Compounds for labeling nucleic acids and uses thereof

Abstract

There is provided a therapeutic diagnostic compound having the formula: (Ia), (Ib) or (Ic), or a salt thereof. # imgabs0 #

Description

Technical Field

[0001] The present disclosure relates generally to theranostic agents and compounds, ie, compounds for labeling DNA and their therapeutic uses. Background Art

[0002] Metabolic labeling of nucleic acids is a powerful technique to decipher the timing and location of DNA synthesis in vivo. The technique has become a cornerstone of cell cycle analysis but has also been expanded for viral detection and elucidation of drug resistance mechanisms. Typically, the nucleoside of interest is functionalized with a synthetic handle that is small enough to retain the original biological function but also allows for chemoselective detection and visualization of the incorporated nucleoside.

[0003] Since conventional immunostaining methods for 5-bromo-2'-deoxyuridine (BrdU) suffer from poor antibody signal intensity and tissue penetration, biorthogonal chemical approaches have been developed. Notably, these involve labeling azide- or ethynyl-modified nucleosides via copper-catalyzed (CuAAC) or strain-promoted azide-alkyne cycloaddition (SPAAC), or alternatively reacting vinyl-nucleosides with tetrazines via the inverse electron-demand Diels-Alder reaction (iEDDA).

[0004] However, several nucleoside analogs such as the widely used 5-ethynyl-2'-deoxyuridine (EdU) may seriously affect nucleic acid metabolism, cell cycle, or worst, be toxic to cells and organisms. In addition, most methods for nucleic acid metabolic labeling still require fixation of biological samples or denaturation of DNA, which hinders many long-term in vivo applications. Therefore, there is a need for improved compounds and methods for nucleic acid metabolic labeling. Summary of the invention

[0005] In one aspect, a compound is provided having the formula:

[0006]

[0007]

[0008] or its salt; wherein: R1 is H, haloalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclic or an optionally substituted group thereof; R2, R 2a , R 2band R3 are independently H, hydroxyl, hydrogen, haloalkyl, cyano, amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, sulfenyl, acyl, sulfinyl, sulfonyl, O-carboxyl, ester, thiocarbonyl, or an optionally substituted group thereof; R4 is an optional group and is H, haloalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, or an optionally substituted group thereof; and Y is an optional linker.

[0009] In one aspect, a method for detecting a nucleic acid containing vinyl-nucleoside is provided, the method comprising: a) contacting a cell or tissue with a compound or salt as described herein; and exposing the cell or tissue of a) to light.

[0010] In another aspect, a kit for detecting nucleic acids in cells or tissues is provided, the kit comprising one or more vinyl-nucleosides; and a compound or salt as described herein.

[0011] In another aspect, provided is a method of treating cancer in a subject who has been administered one or more vinyl-nucleosides, the method comprising administering to the subject a compound or salt as described herein.

[0012] In another aspect, provided herein is a pharmaceutical composition comprising a compound or salt as described herein and an acceptable pharmaceutical carrier.

[0013] In another aspect, there is provided a use of a compound or salt as described herein for preparing a medicament for treating cancer. In yet another aspect, there is provided a use of a compound or salt as described herein or a pharmaceutical composition as described herein for treating cancer.

[0014] In another aspect, there is provided a use of a compound or salt as described herein or a pharmaceutical composition as described herein in combination with one or more vinyl-nucleosides for treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Embodiments of devices, apparatus, methods, and kits are described throughout with reference to the accompanying drawings.

[0016] Figure 1 A scheme for generating vinyl-modified cellular DNA by adding VdU to living cells or animals is shown.

[30] PINK is then added to the cells and it "scans" the DNA for olefin groups through a reversible interaction.

[0017] Figure 2 The PINKπ model stacked on the VdU-dA base pair is shown.

[0018] Figure 3 The photophysical and DNA binding properties of PINK are shown. a) Absorbance change of 10 μM PINK solution in 50 mM NaOAc buffer pH 5.2 (0.2% DMSO) with increasing amount of calf thymus DNA (0–160 μM base pairs; light orange to dark orange). b) Viscosity change of 300 μM calf thymus DNA solution in 50 mM NaOAc buffer pH 5.2 (2.4% DMSO) with increasing amount of PINK (0–120 μM). c) Fluorescence increase of 25 μM PINK solution containing 5 mM VdU in MeCN9:1DMSO over 46 h (excitation: 480 nm). d) Absorbance and fluorescence spectra of 50 μM solutions of PINK-VdU-ox(6) in various solvents and phosphate buffered saline (PBS) (excitation: 500 nm).

[0019] Figure 4 A) The absorbance of a 10 μM PINK solution at 575 nm changes with increasing amounts of CT-DNA (0–160 μM base pairs) in 50 mM NaOAc buffer pH 5.2 (0.2% DMSO), yielding the effective concentration (EC) required to bind 50% of PINK. 50 ) = 50 ± 5.1 μM base pairs. B) Fluorescence change of 20 μM PINK in the presence of 1 mM bpCT-DNA (50 equiv.) followed by addition of 5-norbornene-2-methanol (2 mM, 100 equiv.) in PBS pH 7.4 (10% DMSO) (excitation: 500 nm, emission: 590 nm; 515 nm cutoff filter).

[0020] Figure 5 Shown is a kinetic analysis of the reaction of PINK (100 μM) with VdU (0-5 mM, 0-50 equiv). A) Increase in fluorescence versus time after mixing PINK with VdU. The control sample (grey) does not contain VdU. B) Normalized fluorescence increase. C) Inset of observed rate k' versus PINK concentration. The slope yields the rate constant k.

[0021] Figure 6 Photophysical characterization of PINK is shown. 20 μM PINK in NaOAc buffer pH 5.2 (red) or MeCN (blue); 1% DMSO. A) Total absorbance spectrum. B) Absorbance (solid line) and emission (dashed line; excitation 488 nm, 495 nm cutoff filter).

[0022] Figure 7The photophysical properties of PINK-VdU-ox (50 μM, 0.5% DMSO) in various solvents are shown. A) Absorbance spectra. B) Emission spectra (excitation at 500 nm; 515 nm cutoff filter). [a] PBS pH 7.4 containing 40% PEG200.

[0023] Figure 8 Kinetic measurements of PINK with vinyl-modified ODN are shown. A) Relative fluorescence units (RFU) versus time after reaction of PINK with vinyl-modified ODN. The control sample (dashed line) contained no DNA. B) Normalized fluorescence increase. C) Small plot of observed rate k' versus PINK concentration. The slope yields the rate constant k.

[0024] Fig. 9 DNA metabolic labeling and imaging of live HeLa cells are shown. Control (ctrl) samples were treated with PINK but not VdU. Scale bar represents 40 μm.

[0025] Fig.10 PINK staining kinetics are shown. Scale bar represents 40 μm.

[0026] Fig.11 Live cell metabolic labeling of VdU in U2OS cells is shown.

[0027] Fig.12 Long-term live cell labeling of HeLa cells is shown. The scale bar represents 40 μM.

[0028] Fig.13 Metabolic RNA markers are shown. Scale bars represent 100 μm (a) or 40 μm (b), respectively.

[0029] Fig.14 Shown are a) Pulse-chase labeling with PINK and 5-ethynyl-2'-deoxyuridine in HeLa cells. "No VdU" cells were incubated with 1 μM PINK but not with VdU. The scale bar represents 100 μm. b) Live cell time-lapse imaging of a complete mitotic cycle after labeling with 100 μM VdU. The scale bar represents 20 μm.

[0030] Fig.15 Shown are the absorbance (dashed line) and fluorescence emission (solid line) spectra of AO-495-tet-py before (black) and after (red) reaction with 5-norbornene-2-methanol (in PBS).

[0031] Fig.16Toxicity assessment of PINK (0-12.5 μM; 0.1% DMSO) in HeLa and U2OS cells after 24 and 72 hours is shown. Cellular respiratory activity was assessed by measuring fluorescence emission after addition of resazurin (80 μM).

[0032] Fig.17 Combination toxicity of VdU (0-100 μM) and PINK (0, 5, 10 μM) is shown. Experiments were performed in triplicate; error bars depict standard deviation.

[0033] Fig.18 Combination toxicity of VdU (0-10, 20 μM) and PINK (0-100 μM) is shown. Experiments were performed in triplicate; error bars depict standard deviation.

[0034] Fig.19 Cell viability is shown, based on the metabolic activity of MV4-11 and Jurkat cells resulting in a reduction of resazurin after incubation with different concentrations of VdU, VdA and PINK for 24 and 72 hours. Values ​​are normalized relative to untreated (DMSO+medium) cells. Three technical replicates were performed, and each value is given as mean ± SD.

[0035] Fig. 20 Cell viability is shown, based on the metabolic activity of MV4-11, Jurkat cells resulting in a reduction in resazurin after 48 hours of incubation with different concentrations of VdU / VdA alone and in combination with 1 μM PINK (added 24 hours after nucleoside addition). Values ​​are normalized relative to untreated (DMSO+medium) cells. Three technical replicates were performed, and each value is given as mean ± SD.

[0036] Fig.21 Cell viability is shown, based on the metabolic activity of MV4-11, Jurkat cells resulting in a reduction of resazurin after incubation for 48 hours with different concentrations of VdU / VdA alone and in combination with 1 μM PINK (added on the same day as nucleosides). Values ​​are normalized relative to untreated (DMSO+medium) cells. Three technical replicates were performed, and each value is given as mean ± SD.

[0037] Fig.22A-F shows the relative cell viability and cell concentration of Jurkat, MV4-11 and MOLM-13 treated with VdU, VdA and PINK alone and in combination. Two technical replicates were performed, and each value is given as mean ± SD. A) Jurkat's relative viability plot. B) Jurkat's concentration plot. C) MV4-11's relative viability plot. D) MV4-11's concentration plot. E) MOLM-13's relative viability plot. F) MOLM-13's concentration plot.

[0038] Fig.23A -C shows the Fa-CI graphs of MV4-11, Jurkat and MOLM-13 cells treated with different concentrations of VdU and PINK alone and in combination with a constant 5:1 VdU to PINK ratio. Fa = affected fraction, and CI = combination index. A) Fa-CI graph of MV4-11, where the average CI = 0.157 for 25-35% killed cells. B) Fa-CI graph of Jurkat, where the average CI = 0.486 for 50% killed cells. C) Fa-CI graph of MOLM-13, where the average CI = 0.684 for 50% killed cells.

[0039] Fig.24 The combined effects of 5-VdU and PINK on cell survival and DNA damage induction are shown. (A) Treatment schematic. (B) Confluence growth assay of H1299 cells and (C) mouse KPC cells. Each value is given as mean ± SD. Western blot analysis of DNA damage and cell death markers (D) H1299 cells and (E) mouse KPC cells.

[0040] Fig.25 Shown is FACS analysis of H1299 cells treated with 5-VdU / PINK.

[0041] Fig.26 Shown is the induction of apoptosis by the 5-VdU / PINK combination. Synergistic lethality of 5-VdU and PINK in H1299 cells. (A) Timeline. (B) Percentage of H1299 cells undergoing apoptosis. Each value is given as mean ± SD. (C) Western blot analysis of H1299 treated with 5-VdU and 5 μM PINK at different time points.

[0042] Fig. 27 Shown is the incorporation of 5-VdU in rapidly proliferating cells in vivo. (A) Bone marrow cells, (B) small intestine, and (C) spleen tissue.

[0043] Fig.285-VdU / PINK click chemistry reaction in mouse xenografts is shown. (A) DMSO-injected mice, (B) 5-VdU-injected mice.

[0044] Fig.29 The 5-VdU / PINK combination effect is shown to be independent of the p53 mutation status of the cells. Each value is given as mean ± SD. (A) Treatment schematic, (B) HCT116 p53+ / + cells, (C) isogenic HCT116 p53- / - cell line, (D) Western blot analysis.

[0045] Fig.30 Apotracker analysis of apoptotic cells in H1299 cells at 72 hours is shown. TM Green assay image.

[0046] Fig.31 Schematic diagram of in vivo treatments is shown. (A) Experimental group size n=6, and Figure 7 , experimental group size n=3 (B).

[0047] Fig.32 Shown is the incorporation of 5-VdU in mouse xenograft cells revealed by incubation of tissue sections with PINK. Mice injected with 5-VdU (B) and control mice injected with DMSO (A). DETAILED DESCRIPTION

[0048] Metabolic labeling of nucleic acids without affecting nucleic acid metabolism, cell cycle or toxicity to cells and organisms is challenging. For example, chemical modification of nucleic acids in living cells can be spatially hindered by the close stacking of stacks of bioorthogonal functional groups in chromatin. This article provides a dual enhancement strategy for nucleic acid templated reactions, which utilizes fluorescent intercalators that can react with DNA or RNA containing one or more types of vinyl-nucleosides by inverse electron demand Diels-Alder (IEDDA) reactions. Intercalators with tetrazine functionalized structures are based on acridine orange. The embedding of the agent causes close proximity between the tetrazine moiety and the terminal olefin group of the vinyl-nucleoside. In addition, tetrazine quenches fluorescence by photoinduced electron transfer (PET), making the reaction highly fluorescent.

[0049] definition

[0050] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. In the case where there are multiple definitions of terms used herein, those definitions in this section prevail unless otherwise stated.

[0051] The term "C x” followed by the name of the functional group (e.g. “C 1-6 The term "C6 alkyl" refers to a functional group comprising x carbon atoms (e.g., the term "C6 alkyl" refers to an alkyl group comprising 6 carbon atoms, such as hexyl or 2,3-dimethylbutyl). x-y ” followed by the name of the functional group (e.g. “C 1-6 "alkyl") refers to a functional group (e.g., a "C 1-6 The term "alkyl" refers to an alkyl group comprising 1, 2, 3, 4, 5 or 6 carbon atoms. x " or "C x-y ” followed by a heterofunctional group designation (e.g., “heterocyclyl”) refers to a functional group that includes x or x to y atoms (including heteroatoms), respectively (e.g., the term “C6 heterocyclyl” refers to a ring system that includes 6 ring-forming atoms, wherein the ring-forming atoms include both ring-forming carbon atoms and heteroatoms). If “x” or “y” is not specified for a functional group, the broadest scope described in these definitions is assumed.

[0052] It should be understood that in any compound with one or more chiral centers described herein, if absolute stereochemistry is not clearly indicated, each center can independently have the R configuration or the S configuration or a mixture thereof. Therefore, the compounds provided herein can be enantiomerically pure, enantiomerically enriched, racemic mixtures, diastereomerically pure, diastereomerically enriched or stereoisomeric mixtures. In addition, it should be understood that in any compound with one or more double bonds that generate geometric isomers that can be defined as E or Z described herein, each double bond can independently be E or Z.

[0053] It should be understood that the compounds described herein can be isotopically labeled. Being replaced by isotopes such as deuterium may cause higher metabolic stability, thereby producing certain therapeutic advantages, such as increased half-life in vivo or reduced dosage requirements. Each chemical element represented in the compound structure can include any isotope of the element. For example, in the compound structure, a hydrogen atom can be explicitly disclosed or understood to be present in the compound. Any position where a hydrogen atom may be present in the compound, the hydrogen atom can be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, the compounds mentioned herein encompass all potential isotopic forms, unless the context clearly states otherwise.

[0054] Likewise, it should be understood that in any compound described, all tautomeric forms are also intended to be included.

[0055] As used herein, any "R" group such as but not limited to R1 R2, R 2a , R 2band R3 R4 represent substituents that may be attached to the designated atoms. The R groups may be optionally substituted.

[0056] Whenever a group is described as being "optionally substituted" or "a group optionally substituted therewith", the group may be unsubstituted or substituted with one or more substituents. Similarly, when a group is described as "unsubstituted or substituted (if substituted)", the substituent may be selected from one or more indicated substituents in the indicated substituents. If no substituent is indicated, unless otherwise indicated, it means that the indicated "optionally substituted" or "substituted" group may be substituted with one or more separate and independently selected substituents. Non-limiting examples of possible substituents include alkyl, halogen, alkenyl, alkynyl, alkylene, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclyl, hydroxyl, halide, cyano, amino, thiol, alkoxy, acyl, sulfinyl, sulfenyl, sulfonyl, O-carboxyl, C-carboxyl, thiocarbonyl, pyridyl, pyrimidyl, phenyl, as defined herein.

[0057] As used herein, "alkyl" by itself or as part of another group or substituent refers to a straight or branched hydrocarbon chain including a fully saturated (no double or triple bonds) hydrocarbon group. Typical alkyl groups include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Unless otherwise indicated, alkyl groups may be optionally substituted.

[0058] As used herein, "alkenyl" by itself or as part of another group or substituent refers to a straight or branched hydrocarbon chain including one or more double bonds. Unless otherwise specified, an alkenyl group may be optionally substituted.

[0059] As used herein, "alkynyl" by itself or as part of another group or substituent refers to a straight or branched hydrocarbon chain including one or more triple bonds. Unless otherwise specified, an alkynyl group may be optionally substituted.

[0060] As used herein, "alkylene" by itself or as part of another group or substituent refers to a straight or branched hydrocarbon chain including a fully saturated (no double or triple bonds) hydrocarbon group except that the carbon at the point of attachment forms a double bond with a base molecule. Unless otherwise specified, an alkylene group may be optionally substituted.

[0061] As used herein, "aryl" by itself or as part of another group or substituent refers to a carbocyclic (all-carbon) monocyclic or polycyclic aromatic ring system (a fused ring system comprising two carbon rings sharing a chemical bond) having a completely delocalized π electron system in all rings. The ring-forming carbon atoms of the monocyclic ring may not be replaced by ring-forming heteroatoms. The ring-forming carbon atoms of the fused ring system may be replaced by ring-forming heteroatoms selected from, for example, N, O and S; however, if the fused ring system contains any ring-forming heteroatoms, the ring-forming heteroatoms are not contained in the ring containing the ring-forming carbon atoms, which serve as the connection point to the base molecule. The fused polycyclic ring system comprises a fused ring system comprising an aromatic ring fused to: (i) one or more aryl rings; ii) one or more cycloalkyl rings; (iii) one or more heterocycloalkyl rings; (iv) one or more heteroaryl rings; or (v) any combination or subcombination of (i), (ii), (iii) and (iv). The connection point to the base molecule on the aryl is a ring-forming carbon atom. For greater clarity, where the aryl group is a fused ring system, the point of attachment to the base molecule on the fused ring system is a ring-forming carbon atom of the aromatic ring of the fused ring system, wherein the aromatic ring does not contain any ring-forming heteroatoms. Typical examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, indenyl, and tetrahydronaphthyl. Unless otherwise indicated, aryl groups may be optionally substituted.

[0062] As used herein, "haloalkyl" itself or as a part of another group or substituent refers to a subset of alkyl, wherein one or more hydrogen atoms in the hydrogen atoms in the alkyl are replaced by halogen (for example, monohaloalkyl, dihaloalkyl and trihaloalkyl). Such groups include but are not limited to chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl and 2-fluoroisobutyl. Unless otherwise indicated, haloalkyl can be optionally substituted. Similarly, "haloalkenyl" itself or as a part of another group or substituent refers to a subset of alkenyl, wherein one or two hydrogen atoms in the hydrogen atoms in the alkenyl are replaced by halogen (for example, monohaloalkenyl, dihaloalkenyl). Similarly, "haloalkynyl" itself or as a part of another group or substituent refers to a subset of alkynyl, wherein the hydrogen atoms in the alkynyl are replaced by halogen.

[0063] As used herein, "cycloalkyl" itself or as part of another group or substituent refers to a fully saturated (no double or triple bond) monocyclic or polycyclic hydrocarbon ring system. When composed of two or more rings, the rings can be connected together in a spiral, bridged or fused manner, wherein at least one ring of the ring system is a hydrocarbon ring, wherein all unfused ring-forming carbon atoms are saturated, and contain ring-forming carbon atoms, which serve as a connection point with an alkali molecule. The ring-forming carbon atoms of the monocyclic ring may not be replaced by ring-forming heteroatoms. The ring-forming carbon atoms of the polycyclic system can be replaced by ring-forming heteroatoms selected from, for example, N, O and S; however, if the ring system contains any ring-forming heteroatoms, the ring-forming heteroatoms are not included in the ring containing the ring-forming carbon atoms, which serve as a connection point with an alkali molecule. The fused bicyclic or polycyclic ring system comprises a fused ring system comprising a non-aromatic cycloalkyl ring fused to: (i) one or more cycloalkyl rings; (ii) one or more aryl rings; (iii) one or more heterocycloalkyl rings; (iv) one or more heteroaryl rings; or (v) any combination or subcombination of (i), (ii), (iii) and (iv). The point of attachment to the base molecule on the cycloalkyl is a ring-forming carbon atom. For greater clarity, in the case where the cycloalkyl is a ring system, the point of attachment to the base molecule on the ring system is a ring-forming carbon atom of the non-aromatic cycloalkyl ring of the fused ring system, wherein the non-aromatic cycloalkyl ring does not contain any ring-forming heteroatoms. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl and cyclodecyl. Unless otherwise indicated, the cycloalkyl group may be optionally substituted.

[0064] As used herein, "heteroalkyl" by itself or as part of another group or substituent refers to an alkyl group containing one or more heteroatoms, ie, elements other than carbon, including but not limited to N, O, or S.

[0065] As used herein, "heteroaryl" by itself or as part of another group or substituent refers to a monocyclic or polycyclic aromatic ring system containing one or more heteroatoms (at least one ring in the system contains a fully delocalized π electron system), i.e., an element other than carbon, including but not limited to N, O or S. In addition, any nitrogen in a heterocyclyl group can be quaternized. The term "heteroaryl" includes fused ring systems in which two rings (such as at least one aryl ring and at least one heteroaryl ring or at least two heteroaryl rings) share at least one chemical bond. Fused bicyclic or polycyclic ring systems include fused ring systems that include a heteroaromatic ring fused to: (i) one or more heteroaryl rings; (ii) one or more aryl rings; (iii) one or more cycloalkyl rings; (iv) one or more heterocyclyl rings; or (v) any combination or subcombination of (i), (ii), (iii), (iv) and (v). The point of attachment to the base molecule on the heteroaryl group is a ring-forming atom. For the sake of clarity, when heteroaryl is a fused ring system, the connection point with the base molecule on the fused ring system is the ring-forming atom of the heteroaromatic ring of the fused ring system. The limiting examples of heteroaryl include pyrrolyl, furyl, furazolyl, thienyl, pyrazolyl, benzopyrazolyl, imidazolyl, benzisoxazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, triazolyl. Unless otherwise indicated, heteroaryl can be optionally substituted.

[0066] As used herein, "heterocyclic group" refers to a monocyclic or polycyclic ring system, wherein carbon atoms and heteroatoms together constitute the ring system. However, the heterocyclic ring may optionally contain one or more unsaturated bonds positioned in such a way that there is no complete delocalized π electron system in all rings. Heteroatoms are elements other than carbon, including but not limited to O, S and N. The heterocyclic ring may further contain one or more carbonyl functional groups or thiocarbonyl functional groups, so that the definition includes oxo systems and thio systems, such as lactams, lactones, cyclic imides, cyclic thioimides and cyclic carbamates. When composed of two or more rings, the rings may be joined together in a fused manner. In addition, any nitrogen in the heterocyclic group may be quaternized. Fused polycyclic ring systems include fused ring systems comprising a heterocyclyl ring fused to: (i) one or more heteroaryl rings; (ii) one or more aryl rings; (iii) one or more cycloalkyl rings; (iv) one or more heterocyclyl rings; or (v) any combination or subcombination of (i), (ii), (iii), (iv), and (v). The point of attachment to the base molecule on the heterocyclyl is a ring-forming atom. For greater clarity, where the heterocyclyl is a fused ring system, the point of attachment to the base molecule on the fused ring system is a ring-forming atom of the heterocyclyl ring of the fused ring system. Examples of such "heterocyclic groups" include, but are not limited to, dioxanyl, dioxolanyl, oxathiacyclohexane, succinimidyl, dioxopiperazinyl, hydantoinyl, trioxanyl, imidazolinyl, imidazolidinyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinone, thiazolinyl, thiazolidinyl, morpholinyl, oxirane, piperidinyl, piperazinyl, pyrrolidinyl, pyrrolidinyl, pyrrolidinone, 4-piperidone, pyrazolinyl, pyrazolidinyl. Unless otherwise indicated, heterocyclic groups may be optionally substituted.

[0067] As used herein, the term "hydroxyl" by itself or as part of another group refers to an alcohol functional group. An alcohol functional group can be depicted as "-OH."

[0068] As used herein, the term "halogen atom" or "halogen" by itself or as part of another group refers to any of the radiostable atoms in the 7th column of the periodic table, such as fluorine, chlorine, bromine and iodine. Fluorine can also be depicted as "F", "-F" or "fluoro". Chlorine can also be depicted as "Cl", "-Cl" or "chloro". Bromine can also be depicted as "Br", "-Br" or "bromo". Iodine can also be depicted as "I", "-I" or "iodo".

[0069] As used herein, the term "cyano," by itself or as part of another group, refers to a nitrile functional group. A nitrile functional group can be depicted as "-CN."

[0070] As used herein, the term "amino" by itself or as part of another group refers to an amine functional group. The amine functional group can be depicted as "-NR2", where the R groups can be independently and individually selected from, for example, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclyl, as defined herein. Unless otherwise indicated, the amino group can be optionally substituted.

[0071] As used herein, the term "thiol" by itself or as part of another group refers to a thiol functional group. A thiol functional group can be depicted as "-SH."

[0072] As used herein, "alkoxy" itself or as a part of another group refers to a substituent containing a single bond to an oxygen atom, and the oxygen atom also serves as a point of attachment to a base molecule. Alkoxy can contain a straight chain or a branched chain. Alkoxy can be depicted as "-OR", wherein R is, for example, an alkyl, alkenyl, alkynyl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclic radical, as defined herein. The non-limiting list of alkoxy is methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, phenoxy and benzoyloxy. Unless otherwise indicated, alkoxy can be optionally substituted.

[0073] As used herein, "acyl" by itself or as part of another group refers to a substituent attached to a base molecule via a carbonyl group. Acyl groups may contain straight or branched chains. Acyl groups may be depicted as "-C(=O)R" or "-C(O)R", where R is, for example, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclyl, as defined herein. Examples include formyl, acetyl, propionyl, benzoyl, and acryloyl. Unless otherwise indicated, acyl groups may be optionally substituted.

[0074] As used herein, "sulfinyl" by itself or as part of another group refers to a substituent containing a single bond to a sulfur atom, and the sulfur atom also serves as a point of attachment to a base molecule. The sulfinyl group may contain a straight chain or a branched chain. The sulfinyl functional group may be depicted as "-SR", wherein R is, for example, an alkyl, alkenyl, alkynyl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclyl, as defined herein. Unless otherwise indicated, the sulfinyl group may be optionally substituted.

[0075] As used herein, "sulfenyl" by itself or as part of another group refers to a substituent attached to a base molecule through a sulfur atom which is also doubly bonded to an oxygen atom. A sulfenyl functional group may be depicted as "-S(=O)-R" or "-S(O)R", where R may be the same as defined for a sulfinyl group. Unless otherwise specified, a sulfenyl group may be optionally substituted.

[0076] As used herein, "sulfonyl" by itself or as part of another group refers to a substituent attached to a base molecule through a sulfur atom that is also doubly bonded to each of the two oxygen atoms. A sulfonyl functional group may be depicted as "-S02R," where R may be the same as defined for a sulfinyl group. Unless otherwise specified, a sulfonyl group may be optionally substituted.

[0077] As used herein, "O-carboxyl" by itself or as part of another group refers to a substituent containing an alkoxycarbonyl group, wherein the point of attachment is at the oxygen atom. The O-carboxyl group may be depicted as "RC(=O)O-" or "RC(O)O-" or "RCOO-", wherein R is, for example, hydrogen, halogen, alkyl, alkenyl, alkynyl, aryl, haloalkyl, cycloalkyl, heteroaryl, heterocyclyl, as defined herein. Unless otherwise specified, the O-carboxyl group may be optionally substituted.

[0078] As used herein, the terms "ester" and "C-carboxy" by themselves or as part of another group refer to substituents containing an alkoxycarbonyl group in which the point of attachment is located on the carbon atom. These may be depicted as "-C(=O)OR" or "-C(O)OR" or "-COOR" where R may be the same as defined for O-carboxy. Unless otherwise specified, a C-carboxy group may be optionally substituted.

[0079] As used herein, the term "thiocarbonyl" by itself or as part of another group refers to a functional group similar to acyl in which the oxygen atom is replaced by a sulfur atom. The thiocarbonyl group can be depicted as "-C(=S)R" or "-C(S)R", where R can be the same as defined for acyl. Unless otherwise indicated, the thiocarbonyl group can be optionally substituted.

[0080] As used herein, the term "pyridyl" by itself or as part of another group refers to a substituent consisting of a pyridine molecule in which one of the carbon atoms serves as the point of attachment (and one less hydrogen atom). Pyridyl is considered to be part of a heteroaryl group. Unless otherwise indicated, pyridyl may be optionally substituted.

[0081] As used herein, the term "pyrimidinyl" by itself or as part of another group refers to a substituent consisting of a pyrimidine molecule in which one of the carbon atoms serves as the point of attachment (and one less hydrogen atom). Pyrimidinyl is considered part of a heteroaryl group. Unless otherwise specified, pyrimidinyl may be optionally substituted.

[0082] As used herein, the term "phenyl" by itself or as part of another group refers to a substituent consisting of a benzene molecule in which one of the carbon atoms serves as the point of attachment (and one less hydrogen atom). Phenyl is considered part of an aryl group. Unless otherwise indicated, phenyl can be optionally substituted.

[0083] As used herein, unless otherwise indicated, abbreviations for any protecting groups, amino acids, and other compounds are generally recognized abbreviations according to their common usage or the IUPAC-IUB Commission on Biochemical Nomenclature. The terminology and notation used herein follows that of standard treatises and texts in the art, e.g., Kornberg and Baker, DNA Replication, 2nd ed. (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd ed. (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd ed. (Wiley-Liss, New York, 1999); Eckstein, ed., Oligonucleotides and Analogs: A Practical Approach (Oxford University Press, New York, 1991); Gait, ed., Oligonucleotide Synthesis: A Practical Approach (IRL Press, Oxford, 1993). Press, Oxford), 1984) etc.

[0084] The term "nucleoside" is used herein with the conventional meaning understood by those skilled in the art, and refers to a compound consisting of an optionally substituted pentose moiety or a modified pentose moiety connected to an optionally substituted heterocyclic base or its tautomer, such as connected by the 9 position of a purine base or the 1 position of a pyrimidine base. Examples include, but are not limited to, ribonucleosides including ribose moieties and deoxyribonucleosides including deoxyribose moieties. "Nucleoside" is a monomer that may have a substituted base and / or sugar moiety. As used herein, "vinyl-nucleoside" refers to a nucleoside analog with a vinyl group conjugated to a base moiety. Example vinyl-nucleosides are disclosed in WO2015197655, the entire contents of which are incorporated herein by reference. In addition, nucleosides can be incorporated into larger DNA and / or RNA polymers and oligomers.

[0085] The term "nucleotide" is used herein with the ordinary meaning as understood by those skilled in the art, and refers to a nucleoside having a phosphate bound to a pentose moiety (eg, at the 5' position).

[0086] The terms "subject", "individual" and "patient" are used interchangeably herein to refer to a mammal that is evaluated for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject", "individual" and "patient" encompass, but are not limited to, individuals who suffer from or are at risk of adhesions. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

[0087] The term "diagnosis" is used herein to refer to the identification of a pathological state, disease, or condition, such as the identification and characterization of a cancer or tumor.

[0088] As used herein, "pharmaceutically acceptable salts" include, for example, salts having the desired pharmacological activity of the parent compound (salts that retain the biological effectiveness and / or properties of the parent compound and are not biologically and / or otherwise undesirable). Compounds as described herein that have one or more functional groups capable of forming salts can, for example, be formed into pharmaceutically acceptable salts. Compounds containing one or more basic functional groups can be capable of forming pharmaceutically acceptable salts with, for example, pharmaceutically acceptable organic or inorganic acids.

[0089] "Pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" means an excipient or carrier that can be used to prepare a generally safe, non-toxic and desirable pharmaceutical composition, and includes excipients or carriers that are acceptable for veterinary as well as human pharmaceutical use. Such excipients can be solid, liquid, semisolid, or, in the case of an aerosol composition, a gas.

[0090] The term "pharmaceutically acceptable" referring to compositions, carriers, diluents and agents means that the material can be administered to or on a human without producing undesirable physiological effects to the extent that would prevent administration of the composition.

[0091] In certain embodiments, "in combination with" refers to administering a first therapeutic agent and a second therapeutic agent to a patient simultaneously. When administered in combination, each component can be administered simultaneously at different time points or sequentially in any order. Thus, each component can be administered separately but sufficiently separated in time to provide the desired therapeutic effect.

[0092] Intercalating Agents

[0093] The intercalating agents provided herein are acridine-tetrazine conjugate compounds. As used herein, "acridine moiety" refers to a portion of a compound having a core chemical structure And "tetrazine moiety" refers to a portion of a compound having a core chemical structure Methods and processes for conjugating an acridine moiety to a tetrazine moiety are disclosed, for example, in Yang et al. (Metal-Catalyzed One-Pot Synthesis of Tetrazines Directly from Aliphatic Nitriles and Hydrazine, Angewandte Chemie International Edition, Vol. 51, No. 21, pp. 5222-5225, May 21, 2012), the entire contents of which are incorporated herein by reference.

[0094] In some embodiments, the compound has one of the following formulae (brackets indicate optional groups):

[0095]

[0096] In some embodiments, the compound has formula Ia or Ic, and R4 is absent. In other embodiments, the compound has formula Ia or Ic, and R4 is present, resulting in the formation of an acridinium salt. In some embodiments, the compound has formula Ib and is a salt, wherein X is a counterion. Salts of the compounds of the invention include, but are not limited to, chloride, fluoride, bromide, iodide, or trifluoroacetic acid (TFA) salts; wherein X is Cl, F, Br, I, or TFA. In some embodiments, salts of the compounds of the invention are pharmaceutically acceptable salts.

[0097] In some embodiments, R1 is H, haloalkyl, optionally substituted haloalkyl, alkyl, optionally substituted alkyl, alkenyl, optionally substituted alkenyl, alkynyl, optionally substituted alkynyl, cycloalkyl, optionally substituted cycloalkyl, aryl, optionally substituted aryl, heteroaryl, optionally substituted heteroaryl, or heterocyclyl or optionally substituted heterocyclyl.

[0098] In one embodiment, R1 is H, C 1-6 In one embodiment, R1 is methyl, ethyl, propyl, isopropyl, C4 alkyl, C5 alkyl or C6 alkyl; preferably, R1 is methyl. In one embodiment, R1 is pyridyl, optionally substituted pyridyl, pyrimidinyl, optionally substituted pyrimidinyl, C 6-10 Aryl or optionally substituted C 6-10 In one embodiment, R1 is C6 aryl or phenyl.

[0099] In some embodiments, R2, R 2a , R 2b and R3 are each independently H, hydroxy, hydroxyl, halogen, haloalkyl, optionally substituted haloalkyl, cyano, amino, optionally substituted amino, thiol, optionally substituted thiol, alkyl, optionally substituted alkyl, alkenyl, optionally substituted alkenyl, alkynyl, optionally substituted alkynyl, cycloalkyl, optionally substituted cycloalkyl, aryl, optionally substituted aryl, heteroaryl, optionally substituted heteroaryl, heterocyclyl, optionally substituted heterocyclyl, alkoxy, optionally substituted alkoxy, sulfinyl, optionally substituted sulfinyl, acyl, optionally substituted acyl, sulfenyl, optionally substituted sulfenyl, sulfonyl, optionally substituted sulfonyl, O-carboxy, optionally substituted O-carboxy, ester, optionally substituted ester, thiocarbonyl, or optionally substituted thiocarbonyl.

[0100] In one embodiment, R2, R 2a and R 2b are each independently H, alkyl, optionally substituted alkyl, amino, optionally substituted amino, C 6-10 Cycloalkyl, optionally substituted C 6-10 Cycloalkyl, C 6-10 Aryl, optionally substituted C 6-10 aryl, heteroaryl, optionally substituted heteroaryl, heterocyclyl or optionally substituted heterocyclyl. 2a and R 2b Each independently is H, C 1-6 Alkyl, optionally substituted C 1-6 Alkyl, amino, optionally substituted amino, optionally substituted heterocyclic group, or optionally substituted heterocyclic group forming a ring with the acridine moiety.

[0101] In one embodiment, R2, R 2a and / or R 2b With an acridine moiety as shown below, a julolidine group is formed, which may be further optionally substituted with one or more groups:

[0102]

[0103] In one embodiment, the compound has formula (Ia), and R2 is C 1-6 An alkyl-substituted amino group or an optionally substituted heterocyclic group forming a ring with the acridine portion. In one embodiment, the compound has formula (Ia), and R2 is C 1-6 Alkyl-substituted amino groups are preferably substituted with one or more C 1-3 Alkyl substituted amino, more preferably dimethylamine. In one embodiment, the compound has formula (Ia), and R2 is an optionally substituted heterocyclic group that forms a ring with the acridine part, preferably forms a julolidine group with the acridine part.

[0104] In one embodiment, the compound has formula (Ib) or (Ic), and R 2a and R 2b Independently H, C 1-6 An alkyl-substituted amino group or an optionally substituted heterocyclic group forming a ring with the acridine portion. In one embodiment, the compound has formula (Ib) or (Ic), and R 2a and R 2b One of them is C 1-6 The amino group substituted with alkyl is preferably substituted with one or more C 1-3 In one embodiment, the compound has formula (Ib) or (Ic), and R 2a and R 2b All are C 1-6 The amino group substituted with alkyl is preferably substituted with one or more C 1-3 In one embodiment, the compound has formula (Ib) or (Ic), and R 2a and R 2b One of them is an optionally substituted heterocyclic group that forms a ring with the acridine moiety, preferably forming a julolidine group with the acridine moiety. In one embodiment, the compound has formula (Ib) or (Ic), and R 2a and R 2b Each is an optionally substituted heterocyclic group which forms a ring with the acridine moiety, and preferably forms a julolidine group with the acridine moiety.

[0105] In one embodiment, R3 is H, alkyl, optionally substituted alkyl, heteroalkyl, optionally substituted heteroalkyl, aryl, optionally substituted aryl, heteroaryl, or optionally substituted heteroaryl. In one embodiment, R3 is H, optionally substituted C 1-6 Alkyl, C 1-6 heteroalkyl, optionally substituted 5- or 6-membered heteroaryl, or optionally substituted C 6-10 Aryl.

[0106] In some embodiments, R4 is present and is H, haloalkyl, optionally substituted haloalkyl, alkyl, optionally substituted alkyl, alkenyl, optionally substituted alkenyl, alkynyl, optionally substituted alkynyl, cycloalkyl, optionally substituted cycloalkyl, aryl, optionally substituted aryl, heteroaryl, optionally substituted heteroaryl, heterocyclyl, or optionally substituted heterocyclyl. In one embodiment, R4 is present and is H or C 1-6 Alkyl; preferably H or CH3.

[0107] In some embodiments, the compounds have an optional linker (Y). In some embodiments, the optional linker is absent and the "acridine moiety" is directly conjugated to the "tetrazine moiety". In some embodiments, the compounds have a linker. In one embodiment, Y is alkyl, optionally substituted alkyl, heteroalkyl, optionally substituted heteroalkyl, aryl, optionally substituted aryl, heteroaryl, optionally substituted heteroaryl, alkenyl, optionally substituted alkenyl, alkynyl, optionally substituted alkynyl, alkylthio, or optionally substituted alkylthio. In one embodiment, Y is optionally substituted C 1-6 Alkyl, C 1-6 optionally substituted heteroalkyl, optionally substituted C 6-10 aryl, optionally substituted 5- or 6-membered heteroaryl, optionally substituted C 1-6 Alkenyl, optionally substituted C 1-6 Alkynyl, optionally substituted C 1-6 In one embodiment, Y is C 1-3 Alkyl or C 1-3 Alkylthio.

[0108] The compounds or salts disclosed herein exclude:

[0109]

[0110] In some embodiments, R2, R 2a , R 2b and / or Y is at position 3 or 6. In some embodiments, the compound has one of the following formulae (brackets indicate optional groups):

[0111]

[0112] Among them, R1, R2, R2a , R 2b , R3, R4 and Y are as described above.

[0113] In some embodiments, the compound has Formula IIIb:

[0114] and R1 is as described above.

[0115] Exemplary intercalators of the present disclosure include, but are not limited to, the following compounds and their corresponding salts:

[0116]

[0117]

[0118] In some embodiments, the compounds or salts described herein are probes. In one embodiment, the probes are used to label nucleic acids. In one embodiment, the probes are used to label DNA molecules. In one embodiment, the probes are used to label RNA molecules. In some embodiments, the compounds or salts described herein are in vivo probes. In some embodiments, the compounds or salts described herein are in vitro probes. In some embodiments, the compounds or salts described herein are used to identify or quantify cell growth and / or replication.

[0119] Nucleic acid labeling

[0120] In some embodiments, the acridine-tetrazine conjugate compound or salt provided herein is used to detect nucleic acids containing vinyl-nucleosides. In one embodiment, the compound or salt provided herein is used to detect DNA containing vinyl-nucleosides. In one embodiment, the compound or salt provided herein is used to detect RNA containing vinyl-nucleosides. Exemplary vinyl-nucleosides are disclosed in WO2015197655, the entire contents of which are incorporated herein by reference. In some embodiments, the compound or salt provided herein is used to detect nucleic acids containing one or more of the following: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG). 5-vinyl uridine (VU), 5-vinyl cytidine (VC), 7-vinyl-7-deazaadenosine (VA) and 7-vinyl-7-deaza-2'-deoxyguanosine (VG). In one embodiment, the vinyl-nucleoside is VdU and / or VdA. In one embodiment, the vinyl-nucleoside is VU.

[0121] For nucleic acid labeling, first an organism, tissue or cell is treated or contacted with one or more vinyl-nucleosides, and the one or more vinyl-nucleosides are incorporated into the DNA and / or RNA molecules of the organism, tissue or cell. Then an acridinium-tetrazine conjugate compound provided herein is introduced, which is conjugated with the incorporated vinyl-nucleosides to produce a fluorescent active portion. In some embodiments, a method for detecting nucleic acids containing vinyl-nucleosides comprises: a) contacting cells or tissues with a compound or salt provided herein; and b) exposing the cells or tissues of a) to light. In one embodiment, the method is performed in vitro. In another embodiment, the method is performed in vivo. In some embodiments, a method for detecting nucleic acids containing vinyl-nucleosides is performed in vivo by a) applying a compound or salt provided herein to an organism and b) exposing a target area or excised tissue to light.

[0122] In some embodiments, a kit for detecting nucleic acids in cells or tissues for use in vitro or in vivo is provided. The kit includes one or more vinyl-nucleosides and compounds or salts as provided herein. In one embodiment, the kit includes one or more of the following: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG). 5-vinyl uridine (VU), 5-vinyl cytidine (VC), 7-vinyl-7-deazaadenosine (VA) and 7-vinyl-7-deaza-2'-deoxyguanosine (VG). In one embodiment, the kit includes one or more of VdU, VdC, VdA and VdG. In one embodiment, the kit includes VdU and / or VdA. In one embodiment, the kit includes one or more of VU, VC, VA and VG. In one embodiment, the kit includes VU.

[0123] In some embodiments, nucleic acid markers using acridine-tetrazine conjugate compounds or salts provided herein are used for diagnosis of cancer. In one embodiment, the diagnosis is for tumor growth progression. In some embodiments, nucleic acid markers using acridine-tetrazine conjugate compounds or salts provided herein are used for cell cycle analysis. In one embodiment, acridine-tetrazine conjugate compounds or salts together with the one or more vinyl-nucleosides are markers for DNA synthesis in cell cycle analysis. In one embodiment, acridine-tetrazine conjugate compounds or salts together with the one or more vinyl-nucleosides are S phase markers in cell cycle analysis.

[0124] Treatment Effects

[0125] Vinyl-nucleosides are non-toxic nucleoside analogs. However, when acridine-tetrazine conjugate compounds are conjugated to vinyl-nucleosides, toxicity is generated.

[0126] In some embodiments, a method of treating cancer with an acridinium-tetrazine conjugate compound or salt is provided.A compound or salt described herein is then administered to a subject who has previously been administered one or more vinyl-nucleosides.

[0127] In some embodiments, a pharmaceutical composition comprising an acridine-tetrazine conjugate compound or salt and an acceptable pharmaceutical carrier. In some embodiments, the acridine-tetrazine conjugate compound or salt is used to prepare a medicament for treating cancer. In some embodiments, the acridine-tetrazine conjugate compound or salt, or a pharmaceutical composition comprising an acridine-tetrazine conjugate compound or salt, is used to treat cancer. In some embodiments, a combination of an acridine-tetrazine conjugate compound or salt, or a pharmaceutical composition comprising an acridine-tetrazine conjugate compound or salt and one or more vinyl-nucleosides is used to treat cancer. In one embodiment, the acridine-tetrazine conjugate compound or salt is used to be administered sequentially after administering one or more vinyl-nucleosides.

[0128] In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for sequential administration after administration of one or more of the following: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG). 5-vinyluridine (VU), 5-vinylcytidine (VC), 7-vinyl-7-deazaadenosine (VA) and 7-vinyl-7-deaza-2'-deoxyguanosine (VG). In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for sequential administration after administration of one or more of the following: VdU, VdC, VdA and VdG. In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for sequential administration after administration of VdU and / or VdA. In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for sequential administration after administration of one or more of: VU, VC, VA, and VG. In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for sequential administration after administration of VU. In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for sequential administration after administration of one or more vinyl-nucleosides.

[0129] In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for administration in combination with administration of one or more of the following: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG). 5-vinyluridine (VU), 5-vinylcytidine (VC), 7-vinyl-7-deazaadenosine (VA), and 7-vinyl-7-deaza-2'-deoxyguanosine (VG). In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for administration in combination with administration of one or more of the following: VdU, VdC, VdA, and VdG. In one embodiment, the acridinium-tetrazine conjugate compound or salt is used for administration in combination with administration of VdU and / or VdA. In one embodiment, the acridinium-tetrazine conjugate compound or salt is administered in combination with administration of one or more of: VU, VC, VA, and VG. In one embodiment, the acridinium-tetrazine conjugate compound or salt is administered in combination with administration of VU.

[0130] Examples

[0131] The following examples illustrate certain embodiments and are not intended to limit embodiments described elsewhere in this disclosure.

[0132] Example 1 - Kinetic and fluorescence enhancement strategies for labeling nucleic acids

[0133] Chemical modification of nucleic acids in living cells can be sterically hindered by the close stacking of bioorthogonal functional groups in chromatin. To address this limitation, a dual enhancement strategy for nucleic acid-templated reactions was developed that utilizes fluorescent intercalators capable of inverse electron demand Diels-Alder (IEDDA) reactions with either DNA containing 5-vinyl-2'-deoxyuridine (VdU) or RNA containing 5-vinyl-uridine (VU). Compared to non-templated reactions, the novel acridine-tetrazine conjugate "PINK" (K D =5±1 μM) reversible high affinity intercalation increases the reaction rate of tetrazine-olefin IEDDA on double-stranded DNA by 60,000 times (590 M -1 s -1 ). At the same time, the loss of tetrazine-acridine fluorescence quenching makes the reaction highly fluorescent and detectable under no-wash conditions. This strategy enables live-cell dynamic imaging of acridine-modified nucleic acids in dividing cells.

[0134] Chemical selective modification reactions of biomolecules provide an indispensable tool for biological and biomedical research. [1–4] Specifically, bioorthogonal labeling reactions for chemical biology have been successfully developed for sequence recognition, [5–7] Sensitive detection of viral infections[8–10] , protein cross-linking,

[11] Elucidation of metabolic pathways

[12] and exploration of resistance and sensitivity mechanisms. [13,14] Such bioorthogonal approaches were initially designed for proteins or carbohydrates and subsequently applied to nucleic acids. [15–18] These include copper-catalyzed or strain-promoted azide-alkyne cycloadditions (CuAAC / SPAAC), inverse electron demand Diels-Alder (IEDDA) reactions between olefins and tetrazines, and 1,3-dipole “photoclick” reactions of tetrazoles. [19,20] Despite these advances in vitro, examples of nucleic acid labeling in vivo remain rare and limited to single-stranded RNA [21–25] Or rely on membrane permeabilization and / or phototoxicity methods to achieve efficient reactions within the tightly packed double-stranded (ds) DNA of native chromatin. [26–28] Additionally, previously reported tetrazine-olefin bioorthogonal reactions of cellular nucleic acids were too slow (0.02–0.42 M -1 s -1 ), which cannot visualize highly dynamic processes in living cells. [24,29–31]

[0135] To address these limitations, an intercalator was developed based on the Sondheimer diyne that reacts with two azide groups via a tandem SPAAC cross-linking reaction. [32,33] This method is applicable to the modification of dsDNA and RNA in living cells, but the high background fluorescence of unreacted fluorescent labeling reagents hinders live cell imaging. Probes for imaging nucleoside olefin groups ( P robe for I maging N ucleosidic Al K ene groups, "PINK") to incorporate fluorescent intercalators

[34] In combination with the tetrazine, the tetrazine acts as both a bioorthogonal functional group and a fluorescence quencher. [35,36] PINK is non-fluorescent in nature and exhibits a reversible, intercalating "scan" of double-stranded DNA until it encounters a vinyl-modified nucleotide, which then undergoes a highly and rapidly fluorescent IEDDA reaction ( Figure 1 ). This method provided the first "mix and measure" fluorescence assay for DNA replication in living cells.

[0136] PINK was designed based on crystallographic data and density functional theory (DFT) calculations. The crystal structure of 3,6-bis(dimethylamino)acridine (acridine orange) bound to a CpG dimer revealed a coplanar arrangement between the dimethylamino substituent and the 5-position of the pyrimidine nucleobase.

[37] The 3 position of acridine orange was therefore chosen to install the tetrazine moiety while retaining the dimethylamino group at the 6 position. As previously observed for aryl tetrazines, [38,39] DFT calculations predict that PINK is a planar molecule ( Figure 1 A PINK π model stacked on a VdU-dA base pair was generated by DFT geometry optimization of PINK (LSDA / pBP86 / DN**) and flowed by manual docking of PINK and the DFT geometry-optimized VdU-dA base pair based on the π-stacking arrangement observed in the acridine orange crust structure inserted into a CpG dimer ( Figure 2 ). After embedding, this coplanarity should induce a coplanar arrangement of the 5-vinyl and tetrazine groups, thus promoting a highly rapid reaction between PINK and VdU residues ( Figure 2 ).

[0137] The synthesis of PINK started from aniline 1, which underwent a clean Buchwald-Hartwig cross coupling reaction with aryl bromide 2 (Scheme 1).

[40] The resulting secondary amine spontaneously cyclized upon contact with weakly Lewis-acidic silica gel to provide the new acridinium nitrile 3 in 64% isolated yield. Subsequent installation of the asymmetric methyl tetrazine was accomplished by reaction with hydrazine, Zn(II) and acetonitrile followed by oxidation with NaNO2 to afford PINK (4) as a red solid in 18% overall yield over four steps.

[41]

[0138] Scheme 1. Synthesis of PINK, intermediates and isolated yields. a) 0.1 eq Pd2(dba)3, 0.1 eq dppf, 2.0 eq K2CO3, toluene, 80°C, 17 hours; b) SiO2, low pressure, 64% over two steps. c) 10 eq MeCN, 50 eq N2H4, 0.5 eq Zn(OTf)2, 3 days, 65°C d) 15.5 eq NaNO2, aqueous HCl, 10 minutes, 44% over two steps; e) DMF 3:1 CHCl3, ambient atmosphere, 60°C, 7 days, 73%.

[0139]

[0140] To evaluate the ability of PINK (4) to reversibly intercalate into double-stranded DNA, the absorbance of PINK was monitored after the addition of calf thymus (CT) DNA. Consistent with intercalation, [42–44] After adding CT DNA, a red shift in the absorbance of PINK was observed ( Figure 3, a). To further evaluate the binding mode, viscosity measurements were performed on concentrated DNA solutions in the presence of increasing amounts of PINK. According to the Lerman hypothesis for DNA intercalators,

[45] PINK causes an increase in solution viscosity (η), and the stoichiometric ratio between PINK and DNA base pairs is 1:5 ( Figure 3 , b). Using this stoichiometry and using titration of the absorbance of PINK as a function of increasing DNA concentration, the apparent dissociation constant (K) was estimated. D )=5.1±1.0μM( Figure 3 , a; and Figure 4 , A). By considering the maximum binding stoichiometry of PINK (1 molecule per 5 base pairs; see Figure 1 b), according to K D = [(unbound PINK) * (unbound DNA binding site)] / (bound PINK binding site) to calculate the apparent dissociation constant K D In EC 50 and considering the stoichiometry, this simplifies to K D = [unbound DNA binding site] = [(EC 50 ) / 5]-[unbound PINK]=[(50±5.1μM) / 5]-[5μM]=5.1±1.0μM. This DNA affinity is similar to that of other acridine derivatives. [46–48] This indicates that the tetrazine portion of PINK does not interfere with DNA binding. Prolonging the incubation time of PINK with unmodified DNA resulted in almost no degradation of PINK ( Figure 4 , B), which supports the possibility of a chemically selective reaction between PINK and VdU-containing DNA.

[0141] PINK (4) reacted with VdU (5) in a highly fluorescent and regioselective IEDDA reaction to afford “PINK-VdU-ox” (6) as a single isomer in 76% isolated yield after 7 days at 60 °C (Scheme 1). The slow rate (k app =0.01M -1 s -1 , Figure 5 ) is due to the combination of unstrained terminal olefin and unactivated tetrazine.

[49] During the reaction, an approximately 100-fold increase in fluorescence was observed ( Figure 3 , c). In the quantum yield In the case of PINK (4), it is essentially nonfluorescent in aqueous and aprotic solvents ( Figure 6 ).

[0142] In contrast, the PINK-VdU reaction product (6) showed This depends on the solvent used ( Figure 3 , d; Figure 7 ). High viscosity solvents including glycerol and polyethylene glycol (PEG 200) may induce higher fluorescence of product 6 by restricting bond rotation, thereby suppressing the formation of non-emissive, twisted intramolecular charge transfer (TICT) states.

[50]

[0143] Table 1: Deoxy oligomers investigated. Structures of non-classical nucleosides (V = VdU; X = PINK-VdU-ox).

[0144]

[0145] To characterize the DNA-templated PINK-VdU reaction, a palindromic 17-mer DNA oligonucleotide “ODN1” containing VdU was synthesized (Table 1). The DMT-protected VdU phosphoramidite S4 was synthesized using standard solid-phase supported DNA synthesis (Scheme 2).

[51] ODN1 was purified using reverse phase HPLC and annealed with complementary strands to form duplexes. After mixing PINK with duplexes containing VdU, a highly rapid fluorescence response was observed. By high resolution mass spectrometry, oxidized pyridazine (similar to compound 6) was identified as the major reaction product on DNA. By tracking the fluorescence changes under pseudo-primary conditions ( Figure 8 ), the apparent rate constant k for the reaction between PINK and double-stranded DNA containing VdU was determined to be 590 M -1 s -1 This value is approximately 60,000-fold that of the non-templated reaction between PINK and VdU and is one of the fastest reported bioorthogonal labeling reactions to date. [49,52]

[0146] Scheme 2: Synthesis of PINK, PINK-VdU-ox and DMT protected VdU phosphoramidites. a) 4.0 eq. NBS, 0.1 eq. DBPO, CCl4, reflux, 2 days. b) 1.) Dimethylamine aqueous solution 23.0 eq., 60°C, 90 min; 2.) HCl aqueous solution 50°C, 90 min, 75% (over two steps). c) 0.1 eq Pd2dba3, 0.1 eq dppf, 2.0 eq K2CO3, toluene, 80°C, 17 hours, 64%; d) 1.) 10 eq MeCN, 50 eq N2H4, 0.5 eq Zn(OTf)2, 3 days, 65°C; 2.) 15.5 eq NaNO2, aqueous HCl, 10 minutes, 44%; e) DMF 3:1CHCl3, 60°C, 7 days, 73%; f) 1.2 eq DMT-chloride, pyridine, room temperature, overnight, 93%: g) 2.5 eq DIPEA, 2.0 eq 2-cyanoethyl-N,N-diisopropylphosphoramidite, DCM, 0°C→room temperature, 2.5 hours, 49%.

[0147]

[0148] To assess the ability of PINK to enter living cells and react fluorescently with vinyl groups in native chromatin, HeLa cells were grown in rich medium containing 10 μM VdU for 15 h and subsequently stained with 1–2 μM PINK for 4 h. Total cellular DNA was counterstained with Hoechst 3342 (5 μM; 405 nm laser; emission 410–480 nm) for 10 min prior to imaging of PINK (514 nm laser; emission 570–700 nm). Control (ctrl) samples were treated with PINK but not VdU. All cells were treated and maintained in DMEM medium containing 10% FBS and grown at 37°C in 5% CO2. Live cell images were acquired using confocal microscopy. Live cell fluorescence microscopy revealed a pattern consistent with metabolic labeling, with daughter cell pairs with bright pink fluorescence observed only in VdU-treated cells and excellent colocalization observed between PINK and the non-covalent dye Hoechst 33342 ( Fig. 9 ). Toxicity studies have shown that low micromolar concentrations of PINK are not toxic to cells.

[0149] Next, the intracellular kinetics of PINK staining were investigated. 4Cells were inoculated at a concentration of 10 cells / mL, allowed to stand overnight, and incubated with 100 μM VdU for 24 hours. Cells were stained with 10 μM PINK for 10 minutes to 25 hours and co-stained with Hoechst 33342 (5 μg / mL), and images were acquired on a confocal microscope. After 1 hour of staining, the samples were optimized to obtain an ideal signal-to-noise ratio, showing a sufficiently bright signal at this stage. The Ctrl samples were exposed to PINK for 25 hours, explaining the relatively high background. It was found that a duration of 5 hours was ideal for achieving complete labeling, but a clear signal above background was observed after only 1 hour of incubation time ( Fig.10 ). Labeling is effective in a variety of cell lines. U2OS cells were seeded into 8-well μ-slides in DMEM ( (3.0X 10 in 200 μL 4 cells; 1.5X 10 5 Cells were aspirated and VdU (0-100 μM in 200 μL DMEM) was added. Cells were incubated for 18 hours, aspirated, and PINK (5 μM in 200 μL DMEM, 0.05% DMSO) was added. Cells were incubated for 5 hours, aspirated, and Hoechst 33342 (2 μg / mL in 200 μL DMEM) was added for 15 minutes. Cells were aspirated and the medium was replaced with 200 μL DMEM (-phenol red). Images were acquired on a confocal microscope ( Fig.11 ). 24 hours after removal from the culture medium, the fluorescence signal of PINK remained in living cells. 4 Cells were incubated with 20 μM VdU for 16 hours and stained with 2 μM PINK for 5 hours, aspirated and fresh medium (without phenol red) was added. Control samples received the same treatment but were not exposed to VdU. Before acquiring live cell images on a confocal microscope at t=0, 24 hours, cells were co-stained with Hoechst 33342 (5 μg / mL) for 10 minutes ( Fig.12 ).

[0150] The labeling method was extended to vinyl-modified RNA. HeLa cells were seeded on 8-well plates (5.000 cells per well) and allowed to stand overnight. The cells were then incubated with 5-vinyluridine (VU, 2.5 mM) for 24 hours. After 19 hours, PINK (100 μM final concentration) was added for 5 hours. The cells were incubated for an additional 5 hours. The cells were fixed and optionally treated with an RNase A / T1 mixture (100 μg / mL RNase A; 250 U / mL RNase T1). Total cellular DNA was counterstained with Hoechst 33342 (5 μg / mL). Images were acquired on a confocal microscope at 20x (a) or 63x (b) magnification ( Fig.13 ). Based on successful metabolic RNA labeling of living cells, confocal microscopy revealed a strong fluorescence signal that was greatly attenuated if cells were treated with RNase A and T1 after fixation.

[0151] To investigate the potential effects of VdU-PINK labeling on subsequent DNA synthesis, pulse-chase experiments were performed with 5-ethynyl-2'-deoxyuridine (EdU) ( Fig.14 , a). HeLa cells were grown in cell culture medium containing 20 μM VdU for 16 h, aspirated, treated with 1 μM PINK for 4 h, aspirated and treated with 10 μM EdU for 4 h. After fixation and DNA denaturation, cells were stained with AF488-azide (488 nm laser; emission 495–540 nm), and total cellular DNA was counterstained with Hoechst33342 (405 nm laser; emission 410-480 nm). “No VdU” cells were incubated with 1 μM PINK, but not with VdU. PINK+ and EdU+ “double positive” cells were observed to have colocalized nuclear staining. These striking results indicate that the PINK-VdU reaction product does not cause cell cycle arrest. Therefore, VdU and PINK are used to track DNA synthesis and cell division using live cell time-lapse imaging. For optimal live cell visualization, cells were incubated with 100 μM VdU for 23 h and subsequently labeled with 1 μM PINK for 4 h before imaging to allow recording of a complete mitotic cycle within 2.5 h ( Fig.14 , b). Live cell images were acquired using a confocal microscope equipped with a bench-top incubator at 37°C and 5% CO2. These results provide an example of a "mix and measure" fluorescence assay of DNA replication in live cells.

[0152] In summary, PINK was developed using a dual enhancement strategy, where the acridine portion of PINK acts as both a fluorescent probe and a rate-enhancing intercalator, while the tetrazine acts as a fluorescence quencher and a bioorthogonal functional group. PINK is an example of a fluorescent intercalator that exhibits enhanced fluorescence when covalently reacting with nucleic acids in living cells. Previous studies involving chemical modification of cell-vinyl-modified DNA by bioorthogonal methods relied on cell fixation and DNA denaturation to promote the tetrazine-olefin reaction. [20,30,31,54,55] The new ability to react bioorthogonal functional groups in natural DNA and RNA will open new opportunities in biological and translational research.

[0153] Example 2 - Materials and Methods

[0154] All reagents were purchased from Sigma Aldrich of the highest commercial grade. TM 、Fisher Scientific TM or Fluorochem TM The reaction was obtained and used without further purification. Reactions sensitive to water or air were carried out under an inert gas atmosphere (nitrogen, argon) using anhydrous solvents and standard Schlenk techniques. Analytical thin layer chromatography was performed on pre-coated 250 μm thick silica gel 60 F254 plates and visualized by UV light. Flash column chromatography was performed using 40-63 μm silica gel and compressed air. NMR (for 1 H is 400MHz, for 13 C is 100MHz or for 1 H is 500MHz, for 13 C is 125MHz). 13C spectra were recorded broadband proton decoupled. Chemical shifts (δ) are given in parts per million (ppm) and are reported relative to residual solvent peaks: CDCl3 (δH 7.26, δC 77.0 ppm), methanol-d4 (δH 3.31, δC 49.0 ppm). Coupling constants (J) are given in Hertz (Hz) and the following abbreviations are used to describe multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, quint = quintet, sext = sextet, m = multiplet, dd = doublet-doublet, ddd = doublet-doublet-doublet, dt = doublet-triplet, dq = doublet-quadruplet, br = broad. Mass spectra were recorded on an Advion expression CMS and high resolution mass spectra were obtained on a Bruker MaXis high resolution QTOF or a Thermo QExactive high resolution Orbitrap. Mass is given in m / z.

[0155] Photophysical measurements. Stock solutions (1-10 mM) of the compounds studied were prepared in DMSO and stored at -20°C. After dilution into the corresponding solvent / buffer solution, the DMSO concentration was less than 1%. Measurements were collected in a quartz cuvette with a 1 cm path length on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). EtOH (n=1.3624) was used as a fluorescence standard, and the quantum yield was obtained by excitation at 488 nm. [1] The quantum yield was calculated using Equation I:

[0156]

[0157] in is the quantum yield of the fluorescence standard, Grad X and Grad R is the gradient obtained by plotting the integrated emission of the corresponding optical density at 488 nm. X and n ST are the refractive indices of the sample and reference solvent, respectively.

[0158] Fluorescence switch-on measurements. 25 μM PINK was reacted with 5 mM VdU (200 equiv.) in MeCN 9:1 DMSO in a 1 cm quartz cuvette at 25° C. Fluorescence spectra (excitation: 480 nm; emission: 500-750 nm) were acquired on a Horiba Duetta spectrometer over 46 h.

[0159] PINK stability test. Fluorescence changes of 20 μM PINK were monitored in the presence of 1 mM calf thymus (CT) DNA (50 equiv) base pairs in PBS pH 7.4 (10% DMSO) followed by the addition of 5-norbornene-2-methanol (2 mM, 100 equiv) (excitation: 500 nm, emission: 590 nm; 515 nm cutoff filter). Measurements were performed in a 1 cm path length quartz cuvette on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA).

[0160] Binding affinity measurements of PINK to CT-DNA. Binding of PINK to CT-DNA was assessed by monitoring changes in absorbance collected in a quartz cuvette with a 1 cm path length on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). A small volume of concentrated CT-DNA solution (10 mM base pairs, 0-16 μL) was added to 1 mL of 50 mM NaOAc aqueous solution, pH 5.2 (0.2% DMSO) containing 10 μM PINK. The overall volume change was a maximum of 1.6%. Acidic conditions are required to ensure sufficient solubility of PINK. The binding of PINK to CT-DNA was calculated by plotting the change in absorbance at 575 nm and determining the apparent binding constant (K D ).

[0161] Viscosity measurements of CT-DNA solutions. The viscosity of the DNA solution was determined by measuring the flow time in a Cannon-Manning semi-micro 75 viscometer. The temperature was adjusted to room temperature by immersing the viscometer in a glass beaker filled with water. A separate solution containing 0 to 240 μM PINK and 300 μM base pair CT-DNA was freshly prepared in a 50 mM NaOAc aqueous solution pH 5.2 (2.4% DMSO) and incubated for at least 1 hour to ensure complete equilibrium. The flow time of each solution was measured in triplicate, and the final viscosity data was obtained from three independent experiments. The flow time ranged from 195.5 seconds to 269.1 seconds. Viscosity was calculated as η=t-t0, where t describes the flow time of the corresponding sample, and t0 is the flow time of the buffer alone (135.7 seconds).

[0162] Synthesis and purification of oligodeoxynucleotides. Unmodified sequences were obtained from Sigma Aldrich as HPLC purified products. Standard DNA phosphoramidites, solid supports and other necessary reagents were purchased from LinkTech. VdU-phosphoramidites were freshly synthesized as described below and dissolved in dry MeCN at 25 mg / mL immediately before use. Modified oligodeoxynucleotides were synthesized on a 1.0 μmol scale using a Bioautomation Co. Mermade 4 DNA synthesizer according to a standard trityl removal procedure. Synthesis was monitored by DMT deprotection. Upon completion, the sequence was excised from the solid support and deprotected by treatment with 1.0 mL of 33% aqueous ammonium hydroxide at 55° C. overnight in a 1.5 mL screw cap tube. The resulting solution was filtered through a Whatman FP30 / 0.2CA-S 0.2 μm syringe filter and then washed with deionized water. The resulting solution was lyophilized to dryness. A Varian 140 ProStar HPLC system was used with an analytical C-18 reverse phase column (Waters C18 3. Final purification was performed by HPLC column chromatography on a 4.6 μm × 150 mm column. The following MeCN: 0.1 M aqueous triethylammonium acetate pH 7.4 (TEAA) gradient was applied: 5:95 to 23:77 in 30 minutes at a flow rate of 0.4 ml / min. Elution was monitored by UV absorption at 260 nm. After adding a total of fresh deionized water to completely remove traces of triethylamine and acetic acid, the peaks of several runs were collected and lyophilized to dryness three times in a row.

[0163] Oligodeoxynucleotides were analyzed by LC-MS in negative ESI mode using a Dionex Ultimate 3000UHPLC coupled to a Bruker Maxis Impact QTOF. The samples were run through a Phenomenex Luna C18 (2) -HST (2.5 μM 120A 2.1 × 100 mm) column using a gradient of 90% mobile phase A (100 mM HFIP, 5 mM NEt in H2O) and 10% mobile phase B (MeOH) to 40% mobile phase A and 60% mobile phase B in 20 minutes. The data were processed using Bruker Data Analysis Software Version 4.2 and the spectra were deconvoluted.

[0164] Oligodeoxynucleotide stock solutions were prepared in deionized (milli-Q) water and their concentrations were determined by absorbance at 260 nm using molar extinction coefficients calculated using the nearest neighbor model. [2]Duplex DNA was prepared by mixing 1 equivalent of modified DNA (flank_G) with 1.1 equivalents of complementary sequence (compl_G) in 10 mM NaH2PO4 pH 7. The solution was heated to 95°C for 5 minutes and slowly cooled to room temperature overnight.

[0165] Preparation of PINK modified oligodeoxynucleotide "Flank_G_PINK". Vinyl modified oligodeoxynucleotide (ODN) chains were prepared as described above. 10 μM vinyl-DNA was reacted with 100 μM PINK in 50 mM NaOAc buffer pH 5.2 (2% DMSO) at 37°C overnight. The modified DNA was added using Nap TM -5 column (Catalog No.: 17-0853-01) and purified by elution with ddH2O. Fractions containing DNA were pooled based on A260 absorbance, evaporated and dissolved in 10 mM NaH2PO4 pH 7. The samples were then reannealed as described above. Concentrations were quantified based on A260 values ​​assuming equal molar extinction coefficients for PINK and vinyl-modified ODN.

[0166] Kinetic measurements were performed using the VdU nucleoside. The rate of the second-order reaction between PINK and VdU was determined under pseudo-first-order conditions, in which the dienophile was in great excess over the tetrazine. The emission increase at 590 nm (excitation: 500 nm, 515 nm cutoff filter) was used to monitor the progress of the reaction. The measurements were performed on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA) in 384-well plates (Greiner bio-one, catalog number 871906; 50 μL per well; covered with 20 μL of spectral grade silicone oil to avoid evaporation). The solution contained a final concentration of 100 μM PINK and 1-5 mM VdU (10-50 equivalents) in 50 mM NaOAc aqueous buffer pH 5.2 (27% DMSO). The acidic concentration and high DMSO content were necessary to ensure sufficient solubility of PINK and VdU, respectively. Samples were measured over 18 hours with an interval of 10 minutes. The control condition (0 mM VdU) showed that PINK was completely stable under these conditions and the background signal of PINK was negligible. The reaction rates were calculated by fitting the measurements to a single exponential equation using a pseudo first order approximation. The reported reaction rates are the arithmetic means determined by three independent experiments.

[0167] Kinetic measurements using ODN. The second-order rate constants for the reaction between vinyl-modified ODN and PINK were determined under pseudo-first-order conditions using an excess of tetrazine. The emission increase at 590 nm (excitation 530 nm; 550 nm cutoff filter) was used to monitor the progress of the reaction. The measurements were performed in 384-well plates (Grena First Biochemical Company, catalog number 871906; 40 μL per well) on a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). The solution contained a final solution of 4 μM vinyl-modified ODN (flank_G) and 12-20 μM PINK (3-5 equivalents) in 50 mM NaOAc aqueous buffer pH 5.2 (0.8% DMSO). The acidic concentration is necessary to ensure sufficient solubility of PINK. Samples were measured over 1 hour with an interval of 45 seconds. The control condition (12-20 μM PINK, without vinyl-modified ODN) showed that PINK was completely stable under these conditions and the background signal was negligible. Reaction rates were calculated by fitting the measurements to a single exponential equation using a pseudo-first order approximation. The reported reaction rates are the arithmetic means determined from three independent experiments.

[0168] Eukaryotic cell culture. Eukaryotic cells (HeLa, U2OS) were cultured in DMEM (Gibco) containing 4.5 g / l glucose, 10% FBS (Gibco), 50'000 units of penicillin and 50 mg of streptomycin (Sigma-Aldrich) per L at 37°C, 5% CO2, and 1% MEM non-essential amino acids (Sigma-Aldrich). Cells were grown to confluence using trypsin-EDTA solution (Sigma-Aldrich) and passaged every 2 to 4 days. Cells were counted using an Olympus automated cell counter model R1 to determine seeding density.

[0169] Resazurin assay. HeLa or U2OS cells were seeded at a density of 7,500 cells per well in 96-well plates and incubated overnight. The supernatant was removed and 100 μL of fresh medium containing PINK (0–12.5 μM; 0.1% DMSO) was added. Cells were grown for 24 or 72 h, at which time 10 μL of resazurin (880 μM in PBS; final concentration: 80 μM) was added. After 3 h of incubation, fluorescence emission at 590 nm (excitation 560 nm; cutoff: 575 nm) was measured using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). LC 50 Values ​​were determined from two independent replicates.

[0170] Confocal laser scanning microscopy (CLSM). Confocal laser scanning microscopy (CLSM) was performed on: a) CLSM Leica SP5 Mid UV-VIS (Leica Microsystems) equipped with a HC PLAPO Leica 10x air objective (NA 0.4, WD 2.2), HC PL APO Leica 20x multi-immersion objective (NA 0.7, WD 0.25), HCX PL APO Leica 40x oil immersion objective (NA 1.25, WD 0.1), HCX PL APO Leica 63x oil immersion objective (NA 1.4, WD 0.1) or HCX PL APO 37°C Leica 63x glycerol objective (NA 1.3, WD 0.28); b) CLSM Leica SP8 HC PL FLUOTAR 10x air objective, HC PL APO CS2 20x immersion objective, HC PL APO CS2 63x oil objective or HC PL APO 37°C CS2 63x glycerol objective; c) Leica Stellaris 5LIAchroic (Leica Microsystems) equipped with a HC PL APO 20x multi-immersion objective (0.75IMM CORR CS2) or HC PL APO 63x / 1.40OIL CS2 (FWD: 0.14mm); or d) Nikon Ti2 inverted spinning disk (CREST X-Light V2 L-FOV) confocal device equipped with a Nikon PlanApo 20x0.75NA air objective. DAPI and Hoechst 33342 were excited at 405 nm and the emission between 410 and 480 nm was sampled; Alexa FL488 was excited at 488 nm and the emission between 495 and 540 nm was sampled; PINK was excited at 512, 546, 552 or 561 nm and the emission was recorded between 530 and 700 nm (for 512 nm excitation) or 575 nm and 700 nm (for 546 nm excitation), respectively. A HyD detector was used. Z stacks were recorded with a step size between 0.1 μm and 10 μm. Image analysis was performed using Leica LAS AF Lite 2.6.3 (Leica Microsystems) and Fiji 1.50i (Wayne Rasband, National Institutes of Health, USA).

[0171] Metabolic labeling of living cells. Cells were cultured at 2.5 × 10 4 –1.5×105 The cells were seeded at a density of 100 cells / mL in a μ-slide 8-well chamber ( Catalog number: 80826) and allowed to stand overnight. The cells were aspirated and incubated with different concentrations of VdU. After incubation for different times, the cells were aspirated and incubated with different concentrations of PINK in medium without phenol red for 4-5 hours. Before imaging, total cellular DNA was counterstained with Hoechst 33342 (5 μg / mL) for 10 minutes. All compounds were freshly diluted into DMEM from DMSO stock solutions before addition, ensuring that the final DMSO concentration did not exceed 0.1%.

[0172] Metabolic RNA labeling. HeLa cells were cultured at 2.0×10 4 The cells were seeded at a density of 10 cells / well in a μ-slide 8-well chamber ( Catalog number: 80826) (5.000 cells per well) and allowed to stand overnight. The cells were aspirated and 5-vinyluridine (VU, 2.5 mM, 0.1% DMSO) was added. The cells were incubated for 19 hours, at which time PINK (100 μM; 0.2% DMSO final concentration) was added. After an additional 5 hours, the cells were aspirated, washed with PBS, and fixed with PBS containing 3.7% PFA for 15 minutes. The cells were quenched with 50 mM glycine / NH4Cl for 5 minutes, washed with PBS, and permeabilized twice with PBS containing 0.2% Triton X-100 for 5 minutes each, followed by washing with PBS, 100 mM NaOAc buffer (pH 5.2), PBS containing 10% DMSO, and PBS twice for 5 minutes each. For RNA digestion, the cells were incubated with RNase A / T1 mixture (ThermoScientific TM EN0551; 100 μg / mL RNase A; 250 U / mL RNase T1) was incubated in RNase buffer (10 mM Tris-HCl, 300 mM NaCl, 5 mM EDTA, pH 7.6) for 1 hour. The cells were then washed twice with PBS for 5 minutes. Total cell DNA was counterstained with Hoechst 33342 (5 μg / mL) for 15 minutes, followed by PBS, PBS containing 0.2% Triton X-100, and PBS for 5 minutes each.

[0173] Pulse-chase labeling. A 10 mm diameter coverslip was placed in each well of a 24-well plate. HeLa cells were cultured at 1.2 × 10 cells / mL. 5 Cells were seeded and allowed to stand overnight (22 hours). pulse:The cells were aspirated and incubated with VdU (20 μM) for 16 hours, and again aspirated and incubated with PINK (0, 1, 10 μM, 4 hours). track: The cells were aspirated and treated with DMEM containing EdU (0, 10 μM) for 4 hours. The cells were fixed with 3.7% paraformaldehyde (PFA) for 15 minutes, quenched with 50 mM glycine / NH4Cl for 5 minutes, and washed with PBS for 5 minutes. The cells were permeabilized with PBS containing 0.2% Triton X-100 for 5 minutes at room temperature, on ice for 15 minutes, and washed three times with PBS. DNA was denatured in 2M HCl aqueous solution for 30 minutes, then washed with PBS for 5 minutes, neutralized with 0.1M Na2B4O7·10H2O for 10 minutes, and washed three times with PBS for 5 minutes. Click staining was performed in the dark for 1 hour by incubating the coverslip inverted on a 25 μL solution of PBS containing 10 mM L-sodium ascorbate and 10 μM AF488 azide (Invitrogen, catalog number: A10266), 1 mM CuSO4, 2 mM THPTA, and 10 mM L-sodium ascorbate at a final concentration. The coverslip was washed twice with PBS for 5 minutes, and total cell DNA was counterstained with DAPI (5 μM in PBS) for 15 minutes, washed twice with PBS and H2O, each for 5 minutes. The coverslip was inverted and glued to a microscope slide using Glycergel (11 μL, Dako) and dried overnight. Images were acquired on a confocal microscope. DAPI was excited with a 405 nm laser sampling emission between 410-480 nm, AF488 was excited with a 488 nm laser sampling emission between 495-540 nm, and PINK was excited with a 561 nm laser sampling emission between 600-700 nm.

[0174] Live cell time-lapse imaging. Cells were cultured at 2.0×10 5 The cells were seeded at a density of 10 cells / well in a μ-slide 8-well chamber ( Catalog number: 80826) and allowed to stand overnight. Cells were aspirated and incubated with 100 μM VdU for 23 hours. Cells were aspirated and incubated with 1 μM PINK for 5 hours. Cells were aspirated and fresh DMEM (-phenol red) was added. Images were acquired on a confocal microscope with 30 minute intervals between frames. PINK was excited with a 514 nm laser and the emission was sampled between 525-650 nm.

[0175] Example 3 – Synthetic details

[0176] 3-Bromo-4-(dibromomethyl)benzonitrile (S2)

[0177]

[0178] 3-Bromo-4-(dibromomethyl)benzonitrile was prepared according to a modified literature procedure. [3] 3-bromo-4-methylbenzonitrile (S1, 1.00 g, 5.10 mmol, 1.0 equivalent) is dissolved in CCl4, and NBS (3.63 g, 20.4 mmol, 4.0 equivalent) and DBPO (124 mg, 510 μmol, 0.1 equivalent) are added. The reaction mixture is refluxed for 2 days and filtered. The solid is washed with DCM, and the filtrate is concentrated under reduced pressure to obtain a crude product, which is directly used in the next step. The analytical data is consistent with the literature value. [4]

[0179] 3-Bromo-4-formylbenzonitrile (1)

[0180]

[0181] 3-Bromo-4-formylbenzonitrile was prepared according to a modified literature procedure. [5] Crude 3-bromo-4-(dibromomethyl)benzonitrile (S2, 1.80 g, 5.10 mmol, 1.0 equiv) was suspended in dimethylamine (40% aqueous solution, 15 mL, 13.2 g, 23.0 equiv), and the reaction mixture was stirred at 60 ° C for 90 minutes. The reaction mixture was acidified with HCl (6 M, aqueous solution) and stirred for another 90 minutes at 50 ° C. The aqueous phase was extracted with EtOAc (4 × 8 mL). The combined organic phases were washed with brine (8 mL), dried over MgSO4, filtered and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10% EtOAc / hexane) to obtain 3-bromo-4-formylbenzonitrile (806 mg, 3.84 mmol, 75% over two steps) as a yellow solid. The analytical data were consistent with the literature values. [6]

[0182] 3-Cyano-6-(dimethylamino)acridine (3)

[0183]

[0184] Containing N,N-dimethyl, 1,3-phenylenediamine [7]A flask containing 4-(2-(4-(2-bromo-4-yl)benzonitrile) (2, 50.0 mg, 367 μmol, 1.0 equiv), Pd2(dba)3 (8.40 mg, 9.18 μmol, 0.025 equiv), dppf (10.2 mg, 18.4 μmol, 0.05 equiv) and K2CO3 (102 mg, 794 μmol, 2.0 equiv) was evacuated for 5 minutes and flushed with argon. 1.5 mL of toluene and 3-bromo-4-formylbenzonitrile (1, 92.5 mg, 441 μmol, 1.2 equiv) were added, and the solution was heated to 80° C. for 17 hours. Approximately 1.5 mL of silica gel (SiO2, Silicycle) was added, and the reaction mixture was concentrated under reduced pressure. Purification by flash column chromatography (20→50% EtOAc / hexanes) provided 3-cyano-6-(dimethylamino)acridine (57.7 mg, 233 μmol, 64%) as a red solid.

[0185] 1 H NMR (500MHz, CDCl3) δ [ppm] = 8.47 (s, 1H), 8.38 (s, 1H), 7.89 (d, J = 8.5Hz, 1H), 7.78 (d, J=9.4Hz,1H),7.40(d,J=8.5Hz,1H),7.32(d,J=9.4Hz,1H),7.05(s,1H),3.18(s,6H).

[0186] 13 C NMR (125MHz, CDCl3) δ [ppm] = 152.3, 152.2, 147.9, 135.5, 134.8, 129.9, 129.2, 125.8, 122.9, 122.4, 119.4, 119.3, 113.0, 103.4, 40.5.

[0187] ESI-HRMS:C 16 H 14 N3 + [MH] + Calculated value: 248.1182; experimental value: 248.1176.

[0188] PINK(5)

[0189]

[0190] 3-Cyano-6-(dimethylamino)-acridine (3, 56.7 mg, 229 μmol, 1.0 eq) was dissolved in hydrazine (anhydrous, 1 M in THF, 11.5 mL, 11.5 mmol, 50 eq) and MeCN (312 μL, 2.29 mmol, 10.0 eq) and Zn(OTf)2 (41.7 mg, 115 μmol, 0.5 eq) were added. The reaction mixture was heated to 65 °C for 3 days. NaNO2 (245 mg, 3.55 mmol, 15.5 eq) was added and the pH of the mixture was acidified to 3 with HCl (6 M aqueous solution, 2 mL). (Caution! This step will produce highly toxic nitrous gases). 60 mL of NaHCO3 (saturated aqueous solution) was added and the aqueous phase was extracted with DCM (4×15 mL). The combined organic phases were washed with brine (30 mL), dried over MgSO4, and concentrated under reduced pressure. Purification by flash column chromatography (0→1% MeOH / DCM) gave PINK (4, 32.0 mg, 101 μmol, 44%) as a dark red solid.

[0191] 1 H NMR (500MHz, CDCl3) δ [ppm] = 9.41 (s, 1H), 8.58 (s, 1H), 8.49 (dd, J = 8.8, 1.7Hz, 1H), 8.08 (d, J = 8.7Hz, 1 H), 7.85 (d, J = 9.3Hz, 1H), 7.34 (dd, J = 9.3, 2.5Hz, 1H), 7.18 (d, J = 2.4Hz, 1H), 3.20 (s, 6H), 3.13 (s, 3H).

[0192] 13 C NMR (125MHz, CDCl3) δ [ppm] = 167.3, 164.6, 152.1, 152.0, 149.3, 135.4, 133.0, 129.7, 129.6, 129.2, 126.5, 122.3, 121.1, 119.0, 104.0, 40.5, 21.3.

[0193] ESI-HRMS:C 18 H 17 N6 + [MH] + Calculated value: 317.1509; measured value: 317.1508.

[0194] VdU: 5-vinyl-2'-deoxyuridine (5)

[0195]

[0196] A solution of 5-iodo-2'-deoxyuridine (IdU, 2.00 g, 5.65 mmol, 1.0 equiv), potassium trifluorovinyl borate (vinyl-BF3K, 1.13 g, 8.47 mmol, 1.5 equiv) and K2CO3 (1.56 g, 11.9 mmol, 2.0 equiv) in MeOH (75 mL) was bubbled with argon for 1 hour. Pd(OAc)2 (127 mg, 565 μmol, 0.1 equiv) and triphenylphosphine (PPh3, 296 mg, 1.13 mmol, 0.2 equiv) were added, and the reaction mixture was heated to 70°C for 21 hours. The reaction mixture was concentrated, and VdU was purified by three consecutive rounds of flash column chromatography (0-8% MeOH / DCM). It was obtained as a white solid (971 mg, 3.82 mmol, 68%). The analytical data were consistent with the literature values. [8]

[0197] PINK-VdU-ox(6):

[0198]

[0199] VdU (5, 12.2 mg, 48.0 μmol, 1.0 eq) and PINK (4, 30.2 mg, 95.5 μmol, 2.0 eq) were dissolved in 2 mL (DMF 3:1 CHCl3), and the reaction mixture was heated to 60°C for 7 days. The reaction mixture was concentrated under reduced pressure and purified by flash column chromatography (0-15% MeOH / DCM) to give PINK-VdU-ox (18.9 mg, 35.0 μmol, 73%) as a red solid.

[0200] 1 H NMR (500MHz, methanol-d4) δ [ppm] = 8.72 (s, 1H), 8.26 (s, 1H), 8.05 (s, 1H), 8.02 (d, J = 8.6Hz, 1H), 7.86 (d ,J=9.5Hz,1H),7.74(s,1H),7.63(dd,J=8.6,1.7Hz,1H),7.38(dd,J=9.5,2.5Hz,1H),6.80(s,1H) ,6.19(t,J=6.4Hz,1H),4.15(dt,J=6.8,3.7Hz,1H),3.80(q,J=3.3Hz,1H),3.59(dd,J=13.5,3.1H z,1H),3.19(s,6H),2.79(s,3H),2.13(ddd,J=13.6,6.2,3.8Hz,1H),1.88(dt,J=13.3,6.5Hz,1H).

[0201] 13 C NMR (125 MHz, methanol-d4) δ [ppm] = 163.1, 159.6, 154.4, 151.6, 151.3, 148.2, 144.0, 141.2, 138.6, 134.2, 131.2, 130.9, 130.3, 126.8, 125.7, 123.3, 119.7, 111.4, 101.3, 89.0, 86.7, 71.3, 62.3, 41.7, 40.5, 21.1.

[0202] ESI-HRMS:C 29 H 29 N6O5 + [MH] + Calculated value: 541.2194; measured value: 541.2179.

[0203] DMT-VdU(S3): 5'-O-(4,4-dimethoxytrityl)-5-vinyl-2'-deoxyuridine

[0204]

[0205] 5'-O-(4,4-dimethoxytrityl)-5-vinyl-2'-deoxyuridine was prepared according to a modified literature procedure. [9] 5-vinyl-2'-deoxyuridine (5, 87.0 mg, 342 μmol, 1.0 equivalent) was dissolved in pyridine (2 mL), and DMT-chloride (139 mg, 411 μmol, 1.2 equivalent) was added. The reaction mixture was stirred overnight and concentrated under reduced pressure. Purification by flash column chromatography (30-100% EtOAc / hexane, 1% NEt3) gave 5'-O-(4,4-dimethoxytrityl)-5-vinyl-2'-deoxyuridine (177 mg, 318 μmol, 93%) as an off-white solid.

[0206] The analysis data were consistent with the literature. [9]

[0207] DMT-VdU phosphoramidite (S4): 5'-O-(4,4-dimethoxytrityl)-3'-O-[2-cyanoethyl-(N,N-diisopropylamino)-phosphino]-5-vinyl-2'-deoxyuridine

[0208]

[0209] 5'-O-(4,4-dimethoxytrityl)-5-vinyl-2'-deoxyuridine was prepared according to a modified literature procedure. [9]Freshly distilled DIPEA (63 μL, 46.4 mg, 359 μmol, 2.5 equivalents) was added to a solution of DMT-VdU (S3, 80.0 mg, 144 μmol, 1.0 equivalents) in DCM (1 mL), and the solution was cooled to 0 ° C. 2-cyanoethyl-N, N-diisopropyl chlorophosphoramidite (63 μL, 68.0 mg, 287 μmol, 2.0 equivalents) was added, and the reaction mixture was slowly warmed to room temperature within 2.5 hours. The reaction mixture was directly loaded onto a silica column and purified by flash column chromatography (60% EtOAc / hexane; 0.5% NEt3) to obtain 5'-O-(4,4-dimethoxytrityl)-5-vinyl-2'-deoxyuridine (53.0 mg, 70.0 μmol, 49%) as an off-white solid. The analytical data were consistent with the literature. [9]

[0210] Scheme 3: Synthesis of 10-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)propyl)-3,6-bis(dimethylamino)acridinium trifluoroacetate via 10-(3-cyanopropyl)-3,6-bis(dimethylamino)acridinium iodide intermediate.

[0211]

[0212] 10-(3-Cyanopropyl)-3,6-bis(dimethylamino)acridinium iodide (AO-495-nitrile)

[0213]

[0214] Method 1: the 25mL sealable tube containing 63.5mg acridine orange (239μmol) and stirring bar is evacuated under vacuum and refilled with argon three times.Afterwards, 3.0mL toluene and 52μL 4-iodobutyronitrile (487μmol) are added to the tube.The suspension is refluxed at 120 ℃ for 24 hours and the reaction process is monitored by crude NMR.After the reaction, the mixture is evaporated to dryness under reduced pressure, and washed by 3×8mL DCM (until the supernatant color no longer changes).Precipitate is dried under vacuum to obtain the product (39.9mg, 86.7μmol, 36%) in an orange to red powder.

[0215] Method 2: A 0.5-2 mL microwave reaction tube containing 59.7 mg acridine orange (225 μmol), 1 mL acetonitrile, 50 μL 4-iodobutyronitrile (469 μmol) and a stirring bar was mixed under microwave at 150 ° C for 3 hours. After the reaction, the mixture was evaporated to dryness under reduced pressure and washed with 3×8 mL DCM. The precipitate was dried under vacuum to obtain the product (47.6 mg, 103 μmol, 46%) as a dark red powder.

[0216] 1 H NMR (500MHz, DMSO-d6): δ [ppm] = 8.84 (s, 1H), 7.97 (d, J = 9.3Hz, 2H), 7.31 (dd, J = 9.3, 2.1Hz, 2H), 6.6 8(d,J=2.2Hz,2H),4.81(t,J=8.3Hz,2H),3.30(s,12H),2.89(t,J=6.8Hz,2H),2.21(p,J=7.0Hz,2H)

[0217] 13 C NMR (125MHz, DMSO) δ [ppm] = 156.1, 143.7, 142.8, 133.7, 120.9, 117.0, 114.9, 92.7, 45.8, 40.9, 21.8, 14.6.

[0218] HRMS(ESI):C 21 H 25 N4 + [M] + m / z calculated: 333.2074; found: 333.2073; I - Calculated m / z: 126.9050; found: 126.9037.

[0219] (10-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)propyl)-3,6-bis(dimethylamino)acridine trifluoroacetate) ("AO-495-TET-py")

[0220]

[0221] The 5mL reaction vial containing 20.0mg AO-495- nitrile (43.4μmol), 8.1mg zinc trifluoromethanesulfonate (22μmol), 46.0mg 2- pyridine carbonitrile (442μmol) and stirring bar is evacuated under vacuum and refilled with argon three times. Afterwards, 80μL anhydrous hydrazine (2.6mmol) is added to the pipe with a syringe. The mixture is sealed and stirred overnight and completely dried at 60°C. Another 80μL anhydrous hydrazine is added to the pipe, and stirred for 24 hours at 60°C. The resulting mixture is dissolved with 2mL DMSO and transferred to a conical flask equipped with 15mL 0.5M sodium nitrite. The water containing 5M acetic acid is slowly added to the flask, while stirring, until no longer producing bubbles and pH=3 (note! This step will produce toxic nitrogen oxide gas, this step can only be carried out in a well-ventilated fume hood). Potassium bicarbonate is added to adjust pH to>7. The mixture was extracted three times with 20 mL DCM and evaporated under reduced pressure. The resulting solid was dissolved in water containing 20% ​​acetonitrile, filtered and purified by HPLC (32-40% MeCN, water containing 0.1% TFA). The product was freeze-dried by fractions, and an orange-red solid (2.2 mg, 3.8 μmol, 9%) was obtained.

[0222] 1 H NMR (500MHz, CD3CN) δ [ppm] = 8.94 (d, J = 4.9Hz, 1H), 8.60–8.50 (m, 2H), 8.11 (td, J = 7.8, 1.7Hz, 1H), 7.85 (d, J = 9.3Hz, 2H), 7.68 (ddd, J = 7.6, 4.7, 1.1Hz, 1H), 7.21 (dd, J = 9.3, 2.1Hz, 2H), 6.81 (d, J = 2.2Hz, 2H), 4.81 (t, J = 8.5Hz, 2H), 3.82 (t, J = 6.6Hz, 2H), 3.34 (s, 11H), 2.75 (p, J = 6.6Hz, 2H).

[0223] 13 C NMR (125MHz, CD3CN) δ [ppm] = 164.1, 156.0, 150.7, 143.1, 142.9, 137.7, 133.0, 126.5, 124.0, 117.0, 114.4, 92.5, 46.8, 40.3, 31.3, 21.6.

[0224] HRMS(ESI)C 27 H 29 N8 + [M +] m / z calculated: 465.2510; found: 465.2501

[0225] The absorbance and fluorescence intensity (excitation at 488 nm) of AO-495-TET-py in different solvents (water, PBS and acetonitrile) were measured in a quartz cuvette (light path 1 cm) using a SpectraMax M5 plate reader (Molecular Devices, Sunnyvale, CA, USA). For AO-495-tet-py, samples for photophysical characterization were prepared directly from the stock solution (10 mM in DMSO) by serial dilution. For the reaction of AO-495-tet-py with 5-norbornene-2-methanol, 20 μL of AO-495-tet-py stock solution (in DMSO) was mixed with 2 μL of 5-norbornene-2-methanol (80× excess) at room temperature for 24 h to ensure complete conversion. 3 μL of the reaction mixture was diluted into the specified solvent to make an 8 mM solution for subsequent photophysical characterization. Ethanol containing rhodamine 6G was used as a fluorescence standard See Table 2 and Fig.15 .

[0226] Table 2. Photophysical properties of AO-495-tet-py before and after the reaction with 5-norbornene-2-methanol were obtained.

[0227]

[0228] N,N-Dimethyl-6-(6-(pyridin-2-yl)-1,2,4,5-tetrazine-3-yl)acridin-3-amine (pyridinyl-PINK)

[0229]

[0230] The tube containing 3-cyano-6-(dimethylamino)-acridine (15.0 mg, 60.7 μmol, 1.0 equiv), 2-cyanopyridine (63.2 mg, 58.4 μL, 607 μmol, 10 equiv) and zinc (II) trifluoromethanesulfonate (11.0 mg, 30.3 μmol, 0.5 equiv) was evacuated for 5 minutes and refilled with argon. Hydrazine (1 M in THF, 3.03 mL, 3.03 mmol, 50 equiv) was added, the tube was sealed, and the reaction mixture was heated to 60 ° C for 5 days. The reaction mixture was concentrated under reduced pressure and redissolved in 10 mL H2O 1:1 MeOH. Sodium nitrite (64.9 mg, 940 μmol, 15.5 equiv) was added and the pH of the reaction mixture was acidified to 3 by adding HCl (6M aqueous solution). The pH of the solution was neutralized to 9 by adding NaHCO3. The aqueous phase was extracted with DCM (3×20 mL), and the combined organic phases were washed with brine (20 mL), dried over MgSO 4 and concentrated under reduced pressure. Purification by flash column chromatography (0-2% MeOH / DCM) gave pyridinyl-PINK (8.8 mg, 23.2 μmol, 38%) as a dark purple solid.

[0231] 1 H NMR (500MHz, CDCl3) δ [ppm] = 9.53 (s, 1H), 8.99 (d, J = 3.8Hz, 1H), 8.75 (d, J = 7.8Hz, 1H), 8.62–8.56 (m, 2H), 8.11 (d, J = 8.7Hz, 1H) ,8.04–7.98(m,1H),7.85(d,J=9.2Hz,1H),7.57(ddd,J=7.6,4.7,1.2Hz,1H),7.34(dd,J=9.4,2.4Hz,1H),7.19(d,J=2.4Hz,1H).

[0232] ESI-HRMS:C 22 H 17 N7 + [MH] + Calculated value: 380.1618; measured value: 380.1616.

[0233] 3-(Dimethylamino)-10-methyl-6-(6-methyl-1,2,4,5-tetrazin-3-yl)acridinium-10-cation chloride

[0234]

[0235] PINK (4.50 mg, 14.2 μmol, 1.0 eq) was dissolved in 1 mL of toluene and MeI (221 μL, 3.56 mmol, 250 eq) was added. The reaction mixture was heated to reflux in a sealed pressure flask for 19 hours. The reaction mixture was centrifuged and the supernatant was decanted. The crude product was loaded into 1x4-200 ion exchange resin (700 mg, activated with 1M HCl aqueous solution and balanced with H2O) and eluted with 1:1 H2O / MeOH mixture. The fractions containing the product were concentrated by freeze drying. Purification by column chromatography (5→10% MeOH / DCM) gave XXX (5.00 mg, 13.6 μmol, 96%) as a dark red solid.

[0236] 1 H NMR (500MHz, MeOH-d4): δ [ppm] = 9.39 (s, 1H), 9.18 (s, 1H), 8.79 (d, J = 8.5Hz, 1H), 8.46 (d, J = 8.5Hz, 1H), 8.16(d,J=9.5Hz,1H),7.68(dd,J=9.6,2.2Hz,1H),6.88(s,1H),4.51(s,3H),3.50(s,6H),3.14(s,3H).

[0237] 13 C NMR (125 MHz, methanol-d4) δ [ppm] = 168.3, 163.3, 157.9, 145.3, 143.8, 140.6, 138.4, 133.4, 132.2, 125.1, 123.9, 122.8, 119.1, 115.4, 91.9, 40.2, 35.7, 19.9.

[0238] ESI-HRMS:C 19 H 19 N6 + [M] + Calculated value: 331.1666; measured value: 331.1651.

[0239] 3-(Dimethylamino)-6-(6-methyl-1,2,4,5-tetrazin-3-yl)acridinium-10-cation chloride (PINK-hydrochloride)

[0240]

[0241] PINK (5.00 mg, 15.8 μmol, 1.0 eq) was suspended in 600 μL MeCN and HCl (100 mM aqueous solution, 600 μL, 60 μmol, 3.8 eq) was added, at which point the solution turned purple and formed a precipitate. The supernatant was removed and the residual solvent was removed by lyophilization to obtain PINK-hydrochloride as a dark red solid. The yield was not recorded.

[0242] 1 H NMR (400MHz, DMSO-d6): δ [ppm] = 15.29 (s, 1H), 9.36 (s, 1H), 9.11 (s, 1H), 8.60 (d, J = 8.6Hz, 1H), 8.47 (d, J=8.6Hz,1H),8.19(d,J=9.6Hz,1H),7.68(dd,J=9.6,2.4Hz,1H),6.91(s,1H),3.34(s,6H),3.08(s,3H).

[0243] Example 4 - Therapeutic Uses

[0244] Toxicity studies using resazurin to measure cellular respiratory activity showed that low micromolar concentrations of PINK were not toxic to cells even at longer incubation times ( Fig.16 ). Vinyl nucleosides such as VdU or VdA are also fairly nontoxic to cells. However, if cells are exposed to both compounds at the same time, the products of the click reaction introduce a large number of covalent modifications to the DNA. Similar to classical chemotherapeutic DNA alkylating agents, such modifications can lead to genomic instability and may ultimately lead to cell death.

[0245] A binary chemotherapy approach using vinyl nucleosides such as VdU and VdA and the tetrazine-substituted bioorthogonal intercalator PINK (structure provided below) to form DNA monoadducts was investigated. This approach has been tested for the treatment of acute myeloid leukemia (AML).

[0246]

[0247] AML is a cancer of the blood and bone marrow; it is characterized by the uncontrolled differentiation and proliferation of immature myeloid cells. 13,14 The AML is the most common form of acute leukemia in adults. 15 AML is a highly heterogeneous cancer, making its diagnosis and treatment challenging. 13 Therefore, AML is classified using the French-American-British system (FAB) based on the level at which different DNA abnormalities occur at the level of differentiation of myeloid progenitor cells. 13,16 M0 is at a lower level of differentiation, the myeloblast level, while M7 is at the highest level of differentiation, the megakaryocyte level.13,16 Currently, the first-line treatment for AML is a "7+3" day regimen, which consists of continuous infusion of cytarabine (AraC) for 7 days, followed by continuous infusion of anthracyclines such as daunorubicin or idarubicin for 3 days. 13 Unfortunately, this treatment is mainly administered to patients with good / intermediate-risk prognosis and younger patients, as their risk of treatment-related mortality tends to be lower. 13,17 Elderly AML patients over 65 years of age often tolerate this intensive chemotherapy treatment poorly because they have a worse cytogenetic risk profile (e.g., FLT3 mutation), generally have a worse prognosis, and they tend to be more susceptible to treatment-related toxicities. Therefore, elderly patients often do not have optimal AML treatment. 13 By exploring this DNA monoadduct-forming binary chemotherapy approach, using vinyl-nucleosides (VdU / VdA) in combination with PINK to treat AML, we hope to exploit the selectivity of the bioorthogonal reaction to effectively kill tumors at lower drug concentrations, thereby limiting treatment-related toxicity and, in the future, providing an alternative treatment for patients who are not suitable for the standard "7+3" day cytarabine and anthracycline regimen. This binary approach has great potential, as previous studies by Luedtke's group have shown that VdU is relatively less toxic in several cancer cell lines compared to other modified uridine nucleosides. 18 Furthermore, currently unpublished studies from the Luedtke group have shown that PINK is very efficient at inserting into DNA and has high reaction kinetics with incorporated vinyl-modified nucleosides such as VdU. 19

[0248] Three leukemia cell lines were chosen to test this new dual chemotherapy approach for the treatment of AML: MV4-11, an AML cell line with a FLT3 mutation that promotes overgrowth of abnormal myeloid cells, MOLM-13, and Jurkat cells. 20,21 FLT3 mutations are present in approximately 20% of AML cases and are associated with a poor prognosis. 20,21 MOLM-13 is another AML cell line derived from AML-M5a, known as acute monoblastic leukemia (AMoL). 22,23 AML-M5a accounts for 5-8% of AML cases. 23 An advantage of using MV4-11 and MOLM-13 is that they can also be used as human xenograft models to study AML in mice. 24 Jurkat is a T-acute lymphoblastic leukemia (T-ALL) cell line and was used to evaluate the specificity of the binary approach to treat AML. 25

[0249] Initial experiments were performed in HeLa cells. Cells were treated with increasing concentrations of VdU (0-100 μM) for 16 h, followed by treatment with fixed concentrations of PINK (0, 5, 10 μM) for 5 h. Toxicity was assessed using the Alamar Blue Assay after 24 and 72 h. Fig.17 Cells were treated with a fixed concentration of VdU (0-10, 20 μM) for 16 h, followed by treatment with increasing concentrations of PINK (0-100 μM) for 5 h. Toxicity was assessed using the Alamar Blue assay after 24 and 72 h ( Fig.18 ).

[0250] The resazurin assay was performed to test the effectiveness of VdU, VdA and PINK alone in MV4-11 and Jurkat. The resazurin reduction assay is a colorimetric assay that determines the viability of cells based on their metabolic activity. 26 When cells are alive, resazurin is reduced by NADH through cellular respiration to its resorufin form, which appears pink and is highly fluorescent. 26 When cells die or their metabolism is impaired, resazurin remains in its oxidized form, which is blue and has low fluorescence. 26 The cells can then be imaged using a spectrometer to determine their survival after each treatment. Fig.19 Inhibitory dose response curves were generated as shown. From these dose response curves, the half maximal inhibitory concentration (IC) of each compound in both leukemia cell lines was found after 24 and 72 hours. 50 ), as shown in Table 3.

[0251] Table 3. IC of VdU, VdA and PINK after 24 and 72 h incubation in MV4-11 and Jurkat cells 50 The values ​​were calculated using nonlinear regression (four parameter inhibitory dose response curves) with GraphPad Prism 6 software and interpolated at 50% cell viability.

[0252]

[0253] according to Fig.19 and Table 3 , PINK showed acute toxicity in both MV4-11 and Jurkat, with the average IC 50 For vinyl-nucleosides, VdA showed mild acute toxicity after 24 and 72 h, with IC 50 VdU showed relatively no toxicity in both cell lines, with IC 50Above 1000 μM, except for Jurkat, which showed some slight toxicity after 72 h (IC 50 =52 μM). Overall, VdU is a good candidate for combination therapy with PINK due to its low toxicity in both leukemia cell lines.

[0254] Based on the single toxicity experiments, the optimal subtoxic concentration of PINK for the combined toxicity assay was determined to be 1 μM. To test how effective the vinyl-nucleosides (VdU / VdA) combined with PINK were in killing cells compared to the vinyl-nucleosides alone, an additional resazurin reduction assay was performed. After 48 h, inhibitory dose response curves of the combined and individual vinyl-nucleosides and the corresponding IC 50 ,like Fig. 20 and Table 4 respectively.

[0255] Table 4. IC50 values ​​of VdU / VdA alone and in combination with 1 MPINK (added 24 hours after nucleoside addition) in MV4-11, Jurkat and MOLM-13 cells after 48 hours of incubation. The values ​​were calculated using nonlinear regression (four parameter inhibitory dose response curves) with GraphPad Prism 6 software and interpolated at 50% cell viability.

[0256]

[0257] according to Fig. 20 In MV4-11 and Jurkat, a huge difference in cell viability between VdU alone and in combination with VdU was seen with as little as 1 μM PINK. As shown in Table 4, the IC values ​​of VdU+PINK in MV4-11 and Jurkat were 50 The values ​​were 0.50 μM and 1.1 μM, respectively, which were significantly lower than the IC values ​​of VdU alone in MV4-11 (60 μM) and Jurkat (58 μM). 50 This suggests that the VdU+PINK combination is more effective than VdU alone in killing these two cell lines. A similar toxicity trend of VdU+PINK was observed in MOLM-13, although to a lesser extent than in MV4-11 and Jurkat cells, where the IC 50 The results showed that MOLM-13 cells were more susceptible to VdU than the other two leukemia cell lines. For VdA+PINK, there was almost no difference in cell viability compared with VdA alone. 50 =25 μM) and Jurkat (IC 50=46 μM), MOLM-13 was more susceptible to VdA alone (IC 50 =3 μM). There was little difference in cell viability between VdA and VdA + PINK, suggesting that VdA may not be incorporated into DNA, unlike VdU. Therefore, VdA may induce cytotoxicity through a different mechanism than VdU, such as by depleting the cellular ATP pool, as previously reported for other modified adenosine nucleosides. 27

[0258] To determine whether leukemic cells could be treated with vinyl-nucleosides and PINK simultaneously, rather than adding them sequentially, another resazurin reduction assay was performed using the same concentrations of each compound as in the previous combination experiment, where instead vinyl-nucleosides and PINK were added on the same day, rather than on two consecutive days. After 48 h of incubation, the corresponding IC values ​​of vinyl-nucleosides alone or in combination with 1 μM PINK in MV4-11 and Jurkat cells were calculated. 50 The inhibitory dose response curve was generated by Fig.21 and Table 5 respectively.

[0259] Table 5. IC of VdU / VdA alone and in combination with 1 μM PINK (nucleosides and PINK added on the same day) after 48 h incubation in MV4-11, Jurkat cells 50 The values ​​were calculated using nonlinear regression (four parameter inhibitory dose response curves) with GraphPad Prism 6 software and interpolated at 50% cell viability.

[0260]

[0261] according to Fig.21 , observed that VdU+PINK was less effective in killing MV4-11 and Jurkat cells when VdU and PINK were added simultaneously rather than sequentially ( Fig. 20 ). In addition, as shown in Table 5, when VdU and PINK were added simultaneously, the IC of VdU+PINK in MV4-11 (4.7 μM) and Jurkat (44 μM) was 50 The values ​​were higher than the IC values ​​when they were added sequentially in MV4-11 (0.50 μM) and Jurkat (1.1 μM). 50The values ​​were much larger (Table 4). Adding the two compounds simultaneously may be less effective in killing cells for two reasons: 1) VdU and PINK may react before VdU incorporation, which would not induce DNA alkylation, and 2) because PINK may be cleared from the cells before reacting with VdU to alkylate DNA. Interestingly, the toxicity level of the combination of VdA and PINK remained approximately the same as that of VdA alone in both simultaneous and sequential additions. This further confirms that Fig. 20 4, and supports the hypothesis that VdA is not incorporated into DNA but instead induces cytotoxicity through another mechanism other than VdU+PINK; potentially, by inhibiting enzymes involved in ATP synthesis, leading to depletion of cellular ATP pools.

[0262] To confirm the cytotoxicity results obtained from the resazurin assay, a cell counting assay was performed using trypan blue. Trypan blue is an azo dye that will differentiate live from dead cells, as dead cells will take up the dye, while live cells will remain white. 28 According to the resazurin assay, and after some optimization based on the sensitivity of the cells to vinyl-nucleosides, it was determined that 10 μM and 5 μM vinyl-nucleosides alone and in combination with 1 μM PINK were two interesting concentrations to explore in Jurkat, MV4-11 and MOLM-13 cells. Cells were incubated overnight with 5 μM or 10 μM of each nucleoside (day 0), and 1 μM PINK was added to the combination and PINK alone 24 hours after the nucleoside incubation (day 1). Cells were counted every 24 hours. After continuous daily incubation with the corresponding treatment, a chart of relative cell viability and cell concentration was generated, as shown in Figure 2. Fig.22A Visible in -F.

[0263] according to Fig.22A As expected from the resazurin assay, 10 μM VdU in combination with 1 μM PINK was more effective than VdU alone in killing Jurkat cells, as seen after day 3 (48 h combined incubation). In contrast, 10 μM VdA in combination with 1 μM PINK had relatively low toxicity in Jurkat cells, similar to VdA alone, as demonstrated by the relatively high viability of Jurkat cells. Fig. 22B , it was observed that 10 μM VdU+PINK was very effective in inhibiting cell growth. This suggests that the combination of VdU and PINK appears to cause cell death in Jurkat cells, as expected from the resazurin assay. Interestingly, VdU alone had a greater effect on cell growth than expected from the resazurin assay, suggesting that VdU itself may cause cell cycle arrest, but induces cell death when combined with PINK.

[0264] In MV4-11 cells, according to Fig. 22C , it was observed that 5 μM VdU in combination with 1 μM PINK killed cells more effectively than VdU alone, as seen after day 3. In contrast, 5 μM VdA in combination with 1 μM PINK was as ineffective in killing cells as VdA alone. Fig.22D , noting that 5 μM VdU+PINK was very effective in inhibiting cell growth in MV4-11. This suggests that VdU combined with PINK induces cell death in MV4-11. Surprisingly, VdU significantly inhibited cell growth, suggesting that VdU itself may induce cell cycle arrest in MV4-11 cells. In addition, VdA combined with PINK also inhibited cell growth, suggesting that VdA+PINK may induce cell cycle arrest in MV4-11.

[0265] In MOLM-13 cells, according to Fig.22E , it was observed that 5 μM VdU in combination with 1 μM PINK was more effective in killing cells than VdU alone, as seen after day 3. Interestingly, 5 μM VdA in combination with 1 μM PINK was more effective in killing cells than VdA alone. Fig.22F , noting that both the combination of VdU and PINK and the combination of VdA and PINK significantly inhibited cell growth. This suggests that both VdU+PINK and VdA+PINK appear to induce cell death. Interestingly, both VdU and VdA appear to inhibit cell growth, suggesting that VdU and VdA themselves may induce cell cycle arrest in MOLM-13.

[0266] Overall, in all three cell lines, the combination of VdU with PINK appears to induce cell death. In contrast, the combination of VdA with PINK appears to induce cell cycle arrest in Jurkat and MV4-11, but induces cell death in MOLM-13. Interestingly, in all three leukemia cell lines, it was noted that VdU alone appears to induce cell cycle arrest, but when combined with PINK, induces cell death. More cell counting experiments at smaller / more optimized nucleoside concentrations are needed to confirm these findings. Nonetheless, the dual cytotoxic mechanism of VdU will be an interesting therapeutic feature to explore in AML cell lines that are resistant to AraC treatment due to overexpression of cytidine deaminase, as it converts cytosine bases to uracil bases, which renders the drug ineffective in killing tumors, likely because it induces cell cycle arrest. 29In the future, this resistance mechanism could potentially be used to treat AML patients who have become resistant to AraC therapy. This could be done by first administering VdC, which is fairly inactive on its own, but which could potentially be converted to VdU by cytidine deaminase and then incorporated into DNA, where it reacts with PINK to alkylate the DNA and induce cell death.

[0267] Finally, to determine how synergistic the combination of VdU and PINK was in the three leukemia cell lines, another resazurin reduction assay was performed in which VdU and PINK were maintained at a constant 5:1 ratio. The data generated from this resazurin assay were analyzed using the synergy model developed by Chou and Talalay 30 The synergistic effect model was based on the median effect dose (IC 50 ) and the slope of the drug response curve, as shown in Equation II:

[0268]

[0269] The model quantitatively measures the synergy of drug combinations by generating a combination index (CI). 30 A CI below 1 indicates a synergistic interaction, meaning that the effect of the drug combination is greater than the effect of the two drugs combined alone. 30 A CI equal to 1 indicates an additive interaction, which means that the effect of the drug combination is equal to the effect of the two drugs combined individually. 30 A CI above 1 indicates an antagonistic interaction, meaning that the effect of the drug combination is less than the effect of either drug alone. 30 Fa-CI panels were generated for the VdU+PINK combination in MV4-11, Jurkat, and MOLM-13, where the CI at a specific fraction of affected cells (Fa) was determined for each cell line, as shown in Fig.23A -C. In addition, a dose reduction index (DRI) was generated for each compound and determined at the specific Fa of each cell line, as can be seen in Table 6. Where DRI above 1 is a favorable dose reduction, and DRI below 1 is an unfavorable dose reduction. 30

[0270] Table 6. Dose reduction index (DRI) of specific fraction of affected cells (Fa) for VdU and PINK in MV4-11, Jurkat and MOLM-13 cells when treated with VdU+PINK combination at a constant 5:1 ratio.

[0271] Cell Type Fa Concentration VdU [μM] Concentration PINK [μM] V D DRIPINK MV4-11 [0.25-0.35] 59 3.2 34 times 10 times Jurkat 0.50 3.7 1.1 3 times 5 times MOLM-13 0.50 4.2 1.2 2 times 4 times

[0272] according to Fig.23A, it was observed that VdU combined with PINK was highly synergistic in MV4-11, as there was a significant Fa range of 0.25 to 0.35 (where 25% to 35% of cells were killed), CI = 0.157. Fig. 23B In Jurkat cells, a huge synergistic effect between VdU and PINK was also observed, but to a lesser extent than in MV4-11. At a significant Fa of 0.50 (50% of cells were killed), CI = 0.486. Fig.23C , the synergistic effect of VdU+PINK in MOLM-13 was the smallest, with a CI of 0.684 when the significant Fa was 0.50.

[0273] From Table 6, it is noted that a concentration of 59 μM is required to kill 25% to 35% of MV4-11 with VdU alone, while 3.2 μM is required with PINK alone. The concentrations of VdU and PINK administered can be reduced by 34-fold and 10-fold, respectively, by the VdU+PINK combination. A concentration of 3.7 μM is required to kill 50% of Jurkat cells with VdU alone; however, 1.1 μM is required with PINK alone. The concentrations of VdU and PINK administered can be reduced by 3-fold and 5-fold, respectively, by the VdU+PINK combination. Finally, a concentration of 4.2 μM is required to kill 50% of MOLM-13 cells with VdU alone; while 1.2 μM is required with PINK alone. The concentrations of VdU and PINK administered can be reduced by 2-fold and 4-fold, respectively, by the VdU+PINK combination.

[0274] Overall, the combination of VdU and PINK was found to have significant synergy in all three cell lines, with the strongest in MV4-11 and the smallest in MOLM-13. Compared with other combination treatments (such as AraC and Aplidin) tested for different types of acute lymphoblastic leukemia (ALL), the synergy of VdU and PINK in MV4-11 was significantly more prominent in the tested ALL cell lines, and for Jurkat, it was equal to the synergy of AraC+Aplidin. 31,32 Furthermore, the highly synergistic interaction between VdU and PINK contributed to the large DRIs observed in all three leukemia cell lines tested, with MV4-11 having the highest DRI and MOLM-13 having the lowest DRI for both VdU and PINK. Thus, the combination of VdU and PINK may be beneficial in reducing treatment-related toxicity by reducing the dose of each compound required for clinically effective tumor killing.

[0275] According to these findings, VdU and PINK combination show as a good candidate for the binary chemotherapy method for AML and T-ALL. This is reflected by the huge synergy of VdU and PINK in all three kinds of leukemia cell lines tested, because independent VdU is relatively nontoxic. In addition, the high DRI of the combination can reduce the dosage of PINK and VdU given, and realizes important cell killing simultaneously, which may help to reduce the toxicity related to clinical treatment. In the future, the combination of VdU and PINK should be tested in MV4-11 and MOLM-13 xenograft models and other AML cell lines. Some other experiments should also be carried out to confirm the discovery, such as the cell counting experiment carried out with more optimized nucleoside concentration, the Western blot for screening DNA damage markers and the cell cycle analysis experiment carried out by flow cytometry. In contrast, for the binary chemotherapy method for AML and T-ALL, VdA and PINK combination seem to be less effective candidates. This is reflected as the synergy level between VdA and PINK is low, and the toxicity of VdA itself is significantly higher than the toxicity of VdU. Furthermore, the combination of VdA and PINK did not seem to be effective in killing the leukemia cells tested, as the combination could induce cell cycle arrest in MV4-11 and Jurkat cells.

[0276] Experimental Section

[0277] Cell culture.MV4-11, MOLM-13 and Jurkat cells were cultured in RPMI at 37°C, 5% CO2, supplemented with 10% FBS, 1% MEM non-essential amino acid solution and 1% penicillin-streptomycin. Cells were grown to confluence and passaged once every 2 days (MV4-11) and 3 days (MOLM-13 and Jurkat) at a split ratio of 1:10. Cells were counted using a BIO RADTC20 cell counter to determine seeding density.

[0278] Resazurin single toxicity assay.MV4-11 and Jurkat cells were seeded in 96-well plates at a density of 20 000 cells / well in triplicate. Cells were treated individually with each nucleoside (VdU / VdA) at a concentration of 1000-0 μM diluted in fresh medium in a 1:4 serial dilution (-0.01% DMSO). Cells were treated individually with PINK at a concentration of 100-0 μM diluted in fresh medium in a 1:2 serial dilution (-1% DMSO). Cells were then incubated for 24 and 72 hours. 10 μL of 870 μM Resazurin was added to each well and incubated for 2-5 hours. After incubation, the fluorescence intensity at 590 nm (excitation = 560 nm) was measured using a SpectraMax M5 plate reader.

[0279] Resazurin combination toxicity assay.MV4-11, MOLM-13 and Jurkat cells were seeded in 96-well plates at a density of 20,000 cells / well, 25,000 cells / well and 30,000 cells / well, respectively, in triplicate.For sequential addition, cells were first treated with 500-0 μM nucleosides (VdU / VdA) diluted in fresh culture medium with 1:4 serial dilutions (~0.01% DMSO), and incubated overnight.24 hours after adding nucleosides, cells were treated with 1 μM PINK (~1% DMSO) diluted in fresh culture medium, and incubated for 24-72 hours.As described above, cells were treated with resazurin, incubated and measured.For simultaneous addition, cells were treated with nucleosides and PINK at the same concentrations as described above on the same day, and incubated for 24-72 hours.

[0280] Resazurin Chou-Talalay optimization toxicity assay.MV4-11, MOLM-13 and Jurkat cells were seeded in 96-well plates at a density of 20,000 cells / well, 25,000 cells / well and 30,000 cells / well, respectively, in triplicate.First, cells were treated with 500-0 μM VdU diluted in fresh culture medium with 1:4 serial dilutions (~0.01% DMSO) and in combination with it, and incubated overnight.24 hours after adding nucleosides, cells were treated with 100-0 μM PINK diluted in fresh culture medium with 1:4 serial dilutions (~1% DMSO) or in combination with it, and incubated for 72 hours.The ratio of the VdU and PINK of the combination was a constant 5:1.As described above, cells were treated, incubated and measured with Resazurin. Data were processed and analyzed in the CompuSyn program for running the Chou-Talalay synergy analysis. 33

[0281] Cell counting assay. Jurkat, MV4-11 and MOLM-13 cells were seeded in 12-well plates at a density of 500 000 cells / mL, 175 000 cells / mL and 375 000 cells / mL, respectively, in duplicate. Cells were incubated overnight in fresh culture medium containing 10 μM (Jurkat) or 5 μM (MV4-11 and MOLM-13) each nucleoside (~ 0.01% DMSO). 24 hours after adding nucleoside, 1 μM PINK (~ 0.01% DMSO) was added to the corresponding wells and incubated for 24 hours. Cells were counted using a cell counter (BIO RAD TC20) to determine the percentage and cell concentration of viable cells. Trypan blue was used to count cells every 24 hours at a ratio of 1: 1.

[0282] Example 5-5-VdU and PINK combination study

[0283] The combined effects of 5-VdU (VdU) and PINK on cells were investigated (see Figure 24-32 ). The 5-VdU / PINK combination worked in two additional cell lines, H1299 (a human lung cancer cell line) and KPC (a mouse pancreatic cell line). These studies show that the compound exhibits functionality in cell lines derived from two additional organs and one additional species.

[0284] The synergistic lethality of 5-VdU and PINK was demonstrated in confluent growth assays of H1299 cells and mouse KPC cells, while Western blot analysis showed that 5-VdU and PINK had synergistic effects on DNA damage and cell death markers in these cells. Experiments were performed in triplicate (see Fig.24 ).

[0285] Combined effects of 5-VdU and PINK on the cell cycle. FACS analysis of H1299 cells treated with 5-VdU / PINK showed that cell cycle progression was severely affected upon combined treatment. Time course cell cycle analysis of H1299 cells showed that the cell cycle profile was mostly unperturbed following treatment with either 5-VdU or PINK alone. When the two drugs were combined, an accumulation of cells in the S phase at 48 hours after initial treatment and an accumulation of cells at the G2 / M boundary at 72 hours was observed (see Fig.25 5-VdU and PINK showed synergistic lethality in H1299 cells, and 5-VdU / PINK induced apoptosis, reaching a peak at 72 hours after the start of treatment. (See Fig.26 The percentage of H1299 cells undergoing apoptosis was determined by Apotracker TM Green staining indicated induction after treatment with 1 μM 5-VdU and 10 μM PINK at different time points. H1299 cells were seeded at 20% density and treated with the indicated concentrations of 5-VdU and PINK. One day after the last treatment, the medium was changed. The cells were then stained with ApotrackerTM Green dye and Hoechst. Images were taken 72 hours after the first treatment. The images taken at 72 hours are shown in Fig.30 (C) H1299 cells were treated with 1 μM 5-VdU for 24 h and then with 5 μM PINK for an additional 24 h.

[0286] 5-VdU incorporation in rapidly proliferating cells in vivo. C57BL / 6J mice were intraperitoneally injected with 200 μl 10% DMSO in 1x sterile PBS as a vehicle or 500 mg / kg 5-VdU over the next two days and then sacrificed. The experimental group size was n=6. (A) Bone marrow was explanted, fixed, permeabilized and stained with PINK for 4 hours at room temperature. The nuclei were counterstained with Hoechst33342. When compared to the control, strong nuclear signals can be observed in the PINK-treated samples. (B) Small intestine and (C) spleen tissues were paraffin-embedded and the slides were then stained with PINK for 4 hours at room temperature. The nuclei were counterstained with Hoechst33342. When compared to the control, specific nuclear signals can be seen in the PINK-treated samples. (See Fig. 27 ) High concentrations of 5-VdU are non-toxic to mice and are incorporated into rapidly dividing cells in vivo. These cells can be stained with PINK in FFPE sections = in vivo DNA replication / proliferation reporter gene.

[0287] 5-VdU / PINK click chemistry in mouse xenografts. KPC subcutaneous tumors were generated on both sides of the syngeneic C57BL / 6J mouse model. When the tumor size was approximately 200 mm3, the animals were injected intraperitoneally with 200 μl of 10% DMSO in 1x sterile PBS as vehicle or 500 mg / kg 5-VdU on the following two days, and then 100 μl of 5% DMSO, 30% PEG400 in 1x sterile PBS as vehicle or 20 mg / kg PINK was injected into the tumor, each injected into the left or right tumor of the same animal as an internal control ( Fig.30 , B). The experimental group size was n=3. The animals were sacrificed 6 hours after injection and the transplanted tumors were embedded in paraffin. The nuclei were counterstained with Hoechst33342. PINK reservoirs could be identified at 5x magnification. Specific nuclear signals could be detected only in animals injected with 5-VdU, but not in animals injected with DMSO. (See Fig.28 ) Results showed that tumors from 5-VdU treated mice showed positive nuclei after intratumoral injection of PINK. The reaction was carried out in vivo.

[0288] The combined effect of 5-VdU / PINK was independent of the p53 mutation status of the cells. Fig.29 5-VdU / PINK induces synthetic lethality in HCT116 p53+ / + cells (B) as well as in the isogenic HCT116 p53- / - cell line (C). Experiments were performed in technical triplicate. (D) Western blot analysis showed that pChk1 and γH2AX were exacerbated upon treatment with 5-VdU / PINK in both cell lines.

[0289] A schematic diagram of the in vivo processing is shown in Fig.31 middle.

[0290] Incorporation of 5-VdU in mouse xenograft cells revealed by incubation of tissue sections with PINK. KPC subcutaneous tumors were generated on both sides of a syngeneic C57BL / 6J mouse model. When the tumor size was approximately 200 mm3, the animals were injected intraperitoneally with 200 μl of 10% DMSO in 1x sterile PBS as vehicle or 500 mg / kg 5-VdU on the following two days, followed by injection of 100 μl of 5% DMSO, 30% PEG400 in 1x sterile PBS as vehicle or 20 mg / kg PINK into the tumor, each injected into the left or right tumor of the same animal as an internal control ( Fig.30 , B). The experimental group size was n = 3. The animals were sacrificed 6 hours after injection, and the transplanted tumors were subjected to paraffin embedding. The slides were stained with DMSO or PINK for 4 hours at room temperature, and then the nuclei were counterstained with Hoechst33342. When stained with PINK (B), both DMSO- and PINK-injected tumors showed specific nuclear signals, but not when stained with DMSO (A), indicating that 5-VdU was incorporated into tumor cell DNA (see Fig.32 ). The results support that tumors can be effectively labeled with 5-VdU, allowing for fluorescence-based tumor localization using PINK during cancer surgery.

[0291] Although the embodiments have been described in detail, it should be understood that various changes, substitutions and modifications can be made herein. In addition, the scope of the present application is not intended to be limited to the specific embodiments or examples described in this specification. It is understood that the examples described and shown above are intended to be exemplary only.

[0292] For example, the present invention contemplates that any of the features shown in any of the embodiments described herein may be combined with any of the features shown in any other of the other embodiments described herein and still fall within the scope of the present invention.

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Claims

1. A compound having the formula: or its salts; in: R1 is H, haloalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or an optionally substituted group thereof; R2, R 2a , R 2b and R3 is independently H, hydroxy, hydrogen, haloalkyl, cyano, amino, thiol, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkoxy, sulfenyl, acyl, sulfinyl, sulfonyl, O-carboxyl, ester, thiocarbonyl or an optionally substituted group thereof; R4 is an optional group and is H, haloalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl, heterocyclyl or an optionally substituted group thereof; and Y is an optional linker; Provided that the compound is not 2. The compound or salt according to claim 1, wherein R1 is H, C 1-6 alkyl or optionally substituted aryl or heteroaryl.

3. The compound or salt according to claim 2, wherein R1 is C 1-3 alkyl, optionally substituted pyridyl, optionally substituted pyrimidinyl or optionally substituted C 6-10 Aryl.

4. The compound or salt according to claim 3, wherein R1 is CH3, pyridyl, pyrimidinyl or phenyl.

5. The compound or salt according to claim 4, wherein R1 is CH3.

6. The compound or salt according to any one of claims 1 to 5, wherein R2, R 2a and R 2b are independently H, alkyl, amino, C 6-10 Cycloalkyl, C 6-10 An aryl group, a heteroaryl group, a heterocyclic group or an optionally substituted group thereof.

7. The compound or salt according to claim 6, wherein R2, R 2a and R 2b Independently H, C 1-6 Alkyl, optionally substituted C 1-6 Alkyl, amino, optionally substituted amino, optionally substituted heterocyclic group, or optionally substituted heterocyclic group forming a ring with the acridine moiety.

8. The compound or salt according to claim 7, wherein the compound has formula (Ia), and R2 is C 1-6 an alkyl-substituted amino group or an optionally substituted heterocyclic group which forms a ring with the acridine moiety.

9. The compound or salt according to claim 8, wherein R2 is dimethylamine.

10. The compound or salt according to claim 8, wherein R2 and the acridine moiety form a julolidine group.

11. The compound or salt according to claim 7, wherein the compound has formula (Ib) or (Ic), and R 2a and R 2b Independently H, C 1-6 an alkyl-substituted amino group or an optionally substituted heterocyclic group which forms a ring with the acridine moiety.

12. The compound or salt according to claim 11, wherein R 2a and R 2b One or both of them are dimethylamine.

13. The compound or salt according to claim 11, wherein R 2a and R 2b One or both of them form a ring with the acridine part.

14. The compound or salt according to claim 13, wherein R 2a and R 2b One or both of the acridine moiety and the acridine moiety form a julolidine group.

15. The compound or salt according to any one of claims 1 to 14, wherein R3 is H, alkyl, heteroalkyl, aryl, heteroaryl or an optionally substituted group thereof.

16. The compound or salt according to claim 15, wherein R3 is H, optionally substituted C 1-6 Alkyl, C 1-6 heteroalkyl, optionally substituted 5- or 6-membered heteroaryl, or optionally substituted C 6-10 Aryl.

17. A compound or salt according to any one of claims 1 to 16, wherein R4 is absent.

18. A compound or salt according to any one of claims 1 to 16, wherein R4 is H or C 1-6 alkyl.

19. The compound or salt of claim 18, wherein R4 is H or CH3.

20. The compound or salt of any one of claims 1 to 19, wherein Y is alkyl, heteroalkyl, aryl, heteroaryl, alkenyl, alkynyl, alkylthio, or an optionally substituted group thereof.

21. The compound or salt according to claim 20, wherein Y is an optionally substituted C 1-6 Alkyl, optionally substituted C 1-6 Heteroalkyl, optionally substituted C 6-10 aryl, optionally substituted 5- or 6-membered heteroaryl, optionally substituted C 1-6 Alkenyl, optionally substituted C 1-6 Alkynyl or optionally substituted C 1-6 Alkylthio.

22. The compound or salt according to claim 21, wherein Y is C 1-3 Alkyl or C 1-3 Alkylthio.

23. The compound or salt of any one of claims 1 to 19, wherein Y is absent.

24. A compound or salt according to any one of claims 1 to 23 having the formula:

25. A compound or salt according to any one of claims 1 to 24 having formula (IIIb) 26. The compound or salt according to claim 1, wherein the compound is:

27. A compound or salt according to any one of claims 1 to 26 which is a probe.

28. A method for detecting a nucleic acid containing vinyl-nucleoside, the method comprising: a) contacting a cell or tissue with a compound or salt according to any one of claims 1 to 27; as well as b) exposing the cell or tissue of a) to light.

29. The method of claim 28, wherein the vinyl-nucleoside is: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG), 5-vinyluridine (VU), 5-vinylcytidine (VC), 7-vinyl-7-deazaadenosine (VA), or 7-vinyl-7-deaza-2'-deoxyguanosine (VG).

30. The method of claim 29, wherein the vinyl-nucleoside is VdU and / or VdA.

31. The method of claim 29, wherein the vinyl-nucleoside is VU.

32. The method of any one of claims 28 to 31 performed in vitro.

33. The method of any one of claims 28 to 31 performed in vivo.

34. A kit for detecting nucleic acids in cells or tissues, the kit comprising: one or more vinyl-nucleosides; as well as A compound or salt according to any one of claims 1 to 27.

35. The kit of claim 34, wherein the one or more vinyl-nucleosides include one or more of: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG), 5-vinyluridine (VU), 5-vinylcytidine (VC), 7-vinyl-7-deazaadenosine (VA), and 7-vinyl-7-deaza-2'-deoxyguanosine (VG).

36. A method of treating cancer in a subject who has been administered one or more vinyl-nucleosides, the method comprising administering to the subject a compound or salt according to any one of claims 1 to 27.

37. The method of claim 36, wherein the one or more vinyl-nucleosides include one or more of: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG), 5-vinyluridine (VU), 5-vinylcytidine (VC), 7-vinyl-7-deazaadenosine (VA), and 7-vinyl-7-deaza-2'-deoxyguanosine (VG).

38. A pharmaceutical composition comprising a compound or salt according to any one of claims 1 to 27 and an acceptable pharmaceutical carrier.

39. Use of a compound or salt according to any one of claims 1 to 27 for the preparation of a medicament for the treatment of cancer.

40. Use of a compound or salt according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 38 for the treatment of cancer.

41. Use of a compound or salt according to any one of claims 1 to 27 or a pharmaceutical composition according to claim 38 in combination with one or more vinyl-nucleosides for the treatment of cancer.

42. The use according to claim 41, wherein the one or more vinyl-nucleosides include one or more of: 5-vinyl-2'-deoxyuridine (VdU), 5-vinyl-2'-deoxycytidine (VdC), 7-vinyl-7-deaza-2'-deoxyadenosine (VdA), 7-vinyl-7-deaza-2'-deoxyguanosine (VdG), 5-vinyluridine (VU), 5-vinylcytidine (VC), 7-vinyl-7-deazaadenosine (VA), and 7-vinyl-7-deaza-2'-deoxyguanosine (VG).

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