Patent Application
20240377291
The present invention relates to compounds useful for staining mitochondria, to compositions comprising such compounds, to methods of staining or analysing mitochondria, and to methods of detecting mitochondrial conditions.
Functioning mitochondria underpin many critical cellular processes and mitochondrial dysfunction can therefore be a key factor in disease. Changes of mitochondrial shape, structure and function sometimes occur in response to changes in energy demand and cellular environment and in some animal (including human) diseases. Mitochondrial diseases may occur because of mutations (inherited or acquired), in mtDNA. Some diseases may also arise from the effects of drugs, infections or other causes.
It is helpful to directly image or observe mitochondria to further understand the nature and pathology of disease or to determine mitochondrial location and morphology for medical or research purposes.
Fluorescent dyes for selectively staining mitochondria are widely used in life sciences research, in applications such as fluorescence microscopy, flow cytometry and high-content screening. Most commercially available mitochondrial stains are organic fluorophores that accumulate in the mitochondrial matrix due to the transmembrane potential, for example MitoTracker™ dyes.
Fluorescent mitochondrial markers (or stains) should combine brightness with high photostability and low toxicity. Photostability is particularly important for studying live-cell mitochondrial morphology because mitochondria are dynamic, undergoing fusion and fission and it is desirable to be able to study this attribute over an extended time period without loss of signal or dye-induced toxicity. Overall brightness (typically measured as the product of the extinction coefficient and quantum yield) influences the concentration of stain that can be used and the final image quality. Increased brightness is a beneficial feature for mitochondrial markers. Dyes for use in imaging mitochondria also need to selectively accumulate in the mitochondria.
There is a need for improved stains that combine increased brightness and photostability with low toxicity and can successfully accumulate in both live and fixed cells. Further, it is desirable for certain applications to be able to fix cells prior to imaging whilst preserving specific mitochondrial staining.
It is an aim of the present invention to address this need.
In a first aspect, there is accordingly provided a compound comprising a cationic species of formula (I):
or a solvate, or tautomer thereof; and a counter ion;
wherein:
Such a compound according to formula (I) is greatly advantageous because it may be used to stain mitochondria and provides enhanced photostability with excellent brightness and is able to accumulate in mitochondria that are both live and fixed.
A cationic mitochondrial stain of formula (I) may optionally be generated by oxidation within mitochondria or intracellularly of a compound comprising an alternative, reduced form of formula (I), for example as shown in formula (Ib) below:
Suitably, at least one of Y and Z is a substituted or unsubstituted azetidine group of formula:
Preferably, RA and RB may be independently selected from H, halo, C1 to C8 alkyl, and OR20. Halo may be F.
Thus, in some embodiments, Y or Z may be independently selected from:
In some other embodiments, Y or Z may be independently selected from:
The compound comprises a cationic species of formula (I) and a counter ion. This is advantageous because such a cationic species may be a delocalized lipophilic cation and may selectively accumulate in mitochondria due to the negative potential gradient produced by the mitochondrial membrane.
Thus, suitably such a cationic species may be of formula (II):
Preferably, R8 and R9 may be independently selected from H, halo, C1 to C8 alkyl, and OR20. Halo may be F.
The counter ion will usually result from the method of synthesis of the cationic species. The counter ion may be changed using ion exchange or other methods as known in the art.
Suitably, the counter ion may be a biologically compatible counter ion. A biologically compatible counter ion is not toxic in use and does not have a substantially harmful effect on biomolecules.
The counter ion may be selected from halide, carboxylate, oxalate, trifluoroacetate, sulfate, alkanesulfonate, arylsulfonate, phosphate, perchlorate, tetrafluoroborate, tetraphenylboride, hexafluorophosphate, nitrate and anions of aromatic or aliphatic carboxylic acids. Suitably, the counter ion may be selected from chloro, acetate or trifluoroacetate.
Suitably, the cationic species may be of formula (III):
Preferably, R10 and R11 may be independently selected from H, halo, C1 to C8 alkyl, and OR20. Halo may be F.
Suitably, the cationic species may be of formula (IV):
Structurally, the compound may comprise a species having an azetidine substituted rosamine (or rosamine analogue wherein X is O, S, SO2, Se, NR12, P(O)R12, CR13R14, SiR13R14, Te, or GeR13R14) that may have halo, alkyl or other substituents on the pendant phenyl group and/or elsewhere.
Suitably, the Q group may be in the ortho position on the pendant phenyl ring. Thus, at least one of R1 and R5 may be Q.
Additionally or alternatively, the Q group may be at the para position. Thus, R3 may be Q.
Additionally or alternatively, the Q group may be at one or both meta positions. Thus, at least one of R2 and R4 may be Q
Where present, one or more L groups may comprise an alkylene chain —(CH2)m—, wherein m is 1 to 8, suitably m may be 1 to 6. One or more L groups may be a substituted or unsubstituted phenylene group, for example in NHC(O)-L-CR18R19MB, of formula (if R18 and R19 are each H):
L may be absent.
The preferred MB is halo, more preferably Cl.
Suitably, the compound may comprise a cationic species selected from species of formulae:
or solvates, or tautomers thereof; and a counter ion.
The compound may be isotopically labelled. For example, one or more hydrogens may be replaced with deuterium or tritium, or one or more carbons may be replaced with C-13.
In a second aspect, there is provided a composition for staining mitochondria, the composition comprising a compound as in the first aspect.
The composition may further comprise at least one organic solvent. The at least one organic solvent may be selected from DMSO, acetone, dimethylformamide, acetonitrile, dioxane, and THF.
The concentration of the compound in the composition may be in the range 10 nM to 1 μM, preferably 10 nM to 300 nM.
In a third aspect, there is provided a method for staining mitochondria, the method comprising: providing a sample containing mitochondria, and incubating the sample in a composition comprising a compound as in the first aspect.
Usually, incubating the sample is for a predetermined time, optionally in the range 10 mins to 2 hours and at a predetermined temperature, optionally in the range 20° C. to 39° C.
Usually, the sample containing mitochondria comprises a tissue sample. The sample containing mitochondria may be a plant, animal or fungal tissue sample, a sample of plant, animal or fungal cells or isolated plant, animal or fungal mitochondria. Examples of tissue samples include tissue sections, biopsy, blood draws, cytology samples, etc.
The sample containing mitochondria may comprise a sample containing fixed mitochondria and/or a sample containing mitochondria in fixed cells.
In a fourth aspect, there is provided a method of analysing mitochondria, the method comprising: staining a sample of mitochondria using a compound as in the first aspect, optionally fixing the cells, illuminating the stained sample using light of an appropriate wavelength to fluoresce the compound, and observing or imaging a magnified image of the sample.
The appropriate wavelength may be in the range 400 nm to 800 nm.
In a fifth aspect, there is provided a method of detecting or diagnosing a mitochondrial condition comprising staining a sample of mitochondria as in the third aspect and/or analysing a sample of mitochondria as in the fourth aspect.
Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims, as supported by the description.
“Substituted,” when used in connection with a chemical substituent or moiety (e.g., an alkyl group), means that one or more hydrogen atoms of the substituent or moiety have been replaced with one or more non-hydrogen atoms or groups, provided that valence requirements are met and that a chemically stable compound results from the substitution.
“Optionally substituted” refers to a parent group which may be un-substituted or which may be substituted with one or more substituents. Suitably, unless otherwise specified, when optional substituents are present the optional substituted parent group comprises from one to three optional substituents thus the group may be substituted with 0, 1, 2 or 3 of the optional substituents. Suitably, the group is substituted with 1, 2 or 3 of the optional substituents.
Optional substituents may be selected from C1-8 alkyl, C1-6 alkyl, C2-7 alkenyl, C2-7 alkynyl, C1-12 alkoxy, C5-20 aryl, C3-10 cycloalkyl, C3-10 cycloalkenyl, C3-10 cycloalkynyl, C3-20 heterocyclyl, C3-20 heteroaryl, acetal, acyl, acylamido, acyloxy, amidino, amido, amino, aminocarbonyloxy, azido, carboxy, cyano, ether, formyl, guanidino, halo, hemiacetal, hemiketal, hydroxamic acid, hydroxyl, imidic acid, imino, ketal, nitro, nitroso, oxo, oxycarbonyl, oxycarboyloxy, sulfamino, sulfamyl, sulfate, sulfhydryl, sulfinamino, sulfinate, sulfino, sulfinyl, sulfinyloxy, sulfo, sulfonamido, sulfonamino, sulfonate, sulfonyl, sulfonyloxy, uredio groups. In some aspects, the optional substituents are 1, 2 or 3 optional substituents independently selected from OH, C1-8 alkyl, C1-6 alkyl, OC1-12 alkyl, and halogen. More suitably, the optional substituents are selected from OH, C1-8 alkyl and OC1-12 alkyl; more suitably, the optional substituents are selected from C1-8 alkyl and OC1-12 alkyl.
“Independently” or “Independently selected” is used in the context of statement that, for example, “each R16, R17 is independently H, C1-8 alkyl . . . ” and means that each instance of the functional group, e.g., R16, is selected from the listed options independently of any other instance of R16 or R17 in the compound. Hence, for example, H may be selected for the first instance of R16 in the compound; methyl may be selected for the next instance of R16 in the compound; and ethyl may be selected for the first instance of R17 in the compound.
C1-8 alkyl: refers to straight chain and branched saturated hydrocarbon groups, having from 1 to 8 carbon atoms, and C1-6 alkyl to straight chain and branched saturated hydrocarbon groups, having from 1 to 6 carbon atoms. Suitably a C1-7 alkyl; suitably a C1-6 alkyl; suitably a C1-5 alkyl; more suitably a C1-4 alkyl; more suitably a C1-3 alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent-1-yl, pent-2-yl, pent-3-yl, 3-methylbut-1-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-1-yl, n-hexyl, n-heptyl, n-octyl and the like.
“Alkylene” refers to a divalent radical derived from an alkane which may be a straight chain or branched, as exemplified by —CH2CH2CH2CH2—. The alkylene may have the number of carbons as discussed above for alkyl groups.
“Aryl” refers to fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring. Aryl groups as used herein preferably are preferably “C5-20 Aryl” a fully unsaturated monocyclic, bicyclic and polycyclic aromatic hydrocarbons having at least one aromatic ring and having a specified number of carbon atoms that comprise their ring members (e.g., C5-20 aryl refers to an aryl group having from 5 to 20 carbon atoms as ring members). The aryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements. Suitably, a is selected from a C6-12 aryl, more suitably, a C6-10 aryl. Examples of aryl groups include phenyl.
“Halogen” or “halo”: refers to a group selected from F, Cl, Br, and I. Preferably, the halogen or halo is F or Cl. In some aspects, preferably the halogen is F. In other aspects, suitably the halogen is Cl.
“Maleimidyl” refers to the univalent radical of maleimide of formula:
Attachment to other groups may be through C, or through N as indicated below:
where the wavy line indicate the point of attachment.
“Heteroaryl” refers to unsaturated monocyclic or bicyclic aromatic groups. Preferably heteroaryl is “C5-10 heteroaryl” or “5- to 10-membered heteroaryl” an unsaturated monocyclic or bicyclic aromatic group comprising from 5 to 10 ring atoms, whether carbon or heteroatoms, of which from 1 to 5 are ring heteroatoms. Suitably, any monocyclic heteroaryl ring has from 5 to 6 ring atoms and from 1 to 3 ring heteroatoms. Suitably each ring heteroatom is independently selected from nitrogen, phosphorus, oxygen, sulfur and silicon.
The bicyclic rings include fused ring systems and, in particular, include bicyclic groups in which a monocyclic heterocycle comprising 5 ring atoms is fused to a benzene ring. The heteroaryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.
Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:
Examples of heteroaryl groups which comprise fused rings, include, but are not limited to, those derived from:
As used herein, “solvate” refers to a complex of variable stoichiometry formed by a solute and a solvent. Solvates may be formed for crystalline compounds wherein solvent molecules are incorporated into the crystalline lattice during crystallization. The incorporated solvent molecules can be water molecules or non-aqueous molecules, such as but not limited to, ethanol, isopropanol, dimethyl sulfoxide, acetic acid, ethanolamine, and ethyl acetate molecules.
“Tautomer, “refers to a structural isomer of a compound that readily interconverts to another isomer.
“Fixed cells” refers to cells that have undergone a fixing process to substantially end biochemical reactions within the cells. References to “fixed mitochondria” refer to mitochondria that are or were present in cells that have undergone the fixing process or mitochondria that have undergone a fixing process in order to substantially end biochemical reactions within the mitochondria.
In this specification, “live mitochondria” refers to mitochondria that are functioning in the sense that there is a mitochondrial membrane potential and/or the membrane has not been substantially ruptured.
“Mitochondrial conditions,” as used herein are mitochondrial diseases or conditions involving or that may lead to mitochondrial dysfunction where mitochondria fail to produce enough energy for the body or parts of the body to function properly. Mitochondrial conditions may be chronic, and genetic. Mitochondrial dysfunction occurs when the mitochondria are affected by another disease or condition. Conditions that may lead to such mitochondrial dysfunction include Alzheimer's disease, muscular dystrophy, Lou Gehrig's disease, diabetes and cancer. Mitochondrial conditions/diseases include: Kearns-Sayre syndrome, Leber's hereditary optic neuropathy, Progressive external ophthalmoplegia, Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), and Myoclonic epilepsy with ragged red fibres (MERRF).
The term “subject” as used herein refers to a human or non-human animal, suitably a mammal. Examples of non-human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses.
As used herein the term “comprising” means “including at least in part” and is inclusive or open ended. When interpreting each statement in this specification that includes the term “comprising,” features, elements and/or steps other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. It should be understood that while various aspects in the specification are presented as “comprising,” this includes aspects that “consist essentially of” or “consist of” that aspect.
The term “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. When the phrase “consisting essentially of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause.
The term “consisting of” excludes any element, step, or ingredient not specified in the claim; “consisting of” defined as “closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:
The compounds outlined in
COMPOUND 5 is an excellent mitochondrial stain that localizes specifically to the mitochondria as demonstrated in
In
In
All reagents and solvents were purchased from commercial sources and used without further purification. Nuclear magnetic resonance spectra were recorded on a Bruker Avance III HD spectrometer operating at 400 MHz for 1H NMR and 100 MHz for 13C NMR. 1H NMR and 13C NMR chemical shifts (5) are reported in parts per million (ppm) and are referenced to residual protium in solvent and to the carbon resonances of the residual solvent peak respectively.
Purification by flash chromatography was performed using pre-packed silica gel columns and either a Buchi Reveleris, a Biotage Isolera or a Biotage Selekt system. Analytical thin layer chromatography was performed on glass plates pre-coated with silica gel (Analtech, UNIPLATE™ 250 m/UV254), with visualization being achieved using UV light (254 nm) and/or by staining with alkaline potassium permanganate dip.
Reaction monitoring LC-MS analyses were conducted using Agilent InfinityLab LC/MSD systems. High resolution mass spectral (HRMS) data was collected using an Agilent 6545 LC/Q-TOF system.
Normalized absorption and fluorescence emission spectra were recorded in 10 mM PBS pH 7.3 at the concentration noted for each sample following dilution of a DMSO stock solution. Absorption spectra were recorded with an Agilent Cary 60 UV-Vis spectrophotometer using genuine precision quartz cells from Lovibond with a 1 cm path length. Fluorescence spectra were recorded on an Agilent Cary Eclipse Fluorescence Spectrophotometer using high precision Quartz Suprasil cells from Hellma Analytics and a 1 cm path length.
The invention is further illustrated by the following Examples.
3-Bromoiodobenzene (30 g, 106 mmol), azetidine (7.27 g, 127 mmol) and K3PO4 (67.5 g, 318 mmol) were combined with ethylene glycol (14.2 mL) and 1-butanol (150 mL) in a round bottom flask. The flask was sealed and evacuated/backfilled with nitrogen three times. CuI (2.02 g, 10.6 mmol) was subsequently added and the flask was again sealed and evacuated/backfilled with nitrogen three times. The mixture was then heated at 100° C. under an atmosphere of N2 for 4 h. After cooling to room temperature, a saturated aqueous solution of NH4Cl and EtOAc were added with stirring until there were no solids remaining. The layers were separated and the aqueous was extracted twice with EtOAc. The combined organic layers were washed with brine, then dried (MgSO4) and filtered and the solvent was removed in vacuo. The residue was further dried under high vacuum. The crude product was purified by flash chromatography (0 to 10% Et2O/PE) to give the title compound as a pale-yellow oil (18.2 g, 81%).
1H NMR (CDCl3, 400 MHz) δ 7.04 (1H, t), 6.85-6.80 (1H, m), 6.55 (1H, t), 6.36-6.31 (1H, m), 3.87 (4H, t), 2.37 (2H, p).
A solution of 1-(3-bromophenyl)azetidine (12.6 g, 59.5 mmol) in THF (115 mL) was cooled to −78° C. under nitrogen. A solution of n-butyllithium in hexane (2.5 M, 23.8 mL, 59.5 mmol) was slowly added so that the internal temperature was maintained below −60° C. during the addition. The reaction mixture was subsequently stirred at −78° C. for 30 min. A solution of dichlorodimethylsilane (3.20 g, 24.8 mmol) in THF (10 mL) was then added at a rate such that the internal temperature was kept below −60° C. The cooling bath was removed, and the reaction was stirred at room temperature for 3 h. It was subsequently quenched with saturated aqueous NH4Cl (20 mL), diluted with water, and extracted twice with EtOAc. The combined organic extracts were washed with brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was co-evaporated twice with Et2O and purified by flash chromatography (o to 30% Et2O/PE) to give the title compound as a colourless oil (8.00 g, 84%).
1H NMR (CDCl3, 400 MHz) δ 7.20 (2H, t), 6.90 (2H, d), 6.61 (2H, d), 6.46 (2H, ddd), 3.86 (8H, t), 2.34 (4H, p), 0.51 (6H, s).
N-Bromosuccinimide (7-45 g, 41.9 mmol) was added in portions over 5 minutes to a solution of bis(3-(azetidin-1-yl)phenyl)dimethylsilane (6.75 g, 20.9 mmol) in DMF (120 mL). The resulting mixture was stirred for 5 days. Following removal of the solvent in vacuo, the resulting residue was diluted with water and extracted with EtOAc and then with DCM. The combined organic layers were washed with water and brine, then dried (MgSO4) and filtered and the solvent was removed in vacuo. The crude product was purified by recrystallisation from EtOAc to give the title compound as a white solid (5.69 g, 57%).
1H NMR (CDCl3, 400 MHz) δ 7.31 (2H, d), 6.51 (2H, d), 6.31 (2H, dd), 3.81 (8H, t), 2.36 (4H, p), 0.71 (6H, s).
A solution of t-BuLi in pentane (1.7 M, 10.3 mL) was added dropwise to a cooled (−78° C.) solution of bis(5-(azetidin-1-yl)-2-bromophenyl)dimethylsilane (2.00 g, 4.16 mmol) in THF (180 mL). After stirring for 20 minutes, the reaction mixture was warmed to −10° C. and a solution of MgBr2·OEt2 (2.37 g, 9.18 mmol) in THF (40 mL) was slowly added, keeping the internal temperature below −5° C. The reaction mixture was stirred at −10° C. for 30 minutes and then a solution of phthalide (1.23 g, 9.17 mmol) in THF (25 mL) was added dropwise over 30 minutes. The reaction mixture was allowed to warm to room temperature and was stirred for 60 h. Saturated aqueous NH4Cl and EtOAc were added and the layers were separated, the aqueous was further extracted twice with EtOAc. The combined organic layers were washed with saturated aqueous NaHCO3 and brine, dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (PE to 50% EtOAc/PE to a flush with 50% DCM/EtOAc) to give the title compound as an off-white solid (0.25 g, 14%).
1H NMR (CDCl3, 400 MHz) δ 7.31-7.21 (3H, m), 7.06 (1H, d), 6.95 (2H, d), 6.67 (2H, d), 6.31 (2H, dd), 5.21 (2H, s), 3.87 (8H, t), 2.34 (4H, p), 0.59 (3H, s), 0.52 (3H, s).
Thionyl chloride (0.06 g, 0.50 mmol) was added dropwise to a solution of 1,1′-(5,5-dimethyl-3′H,5H-spiro[dibenzo[b,e]siline-10,1′-isobenzofuran]-3,7-diyl)bis(azetidine) (0.20 g, 0.46 mmol) in DCM (8 mL). After 20 minutes, the reaction mixture was diluted with water and DCM and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (5 to 20% MeOH/DCM) followed by precipitation from DCM/EtOAc to give the title compound as a blue/red solid (0.135 g, 60%).
1H NMR (CDCl3, 400 MHz) δ 7.63-7.61 (1H, m), 7.57-7.52 (1H, m), 7.49-7.45 (1H, m), 7.13 (1H, d), 6.93 (2H, d), 6.89 (2H, d), 6.26 (2H, dd), 4.51-4.36 (8H, m), 4.29 (2H, s), 2.60 (4H, p), 0.63 (3H, s), 0.60 (3H, s).
HRMS (ESI) calcd for C28H30ClN2Si [M]+, 457.1867, found 457.1864.
A solution of t-BuLi in pentane (1.7 M, 2.49 mL) was added dropwise to a cooled (−78° C.) solution of (4-bromophenyl)methoxy-tert-butyldimethylsilane (0.60 g, 2.00 mmol) in THF (8 mL). After stirring for 10 minutes, a portion of this solution (5 mL) was slowly added to a suspension of 3,7-bis(azetidin-1-yl)-5,5-dimethyl-benzo[b][1]benzosilin-10-one (0.58 g, 1.66 mmol) in THF (12.5 mL). The resulting mixture was stirred overnight and was then diluted with both DCM and a saturated aqueous solution of NH4Cl. The layers were separated and the aqueous was extracted three times with additional DCM. The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The resulting residue was dissolved in DCM and 5 drops of 2M aqueous HCl were added. The mixture was subsequently concentrated in vacuo and then co-evaporated with firstly MeOH and then with DCM. The crude product was purified by flash chromatography (5 to 20% MeOH in DCM) to give the title compound as a blue/green solid (0.14 g, 12%).
1H NMR (CDCl3, 400 MHz) δ 7.47-7.437.46 (2H, dm), 7.18-7.13 (2H, m), 7.07 (2H, d), 6.87 (2H, d), 6.23 (2H, dd), 4.86 (2H, s) 4.55-4.32 (8H, m), 2.61 (4H, p), 0.99 (9H, s), 0.60 (6H, s), 0.18 (6H, s).
LC/MS (ES+): m/z 553.4 (100%, M+).
A 1M aqueous solution of HCl (0.4 mL) was added to a solution of 1-(7-(azetidin-1-yl)-10-(4-(((tert-butyldimethylsilyl)oxy)methyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride (0.056 g, 0.095 mmol) in MeOH (8 mL). After stirring for 30 minutes, the reaction mixture was diluted with DCM and water and the layers were separated. The aqueous was further extracted twice with DCM. The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (5 to 25% MeOH in DCM) to give the title compound as a blue/green solid (0.031 g, 71%).
1H NMR (CDCl3, 400 MHz) δ 7.59-7.55 (2H, m), 7.18-7.14 (2H, m), 7.12 (2H, d), 6.80 (2H, d), 6.26 (2H, dd), 4.87 (2H, s) 4.50-4.33 (8H, m), 3.03 (1H, s), 2.61 (4H, p), 0.58 (6H, s).
LC/MS (ES+): m/z 439.2 (100%, M+).
A solution of triphosgene (0.008 g, 0.027 mmol) in DCM (0.5 mL) was added to a solution of 1-(7-(azetidin-1-yl)-10-(4-(hydroxymethyl)phenyl)-5,5-dimethyldibenzo[b,e]silin-3(5H)-ylidene)azetidin-1-ium chloride (0.021 g, 0.044 mmol) in DCM (4 mL). The reaction was monitored by LC/MS and after 30 minutes additional triphosgene/pyridine were added to push the reaction to completion. The mixture was diluted with DCM and washed with a 1M aqueous solution of HCl. The combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (5 to 30% MeOH in DCM) followed by trituration with EtOAc to give the title compound as a blue/green solid (0.08 g, 37%).
1H NMR (CDCl3, 400 MHz) δ 7.57-7.51 (2H, m), 7.24-7.18 (2H, m), 7.02 (2H, d), 6.88 (2H, d), 6.28 (2H, dd), 4.70 (2H, s) 4.52-4.34 (8H, m), 2.62 (4H, p), 0.60 (6H, s).
LC/MS (ES+): m/z 457.3 (100%, M+).
A mixture of phthalic anhydride (3.80 g, 25.7 mmol), 3-bromophenol (8.66 g, 50.0 mmol) and methanesulfonic acid (12.5 mL) was heated at 130° C. for 72 h. After cooling to room temperature, the reaction mixture was poured onto H2O (700 mL) resulting in the formation of a precipitate. The precipitate was collected by filtration, washing with H2O. The solid was subsequently dissolved in DCM (700 mL) with gentle heating and the solution was dried (MgSO4), filtered and solvent was removed in vacuo. The crude product was heated with EtOAc (100 mL) allowed to cool and the solid was collected by filtration, washing with EtOAc to afford the title compound as a pale pink solid (4.20 g, 36%).
1H NMR (CDCl3, 400 MHz) δ 8.06-8.02 (1H, m), 7.71-7.62 (2H, m), 7.50 (2H, d), 7.19 (2H, dd), 7.14-7.10 (1H, m), 6.70 (2H, d).
LC/MS (ES+): m/z 458.9 (100%, [M+H]+).
A solution of LiBH4 in THF (2M, 36.6 mL) was added dropwise to a cooled (o ° C.) suspension of 2-(3,6-dibromo-9H-xanthen-9-yl)benzoic acid (2.68 g, 5.85 mmol) in a mixture of THF (50 mL) and isopropanol (250 mL). The reaction mixture was allowed to warm to room temperature and stirred for 48 h. Subsequently, the reaction mixture was quenched by the cautious addition of saturated aqueous NH4Cl (100 mL) and the product was extracted three times with DCM. The combined organic layers were dried (MgSO4) and filtered and the solvent was removed in vacuo. The crude product was purified by flash chromatography (5 to 10% MeOH in DCM) to give the title compound (2.36 g, 90%) as a white solid.
1H NMR (d6-DMSO, 400 MHz) δ 8.20 (1H, dd), 7.53-7.46 (3H, m), 7.44-7.32 (2H, m), 7.25 (2H, dd), 6.86 (2H, d), 6.77 (1H, s), 4.86 (1H, t), 3.46 (2H, d).
A mixture of (2-(3,6-dibromo-9H-xanthen-9-yl)phenyl)methanol (2.36 g, 5.29 mmol), Pd2(dba)3 (0.484 g, 0.529 mmol), XPhos (0.69 g, 1.59 mmol) and Cs2CO3 (8.27 g, 25.4 mmol) in a round bottom flask was evacuated/backfilled with nitrogen (X3). To this mixture was added azetidine (0.664 g, 11.6 mmol) and 1,4-dioxane (40 mL) and the flask was again evacuated/backfilled three times with nitrogen. The flask was then inserted into a pre-heated metal heating block and stirred at 105° C. overnight. After cooling to room temperature, the reaction mixture was diluted with DCM/water and the layers were separated. The aqueous layer was extracted twice with additional DCM. The combined organic layers were dried (MgSO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash chromatography (2 to 15% MeOH in DCM) followed by trituration with EtOAc to give the title compound as a pale pink solid (0.62 g, 30%).
1H NMR (d6-DMSO, 400 MHz) δ 7.46-7.39 (1H, m), 7.34 (1H, t), 7.23 (1H, t), 6.73 (1H, d), 6.65 (2H, d), 6.15-6.10 (4H, m), 5.16 (2H, s), 3.80 (8H, t), 2.29 (4H, p).
LC/MS (ES+): m/z 397.2 (100%, [M+H]+).
A stock solution of thionyl chloride (300 mg) in DCM (5 mL) was prepared. A portion (0.5 mL) of this stock solution was added dropwise to a solution of 3′,6′-di(azetidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthene] (0.10 g, 0.252 mol) in DCM (10 mL). After 30 minutes, the reaction mixture was diluted with water and DCM and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (6 to 15% MeOH/DCM) followed by trituration with Et2O to give the title compound as a dark red solid (0.114 g, 26%).
1H NMR (CDCl3, 400 MHz) δ 7.70-7.65 (1H, m), 7.62 (1H, t), 7.54 (1H, t), 7.19 (1H, d), 7.06-6.99 (2H, m), 6.58 (2H, dd), 6.49 (2H, d), 4.37 (8H, t), 4.26 (2H, s), 2.60 (4H, p).
HRMS (ESI) calcd for C26H24ClN2O [M]+, 415.1577, found 415.1575.
A mixture of (2-(3,6-dibromo-9H-xanthen-9-yl)phenyl)methanol (1.10 g, 2.47 mmol), Pd2(dba)3 (0.226 g, 0.247 mmol), XPhos (0.321 g, 0.74 mmol) and Cs2CO3 (3.86 g, 11.8 mmol) in a round bottom flask was evacuated/backfilled with nitrogen (X3). To this mixture was added 3-methoxyazetidine hydrochloride (0.67 g, 5.42 mmol) and 1,4-dioxane (20 mL) and the flask was again evacuated/backfilled three times with nitrogen. The flask was then inserted into a pre-heated metal heating block and stirred at 105° C. overnight. After cooling to room temperature, the reaction mixture was diluted with DCM/water and the layers were separated. The aqueous layer was extracted twice with additional DCM. The combined organic layers were dried (MgSO4), filtered and the solvent was removed in vacuo. The crude product was purified by flash chromatography (4 to 15% MeOH in DCM) to give the title compound as a grey solid (0.32 g, 28%).
1H NMR (CDCl3, 400 MHz) δ 7.41-7.34 (2H, m), 7.32-7.24 (2H, m), 6.94 (1H, d), 6.77 (2H, d), 6.25 (1H, d), 6.17 (2H, dd), 5.26 (2H, s), 4.39-4.31 (2H, m), 4.11 (4H, t), 3.79-3.71 (4H, m), 3.35 (6H, s).
LC/MS (ES+): m/z 457.2 (100%, [M+H]+).
A stock solution of thionyl chloride (235 mg) in DCM (10 mL) was prepared. A portion (1.0 mL) of this stock solution was added dropwise to a solution of 3′,6′-bis(3-methoxyazetidin-1-yl)-3H-spiro[isobenzofuran-1,9′-xanthene] (0.10 g, 0.219 mol) in DCM (10 mL). After 20 minutes, the reaction mixture was diluted with water and DCM and the layers were separated. The aqueous layer was extracted twice with DCM and the combined organic layers were dried over anhydrous MgSO4, filtered, and concentrated in vacuo. The crude product was purified by flash chromatography (7 to 15% MeOH/DCM) followed by trituration with Et2O to give the title compound as a dark purple solid (0.086 g, 77%).
1H NMR (CDCl3, 400 MHz) δ 7.70-7.59 (2H, m), 7.58-7.51 (1H, m), 7.19 (1H, d), 7.09-7.03 (2H, m), 6.64-6.55 (4H, m), 4.67-4.58 (4H, m), 4.56-4.49 (2H, m), 4.25 (2H, s), 4.17-4.11 (4H, m), 3.38 (6H, s).
HRMS (ESI) calcd for C28H28ClN2O3 [M]+, 475.1788, found 475.1791.
All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2112198.3 | Aug 2021 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2022/052188 | 8/25/2022 | WO |
