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WO2025172494A1 - Nouvelles sondes fluorescentes réversibles pour cb1 - Google Patents

Nouvelles sondes fluorescentes réversibles pour cb1

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Publication number
WO2025172494A1
WO2025172494A1 PCT/EP2025/053958 EP2025053958W WO2025172494A1 WO 2025172494 A1 WO2025172494 A1 WO 2025172494A1 EP 2025053958 W EP2025053958 W EP 2025053958W WO 2025172494 A1 WO2025172494 A1 WO 2025172494A1
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Prior art keywords
ethyl
methyl
chlorophenyl
dichlorophenyl
ethoxy
Prior art date
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Pending
Application number
PCT/EP2025/053958
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English (en)
Inventor
Uwe Michael GRETHER
Wolfgang Guba
Leonard MACH
Yelena MOSTINSKI
Marc Nazare
Anahid OMRAN
Malgorzata Wasinska-Kalwa
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.)
F Hoffmann La Roche AG
Forschungsverbund Berlin FVB eV
Hoffmann La Roche Inc
Original Assignee
F Hoffmann La Roche AG
Forschungsverbund Berlin FVB eV
Hoffmann La Roche Inc
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Application filed by F Hoffmann La Roche AG, Forschungsverbund Berlin FVB eV, Hoffmann La Roche Inc filed Critical F Hoffmann La Roche AG
Publication of WO2025172494A1 publication Critical patent/WO2025172494A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/06—Hydroxy derivatives of triarylmethanes in which at least one OH group is bound to an aryl nucleus and their ethers or esters
    • C09B11/08—Phthaleins; Phenolphthaleins; Fluorescein
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/28—Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
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    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
    • C09B23/08—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines
    • C09B23/083—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups more than three >CH- groups, e.g. polycarbocyanines five >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/105—The polymethine chain containing an even number of >CH- groups two >CH- groups
    • C—CHEMISTRY; METALLURGY
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    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B69/00—Dyes not provided for by a single group of this subclass
    • C09B69/10—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds
    • C09B69/103—Polymeric dyes; Reaction products of dyes with monomers or with macromolecular compounds containing a diaryl- or triarylmethane dye
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • Novel reversible fluorescent probes for CB1 Field of the Invention relates to organic compounds useful as reversible fluorescent probes for the cannabinoid receptor type 1 (CB1R).
  • the probes are achieved by a novel, convergent synthetic blueprint from a central reactive motif.
  • Background of the Invention Cannabinoid receptor type 1 (CB 1 R) is one of the central elements of the endocannabinoid system regulating a variety of signaling cascades. Extensive efforts on CB 1 R have validated its essential roles in physiology such as appetite regulation, pain perception, memory formation and thermoregulation. Yet, there is a surprising lack of clear understanding of its cellular signaling, distribution and expression dynamics.
  • CB 1 R detection by visualization in real-time is therefore crucial for addressing these open questions in cannabinoid research.
  • Dysregulation of the endocannabinoid system, specifically of signaling pathways encompassed by cannabinoid receptor type 1 (CB1R) has been implicated in a range of diseases including tissue injury, neurodegenerative conditions and inflammation.
  • CB 1 R is a class a GPCR (G-protein coupled receptor), which is predominantly expressed in cells associated with the nervous system and therefore has become an attractive therapeutic target for a vast variety of pathological conditions such as neurodegenerative diseases, inflammatory and metabolic disorders. In spite of the therapeutic potential, no CB 1 R-selective drug has made its way to market permanently.
  • alkylene denotes a linear saturated divalent hydrocarbon group of 1 to 7 carbon atoms or a divalent branched saturated divalent hydrocarbon group of 3 to 7 carbon atoms.
  • alkylene groups include methylene, ethylene, propylene, 2-methylpropylene, butylene, 2-ethylbutylene, pentylene, hexylene.
  • alkenylene alone or in combination, denotes a linear divalent hydrocarbon chain of 2 to 7 carbon atoms or a branched divalent hydrocarbon chain of 3 to 7 carbon atoms with at least one double bond.
  • alkenylene examples include ethenylene, 2,2- dimethylethenylene, propenylene, 2-methylpropenylene, butenylene, and pentenylene.
  • alkynylene alone or in combination, denotes a linear divalent hydrocarbon chain of 2-6 carbon atoms or a branched divalent hydrocarbon chain of 3-6 carbon atoms with at least one triple bond.
  • alkynylene include ethynylene, 2,2- dimethylethynylene, propynylene, 2-methylpropynylene, butynylene, and pentynylene.
  • heteroalkylene denotes an “alkylene” group comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remainder being hydrocarbon.
  • heteroalkenylene alone or in combination, denotes an “alkenylene” group comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remainder being hydrocarbon.
  • heteroalkynylene alone or in combination, denotes an “alkynylene” group comprising 1, 2, or 3 ring heteroatoms selected from N, O and S, the remainder being hydrocarbon.
  • alkyl examples include methyl, ethyl, propyl, 2-propyl (isopropyl), n-butyl, iso-butyl, sec-butyl, tert-butyl, and 2,2- dimethylpropyl.
  • a particularly preferred, yet non-limiting example of alkyl is methyl.
  • pharmaceutically acceptable salt refers to those salts which retain the biological effectiveness and properties of the free bases or free acids, which are not biologically or otherwise undesirable.
  • the salts are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, in particular hydrochloric acid, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, N-acetylcystein and the like.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
  • organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid,
  • occupancy is to be understood in broad term as generally known to the person skilled in the art and as described for instance in ”A Pharmacology Primer (Fourth Edition)”, Kenakin, Terry P. , ISBN 978-0-12-407663-1.
  • agonist denotes a compound that enhances the activity of another compound or receptor site as defined e.g. in Goodman and Gilman's “The Pharmacological Basis of Therapeutics, 7th ed.” in page 35, Macmillan Publ. Company, Canada, 1985.
  • a “full agonist” effects a full response whereas a “partial agonist” effects less than full activation even when occupying the total receptor population.
  • the compounds of formula (I) can contain several asymmetric centers and can be present in the form of optically pure enantiomers, mixtures of enantiomers such as, for example, racemates, optically pure diastereoisomers, mixtures of diastereoisomers, diastereoisomeric racemates or mixtures of diastereoisomeric racemates.
  • the asymmetric carbon atom can be of the "R” or "S” configuration.
  • the abbreviations “CB1” and “CB 1 R” refer to the cannabinoid receptor 1.
  • the invention thus relates to: SPECIFICALLY NUMBERED EMBODIMENTS 1.
  • each n is independently an integer selected from 1, 2, 3, 4, 5, 6, and 7; a wavy line indicates the point of attachment to R a or R b ; and an asterisk indicates the point of attachment of L a or L b to rest of the fragment A.
  • any of R a and/or R b are each independently selected from the following structures or their tautomers: one half of a biotin or biotin derivative binding pair, , , ; to the a b L or L ; preferably wherein any of R a and/or R b are each independently selected from the following structures or their tautomers:
  • R a and/or R b are each independently selected from:
  • 30. A compound according to any one of embodiments 1, 1a, 1b, 3, 5, 7, 9-21, 23 and 25-27 which is an agonist of cannabinoid receptor type 1 (CB1).
  • 31. A compound according to any one of embodiments 1, 1a, 1b, 2, 8, 9-21, 24 and 25- 27 which is an antagonist of cannabinoid receptor type 1 (CB1).
  • 32. A compound according to any one of embodiments 1 to 31, for use in a cannabinoid receptor type 1 (CB1) occupancy study. 35.
  • PG 1 is a suitable protecting group, such as for instance Boc
  • X is a suitable leaving group such as for instance halogen, in particular fluoro and chloro
  • R can be for instance succinimidyl, pentafluorophenyl or sulfodichlorophenyl
  • the acid can be for instance TFA or HCl
  • the solvent can be for instance DCM, dioxane or hexafluoroisopropanol
  • the temperature can be for instance set at between around 0 °C and 150 °C, in particular at between around 0 °C and 65 °C, and more particularly between around 0 °C and room temperature
  • the base in the reaction of step (1)(b)
  • the base can be for instance Huenig’s base
  • Racemic compounds can e.g., be separated into their antipodes via diastereomeric salts by crystallization with optically pure acids or by separation of the antipodes by specific chromatographic methods using either a chiral adsorbent or a chiral eluent. It is equally possible to separate starting materials and intermediates containing stereogenic centers to afford diastereomerically/enantiomerically enriched starting materials and intermediates. Using such diastereomerically/enantiomerically enriched starting materials and intermediates in the synthesis of compounds of formula (I) will typically lead to the respective diastereomerically/enantiomerically enriched compounds of formula (I).
  • the described reactions can take place over a wide range of temperatures, and the precise reaction temperature is not critical to the invention. It is convenient to carry out the described reactions in a temperature range between -78 °C to reflux.
  • the time required for the reaction may also vary widely, depending on many factors, notably the reaction temperature and the nature of the reagents. However, a period of from 0.5 hours to several days will usually suffice to yield the described intermediates and compounds.
  • the reaction sequence is not limited to the one displayed in the schemes, however, depending on the starting materials and their respective reactivity, the sequence of reaction steps can be freely altered. If starting materials or intermediates are not commercially available or their synthesis not described in literature, they can be prepared in analogy to existing procedures for close analogues or as outlined in the experimental section.
  • coupling reagents like N,N'-carbonyl-diimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-bis(dimethylamino)-methylene)-1H-1,2,3-triazolo[4,5- b]pyridinium-3-oxide hexafluorophosphate (HATU), O-benzotriazol-1-yl-N,N,N',N'- tetramethyluronium tetrafluoroborate (TBTU), and O-benzotriazole-N,N,N',N'- tetramethyl-uronium-hexafluoro-phosphate (HBTU) can be employed to affect such a transformation.
  • CDI N,N'-carbonyl-diimidazole
  • DCC N,N'-dic
  • a convenient method is to use for example HATU and a base, such as Huenig’s base, trimethylamine or NMM in a solvent such as N,N-dimethylformamide, DMA, DMSO, DCM, ACN, MeOH or 1,4-dioxane, preferably between 0 °C and room temperature.
  • the substituted heterocyclic carboxylic acids are either commercially available or can be synthesized by a person skilled in the art following procedures described in the literature. (US5624941A, WO2005108393A1, WO2006106054A1, WO2009106982A1, WO0132663A2, US2007293509A1).
  • Compound AC can be converted into compound Ia by I) removal of the protecting group PG 1 and II) subsequent coupling to a suitable reporting unit R a (step c).
  • Methods known in the art for removal of protecting groups comprise the use of TFA in DCM or HCl in dioxane at temperatures between 0 °C and room temperature or the use of hexafluoroisopropanol in a microwave reactor at temperatures between 100 °C and 150 °C (as described for example in “Protective Groups in Organic Chemistry” by T.W. Greene and P.G.M.
  • a reporter group R a such as biotin, fluorescein-carboxylic acid or any other fluorescent dye
  • carboxylic acid functionality can be achieved by amide bond formation.
  • a suitable solvent such as DMF
  • a suitable base such as DIPEA
  • activated esters of fluorescent dyes can be used, such as succinimidyl esters, pentafluorophenyl esters or sulfodichlorophenyl esters to accomplish Ia.
  • Halogenated aryls such as NBD-F, NBD-Cl and other sulfur, selenium or carbon containing congeners, as described by Benson et al. (Angew.Chem.Int.Ed.2019, 58, 6911 –6915), can be used as reporter group R a .
  • Halogenated aryls can react with deprotected primary amines via nucleophilic aromatic substitution in the presence of a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-dimethylformamide, DMA, DCM, ACN, MeOH, H2O or 1,4-dioxane, preferably between 0 °C and 65 °C to achieve Ia.
  • a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-dimethylformamide, DMA, DCM, ACN, MeOH, H2O or 1,4-dioxane, preferably between 0 °C and 65 °C to achieve Ia.
  • a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-d
  • the amine functional group in intermediate AB can be first protected by a suitable protecting group (e.g. Fmoc or Cbz protecting group), separated into their antipodes by specific chromatographic methods using either a chiral adsorbent or a chiral eluent.
  • the intermediate protecting groups can be removed at a later stage of the synthesis using standard methods known in the art.
  • the synthesis of compounds with formulae Ib can, for example, be accomplished according to the following scheme 2.
  • Compound BB can be prepared from the N-PG 2 protected amino acid BA and corresponding linkers containing a terminal amine by suitable amide bond forming reactions (step f).
  • Suitable protecting groups for BA include, but are not limited to, Fmoc or Cbz.
  • Preferred linkers (L b ) for the amide bond formation include, but are not limited to, N-PG 1 protected polyethylene glycol amines of the type PG 1 ⁇ NH ⁇ CH2 ⁇ CH2 ⁇ (O ⁇ CH2 ⁇ CH2)n ⁇ NH2, with n between 0 and 4.
  • N-PG 1 protected polyethylene glycol amines of the type PG 1 ⁇ NH ⁇ CH2 ⁇ CH2 ⁇ (O ⁇ CH2 ⁇ CH2)n ⁇ NH2, with n between 0 and 4.
  • Coupling reagents suited for the a transformation of BA with N-PG 1 protected polyethylene glycol amines to BB are for example SOCl 2 , N,N'- carbonyldiimidazole (CDI), N,N'-dicyclohexylcarbodiimide (DCC), 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1- bis(dimethylamino)-methylene)-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexafluorophosphate (HATU), O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU), and O-benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluorophosphate (HBTU).
  • CDI N,N'-
  • a convenient method is to use for example HATU and a base, such as Huenig’s base, trimethylamine, DMAP or NMM in a solvent such as N,N- dimethylformamide, DMA, DMSO, DCM, ACN, MeOH or 1,4-dioxane, preferably between 0 °C and room temperature.
  • a base such as Huenig’s base
  • trimethylamine such as trimethylamine
  • DMAP or NMM in a solvent
  • a solvent such as N,N- dimethylformamide, DMA, DMSO, DCM, ACN, MeOH or 1,4-dioxane, preferably between 0 °C and room temperature.
  • a solvent such as N,N- dimethylformamide, DMA, DMSO, DCM, ACN, MeOH or 1,4-dioxane, preferably between 0 °C and room temperature.
  • Compounds of formula BA and various protected polyethylene glycol diamines are either commercially available or can
  • Compound BB can be converted to BC by I) selective removal of the protection group PG 2 applying suitable methods known in literature (e.g., a Fmoc group using for example piperidine, DBU or KF in DMF or DCM, as described for example in “Houben-Weyl Methods of Organic Chemistry. Volume E22A. Synthesis of Peptides and Peptidomimetics”, edited by Murray Goodman, Arthur Felix, Luis Moroder, and Claudio Toniolo; Georg Thieme Verlag; Stuttgart, Germany; 2001) and II) a subsequent amide coupling with corresponding substituted heterocyclic carboxylic acids (step g) using suitable amide coupling reagents as described before.
  • suitable methods known in literature e.g., a Fmoc group using for example piperidine, DBU or KF in DMF or DCM, as described for example in “Houben-Weyl Methods of Organic Chemistry. Volume E22A. Synthesis of Peptides and Peptidomimetics”, edited by Murray Goodman, Arthur Felix
  • the amide coupling step might be performed in situ or after purification of the deprotected intermediate amine.
  • the substituted heterocyclic carboxylic acids are either commercially available or can be synthesized by a person skilled in the art following procedures described in the literature. (US5624941A, WO2005108393A1, WO2006106054A1, WO2009106982A1, WO0132663A2, US2007293509A1).
  • Compound BC can be converted into compound Ib by I) removal of the protecting group PG 1 and II) subsequent coupling to a suitable reporting unit (R b ) (step h).
  • Methods known in the art for removal of protecting groups comprise the use of TFA in DCM or HCl in dioxane at temperatures between 0 °C and room temperature or the use of hexafluoroisopropanol in a microwave reactor at temperatures between 100 °C and 150 °C (as described for example in “Protective Groups in Organic Chemistry” by T.W. Greene and P.G.M. Wuts, 4th Ed., 2006, Wiley N.Y.; Choy et al. Synthetic Communications, (2008) 38:21, 3840-3853).
  • protecting groups e.g., tert-butyloxycarbonyl (Boc)
  • a reporter group R b such as biotin, fluorescein-carboxylic acid or any other fluorescent dye with carboxylic acid functionality
  • a reporter group R b such as biotin, fluorescein-carboxylic acid or any other fluorescent dye
  • carboxylic acid functionality can be achieved by amide bond formation.
  • a suitable solvent such as DMF
  • a suitable base such as DIPEA
  • activated esters of fluorescent dyes can be used, such as succinimidyl esters, pentafluorophenyl esters or sulfodichlorophenyl esters to accomplish Ib.
  • Halogenated aryls such as NBD-F, NBD-Cl and other sulfur, selenium or carbon containing congeners, as described by Benson et al. (Angew.Chem.Int.Ed.2019, 58, 6911 –6915), can be used as reporter group R b .
  • Halogenated aryls can react with deprotected primary amines via nucleophilic aromatic substitution in the presence of a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-dimethylformamide, DMA, DCM, ACN, MeOH, H 2 O or 1,4- dioxane, preferably between 0 °C and 65 °C to achieve Ib.
  • a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-dimethylformamide, DMA, DCM, ACN, MeOH, H 2 O or 1,4- dioxane, preferably between 0 °C and 65 °C to achieve Ib.
  • compound BD can be synthesized from BB by I) removal of the protecting group PG 1 and II) subsequent coupling to a suitable reporting unit R b (step h).
  • Methods known in the art for removal of protecting groups comprise the use of TFA in DCM or HCl in dioxane at temperatures between 0 °C and room temperature or the use of hexafluoroisopropanol in a microwave reactor at temperatures between 100 °C and 150 °C (as described for example in “Protective Groups in Organic Chemistry” by T.W. Greene and P.G.M. Wuts, 4th Ed., 2006, Wiley N.Y.; Choy et al. Synthetic Communications, (2008) 38:21, 3840-3853).
  • protecting groups e.g., tert-butyloxycarbonyl (Boc)
  • a reporter group R b such as biotin, fluorescein-carboxylic acid or any other fluorescent dye with carboxylic acid functionality
  • a reporter group R b such as biotin, fluorescein-carboxylic acid or any other fluorescent dye
  • carboxylic acid functionality can be achieved by amide bond formation.
  • a suitable solvent such as DMF
  • a suitable base such as DIPEA
  • activated esters of fluorescent dyes can be used, such as succinimidyl esters, pentafluorophenyl esters or sulfodichlorophenyl esters to accomplish Ib.
  • Halogenated aryls such as NBD-F, NBD-Cl and other sulfur, selenium or carbon containing congeners, as described by Benson et al. (Angew.Chem.Int.Ed.2019, 58, 6911 –6915), can be used as reporter group R b .
  • Halogenated aryls can react with deprotected primary amines via nucleophilic aromatic substitution in the presence of a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-dimethylformamide, DMA, DCM, ACN, MeOH, H 2 O or 1,4-dioxane, preferably between 0 °C and 65 °C to achieve BD.
  • a suitable base such as Huenig’s base, trimethylamine, NMM, NaHCO3 or Cs2CO3 in a solvent such as N,N-dimethylformamide, DMA, DCM, ACN, MeOH, H 2 O or 1,4-dioxane, preferably between 0 °C and 65 °C to achieve BD.
  • Compound BD can be converted to Ib by I) removal of the protection group PG 2 applying suitable methods known in literature (e.g., a Fmoc group using piperidine, DBU or KF in DMF or DCM, as described for example in “Houben-Weyl Methods of Organic Chemistry. Volume E22A. Synthesis of Peptides and Peptidomimetics”, edited by Murray Goodman, Arthur Felix, Luis Moroder, and Claudio Toniolo; Georg Thieme Verlag; Stuttgart, Germany; 2001) and II) a subsequent amide coupling with corresponding substituted heterocyclic carboxylic acids (step g) using suitable coupling reagents as described before.
  • the amide coupling step might be performed in situ or after purification of the deprotected intermediate amine.
  • substituted heterocyclic carboxylic acids are either commercially available or can be synthesized by a person skilled in the art following procedures described in the literature. (US5624941A, WO2005108393A1, WO2006106054A1, WO2009106982A1, WO0132663A2, US2007293509A1).
  • Step h) 5-(4-Chlorophenyl)-1-(2,4-dichlorophenyl)-N-(1-(3',6'-dihydroxy-3-oxo-3H- spiro[isobenzofuran-1,9'-xanthen]-5-yl)-13-ethyl-1,12-dioxo-5,8-dioxa-2,11- diazapentadecan-13-yl)-4-methyl-1H-pyrazole-3-carboxamide and 5-(4-chlorophenyl)-1- (2,4-dichlorophenyl)-N-(1-(3',6'-dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9'-xanthen]- 6-yl)-13-ethyl-1,12-dioxo-5,8-dioxa-2,11-diazapentadecan-13-yl)-4-methyl-1H-pyr
  • Step g) tert-Butyl (2-(2-(2-(5-(2,5-bis(trifluoromethyl)phenyl)-2-methyl-1-(((R)- tetrahydrofuran-2-yl)methyl)-1H-pyrrole-3-carboxamido)-2- ethylbutanamido)ethoxy)ethyl)carbamate
  • the title compound was prepared analogously to Example 3 step g), with the difference that 5-(2,5-bis(trifluoromethyl)phenyl)-2-methyl-1-(((R)-tetrahydrofuran-2-yl)methyl)- 1H-pyrrole-3-carboxylic acid (WO2005108393A1) was used instead of 5-(4- chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxylic acid (CAS RN 162758-35-2).
  • Step g) tert-Butyl (1-(5-(2,5-bis(trifluoromethyl)phenyl)-2-methyl-1-(((R)- tetrahydrofuran-2-yl)methyl)-1H-pyrrol-3-yl)-3,3-diethyl-1,4-dioxo-8,11-dioxa-2,5- diazatridecan-13-yl)carbamate
  • the title compound was prepared analogously to Example 3 step g), with the difference that (9H-Fluoren-9-yl)methyl (16-ethyl-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14- diazaoctadecan-16-yl)carbamate was used instead of (9H-fluoren-9-yl)methyl (13-ethyl- 2,2-dimethyl-4,12-dioxo-3,8-dioxa-5,11-diazapentadecan
  • Step g) tert-Butyl (2-(2-(2-(6-(cyclopropylmethoxy)-5-(4-fluorophenyl)nicotinamido)-2- ethylbutanamido)ethoxy)ethyl)carbamate
  • the title compound was prepared analogously to Example 3 step g), with the difference that 6-(cyclopropylmethoxy)-5-(4-fluorophenyl)nicotinic acid (CAS RN 912454-39-8) was used instead of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3- carboxylic acid (CAS RN 162758-35-2).
  • Step g) tert-Butyl (2-(2-(2-(6-(4-chlorophenyl)-5-(2-methoxyethoxy)pyrazine-2- carboxamido)-2-ethylbutanamido)ethoxy)ethyl)carbamate
  • the title compound was prepared analogously to Example 3 step g), with the difference that 6-(4-chlorophenyl)-5-(2-methoxyethoxy)pyrazine-2-carboxylic acid (CAS RN 960248-07-1) was used instead of 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl- 1H-pyrazole-3-carboxylic acid (CAS RN 162758-35-2).
  • Example 28 tert-Butyl (3,3-diethyl-1-(1-(3-fluorobenzyl)-1H-indazol-3-yl)-1,4-dioxo-8,11-dioxa- 2,5-diazatridecan-13-yl)carbamate
  • Step f) (9H-Fluoren-9-yl)methyl (16-ethyl-2,2-dimethyl-4,15-dioxo-3,8,11-trioxa-5,14- diazaoctadecan-16-yl)carbamate
  • the title compound was prepared analogously to Example 3 step f), with the difference that tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (CAS RN 153086-78-3) was used instead of tert-butyl (2-(2-aminoethoxy)ethyl)carbamate (CAS RN 127828-22- 2).
  • tert-butyl (2-(2-iodoethoxy)ethyl)carbamate (CAS RN 629626-40-0) (325 mg, 1.03 mmol, 1.1 equiv.) solution in anhydrous THF (3 mL) was added dropwise and the mixture was stirred at –78 °C for an additional 15 min. Then it was allowed to gradually warm and stirred overnight. The reaction mixture was diluted with EtOAc and quenched with sat. NH 4 Cl. The organic phase was separated, dried over anhydrous Na 2 SO 4 , filtered and concentrated.
  • Step b) Ethyl 4-(2-((tert-butoxycarbonyl)amino)ethoxy)-2-(5-(4-chlorophenyl)-1-(2,4- dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamido)-2-ethylbutanoate
  • 5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3- carboxylic acid (CAS RN 162758-35-2) (47.0 mg, 123.9 ⁇ mol, 1.1 equiv.) in anhydrous DMF (2 mL) were added anhydrous DIPEA (57.0 ⁇ L, 162.1 ⁇ mol, 3.0 equiv.) and HATU (47 mg, 123.9 ⁇ mol, 1.1 equiv.).
  • the title compound was prepared analogously to Example 33 step b), with the difference that ethyl 14-amino-14-ethyl-2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azapentadecan-15-oate was used instead of ethyl 2-amino-4-(2-((tert-butoxycarbonyl)amino)ethoxy)-2- ethylbutanoate.
  • TR-FRET Time-resolved Förster resonance transfer
  • a mixed population stable line was selected by resistance to blasticidin (TR vector, 5 ⁇ g/ml) and Zeocin; (receptor plasmid, 20 ⁇ g/ml).
  • TR vector 5 ⁇ g/ml
  • Zeocin receptor plasmid, 20 ⁇ g/ml.
  • receptor plasmid 20 ⁇ g/ml.
  • DMEM DMEM containing 1 ⁇ g/ml tetracycline added.24h later cells were labelled with SNAP-Lumi4-Tb (CisBio) and membranes prepared as described in detail below.
  • TR-FRET signals were collected at 665 (acceptor) and 620 nm (donor) when using the red acceptor fluorescent ligand, at 590 (acceptor) and 620 nm (donor) when using the orange acceptor fluorescent ligand or at 520 (acceptor) and 620 nm (donor) when using the green acceptor fluorescent ligand.
  • HTRF ratios were obtained by dividing the acceptor signal by the donor signal and multiplying this value by 10,000. All experiments were analyzed by non-regression using Prism 8.0 (GraphPad Software, San Diego, USA).
  • the cells were then stimulated with compounds diluted in Stimulation Buffer (2.5 ⁇ L/well) for 15 min at room temperature, followed by the addition of 2.5 ⁇ L 5 ⁇ M forskolin. After 15 mins, reactions were stopped by the 1 ⁇ cAMP-d2 conjugate in lysis buffer (5 ⁇ L/well), followed by 1 ⁇ anti-cAMP cryptate conjugate in lysis buffer (5 ⁇ L/well). Following a 1 h incubation at room temperature, the plates were read in a PerkinElmer Envision reader for time- resolved fluorescence resonance energy transfer detection at 620 nm and 665 nm. The HTRF ratio versus compound concentrations was plotted using Prism 8.1 (GraphPad).
  • HTRF Ratio (Signal 665 nm/Signal 620 nm) ⁇ 10 4 All HTRF ratio data sets of test compounds were normalized to the Emax of CP55940 (100%) and obtained the means ⁇ standard error of the mean (SEM) of three independent experiments performed in technical replicates.
  • K d (nM) values obtained for fluorescent examples from saturation assay on HEK293TR cell membranes with SNAP-terbium-hCB 1 R.
  • K i (nM) values obtained from [ 3 H]CP55,940 displacement assays on CHO membranes stably expressing human CB1R.
  • the activity levels (IC 50 ) were measured using cells stably expressing hCB 1 R in homogeneous time- resolved fluorescence (HTRF) cAMP assay.
  • E max in %) was normalized to reference full agonist CP55,940. n.d. is not determined.

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Abstract

La présente invention concerne des sondes fluorescentes réversibles pour le récepteur cannabinoïde de type 1 (CB1) ayant la formule générale (I), A et B étant tels que décrits dans la description, des compositions contenant les composés, des procédés de fabrication des composés et des méthodes d'utilisation des composés.
PCT/EP2025/053958 2024-02-16 2025-02-14 Nouvelles sondes fluorescentes réversibles pour cb1 Pending WO2025172494A1 (fr)

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WO2001032663A2 (fr) 1999-11-03 2001-05-10 Sanofi-Synthelabo Derives tricycliques d'acide pyrazolecarboxylique, leur preparation, les compositions pharmaceutiques en contenant
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US5624941A (en) 1992-06-23 1997-04-29 Sanofi Pyrazole derivatives, method of preparing them and pharmaceutical compositions in which they are present
WO2001032663A2 (fr) 1999-11-03 2001-05-10 Sanofi-Synthelabo Derives tricycliques d'acide pyrazolecarboxylique, leur preparation, les compositions pharmaceutiques en contenant
WO2005108393A1 (fr) 2004-05-10 2005-11-17 F. Hoffmann-La Roche Ag Amides pyrroles ou imidazoles destines au traitement de l'obesite
WO2006106054A1 (fr) 2005-04-06 2006-10-12 F. Hoffmann-La Roche Ag Derives de pyridine-3-carboxamide utilises comme agonistes inverses des cb1
US20070293509A1 (en) 2006-06-19 2007-12-20 Paul Hebeisen Pyrazinecarboxamide derivatives as CB1 antagonists
WO2009106982A1 (fr) 2008-02-29 2009-09-03 Pfizer Inc. Dérivés d'indazole

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