Fluorophore compounds and compositions useful for tyramide signal amplification

Abstract

The present application relates to compounds of formula (Ia) or (Ib): or a salt, solvate, or tautomer thereof; wherein R1, and R2 are independently selected from optionally substituted C1 to C 8 alkyl; or R1 and R2 together with the silicon atom to which they are attached form an optionally substituted 5- or 6-membered ring; R3, and R4 are independently selected from H, optionally substituted C1 to C 8 alkyl; optionally substituted C1 to C 8 haloalkyl; or R3 and R4 together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring; R3', and R4' are independently selected from H, optionally substituted C1 to C8 alkyl; optionally substituted C1 to C 8 haloalkyl; or R3' and R4' together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring;R5 is selected from a negative charge, H, C1 to C 8 alkyl, or optionally substituted aryl; a is 0 or 1; and Z is a divalent linker group; and to fluorophore compositions comprising such compounds, to assay methods and to kits using such fluorophore compositions.

Description

[0001] COMPOUNDS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to compounds, to fluorophore compositions comprising such compounds, to assay methods and to kits.

[0004] BACKGROUND

[0005] Tyramide Signal Amplification (TSA, also known as Catalysed Reporter Deposition, CARD) is a signal amplification system used in immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization (ISH) and diagnostic assays, especially for low abundance samples. Generally, TSA involves binding a primary probe to a sample, binding a HRP (horseradish peroxidase) or other peroxidase enzyme labelled secondary probe to the primary probe, adding a solution of a tyramide-containing fluorophore or a tyramide-containing chromagen which is activated by the HRP in combination with hydrogen peroxide and results in binding multiple activated tyramide molecules to electron-rich moieties such as tyrosine, phenylalanine and tryptophan proximal to the peroxidase, thereby amplifying the signal.

[0006] Although tyramide-containing chromogens have found widespread application, tyramide- containing fluorophores typically provide improved image quality and allow higher levels of multiplexing (i.e. detection of multiple targets in a single experiment), maximising the information gained from a single sample. Consequently, there is an ongoing need for new, high performing tyramide-containing fluorophores which exhibit high levels of stability, particularly at longer wavelengths and when utilised in multiplex assays.

[0007] For optimal performance, a tyramide-containing fluorophore must possess a number of key characteristics which include excellent stability (both chemical and photo) and good aqueous solubility. There are commercially available fluorophores that are intended for use in the TSA process, for example, Opal™ kits and reagents (Akoya Bioscience), Super Boost ™ reagents (Thermo Fischer Scientific) and Tyramide Amplification kits (Biotium Inc.).

[0008] There is, however, a need for fluorophores that can be excited and emit over a range of wavelengths (for example, wavelengths of around 450 nm to 800 nm) and fluorophores that are both photostable, suitable for multiplexing and of increased brightness compared to those that are currently commercially available. Furthermore, specialist technology has been developed for the detection of RNA in tissue samples and there is a need for tyramide- containing fluorophores which are optimised for this application.

[0009] It is an aim of the present invention to address this need and in particular to provide such fluorophores that may be suitable for use in the TSA process, particularly for the detection of RNA, but also in other applications in which peroxidase-labelled detection may be utilized such as IHC.

[0010] SUMMARY

[0011] In a first aspect, there is accordingly provided a compound of formula (la) or (lb): or a salt, solvate, or tautomer thereof; wherein:

[0012] R1, and R2are independently selected from optionally substituted Ci to Cs alkyl; or R1and R2together with the silicon atom to which they are attached form an optionally substituted 5- or 6-membered ring;

[0013] R3, and R4are independently selected from H, optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring;

[0014] R3, and R4are independently selected from H, optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring; R5is selected from a negative charge, H, Ci to Cs alkyl, or optionally substituted aryl; a is 0 or 1; and

[0015] Z is a single bond or a divalent linker group.

[0016] Such a compound is greatly advantageous because it may be used in TSA, similar procedures and in other applications. Furthermore, compounds according to the disclosure have significantly enhanced (photo) stability, including in multiplex assays. This improved performance in complex assays, such as an RNAscope™ assay, make the compounds suitable for the detection of RNA. Improved brightness and stability provide improved, higher quality images when compared with the images obtained using commercially available equivalents, enabling better and higher multiplexing.

[0017] Where the Z group in formula (la) or (lb) is a single bond then the

[0018] In some aspects, R1and R2together with the silicon atom to which they are attached form an optionally substituted 5- or 6-membered ring of the following formula: wherein RA, RB, and Rc, are independently selected from H, or Ci to Cs alkyl. More suitably, RA, RB, and Rc, are the same. More suitably, RA, RB, and Rc, are each H.

[0019] In some aspects, R1and R2together with the silicon atom to which they are attached form an optionally substituted 5-membered ring, thus, suitably the compound may be of formula (Ila) or (lib) :

[0020] wherein RA, RB, and Rc, are independently selected from H, or Ci to Cs alkyl. More suitably, RA, RB, and Rc, are the same. More suitably, RA, RB, and Rc, are each H. In some aspects, R1and R2together with the silicon atom to which they are attached form an optionally substituted 6-membered ring, thus, suitably the compound may be of formula (Illa) or (Illb): wherein RA, RB, and Rc, are independently selected from H, or Ci to Cs alkyl. More suitably, RA, RB, and Rc, are the same. More suitably, RA, RB, and Rc, are each H.

[0021] More suitably, R1, and R2are independently selected from optionally substituted methyl, ethyl, propyl; or R1and R2together with the silicon atom to which they are attached form an optionally substituted 5- or 6-membered ring. More suitably, R1, and R2are the same. Most suitably, R1, and R2are each methyl.

[0022] Suitably, when one of R3, or R4is H, the other of R3, or R4is selected from optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl.

[0023] Suitably, R3, and R4are independently selected from H, optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring of formula: wherein RD, RE and RF are independently selected from H, Ci to Cs alkyl, OR6, C(O)OR6,

[0024] NHC(O)R6, C(O)NHR6, halo, NR6R7, -CN, -NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R6and R7are independently selected from H, and Ci to Cs alkyl.

[0025] Suitably, RD, RE and RF are independently selected from H, halo, Ci to Cs alkyl, and OR6. More suitably, RD, RE and RF are independently selected from H, methyl, ethyl, OCH3,

[0026] OCH2CH3, or halo. Halo may be F.

[0027] Suitably, R3, and R4are independently selected from H, methyl, ethyl, CH2CF3; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring. Suitably, the optionally substituted azetidine ring is selected from:

[0028]

[0029] Suitably the optionally substituted pyrrolidine ring is selected from:

[0030] More suitably, R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine of formula: wherein RD and RE are independently selected from H, Ci to Cs alkyl (e.g. CH3), OR6, C(O)OR6, NHC(O)R6, C(O)NHR6, halo, NR6R7, -CN, -NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R6and R7are independently selected from H, and Ci to Cs alkyl. More suitably, RD and RE are H. Suitably, R3, and R4are independently selected from H, optionally substituted Ci to Cs alkyl; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring of formula: wherein RG, Rn and Ri are independently selected from H, Ci to Cs alkyl, OR8, C(O)OR8, NHC(O)R8, C(O)NHR8, halo, NR8R9, -CN, -NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R8and R9are independently selected from H, and Ci to Cs alkyl.

[0031] Suitably, RG, Ru and Ri are independently selected from H, halo, Ci to Cs alkyl, and OR8. More suitably, RG, RH and Ri are independently selected from H, methyl, ethyl, OCH3, OCH2CH3, or halo. Halo may be F.

[0032] Suitably, when one of R3, or R4is H, the other of R3, or R4is selected from optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl.

[0033] Suitably, R3, and R4are independently selected from H, methyl, ethyl, CH2CF3; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring.

[0034] Suitably, R3, and R4are the same as R3, and R4respectively.

[0035] Suitably, the optionally substituted azetidine ring is selected from:

[0036] Suitably the optionally substituted pyrrolidine ring is selected from:

[0037] More suitably, R3, and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine ring of formula: wherein RG and RH are independently selected from H, Ci to Cs alkyl, OR8, C(O)OR8, NHC(O)R8, C(O)NHR8, halo, NR8R9, -CN, -NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R8and R9are independently selected from H, and Ci to Cs alkyl. More suitably, RG and RH are each H.

[0038] However, preferably, the compound may be of formula (IVa) or (IVb):

[0039] The hydroxyl group on the terminal phenyl ring may be ortho, meta or para, but is preferably para. Thus, suitably, the compound may be of formula (Va)or (Vb): Suitably, R5is selected from a negative charge, H, methyl or ethyl. More suitably, R5is selected from a negative charge, or H. In some aspects, more suitably, R5is H. In some aspects, more suitably R5is a negative charge, in such aspect the -CO2R5group may be represented as -CCh’.

[0040] Suitably, when a is 0, then Z is a single bond.

[0041] Suitably, when a is 1 then Z is a divalent linker group.

[0042] Suitably, when a is 0, then Z is a single bond; and when a is 1 then Z is a divalent linker group.

[0043] Z may be a divalent linker group and may be generally any suitable divalent linker. Optionally, the divalent linker group may be an alkylene group, of formula - (CH2)b -, wherein b is 0, 1, 2, 3, or 4, preferably b is 0, 1 or 2. More preferably Z is a methylene group of formula -CH2-. Thus, Z may be an alkylene group, preferably a methylene group.

[0044] Suitably, Z is a single bond, or wherein b is 0, 1 or 2; c, d, e and f are each independently selected from an integer from 0 to 20; g and h are independently 0 or 1 ; each Xi and X2 are independently selected from a triazolyl, optionally substituted arylene, NR10, O, or -O-CH2-CH(O(CH2)I-R10)-CH2-O-, where each 1 is independently 0 or 1;

[0045] R10is H or is R11; where each i and j are each independently selected from an integer from 0 to 20; each k is 0, 1 or 2; each X3 is independently selected from a triazolyl, optionally substituted arylene, NH, or O; and

[0046] L is selected from: a straight or branched C1-12 alkylene chain which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain

[0047] - (CH2CH2)n(CH2)m(OCH2)s(O)w(CH2CH2)p(CH2)q- , a polyethylene glycol chain

[0048] - (CH2CH2)n(CH2)m(OCH2CH2)v(O)w(CH2CH2)p(CH2)q -, which chains may optionally be

[0049] — N ) - interrupted by one, two or three groups selected from -O-, -S-, -NH-, \ '

[0050] N— , C3-8 cycloalkyl, C5-10 heteroarylene and / or phenylene; wherein n, m, p and q are independently 0, 1 or 2; w is 0 or 1; s and v are independently 1 to

[0051] 12, optionally 1 to 8, optionally 1 to 6.

[0052] The group -O-CH2-CH(O(CH2)I-R10)-CH2-O- is of formula:

[0053] More suitably, each Xi, and X2, containing sub-unit may be selected from:

[0054] More suitably, the compound is of formula (Via) or (VIb):

[0055] or a salt, solvate or tautomer thereof.

[0056] Suitably, in some aspects, the compound is of formula (Vila) or (Vllb):

[0057]

[0058] (Vllb); or a salt, solvate or tautomer thereof; wherein f is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; j is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; and v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

[0059] Suitably, in some aspects, the compound is of formula (Villa) or (Vlllb) is:

[0060]

[0061] (Vlllb) or a salt, solvate or tautomer thereof; wherein f is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; j is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20;

[0062] 1 is 0 or 1; and v is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12. The hydroxyl group on the terminal phenyl ring(s) of formulas (la), (lb), (Ila), (lib), (Illa), (Illb), (IVa), (IVb), (Via), (VIb), (Vila) or (Vllb) may be ortho, meta or para, but is preferably para.

[0063] Suitably, the compound may be of formula: or salts, solvates, or tautomers thereof. Preferred embodiments of the disclosure may be such that the compound may be of formula:

[0064] or salts, solvates, or tautomers thereof.

[0065] 10 Thus, the present invention provides a compound of formula: or a salt, solvate, or tautomer thereof.

[0066] The present invention also provides a compound of formula:

[0067] (TY60) , or a salt, solvate, or tautomer thereof; or (TY10), or a salt, solvate, or tautomer thereof.

[0068] 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.

[0069] In a second aspect, there is provided a fluorophore composition, the composition comprising a compound as in the first aspect.

[0070] The fluorophore composition may further comprise at least one enhancer. Suitably, the at least one enhancer comprises NaCl, MgCh, KC1, CaCh, sodium phosphate, sodium acetate, ammonium acetate, ammonium sulfate, 4-fluorophenyl boronic acid, 3 -fluorophenyl boronic acid, 4-chlorophenyl boronic acid, 3 -chlorophenyl boronic acid, 4-bromophenyl boronic acid,

[0071] 3 -iodophenyl boronic acid, 4-iodophenyl boronic acid, 4-acetylphenyl boronic acid, 4- thioanisole boronic acid, 4-hydroxy-4’-iodobiphenyl, 4-iodophenol, 4-bromophenol, 4- (imidazol-l-yl)phenol, 4-methoxyphenol, 2-iodophenol, l-bromo-4-iodobenzene, 3- iodophenol, 4-phenylphenol, 4-4'-diiodobiphenyl, or mixtures thereof.

[0072] In some aspects, the at least one enhancer comprises an inorganic salt that is NaCl, MgCh, KC1, CaCh, sodium phosphate, sodium acetate, ammonium acetate, ammonium sulfate, or mixtures thereof.

[0073] In other aspects, the at least one enhancer comprises an organic enhancer that is 4-hydroxy- 4’ -iodobiphenyl, 4-iodophenol, 4-bromophenol, 4-(imidazol-l-yl)phenol, 4-methoxyphenol, 2-iodophenol, l-bromo-4-iodobenzene, 3 -iodophenol, 4-phenylphenol, 4-4'-diiodobiphenyl, or mixtures thereof.

[0074] More suitably, the at least one enhancer comprises an organic enhancer that is 4-fluorophenyl boronic acid, 3-fluorophenyl boronic acid, 4-chlorophenyl boronic acid, 3 -chlorophenyl boronic acid, 4-bromophenyl boronic acid, 3 -iodophenyl boronic acid, 4-iodophenyl boronic acid, 4-acetylphenyl boronic acid, 4-thioanisole boronic acid, or mixtures thereof. More suitably, the at least enhancer comprises an organic enhancer that is 4-fluorophenyl boronic acid, 4-chlorophenyl boronic acid, 4-bromophenyl boronic acid, 4-iodophenyl boronic acid,

[0075] 4-acetylphenyl boronic acid, 4-thioanisole boronic acid, or mixtures thereof. For example, the enhancer may comprise 4-halophenyl boronic acid, i.e. 4-fluorophenyl boronic acid, 4- chlorophenyl boronic acid, 4-bromophenyl boronic acid, 4-iodophenyl boronic acid, or mixtures thereof. More suitably, the enhancer is 4-iodophenylboronic acid.

[0076] Suitably, the equivalents ratio (e.g. molar ratio) of enhancer to the compound may be 6 or higher, optionally 8 or higher, optionally 10 or higher, optionally 12 or higher, optionally 15 or higher, optionally 20 or higher, optionally 24 or higher.

[0077] The fluorophore 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.

[0078] The concentration of the compound in the composition may be in the range 10 nM to 10 pM, preferably 50 nM to 1 pM. The fluorophore may fluoresce at a wavelength in the range 660 nm to 720 nm, preferably 670 nm to 710 nm, more preferably 680 nm to 700 nm, most preferably about 685 nm.

[0079] In a third aspect, there is provided an assay method comprising providing a sample, providing a fluorophore composition according to the second aspect, contacting the sample with the fluorophore composition in the presence of peroxidase and peroxide, and detecting a signal at a wavelength in the range 660 nm to 720 nm.

[0080] In a fourth aspect, there is provided a kit for use in an assay, the kit comprising: a first vessel containing a fluorophore composition according to the second aspect, and a second vessel containing an organic solvent selected from DMSO, acetone, dimethylformamide, acetonitrile, dioxane, THF, and mixtures thereof.

[0081] Suitably, in the fourth aspect the kit is for use in a peroxidase-labelling assay.

[0082] In a fifth aspect, there is provided a kit for use in a Tyramide Signal Amplification method, the kit comprising: a first vessel containing a fluorophore composition according to the second aspect, and a second vessel containing an organic solvent selected from DMSO, acetone, dimethylformamide, acetonitrile, dioxane, THF, and mixtures thereof.

[0083] 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.

[0084] DEFINITIONS

[0085] “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.

[0086] “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.

[0087] Optional substituents may be selected from Ci-s alkyl, Ci-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, sulfmamino, 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, Ci-s alkyl, C1-6 alkyl, OC1-12 alkyl, and halogen. More suitably, the optional substituents are selected from OH, Ci-s alkyl and OC1-12 alkyl; more suitably, the optional substituents are selected from Ci-s alkyl and OC1-12 alkyl.

[0088] “Independently” or “Independently selected” is used in the context of statement that, for example, “each Ri6, R17 is independently H, Ci-s alkyl. . .” and means that each instance of the functional group, e.g., Ri6, is selected from the listed options independently of any other instance of Ri6 or R17 in the compound. Hence, for example, H may be selected for the first instance of Ri6 in the compound; methyl may be selected for the next instance of Ri6 in the compound; and ethyl may be selected for the first instance of R17 in the compound.

[0089] 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 Ci-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-l-yl, pent-2- yl, pent-3 -yl, 3-methylbut-l-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth-l-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.

[0090] “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 Ce-io aryl. Examples of aryl groups include phenyl.

[0091] “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.

[0092] “Ci to Cs haloalkyl” refers to a straight chain and branched saturated hydrocarbon groups, having from 1 to 8 carbon atoms, and having at least one halogen substituent. Suitably, a C1-6 haloalkyl to straight chain and branched saturated hydrocarbon groups, having from 1 to 6 carbon atoms. Suitably a C1-7 haloalkyl; suitably a C1-6 haloalkyl; suitably a C1-5 haloalkyl; more suitably a Ci-4 haloalkyl; more suitably a C1-3 haloalkyl. Suitably, the optionally substituted Ci to Cs haloalkyl is an optionally substituted -CH2X, -CHX2, -CX3, -CH2CH2X, - CH2CHX2, -CH2CX3, -CHXCH3, -CHXCH2X, -CHXCHX2, -CHXCX3, -CX2CH3, - CX2CH2X, -CX2CHX2, or -CX2CX3, wherein X is halo. Suitably, the optionally substituted Ci to Cs haloalkyl is -CH2CX3. In some aspects, preferably X is F. In other aspects, suitably X is Cl.

[0093] “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.

[0094] As used herein, “salt” refers to an electronically neutral compound consisting of cations and anions connected by an ionic bond. Suitably, the compound of the disclosure or salt, solvate, or tautomer thereof, comprises salts of the compounds of the disclosure. These salts include nontoxic acid addition salts (including di-acids) and base salts.

[0095] If the compound is cationic or has a functional group which may be cationic (e.g. -NHz may be -NH3+), then an acid addition salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acids. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride / chloride, hydrobromide / bromide, hydroiodide / iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methyl sulfonate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

[0096] For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -RCOOH may be -RCOO"), then a base salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations, such as an alkali or alkaline earth metal cation, ammonium and substituted ammonium cations, as well as amines. Examples of suitable metal cations include sodium (Na+) potassium (K+), magnesium (Mg2+), calcium (Ca2+), zinc (Zn2+), and aluminum (Al3+). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4+) and substituted ammonium ions (e.g. NEER+, NH2R2+, NHR3+, NRG). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)G. Examples of suitable amines include arginine, N,N'-dibenzylethylene-diamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, olamine, 2-amino-2-hydroxymethyl-propane- 1,3 -diol, and procaine. For a discussion of useful acid addition and base salts, see S. M. Berge et al., J. Pharm. Sci. (1977) 66: 1-19; see also Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection, and Use (2011)

[0097] Salts may be prepared using various methods. For example, one may react a compound of the disclosure with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of the disclosure with an acid or base to remove an acid- or base- labile protecting group or to open a lactone or lactam group of the precursor. Additionally, one may convert a salt of the compound of the disclosure to another salt through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, one may then isolate the salt by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.

[0098] 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.

[0099] “Tautomer” refers to a structural isomer of a compound that readily interconverts to another isomer. 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.

[0100] 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.

[0101] 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.

[0102] BRIEF DESCRIPTION OF THE DRAWINGS

[0103] Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which:

[0104] Figure 1 Representative images of HeLa cell pellets (DAPI used as a counterstain) obtained from the ACD RNAscope™ V2 assay (with Polr2a as the target) comparing the performance of TY1 and TY10, both with optimal levels of enhancer.

[0105] Figure 2 Representative image of HeLa cell pellets obtained from the ACD RNAscope™ V2 assay (with Polr2a as the target) with TY60 and an optimal level of enhancer.

[0106] Figure 3 shows results of formulation investigations with TY1 and the enhancer reagent in the ACD RNAscope V2 assay (with Polr2a as the target). Figure 4 Images of mouse brain tissue (A, staining for POLR2A) and human pancreatic tissue (B, staining for PPIB and C, staining for GAPDH) obtained using the ACD RNAscope V2 assay and TY 1 with an optimum level of enhancer

[0107] Figure 5 shows results comparing the performance of compounds according to the disclosure against the Comparator 690 (commercially available 690 kit) in the ACD RNAscope V2 assay run as a 3-plex, with the Comparator 690 or Example 1 690 applied either first (Pl) or third (P3). Experiment performed using HeLa cell pellets, staining for either Polr2A (top panel) or GAPDH (lower panel).

[0108] Figure 6 shows normalized absorption and fluorescence emission spectra for TY1, 2-(3- (azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10- yl)-3,5,6-trifluoro-4-((2-((4-hydroxyphenethyl)amino)-2-oxoethyl)thio)benzoate. kma\ abs 67 l nnr kmax em 684 nm.

[0109] Figure 7 shows normalized absorption and fluorescence emission spectra for TY10 2-(3- (azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10- yl)-3,5,6-trifluoro-4-((18-(4-hydroxyphenyl)-2,15-dioxo-6,9,12-trioxa-3,16- diazaoctadecyl)thio)benzoate. ma\ abs 671 nm; . kmax em 685 nm.

[0110] Figure 8 shows normalized absorption and fluorescence emission spectra for TY60 2-(3- (azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5-dihydrodibenzo[b,e]silin-10- yl)-3,5,6-trifluoro-4-((4-hydroxyphenethyl)carbamoyl)benzoate. A,max abs 671 nm; . Zmax em 686 nm.

[0111] DESCRIPTION OF THE EMBODIMENTS

[0112] General Chemistry Methods

[0113] 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 forXH NMR, 100 MHz for13C NMR and 376 MHz for19F NMR.XH NMR and13C NMR chemical shifts (6) 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.19F NMR chemical shifts are reported in ppm and are uncorrected. Purification by flash chromatography was performed using pre-packed silica gel columns and either a Buchi Reveleris, Buchi Pure, Biotage Isolera or a Biotage Selekt system. Analytical thin layer chromatography was performed on glass plates pre-coated with silica gel (Analtech, UNIPLATE™ 250 pm / UV254), with visualization being achieved using UV light (254 nm) and / or by staining with alkaline potassium permanganate dip.

[0114] Reactions were performed at room temperature unless otherwise indicated. 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.

[0115] 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.

[0116] Examples

[0117] The invention is further illustrated by the following Examples.

[0118] Example 1 - TY1 - 2-(3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5- dihydrodibenzo[b,e]silin-10-yl)-3,5,6-trifluoro-4-((2-((4-hydroxyphenethyl)amino)-2- oxoethyl)thio)benzoate

[0119] Step 1: Synthesis of 2-(3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5- dihydrodibenzo[b,e]silin-10-yl)-3,5,6-trifluoro-4-((2-((4-hydroxyphenethyl)amino)-2- oxoethyl)thio)benzoate 2-(4-Hydroxyphenyl)ethylamine (30 mg, 0.22 mmol) was added to a solution of 2-[3- (azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-benzo[b][l]benzosilin-10-yl]-4-[2- (2,5-dioxopyrrolidin-l-yl)oxy-2-oxo-ethyl]sulfanyl-3,5,6-trifluoro-benzoate (51 mg, 0.073 mmol) in DMF (1 mL). The resulting solution was stirred overnight before being coevaporated with a mixture of DCM / heptane to remove DMF. The crude product was purified by flash chromatography (50 to 100 % EtOAc / Petroleum ether) to give the title compound as a blue solid (7 mg, 13%).

[0120] ’H NMR (de-DMSO, 400 MHz) 8 9.17 (1H, s), 8.19 (1H, t, J = 5.7 Hz), 6.92 (2H, AA’BB’), 6.78 (2H, dd, J = 8.6, 1.4 Hz), 6.72 (2H, d, J = 2.6 Hz), 6.65 (2H, AA’BB’), 6.25 (2H, dd, J = 8.7, 2.6 Hz), 3.81 (8H, t, J = 7.8 Hz), 3.67 (2H, s), 3.17-3.10 (2H, m), 2.54 - 2.45 (2H, m), 2.34 - 2.24 (4H, m), 0.57 (3H, s), 0.47 (3H, s).

[0121] 19F NMR (de-DMSO, 376 MHz) 6 -110.12 (d, IF), -125.32 (d, IF), -144.31 (t, IF).

[0122] HRMS (ESI) calcd for C3sH36F3N3O4SSi [M]+, 715.2148, found 715.2150.

[0123] Example 2 - TY10 - 2-(3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5- dihydrodibenzo[b,e]silin-10-yl)-3,5,6-trifluoro-4-((18-(4-hydroxyphenyl)-2,15-dioxo- 6,9,12-trioxa-3,16-diazaoctadecyl)thio)benzoate

[0124] Step 1: Synthesis of tert-butyl (15-(4-hydroxyphenyl)-12-oxo-3,6,9-trioxa-13- azapentadecyl)carbamate

[0125] 2-(4-Hydroxyphenyl)ethylamine (0.39 g, 2.86 mmol) was added to a solution of (2,5- dioxopyrrolidin-l-yl) 3-[2-[2-[2-(tert-butoxycarbonylamino)ethoxy]ethoxy]ethoxy]- propanoate (1.00 g, 2.39 mmol) in DMF (4 mL) and the resulting solution was stirred overnight. The reaction mixture was subsequently co-evaporated with a mixture of DCM and heptane to remove DMF. The crude product was purified by flash chromatography (0 to 20% MeOH / EtOAc) to give the title compound as a colourless oil (0.78 g, 74%).

[0126] 'H NVIR (CDCh, 400 MHz) 8 7.04 (2H, AA’BB’), 6.80 (2H, AA’BB’), 6.47 (1H, brs), 5.03 (1H, brs), 3.66 -3.45 (14H, m), 3.36-3.24 (2H, m), 2.73 (2H, t, J = 6.6 Hz), 2.44 (2H, t, J = 5.6 Hz), 1.45 (9H, s).

[0127] Step 2: Synthesis of 15-(4-hydroxyphenyl)-12-oxo-3,6,9-trioxa-13-azapentadecan-l- aminium chloride

[0128] A solution of HC1 in dioxane (4M, 3.40 mL, 13.6 mmol) was slowly added to a solution of tert-butyl N-[2-[2-[2-[3-[2-(4-hydroxyphenyl)ethylamino]-3-oxo-propoxy]ethoxy]ethoxy]- ethyl]carbamate (0.60 g, 1.36 mmol) in dioxane (15 mL). The resulting solution was stirred overnight. The reaction mixture was subsequently co-evaporated with EtOAc and then with a mixture of DCM and methanol to give the title compound as a colourless oil (0.48 g, 94%). 'H NVIR (de-DMSO, 400 MHz) 8 9.20 (1H, brs), 7.93 (4H, brs), 6.99 (2H, AA’BB’), 6.68 (2H, AA’BB’), 3.65 - 3.46 (14H, m), 3.00 - 2.92 (2H, m), 2.63 - 2.54 (2H, m), 2.30 (2H, t, J = 6.5 Hz ).

[0129] Step 3: Synthesis of 2-(3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5- dihydrodibenzo[b,e]silin-10-yl)-3,5,6-trifluoro-4-((18-(4-hydroxyphenyl)-2,15-dioxo- 6,9,12-trioxa-3,16-diazaoctadecyl)thio)benzoate

[0130] A solution of 3-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]-N-[2-(4-hydroxyphenyl)ethyl]- propenamide hydrochloride (27 mg, 72.7 pmol) in a mixture of DMF (2 mL) and TEA (18.4 mg, 181.8 pmol) was added to 2-[3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5- dimethyl-benzofb]

[0001] benzosilin- 10-y 1 ] -4- [2-(2, 5-dioxopyrrolidin- 1 -yl)oxy-2-oxo- ethyl]sulfanyl-3,5,6-trifluoro-benzoate (42 mg, 60.5 pmol). The resulting solution was stirred overnight before being co-evaporated with a mixture of DCM / heptane to remove DMF. The crude product was purified by flash chromatography (0 to 5 % MeOH / EtOAc) to give the title compound as a blue / green solid (42 mg, 76%).

[0131] XH NMR (de-DMSO, 400 MHz) 8 9.15 (1H, s), 8.21 (1H, t, J = 5.5 Hz), 7.85 (1H, t, J = 5.3 Hz), 6.97 (2H, AA’BB’), 6.79 - 6.74 (2H, m), 6.73 (2H, d, J = 2.6 Hz), 6.66 (2H, AA’BB’), 6.28 (2H, dd, J = 8.7, 2.6 Hz), 3.85 (8H, t), 3.69 (2H, s), 3.56 (2H, t, J = 6.5 Hz), 3.49 - 3.41 (8H, m), 3.22- 3.11 (4H, m), 2.60 - 2.54 (2H, m), 2.36 - 2.23 (8H, m), 0.57 (3H, s), 0.47 (3H, s).

[0132] 19F NMR (de-DMSO, 376 MHz) 8 -110.30 (d, IF), -125.40 (d, IF), -144.27 (t, IF).

[0133] HRMS (ESI) calcd for C47H53F3N4OsSSi [M]+, 918.3305, found 918.331.

[0134] Exam ple 3 - TY60 - 2-(3-(azetidin-l-ium -l-ylidene)-7-(azetidin-l-yl)-5,5- dimethyl-3,5-dihydrodibenzo[b,e]silin-10 -yl) -3, 5, 6 -trifluoro -4 -((4- hydroxyphenethyl)carbam oyl) benzoate

[0135] TY60

[0136] Step 1: Synthesis of 2-(3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5-dimethyl-3,5- dihydrodibenzo[b,e]silin-10-yl)-3,5,6-trifluoro-4-((4-hydroxyphenethyl)carbamoyl)- benzoate

[0137] A solution of 2-(methoxymethoxy)propanedinitrile (48 mg, 0.38 mmol) (prepared as described in J. Org. Chem. 1990, 55, 4515) in DMF (2.8 mL) was added dropwise over 15 minutes to a rapidly stirred solution of 2-[3-(azetidin-l-ium-l-ylidene)-7-(azetidin-l-yl)-5,5- dimethyl-benzo[b][l]benzosilin-10-yl]-3,4,5,6-tetrafluoro-benzoate (0.20 g, 0.38 mmol) and DIPEA (200 pL, 2.00 mmol). After stirring overnight, the DMF was removed by repeated co-evaporation with DCM / heptane. The resulting residue was dissolved in DCM and triethylsilane (2 mL) and TFA (4 mL) were sequentially added. After stirring for 6 h, the mixture was co-evaporated with toluene (X 3) and the resulting reside was dissolved in DCM (4 mL). A solution of tyramine (80 mg, 0.58 mmol) and DIPEA (0.64 mL) in DMF (16 mL) was added to dissolve the residue and the mixture was stirred overnight before being coevaporated with heptane to remove DMF. The crude product was purified by flash chromatography (0 to 30 % MeOH / DCM) to give the title compound as a blue / green solid (20 mg, 8%).

[0138] ’H NMR (de-DMSO, 400 MHz) 8 9.17 (1H, s), 9.03 (1H, t, J = 5.7 Hz), 7.00 (2H, AA’BB’), 6.80 (2H, d, J = 8.5 Hz ), 6.72 (2H, d, J = 2.6 Hz), 6.65 (2H, AA’BB’), 6.35 (2H, dd, J = 8.7, 2.6 Hz), 3.87 (8H, t, J = 7.3 Hz), 3.46 - 3.36 (2H, m), 2.70 - 2.65 (2H, m), 2.37 - 2.27 (4H, m), 0.54 (3H, s), 0.48 (3H, s).

[0139] 19F NMR (de-DMSO, 376 MHz) 8 -120.54 (d, IF), -135.20 (d, IF), -143.81 (t, IF).

[0140] HRMS (ESI) calcd for C37H34F3N3O4Si [M]+, 669.2271, found 669.2260.

[0141] Testing / imaging protocol with RNAscope™ Multiplex Fluorescent Reagent Kit v2 Assay

[0142] For tissue testing, the protocol described in the ACD user manual for the RNAscope Multiplex Fluorescent Reagent Kit v2 Assay was followed with the novel fluorophore substituted into the workflow where appropriate. Imaging was performed using an Akoya Bioscience PhenoImager HT and sample exposures were determined utilizing the instrument software’s autoexposure feature for each unique sample type with the Opal 690 filter.

[0143] Formulation Studies and Determination of Properties

[0144] The compounds according to the disclosure are exceptionally bright dyes that offer an effective way to boost signal intensity and detect low-abundance targets in spatial biology applications. They are suitable for use in ISH, IHC and ICC experiments where in situ detection of target protein or nucleic acid sequences is required. They are suitable for multiplexing and can be combined with DAPI for counter-staining.

[0145] The compounds may be used in the TSA process and may also be used in the other applications.

[0146] Test were conducted using the RNAscope V2 assay (ACD). The detection step in this assay can either be chromogenic or fluorogenic (using TSA). For fluorogenic detection, a vial containing a mixture of a compound according to the disclosure as fluorophore and an enhancer reagent is required. In these studies, the enhancer reagent used was 4- iodophenylboronic acid. The enhancer is advantageous because its use reduces the quantity of fluorophore needed to produce images in the assay. The enhancer catalyzes formation of activated tyramide which increases the efficiency of the amplification process and therefore leads to the ability to reduce exposure time which reduces background signal.

[0147] Figure 1 shows images of HeLa cell pellets obtained from the ACD RNAscope V2 assay (with Polr2a as the target). Identical microscopic settings were used in the generation of both of the images. Image A was obtained when TY 1 (and an optimal quantity of enhancer)) was used and image B was obtained when TY10 (and an optimal quantity of enhancer) was used. Both candidates TY1 and TY10 generate clear images in the assay, however, TY1 outperforms TY10 as some faint dots are observed with the former.

[0148] Figure 2 shows images of HeLa cell pellets obtained from the ACD RNAscope V2 assay (with Polr2a as the target) when TY60 (and enhancer) was used. Clear, punctate dots were obtained with TY60 , although some bleed-through was observed into other channels making TY 1 the preferred candidate.

[0149] Formulation experiments

[0150] Formulation experiments were undertaken to explore the impact the amount of enhancer reagent present in the fluorophore composition has on the quality of the images obtained. The results are shown in Figure 2.

[0151] Figure 2 illustrates the impact that the quantity of enhancer present in the enhancer / fluorophore formulation has on the staining of the sample when the quantity of fluorophore is fixed. Experiments were undertaken with the ACD RNAscope V2 assay with Polr2a as the target. Quantities of enhancer mixed with the fluorophore increase from left to right across the figure. The imaging exposure time was kept consistent across all experiments. The signal reduces significantly when less than an optimal quantity of enhancer is used (Figure 2, A-D). An increase in enhancer quantity beyond an optimal level furnishes little extra benefit in terms of signal strength (Figure 2, E-F)

[0152] In the studies detailed below, TY 1 was formulated with an optimal quantity of enhancer (as per Figure 2, E). Tissue Testing

[0153] Extensive tissue testing in both singleplex and multiplex forms with the RNAscope V2 assay has been undertaken with TY 1 (and an optimal quantity of enhancer) to assess performance in a range of different tissue types detecting targets with different levels of expression in the sample. Some of these experiments have been performed in comparison with the Comparator 690. Shown in Figure 3, are high quality images with bright, defined dots obtained from the RNAscope V2 assay with TY1 and two different tissue types; mouse brain tissue (A, staining for POLR2A, exposure time 490 ms) and human pancreatic tissue (B, staining for PPIB, low expressor in the tissue sample, exposure time 300 ms) and C, staining for GAPDH, highly expressed in sample, exposure time 400 ms).

[0154] Real -world multiplexing experiments (TY 1 and Comparator 690, commercially available 620 and commercially available 570 kits) with HeLa cell pellets (staining for Polr2A, top series of images and GAPDH, bottom series of images) reveal an important advantage for TY 1 over the commercial Comparator 690 kit. The results are shown in Figure 4 - note, a consistent exposure time and fluorophore concentration was used across each individual series of images.

[0155] In Figure 4, ‘P’ refers to the order of staining within the multiplex experiment (Pl is first). Consistent staining is observed with TY 1 (and enhancer) in either Pl or P3 positions. In contrast, Comparator 690 shows a drop in signal intensity in the Pl position compared to the P3 position. This is observed for both Polr2A and GAPDH but is most clearly observed for GAPDH staining.

[0156] A visual reduction in signal intensity is observed for the Comparator 690 fluorophore when applied first compared with third in the multiplex whereas TY1 gives a much more consistent staining intensity when applied to the sample either first or third in the multiplex. Consequently, the current invention is advantageous over the comparator in that it can be utilised in any position in a multiplex assay, thus enabling increased capabilities during assay optimization.

[0157] Other applications

[0158] Compounds according to the disclosure may be used for detecting and visualising targets in other detection modalities labelled with HRP (most typically primary and secondary antibodies). Examples of these applications include but are not limited to immunohistochemistry (IHC), in situ hybridization (ISH), immunocytochemistry (ICC), flow cytometry and ELISA.

[0159] References

[0160] “General Synthetic Method for Si-Fluoresceins and Si-Rhodamines” ACS Cent. Sci., 2017, 3, 975-985.

[0161] “Optimized Red-Absorbing Dyes for Imaging and Sensing” J. Am. Chem. Soc. 2023, 145, 42, 23000-23013.

[0162] US 2021 / 0171490

[0163] WO2015153813 (and corresponding patent documents).

[0164] 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.

Claims

CLAIMS1. A compound of formula (la) or (lb):or a salt, solvate, or tautomer thereof; wherein:R1, and R2are independently selected from optionally substituted Ci to Cs alkyl; or R1and R2together with the silicon atom to which they are attached form an optionally substituted 5- or 6-membered ring;R3, and R4are independently selected from H, optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring;R3, and R4are independently selected from H, optionally substituted Ci to Cs alkyl; optionally substituted Ci to Cs haloalkyl; or R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine or pyrrolidine ring;R5is selected from a negative charge, H, Ci to Cs alkyl, or optionally substituted aryl; a is 0 or 1; andZ is a divalent linker group.

2. A compound as claimed in claim 1, wherein R3and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine group of formula:wherein RD and RE are independently selected from H, Ci to Cs alkyl, OR6, C(O)OR6, NHC(O)R6, C(O)NHR6, halo, NR6R7, -CN, -NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R6and R7are independently selected from H, and Ci to Cs alkyl.

3. A compound as claimed in either claim 1 or claim 2, wherein R3, and R4together with the nitrogen atom to which they are attached form an optionally substituted azetidine ring of formula:wherein RG and RH are independently selected from H, Ci to Cs alkyl, OR8, C(O)OR8, NHC(O)R8, C(O)NHR8, halo, NR8R9, -CN, -NC, optionally substituted aryl or optionally substituted heteroaryl; wherein R8and R9are independently selected from H, and Ci to Cs alkyl.

4. A compound as claimed in any one of the preceding claims, wherein the compound is of formula (Va) or (Vb):

5. A compound as claimed in any one of the preceding claims, wherein Z is a single bond or:wherein b is 0, 1 or 2; c, d, e and f are each independently selected from an integer from 0 to 20; g and h are independently 0 or 1 ; each Xi and X2 are independently selected from a triazolyl, optionally substituted arylene, NR10, O, or -O-CH2-CH(O(CH2)I-R10)-CH2-O-, where each 1 is independently 0 or 1;R10is H or Ru;where each i and j are independently selected from an integer from 0 to 20; each k is 0, 1 or 2; each X3 is independently selected from a triazolyl, optionally substituted arylene, NH, or O; andL is selected from: a straight or branched C1-12 alkylene chain which may contain one or more carbon-carbon double or triple bonds, a paraformaldehyde chain- (CH2CH2)n(CH2)m(OCH2)s(O)w(CH2CH2)p(CH2)q- , a polyethylene glycol chain- (CH2CH2)n(CH2)m(OCH2CH2)v(O)w(CH2CH2)p(CH2)q -, which chains may be interrupted by one, two or three groups selected from -O-, -S-, -NH-,, , C3-8 cycloalkyl, C5-10 heteroarylene and / or phenylene; wherein n, m, p and q are independently 0, 1 or 2; w is 0 or 1; s and v are independently 1 to 12, optionally 1 to 8, optionally 1 to 6.

6. A compound as claimed in any one of the preceding claims, wherein a is 0.

7. A compound as claimed in any one of the preceding claims, wherein the compound is of formula:or salts, solvates, or tautomers thereof.

8. A compound of formula:or a salt, solvate, or tautomer thereof.

9. A fluorophore composition, the composition comprising a compound as claimed in any one of claims 1 to 8.

10. A fluorophore composition as claimed in claim 9, further comprising at least one enhancer.

11. A fluorophore composition as claimed in claim 10, wherein the at least one enhancer comprises NaCl, MgCh, KC1, CaCh, sodium phosphate, sodium acetate, ammonium acetate, ammonium sulfate, 4-fluorophenyl boronic acid, 3 -fluorophenyl boronic acid, 4-chlorophenyl boronic acid, 3 -chlorophenyl boronic acid, 4-bromophenyl boronic acid, 3 -iodophenyl boronic acid, 4-iodophenyl boronic acid, 4-acetylphenyl boronic acid, 4-thioanisole boronicacid, 4-hydroxy -4’ -iodobiphenyl, 4-iodophenol, 4-bromophenol, 4-(imidazol-l-yl)phenol, 4- m ethoxyphenol, 2-iodophenol, l-bromo-4-iodobenzene, 3 -iodophenol, 4-phenylphenol, 4-4'- diiodobiphenyl, or mixtures thereof.

12. A fluorophore composition as claimed in any one of claims 9 to 11, further comprising at least one organic solvent.

13. A fluorophore composition as claimed in claim 12, wherein the organic solvent is selected from DMSO, acetone, dimethylformamide, acetonitrile, dioxane, THF, and mixtures thereof.

14. A fluorophore composition as claimed in any one of claims 9 to 13, wherein the concentration of the compound in the composition is in the range 10 nM to 10 pM.

15. A fluorophore composition as claimed in any one of claims 9 to 14, wherein the fluorophore fluoresces at a wavelength in the range 660 nm to 720 nm, preferably 670 nm to 710 nm, more preferably 680 nm to 700 nm, most preferably about 685 nm.

16. An assay method comprising providing a sample, providing a fluorophore composition as claimed in any one of claims 9 to 15, contacting the sample with the fluorophore composition in the presence of peroxidase and peroxide, and detecting a signal at a wavelength in the range 660 nm to 720 nm.

17. A kit for use in a peroxidase-labelling assay, the kit comprising: a first vessel containing a fluorophore composition as claimed in any one of claims 9 to 15, anda second vessel containing an organic solvent selected from DMSO, acetone, dimethylformamide, acetonitrile, dioxane, THF, and mixtures thereof.

Images