Fluorescent Nucleoside Phosphates

Abstract

This specification discloses a novel phosphate compound of formula (I) that is useful for labeling RNA in cellulo.The phosphate can be spontaneously taken up by cells and used as a substrate for RNA synthesis after passing through cell membrane.Therefore, this technology provides a general route to understand the biological behavior of RNA of interest, including RNA-based drugs.

Description

[Technical field]

[0001] This document relates to modified fluorescent nucleoside phosphates and their use to elucidate biological mechanisms. [Background technology]

[0002] RNA plays a fundamental role in biology: it is a protagonist of the central dogma of biochemistry and a key regulator of gene expression, for example, via microRNAs and small interfering RNAs, as well as via endogenous catalytic activity. For these reasons, RNA therapy has also emerged as a highly promising and versatile new drug treatment: the potential to modify cellular functions at the translational level by RNA therapy may open new opportunities to address targets that were previously undruggable.

[0003] Therefore, improving molecular and mechanistic knowledge of biological processes involving RNA is crucial for understanding and treating diseases. For example, growing evidence suggests that understanding the processes of cellular uptake and endosomal release is key to realizing the full potential of RNA-based drugs (Dowdy, SF, Nat. Biotechnol. 35, 222-229,

[2017] ). Regardless of the mechanism of endocytosis, delivery of nucleic acid cargo to the cytoplasm always relies on endosomal escape, the understanding of which remains elusive despite extensive research (Crooke, ST et al., Nat. Biotechnol. 35, 230

[2017] ; Pei, D. & Buyanova, M., Bioconjugate Chem.

[2018] ). In this context, tracking endogenous and exogenous (therapeutic) RNAs in cells, including their translocation, localization, splicing, and degradation, is of great importance.

[0004] Recent advances have led to the development of a wide range of tools and probes that can analyze and quantify RNA, but they generally contain highly modified oligonucleotides with properties that are significantly different from their natural counterparts, which may result in loss of their ability to be recognized and processed by the cellular enzymatic machinery. For example, a drawback of existing fluorescence-based techniques to study the subcellular localization of RNA is that they mainly rely on highly amphiphilic and / or bulky external fluorescent constructs that can impair motility and disrupt RNA localization and (for example) molecular interactions with membrane components. Moreover, the majority of these techniques are not compatible with live cell imaging (Li, Y., Ke, K. & Spitale, RC, Biochemistry 58, 379-386

[2019] ).

[0005] To overcome such problems and provide an improved method for investigating RNA-mediated mechanisms, fluorescent nucleoside phosphates are disclosed herein that are non-cytotoxic and therefore suitable for use in cells. After being incorporated via an energy-dependent pathway in cultured human cells, the phosphates spontaneously accumulate in different cellular localizations (e.g., some phosphates accumulate preferentially in the nucleus, while others accumulate in the cytosol) depending on the molecular structure of the nucleobase. This allows control of the endogenous labeling process of downstream cells.

[0006] To the inventors' knowledge, this combination of properties has not previously been observed with other fluorescent nucleotides.

[0007] Once inside the cell, the enzyme accepts the synthetic phosphate as a standard substrate and eventually incorporates the synthetic phosphate into endogenous cellular RNA without the need for external transfection techniques. Once incorporated, the labeled residue is minimally disrupted, allowing mechanistic studies with minimal effects of labeling on the process under observation. Since it is possible to subsequently isolate RNA from treated cells thus labeled, the technique described herein offers the unique opportunity to fluorescently label RNA "in-cellulo" (i.e., within cells, including living cells).

[0008] Thus, in summary, we provide a non-invasive, non-genetic method to fluorescently label endogenous RNA, which can be used to visualize biochemical reactions involving RNA production, transport, processing, secretion, and protein interactions in living cells, opening new avenues for the research and development of novel nucleic acid-based therapeutics. Summary of the Invention

[0009] A primary objective of the present invention is to provide modified nucleoside phosphates that can be used to conveniently and endogenously prepare fluorescently labeled RNA in-cellulo.

[0010] Accordingly, this specification describes, in part, compounds of formula (I) as set forth in claim 1, physiologically cleavable precursors or salts thereof.

[0011] This specification also describes, in part, a method for preparing a compound of formula (I) as set forth in claim 9, a physiologically cleavable precursor or a salt thereof.

[0012] This specification also describes, in part, a composition for preparing a labeled RNA molecule according to claim 18.

[0013] This specification also describes, in part, the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, for preparing a labeled RNA molecule according to claim 19.

[0014] This specification also describes, in part, a method for preparing an in vitro labeled RNA molecule according to claim 24.

[0015] This specification also describes, in part, a method for preparing labeled RNA molecules in cellulo according to claim 25.

[0016] Exemplary Implementations The invention detailed herein should not be construed as limited to the cited embodiments or examples, as other embodiments will be readily apparent to those skilled in the art.

[0017] General definitions "A" or "an" means "at least one." In any embodiment where "a" or "an" is used to refer to a given material or element, "a" or "an" can mean one. In any embodiment where "a" or "an" is used to refer to a given material or element, "a" or "an" can mean 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1000, 10000, 100000, or 1 million.

[0018] When an embodiment includes "a" or "an" feature X, a subsequent reference to "the" feature X does not imply that only one of the features is present. Instead, the above interpretation of "a" or "an" continues to apply, with "the" also meaning "at least one." In other words, an embodiment that includes "feature X, where feature X is . . . " should be interpreted as "at least one feature X, where at least one feature X is . . . "

[0019] By "comprising," it is meant that a given embodiment may include other features. For example, in any embodiment where a material "comprising" a particular material or element is referred to, the given material may be made up of at least 10% w / w, at least 20% w / w, at least 30% w / w, or at least 40% w / w of that material or element (or combination of materials or elements).

[0020] In any embodiment where "comprising" is mentioned, "comprising" can also mean "consisting of" (or "consists of") or "consisting essentially of" (or "consists essentially of").

[0021] With respect to material embodiments, "consisting of" or "consists of" means that the material or element is formed entirely of that material or element (or combination of materials or elements). In embodiments described as "consisting of" or "consists of," a given material or element may be formed of 100% w / w of that material or element.

[0022] With respect to material embodiments, "consisting essentially of" or "consists essentially of" means that a given material or element consists almost entirely of that material or element (or combination of materials or elements). In any embodiment where "consisting essentially of" or "consists essentially of" is referred to, a given material or element may be made up of at least 50% w / w, at least 60% w / w, at least 70% w / w, at least 80% w / w, at least 90% w / w, at least 95% w / w, or at least 99% w / w of the material or element.

[0023] In any embodiment where "is" or "may be" is used to define a material or element, "is" or "may be" can mean that the material or element "consists of" or "consists essentially of".

[0024] When an element is referred to as being present "in some embodiments," the element may be present in any suitable embodiment in any part of the specification, not just in any suitable embodiment in the same part or text region of the specification.

[0025] When "selected" from a list, the feature is selected from a list consisting of the specified options (i.e., the specified list of options and nothing else).

[0026] The claims are embodiments. Modified Nucleoside Monophosphates, Modified Nucleoside Diphosphates and Modified Nucleoside Triphosphates

[0027] In one embodiment, a compound of formula (I): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a 6-membered carboaromatic ring, and R 3 is selected from -P(O)(OH), -P(O)(OH)-OP(O)(OH) and -P(O)(OH)-OP(O)(OH)-OP(O)(OH). The present invention provides a compound of the formula:

[0028] A "hydro" group corresponds to a hydrogen atom. The atom to which the hydro group is attached may be considered unsubstituted.

[0029] "R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring," this is understood to include the following scheme: [ka] is taken to mean a phenyl ring fused to a tetracyclic heteroaromatic system.

[0030] Formula (I) [wherein, R 3 is -P(O)(OH)2] has the following structure: [ka]

[0031] Formula (I) [wherein, R 3 is -P(O)(OH)-OP(O)(OH)2] has the following structure: [ka]

[0032] Formula (I) [wherein, R 3 is -P(O)(OH)-OP(O)(OH)-OP(O)(OH)2] has the following structure: [ka]

[0033] In one embodiment, a compound of formula (I): [ka] [In the formula, R 1 is hydro, and R 2 is cyano, or R 1 and R 2 form, together with the atoms to which they are attached, a 6-membered carboaromatic ring, and R 3 is selected from -P(O)(OH), -P(O)(OH)-OP(O)(OH) and -P(O)(OH)-OP(O)(OH)-OP(O)(OH). The present invention provides a compound of the formula:

[0034] In one embodiment, a compound of formula (II): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring. The present invention provides a compound of the formula:

[0035] In one embodiment, a compound of formula (III): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring. The present invention provides a compound of the formula:

[0036] In one embodiment, a compound of formula (IV): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring. The present invention provides a compound of the formula:

[0037] In one embodiment, a compound of formula (IV): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring. or a salt thereof.

[0038] In any embodiment where it is stated that a compound of Formula (I) may have certain characteristics or properties, those characteristics or properties may also apply to a compound of Formula (II), (III) or (IV).

[0039] In some embodiments, R 1 is hydro and R 2 is cyano.

[0040] In some embodiments, R 1 and R 2 together with the atoms to which they are attached form a 6-membered carboaromatic ring.

[0041] In some embodiments, R 3 is selected from -P(O)(OH)2 and -P(O)(OH)-OP(O)(OH)2.

[0042] In some embodiments, R 3 is selected from -P(O)(OH)2 and -P(O)(OH)-OP(O)(OH)-OP(O)(OH).

[0043] In some embodiments, R 3 is selected from -P(O)(OH)-OP(O)(OH) and -P(O)(OH)-OP(O)(OH)-OP(O)(OH).

[0044] In some embodiments, R 3 is -P(O)(OH).

[0045] In some embodiments, R 3 is -P(O)(OH)-OP(O)(OH).

[0046] In some embodiments, R 3 is -P(O)(OH)-OP(O)(OH)-OP(O)(OH).

[0047] In one embodiment, ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate 7a (compound 7a, 2CNqAMP), a physiologically cleavable precursor or salt thereof, ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate 7a (compound 7a, 2CNqAMP), The present invention provides a compound of formula (I) selected from ((2R,3S,4R,5R)-5-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (compound 17, pATP), a physiologically cleavable precursor or salt thereof.

[0048] "2CNAqMP" refers to the monophosphate depicted as compound 7a. "2CNAqDP" refers to the diphosphate analog of compound 7a (i.e., R 3 "2CNAqTP" refers to the triphosphate depicted as compound 7. The labeled residue derived from the incorporation of these compounds into RNA is the "2CNqA label."

[0049] "pATP" refers to the triphosphate depicted as compound 17. "pAMP" refers to the monophosphate analog of compound 17 (i.e., R 3 "pADP" refers to the diphosphate of compound 17 (i.e., the analogous compound of formula (I) where R is -P(O)(OH)-OP(O)(OH)). The labeled residue derived from the incorporation of these compounds into RNA is the "pA label."

[0050] In one embodiment, there is provided a compound according to formula (I) selected from ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (compound 7, 2CNqATP) or a salt thereof and ((2R,3S,4R,5R)-5-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (compound 17, pATP) or a salt thereof.

[0051] In one embodiment, there is provided a compound according to formula (I) which is ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate, or a salt thereof.

[0052] In one embodiment, there is provided a compound according to formula (I) or a salt thereof, which is ((2R,3S,4R,5R)-5-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate or a salt thereof.

[0053] In one embodiment, there is provided a compound according to formula (I) which is ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate.

[0054] In one embodiment, there is provided a compound of formula (I) which is ((2R,3S,4R,5R)-5-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate.

[0055] In one embodiment, [ka] or a physiologically cleavable precursor or salt thereof.

[0056] In one embodiment, [ka] or a physiologically cleavable precursor or salt thereof.

[0057] In one embodiment, [ka] In accordance with the present invention, there is provided a physiologically cleavable precursor of a compound of formula (I),

[0058] In one embodiment, [ka] In accordance with the present invention, there is provided a physiologically cleavable precursor of a compound of formula (I),

[0059] In one embodiment, [ka]

[0023] There is provided a salt of a compound of formula (I)

[0060] In one embodiment, [ka]

[0023] There is provided a salt of a compound of formula (I)

[0061] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0062] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0063] In one embodiment [ka] The present invention provides a compound of formula (I):

[0064] In one embodiment, [ka] or a physiologically cleavable precursor or salt thereof.

[0065] In one embodiment, [ka] In accordance with the present invention, there is provided a physiologically cleavable precursor of a compound of formula (I),

[0066] In one embodiment, [ka] In accordance with the present invention, there is provided a physiologically cleavable precursor of a compound of formula (I),

[0067] In one embodiment, [ka]

[0023] There is provided a salt of a compound of formula (I)

[0068] In one embodiment, [ka]

[0023] There is provided a salt of a compound of formula (I)

[0069] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0070] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0071] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0072] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0073] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0074] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0075] In one embodiment, [ka] In accordance with the present invention, there is provided a physiologically cleavable precursor of a compound of formula (I),

[0076] In one embodiment, [ka] In accordance with the present invention, there is provided a physiologically cleavable precursor of a compound of formula (I),

[0077] In one embodiment, [ka]

[0023] There is provided a salt of a compound of formula (I)

[0078] In one embodiment, [ka]

[0023] There is provided a salt of a compound of formula (I)

[0079] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0080] In one embodiment, [ka] The present invention provides a compound of formula (I):

[0081] The compounds and salts described herein may exist as a mixture of tautomers (structural isomers resulting from the migration of a hydrogen atom that exists in equilibrium). Related embodiments include all tautomers of the compounds of formula (I) or salts thereof.

[0082] The atoms of the compounds and salts described herein may be present in their isotopes. In embodiments, all compounds of formula (I) (e.g., where one or more carbon atoms are substituted with one or more isotopes) may be present. 11 C or 13 Compounds of formula (I) in which one or more hydrogen atoms are 2 H or 3 H isotope).

[0083] A physiologically cleavable precursor of a compound of formula (I) is, for example, one in which the monophosphate, diphosphate or triphosphate group attached to the nucleoside portion of the molecule is masked with a suitable protecting group (e.g., a group attached to the oxygen or phosphorus atom of the phosphate group) that can be removed under physiological conditions.When used in the processes described herein (e.g., when provided to a cell), a physiologically cleavable precursor of a compound of formula (I) is converted (e.g., by metabolism) to a compound of formula (I), which can then participate in intracellular processes (such as cellular localization and RNA synthesis).

[0084] In some embodiments, the physiologically cleavable precursor has the formula (I), 3 is -P(O)(OH)2.

[0085] In some embodiments, the physiologically cleavable precursor has the formula (I), 3 is a compound represented by the formula: -P(O)(OH)-OP(O)(OH)2.

[0086] In some embodiments, the physiologically cleavable precursor has the formula (I), 3 is a compound represented by the formula: -P(O)(OH)-OP(O)(OH)-P(O)(OH)2.

[0087] Suitable physiologically cleavable precursors of the compounds of formula (I) are described, for example, in Pradere, U. et al., Chem. Rev. 2014, 114, 18, 9154-9218 (for example in Figure 3); Wiemer, AJ et al., Top. Curr. Chem. 2015 (for example in Table 1); 360: 115-160 and / or Wiemer, AJ; ACS Pharmacol. Transl. Sci. 2020, 3, 4, 613-626, These are any of the groups used to prepare nucleoside phosphate and / or phosphonate prodrugs, the contents of which are incorporated herein by reference.

[0088] Suitable salts of the compound of formula (I) are, for example, base addition salts. Base addition salts are formed by contacting the compound of formula (I) with a suitable organic or inorganic base. Base addition salts can be formed, for example, with a suitable organic base, such as a nitrogen base, ammonia or a trialkylamine, such as triethylamine. Base addition salts can also be formed, for example, with a suitable inorganic base, such as an alkali metal or rare earth hydroxide, for example potassium hydroxide, sodium hydroxide, magnesium hydroxide or manganese hydroxide.

[0089] In one embodiment, there is provided a compound of Formula (I) which is the free acid.

[0090] In one embodiment, there is provided a compound of formula (I) that is a salt.

[0091] In one embodiment, there is provided a compound of formula (I) that is a sodium, potassium, magnesium or ammonium salt.

[0092] In one embodiment, there is provided a compound of formula (I) that is a sodium, potassium or ammonium salt.

[0093] In one embodiment, there is provided a compound of formula (I) that is a sodium or ammonium salt.

[0094] In one embodiment, there is provided a compound of Formula (I) which is a sodium salt.

[0095] In one embodiment, there is provided a compound of formula (I) that is a monopotassium salt, a dipotassium salt, a tripotassium salt, a tetrapotassium salt, a monosodium salt, a disodium salt, a trisodium salt, a tetrasodium salt, a monoammonium salt, a diammonium salt, a triammonium salt or a tetraammonium salt.

[0096] In one embodiment, there is provided a compound of formula (I) that is a monosodium salt, a disodium salt, a trisodium salt, a tetrasodium salt, a monoammonium salt, a diammonium salt, a triammonium salt or a tetraammonium salt.

[0097] In one embodiment, there is provided a compound of formula (I) that is a monosodium salt, a disodium salt, a trisodium salt or a tetrasodium salt.

[0098] In one embodiment, there is provided a compound of formula (I) that is a monosodium salt.

[0099] In one embodiment, there is provided a compound of formula (I) that is a disodium salt.

[0100] In one embodiment, there is provided a compound of Formula (I) that is a trisodium salt.

[0101] In one embodiment, there is provided a compound of formula (I) that is a monoammonium salt, a diammonium salt, a triammonium salt or a tetraammonium salt.

[0102] In one embodiment, there is provided a compound of Formula (I) that is a monoammonium salt.

[0103] In one embodiment, there is provided a compound according to Formula (I) which is a diammonium salt.

[0104] In one embodiment, there is provided a compound of Formula (I) which is a triammonium salt.

[0105] In one embodiment, there is provided a compound of formula (I) as disclosed in the Examples, a physiologically cleavable precursor or a salt thereof.

[0106] Synthesis Process In one embodiment, there is provided a method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, comprising the steps of: i. Formula (V): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form together with the atoms to which they are attached a 6-membered carboaromatic ring, and PG 1 is a suitable protecting group] or a salt thereof; ii. immobilizing the compound of formula (II) or a salt thereof by binding one of its secondary alcohol groups to a suitable support; iii. The remaining secondary alcohol groups are protected by a suitable protecting group, PG 2 Capping with; iv. Protecting group PG 1 Removal of; v. Reconstituting the exposed primary alcohol groups with a compound of formula (VI): [ka] [In the formula, R 4 is a hydro group and C 1-3 alkyl groups] reacting with a compound of the formula: vi. oxidizing the resulting phosphorus(III) compound to a phosphorus(V) compound; Optionally, vii. reacting the resulting phosphorus(V) compound with a tetraalkylammonium pyrophosphate to produce the triphosphate; viii. Protecting group PG 2 Removal of; ix. cleaving the resulting phosphate from the support to produce a compound of formula (I) or a salt thereof; and, optionally, x. forming the free acid, physiologically cleavable precursors or various salts of the compound of formula (I); A method is provided, comprising:

[0107] In one embodiment, there is provided a method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, comprising the steps of: i. Formula (V): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring, and PG 1 is a suitable protecting group] or a salt thereof; ii. immobilizing the compound of formula (II) or a salt thereof by binding one of its secondary alcohol groups to a suitable support; iii. The remaining secondary alcohol groups are protected by a suitable protecting group, PG 2 Capping with; iv. Protecting group PG 1 Removal of; v. Reconstituting the exposed primary alcohol groups with a compound of formula (VI): [ka] [In the formula, R 4 is a hydro group and C 1-3 alkyl groups] reacting with a compound of the formula: vi. oxidizing the resulting phosphorus(III) compound to a phosphorus(V) compound; vii. reacting the resulting phosphorus(V) compound with a tetraalkylammonium pyrophosphate to produce the triphosphate; viii. Protecting group PG 2 Removal of; ix. cleaving the resulting phosphate from the support to produce a compound of formula (I) or a salt thereof; and, optionally, xi. forming the free acid, physiologically cleavable precursor or various salts of the compound of formula (I); A method is provided, comprising:

[0108] Protective groups ("PG", e.g., PG 1 or PG 2 ) is any group suitable for temporary protection of the reactive center, such as a hydroxyl group. Suitable protecting groups for the reactive centers disclosed herein can be found, for example, in "Greene's Protective Groups in Organic Synthesis, Fourth Edition", Greene TW, Wuts PGM; John Wiley & Sons, Inc. 2007, doi: 10.1002 / 0470053488), the entire contents of which are incorporated herein by reference.

[0109] "C 1-3 An "alkyl group" is a linear or branched saturated alkyl group having the indicated number of carbon atoms.1-3 The alkyl groups include methyl, ethyl, propyl and isopropyl.

[0110] In step iii) above, the secondary alcohol that is capped may be one in the ribose portion of the molecule.

[0111] This overall process is an advantageous method for the preparation of compounds of formula (I) for several reasons: - no need for pre-protection of secondary alcohol groups on the starting protected nucleoside; - After step 1, the unreacted nucleoside can be recovered, which minimizes the loss of material; -Solid-supported nucleosides can be stored for up to 3 months without degradation; -This route is suitable for automated synthesis; -The process is robust and has high overall reproducibility; - A clean crude phosphate product is produced and is easy to purify. -High phosphation yields (usually about 60% from nucleoside-loaded resin) are obtained.

[0112] In some embodiments, R 4 can be a hydro group.

[0113] In some embodiments, R 4 is C 1-3 R may be an alkyl group. 1 C 1-3 When R is an alkyl group, the preparation of the phosphoramidite reagent is easier and in higher yields, but at least 4 It has been observed that this functions similarly to step v above, where is a hydro group.

[0114] In some embodiments, R 4 can be methyl.

[0115] In one embodiment, the compound of formula (VI): [ka] [In the formula, R 4 is C 1-3 is an alkyl group. The compound is provided:

[0116] In one embodiment, the compound of formula (VIa): [ka] The compound is provided:

[0117] In some embodiments, the support can be a solid polymer.

[0118] In some embodiments, the support can be a solid polymer selected from controlled-porosity glass and polystyrene.

[0119] In some embodiments, the support can be polystyrene.

[0120] In some embodiments, the support can be a controlled-porosity glass.

[0121] In some embodiments, the support may be functionalized with primary amino groups, which can form reactive points for attachment to the support.

[0122] In some embodiments, the support is a controlled-porosity glass functionalized with primary amino groups (e.g., Amino-SynBase TM ).

[0123] In some embodiments, PG 1 may be selected from trityl, dimethoxytrityl and trimethoxytrityl.

[0124] In some embodiments, PG 2 is selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, tert-butyldimethylsilyl, and methoxyethyl.

[0125] In some embodiments, PG 2 is acetyl. If the immobilized molecule is base labile, this allows 2 This allows for an efficient synthesis in which removal of the group and cleavage from the resin can be accomplished in one step.

[0126] In some embodiments, PG 1 can be dimethoxytrityl, PG 2 can be acetyl.

[0127] In some embodiments, immobilization of the compound of Formula (II) in step i) may occur primarily at the 2'-hydroxy position.

[0128] When immobilization occurs primarily at the 2'-hydroxy position, this can be more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or 100% of the total immobilization (i.e., all covalent attachments of both secondary hydroxyl groups to the support).

[0129] In some embodiments, the tetraalkylammonium pyrophosphate can be tetrabutylammonium pyrophosphate.

[0130] In one embodiment, there is provided a method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, comprising the steps of: i. Formula (II): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring, and PG 1 is selected from trityl, dimethoxytrityl and trimethoxytrityl, or a salt thereof; ii. immobilizing the compound of formula (V) or a salt thereof by attaching one of its secondary alcohol groups to a controlled-porosity glass support; iii. Protecting the remaining secondary alcohol groups with a protecting group PG selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, tert-butyldimethylsilyl, and methoxyethyl. 2 Capping with; iv. Protecting group PG 1 Removal of; v. Reconstituting the exposed primary alcohol groups with a compound of formula (VI): [ka] [In the formula, R 4 is C 1-3 wherein the alkyl group is an alkyl group; vi. oxidizing the resulting phosphorus(III) compound to a phosphorus(V) compound; Optionally, vii. reacting the resulting phosphorus(V) compound with tetrabutylammonium pyrophosphate to produce the triphosphate; viii. Protecting group PG 2 Removal of; ix. cleaving the resulting phosphate from the support to produce a compound of formula (I) or a salt thereof; and, optionally, x. forming the free acid, physiologically cleavable precursors or various salts of the compound of formula (I); A method is provided, comprising:

[0131] In one embodiment, there is provided a method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, comprising the steps of: i. Formula (V): [ka] [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form, together with the atoms to which they are attached, a six-membered carboaromatic ring, and PG 1 is dimethoxytrityl] or a salt thereof; ii. immobilizing the compound of formula (II) or a salt thereof by attaching one of its secondary alcohol groups to a controlled-porosity glass support; iii. Protecting any remaining secondary alcohol groups with the protecting group PG, which is acetyl 2 Capping with; iv. Protecting group PG 1 Removal of; v. Reconstituting the exposed primary alcohol groups with a compound of formula (VI): [ka] [In the formula, R 4 is a methyl group; vi. oxidizing the resulting phosphorus(III) compound to a phosphorus(V) compound; optionally, vii. reacting the resulting phosphorus(V) compound with tetrabutylammonium pyrophosphate to produce the triphosphate; viii. Protecting group PG 2 Removal of; ix. cleaving the resulting phosphate from the support to produce a compound of formula (I) or a salt thereof; and, optionally, x. forming the free acid, physiologically cleavable precursors or various salts of the compound of formula (I); A method is provided, comprising:

[0132] Suitable conditions and reagents for carrying out each of the above steps i) to ix) are known to those skilled in the art or are described in the detailed description.

[0133] In some embodiments, immobilization of the compound of Formula (II) or a salt thereof in step ii) can be achieved by a coupling agent (e.g., succinic anhydride catalyzed by dimethylaminopyridine when the support is functionalized with a primary amino group).

[0134] In some embodiments, the reaction of exposed primary alcohol groups with a compound of Formula (III) can be accomplished using an activator (e.g., BTT activator or Activator 4®).

[0135] In some embodiments, in step vi), the phosphorus(III) compound can be oxidized to the phosphorus(V) compound using aqueous pyridine and iodine.

[0136] In some embodiments, cleavage of the triphosphate from the support can be accomplished using basic conditions (e.g., by treatment with AMA). 2 If a base-labile protecting group is chosen for , then these conditions allow for simultaneous deprotection and cleavage.

[0137] RNA synthesis composition The compounds of formula (I) may be used as substrates for RNA synthesis together with other natural and synthetic RNA building blocks.

[0138] In one embodiment, a composition for preparing a labeled RNA molecule is provided, comprising a compound of Formula (I) and natural ribonucleotides.

[0139] A "naturally occurring ribonucleotide" is a ribonucleotide that has a phosphate group attached to the 5' hydroxy position (e.g., R 3 In some embodiments, "natural ribonucleotide" refers to a natural ribonucleoside triphosphate.

[0140] In some embodiments, the natural ribonucleotides (e.g., natural ribonucleoside triphosphates) can be selected from cytidine 5'-triphosphate, uridine 5'-triphosphate, adenosine 5'-triphosphate and guanidine 5'-triphosphate. The natural ribonucleotide composition (e.g., the natural nucleoside triphosphate composition) can include a combination of various amounts of these components in sufficient amounts to construct a target RNA molecule (e.g., as provided in an NTP mix).

[0141] Synthesis of labeled RNA In one embodiment, there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, for preparing a labeled RNA molecule (eg, a 2CNqA or PA labeled RNA molecule).

[0142] The labeled RNA molecules contain at least one modified fluorescent residue (e.g., a residue derived from a compound of formula (I) where the modified residue is a 2CNqA or PA residue), but are otherwise similar to naturally occurring RNA molecules (i.e., have an unmodified adenosine residue in the same position as the 2CnqA or pA residue).

[0143] In some embodiments, the labeled RNA molecule can be a 2CNqA or pA labeled mRNA (messenger RNA) molecule.

[0144] In one embodiment, a method for preparing a 2CNqA-labeled RNA molecule is provided, comprising the steps of: 1 Hydro; R 2 is selected from cyano; or a physiologically cleavable precursor or salt thereof.

[0145] In one embodiment, a method for preparing a pA-labeled RNA molecule is provided, comprising the steps of: 1 and R 2 together with the atom to which they are attached form a 6-membered carboaromatic ring; or a physiologically cleavable precursor or salt thereof.

[0146] In one embodiment, there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, for the enzymatic preparation of a labeled RNA molecule.

[0147] In one embodiment, there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, for the enzymatic preparation of labeled RNA molecules in vitro.

[0148] In one embodiment, there is provided a method for preparing a labeled RNA molecule in vitro, comprising: i. providing a DNA template to a composition comprising a compound of formula (I), a physiologically cleavable precursor or a salt thereof, and natural ribonucleotides; ii. treating the resulting mixture with RNA polymerase; optionally, iii. monitoring the labeled RNA molecules using a microscope; and, optionally, iv. isolating the labeled RNA molecules; A method is provided, comprising:

[0149] In one embodiment, there is provided a method for preparing a labeled RNA molecule in vitro, comprising: v. providing a DNA template to a composition comprising a compound of formula (I), a physiologically cleavable precursor or a salt thereof, and natural ribonucleotides; vi. treating the resulting mixture with RNA polymerase; vii. monitoring the labeled RNA molecules using a microscope; and viii. isolating the labeled RNA molecules; A method is provided, comprising:

[0150] In some embodiments, the microscope may be a confocal laser scanning fluorescence microscope.

[0151] In some embodiments, the method of preparing labeled RNA molecules in vitro may be carried out in the presence of a transcription buffer (e.g., 5X transcription buffer), a magnesium salt (e.g., magnesium(II) chloride) and / or an RNase inhibitor (e.g., Ribolock).

[0152] In one embodiment, there is provided a kit for preparing a labeled RNA molecule, comprising: i. a compound represented by formula (I); ii. the composition of naturally occurring ribonucleotides; iii. RNA polymerase; optionally iv. a DNA template; and, optionally v. Instructions for use; A kit is provided comprising:

[0153] In one embodiment, there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, for preparing an endogenous labeled RNA molecule.

[0154] In one embodiment, there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof in a living cell for preparing endogenous labeled RNA molecules.

[0155] In one embodiment, there is provided the use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, for preparing labeled RNA molecules in-cellulo.

[0156] In one embodiment, there is provided a method for preparing a labeled RNA molecule in-cellulo, comprising: i. providing a compound of formula (I), a physiologically cleavable precursor or a salt thereof to a eukaryotic or prokaryotic cell; ii. causing the organism to spontaneously internalize the compound; iii. causing the organism to prepare labeled RNA molecules; optionally, iv. monitoring the labeled RNA molecules using a microscope; and, optionally, v. isolating the labeled RNA molecules; A method is provided, comprising:

[0157] In one embodiment, there is provided a method for preparing a labeled RNA molecule in-cellulo, comprising: vi. providing a compound of formula (I), a physiologically cleavable precursor or a salt thereof to a eukaryotic or prokaryotic cell; vii. causing the organism to spontaneously internalize the compound; viii. causing the organism to prepare labeled RNA molecules; ix. monitoring the labeled RNA molecules using a microscope; and x. isolating the labeled RNA molecules; A method is provided, comprising:

[0158] In some embodiments, the eukaryotic cell may be contained in C. elegans or zebrafish.

[0159] In some embodiments, the labeled RNA molecule can include more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90% or 100% modified residues (e.g., derived from compounds of Formula (I) where the modified residues are 2CNqA or pA residues) in place of unmodified adenosine residues.

[0160] In some embodiments, the labeled RNA molecule can contain 10% to 20%, 10% to 30%, 10% to 40%, 20 to 50%, 30 to 60%, 40 to 70%, 50% to 80%, or 50% to 90% modified residues (e.g., derived from a compound of Formula (I) where the modified residues are 2CNqA or pA residues) in place of unmodified adenosine residues.

[0161] In some embodiments, the labeled RNA molecule may be selected from mRNA and ribosomal RNA.

[0162] In one embodiment, a method is provided for preparing labeled RNA molecules in-cellulo, essentially as described in the detailed description. [Brief description of the drawings]

[0163] [Figure 1] General scheme for the preparation of compounds of formula (I).

[0164] [Diagram 2] Preparation of nucleosides.

[0165] [Diagram 3] Cytotoxicity assessment. (A) Cell viability measured as a decrease in metabolic activity using the alamarBlue assay. (B) Cell membrane integrity measured by lactate dehydrogenase (LDH) leakage assay. Huh-7 cells were treated with the indicated doses of compounds for 24 h. Error bars represent the standard deviation of three independent experiments.

[0166] [Figure 4]Confocal fluorescence microscopy images of live Huh-7 cells exposed to 2.5 μM 2CNqATP or pATP in complete cell culture medium for (A) 20 h at 37 °C, (B) 1.5 h at 37 °C, or (C) 1.5 h at 4 °C.

[0167] [Diagram 5] Measurement of mean fluorescence intensity (MFI) of 2CNqATP or pATP in single live cells after exposure to 2.5 μM pATP or 2CNqATP for the indicated times. Cells were washed, trypsinized, and analyzed using flow cytometry with excitation at 405 nm. A) MFI distribution of cell samples measured for untreated cells and cells treated with 2CNqATP and pATP. B) Mean MFI of 2CNqATP in single live cells plotted against exposure time. C) Mean MFI of pATP in single live cells plotted against exposure time. Lines indicate guide to the view.

[0168] [Figure 6] Dose response of cellular uptake measured as normalized fluorescence intensity in cell lysates harvested from Huh-7 cell cultures exposed for 24 h to various concentrations of A) 2CNqATP or B) pATP. 2CNqATP was excited at 355 nm with a bandpass filter and emission was detected at 460 nm. For pATP, bandpass filters for excitation at 380 nm and emission at 410 nm were used. Error bars represent standard deviation.

[0169] [Figure 7] Normalized mean fluorescence intensity of live Huh-7 cells after exposure to 2.5 μM 2CNqATP (top graph) or pATP (bottom graph) in the presence of increasing concentrations of ATP (black solid line) or adenosine (grey dotted line). Cells were exposed for 4 h, washed, and analyzed by flow cytometry using a 405 nm laser for excitation. Lines are guided by gaze; error bars represent standard deviation of three independent experiments. Lines are guided by gaze.

[0170] [Figure 8] Fluorescence emission spectra of cell-extracted and purified RNA from Huh-7 cells treated for 24 h with 2.5 μM (A) 2CNqATP or (B) pATP indicate that the cellular machinery is active and capable of incorporating specific nucleotide analogs into endogenously produced RNA. The solid black line represents labeled RNA extracted from cells; the dotted grey lines represent the following controls: (light black dashed line) compounds added to cell lysates of untreated cells prior to RNA purification; (dark grey dotted line) compounds added to RNA prior to final column purification; (light grey dashed line) compounds added directly to the RNA purification column. Spectra reflect the total RNA concentration in solution and are normalized to the corresponding absorbance at 260 nm.

[0171] [Figure 9] Spectral comparison of RNA extracted from Huh-7 cells treated with 2CNqATP with 2CNqATP and in vitro 2CNqA-modified RNA strands. A) Normalized excitation spectrum (black thick line) of RNA extracted from Huh-7 cells exposed to 2.5 μM 2CNqATP for 24 h overlaid with absorption spectra of 2CNqATP (grey dotted line) and 2CNqA incorporated into 25:mer (5'-CGA CAA AAU CAA [2CNqA]AU GCG UGA UUG G -3') of ssRNA (black thin line) or dsRNA with hybridized complementary strand (grey thin line). Each spectrum is normalized to the peak maximum. The emission wavelength was fixed at 443 nm to record the excitation spectra. B) Normalized emission spectra of RNA extracted from cells exposed to 2.5 μM 2CNqATP (thick black line), 2CNqATP (dashed grey line), 25:mer ssRNA (as in A) (thin black line) and 2CNqA incorporated into dsRNA (thin grey line).

[0172] [Figure 10]Cytotoxicity assessment using 2CNqAMP. (A) Cell viability measured as a decrease in metabolic activity using the alamarBlue assay. (B) Cell membrane integrity assessed by lactate dehydrogenase (LDH) leakage assay. Huh-7 cells were treated with the indicated doses of compounds for 24 h. Error bars represent the standard deviation of triplicate exposures.

[0173] [Figure 11] Confocal fluorescence microscopy images of live Huh-7 cells exposed to 2.5 μM 2CNqAMP in complete cell culture medium or DPBS (as a control) for (A) 24 h at 37 °C, (B) 1.5 h at 37 °C, and (C) 1.5 h at 4 °C.

[0174] [Figure 12] Measurement of the mean fluorescence intensity (MFI) of 2CNqAMP in single live cells after exposure to 2.5 μM 2CNqAMP for the indicated times. Cells were washed, trypsinized, and analyzed using flow cytometry with excitation at 405 nm. The mean MFI of 2CNqAMP in single live cells was plotted against exposure time. Two independent experiments are shown (blank / filled squares). Error bars represent the standard deviation of triplicate exposures.

[0175] [Figure 13]Spectral readout of cell-extracted and purified RNA from Huh-7 cells treated with 2.5 μM 2CNqAMP for 24 h at 37 °C indicates that the cellular machinery is active and capable of incorporating specific nucleotide analogs into endogenously produced RNA. (A) Spectral comparison of RNA extracted from Huh-7 cells treated with 2CNqAMP with 2CNqATP, RNA from Huh-7 cells treated with 2CNqATP, and in vitro 2CNqA modified RNA strands. Excitation spectrum of RNA extracted from Huh-7 cells exposed to 2.5 μM 2CNqAMP for 24 h (solid black line) overlaid with absorption spectra of 2CNqA incorporated into the 25:mer (5'-CGA CAA AAU CAA [2CNqA]AU GCG UGA UUG G -3') of 2CNqATP (dashed black line), ssRNA (dashed grey line) or dsRNA with hybridized complementary strand (dashed grey line), and excitation spectrum of RNA from Huh-7 cells treated with 2CNqATP (solid grey line). Each spectrum is normalized to the peak maximum. To record the excitation spectrum, the emission wavelength was fixed at 443 nm. (B) Fluorescence emission spectrum of cell-extracted and purified RNA from Huh-7 cells treated with 2.5 μM 2CNqAMP for 24 h. The solid black line represents the labeled RNA extracted from cells; the solid grey line represents the control where compounds are added to cell lysates of untreated cells before RNA purification. The spectra are normalized to the corresponding absorbance at 260 nm, reflecting the total RNA concentration in the solution. The dashed light grey line shows the emission spectrum of ultrapure water (milliQ) for comparison.

[0176] Detailed Description Compounds of formula (I) can be prepared according to the synthetic scheme shown in Figure 1 and the methods described below. Unless otherwise stated, reagents were commercially available and used without further purification. The following reagents used for triphosphate synthesis were purchased from Sigma-Aldrich: DCA deblock for AKTA, CAP A for AKTA, CAP B1 and B2 for AKTA, BTT activator. 1H(500MHz) and 13 C (126 MHz) NMR spectra were recorded at 300 K on a Bruker 500 MHz system equipped with a CryoProbe. 31 P (202 MHz) NMR spectra were recorded on a Bruker 500 MHz system at 300 K. All shifts are reported in ppm relative to the deuterated solvent (DMSO-d6, CDCl3 or D2O).

[0177] Example 1: Synthesis of modified nucleoside phosphate-2CNqATP 2-(5-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl)-2,6-dihydro-2,3,5,6-tetraazaaceanthrylene-8-carbonitrile 1 Compound 1 was prepared according to the literature (Wypijewska del Nogal et al., Nucleic Acids Research 48, 7640-7652

[2020] ). Analytical data were consistent with those reported.

[0178] CPG solid support 3 Amino-SynBase from LinkTech TM CPG 500 / 110 (LCAA)2 (Nu.1397-C025, 1 g, 0.08 mmol) was activated by shaking in 3% trichloroacetic acid in DCE (8 mL, 0.08 mmol) for 18 h. The activated support was then filtered off and washed with 9:1 triethylamine:diisopropylethylamine (20 mL), dichloromethane (20 mL) and diethyl ether (20 mL). The activated support was dried under vacuum for 2 days before use. The support (1 g, 0.08 mmol), succinic anhydride (0.345 g, 3.44 mmol) and N,N-dimethylpyridin-4-amine (0.070 g, 0.57 mmol) were then added to N 2The support was suspended in dry pyridine (3 mL) under reduced pressure. The reaction mixture was gently shaken at RT for 4 h. After 4 h, the solvent was filtered off and the support was washed successively with pyridine (20 mL), dichloromethane (20 mL), diethyl ether (20 mL) and air-dried. A small portion of the support gave a negative ninhydrin test, proving complete succinylation. Succinylated CPG3 could then be stored at room temperature for several months.

[0179] CPG-supported 2-(5-O-(4,4'-dimethoxytrityl)-PD-ribofuranosyl)-2,6-dihydro-2,3,5,6-tetraazaaceanthrylene-8-carbonitrile 4 In a 10 mL syringe equipped with a PTFE filter, succinylated support 3 (0.400 g, 82 gmol / g, 0.03 mmol), DMAP (8 mg, 0.07 mmol), DIC (203 μl, 1.31 mmol), nucleoside 1 (0.022 g, 0.03 mmol) and triethylamine (14 μl, 0.10 mmol) were suspended in pyridine (3 mL). The mixture was gently shaken at RT for 18 h. After 18 h, the syringe was purged and the support was washed with pyridine (5 ml), dichloromethane (5 mL) and diethyl ether. Then, in the same syringe, DMAP (8 mg, 0.07 mmol), DIC (203 μl, 1.31 mmol), triethylamine (14 μl, 0.10 mmol) and 2,3,4,5,6-pentachlorophenol (0.87 g, 0.33 mmol) were added to the support and suspended in pyridine (3 mL). The mixture was gently shaken for 4 h at RT, after which a piperidine solution (2 mL, 20% in DMF - for capping unreacted carboxylic acids on the support) was added for 1 min (longer exposure times result in reduced loading as piperidine cleaves the ester bond with the nucleoside), followed by a quick wash with DMF (3x5 mL), dichloromethane (5 mL) and diethyl ether (5 mL). Finally, the resin was shaken under argon atmosphere in a mixture of CAP A + CAP B (50 / 50 v / v) for 2 h, then washed with DMF (5 ml), dichloromethane (5 ml), diethyl ether (5 mL) and dried with argon (final loading: 16 μmol / g - determined by reading the optical density of the DMT solution cleaved from a weighed amount of support - ε = 70000 M-1.cm-1 at 498 nm). The final loading can be increased by performing a second coupling with 1 under the same conditions before capping (typical loading after the second coupling is 20-25 μmol / g). Concentration of the reaction mixture and multiple washing of the residue with water and diethyl ether allows the recovery of almost 85% of the unreacted nucleoside 1.

[0180] 6-Chloro-N,N-diisopropyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 5 Compound 5 was prepared according to the literature (Ducho, C. et al., J. Med. Chem. 50, 1335-1346

[2007] ). Briefly, 5-chlorosalicylic acid was reduced with LAH (0.5 equiv.) at −20° C., and the resulting 5-chlorosalicyl alcohol was cyclized to 2,6-dichloro-4H-benzo[d][1,3,2]dioxaphosphinine with PCl3 (1.2 equiv.) and triethylamine (2.3 equiv.) at −20° C. under argon. The low temperature and the use of triethylamine as base were crucial to avoid the rapid and quantitative Arbuzov rearrangement of the desired product to the more stable 2,5-dichloro-3H-benzo[d][1,2]oxaphosphole 2-oxide. The crude 2,6-dichloro-4H-benzo[d][1,3,2]dioxaphosphinine was then treated with diisopropylamine (3 equiv.) at room temperature for 2 h. The mixture was then filtered under argon, concentrated to dryness, and taken up in 20% diisopropylamine in heptane. A quick filtration through a small silica gel plug afforded the desired compound 5 as a colorless oil that crystallized over time at -20 °C. A more exhaustive column chromatography attempt on compound 5 would have led to a quantitative Arbuzov rearrangement.

[0181] 1 H NMR (500 MHz, DMSO-d6) δ = 7.23 (dd, J = 8.6, 2.6 Hz, 1H), 7.20 (d, J = 2.4 Hz, 1H), 6.92 (d,J = 8.6 Hz, 1H), 5.06 (dd, J = 14.7, 5.2 Hz, 1H), 4.89 (dd, J = 19.6, 14.8 Hz, 1H), 3.53 - 3.63 (m, 2H), 1.15 - 1.19 (dd, J = 8.0, 7.0 Hz, 12H). 31 P NMR (202 MHz, DMSO-d6) δ = 136.00 (s, 1P).

[0182] Bis(tetrabutylammonium) dihydrogen diphosphate 6 Compound 6 was prepared according to the literature (Warnecke, S. & Meier, C., J. Org. Chem. 74, 3024-3030

[2009] ).

[0183] 1 H NMR (500 MHz, D2O) δ 3.04 - 3.13 (m, 16H), 1.53 (bs, 16H), 1.24 (h, J=7.3, 16H), 0.83 (t, J=7.4, 24H). 31 P NMR (202 MHz, D2O) δ = -10.78 (s, 2P).

[0184] ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (2CNqATP)7 The reaction was carried out in a 5 mL syringe with a PTFE filter loaded with CPG-linked 2CNaA nucleoside 4 (400 mg, 0.0064 mmol) under argon atmosphere with shaking. The procedure was as follows: a. Removal of 5'-DMT: The support was washed with DCA deblock until the filtrate was colorless, then washed with ACN (5 x 5 mL). b. Coupling: N,N-diisopropyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 5 (345 mg, 1.36 mmol) was dissolved in 4.8 ml-ACN and reacted partially with the support (three equal couplings with reaction times of 60 s-60 s-90 s each). To each coupling, BTT activator (2.4 mL) was also added. The support was then washed with ACN (3 x 5 mL). c. Oxidation: Pyridine / water / rosin (9 / 1 / 12.7 v / v / w, 5 mL) for 45 seconds, followed by ACN washes (3×5 mL) and drying the support in a stream of argon. d. Triphosphate: Bis(tetrabutylammonium) dihydrogen diphosphate 6 (0.5 M, 5 mL) was injected twice for 15 min and 18 h, respectively. The support was then washed with DMF (5 mL), water (3×5 mL), ACN (5 mL), and then dried under a stream of argon. e. Cleavage and purification: Cleavage of the triphosphate was carried out with AMA (50 / 50 v / v mixture of 23% aq. NH40H and 40% aq. methylamine, 5 mL) at room temperature for 2 h. After 2 h, the AMA filtrate was purged in the round-bottom flask and the support was rinsed three times with 23% aq. NH40H solution. The mixture was lyophilized and then purified by HPLC (Waters Acquity HSS T3 column, 2.1 x 50 mm, 0.4 mL / min, 2-99% 50 mM NH40Ac in water 80:20 EtOH) to give 2CNqATP (2.3 mg, 55% as determined by UV absorbance) as a pale yellow solid (ammonium salt).

[0185] HRMS (ESI-TOF) m / z calcd. for C18H19N5O13P3 [M+H]+: 606.0192, found: 606.0170.

[0186] ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate can also be prepared by a slightly modified route in which coupling step (b above) is performed with a modified phosphoramidite such as 6-chloro-N,N-diisopropyl-4-methyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 8 (compound (IIIa) above). This reagent has proven to be easier to prepare: compound 8 is obtained in 60% yield compared to about 3-10% yield for compound 5 when prepared under the conditions herein.

[0187] Example 1a: Synthesis of modified nucleoside phosphate-2CNqAMP ((2R,3S,4R,5R)-5-(8-cyano-2,3,5,6-tetraazaaceanthrylene-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl dihydrogen phosphate 7a The reaction was carried out in a 5 ml syringe with a PTFE filter loaded with the CPG-linked 2CNaA nucleoside 4 (400 mg, 0.0064 mmol) prepared above, under an argon atmosphere, with shaking. The procedure was as follows: a. Removal of 5'-DMT: The support was washed with DCA deblock until the filtrate was colorless, then washed with ACN (5 x 5 mL). b. Coupling: N,N-diisopropyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 5 (345 mg, 1.36 mmol) was dissolved in 4.8 ml-ACN and reacted partially with the support (three equal couplings with reaction times of 60 s-60 s-90 s each). To each coupling, BTT activator (2.4 mL) was also added. The support was then washed with ACN (3 x 5 mL). c. Oxidation: Pyridine / water / rosin (9 / 1 / 12.7 v / v / w, 5 mL) for 45 seconds, followed by ACN washes (3×5 mL) and drying the support in a stream of argon. d. Cleavage and purification: Cleavage of the triphosphate was carried out with AMA (50 / 50 v / v mixture of 23% aq. NH40H and 40% aq. methylamine, 5 mL) at room temperature for 2 h. After 2 h, the AMA filtrate was purged in a round-bottom flask and the support was washed three times with 23% aq. NH40H solution. The mixture was lyophilized and then purified by HPLC (Waters Acquity HSS T3 column, 2.1x50 mm, 0.4 mL / min, 2-99% 50 mM NH4OAc in water 80:20 EtOH) to give 2CNqAMP (2.3 mg, 55% as determined by UV absorbance) as a pale yellow solid (ammonium salt).

[0188] 6-Chloro-N,N-diisopropyl-4-methyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 8 5-Chloro-2-hydroxybenzaldehyde was reacted with methylmagnesium bromide (2.5 equiv.) at -20 °C, and the resulting 4-chloro-2-(1-hydroxyethyl)phenol was cyclized to 2,6-dichloro-4-methyl-4H-benzo[d][1,3,2]dioxaphosphinine with PC13 (1.2 equiv.) and triethylamine (2.3 equiv.) at -20 °C under argon. The crude 2,6-Dichloro-4-methyl-4H-benzo[d][1,3,2]dioxaphosphinine was treated with diisopropylamine (3 equiv.) at room temperature for 2 h. The mixture was then filtered under argon, concentrated to dryness, and taken up in 20% diisopropylamine in heptane. Quick filtration through a small silica gel plug afforded the desired compound 8 as a colorless oil.

[0189] 1 H NMR (500 MHz, DMSO-d6) δ = 6.96 (d, J = 8.5 Hz, 1H), 6.87 (d, J = 8.5 Hz, 1H), 6.74 (d, J =8.4 Hz, 1H), 5.19 - 5.26 (m, 1H), 5.16 (dq, J = 10.4, 6.6 Hz, 1H), 3.57 (tdt, J = 13.6, 10.6, 6.8 Hz, 2H), 1.63(d, J = 6.6 Hz, 3H), 1.55 (d, J = 6.4 Hz, 2H), 1.16 - 1.19 (m, 24H). 31 P NMR (202 MHz, DMSO-d6) δ = 137.63 (s, 1P), 127.90 (s, 1P).

[0190] Example 2: Synthesis of modified nucleoside phosphate-pATP (2R,3R,4R,5R)-2-((benzoyloxy)methyl)-5-(4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate 9 4-Chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (20 g, 71.6 mmol) was dissolved in MeCN (480 mL) and trimethylsilyl (E)-N-(trimethylsilyl)acetimidate (19.5 mL, 78.7 mmol) was added dropwise. The mixture was stirred at RT for 20 min. (2S,3R,4R,5R)-2-acetoxy-5-((benzoyloxy)methyl)tetrahydrofuran-3,4-diyldibenzoate (46.9 g, 93.0 mmol) was added in one portion followed by the dropwise addition of trimethylsilyl trifluoromethanesulfonate (15.2 mL, 78.7 mmol). The reaction mixture was stirred at 80° C. for 2 h. The reaction mixture was allowed to cool to room temperature and diluted with EtOAc (200 mL). The organic phase was washed with aq.satd.NaHC03 (100 mL) and brine (100 mL), dried over anhydrous Na2S04, filtered, concentrated under reduced pressure, absorbed onto Celite and purified by flash column chromatography (Hept:EtOAc 90:10 to 70:30, KP-Sil 330 g) to give 3 (31.0 g, 60%) as a white solid.

[0191] 1 H NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 8.09 - 8.13 (m, 2H), 7.99 (dd, J = 8.3, 1.2 Hz, 2H), 7.92 (dd, J = 8.4, 1.2 Hz, 2H), 7.49 - 7.64 (m, 6H), 7.39 (dt, J = 22.8, 7.8, 7.8 Hz, 4H), 6.67 (d, J = 5.4 Hz, 1H), 6.09 - 6.17 (m, 2H), 4.90 (dd, J = 12.3, 3.1 Hz, 1H), 4.80 (q, J = 3.5, 3.5, 3.4 Hz, 1H), 4.68 (dd, J = 12.3, 3.6 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 166.2, 165.5, 165.2, 153.3, 151.4, 151.1, 133.94, 133.91, 133.7, 132.1, 129.98, 129.96, 129.8, 129.4, 128.9, 128.8, 128.71, 128.66, 128.5, 117.9, 86.9, 80.8, 74.3, 71.6, 63.6, 53.8. HRMS (ESI-TOF) m / z calcd. H]+ : 724.0347, found: 724.0384.

[0192] (2R,3R,4R,5R)-2-((benzoyloxy)methyl)-5-(4-chloro-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate 10 Compound 9 (31 g, 42.8 mmol) and tetrakis(triphenylphosphine)palladium(0) (0.99 g, 0.86 mmol) were dissolved in THF (360 mL) to which triethylamine (59 mL, 428 mmol) was added. The reaction mixture was cooled to -78°C and stirred for 5 min, after which 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (9.3 mL, 64.2 mmol) was added dropwise to the mixture. The reaction mixture was then warmed to RT and continuously heated to 80°C for 36 h. The reaction mixture was allowed to cool to RT, concentrated under reduced pressure, absorbed onto Celite, and purified by flash chromatography (KP-Sil, 330 g, Hept:EtOAc, 95:5 to 70:30) to give the desired compound (10, 23.5 g, 76%) as a white solid.

[0193] 1H NMR (500 MHz, DMSO) δ 8.58 (s, 1H), 8.33 (s, 1H), 7.98 (ddd, J = 11.8, 8.3, 1.2 Hz, 4H), 7.83 (dd, J = 8.3, 1.2 Hz, 2H), 7.59 - 7.69 (m, 3H), 7.50 (dt, J = 9.8, 8.1, 8.1 Hz, 4H), 7.39 - 7.43 (m, 2H), 6.76 (d, J = 5.4 Hz, 1H), 6.35 - 6.39 (m, 1H), 6.14 - 6.18 (m, 1H), 4.89 (m, 2H), 4.69 (dd, J = 12.1, 5.0Hz, 1H), 1.28 (s, 12H). 13 C NMR (126 MHz, DMSO) δ 165.4; 129.17, 128.75, 128.72, 128.71, 128.6, 128.2, 119.8, 86.7, 83.6, 79.3, 73.3, 70.8, 63.5, 54.9, HRMS (ESI-TOF) m / z calcd. for C38H35BClN3O9[M+H]+: 724.2233, found: 724.2245.

[0194] tert-chain(3-chain-2-chain)chain11 An oven-dried, 3-necked, 1 L round-bottom flask equipped with a magnetic stir bar was charged with 3-iodo-2-naphthoic acid (24.8 g, 83.2 mmol) dissolved in toluene (320 mL) and triethylamine (14 mL, 100.0 mmol) was added. The flask was fitted with a reflux condenser and an addition funnel. The reaction was heated to reflux and diphenylphosphorazidate (21.6 mL, 100.0 mmol) in toluene (80 mL) was added dropwise to the reaction mixture over a total of 60 minutes. The addition rate was kept at 1 drop every 2-5 seconds and bubbles were observed 5 minutes after addition. The addition funnel was rinsed with additional toluene (20 mL). After stirring at reflux for 15 minutes, bubble formation ceased. After an additional 15 min, the addition funnel was charged with 2-methylpropan-2-ol (40 mL, 416.0 mmol) in toluene (60 mL) and this was carefully (Caution: the intermediate formed is very reactive and must be handled with care) added dropwise to the reaction mixture at reflux. The reaction was stirred at reflux for an additional 3 h. The reaction mixture was allowed to cool to room temperature, transferred to a separatory funnel and the material was washed successively with water (3 x 500 mL), aq.satd.NaHC03 (3 x 250 mL) and brine (1 x 500 mL). The resulting orange solution was dried over MgSO4, filtered and concentrated under reduced pressure to give 5 (20.0 g, 65%) as a beige solid.

[0195] 1 H NMR (500 MHz, DMSO) δ 8.63 (s, 1H), 8.52 (s, 1H), 7.94 (s, 1H), 7.86 (dd, J = 14.9, 8.1 Hz, 2H), 7.46 - 7.54 (m, 2H), 1.48 (s, 9H). 13 C NMR (126 MHz, DMSO) δ 153.4, 138.1, 136.1, 132.7, 132.2, 127.3, 126.8, 126.4, 126.1, 123.5, 96.7, 79.2, 28.1. HRMS (ESI-TOF) m / z calcd. for C15H16INO2 [M + H]+ : 370.0304, found: 370.0295.

[0196] (2R,3R,4R,5R)-2-((benzoyloxy)methyl)-5-(5-(3-((tert-butoxycarbonyl)amino)naphthalen-2-yl)-4 chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate 12 Compound 10 (22 g, 30.4 mmol), tert-butyl (3-iodonaphthalen-2-yl)carbamate 11 (10.7 g, 29.0 mmol), bis(triphenylphosphine)palladium(II) dichloride (1.02 g, 1.45 mmol) and potassium carbonate (10.0 g, 72.4 mmol) were dissolved in DME (300 ml) and the reaction was stirred for 24 h at 80° C. The reaction mixture was concentrated under reduced pressure, absorbed onto Celite and purified by flash-chromatography (KP-Sil 330 g, Hept:EtOAc 90:10 to 40:60) to give 6 (18.2 g, 75%) as a yellow solid.

[0197] 1 H NMR (500 MHz, DMSO) δ 8.64 (s, 1H), 8.40 (s, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 7.99 (d, J = 7.3 Hz, 2H), 7.88 - 7.94 (m, 5H), 7.84 (d, J = 6.6 Hz, 2H), 7.63 - 7.68 (m, 2H), 7.57 (t, J = 7.3, 7.3 Hz, 1H), 7.4 - 7.53 (m, 8H), 6.87 (d, J = 4.7 Hz, 1H), 6.39 (t, J = 5.1, 5.1 Hz, 1H), 6.23 (t, J = 5.9, 5.9 Hz, 1H), 4.91 (q, J = 5.1, 5.0, 5.0 Hz, 1H), 4.82 (dd, J = 12.1, 3.5 Hz, 1H), 4.71 (dd, J = 12.1, 5.0 Hz, 1H), 1.27 (s, 9H). 13C NMR (125 MHz, DMSO) δ 165.4, 164.6, 164.5, 153.2, 151.5, 151.3, 150.5, 135.6, 134.0, 133.9, 133.5, 133.1, 130.6, 129.5, 129.4, 129.3, 129.2, 129.17, 128.9, 128.8, 128.7, 128.6, 128.5, 128.3, 127.4, 127.0, 126.5, 125.5, 125.1, 116.6, 112.9, 86.6, 78.8, 73.8, 31.2, 27.9, 22.1, 13.9. HRMS (ESI-TOF) m / z calcd. for C47H39ClN4O9 [M + H]+ : 839.2484, found: 839.2485.

[0198] (2R,3R,4R,5R)-2-((benzoyloxy)methyl)-5-(5-(3-((tert-butoxycarbonyl)amino)naphthalen-2-yl)-4 chloro-7H-pyrrolo[2,3-d]pyrimidin-7-yl)tetrahydrofuran-3,4-diyl dibenzoate 13 Compound 10 (22 g, 30.4 mmol), tert-butyl (3-iodonaphthalen-2-yl)carbamate 11 (10.7 g, 29.0 mmol), bis(triphenylphosphine)palladium(II) dichloride (1.02 g, 1.45 mmol) and potassium carbonate (10.0 g, 72.4 mmol) were dissolved in DME (300 ml) and the reaction was stirred for 24 h at 80° C. The reaction mixture was concentrated under reduced pressure, absorbed onto Celite and purified by flash chromatography (KP-Sil 330 g, Hept:EtOAc 90:10 to 40:60) to give 6 (18.2 g, 75%) as a yellow solid.

[0199] 1H NMR (500 MHz, DMSO) δ 8.64 (s, 1H), 8.40 (s, 1H), 8.15 (s, 1H), 8.10 (s, 1H), 7.99 (d, J = 7.3Hz, 2H), 7.88 - 7.94 (m, 5H), 7.84 (d, J = 6.6 Hz, 2H), 7.63 - 7.68 (m, 2H), 7.57 (t, J = 7.3, 7.3 Hz, 1H), 7.4- 7.53 (m, 8H), 6.87 (d, J = 4.7 Hz, 1H), 6.39 (t, J = 5.1, 5.1 Hz, 1H), 6.23 (t, J = 5.9, 5.9 Hz, 1H), 4.91 (q, J = 5.1, 5.0, 5.0 Hz, 1H), 4.82 (dd, J = 12.1, 3.5 Hz, 1H), 4.71 (dd, J = 12.1, 5.0 Hz, 1H), 1.27 (s, 9H). 13 C NMR (125 MHz, DMSO) δ 165.4, 164.6, 164.5, 153.2, 151.5, 151.3, 150.5, 135.6, 134.0, 133.9, 133.5, 133.1, 130.6, 129.5, 129.4, 129.3, 129.2, 129.17, 128.9, 128.8, 128.7, 128.6, 128.5, 128.3, 127.4, 127.0, 126.5, 125.5, 125.1, 116.6, 112.9, 86.6, 78.8, 73.8, 31.2, 27.9, 22.1, 13.9. HRMS (ESI-TOF) m / z calcd for C47H39ClN4O9 [M + H]+ : 839.2484, found: 839.2485.

[0200] (2R,3R,4S,5R)-2-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol 14 Compound 6 (7 g, 8.3 mmol) was dissolved in DMF (18 ml) to which 1,4-diazabicyclo[2.2.2]octane (7.5 mL, 16.7 mmol) and 2,3,4,6,7,8,9,10-octahydropyrimido[1,2-a]azepine (2.5 mL, 16.7 mmol) were added. The reaction mixture was stirred at 75° C. for 17 h. The reaction mixture was allowed to cool to RT and then coevaporated with toluene (5×15 mL). The crude product was purified by flash chromatography (KP-Sil 100 g, DCM:MeOH 100:0 to 95:5) to give a mixture of products consisting of Boc- and de-Boc-protected products (7a and 7b, 3.1 g). The obtained material was used in the next step without further purification. A mixture of compounds 7a and 7b (2.85 g) was dissolved in MeCN (32 mL) and sodium methanolate (3.9 ml, 21.3 mmol) was added. The reaction mixture was stirred at RT for 1 h. The reaction mixture was concentrated under reduced pressure, absorbed onto Celite, and purified by flash chromatography (KP-Sil 25 g, DCM:MeOH 100:0 to 90:10) to give 8 (0.55 g, 17% over two steps) as a white solid.

[0201] 1 H NMR (500 MHz, DMSO) δ 10.90 (s, 1H), 8.19 (s, 1H), 8.15 (s, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.1 Hz, 1H), 7.70 (s, 1H), 7.55 (s, 1H), 7.33 - 7.43 (m, 2H), 5.95 (d, J = 6.4 Hz, 1H), 5.40 (d, J = 6.5 Hz, 1H), 5.18 (d, J = 4.5 Hz, 1H), 4.55 (q, J = 6.3, 6.3, 6.3 Hz, 1H), 4.15 (q, J = 4.6, 4.6, 4.6 Hz, 1H),3.97 (q, J = 3.8, 3.8, 3.8 Hz, 1H), 3.68 (d, J = 12.0 Hz, 1H), 3.55 - 3.61 (m, 1H), 2.54 (s, 1H).13 C NMR (126 MHz, DMSO) δ 155.2, 153.6, 147.2, 137.4, 132.6, 129.6, 129.2, 128.5, 127.2, 126.6, 126.3, 124.7, 122.4, 120.3, 113.4, 113.3, 110.4, 106.7, 88.5, 85.6, 74.1, 70.9, 61.9. HRMS (ESI-TOF) m / z calcd. for C21H18N4O4 [M + H]+ : 391.1406, found: 391.1405.

[0202] (2R,3R,4S,5R)-2-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3,4-diol 15 (2R,3R,4S,5R)-2-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol (150 mg, 0.38 mmol) was coevaporated three times with pyridine (25 mL), then pyridine (3 mL) was added and the flask was placed in an ice bath (0 °C). DMTr-Cl (143 mg, 0.42 mmol) was then added once and the reaction mixture was stirred for 5 min before being allowed to warm to RT and stirred for 18 h. Methanol (3 mL) was then added and the reaction mixture was extracted with EtOAc (50 mL) and washed with water (20 mL) and brine (10 mL). The organic layer was dried over MgSO4, concentrated to dryness, absorbed onto Celite and purified by flash chromatography (KP-Sil 25 g, flushed with 2% Et3N in DCM before use, EtOAc in DCM: 0-50%) to give compound 9 (60 mg, 23%) as a light brown solid.

[0203] 1H NMR (500 MHz, DMSO-d6) δ 10.90 (bs, 1H), 8.19 (s, 1H), 7.74 (s, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.66 (d, J = 9.9 Hz, 2H), 7.52 (s, 1H), 7.40 (d, J = 7.5 Hz, 3H), 7.33 - 7.39 (m, 2H), 7.28 (t, J = 8.3 Hz, 6H), 7.20 (t, J = 7.3 Hz, 1H), 6.84 (dd, J = 8.9, 6.9 Hz, 4H), 6.03 (d, J = 4.5 Hz, 1H), 5.57 (bs, 1H), 4.67 (t, J = 4.8 Hz, 1H), 4.39 (t, J = 5.0 Hz, 1H), 4.07 (q, J = 4.4 Hz, 1H), 3.66 (s, 3H), 3.65 (s, 3H), 3.22 (m, 2H). 13 C NMR (126 MHz, DMSO-d6) δ 158.1, 158.0, 155.5, 153.6, 147.6, 144.8, 137.6, 135.7, 135.6, 132.7, 129.8, 129.7, 129.5, 127.9, 127.8, 127.1, 126.7, 126.6, 126.4, 124.7, 122.2, 120.3, 113.3, 113.2, 112.8, 110.7, 106.5, 88.4, 85.6, 82.8, 74.2, 70.6, 63.7, 55.0, 54.9. HRMS (ESI-TOF) m / z calcd. for C42H36N4O6 [M+H]+: 693.2713, found: 693.2729.

[0204] CPG-supported (2R,3R,4S,5R)-2-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)tetrahydrofuran-3,4-diol 16 In a 10 mL syringe with a PTFE filter, succinylated support 3 (400 mg, 82 gmol / g, 0.03 mmol), DMAP (0.008 g, 0.07 mmol), DIC (203 μl, 1.31 mmol) and tetraazacyclopenta[de]tetracen-2(6H)-yl)-5-(bis(4-methoxyphenyl(phenyl)methoxy)methyl)tetrahydrofuran-3.4-diol (0.023 g, 0.03 mmol) and triethylamine (14 μl, 0.10 mmol) were suspended in pyridine (5 mL). The mixture was gently shaken at RT for 18 h. After 18 h, the syringe was purged and the support was washed with pyridine (5 mL), dichloromethane (5 mL) and diethyl ether. A second coupling was then performed under exactly the same conditions as the first to increase the loading on the solid support. Then, with the same syringe, DMAP (0.008 g, 0.07 mmol), DIC (203 μl, 1.31 mmol), triethylamine (14 μl, 0.10 mmol) and 2,3,4,5,6-pentachlorophenol (0.087 g, 0.33 mmol) were added to the support, suspended in pyridine (4 mL). The mixture was gently shaken for 4 h at RT before a solution of piperidine (4 mL, 20% in DMF - for capping unreacted carboxylic acids on the support) was added for 1 min (longer exposure times result in reduced loading, as piperidine cleaves the ester bond with the nucleoside) and quickly washed with DMF (3 × 5 mL), dichloromethane (5 mL) and diethyl ether (5 mL). Finally, the resin was shaken in a CAP A + CAP B mixture (50 / 50 v / v) under argon for 2 h, washed with DMF (5 mL), dichloromethane (5 mL), diethyl ether (5 mL) and dried under argon (final loading: 18 μmol / g - determined by reading the optical density of the DMT solution cleaved from the weighed support - ε = 70000 M -1 .cm -1 (at 498 nm).

[0205] ((2R,3S,4R,5R)-5-(2,3,5,6-tetraazacyclopenta[de]tetracen-2(6H)-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl hydrogen triphosphate (pATP) 17 The reaction was carried out in a 5 mL syringe with a PTFE filter loaded with CPG-linked pA nucleoside (400 mg, 0.007 mmol) under argon atmosphere with shaking. The procedure was as follows: a. Removal of 5'-DMT: The support was washed with DCA deblock until the filtrate was colorless, then washed with ACN (5 x 5 mL). b. Coupling: N,N-diisopropyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 5 (345 mg, 1.36 mmol) was dissolved in 4.8 mL of ACN and partially reacted with the support (three equal couplings with reaction times of 60 s-60 s-90 s each). To each coupling, BTT activator (2.4 mL) was also added. The support was then washed with ACN (3 x 5 mL). c. Oxidation: Pyridine / water / rosin (9 / 1 / 12.7 v / v / w, 5 mL) for 45 seconds, followed by ACN washes (3×5 mL) and drying the support in a stream of argon. d. Triphosphate: Bis(tetrabutylammonium) dihydrogen diphosphate 6 (0.5 M, 5 mL) was injected twice for 15 min and 18 h, respectively, after which the support was washed with DMF (5 mL), water (3×5 ml), ACN (5 ml), and then dried under a stream of argon. e. Cleavage and purification: Cleavage of the triphosphate was carried out with AMA (50 / 50 v / v mixture of 23% aq. NH40H and 40% aq. methylamine, 5 mL) at room temperature for 2 h. After 2 h, the AMA filtrate was purged in the round-bottom flask and the support was rinsed three times with 23% aq. NH40H solution. The mixture was lyophilized, followed by purification by HPLC (Waters Acquity HSS T3 column, 2.1 x 50 mm, 0.4 mL / min, 2-99% 50 mM NH40Ac in 80:20 EtOH) to give pATP (2.7 mg, 60% as determined by UV absorbance) as a pale yellow solid (ammonium salt).

[0206] 31 P NMR (202 MHz, D2O) δ -22.88 (t, J=17.7 Hz, 1P), -11.14 (d, J=19.7 Hz, 1P), -10.21 (bs, 1P). HRMS (ESI-TOF) m / z calcd. for C21H21N4O13P3 [M+H]+: 631.0396, found: 631.0416.

[0207] pATP17 can also be prepared by a slightly modified route in which the coupling step (b above) is carried out with a modified phosphoramidite such as 6-chloro-N,N-diisopropyl-4-methyl-4H-benzo[d][1,3,2]dioxaphosphinine-2-amine 8 (compound (IIIa) above).

[0208] Example 3: Physical Characterization Absorption spectra were recorded on a Cary 4000 spectrometer (Agilent Technologies). Emission spectra were measured on a SPEX Fluorolog 3 spectrofluorometer (Jobin Yvon Horiba). -1Three consecutive emission spectra recorded at a scan rate of 100 Hz were collected and averaged. Spectra of compounds 7 and 17 in DPBS were recorded from 360 to 695 nm with excitation at 355 nm. To avoid bleaching, the monochromator slits were set at 0.8 nm on the excitation side and 2.5 nm on the emission side.

[0209] Molar absorbance spectrum (E unit: M -1 cm -1 ) is expressed as Equation 1:

number

[0210] The fluorescence quantum yield is the quantum yield of quinine sulfate in 0.5 M H2SO4 (φF = 0.546 42 , referred to as the reference), and is calculated using Equation 2:

number

[1988] ).

[0211] The spectral properties of compounds 7 and 17 were measured under physiological conditions to determine the experimental readiness for cellular studies; absorption and emission spectra were recorded in DPBS to mimic the ionic strength and salt composition in mammalian cells. To record emission spectra, the excitation wavelength was set at 355 nm and measurements were performed at room temperature (approximately 22 °C). The molar absorbance (ε) was calculated using the Beer-Lambert law.

[0212] Apart from the absorption of common nucleobases (<300 nm), compounds 7 and 17 absorb in the range of 350-400 nm, with 2CNqATP showing an absorption maximum at 352 nm (Table 2). On the other hand, the pATP absorption band contains two peaks at 370 nm and 386 nm, respectively, the latter being slightly more intense. The emission peaks are centered at 463 nm and 420 nm for 2CNqATP and pATP, respectively. FBA stands for "fluorescent base analogue". Table 1: Spectroscopic parameters of compounds 7 and 17 in DPBS at room temperature. [Table 1]

[0213] Example 4: Cell Culture The Huh-7 human hepatocyte cell line was used. Huh-7 is a well-differentiated human hepatocyte cell line with epithelial-like morphology. 43 Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM GlutaMax with phenol red, Gibco) containing 4.5 g / l glucose, supplemented with 10% fetal bovine serum (FBS, Gibco, country of origin Brazil), 2 mM L-glutamine, and 1 mM sodium pyruvate (hereafter referred to as CCM, complete cell culture medium) at 37 °C under 5% CO2. For subculture, adherent cells were washed twice with DPBS without calcium or magnesium and detached with 0.25% trypsin ethylenediaminetetraacetic acid 1x (trypsin-EDTA, Gibco, with phenol red). For seeding, cells were counted after trypsin neutralization and diluted to the desired cell number (Table 2), and then incubated at 37 °C with 5% CO2 for 24 h before the experiment. Table 2: Reservoirs, cell numbers and working volumes used for seeding Huh-7 cells. [Table 2]

[0214] Example 5: Cytotoxicity To study the cytotoxicity of compounds 7 and 17, Huh-7 cells were seeded in 96-well plates as described above. Prior to treatment, conditioned medium was removed from Huh-7 cells and compounds (stock solutions in DPBS diluted in CCM) were added to the cells for various exposure times. Unexposed cells were treated similarly, with the same volume of DPBS added to the CCM instead of compounds. Two types of cytotoxicity assays (alamarBlue to measure metabolic activity and LDH leakage to measure cell membrane integrity) were performed in parallel with cells and culture medium from each sample, respectively. Treatments were performed in triplicate and experiments were repeated twice.

[0215] After 24 h exposure to compounds 7 or 17, the conditioned medium was removed from the cells and used for LDH leakage assays (see below). Freshly prepared alamarBlue cell viability reagent (Invitrogen, diluted 1:10 in CCM) was added and then incubated at 37°C, 5% CO2 for 3 h. The resulting resorufin fluorescence was read at Ex: 544 nm / Em: 590 nm using an Optima Fluostar plate reader (BMG Labtech). Dimethylsulfoxide 5% and CCM alone (i.e., compounds 7 and 17 0 μM) were used as positive and negative controls for cell death, respectively.

[0216] Lactate dehydrogenase (LDH) leakage assay: To examine released LDH, a CyQUANT LDH Cytotoxicity Assay Kit (Invitrogen) was used. Reaction mix and enzyme controls (LDH, pure enzyme) were prepared according to the manufacturer's instructions. Maximum LDH release was determined by treating cells with a 1:10 dilution of lysis buffer in serum-free medium for 30 min at 37°C, 5% CO2.

[0217] Results: The alamarBlue assay reports the metabolic activity of cells and is therefore informative about cell viability. The LDH leakage assay reports cell membrane integrity and constitutes a complementary readout to cytotoxicity (membrane damage). Figure 3 shows that 2CNqATP and pATP induced up to 20% cell death at 10 μM, the two highest concentrations tested (Figure 3A). Comparison of cell viability at lower doses suggests that 2CNqATP is slightly more toxic than pATP. The level of released LDH was around 10% in all exposure conditions and was comparable to the values ​​obtained in untreated control cells, indicating that cell integrity was preserved. Based on this result, cellular uptake experiments were performed at a concentration of 2.5 μM, which gave a reliable intracellular fluorescent signal and in-cellulo RNA uptake with low impact on cell viability after 24 h exposure.

[0218] As an additional observation regarding viability, there were no morphological changes or other signs of cell death (such as detachment from the vascular surface) during live cell imaging, indicating that the excitation light used to track compounds inside live cells does not harm the cells under the conditions tested.

[0219] Example 6: Monitoring cellular uptake using fluorescence microscopy Screening of cellular uptake of compounds 7 and 17 was carried out as follows.

[0220] Quantification in cell lysates: For dose-response experiments of compound 7 and 17 uptake, whole cell lysates of exposed cells were collected and measured on an Optima Blue Fluostar plate reader (BMG Labtech) with lysis buffer as background. 2CNqATP was excited using a 355 nm (±20 nm) filter and emission was collected with a bandpass filter centered at 460 nm (±12 nm). pATP was excited with a 380 nm (±5 nm) filter and emission was collected with a bandpass filter centered at 410 nm (±5 nm). Lysis buffer fluorescence was subtracted as background and triplicate fluorescence intensities were averaged. Fluorescence intensities were normalized to the highest added compound concentration (10 μM).

[0221] Confocal laser scanning fluorescence microscopy: Images were taken using a Nikon eclipse Ti microscope equipped with a Nikon C2plus scanner, two parallel gallium arsenide phosphide (GaAsP) detectors and an Apo 60x Oil λS DIC N2 objective. A 405 nm laser line was used to excite fluorescent nucleoside triphosphates, and emission was collected at 407–607 nm. Laser power was 2% and detector gain was 4% to optimize signal while avoiding compound photobleaching. For live cell imaging, Huh-7 cells were seeded as described above in glass-bottom four-compartment dishes (CELLview dishes, Greiner Bio-One). During imaging, dishes were kept in a stage-top incubation chamber (OKO lab) maintained at 37 °C and 5% CO2.

[0222] Time-lapse imaging: To image the uptake of compounds 7 and 17 over time, the time-lapse setup of the NIS software was used. One field per compartment of the dish was chosen from which images were captured every 15 min for the first 2 h and then every hour for 18 h (i.e., 20 h in total). The exposure period began with the addition of pre-warmed (37 °C) CCM containing 2.5 μM FBA-TP to the cells. Fresh CCM for control cells contained an equal volume of DPBS instead of compound solution.

[0223] Results: To investigate the cellular uptake of compounds 7 and 17, they were added to Huh-7 cells in CCM and the uptake was monitored as a function of time using confocal fluorescence microscopy (Figure 4). Control cells exposed to CCM only were also imaged with the same microscope settings and recorded as a reference to probe the cellular autofluorescence levels. After 20 h of continuous continuous uptake, clear differences in compound accumulation and localization were observed.

[0224] 2CNqATP (compound 7) is seemingly distributed homogeneously in the cytosol and cell nucleus. Bright spots inside individual nuclei further indicate that it also accumulates in the nucleoli. In contrast, pATP accumulates in the cytosol but not in the cell nucleus. Furthermore, its distribution in the cytoplasm is not homogeneous, but appears to be preferentially localized to intracellular structures close to the nucleus, which may be part of the endoplasmic reticulum or Golgi apparatus.

[0225] No uptake of the compound was observed when Huh-7 cells were kept at 4°C, indicating that the modified triphosphate enters the cells by an energy-dependent mechanism rather than by passive diffusion across the plasma membrane.

[0226] Example 7: Monitoring uptake kinetics by flow cytometry For flow cytometry experiments, cells were seeded in 48-well plates as described above. For uptake kinetics experiments, a solution of 2.5 μM of compound 7 or 17 in CCM was added. Control samples were CCM containing an equal volume of DPBS instead of FBA-TP. All treatments were performed in triplicate. The experiment was set up so that all samples could be collected simultaneously at the end of the experiment. Thus, FBA-TP solution was added at different time points. At the end of the experiment, cells were washed twice with DPBS, after which cells were harvested with the addition of 0.25% trypsin-EDTA. Trypsin was neutralized by adding DPBS with 2% FBS, and samples were transferred to 96 U-bottom well plates for high-throughput reading.

[0227] Measurements were typically performed using a BD LSRFortessa flow cytometer with a BD high-throughput sampler (HTS). The system was interfaced with BD FACSDiva software. Samples were excited with a 405 nm laser and emission was passed through a bandpass filter centered at 450 nm (± 20 nm). Uptake kinetics experiments were repeated twice. For monophosphate compounds, a Luminex CellStream flow cytometer with a high-throughput sampler (HTS) interfaced with CellStream Acquisition software was used. The excitation wavelength was 405 nm and emission was passed through a 456 / 51 nm bandpass filter.

[0228] Results: Cells were exposed to compounds 7 and 17 for set times ranging from 5 min to 4 h. Before flow cytometry analysis, cells were washed and trypsinized to remove potential extracellular compounds 7 and 17. For each time point and treatment condition, the intracellular fluorescence intensity of individual cells was recorded (Figure 5A). Intensity histograms show a continuous increase in intracellular fluorescence for both compounds, although significantly slower for pATP compared to 2CNqATP. Uptake kinetic traces (Figure 5B,C) revealed that 2CNqATP fluorescence already significantly exceeded the initial (t=0) level after 5 min, after which uptake rapidly increased and reached a plateau after 2 h of exposure time (Figure 5B). pATP uptake showed a lag phase during the first 15 min, followed by internalization at a nearly constant rate over the next 3 h (Figure 5C). pATP uptake did not reach saturation within the 4 h experimental time frame. This observation clearly indicates a difference in the uptake kinetics of compounds 7 and 17.

[0229] To investigate the concentration dependence of intracellular FBA-TP uptake, a dose-response experiment was performed at a selected time point (24 h). After the end of the exposure, the cells were washed and lysed. The fluorescence of the compound in the lysate, reflecting the internalized FBA, was measured using a plate reader. For pATP and 2CNqATP, an increase in the fluorescence emission in the lysate was observed with increasing concentrations of the added compound (Figure 6). Compounds 7 and 17 showed quite different uptake characteristics. The cellular uptake of 2CNqATP increased dramatically with small concentration increases, and a maximum value indicating uptake saturation was already reached when 2.5 μM FBA-TP was added to the cells. In contrast, the increase in pATP emission from 0 to 2.5 μM was very small, only 4% of that of 2CNqATP, but increased by about 7-fold when the FBA-TP concentration was doubled. The addition of 10 μM FBA-TP increased the fluorescence by about 25-fold compared to the addition of 2.5 μM of the compound.

[0230] Example 8: Uptake competition between standard nucleoside triphosphates and nucleosides Uptake of 2CNqATP and pATP was compared to natural adenosine triphosphate (ATP) or adenosine (both purchased from Sigma-Aldrich). Readout was based on the fluorescence of the compounds detected in live cells by flow cytometry or in cell lysates using a microplate reader after co-administration with increasing concentrations of natural ATP or adenosine.

[0231] Cells were seeded in 96-well plates. ATP or adenosine treatments were prepared by serial dilution in CCM to the concentrations shown in Figure 7. 2CNqATP or pATP was added at a concentration of 2.5 μM. Cells were incubated at 37°C, 5% CO2 for 4 hours. Cell morphology was checked by light microscopy.

[0232] For flow cytometry, treated cells were washed three times with DPBS, detached with 0.25% trypsin-EDTA, and then neutralized with CCM. Samples were transferred to 96 U-well plates and read on a BD LSRFortessa flow cytometer for high-throughput. Samples were excited with a 405 nm laser and emission was passed through a bandpass filter centered at 450 nm (± 20 nm).

[0233] To read the lysates, cells were lysed in 5x passive lysis buffer (Promega) for 1 h at room temperature (approximately 22°C) with rocking. Fluorescence was recorded with an Optima Blue Fluostar plate reader (BMG Labtech). 2CNqATP was excited at 355 nm (± 20 nm) and emission was detected using a bandpass filter centered at 460 nm (± 12 nm). pATP had an excitation wavelength of 380 nm (± 5 nm) and an emission wavelength of 410 nm (± 5 nm). The mean emission of duplicate samples was averaged and normalized to the fluorescence intensity of cells without added competitor (ATP or adenosine).

[0234] Results: The results are shown in Figure 7. The cell (or lysate) fluorescence of the compounds decreases with increasing (superstoichiometric) addition of ATP or adenosine as competitors, but the responses are different. 2CNqATP competes with both ATP and adenosine, and at lower concentrations shows a stronger competitive effect on ATP. pATP uptake also competes with ATP and adenosine, but its effect is much weaker.

[0235] The results suggest that this compound partially shares an uptake pathway with conventional ATP and adenosine.

[0236] Example 9: Cellular incorporation into RNA RNA extraction: Cells were seeded in 12-well plates as described above. Treatment solutions were prepared by diluting compounds 7 and 17 in CCM to a concentration of 2.5 μM or 5 μM. Culture supernatant was removed from the cells and treatment solutions were added. Control cells received only CCM. Cells were incubated at 37°C, 5% CO2 for 24 h. RNA extraction and purification was performed using the QIAGEN RNeasy Mini Kit, following the manufacturer's protocol. Briefly, cells were washed with DPBS, lysed, and homogenized by passing through a 20-gauge needle (0.9 mm outer diameter) 12 times, and an equal volume of ethanol was added. The resulting solution was transferred to an RNeasy spin column and centrifuged. Binding buffer was then added to the top of the column, followed by centrifugation again. The column-bound RNA was washed five times with wash buffer, with a centrifugation step between each addition. To elute the extracted RNA, 30 μL of the provided RNase-free water was added to the column and centrifuged. This step was repeated twice to increase the yield of extracted RNA.

[0237] Three control samples were prepared to test for possible interference of FBA-TP with the extraction procedure and for non-covalent binding between FBA-TP and RNA. 1) FBA-TP was added to the kit's lysate buffer to a concentration of 2.5 μM and treated similarly starting from the homogenization step. 2) Unexposed cells were seeded and lysed as above, after which FBA-TP was added directly to the lysate to a final concentration of 2.5 μM. 3) RNA previously extracted from unexposed cells was included with the addition of FBA-TP. The concentration and purity of RNA extracted from unexposed cells was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific) and FBA-TP was added to a final concentration of 2.5 μM in solution. All control samples were applied to RNeasy spin columns and treated similarly to the compound-exposed cell samples following the purification protocol described above.

[0238] Absorption and emission readout of cellular RNA: Absorption spectra of extracted and purified RNA samples and controls were recorded from 200 nm to 600 nm using a Cary 4000 Readout (Agilent Technologies). Emission spectra were measured on a SPEX Fluorolog 3 spectrofluorometer (Jobin Yvon Horiba). 2CNqA was excited at 350 nm and pA was excited at 370 nm. The excitation and emission bandpasses were 2 nm and 3 nm, respectively. The emission wavelength of the excitation spectrum was set in nm.

[0239] Results: Compound uptake into cellular endogenous RNA was tested by harvesting RNA from exposed cells using a commercially available RNA extraction and purification kit. Control samples were prepared to exclude interference of the readout with non-uptaken compounds (due to passive interactions with purified RNA).

[0240] After purification, the emission and absorption spectra of the extracted intracellular RNA were recorded (Figure 8). The emission spectra of the compounds were normalized to their corresponding absorption values ​​at 260 nm to correct for variations in RNA concentration. All control samples showed very low emission, indicating little non-covalent interaction of compounds 7 and 17 with RNA or binding to the RNA purification column material. Furthermore, this purification method effectively separates RNA and free triphosphates.

[0241] The emission of RNA samples from cells exposed to pATP was at the same level as the control and showed no clear peak (Fig. 8, right graph). The RNA samples from cells exposed to 2CNqATP showed a prominent emission peak with a maximum at 460 nm when excited at 350 nm (Fig. 8, left graph), demonstrating in-cellulo labeling of RNA.

[0242] The results of incorporating 2CNqATP were verified by analyzing the difference between the spectra of RNA-incorporated 2CNqA and free 2CNqATP (Figure 9). The excitation / absorption spectra (Figure 9A) show that a clear red shift of about 10 nm is observed for extracted 2CNqA-RNA compared to free 2CNqATP (Figure 9A; thick black line vs. dashed gray line), which overlaps well with the absorption of single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) solid-phase synthesis 25-mers containing 2CNqA (Figure 9A; thin black and gray lines).

[0243] The emission spectra of extracted 2CNqA-RNA, free 2CNqATP, and artificial RNA were also compared (Figure 9B). The emission maximum of extracted 2CNqA-RNA (Figure 9B; thick black line) is blue-shifted by approximately 20 nm compared to free 2CNqATP (Figure 9B; dashed gray line). The peaks of synthetic short ssRNA and dsRNA are also blue-shifted, but more so than 2CNqA-RNA extracted from cells (Figure 9B; thin black and thin gray lines, respectively). The emission of 2CNqA-RNA extracted from cells matches most closely with that of ssRNA, indicating that it is likely single-stranded.

[0244] Example 10: Monophosphate Experiments Similar experiments were also performed using compound 7a (2CNqAMP) as the substrate instead of compounds 7 and / or 17 (see Figures 10-13), demonstrating that the equivalent monophosphate compounds are also non-toxic and exhibit similar properties to their triphosphate counterparts with regard to spontaneous cellular uptake and RNA uptake.

[0245] conclusion Compounds of formula (I) disclosed herein have been shown to spontaneously internalize into living cells, resulting in in-cellulo incorporation into endogenous RNA. In cytotoxicity studies using the alamarBlue assay, compounds of formula (I) show approximately a 20% reduction in cellular metabolic activity at the highest concentration measured (10 μM), while the LDH assay showed no disruption of cell membrane integrity compared to untreated cells. This allows easy imaging of intracellular locations with a confocal fluorescence microscope, indicating that the compounds at the concentration required to achieve in-cellulo RNA incorporation (2.5 μM) are suitable for experiments in a cellular context.

[0246] Both compounds 7 and 17 produced strong intracellular fluorescence after 20 h exposure in Huh-7 cells. No cellular uptake was observed at 40 °C, indicating that uptake into cells proceeds by an active process, such as involving protein transporters, endocytosis, or similar energy-dependent mechanisms, rather than simple passive diffusion through the phospholipid bilayer membrane. Compound uptake is partially competed by natural ATP / adenosine. Confocal microscopy images (Figure 4) show that compounds 7 and 17 are distributed uniformly throughout the cytosol, making uptake unlikely to be vesicle-mediated. The results also indicate that chemical modification of FBA enhances FBA uptake compared to standard nucleotides.

[0247] pATP and 2CNqATP accumulate in different locations within the cell. Whereas pATP accumulates preferentially in the cytosol (with prominent localization in perinuclear subcellular structures), 2CNqATP is seemingly distributed equally between the cytosol and the nucleus. Thus, in addition to crossing the cell membrane by an active mechanism, 2CNqATP is effectively retained in the nucleus, with a particular concentration in the nucleolus. Since the nucleolus is the site of ribosome biogenesis (transcription, formation and maturation of ribosomal RNA; see Hadjiolov, AA "The Nucleolus and Ribosome Biogenesis" vol. 12 (Springer Vienna, 1985)), it was suggested that 2CNqATP, if spontaneously incorporated into RNA synthesized in the cell, could be used to fluorescently label ribosomal RNA.

[0248] Similar results were obtained when monophosphate compounds such as 7a (2CNqAMP) were used rather than the triphosphate analogs.

[0249] The results summarized above show that compounds of formula (I) are unexpectedly taken up spontaneously (without the need for formulations or transfection reagents) into suitable cells. Their uptake and processing properties depend critically on the structural modifications of the base portion of the molecule, with the compounds differing in their behavior in subcellular localization, uptake kinetics, dose response, and competition with the corresponding canonical nucleosides. As it is possible to isolate RNA from cells treated with labels derived from compounds of formula (I), this technique offers a unique opportunity for novel fluorescent labeling of endogenous RNA in cells.

Claims

1. Formula (I): 【Chemistry 1】 [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form together with the atoms to which they are attached a 6-membered carboaromatic ring, and R 3 is -P(O)(OH) 2 , -P(O)(OH)-OP(O)(OH) 2 and -P(O)(OH)-OP(O)(OH)-OP(O)(OH) 2 Selected from or a physiologically cleavable precursor or a salt thereof.

2. R 1 is hydro and R 2 2. The compound of formula (I) according to claim 1, or a physiologically cleavable precursor or salt thereof, wherein is cyano.

3. R 1 and R 2 A compound of formula (I) or a physiologically cleavable precursor or salt thereof according to claim 1, wherein, together with the atom to which they are attached, form a 6-membered carboaromatic ring.

4. R 3 -P(O)(OH) 2 and -P(O)(OH)-OP(O)(OH)-OP(O)(OH) 2 2. A compound of formula (I) according to claim 1, a physiologically cleavable precursor or a salt thereof, selected from:

5. R 3 is -P(O)(OH)-OP(O)(OH)-OP(O)(OH) 2 5. A compound of formula (I) according to claim 4, a physiologically cleavable precursor or a salt thereof,

6. A compound of formula (I) according to claim 1.

7. 2. The compound of formula (I) according to claim 1, which is a sodium salt, a potassium salt or an ammonium salt.

8. 8. The compound of formula (I) according to claim 7, which is a monosodium salt, a disodium salt, a trisodium salt, a monoammonium salt, a diammonium salt or a triammonium salt.

9. A method for preparing a compound of formula (I) according to claim 1, or a physiologically cleavable precursor or salt thereof, comprising the steps of: i. Formula (V): 【Chemistry 2】 [In the formula, R 1 is selected from hydro, and R 2 is selected from cyano; or R 1 and R 2 form together with the atoms to which they are attached a 6-membered carboaromatic ring, and PG 1 is a suitable protecting group. or a salt thereof; ii. immobilizing the compound of formula (V) or a salt thereof by binding one of its secondary alcohol groups to a suitable support; iii. Protect the free secondary alcohol group by a suitable protecting group, PG 2 Capping with; iv. Protecting group PG 1 Removal of; v. Reconstituting the exposed primary alcohol groups with a compound of formula (VI): 【Chemistry 3】 [In the formula, R 4 is a hydro group and C 1-3 alkyl groups] with a compound of formula (I); vi. oxidizing the resulting phosphorus(III) compound to a phosphorus(V) compound; And, optionally, vii. reacting the resulting phosphorus(V) compound with tetraalkylammonium pyrophosphate to produce the triphosphate; viii. Protecting group PG 2 Removal of; ix. cleaving the resulting phosphate from the support to produce a compound of formula (I) or a salt thereof; and, optionally, x. forming the free acid, physiologically cleavable precursors or various salts of the compound of formula (I); The method includes:

10. R 4 A method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 9, wherein is a methyl group.

11. 10. A method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 9, wherein the support is a solid polymer.

12. A method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 11, wherein the support is selected from controlled pore glass and polystyrene.

13. A method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 12, wherein the support is controlled pore glass.

14. PG 1 A process for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 9, wherein is selected from trityl, dimethoxytrityl and trimethoxytrityl.

15. PG 2 10. A process for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 9, wherein is selected from acetyl, benzoyl, 2,2,2-trichloroethylcarbonyl, paramethoxybenzyl, methyl, tetrahydropyranyl, triethylsilyl, triisopropylsilyl, trimethylsilyl, tert-butyldimethylsilyl and methoxyethyl.

16. PG 1 is dimethoxytrityl, and PG 2 10. A method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 9, wherein is acetyl.

17. 10. A method for preparing a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 9, wherein the tetraalkylammonium pyrophosphate is tetrabutylammonium pyrophosphate.

18. A composition for preparing a labeled RNA molecule, comprising a compound of formula (I) according to claim 9, a physiologically cleavable precursor or salt thereof, and natural ribonucleotides.

19. 10. Use of a compound of formula (I) according to claim 9, or a physiologically cleavable precursor or a salt thereof, for preparing a labeled RNA molecule.

20. R 1 is selected from hydro; R 2 20. The use of a compound of formula (I), its physiologically cleavable precursor or salt thereof, according to claim 19, wherein is selected from cyano and said RNA molecule is labeled with 2CNqA.

21. R 1 and R 2 together with the atom to which they are attached form a 6-membered carboaromatic ring, and the RNA molecule is labeled with pA. Use of a compound of formula (I), or a physiologically cleavable precursor or salt thereof, as claimed in claim 19.

22. 20. The use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to claim 19, wherein the RNA molecule is selected from mRNA and ribosomal RNA.

23. Use of a compound of formula (I), a physiologically cleavable precursor or a salt thereof, according to any one of claims 19 to 22, wherein the RNA molecule is prepared in cellulo.

24. 1. A method for preparing a labeled RNA molecule in vitro, comprising: i. providing a DNA template to a composition comprising a compound of formula (I) according to any one of claims 1 to 8, its physiologically cleavable precursor or salt and natural ribonucleotides; ii. treating the resulting mixture with RNA polymerase; Optionally, iii. monitoring the labeled RNA molecules using a microscope; and Optionally, iv. isolating the labeled RNA molecules; A method comprising:

25. 1. A method for preparing labeled RNA molecules in cellulo, comprising: i. providing a compound of formula (I) according to any one of claims 1 to 8, a physiologically cleavable precursor or a salt thereof to a eukaryotic or prokaryotic cell; ii. causing the organism to spontaneously internalize the compound; iii. causing the organism to prepare labeled RNA molecules; optionally, iv. monitoring the labeled RNA molecules using a microscope; and, optionally, v. isolating the labeled RNA molecules; A method comprising:

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