLRP3 inflammasome can modulate both acute kidney injury (AKI) and chronic kidney disease (CKD); mice deficient in NLRP3 inflammasome components and its downstream mediators can be protected from renal injury in experimental models of both AKI and CKD (Hutton HL et al. Nephrology. 2016 21(9):736-744). Inflammation plays a key role in the pathogenesis of AKI; after an initial ischaemic, septic or nephrotoxic trigger, release of inflammatory cytokines and chemokines by renal endothelial cells and tubular epithelium can result in leukocyte recruitment and subsequent renal injury.
  • AKI acute kidney injury
  • CKD chronic kidney disease
  • Nonalcoholic fatty liver disease is defined as excess liver fat accumulation (fatty liver) greater than 5% induced by causes other than alcohol intake.
  • Fatty liver progresses to nonalcoholic steatohepatitis (NASH) with or without fibrosis in a variable proportion of individuals, ultimately leading to liver cirrhosis, liver failure and hepatocellular carcinoma in susceptible individuals (Friedman et al Nat Med. 2018 Jul;24(7): 908-922).
  • Inflammation including the NLRP3 inflammasome contributes to the pathogenesis of most acute and chronic liver diseases including NAFLD, NASH, alcoholic steatohepatitis, chronic hepatitis C virus (HCV) infection, ischaemia-reperfusion injury and paracetamol-induced liver injury (Szabo et al Nat Rev Gastroenterol Hepatol 2015; 12:387-400).
  • Hepatic NLRP3 and down-stream target mRNA levels are increased in NASH and correlate with liver collagen expression levels in humans.
  • NLRP3 inducible activation increases liver fibrosis in mice and NLRP3 knock-out mice are protected from experimentally induced NASH including liver inflammation and fibrosis (Wree et al J Mol Med, 2014, DOI: 10.1007/s00109-014-l 170-1).
  • NLRP3 inflammasome inhibition using a small molecule inhibitor (MCC950) reduces liver inflammation and fibrosis in experimental models of NASH where mice were fed a high fat diet or a methionine and choline deficient diet (Mridha et al Journal of Hepatology, 2017, DOI: 10.1016/j.jhep.2017.01.022).
  • MCC950 small molecule inhibitor
  • NLRP3 inflammasome inhibition can protect against liver diseases including NAFLD and NASH.
  • Cryopyrin-associated periodic syndromes include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome or neonatal onset multi-system inflammatory disease (NOMID) (Booshehri ML et al. J Clin Immunol. 2019 Apr;39(3):277-286).
  • FCAS familial cold autoinflammatory syndrome
  • MFS Muckle-Wells syndrome
  • CINCA chronic infantile neurologic cutaneous articular
  • NOMID neonatal onset multi-system inflammatory disease
  • the NLRP3 inflammasome has also been indicated in gout and pseudo gout since monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD), both crystals found in gout, are activators of the NLRP3 inflammasome (Martinon F et al. Nature 440: 237-241, 2006).
  • MSU monosodium urate
  • CPPD calcium pyrophosphate dihydrate
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • Addison s disease
  • celiac disease celiac disease
  • SLE systemic lupus erythematous
  • vitiligo Shaw PJ et al. Trends Mol Med. 2011 Feb; 17(2): 57-64.
  • NLRP3 may be involved in the progression of several chronic pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma (De Nardo D. et al. Am J Pathol. 2014 Jan;184(l):42-54).
  • IPF idiopathic pulmonary fibrosis
  • COPD chronic obstructive pulmonary disease
  • asthma De Nardo D. et al. Am J Pathol. 2014 Jan;184(l):42-54.
  • IBD inflammatory bowel disease
  • inhibitors of the NLRP3 inflammasome may be useful in the treatment of the diseases and conditions described herein which are linked to NLRP3 inflammasome activation.
  • no small-molecule synthetic inhibitor of the NLRP3 inflammasome has been approved for medical use.
  • This specification also describes, in part, a crystalline form of Compound 1 which is crystalline Form A.
  • This specification also describes, in part, a crystalline form of 2-(4-(((lR,2R)-2- hydroxycyclohexyl)amino)pyrido[3,4-d]pyridazin-l-yl)-5-(trifluoromethyl)phenol (Compound 2), hydrochloride salt.
  • composition comprising a crystalline form as disclosed herein, and a pharmaceutically acceptable excipient.
  • This specification also describes, in part, a crystalline form or pharmaceutical composition as disclosed herein for use in therapy.
  • This specification also describes, in part, a crystalline form or pharmaceutical composition as disclosed herein for use in the treatment of a subject with a disease or condition in which NLRP3 inflammasome activity is implicated.
  • This specification also describes, in part, a method of inhibiting NLRP3 inflammasome activity in a subject in need thereof, comprising administering a crystalline form or pharmaceutical composition as disclosed herein to the subject.
  • This specification also describes, in part, a process for the preparation of a compound of Formula (Ic), or a salt thereof, comprising the steps:
  • This specification also describes, in part, a process for the preparation of 4-(2-hydroxy-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, comprising the steps:
  • This specification also describes, in part, a process for the preparation of 2-(4- chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, comprising the steps:
  • This specification also describes, in part, a compound that is
  • This specification also describes, in part, a compound that is salt thereof.
  • This specification also describes, in part, a compound that i
  • This specification also describes, in part, a compound that i salt thereof, wherein X is CH or N.
  • This specification also describes, in part, a compound that is salt thereof, wherein X is CH or N.
  • This specification also describes, in part, a compound that i salt thereof, wherein X is CH or N. This specification also describes, in part, a compound that is salt thereof.
  • This specification also describes, in part, a compound that is salt thereof.
  • This specification also describes, in part, a compound that , or a salt thereof.
  • This specification also describes, in part, a compound that i
  • Figure 1 is an X-Ray Powder Diffraction Pattern of Form A of Compound 1.
  • Figure 2 shows the TG/DTA output from analysis of Form A of Compound 1.
  • Compound 1 is a compound with chemical name (2S)-3-[[4-[2-hydroxy-4- (trifluoromethyl)phenyl]phthalazin-l-yl]amino]propane-l,2-diol, and the following chemical structure:
  • Compound 2 is a compound with chemical name 2-(4-(((lR,2R)-2- hydroxy cyclohexyl)amino)pyrido[3,4-d]pyridazin-l-yl)-5-(trifluoromethyl)phenol, and the following chemical structure:
  • Compound 1 and Compound 2 are inhibitors of the NLRP3 inflammasome, and may therefore be useful in the treatment of disease states in which inhibition of the NLRP3 inflammasome is beneficial.
  • “About” may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Embodiments described herein as “comprising” one or more features may also be considered as disclosure of the corresponding embodiments “consisting of' such features.
  • solvated forms may be a hydrated form, such as a hemi -hydrate, a mono-hydrate, a di-hydrate, a tri-hydrate or an alternative quantity thereof. All such solvated and unsolvated forms of compounds described herein are encompassed herein.
  • Atoms of the compounds and salts described in this specification may exist as their isotopes. All compounds described herein where an atom is replaced by one or more of its isotopes (for example a compound described herein where one or more carbon atom is an "C or 13 C carbon isotope, or where one or more hydrogen atoms is a 2 H or 3 H isotope) are encompassed herein.
  • Compounds described herein may exist in one or more geometrical, optical, enantiomeric, and diastereomeric forms, including, but not limited to, cis- and trans-forms, E- and Z-forms, and R-, S- and meso-forms. Unless otherwise stated a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g. chromatographic techniques and recrystallisation techniques). Where appropriate such isomers can be prepared by the application or adaptation of known methods.
  • the compounds described herein may include one or more chiral centres.
  • a structure or chemical name in this specification does not indicate chirality, the structure or name is intended to encompass any single stereoisomer corresponding to that structure or name, as well as any mixture of stereoisomers (e.g. a racemate).
  • bonds drawn as solid and hashed wedges i.e. " and )
  • a single stereoisomer can be obtained by isolating it from a mixtures of isomers (e.g. a racemate) using, for example, chiral chromatographic separation.
  • a single stereoisomer is obtained through direct synthesis from, for example, a chiral starting material.
  • the compounds described herein are provided as a single enantiomer being in enantiomer excess (%ee) of > 95%, > 98%, or > 99%. Conveniently a single enantiomer is present in an enantiomer excess of > 99%.
  • a compound described herein is provided as a single enantiomer being in enantiomer excess (%ee) in the range 95 to 100%.
  • Compounds described herein may exist in one or more tautomeric forms, including, but not limited to, keto-, and enol-forms.
  • a reference to a particular compound includes all tautomeric forms, including mixtures thereof. Accordingly, a structure depicted herein as one tautomer is intended to also include other tautomers.
  • the different physical properties of the crystalline forms with respect to each other and with respect to the non-crystalline state may influence markedly the chemical and pharmaceutical processing of a compound, particularly when the compound is prepared or used on an industrial scale.
  • the drug substance, and compositions containing it should be capable of being effectively stored over appreciable periods of time, without exhibiting a significant change in the active component’s physico-chemical characteristics (e.g. its chemical composition, density, hygroscopicity and solubility).
  • Compound 1 may be prepared in a crystalline form.
  • This crystalline form may be characterised as being a particular polymorphic form.
  • the degree of crystallinity may be greater than about 60%, optionally greater than about 80%, greater than about 90% or greater than about 95%. In one embodiment, the degree of crystallinity is greater than about 98%.
  • an X-ray powder diffraction pattern may be obtained which has one or more measurement errors depending on measurement conditions (such as equipment or machine used).
  • intensities in an X-ray powder diffraction pattern may fluctuate depending on measurement conditions. Therefore it should be understood that the crystalline forms described herein are not limited to the crystals that provide X-ray powder diffraction patterns that are identical to the X-ray powder diffraction pattern shown in the Figures, and any crystals providing X-ray powder diffraction patterns substantially the same as those shown in the Figures fall within the scope of the embodiments described herein. A person skilled in the art of X-ray powder diffraction is able to judge the substantial identity of X-ray powder diffraction patterns.
  • a measurement error of a diffraction angle in an X-ray powder diffractogram is about 5% or less, in particular plus or minus 0.2° 2-theta, and such degree of a measurement error should be taken into account when considering the X-ray powder diffraction patterns in the Figures herein and when reading the data described herein. Furthermore, it should be understood that intensities may fluctuate depending on experimental conditions and sample preparation (preferred orientation).
  • a useful crystalline polymorphic form of Compound 1 has been produced using the conditions described in Example 1.
  • the peaks of the X-ray diffraction patterns are measured using Cu K Directory radiation.
  • This polymorphic form may be characterised in that it provides at least one of the following 29 values measured using Cu Li radiation: 13.0, 19.3, 19.8, 22.7, and 24.1°.
  • Polymorphic Form A of Compound 1 is characterised in providing an X-ray powder diffraction pattern, substantially as shown in Figure 1.
  • polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 1.
  • crystalline Form A of Compound 1 is unsolvated.
  • a useful crystalline polymorphic form of the hydrochloride salt of Compound 2 has been produced using the conditions described in Example 3.
  • Compound 1 is an inhibitor of the NLRP3 inflammasome
  • pharmaceutical compositions comprising crystalline Compound 1, in particular polymorphic Form A of Compound 1 (as described herein), and a pharmaceutically acceptable excipient are expected to be useful in the treatment of disease states in which inhibition of the NLRP3 inflammasome is beneficial.
  • a pharmaceutical composition comprising Compound 1 in crystalline form, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising Compound 1 in crystalline form and a pharmaceutically acceptable excipient, wherein the crystalline form is Form A.
  • a pharmaceutical composition comprising polymorphic Form A of Compound 1, and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 1, and a pharmaceutically acceptable excipient.
  • compositions comprising crystalline Compound 2, in particular the hydrochloride salt of Compound 2, and a pharmaceutically acceptable excipient are expected to be useful in the treatment of disease states in which inhibition of the NLRP3 inflammasome is beneficial.
  • a pharmaceutical composition comprising the hydrochloride salt of Compound 2 in crystalline form, and a pharmaceutically acceptable excipient.
  • the pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients.
  • the excipient(s) selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to persons skilled in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, Sixth edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian.
  • compositions may function as, for example, adjuvants, diluents, carriers, stabilisers, flavourings, colorants, fillers, binders, disintegrants, lubricants, glidants, thickening agents and coating agents.
  • certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the composition and what other excipients are present in the composition.
  • the amount of pharmaceutically acceptable excipient in the pharmaceutical composition is greater than or equal to 1 mg. In a further embodiment, the amount of pharmaceutically acceptable excipient in the pharmaceutical composition is greater than or equal to 10 mg. In a further embodiment, the amount of pharmaceutically acceptable excipient in the pharmaceutical composition is greater than or equal to 100 mg.
  • compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, or dispersible powders or granules), for topical use (for example as creams, ointments, or aqueous or oily suspensions), for administration by inhalation (for example as a finely divided powder), for administration by insufflation (for example as a finely divided powder), or as a suppository for rectal dosing.
  • the compositions may be obtained by conventional procedures well known in the art.
  • Compositions intended for oral use may contain additional components, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
  • pharmaceutically acceptable is used to specify that an object (for example an excipient) is suitable for use in patients.
  • An example list of pharmaceutically acceptable salts can be found in the Handbook of Pharmaceutical Salts: Properties, Selection and Use, P. H. Stahl and C. G. Wermuth, editors, Weinheim/Zurich:Wiley-VCH/VHCA, 2002.
  • the crystalline forms and pharmaceutical compositions described herein that comprise Compound 1 or Compound 2 are expected to be useful in therapy.
  • the term “therapy” is intended to have its normal meaning of dealing with a disease or condition in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology.
  • the term “therapy” also includes “prophylaxis” unless there are specific indications to the contrary.
  • the terms “therapeutic” and “therapeutically” should be interpreted in a corresponding manner.
  • prophylaxis is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease or condition and secondary prophylaxis whereby the disease or condition has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or condition, or the development of new symptoms associated with the disease or condition.
  • treatment is used synonymously with “therapy”.
  • treat can be regarded as “applying therapy” where “therapy” is as defined herein.
  • a method for treating a disease or condition in which NLRP3 inflammasome activity is implicated, in a subject in need of such treatment which comprises administering to said subject a therapeutically effective amount of a crystalline form or pharmaceutical composition as described herein.
  • kidney diseases such as acute kidney injury, chronic kidney disease, and diabetic kidney disease
  • cardiovascular diseases such as coronary atherosclerotic heart disease, cardiomyopathy, myocardial infarction, cardiac hypertrophy, and ischaemia-reperfusion injury
  • liver diseases such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, alcoholic steatohepatitis, chronic hepatitis C virus infection, and paracetamol-induced liver injury
  • inflammatory diseases such as autoinflammatory disorders, Cryopyrin-associated periodic syndromes, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome, and neonatal onset multi-system inflammatory disease (NOMID); inflammatory skin diseases such as acne vulgaris, and hidradenitis suppurativa; inflammatory bowel diseases such as ulcerative colitis (UC), and Crohn’s disease; autoimmune diseases such as
  • a disease or condition selected from acute kidney injury, chronic kidney disease, diabetic kidney disease, coronary atherosclerotic heart disease, cardiomyopathy, myocardial infarction, cardiac hypertrophy, ischaemia-reperfusion injury, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, alcoholic steatohepatitis, chronic hepatitis C virus infection, paracetamol-induced liver injury, autoinflammatory disorders, Cryopyrin-associated periodic syndromes, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome, neonatal onset multi-system inflammatory disease (NOMID), acne vulgaris, hi dradenitis suppurativa, ulcerative colitis (UC), Crohn’s disease, gout, pseudo gout, rheumatoid arthritis (RA), multiple sclerosis (MS), Addison’s disease, celiac disease, system
  • a crystalline form or pharmaceutical composition as described herein, for use in therapy in one embodiment there is provided a crystalline form or pharmaceutical composition as described herein, for use in therapy.
  • a crystalline form or pharmaceutical composition as described herein for use in a method for treating a disease or condition as described herein.
  • a crystalline form or pharmaceutical composition as described herein in the manufacture of a medicament for a disease or condition as described herein.
  • therapeutically effective amount refers to an amount of a crystalline form or pharmaceutical composition as described in any of the embodiments herein which is effective to provide “therapy” in a subject, or to “treat” a disease or condition in a subject.
  • the therapeutically effective amount may cause any of the changes observable or measurable in a subject as described in the definition of “therapy”, “treatment” and “prophylaxis” above.
  • effective amounts may vary depending on route of administration, excipient usage, and co-usage with other agents.
  • the amount of the crystalline form or pharmaceutical composition described in this specification and the amount of the other pharmaceutically active agent(s) are, when combined, jointly effective to treat a targeted disorder or condition in the subject.
  • the combined amounts are in a “therapeutically effective amount” if they are, when combined, sufficient to decrease the symptoms of a disease or condition responsive to inhibition of the NLRP3 inflammasome as described above.
  • such amounts may be determined by one skilled in the art.
  • Subjects include, for example, mammals, for example, humans.
  • Compounds 1 and 2 may be prepared by a Suzuki-Miyaura coupling with an appropriate boronic acid or boronate ester (for example, see Scheme 2 and the synthesis described in Example 1). Similar routes have also been used in the synthesis of other known inhibitors of the NLRP3 inflammasome (see WO2022/135567, for example). However, such a synthetic procedure requires additional steps to synthesise the boronic acid/boronate ester intermediate, and starting materials for synthesis of boronic acids/boronate esters tend to be halogenated, which is undesirable in view of the halogenation steps required to prepare such starting materials. Furthermore, the use of palladium catalysts in the synthesis of pharmaceuticals is undesirable since such residual palladium may contaminate reaction products, and the level of residual palladium in the pharmaceutical product must be controlled.
  • Suitable solvents for steps (a)-(c) include ethers, for example THF.
  • the alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi.
  • Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C.
  • step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C.
  • steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine.
  • magnesium chloride is added in step (b).
  • the hydrazine used in step (d) may be in the form of hydrazine hydrate. Suitable solvents for step (d) include THF and EtOH.
  • Step (d) may be performed at a temperature of -40 °C to 40 °C, such as -30 °C to 30 °C, -30 °C to 10 °C, and -30 °C to 0 °C.
  • the acid used in step (d) may be a carboxylic acid, such as acetic acid.
  • tri fl uoromethy I phenyl (phthal azin- 1 (2H)-one. or a salt thereof (which is of use in the synthesis of Compound 1), in a more efficient manner.
  • Steps (a)-(c) Suitable solvents for steps (a)-(c) include ethers, for example THF.
  • the alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi.
  • Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C.
  • step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C.
  • steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine.
  • magnesium chloride is added in step (b).
  • the aqueous acid is hydrochloric acid.
  • Step (d) may be performed at temperatures of 0 °C to 70 °C, such as 10 °C to 60 °C, 20 °C to 50 °C, and 30 °C to 40 °C.
  • Step (e): The hydrazine used in step (e) may be in the form of hydrazine hydrate.
  • Suitable solvents for step (e) include THF.
  • Step (e) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
  • the compound of Formula (Ic), or salt thereof may by isolated by precipitation and filtration, such as precipitation from a mixture of THF and heptane, optionally 2:1 THF/heptane.
  • a process for the preparation of 4-(2- hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof comprising the steps: (a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)tithium;
  • a phenol protecting group is not required for chlorination of 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one with a suitable chlorinating agent such as POCh.
  • a suitable chlorinating agent such as POCh.
  • the reaction of step (a) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
  • the 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, used in step (a) is prepared by a process described herein.
  • Suitable solvents for steps (a)-(c) include ethers, for example THF.
  • the alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi.
  • Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C.
  • step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C.
  • steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine.
  • magnesium chloride is added in step (b).
  • the aqueous acid is hydrochloric acid.
  • Step (d) may be performed at temperatures of 0 °C to 70 °C, such as 10 °C to 60 °C, 20 °C to 50 °C, and 30 °C to 40 °C.
  • Step (e): The hydrazine used in step (e) may be in the form of hydrazine hydrate.
  • Suitable solvents for step (e) include THF.
  • Step (e) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
  • Step (1) A suitable solvent for step (1) is MeCN.
  • the reaction of step (1) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
  • the reaction of step (g) may be performed at a temperature of 20 °C to 150 °C, such as 60 °C to 140 °C, and 100 °C to 140 °C.
  • Diol-protected (2S)-3-amino-l,2-propanediol refers to a compound in which the two alcohol groups of (2S)-3-amino-l,2-propanediol are protected using known protecting groups suitable for 1,2-diols. Such protection may be provided by forming a 5-membered ketal, such as a 5-membered acetal, from the 1 ,2-diol. Therefore, in one embodiment, the diol-protected (2S)- 3-amino-l,2-propanediol is (S)-(2,2-dimethyl-l,3-dioxolan-4-yl)methanamine.
  • the removal of the diol-protecting group may be performed according to known methods.
  • the diol protecting group is removed using aqueous acid or alcoholic acid.
  • the diol protecting group is removed using hydrochloric acid in an alcohol solvent, such as isopropyl alcohol.
  • Step (h): Isolation of Compound 1, or a salt thereof, may be performed by addition of water to a solution of Compound 1, or a salt thereof, to precipitate Compound 1, or a salt therof.
  • a process for the preparation of Compound 1, or a salt thereof comprising the steps: (a) reacting 2-(3-(trifluoromethyl)phenoxy)tetiahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)tithium;
  • Suitable solvents for steps (a)-(c) include ethers, for example THF.
  • the alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi.
  • Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C.
  • step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C.
  • steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine.
  • magnesium chloride is added in step (b).
  • the hydrazine used in step (d) may be in the form of hydrazine hydrate. Suitable solvents for step (d) include THF and EtOH.
  • Step (d) may be performed at a temperature of -40 °C to 40 °C, such as -30 °C to 30 °C, -30 °C to 10 °C, and -30 °C to 0 °C.
  • the acid used in step (d) may be a carboxylic acid, such as acetic acid.
  • the coupling reagent is a reagent suitable for activating a carboxyl group for amide synthesis.
  • Such coupling agents are known in the art and include, for example, ((1H- benzo [d] [ 1 ,2,3]triazol- 1 -y l)oxy )tri(py rrolidin- 1 -y l)phosphonium hexafluorophosphate(V).
  • the reaction of step (e) may be performed in the presence of a base, such as DBU.
  • the reaction of step (e) may be performed in the presence of molecular sieves to capture water, for example molecular sieve 0.3 nm.
  • Suitable solvents for step (e) include polar apotic solvents, such as MeCN.
  • Step (e) may be performed at a temperature of 20 °C to 70 °C, such as 30 °C to 60 °C.
  • the removal of the THP protecting group and diol protecting group may be performed according to known methods.
  • the THP protecting group and diol protecting group are removed using aqueous acid or alcoholic acid.
  • the THP protecting group and diol protecting group are removed using hydrochloric acid in an alcohol solvent, such as isopropyl alcohol.
  • a process for the preparation of Compound 2, or a salt thereof comprising the steps:
  • Suitable solvents for steps (a)-(c) include ethers, for example THF.
  • the alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi.
  • Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C.
  • step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C.
  • steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine.
  • magnesium chloride is added in step (b).
  • the hydrazine used in step (d) may be in the form of hydrazine hydrate.
  • Suitable solvents for step (d) include THF and EtOH.
  • Step (d) may be performed at a temperature of -40 °C to 40 °C, such as -30 °C to 30 °C, -30 °C to 10 °C, and -30 °C to 0 °C.
  • the acid used in step (d) may be a carboxylic acid, such as acetic acid.
  • the coupling reagent is a reagent suitable for activating a carboxyl group for amide synthesis.
  • Such coupling agents are known in the art and include, for example, ((1H- benzo [d] [ 1 ,2,3]triazol- 1 -y l)oxy )tri(py rrolidin- 1 -y l)phosphonium hexafluorophosphate(V).
  • the reaction of step (e) may be performed in the presence of a base, such as DBU.
  • the reaction of step (e) may be performed in the presence of molecular sieves to capture water, for example molecular sieve 0.3 nm.
  • Suitable solvents for step (e) include polar apotic solvents, such as MeCN.
  • Step (e) may be performed at a temperature of 20 °C to 70 °C, such as 30 °C to 60 °C.
  • Step (I): The removal of the THP protecting group may be performed according to known methods.
  • the THP protecting group is removed using aqueous acid or alcoholic acid.
  • the THP protecting group is removed using hydrochloric acid in an alcohol solvent, such as isopropyl alcohol.
  • novel chemical intermediates that, as described herein, are useful in the synthesis of inhibitors of the NLRP3 inflammasome such as Compound 1 and Compound 2.
  • a compound that i or a salt thereof In one embodiment there is provided a compound that i or a salt thereof. In one embodiment there is provided a compound that i a salt thereof.
  • a compound that i a salt thereof there is provided a compound that i a salt thereof.
  • a chemical intermediate as described in the Examples herein, or a salt thereof there is provided.
  • Salts of compounds described herein may be, for example, acid-addition salts or baseaddition salts.
  • An acid addition salt of a compound described herein may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person.
  • An acid addition salt may for example be formed using an inorganic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid.
  • An acid addition salt may also be formed using an organic acid selected from the group consisting of trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
  • an organic acid selected from the group consisting of trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
  • a base-addition salt of a compound described herein may be formed by bringing the compound into contact with a suitable inorganic or organic base under conditions known to the skilled person.
  • a suitable inorganic or organic base under conditions known to the skilled person.
  • an alkali metal such as sodium, potassium, or lithium
  • an alkaline earth metal such as a calcium
  • an alkali metal or alkaline earth metal hydroxide or alkoxide e.g., an ethoxide or methoxide
  • a suitably basic organic amine e.g., a choline or meglumine
  • Flash chromatography was performed using either normal phase silica FLASH+® (40M, 25M or 12M), Biotage® SNAP Cartridges KP-Sil (340, 100, 50 or 10), Biotage® SNAP Cartridges KP- NH (340, 100, 50 or 10), or Agela® Flash Column Silica-CS Cartridges (330, 180, 120, 80) unless otherwise stated.
  • Reversed phase flash chromatography was performed using Agela® C-18 spherical 20-35 pm 100A cartridges unless otherwise stated.
  • DIPEA N,N-Diisopropylethylamine
  • DMSO-d6 Hexadeuterodimethyl sulfoxide
  • IPE isopropyl ether
  • iPrOAc Isopropyl acetate
  • NMP N-Methyl-2-pyrrolidone
  • Step 1 Intermediate 1: 4-chloro-N-[[(4S)-2,2-dimethyl-l,3-dioxolan-4-yl]methyl]phthalazin-l- amine
  • Step 2 Intermediate 2: 2-[4-[[(4S)-2,2-dimethyl-l,3-dioxolan-4-yl]methylamino]phthalazin-l- yl] -5-(trifluoromethyl)phenol
  • Step 3 Compound 1: (2S)-3-[[4-[2-hydroxy-4-(trifluoromethyl)phenyl]phthalazin-l- yl]amino]propane-l,2-diol (Form A)
  • X-ray powder diffraction (XRPD) data is measured with Corundum as an internal reference.
  • the XRPD pattern is determined by mounting a sample on a zero-background holder, single silicon crystal, and spreading out the sample into a thin layer.
  • the powder X-ray diffraction is recorded with a Theta-Theta PANalytical X’Pert PRO (wavelength of X-rays 1.5418 A nickel-filtered Cu radiation, Voltage 45 kV, filament emission 40 mA). Variable divergence and anti-scatter slits and incident and diffracted soller slit 0.04° are used.
  • the sample is rotated during measurement. Sample is scanned from 2.4 - 50°2Theta using a 0.013° step width and a 115.770 s count time together with a PIXcellD detector (active length 3.347° 2Theta).
  • the XRPD pattern in this was obtained in Bragg-Brentano geometry.
  • the XRPD pattern of Form A of Compound 1 is shown in Figure 1.
  • the most prominent peaks in the XRPD pattern are listed in Table 1.
  • the peak intensities are described herein as vs (very strong), s (strong), m (medium) and w (weak) and correspond to % relative intensity (based on the most intense peak) of 25-100%, 10-25%, 3-10% and 1-3%, respectively.
  • TG/DTA Thermo Gravimetry / Differential Thermal Analysis
  • the output was introduced into a reaction tube to mix with a room temperature pumped solution of magnesium chloride (251g, 2.63 mol) in THF (4.78 L) @ 13.9g min' 1 and this output flowed to a reaction tube where it was quenched with a pumped solution of phthalic anhydride (651g, 4.40 mol) in THF (3.70 L) @12.0g min'l in a helical mixer.
  • the solution was allowed to cool and treated with a 3M hydrochloric acid solution (1021g), the organics were separated and then saturated brine (556g) was added, some precipitation occurred which was dissolved by diluting with THF (1037 g) and heating to 50°C, the organic layer was separated and washed with saturated brine (800g x 2), organic layer partially evaporated and heated to 65°C and heptane (1757 g) was added slowly.
  • Step 2 Intermediate 6: 2-(4-chl oro-1 -phthalazinyl)-5-(trifluoromethyl)phenol
  • Step 1 Intermediate 8: 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)pheny l)phthalazin- 1 (2H)-one o Intermediate 7
  • 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran (56.3 g, 228.78 mmol) andNl,Nl,N2,N2- tetramethylethane-l ,2-diamine (37.6 mL, 250.57 mmol) were mixed in THF (200 mL) at rt under N2 and cooled to -5 °C.
  • Butyllithium (2.5 M in hexane, 104 mL, 250.57 mmol) was added over 10 min.
  • a solution of tert-butyl methyl phthalate 40g, 167.61 mmol
  • THF 200 mL
  • hydrazine hydrate 16.31 mL, 335.21 mmol
  • a further 2 mL of water was added, followed by acetic acid.
  • the reaction mixture was stirred overnight at -20 °C.
  • Step 2 Intermediate 9: N-(((S)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-4-(2-((tetrahydro-2H- py ran-2-yl)oxy )-4-(trifluoromethy l)pheny l)phthalazin- 1 -amine
  • Step 1 Intermediate 10: l-bromo-2-((4-methoxybenzyl)oxy)-4-(trifluoromethyl)benzene
  • Step 2 Intermediate 11: 3-(tert-butyl) 4-methyl pyridine-3,4-dicarboxylate
  • Step 3 Intermediate 12: tert-butyl 4-[2-[(4-methoxyphenyl)methoxy]-4- (trifluoromethyl)benzoyl]pyridine-3-carboxylate
  • Step 4 Intermediate 13: l-[2-[(4-methoxyphenyl)methoxy]-4-(trifluoromethyl)phenyl]-3H- pyrido[3,4-d]pyridazin-4-one
  • Step 5 Intermediate 14: 4-chloro-l-[2-[(4-methoxyphenyl)methoxy]-4- (trifluoromethyl)phenyl]pyrido[3,4-d]pyridazine
  • Step 7 Compound 2: 2-(4-(((lR,2R)-2-hydroxycyclohexyl)amino)pyrido[3,4-d]pyridazin-l- yl)-5-(trifluoromethyl)phenol
  • Step 1 Intermediate 17: l-[2-tetrahydropyran-2-yloxy-4-(trifhioromethyl)phenyl]-3H- pyrido[3,4-d]pyridazin-4-one
  • Step 2 Intermediate 18: (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan-l-ol
  • EtOH absolute; 300 mL
  • EtOH absolute; 50 mL
  • water 250 mL
  • the suspension is cooled from 60 °C to 20 °C within 1 h and is stirred for 1 h at 20 °C before isolation by filtration.
  • the product cake is washed with water (8 L).
  • the product is dried overnight on the nutsche in vacuum with aNi current (20 L/min) through the cake.
  • the pure product (186 g) is obtained as an off-white, slightly yellow solid.
  • the material is ground and homogenized in the nutsche during off-loading and is further dried in a vacuum oven ( ⁇ 10 mbar) at elevated temperature (50 °C).
  • the pure, dry title compound (153 g, 50 %) hydrochloride salt is obtained as an off-white, slightly yellow, crystalline solid.
  • ASC-GFP Reporter Monocytes (InvivoGen #thp- ascgfp) was employed.
  • the assay is based on NF-kB dependent expression of the ASC::GFP fusion protein. LPS-priming of cells increases ASC::GFP expression and Nigericin recruits ASC::GFP, pro-caspase-1 and NLRP3 to form micrometer-sized complexes, ASC-specks, that are quantified by fluorescence microscopy.
  • Assay medium RPMI 1640 (Gibco #72400-021) supplemented with 10% heat inactivated FBS (Gibco #10270)
  • THP-ASC-GFP were cultured in RPMI 1640 (Gibco #72400-021) supplemented with 10% heat inactivated FBS (Gibco #10270) and lOOpg/mL Zeocin (Life Technologies #46- 0072) (every other passage) to maintain ASC::GFP expression.
  • test compound in DMSO were prepared in concentration response curves and diluted with 20 pl assay medium supplemented with 68 pM ZVAD-FMK (Promega #7231) in polypropylen 384 well plates (Greiner # 781280)
  • Compounds were profiled for NLRP3 antagonist activity with respect to inhibition of Nigericin triggered IL-ip release from THP-1 human monocytes. Quantification was performed using a commercially available human IL-ip HTRF detection kit (CisBio, 62HIL1BPEH). The assay uses two anti-IL-ip antibodies in a sandwich assay format. One labeled with a donor fluorophore (Eu cryptate), a second with an acceptor (XL).
  • Eu cryptate donor fluorophore
  • XL acceptor
  • Immune-complexes containing the two antibodies bound to the same IL-ip molecule allows fluorescence resonance energy transfer (FRET) between the donor and acceptor after excitation of the donor with a light source, subsequently resulting in fluorescence at 665 nM from the acceptor.
  • FRET fluorescence resonance energy transfer
  • the fluorescence signal intensity is proportional to the IL-ip concentration in the sample.
  • Cells THP human monocytic leukemia cell line. Cells generally passaged every 2-3 days with density kept from 0.2 to 0.4*10 A 6 cells/mL.
  • IL-ip standard reconstituted IL-ip standard provided in the CisBio kit was diluted in assay medium to a top final concentration of 2 ng/mL in the assay.
  • HTRF detection reagents cAMP-d2 and anti-cAMP cryptate were reconstituted according to CisBio kit instructions. Just prior to use, reagents were combined using the following proportions: 10/24 Detection buffer (provided with the kit), 14/24 PBS (Gibco, 10010), 1/120 IL-ip Eu-cryptate Antibody and 1/120 IL-ip XL Antibody.
  • test compounds dissolved in DMSO were aquostically dispensed (Labcyte Echo) to white 384-well plates (Greiner; 784075), sealed and stored at rt until assayed.
  • control compound 20 nL 50 pM of a control compound in DMSO (250 nM final concentration) was added to 100% inhibition control wells and 20 nL DMSO added to 0% control wells with Echo dispenser.
  • the control compound may be selected from MCC950 (N-[[(l,2,3,5,6,7- hexahydro-s-indacen-4-yl)amino]carbonyl]-4-(l-hydroxy-l-methylethyl)-2- furansulfonamide) or any other compound that acts as a full antagonist in the assay.
  • LPS (Sigma; L2654) was added to a final concentration of 1 pg/mL.
  • Cell were LPS-primed in bulk in a 50 mL tube by incubating at 37°C, 5% CO2 and 95% humidity for 3 h.
  • HTRF Homogenous Time-Resolved Fluorescence
  • HTRF data was converted to amount IL-ip produced in the samples which was subsequentially used for calculation of concentration responses.
  • Concentration response data were analyzed with Screener (Genedata) and fitted with a four parameter logistic fit. The results from the assay are reported in Table 2 as ICso (pM).
  • IC50 is defined as the concentration at which the inhibitory activity reaches 50% of its maximum level. Where the assay was run multiple times for the same compound, the geometric mean is reported. To facilitate comparison of efficacy data, efficacy was normalized to % inhibitory effect of the test compound compared to the inhibition caused by a saturating concentration of the control compound (250 nM).
  • BzATP triggered (human NLRP3) IL-IP assay (Test C)
  • compounds were tested for their ability to inhibit BzATP (2'(3')-O-(4-Benzoylbenzoyl)adenosine 5 '-triphosphate) triggered IL-ip release from THP-1 human monocytes.
  • BzATP 2'(3')-O-(4-Benzoylbenzoyl)adenosine 5 '-triphosphate
  • quantification was performed using a human IL-10 HTRF detection kit (CisBio, 62HIL1BPEH).
  • Cell culture medium RPMI 1640 (Gibco, 11875-119) supplemented with 10% FBS (Sigma, 171012) and Penicillin-streptomycin (Thermo Fisher, 15140-122).
  • RPMI 1640 Gibco, 22400-105 supplemented with 1% FBS (Sigma, 171012).
  • BzATP Sigma, B6396
  • nigericin instead of nigericin
  • HBSS Hepes-buffered saline solution
  • the internal patch clamp solution was KF 120 mM, KC120 mM, HEPES 10 mM, EGTA 10 mM, and 25 pM Escin (pH7.2).
  • a seal enhancer solution comprising HBSS supplemented with 10 mM CaCh and ImM MgCh was applied to cells.
  • the external solution was then exchanged (4 times) for external patch clamp solution comprising NaCl 80 mM, KC1 4 mM, HEPES 10 mM, CaCh 2 mM, MgCh 1 mM, glucose 5 mM, and NMDG 60 mM (pH 7.4). All solutions were stored at rt, except Escin, which was stored at 4 °C.
  • the assay was conducted according to the Solubility Assay described in pages 164-167 of Wemevik, J. et al., “A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling”, Assay and Drug Development Technologies, 2020, 18(4), 157- 179. Data are reported in Table 2 as solubility (pM). Where the assay was run multiple times for the same compound, the arithmetic mean is reported.
  • disodium hydrogenphosphate-citric acid buffer solution (Diluted Mcllvaine buffer, pH6.5) was added to dilute 100 fold. Under these conditions, the theoretical maximum concentration of the test compound was 200 pM.
  • the buffer was sonicated, shaken, and held at 25 °C for 24 to 72 h.
  • the buffer sample was filtered and the filtrate was diluted with an equal volume of acetonitrile/methanol (1:1, v/v) in a 96 well plate.
  • a 20 mM DMSO solution containing the test compound was diluted 100 fold with acetonitrile/methanol (1:1, v/v) and the same amount of Mcllvaine buffer (pH 6.5) was added to use as the standard solution.
  • the standard and test samples were transferred to a 384 well plate and analyzed by HPLC. The results of the solubility assay are reported in Table 2 in pg/mL.
  • mice Male 7-week-old BALB/cAJcl mice were intraperitoneally administered 0.5 mL of 4 pg/mL LPS (Sigma-Aldrich Co. LLC, L2630) solution in PBS (Thermo Fisher Scientific Inc., 10010). One hour later, the test article suspension in 0.5% (w/v) CMC sodium (Nacalai tesque INC., 07326-95) aqueous solution was orally administered at a volume of 10 mL/kg. One hour after the article administration, 0.5 mL of 30 pmol/L ATP (Sigma- Aldrich Co. LLC, A7699) solution in PBS was intraperitoneally administered.

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Abstract

The specification generally relates to crystalline forms of inhibitors of the NLRP3 inflammasome, pharmaceutical compositions comprising such crystalline forms, chemical processes useful for the preparation of inhibitors of the NLRP3 inflammasome, and intermediate compounds useful in such processes. Such crystalline forms and pharmaceutical compositions are useful in inhibiting NLRP3 inflammasome activity and may be useful as therapeutic agents.

Description

CRYSTALLINE FORMS OF NLRP3 INFLAMMASOME INHIBITORS, CHEMICAL PROCESSES AND CHEMICAL COMPOUNDS
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
This application claims the benefit of U.S. Provisional Patent Application No. 63/477,516, filed December 28, 2022, the entirety of which is incorporated by reference herein.
FIELD
Described in this specification are crystalline forms of inhibitors of the NLRP3 inflammasome, pharmaceutical compositions comprising such crystalline forms, chemical processes useful for the preparation of inhibitors of the NLRP3 inflammasome, and intermediate compounds useful in such processes.
BACKGROUND
The NLRP3 inflammasome is a multi-protein complex consisting of the NLR family pyrin domain containing 3 (NLRP3) protein, PYD and CARD domain containing (ASC, also known as PYCARD), and caspase 1 (CASP1), and is a stress sensing pathway leading to an inflammatory response (Swanson KV et al. Nat Rev Immunol. 2019 Aug;19(8):477-489). When activated, these three proteins condense into a large multiprotein complex; a speck.
The NLRP3 protein consists of three domains, PYD, NACHT and LRR (Sharif H et al. Nature. 2019 Jun;570(7761):338-343). The aminoterminal PYD domain is thought to be important in the binding of NLRP3 to the PYD domain of ASC, the NACHT domain has ATPase activity suggested to regulate the oligomerization, potentially through conformational change of the LRR domain, and the LRR domain is considered to induce autoinhibition by folding onto the NACHT domain. The activity of the NLRP3 protein is further regulated by a multitude of posttranslational modifications including phosphorylations and ubiquitinylations.
A multitude of cellular stressors such as pathogen associated molecular patterns (PAMP’s), endogenous danger signals (DAMP’s) and environmental irritants have been shown to lead to the condensation of the inflammasome into a speck. It is considered that the activation of the inflammasome requires two steps (McKee CM et al. J Leukoc Biol. 2020 Sep;108(3):937- 952). The initial priming step serves to increase the levels of inflammasome components and can be initiated by for example lipopolysaccharide (LPS, a common PAMP). LPS is detected through toll-like receptors resulting in NF-kB driven transcription of NLRP3 and IL1B. A secondary insult initiates rapid oligomerization of the inflammasome components into a speck, producing activated caspase 1.
In addition to this two step process a very high induction of NLRP3 transcription has been demonstrated to drive the inflammasome activation in a single step, typically through prolonged LPS exposure.
Downstream effects of an activated NLRP3 inflammasome is further expanded through caspase- 1 mediated cleavage and hence activation of gasdermin D. When activated, gasdermin D forms a large pore leading to a regulated form of lytic cell death called pyroptosis (Kovacs SB et al. Trends Cell Biol. 2017 Sep;27(9):673-684). In effect, pyroptosis amplifies inflammation through release of cellular contents subsequently leading to the recruitment and influx of additional immune cells.
It is likely that a dysregulated inflammasome drive can, even at low levels over several years, lead to tissue damage and chronic disease. This is proven for cryopyrin-associated periodic syndromes 1, 2 and 3 where causative genetic lesions in NLRP3 have been identified (Kacar M et al. Rheumatology (Oxford). 2019 Nov l;58(Suppl 6):vi31-vi43).
NLRP3 inflammasome activation has been linked to multiple indications (as discussed herein) often with demonstrated presence or activity in the affected tissue, and inhibition of the NLRP3 inflammasome will therefore resolve unfavorable inflammation.
The NLRP3 inflammasome can modulate both acute kidney injury (AKI) and chronic kidney disease (CKD); mice deficient in NLRP3 inflammasome components and its downstream mediators can be protected from renal injury in experimental models of both AKI and CKD (Hutton HL et al. Nephrology. 2016 21(9):736-744). Inflammation plays a key role in the pathogenesis of AKI; after an initial ischaemic, septic or nephrotoxic trigger, release of inflammatory cytokines and chemokines by renal endothelial cells and tubular epithelium can result in leukocyte recruitment and subsequent renal injury. The role of the inflammasome in this process is evident in both studies on biomarkers and experimental models of AKI (Andersen K et al. Kidney Int. 2014 Nov;86(5):965-78). Increasing evidence from clinical and experimental studies indicates that both systemic and local renal inflammation have crucial roles in the development and progression of diabetic kidney disease (DKD) (Tang SCW et al. Nat Rev Nephrol. 2020 Apr;16(4):206-222). Specifically, the NLRP3 inflammasome links sensing of metabolic stress in the diabetic kidney to activation of pro-inflammatory cascades via the induction of IL-ip and IL-18 leading to chronic injury and kidney functional decline in CKD/DKD (Shahzad K et al. J Am Soc Nephrol. 2016 Aug;27(8):2270-5). Studies have implicated the NLRP3 inflammasome in cardiovascular diseases (An N et al. Front Immunol. 2019 Jul 10; 10: 1592). The relationship between the NLRP3 inflammasome and coronary atherosclerotic heart disease through cholesterol crystals/monosodium glutamate and downstream factors and vascular injury is well described (Jin Y et al. J Am Heart Assoc. 2019 Jun 18;8(12):e012219). In addition, the NLRP3 inflammasome may also be involved in the pathological mechanism of cardiomyopathies, including myocardial infarction (MI), cardiac remodelling and cardiac hypertrophy (An N et al. Front Immunol. 2019 Jul 10;10: 1592).
Nonalcoholic fatty liver disease (NAFLD) is defined as excess liver fat accumulation (fatty liver) greater than 5% induced by causes other than alcohol intake. Fatty liver progresses to nonalcoholic steatohepatitis (NASH) with or without fibrosis in a variable proportion of individuals, ultimately leading to liver cirrhosis, liver failure and hepatocellular carcinoma in susceptible individuals (Friedman et al Nat Med. 2018 Jul;24(7): 908-922). Inflammation including the NLRP3 inflammasome contributes to the pathogenesis of most acute and chronic liver diseases including NAFLD, NASH, alcoholic steatohepatitis, chronic hepatitis C virus (HCV) infection, ischaemia-reperfusion injury and paracetamol-induced liver injury (Szabo et al Nat Rev Gastroenterol Hepatol 2015; 12:387-400). Hepatic NLRP3 and down-stream target mRNA levels are increased in NASH and correlate with liver collagen expression levels in humans. In addition, NLRP3 inducible activation increases liver fibrosis in mice and NLRP3 knock-out mice are protected from experimentally induced NASH including liver inflammation and fibrosis (Wree et al J Mol Med, 2014, DOI: 10.1007/s00109-014-l 170-1). NLRP3 inflammasome inhibition using a small molecule inhibitor (MCC950) reduces liver inflammation and fibrosis in experimental models of NASH where mice were fed a high fat diet or a methionine and choline deficient diet (Mridha et al Journal of Hepatology, 2017, DOI: 10.1016/j.jhep.2017.01.022). Thus, NLRP3 inflammasome inhibition can protect against liver diseases including NAFLD and NASH.
Several overactivating mutations in NLRP3 have been linked to autoinflammatory disorders leading to inappropriate release of inflammatory cytokines including IL-ip and inflammatory symptoms. Cryopyrin-associated periodic syndromes, CAPS, include familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome or neonatal onset multi-system inflammatory disease (NOMID) (Booshehri ML et al. J Clin Immunol. 2019 Apr;39(3):277-286).
The NLRP3 inflammasome has also been indicated in gout and pseudo gout since monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD), both crystals found in gout, are activators of the NLRP3 inflammasome (Martinon F et al. Nature 440: 237-241, 2006). In sarcoidosis, the NLRP3 inflammasome has been identified as one of the key cellular pathways (RiteauN et al. Eur Respir J. 2020; 55(3):2000149) and increased activity has been demonstrated in the lungs of sarcoid patients.
Evidence suggest that inflammasomes play a role in auto-immune diseases and inhibition of the NLRP3 inflammasome may have a positive effect in rheumatoid arthritis (RA), multiple sclerosis (MS), Addison’s disease, celiac disease, systemic lupus erythematous (SLE) and vitiligo (Shaw PJ et al. Trends Mol Med. 2011 Feb; 17(2): 57-64).
In inflammatory skin diseases, NLRP3 inflammasome activation has been demonstrated in acne vulgaris (Li ZJ et al. J Invest Dermatol. 2014 Nov;134(l l):2747-2756) and hidradenitis suppurativa (Kelly G et al. Br J Dermatol. 2015 Dec;173(6):1431-9).
Emerging evidence suggest that persistant activation of NLRP3 may be involved in the progression of several chronic pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD) and asthma (De Nardo D. et al. Am J Pathol. 2014 Jan;184(l):42-54).
In inflammatory bowel disease (IBD) there is evidence showing that inflammasome- driven IL-ip and IL- 18 play a role in IBD pathology and that NLRP3 inflammasome inhibitors may be efficacious in ulcerative colitis (UC) and Crohn’s disease. (Zhen Y et al. Front Immunol. 2019 Feb 28;10:276).
Accordingly, inhibitors of the NLRP3 inflammasome may be useful in the treatment of the diseases and conditions described herein which are linked to NLRP3 inflammasome activation. However, to date, no small-molecule synthetic inhibitor of the NLRP3 inflammasome has been approved for medical use.
Small-molecule inhibitors of the NLRP3 inflammasome have been previously discussed, for example, in WO2020/234715 Al, WO2022/135567 Al and WO2022/216971 Al, but, despite the foregoing, a need continues to exist for further compounds that are inhibitors of the NLRP3 inflammasome which may make the compounds especially promising for development as therapeutic agents, and processes to make such compounds.
Compound 1 and Compound 2, as described herein, are described in International application no. PCT/EP2022/068292, and are inhibitors ofthe NLRP3 inflammasome. Stable, crystalline forms of such compounds, and pharmaceutical compositions comprising such stable crystalline forms are desired which may be useful in the commercial manufacture of pharmaceutical compositions. Furthermore, efficient processes to make such compounds are desired, along with new chemical intermediates that are useful in such synthetic processes. SUMMARY
This specification describes, in part, a crystalline form of (2S)-3-[[4-[2-hydroxy-4- (trifluoromethyl)phenyl]phthalazin-l-yl]amino]propane-l,2-diol (Compound 1).
This specification also describes, in part, a crystalline form of Compound 1 which is crystalline Form A.
This specification also describes, in part, a crystalline form of 2-(4-(((lR,2R)-2- hydroxycyclohexyl)amino)pyrido[3,4-d]pyridazin-l-yl)-5-(trifluoromethyl)phenol (Compound 2), hydrochloride salt.
This specification also describes, in part, a pharmaceutical composition comprising a crystalline form as disclosed herein, and a pharmaceutically acceptable excipient.
This specification also describes, in part, a crystalline form or pharmaceutical composition as disclosed herein for use in therapy.
This specification also describes, in part, a crystalline form or pharmaceutical composition as disclosed herein for use in the treatment of a subject with a disease or condition in which NLRP3 inflammasome activity is implicated.
This specification also describes, in part, a method of inhibiting NLRP3 inflammasome activity in a subject in need thereof, comprising administering a crystalline form or pharmaceutical composition as disclosed herein to the subject.
This specification also describes, in part, a process for the preparation of a compound of Formula (Ic), or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium; (b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with a compound of Formula (la)
Formula (la) to form a compound of Formula (lb), or a salt thereof
Formula (lb)
(d) reacting the compound of Formula (lb), or a salt thereof, with hydrazine, optionally in the presence of an acid such as acetic acid, to form a compound of Formula (Ic), or a salt thereof; and
(e) isolating the compound of Formula (Ic), or a salt thereof; and wherein X = CH or N in Formula (la), Formula (lb) and Formula (Ic).
This specification also describes, in part, a process for the preparation of 4-(2-hydroxy-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof; (d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with aqueous acid to form 2-[2-hydroxy-4- (trifluoromethyl)benzoyl] benzoic acid, or a salt thereof;
(e) reacting 2-[2-hydroxy-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(f) isolating 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof.
This specification also describes, in part, a process for the preparation of 2-(4- chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, comprising the steps:
(a) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)- 5-(trifluoromethyl)phenol, or a salt thereof; and
(b) isolating 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof.
This specification also describes, in part, a process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with aqueous acid to form 2-[2-hydroxy-4- (trifluoromethyl)benzoyl] benzoic acid, or a salt thereof;
(e) reacting 2-[2-hydroxy-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(f) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)- 5-(trifluoromethyl)phenol, or a salt thereof;
(g) reacting 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, with (1) (2S)-3-amino-l,2-propanediol, or a salt thereof, to form Compound 1, or a salt thereof, or (2) diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, followed by deprotection of the resulting product to form Compound 1, or a salt thereof; and
(h) isolation of Compound 1, or a salt thereof.
This specification also describes, in part, a process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with tert-butyl methyl phthalate to form tert-butyl 2- (2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)benzoyl)benzoate;
(d) reacting tert-butyl 2-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)benzoate with hydrazine, optionally in the presence of an acid such as acetic acid, to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, with diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, in the presence of a coupling reagent, followed by removal of the THP protecting group and diol protecting group from the resulting product by reaction with an acid, to form Compound 1, or a salt thereof; and
(f) isolation of Compound 1, or a salt thereof.
This specification also describes, in part, a process for the preparation of Compound 2, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with 3 -(tert-butyl) 4-methyl pyridine-3,4- dicarboxylate to form tert-butyl 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)nicotinate, or a salt thereof; (d) reacting tert-butyl 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)nicotinate, or a salt thereof, with hydrazine, optionally in the presence of an acid such as acetic acid, to form l-[2-tetrahydropyran-2-yloxy-4- (trifluoromethyl)phenyl]-3H-pyrido[3,4-d]pyridazin-4-one, or a salt thereof;
(e) reacting l-[2-tetrahydropyran-2-yloxy-4-(trifluoromethyl)phenyl]-3H-pyrido[3,4- d]pyridazin-4-one with (lR,2R)-2-aminocyclohexan-l-ol, or a salt thereof, in the presence of a coupling reagent, to form (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran-2- yl)oxy)-4-(trifhioromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan-l- ol, or a salt thereof;
(f) removal of the THP protecting group from (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran- 2-yl)oxy)-4-(trifluoromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan- l-ol, or a salt thereof, by reaction with an acid to form Compound 2, or a salt thereof; and
(g) isolation of Compound 2, or a salt thereof.
This specification also describes, in part, a compound that is
This specification also describes, in part, a compound that is salt thereof.
This specification also describes, in part, a compound that i
, or a salt thereof. This specification also describes, in part, a compound that i
, or a salt thereof.
This specification also describes, in part, a compound that i salt thereof, wherein X is CH or N.
This specification also describes, in part, a compound that is salt thereof, wherein X is CH or N.
This specification also describes, in part, a compound that i salt thereof, wherein X is CH or N. This specification also describes, in part, a compound that is salt thereof.
This specification also describes, in part, a compound that is salt thereof.
This specification also describes, in part, a compound that , or a salt thereof.
This specification also describes, in part, a compound that i
, or a salt thereof.
Further aspects of the disclosure will be apparent to one skilled in the art from reading this specification.
LIST OF FIGURES
Figure 1 is an X-Ray Powder Diffraction Pattern of Form A of Compound 1.
Figure 2 shows the TG/DTA output from analysis of Form A of Compound 1.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS “Compound 1”, as used herein, is a compound with chemical name (2S)-3-[[4-[2-hydroxy-4- (trifluoromethyl)phenyl]phthalazin-l-yl]amino]propane-l,2-diol, and the following chemical structure:
Compound 1
“Compound 2”, as used herein, is a compound with chemical name 2-(4-(((lR,2R)-2- hydroxy cyclohexyl)amino)pyrido[3,4-d]pyridazin-l-yl)-5-(trifluoromethyl)phenol, and the following chemical structure:
Compound 2
As described in the Examples herein, Compound 1 and Compound 2 are inhibitors of the NLRP3 inflammasome, and may therefore be useful in the treatment of disease states in which inhibition of the NLRP3 inflammasome is beneficial.
Terms not specifically defined herein should be understood to have the meanings that would be given to them by one of skill in the art in light of the disclosure and the context.
"About" may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Embodiments described herein as "comprising" one or more features may also be considered as disclosure of the corresponding embodiments "consisting of' such features.
Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
The chemical names of compounds described in this specification were generated using ChemDraw® Professional version 19.0.0.22 from PerkinElmer®. The skilled person will understand that different chemical naming software may generate different chemical names for a particular compound. In case a compound described herein is depicted in form of a chemical name and as a formula, the formula shall prevail in case of any discrepancy.
Compounds and salts described in this specification may exist in solvated forms and unsolvated forms. For example, a solvated form may be a hydrated form, such as a hemi -hydrate, a mono-hydrate, a di-hydrate, a tri-hydrate or an alternative quantity thereof. All such solvated and unsolvated forms of compounds described herein are encompassed herein.
Atoms of the compounds and salts described in this specification may exist as their isotopes. All compounds described herein where an atom is replaced by one or more of its isotopes (for example a compound described herein where one or more carbon atom is an "C or 13C carbon isotope, or where one or more hydrogen atoms is a 2H or 3H isotope) are encompassed herein.
Compounds described herein may exist in one or more geometrical, optical, enantiomeric, and diastereomeric forms, including, but not limited to, cis- and trans-forms, E- and Z-forms, and R-, S- and meso-forms. Unless otherwise stated a reference to a particular compound includes all such isomeric forms, including racemic and other mixtures thereof. Where appropriate such isomers can be separated from their mixtures by the application or adaptation of known methods (e.g. chromatographic techniques and recrystallisation techniques). Where appropriate such isomers can be prepared by the application or adaptation of known methods.
The compounds described herein may include one or more chiral centres. To the extent a structure or chemical name in this specification does not indicate chirality, the structure or name is intended to encompass any single stereoisomer corresponding to that structure or name, as well as any mixture of stereoisomers (e.g. a racemate). Where a structure in this specification includes bonds drawn as solid and hashed wedges (i.e. " and ), it is intended that the solid and hashed wedges indicate the absolute configuration of a chiral centre.
It is well-known in the art how such optically-active forms can be separated. For example, a single stereoisomer can be obtained by isolating it from a mixtures of isomers (e.g. a racemate) using, for example, chiral chromatographic separation. In other embodiments, a single stereoisomer is obtained through direct synthesis from, for example, a chiral starting material.
According to one embodiment, the compounds described herein are provided as a single enantiomer being in enantiomer excess (%ee) of > 95%, > 98%, or > 99%. Conveniently a single enantiomer is present in an enantiomer excess of > 99%.
According to one embodiment, a compound described herein is provided as a single enantiomer being in enantiomer excess (%ee) in the range 95 to 100%.
Compounds described herein may exist in one or more tautomeric forms, including, but not limited to, keto-, and enol-forms. A reference to a particular compound includes all tautomeric forms, including mixtures thereof. Accordingly, a structure depicted herein as one tautomer is intended to also include other tautomers.
Crystalline Forms
In the formulation of drug compositions, it is important for the drug substance to be in a form in which it can be conveniently handled and processed. This is of importance, not only from the point of view of obtaining a commercially viable manufacturing process, but also from the point of view of subsequent manufacture of pharmaceutical formulations (e.g. oral dosage forms such as tablets) comprising the active compound.
The different physical properties of the crystalline forms with respect to each other and with respect to the non-crystalline state may influence markedly the chemical and pharmaceutical processing of a compound, particularly when the compound is prepared or used on an industrial scale.
Further, in the manufacture of oral drug compositions, it is important that a reliable and reproducible plasma concentration profile of drug is provided following administration to a patient. Inter-patient variability in the absorption profile of a drug within the stomach, intestine or bloodstream can have an effect on drug safety and efficacy.
Chemical stability, solid state stability and “shelflife” of the active ingredients are also very important factors. The drug substance, and compositions containing it, should be capable of being effectively stored over appreciable periods of time, without exhibiting a significant change in the active component’s physico-chemical characteristics (e.g. its chemical composition, density, hygroscopicity and solubility).
Moreover, it is also important to be able to provide drug in a form which is as chemically pure as possible.
Amorphous materials may present problems in this regard. For example, such materials are typically difficult to handle and to formulate, provide for unreliable solubility, and are often found to be unstable and chemically impure.
The skilled person will appreciate that, if a drug can be readily obtained in a stable crystalline form, the above problems may be solved.
Thus, in the manufacture of commercially viable, and pharmaceutically acceptable, drug compositions, it is important, wherever possible, to provide drug in a crystalline, and stable, form.
It is to be noted, however, that this goal is not always achievable. Indeed, typically, it is not possible to predict, from molecular structure alone, what the crystallisation behaviour of a compound, either as such or in the form of a salt, will be. This can only be determined empirically.
In one embodiment, Compound 1 may be prepared in a crystalline form. This crystalline form may be characterised as being a particular polymorphic form. When it is stated that an embodiment relates to a crystalline form, the degree of crystallinity may be greater than about 60%, optionally greater than about 80%, greater than about 90% or greater than about 95%. In one embodiment, the degree of crystallinity is greater than about 98%.
The specific crystalline forms described herein provide X-ray powder diffraction patterns substantially the same as the X-ray powder diffraction patterns shown in the Figures and have the various 2-theta values as described herein. It will be understood that the 2-theta values of a X-ray powder diffraction pattern may vary slightly from one machine to another or from one sample to another, and so the values quoted are not to be construed as absolute.
It is known that an X-ray powder diffraction pattern may be obtained which has one or more measurement errors depending on measurement conditions (such as equipment or machine used). In particular, it is generally known that intensities in an X-ray powder diffraction pattern may fluctuate depending on measurement conditions. Therefore it should be understood that the crystalline forms described herein are not limited to the crystals that provide X-ray powder diffraction patterns that are identical to the X-ray powder diffraction pattern shown in the Figures, and any crystals providing X-ray powder diffraction patterns substantially the same as those shown in the Figures fall within the scope of the embodiments described herein. A person skilled in the art of X-ray powder diffraction is able to judge the substantial identity of X-ray powder diffraction patterns.
Persons skilled in the art of X-ray powder diffraction will realise that the relative intensity of peaks can be affected by, for example, grains above 30pm in size and non-unitary aspect ratios, which may affect analysis of samples. The skilled person will also realise that the position of reflections can be affected by the precise height at which the sample sits in the diffractometer and the zero calibration of the diffractometer. The surface planarity of the sample may also have a small effect. Hence the diffraction pattern data presented are not to be taken as absolute values. (Jenkins, R & Snyder, R.L. ‘Introduction to X-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C.W. (1948), Chemical Crystallography, Clarendon Press, London; Klug, H. P. & Alexander, L. E. (1974), X-Ray Diffraction Procedures). The peak intensities are described herein as vs (very strong), s (strong), m (medium) and w (weak) and correspond to % relative intensity (based on the most intense peak) of 25-100%, 10-25%, 3-10% and 1-3%, respectively. The relative intensities are derived from diffractograms measured with fixed slits.
Generally, a measurement error of a diffraction angle in an X-ray powder diffractogram is about 5% or less, in particular plus or minus 0.2° 2-theta, and such degree of a measurement error should be taken into account when considering the X-ray powder diffraction patterns in the Figures herein and when reading the data described herein. Furthermore, it should be understood that intensities may fluctuate depending on experimental conditions and sample preparation (preferred orientation).
A useful crystalline polymorphic form of Compound 1 has been produced using the conditions described in Example 1. In all of the embodiments relating to crystalline forms recited herein, the peaks of the X-ray diffraction patterns are measured using Cu K„ radiation.
Thus in one embodiment, there is provided Compound 1 in crystalline form.
In one embodiment, there is provided Compound 1 in crystalline form, wherein the crystalline form is Form A.
Therefore in one embodiment there is provided polymorphic Form A of Compound 1. This polymorphic form may be characterised in that it provides at least one of the following 29 values measured using Cu Li radiation: 13.0, 19.3, 19.8, 22.7, and 24.1°.
Polymorphic Form A of Compound 1 is characterised in providing an X-ray powder diffraction pattern, substantially as shown in Figure 1.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 19.3°. In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 13.0°.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 13.0 and 19.3°.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least five specific peaks at about 2-theta = 13.0, 19.3, 19.8, 22.7 and 24.1°.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with specific peaks at about 2-theta = 10.7, 11.6, 13.0, 16.1, 19.3, 19.8, 22.7, 24.1, 26.3, 29.5 and 30.9°.
In one embodiment there is provided polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 1.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 19.3° ± 0.2° 2- theta.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 13.0° ± 0.2° 2- theta.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least two specific peaks at 2-theta = 13.0 and 19.3°, wherein said values are each ± 0.2° 2-theta.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least five specific peaks at 2-theta = 13.0, 19.3, 19.8, 22.7 and 24.1°, wherein said values are each ± 0.2° 2-theta.
In one embodiment there is provided the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with specific peaks at 2-theta = 10.7, 11.6, 13.0, 16.1, 19.3, 19.8, 22.7, 24.1, 26.3, 29.5 and 30.9°, wherein said values are each ± 0.2° 2-theta.
In one embodiment, crystalline Form A of Compound 1 is unsolvated. A useful crystalline polymorphic form of the hydrochloride salt of Compound 2 has been produced using the conditions described in Example 3. Thus in one embodiment, there is provided the hydrochloride salt of Compound 2 in crystalline form.
Pharmaceutical Compositions
Since Compound 1 is an inhibitor of the NLRP3 inflammasome, pharmaceutical compositions comprising crystalline Compound 1, in particular polymorphic Form A of Compound 1 (as described herein), and a pharmaceutically acceptable excipient are expected to be useful in the treatment of disease states in which inhibition of the NLRP3 inflammasome is beneficial.
Therefore, in one embodiment, there is provided a pharmaceutical composition comprising Compound 1 in crystalline form, and a pharmaceutically acceptable excipient.
In one embodiment, there is provided a pharmaceutical composition comprising Compound 1 in crystalline form and a pharmaceutically acceptable excipient, wherein the crystalline form is Form A.
In one embodiment there is provided a pharmaceutical composition comprising polymorphic Form A of Compound 1, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 19.3°, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at about 2-theta = 13.0°, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least two specific peaks at about 2-theta = 13.0 and 19.3°, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least five specific peaks at about 2-theta = 13.0, 19.3, 19.8, 22.7 and 24.1°, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with specific peaks at about 2-theta = 10.7, 11.6, 13.0, 16.1, 19.3, 19.8, 22.7, 24.1, 26.3, 29.5 and 30.9°, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern substantially the same as the X-ray powder diffraction pattern shown in Figure 1, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 19.3° ± 0.2° 2-theta, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least one specific peak at 2-theta = 13.0° ± 0.2° 2-theta, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least two specific peaks at 2-theta = 13.0 and 19.3°, wherein said values are each ± 0.2° 2-theta, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with at least five specific peaks at 2-theta = 13.0, 19.3, 19.8, 22.7 and 24.1°, wherein said values are each ± 0.2° 2-theta, and a pharmaceutically acceptable excipient.
In one embodiment there is provided a pharmaceutical composition comprising the polymorphic Form A of Compound 1 which has an X-ray powder diffraction pattern with specific peaks at 2-theta = 10.7, 11.6, 13.0, 16.1, 19.3, 19.8, 22.7, 24.1, 26.3, 29.5 and 30.9°, wherein said values are each ± 0.2° 2-theta, and a pharmaceutically acceptable excipient.
Since Compound 2 is an inhibitor of the NLRP3 inflammasome, pharmaceutical compositions comprising crystalline Compound 2, in particular the hydrochloride salt of Compound 2, and a pharmaceutically acceptable excipient are expected to be useful in the treatment of disease states in which inhibition of the NLRP3 inflammasome is beneficial.
Therefore, in one embodiment, there is provided a pharmaceutical composition comprising the hydrochloride salt of Compound 2 in crystalline form, and a pharmaceutically acceptable excipient. The pharmaceutical compositions described herein may include one or more pharmaceutically acceptable excipients. The excipient(s) selected for inclusion in a particular composition will depend on factors such as the mode of administration and the form of the composition provided. Suitable pharmaceutically acceptable excipients are well known to persons skilled in the art and are described, for example, in the Handbook of Pharmaceutical Excipients, Sixth edition, Pharmaceutical Press, edited by Rowe, Ray C; Sheskey, Paul J; Quinn, Marian. Pharmaceutically acceptable excipients may function as, for example, adjuvants, diluents, carriers, stabilisers, flavourings, colorants, fillers, binders, disintegrants, lubricants, glidants, thickening agents and coating agents. As persons skilled in the art will appreciate, certain pharmaceutically acceptable excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the composition and what other excipients are present in the composition.
In one embodiment the amount of pharmaceutically acceptable excipient in the pharmaceutical composition is greater than or equal to 1 mg. In a further embodiment, the amount of pharmaceutically acceptable excipient in the pharmaceutical composition is greater than or equal to 10 mg. In a further embodiment, the amount of pharmaceutically acceptable excipient in the pharmaceutical composition is greater than or equal to 100 mg.
The pharmaceutical compositions may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, or dispersible powders or granules), for topical use (for example as creams, ointments, or aqueous or oily suspensions), for administration by inhalation (for example as a finely divided powder), for administration by insufflation (for example as a finely divided powder), or as a suppository for rectal dosing. The compositions may be obtained by conventional procedures well known in the art. Compositions intended for oral use may contain additional components, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
The term “pharmaceutically acceptable” is used to specify that an object (for example an excipient) is suitable for use in patients. An example list of pharmaceutically acceptable salts can be found in the Handbook of Pharmaceutical Salts: Properties, Selection and Use, P. H. Stahl and C. G. Wermuth, editors, Weinheim/Zurich:Wiley-VCH/VHCA, 2002.
Therapeutic use of crystalline forms and pharmaceutical compositions
As a result of their NLRP3 inflammsome inhibitory activity, the crystalline forms and pharmaceutical compositions described herein that comprise Compound 1 or Compound 2 are expected to be useful in therapy. The term “therapy” is intended to have its normal meaning of dealing with a disease or condition in order to entirely or partially relieve one, some or all of its symptoms, or to correct or compensate for the underlying pathology. The term "therapy" also includes "prophylaxis" unless there are specific indications to the contrary. The terms "therapeutic" and "therapeutically" should be interpreted in a corresponding manner.
The term “prophylaxis” is intended to have its normal meaning and includes primary prophylaxis to prevent the development of the disease or condition and secondary prophylaxis whereby the disease or condition has already developed and the patient is temporarily or permanently protected against exacerbation or worsening of the disease or condition, or the development of new symptoms associated with the disease or condition.
The term “treatment” is used synonymously with “therapy”. Similarly the term “treat” can be regarded as “applying therapy” where “therapy” is as defined herein.
In one embodiment there is provided a method for treating a disease or condition in which NLRP3 inflammasome activity is implicated, in a subject in need of such treatment, which comprises administering to said subject a therapeutically effective amount of a crystalline form or pharmaceutical composition as described herein.
In one embodiment there is provided a method for treating a disease or condition selected from kidney diseases such as acute kidney injury, chronic kidney disease, and diabetic kidney disease; cardiovascular diseases such as coronary atherosclerotic heart disease, cardiomyopathy, myocardial infarction, cardiac hypertrophy, and ischaemia-reperfusion injury; liver diseases such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, alcoholic steatohepatitis, chronic hepatitis C virus infection, and paracetamol-induced liver injury; inflammatory diseases such as autoinflammatory disorders, Cryopyrin-associated periodic syndromes, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome, and neonatal onset multi-system inflammatory disease (NOMID); inflammatory skin diseases such as acne vulgaris, and hidradenitis suppurativa; inflammatory bowel diseases such as ulcerative colitis (UC), and Crohn’s disease; autoimmune diseases such as gout, pseudo gout, rheumatoid arthritis (RA), multiple sclerosis (MS), Addison’s disease, celiac disease, systemic lupus erythematous (SLE), and vitiligo; and respiratory diseases such as chronic pulmonary diseases, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and asthma, in a subject in need of such treatment, which comprises administering to said subject a therapeutically effective amount of a crystalline form or pharmaceutical composition as described herein. In one embodiment there is provided a method for treating a disease or condition selected from acute kidney injury, chronic kidney disease, diabetic kidney disease, coronary atherosclerotic heart disease, cardiomyopathy, myocardial infarction, cardiac hypertrophy, ischaemia-reperfusion injury, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, alcoholic steatohepatitis, chronic hepatitis C virus infection, paracetamol-induced liver injury, autoinflammatory disorders, Cryopyrin-associated periodic syndromes, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome, neonatal onset multi-system inflammatory disease (NOMID), acne vulgaris, hi dradenitis suppurativa, ulcerative colitis (UC), Crohn’s disease, gout, pseudo gout, rheumatoid arthritis (RA), multiple sclerosis (MS), Addison’s disease, celiac disease, systemic lupus erythematous (SLE), vitiligo, chronic pulmonary diseases, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and asthma, in a subject in need of such treatment, which comprises administering to said subject a therapeutically effective amount of a crystalline form or pharmaceutical composition as described herein.
In one embodiment there is provided a crystalline form or pharmaceutical composition as described herein, for use in therapy.
In one embodiment there is provided a crystalline form or pharmaceutical composition as described herein, for use in a method for treating a disease or condition as described herein.
In one embodiment there is provided a use of a crystalline form or pharmaceutical composition as described herein, in the manufacture of a medicament for a disease or condition as described herein.
The term "therapeutically effective amount" refers to an amount of a crystalline form or pharmaceutical composition as described in any of the embodiments herein which is effective to provide “therapy” in a subject, or to “treat” a disease or condition in a subject. The therapeutically effective amount may cause any of the changes observable or measurable in a subject as described in the definition of “therapy”, “treatment” and “prophylaxis” above. As recognized by those skilled in the art, effective amounts may vary depending on route of administration, excipient usage, and co-usage with other agents. For example, where a combination therapy is used, the amount of the crystalline form or pharmaceutical composition described in this specification and the amount of the other pharmaceutically active agent(s) are, when combined, jointly effective to treat a targeted disorder or condition in the subject. In this context, the combined amounts are in a “therapeutically effective amount” if they are, when combined, sufficient to decrease the symptoms of a disease or condition responsive to inhibition of the NLRP3 inflammasome as described above. Typically, such amounts may be determined by one skilled in the art.
“Subjects” include, for example, mammals, for example, humans.
Processes for the preparation of Compound 1 and Compound 2
Synthetic routes to Compound 1 and Compound 2 from our previous work have used Li- halogen exchange of a benzyl, PMB or methoxy-protected bromophenol compound, followed by reaction of the lithiated compound with an ester (for example, see Scheme 1 and Comparative Example 1). Similar routes have also been used in the synthesis of other known inhibitors of the NLRP3 inflammasome (see US11,319,319, for example). Such a lithium-halogen exchange requires cryogenic temperatures due to the nature of the reaction and instability of the resulting lithiated compound, and such cryogenic conditions are not desired in larger scale synthesis. Furthermore, the use of a brominated starting material is not desired from a commercial perspective in view of the bromination steps required to prepare such material.
Scheme 1
Alternatively, Compounds 1 and 2 may be prepared by a Suzuki-Miyaura coupling with an appropriate boronic acid or boronate ester (for example, see Scheme 2 and the synthesis described in Example 1). Similar routes have also been used in the synthesis of other known inhibitors of the NLRP3 inflammasome (see WO2022/135567, for example). However, such a synthetic procedure requires additional steps to synthesise the boronic acid/boronate ester intermediate, and starting materials for synthesis of boronic acids/boronate esters tend to be halogenated, which is undesirable in view of the halogenation steps required to prepare such starting materials. Furthermore, the use of palladium catalysts in the synthesis of pharmaceuticals is undesirable since such residual palladium may contaminate reaction products, and the level of residual palladium in the pharmaceutical product must be controlled.
Accordingly, improved processes for the synthesis of inhibitors of the NLRP3 inflammasome, such as Compounds 1 and 2, are desired which are more compatible with production on a large scale and offer improved efficiency.
It has been found that utilising a tetrahydropyran(THP)-protected phenol enables efficient ortho-lithiation at non-cryogenic temperatures, and thus avoids the use of a bromine-containing starting material. Reaction of the lithiated intermediate with tert-butyl methyl phthalate or 3- (tert-butyl) 4-methyl pyridine-3,4-dicarboxylate (Formula (la)) results in an intermediate (Formula (lb)) that may be telescoped through to a cyclisation with hydrazine to form a useful intermediate (Formula (Ic)) in the synthesis of Compound 1 and Compound 2. (See Scheme 3)
Scheme 3
Formula (la)
X = CH or N Accordingly, in one embodiment there is provided a process for the preparation of a compound of Formula (Ic), or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with a compound of Formula (la) to form a compound of Formula (lb), or a salt thereof;
(d) reacting the compound of Formula (lb), or a salt thereof, with hydrazine, optionally in the presence of an acid such as acetic acid, to form a compound of Formula (Ic), or a salt thereof; and
(e) isolating the compound of Formula (Ic), or salt thereof; and wherein X = CH or N in Formula (la), Formula (lb) and Formula (Ic).
In one embodiment, X = CH. In one embodiment, X = N.
Steps (a)-(c): Suitable solvents for steps (a)-(c) include ethers, for example THF. The alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi. Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C. Optionally, step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C. Optionally, steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine. In one embodiment, magnesium chloride is added in step (b).
Step (d): In one embodiment, the reaction mixture from step (c) is used directly in step (d) without further purification. The hydrazine used in step (d) may be in the form of hydrazine hydrate. Suitable solvents for step (d) include THF and EtOH. Step (d) may be performed at a temperature of -40 °C to 40 °C, such as -30 °C to 30 °C, -30 °C to 10 °C, and -30 °C to 0 °C. The acid used in step (d) may be a carboxylic acid, such as acetic acid.
Step (e): In one embodiment, the compound of Formula (Ic), or salt thereof, may by isolated by precipitation and filtration.
Surprisingly, it has been found that the cyclisation step with hydrazine to form a compound of Formula (Ic) (see Scheme 3) can be conducted without protection of the phenol (such as with a THP group), and with a carboxylic acid group instead of the tert-butyl ester present in the compound of Formula (lb), as shown in Scheme 4, thus leading to 4-(2-hydroxy-4-
(tri fl uoromethy I (phenyl (phthal azin- 1 (2H)-one. or a salt thereof (which is of use in the synthesis of Compound 1), in a more efficient manner.
Scheme 4
Accordingly, in one embodiment, there is provided a process for the preparation of 4-(2- hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with aqueous acid to form 2-[2-hydroxy-4- (trifluoromethyl)benzoyl] benzoic acid, or a salt thereof;
(e) reacting 2-[2-hydroxy-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(f) isolating 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof. Steps (a)-(c): Suitable solvents for steps (a)-(c) include ethers, for example THF. The alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi. Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C. Optionally, step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C. Optionally, steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine. In one embodiment, magnesium chloride is added in step (b).
Step (d): A suitable solvent for step (d) is THF. Optionally the aqueous acid is hydrochloric acid. Step (d) may be performed at temperatures of 0 °C to 70 °C, such as 10 °C to 60 °C, 20 °C to 50 °C, and 30 °C to 40 °C.
Step (e): The hydrazine used in step (e) may be in the form of hydrazine hydrate. Suitable solvents for step (e) include THF. Step (e) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
Step (f): In one embodiment, the compound of Formula (Ic), or salt thereof, may by isolated by precipitation and filtration, such as precipitation from a mixture of THF and heptane, optionally 2:1 THF/heptane.
Alternatively, as shown in Scheme 5, the order of the hydrazine cyclisation and removal of THP protecting group may be reversed:
Scheme 5
Accordingly, in one embodiment, there is provided a process for the preparation of 4-(2- hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, comprising the steps: (a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)tithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazine- l(2H)-one, or a salt thereof, with an aqueous acid to form 4-(2-hydroxy-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(f) isolating 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof.
Furthermore, it was found that a phenol protecting group is not required for chlorination of 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one with a suitable chlorinating agent such as POCh. This is helpful in the commercial synthesis of NLRP3 inhibitors such as Compound 1 and 2 since the THP phenol protecting group can undergo side reactions with the chlorinating agent (such as POCI3) leading to alkyl chloride impurities which are undesirable in such commercial processes.
Accordingly, there is provided a process for the preparation of 2-(4-chlorophthalazin-l- yl)-5-(trifluoromethyl)phenol, or a salt thereof, comprising the steps:
(a) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCI3 to form 2-(4-chlorophthalazin-l-yl)-
5-(trifluoromethyl)phenol, or a salt thereof; and
(b) isolating 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof.
Step (a): A suitable solvent for step (a) is MeCN. The reaction of step (a) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C. In one embodiment, the 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, used in step (a) is prepared by a process described herein. Step (b): In one embodiment, the 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof is isolated by precipitation, such as precipitation from a mixture of MeCN and water.
There are also provided processes for the preparation of Compounds 1 and 2, or salts thereof, that utilise the efficient syntheses of intermediates as described herein.
Accordingly, in one embodiment, there is provided a process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with aqueous acid to form 2-[2-hydroxy-4- (trifluoromethyl)benzoyl] benzoic acid, or a salt thereof;
(e) reacting 2-[2-hydroxy-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(f) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)- 5-(trifluoromethyl)phenol, or a salt thereof;
(g) reacting 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, with (1) (2S)-3-amino-l,2-propanediol, or a salt thereof, to form Compound 1, or a salt thereof, or (2) diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, followed by deprotection of the resulting product to form Compound 1, or a salt thereof; and
(h) isolation of Compound 1, or a salt thereof.
Steps (a)-(c): Suitable solvents for steps (a)-(c) include ethers, for example THF. The alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi. Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C. Optionally, step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C. Optionally, steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine. In one embodiment, magnesium chloride is added in step (b).
Step (d): A suitable solvent for step (d) is THF. Optionally the aqueous acid is hydrochloric acid. Step (d) may be performed at temperatures of 0 °C to 70 °C, such as 10 °C to 60 °C, 20 °C to 50 °C, and 30 °C to 40 °C.
Step (e): The hydrazine used in step (e) may be in the form of hydrazine hydrate. Suitable solvents for step (e) include THF. Step (e) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
Step (1): A suitable solvent for step (1) is MeCN. The reaction of step (1) may be performed at a temperature of 0 °C to 80 °C, such as 20 °C to 70 °C, and 30 °C to 60 °C.
Step (g): A suitable solvent for step (g) is a polar aprotic solvent such as NMP or DMF, optionally NMP. The reaction of step (g) may be performed at a temperature of 20 °C to 150 °C, such as 60 °C to 140 °C, and 100 °C to 140 °C.
Diol-protected (2S)-3-amino-l,2-propanediol refers to a compound in which the two alcohol groups of (2S)-3-amino-l,2-propanediol are protected using known protecting groups suitable for 1,2-diols. Such protection may be provided by forming a 5-membered ketal, such as a 5-membered acetal, from the 1 ,2-diol. Therefore, in one embodiment, the diol-protected (2S)- 3-amino-l,2-propanediol is (S)-(2,2-dimethyl-l,3-dioxolan-4-yl)methanamine.
The removal of the diol-protecting group may be performed according to known methods. In one embodiment, the diol protecting group is removed using aqueous acid or alcoholic acid. In one embodiment, the diol protecting group is removed using hydrochloric acid in an alcohol solvent, such as isopropyl alcohol.
Step (h): Isolation of Compound 1, or a salt thereof, may be performed by addition of water to a solution of Compound 1, or a salt thereof, to precipitate Compound 1, or a salt therof. For example, addition of water to a solution of Compound 1, or a salt thereof, in NMP to precipitate Compound 1, or a salt thereof.
Alternatively, the order of the hydrazine cyclisation and removal of THP protecting group may be reversed. Accordingly, in one embodiment, there is provided a process for the preparation of Compound 1, or a salt thereof, comprising the steps: (a) reacting 2-(3-(trifluoromethyl)phenoxy)tetiahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)tithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazine- l(2H)-one, or a salt thereof, with an aqueous acid to form 4-(2-hydroxy-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(1) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)-
5-(trifluoromethyl)phenol, or a salt thereof;
(g) reacting 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, with (1) (2S)-3-amino-l,2-propanediol, or a salt thereof, to form Compound 1, or a salt thereof, or (2) diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, followed by deprotection of the resulting product to form Compound 1, or a salt thereof; and
(h) isolation of Compound 1, or a salt thereof.
In one embodiment, there is provided a process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with tert-butyl methyl phthalate to form tert-butyl 2- (2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)benzoyl)benzoate;
(d) reacting tert-butyl 2-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)benzoate with hydrazine, optionally in the presence of an acid such as acetic acid, to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, with diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, in the presence of a coupling reagent, followed by removal of the THP protecting group and diol protecting group from the resulting product by reaction with an acid, to form Compound 1, or a salt thereof; and
(f) isolation of Compound 1, or a salt thereof.
Steps (a)-(c): Suitable solvents for steps (a)-(c) include ethers, for example THF. The alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi. Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C. Optionally, step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C. Optionally, steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine. In one embodiment, magnesium chloride is added in step (b).
Step (d): In one embodiment, the reaction mixture from step (c) is used directly in step (d) without further purification. The hydrazine used in step (d) may be in the form of hydrazine hydrate. Suitable solvents for step (d) include THF and EtOH. Step (d) may be performed at a temperature of -40 °C to 40 °C, such as -30 °C to 30 °C, -30 °C to 10 °C, and -30 °C to 0 °C. The acid used in step (d) may be a carboxylic acid, such as acetic acid.
Step (e): The coupling reagent is a reagent suitable for activating a carboxyl group for amide synthesis. Such coupling agents are known in the art and include, for example, ((1H- benzo [d] [ 1 ,2,3]triazol- 1 -y l)oxy )tri(py rrolidin- 1 -y l)phosphonium hexafluorophosphate(V). Optionally the reaction of step (e) may be performed in the presence of a base, such as DBU. Optionally the reaction of step (e) may be performed in the presence of molecular sieves to capture water, for example molecular sieve 0.3 nm. Suitable solvents for step (e) include polar apotic solvents, such as MeCN. Step (e) may be performed at a temperature of 20 °C to 70 °C, such as 30 °C to 60 °C.
The removal of the THP protecting group and diol protecting group may be performed according to known methods. In one embodiment, the THP protecting group and diol protecting group are removed using aqueous acid or alcoholic acid. In one embodiment, the THP protecting group and diol protecting group are removed using hydrochloric acid in an alcohol solvent, such as isopropyl alcohol. In one embodiment, there is provided a process for the preparation of Compound 2, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with 3 -(tert-butyl) 4-methyl pyridine-3,4- dicarboxylate to form tert-butyl 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)nicotinate, or a salt thereof;
(d) reacting tert-butyl 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)nicotinate, or a salt thereof, with hydrazine, optionally in the presence of an acid such as acetic acid, to form l-[2-tetrahydropyran-2-yloxy-4- (trifluoromethyl)phenyl]-3H-pyrido[3,4-d]pyridazin-4-one, or a salt thereof;
(e) reacting l-[2-tetrahydropyran-2-yloxy-4-(trifluoromethyl)phenyl]-3H-pyrido[3,4- d]pyridazin-4-one with (lR,2R)-2-aminocyclohexan-l-ol, or a salt thereof, in the presence of a coupling reagent, to form (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran-2- yl)oxy)-4-(trifhioromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan-l- ol, or a salt thereof;
(f) removal of the THP protecting group from (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran- 2-yl)oxy)-4-(trifluoromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan- l-ol, or a salt thereof, by reaction with an acid to form Compound 2, or a salt thereof; and
(g) isolation of Compound 2, or a salt thereof.
Steps (a)-(c): Suitable solvents for steps (a)-(c) include ethers, for example THF. The alkyl lithium may be n-BuLi or t-BuLi, optionally n-BuLi. Steps (a)-(c) may be performed at temperatures of -80 °C to 20 °C, such as -80 °C to 10 °C, -80 °C to 0 °C, -40 °C to 10 °C, and - 20 °C to 5 °C. Optionally, step (c) is performed at a temperature of -80 °C to -60 °C, such as -80 °C to -70 °C, or about -78 °C. Optionally, steps (a)-(c) may be be performed in the presence of a coordinating ligand such as N,N,N',N' -tetramethyl- 1,2-ethanediamine. In one embodiment, magnesium chloride is added in step (b). Step (d): In one embodiment, the reaction mixture from step (c) is used directly in step (d) without further purification. The hydrazine used in step (d) may be in the form of hydrazine hydrate. Suitable solvents for step (d) include THF and EtOH. Step (d) may be performed at a temperature of -40 °C to 40 °C, such as -30 °C to 30 °C, -30 °C to 10 °C, and -30 °C to 0 °C. The acid used in step (d) may be a carboxylic acid, such as acetic acid.
Step (e): The coupling reagent is a reagent suitable for activating a carboxyl group for amide synthesis. Such coupling agents are known in the art and include, for example, ((1H- benzo [d] [ 1 ,2,3]triazol- 1 -y l)oxy )tri(py rrolidin- 1 -y l)phosphonium hexafluorophosphate(V). Optionally the reaction of step (e) may be performed in the presence of a base, such as DBU. Optionally the reaction of step (e) may be performed in the presence of molecular sieves to capture water, for example molecular sieve 0.3 nm. Suitable solvents for step (e) include polar apotic solvents, such as MeCN. Step (e) may be performed at a temperature of 20 °C to 70 °C, such as 30 °C to 60 °C.
Step (I): The removal of the THP protecting group may be performed according to known methods. In one embodiment, the THP protecting group is removed using aqueous acid or alcoholic acid. In one embodiment, the THP protecting group is removed using hydrochloric acid in an alcohol solvent, such as isopropyl alcohol.
Chemical Intermediates
Provided herein are novel chemical intermediates that, as described herein, are useful in the synthesis of inhibitors of the NLRP3 inflammasome such as Compound 1 and Compound 2.
Accordingly, in one embodiment there is provided a compound that is salt thereof.
In one embodiment there is provided a compound that i or a salt thereof. In one embodiment there is provided a compound that i a salt thereof.
In one embodiment there is provided a compound that a salt thereof.
In one embodiment there is provided a compound that thereof, wherein X is CH or N.
In one embodiment there is provided a compound that or a salt thereof, wherein X is CH or N.
In one embodiment there is provided a compound that i salt thereof, wherein X is CH or N.
In one embodiment there is provided a compound that i or a salt thereof.
In one embodiment there is provided a compound that i or a salt thereof.
In one embodiment there is provided a compound that i a salt thereof.
In one embodiment there is provided a compound that i a salt thereof. In one embodiment there is provide a chemical intermediate as described in the Examples herein, or a salt thereof.
In one embodiment there is provided Compound 1 or a salt thereof, obtainable by any of the processes described herein.
In one embodiment there is provided Compound 1 or a salt thereof, obtained by any of the processes described herein.
In one embodiment there is provided Compound 2 or a salt thereof, obtainable by any of the processes described herein.
In one embodiment there is provided Compound 2 or a salt thereof, obtained by any of the processes described herein.
Salts of compounds described herein may be, for example, acid-addition salts or baseaddition salts. An acid addition salt of a compound described herein may be formed by bringing the compound into contact with a suitable inorganic or organic acid under conditions known to the skilled person. An acid addition salt may for example be formed using an inorganic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid. An acid addition salt may also be formed using an organic acid selected from the group consisting of trifluoroacetic acid, citric acid, maleic acid, oxalic acid, acetic acid, formic acid, benzoic acid, fumaric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, methanesulfonic acid, benzenesulfonic acid and para-toluenesulfonic acid.
A base-addition salt of a compound described herein may be formed by bringing the compound into contact with a suitable inorganic or organic base under conditions known to the skilled person. For example, it may be possible to make an alkali metal (such as sodium, potassium, or lithium) or an alkaline earth metal (such as a calcium) salt by treating a compound with an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g., an ethoxide or methoxide) or a suitably basic organic amine (e.g., a choline or meglumine) in an aqueous medium.
In the processes described herein, it may be necessary to protect reactive functional groups (e.g. hydroxy, dihydroxy) in intermediates described in the syntheses herein to avoid their unwanted participation in a reaction leading to the formation of desired products. Conventional protecting groups, for example those described by P. G. M. Wuts in “Greene’s Protective Groups in Organic Synthesis”, Fifth Edition., John Wiley & Sons Inc., 2014, may be used. For example, where a phenolic hydroxy group is protected as a methyl ether, the protecting group may be removed by using BBn in dichloromethane. Benzyl protecting groups may be removed by hydrogenation over a palladium catalyst, and paramethoxybenzyl groups may be removed using HC1 in an alcohol. Acetal protecting groups of diols may be removed by treatment with an acid (for example ACOH/H2O, or HC1 in 1,4-di oxane).
EXAMPLES
The crystalline forms, chemical processes and intermediates used in such processes described in this specification are further illustrated in the following Examples. These Examples are given by way of illustration only and are non-limiting.
In the examples, high resolution mass spectra were recorded on a Micromass LCT mass spectrometer equipped with an electrospray interface (LC-HRMS).
JH NMR measurements were performed on Bruker Avance III 300, 400, 500 and 600 spectrometers, operating at JH frequencies of 300, 400, 500 and 600 MHz, respectively. The experiments were typically recorded at 25 °C. Chemical shifts are given in ppm with the solvent as internal standard. Protons on heteroatoms such as NH and OH protons are only reported when detected in NMR and can therefore be missing. The following abbreviations have been used (and derivatives thereof, e.g. dd, doublet of doublets, etc.): s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad; qn, quintet; p, pentet.
Flash chromatography was performed using either normal phase silica FLASH+® (40M, 25M or 12M), Biotage® SNAP Cartridges KP-Sil (340, 100, 50 or 10), Biotage® SNAP Cartridges KP- NH (340, 100, 50 or 10), or Agela® Flash Column Silica-CS Cartridges (330, 180, 120, 80) unless otherwise stated.
Reversed phase flash chromatography was performed using Agela® C-18 spherical 20-35 pm 100A cartridges unless otherwise stated.
Purifications were performed by preparative HPLC, preparative SFC or reversed phase flash chromatography on a standard equipment, using MS or UV triggered fraction collection, and using stated conditions. In general, all solvents used were commercially available and of analytical grade. Anhydrous solvents were routinely used for reactions.
The Intermediates and Examples named below were named using ChemDraw Professional version 19.0.0.22 from PerkinElmer or Biovia Draw 2020 EE. The skilled person will understand that different chemical naming software may generate different chemical names for a particular compound.
List of abbreviations
AcOH = acetic acid aq. = aqueous d = days
DBU = l,8-Diazabicyclo[5.4.0]undec-7-ene
DCM = Dichloromethane
DIPEA = N,N-Diisopropylethylamine
DMAP = Dimethylaminopyridine
DMF = Dimethylformamide
DMSO = Dimethylsulfoxide
DMSO-d6 = Hexadeuterodimethyl sulfoxide
Et20 = Diethyl ether
EtOAc = Ethyl acetate
EtOH = Ethanol h = hours
HPLC = High Performance Liquid Chromatography
IPA = 2-propanol
IPE = isopropyl ether iPrOAc = Isopropyl acetate
LCMS = Liquid Chromatography Mass Spectrometry
MeCN = acetonitrile
MeOH = Methanol min = minutes
MS (ESI)/ HRMS (ESI) = Mass spectrometry (electrospray ionisation) / High resolution mass spectrometry
MTBE = /c/'Z-Butyl methylether n-BuLi = 1 -Butyl lithium
NMP = N-Methyl-2-pyrrolidone
Pd(dppf C12'CH2C12 = [l,T-Bis(diphenylphosphino)ferrocene]dichloropalladium(II), complex with dichloromethane rt = room temperature
RT = retention time sat. = saturated
SFC = Supercritical Fluid Chromatography
THF = tetrahydrofuran
Examples
Example 1: Preparation of Compound 1, Form A
Step 1: Intermediate 1: 4-chloro-N-[[(4S)-2,2-dimethyl-l,3-dioxolan-4-yl]methyl]phthalazin-l- amine
To a solution of 1,4-di chlorophthalazine (177 g, 889 mmol, 1.0 eq) in anhydrous NMP (450 mL) were added DIPEA (310 mL, 1.78 mmol, 2.0 eq) and (S)-(2,2-dimethy 1-1,3 -dioxolan-4- yl)methanamine (123 g, 938 mmol, 1.05 eq) at rt and the mixture was stirred at 110 °C for 5 h. The reaction mixture was cooled to rt and poured into H2O. The mixture was extracted with EtOAc/hexane=l : 1 and washed by H2O. The organic layer was evaporated under reduced pressure. The residue was triturated with IPE and filtered to give the title compound (212 g, 81%) as a pale yellow powder. MS(ESI): m/z 294.1/296.1 [M+H]+. 'H NMR (400 MHz, DMSO- d6) 6 1.28 (s, 3H), 1.38 (s, 3H), 3.50 - 3.77 (m, 3H), 4.00 - 4.20 (m, 1H), 4.40 - 4.48 (m, 1H), 7.83 - 7.91 (m, 1H), 7.97 - 8.04 (m, 2H), 8.05 - 8.11 (m, 1H), 8.35 - 8.41 (m, 1H).
Step 2: Intermediate 2: 2-[4-[[(4S)-2,2-dimethyl-l,3-dioxolan-4-yl]methylamino]phthalazin-l- yl] -5-(trifluoromethyl)phenol
To a suspension of the Intermediate 1 (203 g, 691 mmol, 1.0 eq) and (2-hydroxy-4- (trifluoromethyl)phenyl)boronic acid (213 g, 1.03 mol 1.5 eq) in 1,4-dioxane (1.7 L) and 2.0 M aq. NazCCh (1.04 L, 2.08 mol, 3.0 eq) was added Pd^ppQCh CThCh (11.3 g, 13.8 mmol, 0.02 eq) and the mixture was stirred and refluxed under argon atmosphere for 6 h. To the reaction mixture were added (2-hydroxy-4-(trifluoromethyl)phenyl)boronic acid (28.5 g, 138 mmol) and Pd(dppf )Ch- CH2CI2 (11.3 g, 13.8 mmol, 0.02 eq). After 3 h, the mixture was cooled to rt and poured into H2O and EtOAc. To the solvent was added activated carbon and the mixture was stirred and filtered through Celite®. The filtrate was extracted with EtOAc and the organic layer was evaporated under reduced pressure. The crude mixture was purified by flash chromatography (silica; CHC13/MeOH=100/0-19/l-8/2). The collected fractions were further purified by flash chromatography (NH-silica; CHC13/MeOH=100/0-39/l-8/2) to give the title compound (112 g, 39%) as a brown solid. MS(ESI) m/z 420.2 [M+H]+. JH NMR (400 MHz, DMSO-d6) 6 1.29 (s, 3H), 1.40 (s, 3H), 3.60 - 3.73 (m, 1H), 3.76 - 3.87 (m, 2H), 4.01 - 4.10 (m, 1H), 4.46 - 4.55 (m, 1H), 7.25 - 7.33 (m, 2H), 7.41 - 7.57 (m, 2H), 7.69 - 7.91 (m, 3H) 8.32 - 8.38 (m, 1H), 10.3 (br s, 1H).
Step 3: Compound 1: (2S)-3-[[4-[2-hydroxy-4-(trifluoromethyl)phenyl]phthalazin-l- yl]amino]propane-l,2-diol (Form A)
To a suspension of the Intermediate 2 (112 g, 267 mmol) in AcOH (240 mL) was added H2O (80 mL) and the mixture was stirred at 80 °C for 5 h. The mixture was cooled to rt and evaporated under reduced pressure. The crude mixture was purified by flash chromatography (silica; CHC13/MeOH=100/0-90/10-80/20) to give the title compound (65.3 g, 57%) as a colorless solid. The residue (65.3 g+7.51 g (the residue of a previous batch)) was triturated with MeOH and filtered. To the obtained solid was added EtOH, and the solvent was evaporated under reduced pressure to give the title compound (62.7 g, 86%) as a colorless crystalline solid (Form A). MS(ESI): m/z 380.1 [M+H]+. 'H NMR (400 MHz, DMSO-d6) 63.39 - 3.49 (m, 2H), 3.51 - 3.61 (m, 1H), 3.70 - 3.80 (m, 1H), 3.82 - 3.91 (m, 1H), 4.80 - 4.90 (m, 1H), 5.21 - 5.29 (m, 1H), 7.26 - 7.32 (m, 2H), 7.43 - 7.48 (m, 1H), 7.49 - 7.54 (m, 1H), 7.71 (br s, 1H), 7.77 - 7.83 (m, 1H), 7.85 - 7.92 (m, 1H), 8.32 - 8.41 (m, 1H) , 10.37 (br s, 1H).
X-ray diffraction analysis of Form A of Compound 1
The X-ray diffraction analysis of Form A of Compound 1 is performed according to standard methods, which can be found for example in e.g. Kitaigorodsky, A.I. (1973), Molecular Crystals and Molecules, Academic Press, New York; Bunn, C.W. (1948), Chemical Crystallography, Clarendon Press, London; or Klug, H.P. & Alexander, L.E. (1974), X-ray Diffraction Procedures, John Wiley & Sons, New York.
X-ray powder diffraction (XRPD) data is measured with Corundum as an internal reference. The XRPD pattern is determined by mounting a sample on a zero-background holder, single silicon crystal, and spreading out the sample into a thin layer.
The powder X-ray diffraction is recorded with a Theta-Theta PANalytical X’Pert PRO (wavelength of X-rays 1.5418 A nickel-filtered Cu radiation, Voltage 45 kV, filament emission 40 mA). Variable divergence and anti-scatter slits and incident and diffracted soller slit 0.04° are used. The sample is rotated during measurement. Sample is scanned from 2.4 - 50°2Theta using a 0.013° step width and a 115.770 s count time together with a PIXcellD detector (active length 3.347° 2Theta).
The XRPD pattern in this was obtained in Bragg-Brentano geometry.
The XRPD pattern of Form A of Compound 1 is shown in Figure 1. The most prominent peaks in the XRPD pattern are listed in Table 1. The peak intensities are described herein as vs (very strong), s (strong), m (medium) and w (weak) and correspond to % relative intensity (based on the most intense peak) of 25-100%, 10-25%, 3-10% and 1-3%, respectively.
Table 1 - Most prominent peaks in the XRPD pattern of Form A of Compound 1
TG/DTA (Thermo Gravimetry / Differential Thermal Analysis) measurements were performed using a TG/DTA 7200 (SII NanoTechnology Inc.). 3.904 mg of Form A of Compound 1 was weighed into an aluminum pan. The sample was then heated from 30 °C to 300 °C, with a heating rate of 10 °C/min under a nitrogen purge of 200 mL/min. An empty aluminum pan was used as a reference. The output from the TG/DTA analysis is shown in Figure 2. Solid line = DTA analysis, Long dashed line = Temperature, Short dashed line = TG analysis. No weight loss from the sample was observed prior to melting. Storage of Form A of Compound 1 at 60 °C/75% relative humidity (RH) (Open container) for 1 week, and 60 °C (Closed container, ambient RH) for 1 week, showed no weight change, and no change in XRPD pattern.
Example 2 - THP-protected route to Compound 1 Step 1: Intermediates 3, 4 and 5:
A solution containing 2-[3-(trifluoromethyl)phenoxy]tetrahydro-2H-pyran (534g, 2.17 mol), N,N,N',N'-tetramethyl-l,2-ethanediamine (266 g, 2.29 mol) in THF (3.2 L) was pumped into a reaction tube at -10°C @10g min'1. Separately n-butyl lithium (1.41 L, 1.96 mol, 1.4M in hexanes) was pumped into the reaction tube at -10°C @3.9 ml min'1 and allowed to mix in a helical chamber. The output was introduced into a reaction tube to mix with a room temperature pumped solution of magnesium chloride (251g, 2.63 mol) in THF (4.78 L) @ 13.9g min'1 and this output flowed to a reaction tube where it was quenched with a pumped solution of phthalic anhydride (651g, 4.40 mol) in THF (3.70 L) @12.0g min'l in a helical mixer. The output was collected and evaporated to approximately half weight and then treated with a IM sodium hydroxide solution (6.4 Kg) and stirred at 10°C for 40 minutes, the layers were separated, and the aqueous layer was extracted with THF (I L) and then the organics were combined to afford a THF solution of Intermediate 3 (2-[2-(tetrahydro-2H-pyran-2-yloxy)-4- (trifluoromethyl)benzoyl]benzoic acid) (13.98 kg), MS (ESI): m/z [M+Na+H2O]+ 435.2.
The THF solution of Intermediate 3 was heated to 35°C and treated with portions of aqueous hydrochloric acid, after stirring for 48 hours aqueous brine (564 g) was added and the phases separated, the organic layer was washed with brine (1 kg x 2), organic layer separated and partially evaporated to afford a THF solution of Intermediate 4 (2-[2-hydroxy-4- (trifluoromethyl)benzoyl]benzoic acid) (1.4 Kg, 24.5%wt by NMR assay, 50.4% yield). 'H NMR (300 MHz, CDC13) 8 8.10 - 8.15 (m, 1H), 7.56 - 7.70 (m, 2H), 7.32 - 7.35 (m, 1H), 7.26 (d, 1H), 7.15 - 7.20 (m, 1H), 6.93 - 6.97 (m, 1H); MS (ESI): m/z [M+Na]+ 333.1. The solution of Intermediate 4 in THF (1.4 kg, 345 g) was diluted with THF (860 mL) then hydrazine hydrate was added (152 g, 3.1 mol) and stirred at 50°C for 5 hours. The solution was allowed to cool and treated with a 3M hydrochloric acid solution (1021g), the organics were separated and then saturated brine (556g) was added, some precipitation occurred which was dissolved by diluting with THF (1037 g) and heating to 50°C, the organic layer was separated and washed with saturated brine (800g x 2), organic layer partially evaporated and heated to 65°C and heptane (1757 g) was added slowly. When the final approximate ratio of THF:heptane (1:1) was reached the mixture was cooled to 20°C and the solids were filtered, washed with THF:heptane (2: 1, 2x 500 mL) and dried to afford Intermediate 5 (4-[2-hydroxy-4- (trifluoromethyl)phenyl]-l(2H)-phthalazinone) (322.5 g, 93%).
'H NMR (300 MHz, CDC13) 8 12.85 (s, 1H), 10.45 (s, 1H), 8.29 - 8.32 (m, 1H), 7.84 - 7.87 (m, 2H), 7.53 (d, 1H), 7.20 - 7.35 (m, 3H); MS (ESI): m/z [M+H]+ 307.0.
Step 2: Intermediate 6: 2-(4-chl oro-1 -phthalazinyl)-5-(trifluoromethyl)phenol
A mixture of Intermediate 5 (122g, 392 mmol) and acetonitrile (1200 mL) were stirred at room temperature before adding phosphoryl chloride (72.8 mL, 784 mmol) and heated to 50°C for 22 hours. The mixture was cooled to 0-5°C and water was added (600 mL) while maintaining the temperature below 10°C then a solution of dipotassium phosphate (204.8g, 1176 mmol in water 1200 mL) was added and the mixture warmed to 20°C and the solids filtered and washed with water (2 x 250 mL) and then acetonitrile ( 4 x 500 mL), dried in vacuo at 50°C to afford the title compound (112.9 g, 71% @80% w/w).
'H NMR (DMSO-de) 8: 10.13-11.11 (br s, 1H), 8.35 (ddd, J=8.1, 1.0 Hz, 1H), 8.16 (ddd, J=8.3, 7.1, 1.2 Hz, 1H), 8.06 (ddd, J=8.3, 7.1, 1.2 Hz, 1H), 7.73 (ddd, J=8.1, 1.0 Hz, 1H), 7.57-7.66 (m, 1H), 7.32-7.40 (m, 2H); MS (ESI): m/z [M+H]+ 325.0/327.0.
Step 3: Compound 1:
To a stirred mixture of (2S)-3-amino-l,2-propanediol (CAS#61278-21-5) andN-methyl-2- pyrrolidone (160 mL) at 125°C was added Intermediate 6 (80 g, 197mmol @ 80% w/w) in N- methyl-2-pyrrolidone (400 mL) over 3 hours and then stirred at 120°C for 2 hours. The mixture was cooled to 20°C and stirred for 16 hours and then water (320 mL) was added over 30 minutes, seed (400 mg, Form A) was added and stirred for 30 minutes and then water (800 mL) was added over 90 minutes, the resulting mixture was stirred for 18 hours and then the solids were filtered, washed with water: isopropyl alcohol (1:1, 400 mL) and then water (2 x 400 mL), dried at 60°C to afford Compound 1 (62.25 g, 81.7% @ 98.0% w/w). XH NMR (DMSO-d6) 8: 10.22-10.46 (br s, 1H), 8.35 (d, J=8.1 Hz, 1H), 7.87 (ddd, J=8.2, 7.1, 1.3 Hz, 1H), 7.78 (ddd, J=7.6, 1.1 Hz, 1H), 7.62 (t, J=5.6 Hz, 1H), 7.51 (d, J=7.6 Hz, 1H), 7.44 (dd, J=8.2, 0.7 Hz, 1H), 7.25-7.31 (m, 2H), 5.24 (br s, 1H), 4.81 (br t, J=5.8 Hz, 1H), 3.81-3.89 (m, 1H), 3.73 (m, 1H), 3.50-3.60 (m, 1H), 3.39-3.45 (m, 2H); MS (ESI): m/z [M+H]+ 380.1. Example 3 - Alternative THP-protected route to Compound 1
Step 1: Intermediate 8: 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)pheny l)phthalazin- 1 (2H)-one o Intermediate 7 Intermediate 8 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran (56.3 g, 228.78 mmol) andNl,Nl,N2,N2- tetramethylethane-l ,2-diamine (37.6 mL, 250.57 mmol) were mixed in THF (200 mL) at rt under N2 and cooled to -5 °C. Butyllithium (2.5 M in hexane, 104 mL, 250.57 mmol) was added over 10 min. To this mixture was added a solution of tert-butyl methyl phthalate (40g, 167.61 mmol) in THF (200 mL) cooled to -78 °C, over 10 min. The mixture was stirred at -78 °C for another 2 h. The reaction mixture was allowed to warm to -20 °C, then hydrazine hydrate (16.31 mL, 335.21 mmol) was added. A further 2 mL of water was added, followed by acetic acid. The reaction mixture was stirred overnight at -20 °C. The mixture was partitioned between water (200 mL) and MeTHF (200 mL), the layers separated and the aqueous extracted with further MeTHF (100 mL). The organic layers were combined, and concentrated in vacuo. The residue was dissolved in TBME (40 mL) and heptane (160 mL) was slowly added. The precipitate was isolated by filtration to give crude title compound as a white solid (22.5 g).
Step 2: Intermediate 9: N-(((S)-2,2-dimethyl-l,3-dioxolan-4-yl)methyl)-4-(2-((tetrahydro-2H- py ran-2-yl)oxy )-4-(trifluoromethy l)pheny l)phthalazin- 1 -amine
Intermediate 8 (2.00 g, 5.12 mmol) and (S)-(2,2-dimethyl-l,3-dioxolan-4-yl)methanaminium chloride (3.44 g, 20.49 mmol) was mixed in acetonitrile (20 mL). Addition of DBU (6.12 mL, 40.99 mmol) and molecular sieve 0.3 nm (water abs. capacity >=20%) (Merck EMD Millipore Corporation). The mixture is stirred for ca. 1 h at rt. ((lH-benzo[d][l,2,3]triazol-l- yl)oxy)tri(pyrrolidin-l-yl)phosphonium hexafluorophosphate(V) (5.33 g, 10.25 mmol) is added, and the reaction mixture is heated to 50 °C and stirred at 50 °C for several hours. The conversion is checked by IPC. The reaction mixture is filtered to remove the molecular sieves; the molecular sieves are rinsed with acetonitrile, and the filtrate is evaporated to dryness at 40 °C under vacuum. The residue on evaporation (18.044 g), containing the product, is obtained as an orange oil. It is taken up in EtOAc (150 mL) and aq. 10% citric acid (100 mL). After phase separation, the organic phase is washed with additional aq. 10% citric acid (2 x 100 mL), then with aq. sat. NaHCOs (2 x 100 mL), and then dried with MgSO4. The MgSO4 is removed by filtration and is thoroughly washed, and the filtrate is reduced by evaporation at 40 °C under vacuum. The crude product (4.00 g) is obtained as a yellow to light orange foam. MS (ESI): m/z [M+H]+ 504.5.
Step 3: Compound 1:
Intermediate 9 is mixed with 5-6 M HC1 in IPA (4.66 mL, 25.62 mmol) and iPrOH (20 mL) at 40 °C for ca. 10 min. An orange solution is present which is evaporated to dryness. The crude product (4.578 g) is obtained as an orange resin. 200 mg of crude product was triturated in ethyl acetate/ethanol (1/1 vol/vol). The product is isolated by filtration, washed with EtOAc, and dried The product (105 mg, HC1 salt) is obtained as an off-white solid.
'H NMR (500 MHz, DMSO) 6 10.91 (br, 1H), 10.29 (br, 1H), 9.00 (d, 1H), 8.21-8.02 (m, 2H), 7.70-7.53 (m, 2H), 7.43 (s, 1H), 7.39-7.33 (m, 1H), 4.00 (br, 2H, OH), 4.00-3.92 (m, 2H), 3.85- 3.69 (m, 2H), 3.56-3.46 (m, 2H), 2.50 (DMSO). MS (ESI): m/z [M+H]+ 380.3.
Comparative Example 1: Preparation of Compound 2
Step 1: Intermediate 10: l-bromo-2-((4-methoxybenzyl)oxy)-4-(trifluoromethyl)benzene
2-bromo-5-(trifluoromethyl)phenol (3.0 g, 12.45 mmol) and l-(bromomethyl)-4- methoxybenzene (2.53 g, 12.57 mmol) was dissolved in MeCN (30 mL) and potassium carbonate (1.892 g, 13.69 mmol) was added in one portion (no or very weak exotherm). The reaction mixture turns yellow. The reaction mixture was stirred at rt overnight. The reaction was complete after 16 h according to NMR. Water and EtOAc were added and the phases were separated. The aqueous phase was extracted with EtOAc and the combined organic extract was washed with brine and evaporated. This gave a pale orange oil that did not crystallize from IPA (approximately 15 mL). The oil was instead purified by column chromatography (silica gel, heptane/EtOAc:20/l as eluent) to yield 3.58 g (80%) of the title compound as a colorless oil that crystallized upon standing. 'H NMR (500 MHz, DMSO-d6) 6 3.76 (s, 3H), 5.23 (s, 2H), 6.95 - 7.00 (m, 2H), 7.22 - 7.28 (m, 1H), 7.42 (d, 2H), 7.51 (d, 1H), 7.80 - 7.86 (m, 1H).
Step 2: Intermediate 11: 3-(tert-butyl) 4-methyl pyridine-3,4-dicarboxylate
Tert-butanol (200 mL) was added to 4-(methoxycarbonyl)nicotinic acid (25.0 g, 138 mmol) followed by the addition of di-tert-butyl di carbonate (60.2 g, 276 mmol) and pyridine (25 mL). DMAP (100 mg, cat.) was added and the reaction stirred at 35 °C overnight. Water and iPrOAc were added and the two phases separated. The organic extract was washed with two portions of water, evaporated and the residue was evaporated two times with toluene. The residue was filtered through a column of silica using 40% MTBE in heptane as mobile phase to afford the title compound (27.7 g, 84%) as a pale yellow oil. 'H NMR (500 MHz, DMSO-d6) 6 1.52 (s, 9H), 3.88 (s, 3H), 7.65 (dd, 1H), 8.87 (d, 1H), 8.96 (d, 1H).
Step 3: Intermediate 12: tert-butyl 4-[2-[(4-methoxyphenyl)methoxy]-4- (trifluoromethyl)benzoyl]pyridine-3-carboxylate
Intermediate 11 (7.0 g, 29.6 mmol) was dissolved in THF (50 mL) and cooled to -78 °C. In another flask, Intermediate 10 (10.7 g, 29.6 mmol) was dissolved in THF (50 mL) and n-BuLi (19.4 mL, 31.1 mmol, 1.6 M in hexanes) was added at -78 °C. The lightyellow solution was stirred at -78 °C for 15 seconds before added dropwise via cannula to the first solution. The reaction mixture was stirred at -78 °C for 10 min, then AcOH (1.9 mL in 100 mL water) was added followed by the addition of EtOAc. The reaction mixture was allowed to reach rt and the two phases were separated. The organic extract was washed with water and evaporated to afford the title compound (14.4 g, quant.) as an orange oil. Used in the next step without further purification. MS (ESI): m/z [M+H]+ 488.3.
Step 4: Intermediate 13: l-[2-[(4-methoxyphenyl)methoxy]-4-(trifluoromethyl)phenyl]-3H- pyrido[3,4-d]pyridazin-4-one
Intermediate 12 (41.4 g, 84.9 mmol) was dissolved in THF (300 mL), hydrazine monohydrate (21.1 mL, 340 mmol, 50% in water) was added and the reaction mixture was stirred at 60 °C for 16 h. Water (100 mL) was added and the mixture was stirred at rt before being poured into water (600 mL). The solid was filtered off and washed with water and MTBE. The product was slurried in refluxing EtOAc (1 L), cooled to rt and filtered to afford the title compound (17.3 g, 48%) as an off-white solid. MS (ESI): m/z [M+H]+ 428.2. 'H NMR (500 MHz, DMSO-d6) 6 3.68 (s, 3H), 5.15 (s, 2H), 6.75 (d, 2H), 7.02 (d, 2H), 7.27 (d, 1H), 7.51 (d, 1H), 7.65 (d, 2H), 8.93 (d, 1H), 9.48 (s, 1H), 13.23 (s, 1H).
Step 5: Intermediate 14: 4-chloro-l-[2-[(4-methoxyphenyl)methoxy]-4- (trifluoromethyl)phenyl]pyrido[3,4-d]pyridazine
Intermediate 13 (6.0 g, 14.0 mmol) was slurried in 1,4-dioxane (55 mL). Pyridine (9.9 mL, 122 mmol) and phosphoryl trichloride (4.6 mL, 48.9 mmol) were added and the reaction stirred at 60 °C for 19 h. The mixture was cooled to rt and then added to tri-sodium citrate (180 mL, aq., 1 M). The precipitated product was filtered off, washed with water (2 x 50 mL) and dried under vacuum give a tan solid. The crude was slurried in MeCN (80 mL) and heated to 80 °C until dissolved. The mixture was cooled to rt and the formed precipitate was filtered off, washed with MeCN (2 x 15 mL) and dried to afford the title compound (2.57 g, 41%) as a tan solid. MS (ESI): m/z [M+H]+ 446.3. *H NMR (500 MHz, DMSO-d6) 63.66 (s, 3H), 5.15 (s, 2H), 6.73 (d, 2H), 6.99 (d, 2H), 7.58 (d, 1H), 7.65 (dd, 1H), 7.72 (d, 2H), 9.10 (d, 1H), 9.69 - 9.82 (m, 1H).
Step 6: Intermediate 15: (lR,2R)-2-[[l-[2-[(4-methoxyphenyl)methoxy]-4-
(trifluoromethyl)phenyl]pyrido[3,4-d]pyridazin-4-yl]amino]cyclohexanol
Intermediate 14 (2.6 g, 5.7 mmol), (lR,2R)-2-aminocyclohexan-l-ol (1.1 g, 9.2 mmol) and NaHCCh (2.4 g, 28.7 mmol) were mixed in IPA (22 mL) and stirred at 80 °C for 3 d. The reaction mixture was poured into water (100 mL) and stirred to rt for 2 h. The solid was filtered off, washed with water and dried under vacuum at 40°C to afford the title compound (2.9 g, 96%) as a tan solid. MS (ESI): m/z [M+H]+ 535.6. 'H NMR (500 MHz, DMSO-d6) 6 1.30 (s, 4H), 1.69 (d, 2H), 1.97 (d, 1H), 2.12 (s, 1H), 3.61 (d, 1H), 3.66 (s, 3H), 4.19 (s, 1H), 4.83 (s, 1H), 5.11 (s, 2H), 6.74 (d, 2H), 7.03 (d, 2H), 7.26 (d, 1H), 7.48 (d, 1H), 7.60 (s, 2H), 7.67 (d, 1H), 8.81 (d, 1H), 9.76 (s, 1H).
Step 7: Compound 2: 2-(4-(((lR,2R)-2-hydroxycyclohexyl)amino)pyrido[3,4-d]pyridazin-l- yl)-5-(trifluoromethyl)phenol
Intermediate 15 (2.9 g, 5.5 mmol) was slurried in absolute EtOH (99.5%, 7 mL), HC1 (4 M in 1,4-dioxane, 20.7 mL, 82.9 mmol) was added and the reaction stirred at rt for 2 h. The mixture was added dropwise to Et2O (150 mL) under stirring to give a precipitate which was filtered off, washed with Et2O and dried to give a light yellow solid (HCl-salt). The solid was slurried in water (50 mL), made basic (pH=9) with sat. aq. NaHCCL and extracted with DCM:MeOH = 9:1 (multiple times). The combined organic extracts were filtered through a phase separator and evaporated to give 1.85 g orange semi-solid. This crude was dissolved in MeCN (20 mL) and IPA (0.5 mL) at 70 °C, cooled to rt, filtered, washed with MeCN and dried under vacuum at 40 °C to afford the title compound (1.25 g, 56%) as an yellow solid. MS (ESI): m/z [M+H]+ 405.3. HRMS (ESI): m/z [M+H]+ calcd for C20H19F3N4O2: 405.1538, found: 405.1538. 'H NMR (500 MHz, DMSO-d6) 6 1.25 - 1.41 (m, 4H), 1.72 (d, 2H), 1.99 (d, 1H), 2.13 (s, 1H), 3.56 - 3.69 (m, 1H), 4.17 - 4.28 (m, 1H), 7.24 - 7.36 (m, 3H), 7.55 (d, 1H), 7.71 (d, 1H), 8.84 (d, 1H), 9.80 (s, 1H), 10.46 (s, 1H).
Example 3: Preparation of Compound 2 via THP-protected route
Step 1: Intermediate 17: l-[2-tetrahydropyran-2-yloxy-4-(trifhioromethyl)phenyl]-3H- pyrido[3,4-d]pyridazin-4-one
2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran (45.9 g, 186.6 mmol) and Nl,Nl,N2,N2- tetramethylethane-l,2-diamine (25.9 mL, 173 mmol) were mixed in THF (250 mL) at rt under N2 and cooled to 0 °C. Butyllithium (2.4 M in hexane, 71.9 mL, 173 mmol) was added over 30 min keeping the temperature below 5 °C. This mixture was transferred via teflon tube to 3-(tert- butyl) 4-methyl pyridine-3,4-dicarboxylate (0.5 M in THF, 300 ml, 150 mmol) under N2 and cooled to -78 °C over 30 min, keeping the temperature below -70 °C. The mixture was stirred at -78 °C for another 30 min (resulting in the formation of Intermdiate 16). (Intermediate 16: 'H NMR (500 MHz, CDC13) 6 9.17 (d, 1H), 8.81 (d, 1H), 8.19 (d, 1H), 7.54 (s, 1H), 7.40 (dd, 1H), 7.28 (CDC13), 7.25 (dd, 1H), 5.31 (t, 1H), 3.64-3.50 (m, 2H), 1.59-1.45 (m, 2H), 1.45-1.31 (m, 2H), 1.39 (s, 9H), 1.02-0.93 (m, 1H), 0.88-0.76 (m, 1H). MS (ESI): m/z [M+H]+ 452.5.) Hydrazine hydrate (14.6 mL, 300 mmol) was added over 4 min at -78 °C, then the cooling bath was removed and the reaction stirred to rt for 1 h. The reaction mixture was heated to 30 °C and acetic acid (42.9 mL, 750 mmol) in absolute EtOH (43 mL) was added via dropping funnel over 15 min and the mixture diluted with more EtOH (100 mL) and water (50 mL). This mixture was evaporated to a volume of 200 mL to give a thick suspension which was cooled to 10 °C, then filtered. The solid was washed with cooled EtOH: water 1:1 (110 mL) and cooled water (100 mL), air dried for 30 min, then transferred to a flask and dried under vacuum over night to afford the title compound (40.1 g, 68%) as a tan solid. 'H NMR (500 MHz, DMSO) 60.8 - 1.62 (6H, m), 3.41 - 3.69 (2H, m), 5.72 (1H, d), 7.25 - 7.39 (1H, m), 7.49 - 7.76 (3H, m), 8.96 (1H, dd), 9.52 (1H, s), 13.28 (1H, s). MS (ESI): m/z [M+H]+ 392.2.
Step 2: Intermediate 18: (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan-l-ol
(lR,2R)-2-aminocyclohexan-l-ol hydrochloride (291 g, 1916 mmol), ((1H- benzo[d][l,2,3]triazol-l-yl)oxy)tri(pyrrolidin-l-yl)phosphonium hexafluorophosphate(V) (665 g, 1278 mmol) and DBU (382 ml, 2555 mmol) are mixed in acetonitrile (1.25 L) in a 10 L reactor. The reaction mass is agitated until a solution is present. Addition of a solution of Intermediate 17 (250 g, 639 mmol) and DBU (191 ml, 1278 mmol) in THF (1000 mL) [1.4 L, orange solution] within 3 h. An IPC after an additional hour shows complete conversion. Work-up: Addition of EtOAc (2.5 L) and washing with aq. 10% citric acid (2 x 2.5 L). The aqueous phase is back- extracted with EtOAc (2.5 L), and the combined organic phases are washed with aq. sat. NaHCOs (2 x 2.5 L). The organic phase is reduced by distillation in vacuum at elevated temperature (ca. 70 °C) until a target volume of 1.25 L remains. The concentrated phase of the title compound is cooled to 20 °C. MS (ESI): m/z [M+H]+ 489.4.
A 5 to 6N solution of hydrogen chloride in 2-propanol (532 ml, 3194 mmol) is added to the concentrated phase of Intermediate 18. A suspension starts to forms, and an IPC after 0.5 h shows complete conversion. Acetonitrile (6 L) is added and an orange suspension is present which is agitated for 2 h. The product is isolated by filtration, washed with acetonitrile (1 L) in 2 portions, and dried on the nutsche in vacuum with a N2 current through the cake. The crude product (291 g) is obtained as a yellow solid. The crude material is mixed with water (1500 mL) and a yellow, foamy, thin suspension forms. EtOH (absolute; 300 mL) is added and the resulting reaction mass is heated to 60 °C to form a yellow suspension; addition of more EtOH (absolute; 50 mL) and water (250 mL). The suspension is cooled from 60 °C to 20 °C within 1 h and is stirred for 1 h at 20 °C before isolation by filtration. The product cake is washed with water (8 L). The product is dried overnight on the nutsche in vacuum with aNi current (20 L/min) through the cake. The pure product (186 g) is obtained as an off-white, slightly yellow solid. The material is ground and homogenized in the nutsche during off-loading and is further dried in a vacuum oven (<10 mbar) at elevated temperature (50 °C). The pure, dry title compound (153 g, 50 %) hydrochloride salt is obtained as an off-white, slightly yellow, crystalline solid.
Biological and Physicochemical Data
Human NLRP3 speck formation assay (Test A)
To profile compounds for NLRP3 antagonist activity with respect to inhibition of Nigericin triggered speck formation, the ASC-GFP Reporter Monocytes (InvivoGen #thp- ascgfp) was employed. The assay is based on NF-kB dependent expression of the ASC::GFP fusion protein. LPS-priming of cells increases ASC::GFP expression and Nigericin recruits ASC::GFP, pro-caspase-1 and NLRP3 to form micrometer-sized complexes, ASC-specks, that are quantified by fluorescence microscopy. Preparation of assay reagents:
Assay medium: RPMI 1640 (Gibco #72400-021) supplemented with 10% heat inactivated FBS (Gibco #10270)
Cells: THP-ASC-GFP were cultured in RPMI 1640 (Gibco #72400-021) supplemented with 10% heat inactivated FBS (Gibco #10270) and lOOpg/mL Zeocin (Life Technologies #46- 0072) (every other passage) to maintain ASC::GFP expression.
Step by step protocol for running the assay:
Day 1
1. Cells were counted with a CEDEX (Innovartis) and diluted with assay medium supplemented with lOOnM Phorbol 12-myristate 13 acetate (Sigma #P8139) to 375000 cells/mL.
2. 20 pl cell mix above were dispensed into black pclear TC-treated (Greiner #781091) 384 well plates with Multidrop Combi (ThermoFisher).
3. Plates were incubated at 37°C, 5% CO2 for 20h.
Day 2
1. 10 pl LPS (Sigma #L2654) were dispensed with Multidrop Combi (ThermoFisher) for 1 pg/mL.
2. Plates were incubated at 37°C, 5% CO2 for 3h.
3. 80 nl test compound in DMSO were prepared in concentration response curves and diluted with 20 pl assay medium supplemented with 68 pM ZVAD-FMK (Promega #7231) in polypropylen 384 well plates (Greiner # 781280)
4. 10 pl above test compound solution were transferred to cell plate with Bravo (Agilent).
5. Plates were incubated at 37°C, 5% CO2 for 30 min
6. 15 pl Nigericin (Sigma #SML1779) at 75 pM were dispensed to cell plates with Certus (Gyger)
7. Plates were incubated at 37°C, 5% CO2 for Ih
8. 15 pl 17.3 % Formaldehyde (Sigma #F8775) supplemented with Hoechst nucleic acid stain (Life Technologies #H3570) diluted 1:5000 were added with Multidrop (ThermoFisher)
9. Plates were incubated at RT for 15 min
10. Plates were washed two times with 40pl PBS (Gibco #100100) with Bluewasher (BlueCatBio)
11. Plates were imaged using ImageXpress (Molecular Devices) Image data was processed using Columbus software (Perkin Elmer) using nucei stain to identify cells and spot detection to identify the ASC-specks within the cells. Screener (Genedata AG) was used to further process data. Concentration response data of number of specks per cell were fitted using a four parameter logistic fit and EC 50 values reported in Table 2.
Nigericin triggered (human NLRP3) IL-1B assay (Test B)
Compounds were profiled for NLRP3 antagonist activity with respect to inhibition of Nigericin triggered IL-ip release from THP-1 human monocytes. Quantification was performed using a commercially available human IL-ip HTRF detection kit (CisBio, 62HIL1BPEH). The assay uses two anti-IL-ip antibodies in a sandwich assay format. One labeled with a donor fluorophore (Eu cryptate), a second with an acceptor (XL). Immune-complexes containing the two antibodies bound to the same IL-ip molecule allows fluorescence resonance energy transfer (FRET) between the donor and acceptor after excitation of the donor with a light source, subsequently resulting in fluorescence at 665 nM from the acceptor. The fluorescence signal intensity is proportional to the IL-ip concentration in the sample.
Preparation of assay reagents:
Cells: THP human monocytic leukemia cell line. Cells generally passaged every 2-3 days with density kept from 0.2 to 0.4*10A6 cells/mL.
Culture and assay medium: RPMI 1640 (Gibco, 72400-021) supplemented with 10% FBS (Sigma, F2442)
IL-ip standard: reconstituted IL-ip standard provided in the CisBio kit was diluted in assay medium to a top final concentration of 2 ng/mL in the assay.
HTRF detection reagents: cAMP-d2 and anti-cAMP cryptate were reconstituted according to CisBio kit instructions. Just prior to use, reagents were combined using the following proportions: 10/24 Detection buffer (provided with the kit), 14/24 PBS (Gibco, 10010), 1/120 IL-ip Eu-cryptate Antibody and 1/120 IL-ip XL Antibody.
Step by step protocol for running the assay:
Day 1
1. 20 nL test compounds dissolved in DMSO were aquostically dispensed (Labcyte Echo) to white 384-well plates (Greiner; 784075), sealed and stored at rt until assayed.
2. 20 nL 50 pM of a control compound in DMSO (250 nM final concentration) was added to 100% inhibition control wells and 20 nL DMSO added to 0% control wells with Echo dispenser. The control compound may be selected from MCC950 (N-[[(l,2,3,5,6,7- hexahydro-s-indacen-4-yl)amino]carbonyl]-4-(l-hydroxy-l-methylethyl)-2- furansulfonamide) or any other compound that acts as a full antagonist in the assay.
3. An aliquot of cells was taken out from cells grown in continuous culture and counted with a CEDEX (Innovatis).
4. The number of cells needed for an experiment were centrifuged for 5 min at 250 xg and resuspend to 1.0*10A6 cells/mL with 37°C assay medium.
5. LPS (Sigma; L2654) was added to a final concentration of 1 pg/mL.
6. Cell were LPS-primed in bulk in a 50 mL tube by incubating at 37°C, 5% CO2 and 95% humidity for 3 h.
7. 4 pL cell solution at 1.0*10A6 cells/mL was dispensed with Multidrop Combi (Thermo Fisher) to white 384-well small volume plates (Greiner; 784075) to give 4000 cells/well.
8. 30 min incubation at 37°C, 5% CO2 and 95% humidity.
9. 4 pL of 40 pM nigericin in assay medium was added with Certus (Gyger) to a final concentration of 20 pM.
10. 1 h incubation at 37°C, 5% CO2 and 95% humidity.
11. 4 pL HTRF detection reagents was added with Multidrop Combi.
12. 3 h incubation at rt protected from light.
13. Homogenous Time-Resolved Fluorescence (HTRF) signal was detected with an Envision (PerkinElmer) or Pherastar (BMG Labtech) reader (Lex = 340 nm, Lem = 665 and 615 nm).
Using an IL-ip standard curve, HTRF data was converted to amount IL-ip produced in the samples which was subsequentially used for calculation of concentration responses. Concentration response data were analyzed with Screener (Genedata) and fitted with a four parameter logistic fit. The results from the assay are reported in Table 2 as ICso (pM).
IC50 is defined as the concentration at which the inhibitory activity reaches 50% of its maximum level. Where the assay was run multiple times for the same compound, the geometric mean is reported. To facilitate comparison of efficacy data, efficacy was normalized to % inhibitory effect of the test compound compared to the inhibition caused by a saturating concentration of the control compound (250 nM).
BzATP triggered (human NLRP3) IL-IP assay (Test C) In a variant of the IL-ip assay, compounds were tested for their ability to inhibit BzATP (2'(3')-O-(4-Benzoylbenzoyl)adenosine 5 '-triphosphate) triggered IL-ip release from THP-1 human monocytes. Like the nigericin triggered assay, quantification was performed using a human IL-10 HTRF detection kit (CisBio, 62HIL1BPEH).
There were some differences between the nigericin triggered assay (Test B) and the BzATP triggered assay. Conditions in the BzATP triggered assay with relevant differences compared to the nigericin triggered assay include:
Cell culture medium: RPMI 1640 (Gibco, 11875-119) supplemented with 10% FBS (Sigma, 171012) and Penicillin-streptomycin (Thermo Fisher, 15140-122).
- Assay medium: RPMI 1640 (Gibco, 22400-105) supplemented with 1% FBS (Sigma, 171012).
Cells were primed with LPS (Sigma, L2630) at a final concentration of 2 pg/mL for 24 h (instead of 1 pg/mL for 3 h).
- IL-ip production was triggered by addition of BzATP (Sigma, B6396) (instead of nigericin) at a final concentration of 1 mM followed by 30 min incubation at 37°C, 5% CO2 and 95% humidity.
The results from the assay are reported in Table 2 as IC50 (pM). hERG assay (Test D)
Experiments were performed on the SyncroPatch 384PE (Nanion Technologies) high throughput patch clamp platform at rt and used medium resistance chips with 4 patch holes per site. hERG-expressing Chinese hamster ovary KI (CHO) cell line were used in assay-ready format and kept in liquid nitrogen until use. 2 vials of cells (10 x 106 cells per vial) were thawed and added to 20 mL Hepes-buffered saline solution (HBSS). HBSS comprised 140 mM NaCl, 4 mM KC1, 10 mM HEPES and 5 mM Glucose (pH 7.4). The internal patch clamp solution was KF 120 mM, KC120 mM, HEPES 10 mM, EGTA 10 mM, and 25 pM Escin (pH7.2). After the initial sealing process was complete, a seal enhancer solution comprising HBSS supplemented with 10 mM CaCh and ImM MgCh was applied to cells. The external solution was then exchanged (4 times) for external patch clamp solution comprising NaCl 80 mM, KC1 4 mM, HEPES 10 mM, CaCh 2 mM, MgCh 1 mM, glucose 5 mM, and NMDG 60 mM (pH 7.4). All solutions were stored at rt, except Escin, which was stored at 4 °C. All compounds were dispensed in greiner-bio 384 well plates and tested in a 6 point cumulative assay (final DMSO concentration 0.33%). Only wells that passed acceptance criteria (30 MegaOhm seal resistance, Z prime >0.4 and current size >0.2 nA) were used in this analysis. The IC50 (pM) results of the hERG assay are reported in Table 2.
Solubility (Test E)
The assay was conducted according to the Solubility Assay described in pages 164-167 of Wemevik, J. et al., “A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling”, Assay and Drug Development Technologies, 2020, 18(4), 157- 179. Data are reported in Table 2 as solubility (pM). Where the assay was run multiple times for the same compound, the arithmetic mean is reported.
Solubility (Test F)
After drying a 20 mM DMSO solution containing the test compound, disodium hydrogenphosphate-citric acid buffer solution (Diluted Mcllvaine buffer, pH6.5) was added to dilute 100 fold. Under these conditions, the theoretical maximum concentration of the test compound was 200 pM. The buffer was sonicated, shaken, and held at 25 °C for 24 to 72 h. The buffer sample was filtered and the filtrate was diluted with an equal volume of acetonitrile/methanol (1:1, v/v) in a 96 well plate. A 20 mM DMSO solution containing the test compound was diluted 100 fold with acetonitrile/methanol (1:1, v/v) and the same amount of Mcllvaine buffer (pH 6.5) was added to use as the standard solution. The standard and test samples were transferred to a 384 well plate and analyzed by HPLC. The results of the solubility assay are reported in Table 2 in pg/mL.
Table 2 - Assay data
LPS/ATP test
Male 7-week-old BALB/cAJcl mice were intraperitoneally administered 0.5 mL of 4 pg/mL LPS (Sigma-Aldrich Co. LLC, L2630) solution in PBS (Thermo Fisher Scientific Inc., 10010). One hour later, the test article suspension in 0.5% (w/v) CMC sodium (Nacalai tesque INC., 07326-95) aqueous solution was orally administered at a volume of 10 mL/kg. One hour after the article administration, 0.5 mL of 30 pmol/L ATP (Sigma- Aldrich Co. LLC, A7699) solution in PBS was intraperitoneally administered. Twenty minutes later, the animals were euthanized by cervical dislocation under sevoflurane anesthesia. Immediately after euthanasia, peritoneal cavity of each animal was washed with 3 mL of ice-cold PBS intraperitoneally injected. Then, the PBS was collected, and the concentrations of IL-1B were determined using ELISA kit (R&D Systems Inc., MLBOOC). The results of the test are shown in Table 3.
Table 3 - LPS/ATP test data
Those skilled in the art will appreciate that the biological assays described above may be performed using alternative equipment and minor variations to the protocol without significantly affecting the results.
Any publications disclosed within the specification are hereby incorporated by reference.

Claims

1. A crystalline form of (2S)-3-[[4-[2-hydroxy-4-(trifluoromethyl)phenyl]phthalazin-l- yl]amino]propane-l,2-diol (Compound 1).
2. The crystalline form of claim 1, which is crystalline Form A.
3. The crystalline form of claim 1, which has an X-ray powder diffraction pattern (Cu K„ radiation) with at least five specific peaks at about 2-theta = 13.0, 19.3, 19.8, 22.7 and 24.1°.
4. The crystalline form of claim 1, which has an X-ray powder diffraction pattern (Cu K„ radiation) with at least five specific peaks at 2-theta = 13.0, 19.3, 19.8, 22.7 and 24.1°, wherein said values are each ± 0.2° 2-theta.
5. The crystalline form of claim 1, which has an X-ray powder diffraction pattern (Cu K„ radiation) substantially the same as the X-ray powder diffraction pattern shown in Figure 1.
6. A crystalline form of 2-(4-(((lR,2R)-2-hydroxycyclohexyl)amino)pyrido[3,4- d]pyridazin-l-yl)-5-(trifluoromethyl)phenol (Compound 2), hydrochloride salt.
7. A pharmaceutical composition comprising the crystalline form of any one of the preceding claims, and a pharmaceutically acceptable excipient.
8. The crystalline form or the pharmaceutical composition of any one of claims 1-7 for use in therapy.
9. The crystalline form or the pharmaceutical composition of any one of claims 1-7 for use in the treatment of a subject with a disease or condition in which NLRP3 inflammasome activity is implicated.
10. The crystalline form or the pharmaceutical composition of any one of claims 1-7 for use in the treatment of a subject with a disease or condition selected from the group consisting of kidney diseases such as acute kidney injury, chronic kidney disease, and diabetic kidney disease; cardiovascular diseases such as coronary atherosclerotic heart disease, cardiomyopathy, myocardial infarction, cardiac hypertrophy, and ischaemiareperfusion injury; liver diseases such as nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, alcoholic steatohepatitis, chronic hepatitis C virus infection, and paracetamol-induced liver injury; inflammatory diseases such as autoinflammatory disorders, Cryopyrin-associated periodic syndromes, familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), chronic infantile neurologic cutaneous articular (CINCA) syndrome, and neonatal onset multi-system inflammatory disease (NOMID); inflammatory skin diseases such as acne vulgaris, and hidradenitis suppurativa; inflammatory bowel diseases such as ulcerative colitis (UC), and Crohn’s disease; autoimmune diseases such as gout, pseudo gout, rheumatoid arthritis (RA), multiple sclerosis (MS), Addison’s disease, celiac disease, systemic lupus erythematous (SLE), and vitiligo; and respiratory diseases such as chronic pulmonary diseases, idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disease (COPD), and asthma.
11. A method of inhibiting NLRP3 inflammasome activity in a subject in need thereof, comprising administering the crystalline form or the pharmaceutical composition of any one of claims 1-7 to the subject.
12. A process for the preparation of a compound of Formula (Ic), or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a); (c) reacting the product of (a) or (b) with a compound of Formula (la)
Formula (la) to form a compound of Formula (lb), or a salt thereof
(d) reacting the compound of Formula (lb), or a salt thereof, with hydrazine, optionally in the presence of an acid such as acetic acid, to form a compound of Formula (Ic), or a salt thereof; and
(e) isolating the compound of Formula (Ic), or a salt thereof; and wherein X = CH or N in Formula (la), Formula (lb) and Formula (Ic).
13. A process for the preparation of 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with aqueous acid to form 2-[2-hydroxy-4- (trifluoromethyl)benzoyl] benzoic acid, or a salt thereof; (e) reacting 2-[2-hydroxy-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(f) isolating 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof.
14. A process for the preparation of 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazine- l(2H)-one, or a salt thereof, with an acid to form 4-(2-hydroxy-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(f) isolating 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof.
15. A process for the preparation of 2-(4-chlorophthalazin- 1 -yl)-5-(trifluoromethyl)phenol, or a salt thereof, comprising the steps:
(a) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)-
5-(trifluoromethyl)phenol, or a salt thereof; and
(b) isolating 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof.
16. The process of claim 15, wherein the 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, used in step (a) is prepared by the process of claim 13.
17. The process of claim 15, wherein the 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, used in step (a) is prepared by the process of claim 14.
18. A process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with aqueous acid to form 2-[2-hydroxy-4- (trifluoromethyl)benzoyl] benzoic acid, or a salt thereof;
(e) reacting 2-[2-hydroxy-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(1) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)- 5-(trifluoromethyl)phenol, or a salt thereof;
(g) reacting 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, with (1) (2S)-3-amino-l,2-propanediol, or a salt thereof, to form Compound 1, or a salt thereof, or (2) diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, followed by deprotection of the resulting product to form Compound 1, or a salt thereof; and
(h) isolation of Compound 1, or a salt thereof.
19. A process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a); (c) reacting the product of (a) or (b) with phthalic anhydride to form 2-[2-(tetrahydro- 2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid or a salt thereof;
(d) reacting 2-[2-(tetrahydro-2H-pyran-2-yloxy)-4-(trifluoromethyl)benzoyl]benzoic acid, or a salt thereof, with hydrazine to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-
4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazine- l(2H)-one, or a salt thereof, with an aqueous acid to form 4-(2-hydroxy-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof;
(f) reacting 4-(2-hydroxy-4-(trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof, with a chlorinating agent such as POCh to form 2-(4-chlorophthalazin-l-yl)-
5-(trifluoromethyl)phenol, or a salt thereof;
(g) reacting 2-(4-chlorophthalazin-l-yl)-5-(trifluoromethyl)phenol, or a salt thereof, with (1) (2S)-3-amino-l,2-propanediol, or a salt thereof, to form Compound 1, or a salt thereof, or (2) diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, followed by deprotection of the resulting product to form Compound 1, or a salt thereof; and
(h) isolation of Compound 1, or a salt thereof.
20. A process for the preparation of Compound 1, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with tert-butyl methyl phthalate to form tert-butyl 2- (2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)benzoyl)benzoate;
(d) reacting tert-butyl 2-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)benzoate with hydrazine, optionally in the presence of an acid such as acetic acid, to form 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)phthalazin-l(2H)-one, or a salt thereof; and
(e) reacting 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4-(trifluoromethyl)phenyl)phthalazin- l(2H)-one, or a salt thereof, with diol-protected (2S)-3-amino-l,2-propanediol, or a salt thereof, in the presence of a coupling reagent, followed by removal of the THP protecting group and diol protecting group from the resulting product by reaction with an acid, to form Compound 1, or a salt thereof; and
(f) isolation of Compound 1, or a salt thereof.
21. A process for the preparation of Compound 2, or a salt thereof, comprising the steps:
(a) reacting 2-(3-(trifluoromethyl)phenoxy)tetrahydro-2H-pyran with an alkyl lithium, optionally n-BuLi, to form (2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)phenyl)lithium;
(b) optionally adding magnesium chloride or magnesium bromide to the reaction product of (a);
(c) reacting the product of (a) or (b) with 3 -(tert-butyl) 4-methyl pyridine-3,4- dicarboxylate to form tert-butyl 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)nicotinate, or a salt thereof;
(d) reacting tert-butyl 4-(2-((tetrahydro-2H-pyran-2-yl)oxy)-4- (trifluoromethyl)benzoyl)nicotinate, or a salt thereof, with hydrazine, optionally in the presence of an acid such as acetic acid, to form l-[2-tetrahydropyran-2-yloxy-4- (trifluoromethyl)phenyl]-3H-pyrido[3,4-d]pyridazin-4-one, or a salt thereof;
(e) reacting l-[2-tetrahydropyran-2-yloxy-4-(trifluoromethyl)phenyl]-3H-pyrido[3,4- d]pyridazin-4-one with (lR,2R)-2-aminocyclohexan-l-ol, or a salt thereof, in the presence of a coupling reagent, to form (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran-2- yl)oxy)-4-(trifhioromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan-l- ol, or a salt thereof;
(f) removal of the THP protecting group from (lR,2R)-2-((l-(2-((tetrahydro-2H-pyran- 2-yl)oxy)-4-(trifluoromethyl)phenyl)pyrido[3,4-d]pyridazin-4-yl)amino)cyclohexan- l-ol, or a salt thereof, by reaction with an acid to form Compound 2, or a salt thereof; and
(g) isolation of Compound 2, or a salt thereof. salt thereof. is CH or N. 27. A compound that salt thereof, wherein X is CH or
N.
28. salt thereof, wherein X is CH or N.
29.
30.
31. 32.
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