Compositions and methods for decrosslinking biological samples

Abstract

This specification discloses compositions and methods for decrosslinking formaldehyde-crosslinked biological samples, such as formalin-fixed paraffin-embedded (FFPE) tissue samples.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 500,180, filed on 4 May 2023, which is incorporated herein by reference in its entirety.

[0002] This specification provides compositions and methods for decrosslinking formaldehyde-crosslinked biological samples, such as formalin-fixed paraffin-embedded (FFPE) tissue samples. [Background technology]

[0003] Formalin-fixed, paraffin-embedded (FFPE) tissue specimens are valuable clinical samples containing a vast amount of patient information. When such samples are prepared, formalin acts as a preservative by forming extensive molecular crosslinks. These crosslinks must be reversed before nucleic acids, proteins, and other biomolecules can be extracted and used in downstream assays, requiring considerable pretreatment time. Typically, after deparaffinization and lysis, tissues are decrosslinked at high temperatures (60-90°C) for extended periods (several hours to several days). While this decrosslinking step is necessary, it can lead to extraction failures and reduced sample quality, posing significant challenges for clinical and research applications. [Overview of the project]

[0004] In one embodiment, the Specified Method describes using an effective amount of the compound of formula (I) as a sample. [ka] A method for decrosslinking a formaldehyde-crosslinked biological sample is disclosed, which includes contact with a salt thereof. (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2is selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl, R 3 is -X-R 4 wherein X is selected from -C(O)- and -SO2-, and R 4 is selected from C1-C6-alkyl, aryl, and heteroaryl, each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester, each alkyl is unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl, and optionally substituted 3-6 member heterocyclyl, or (ii)R 1 is selected from H, C1-C6 alkyl, and carboxy-C1-C4-alkyl, R 2 is selected from C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -Y-R 5 wherein Y is selected from a bond, -C(O)-, and -SO2-, and R 5 is selected from C1-C6 alkyl, aryl, and heteroaryl, R 3 is H, each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (iii)R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0005] In some embodiments, R 1 H is R 2 R is selected from C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, 3 -XR 4 And in the formula, X is -C(O)- and R 4 The alkyl groups are selected from C1-C6 alkyl, aryl, and heteroaryl groups, where each alkyl group is independently unsubstituted or substituted with one substituent selected from hydroxy and carboxyl groups, and each aryl and heteroaryl group is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxyl groups.

[0006] In some embodiments, R 1 H is R 2 R is a C1-C6 alkyl group that is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl, 3 -XR 4 And in the formula, X is -C(O)-, and R4 This is either an unsubstituted phenyl compound or a phenyl compound substituted with one or two substituents independently selected from methoxy, hydroxy, and halo.

[0007] In some embodiments, R 1 H is R 2 R is selected from aryl-C1~C4-alkyl and heteroaryl-C1~C4-alkyl, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. 3 H is H.

[0008] In some embodiments, R 1 and R 2 Together with the nitrogen atom to which they are bonded, they form a saturated 4-6 membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo, R 3 H is H.

[0009] In some embodiments, R 1 H is R 2 and R 3 These atoms, together with the atoms to which they are bonded, form a saturated 4- to 7-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo.

[0010] In some embodiments, the compound of formula (I) is [ka] [ka] And selected from those salts.

[0011] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0012] In some embodiments, the compound of formula (I) is in the form of a salt. In some embodiments, the compound of formula (I) is in the form of a hydrochloride salt.

[0013] In some embodiments, the method involves contacting a sample with an effective amount of at least two different compounds of formula (I) or salts thereof. In some embodiments, at least two different compounds of formula (I) or salts thereof are added to the sample simultaneously. In some embodiments, at least two different compounds of formula (I) or salts thereof are added to the sample sequentially.

[0014] In some embodiments, the sample is a formalin-fixed, paraffin-embedded tissue sample. In some embodiments, the method further includes the step of deparaffinizing the sample before contacting the sample with the compound of formula (I) or a salt thereof. In some embodiments, the method further includes the step of contacting the sample with a protease before contacting the sample with the compound of formula (I) or a salt thereof.

[0015] In some embodiments, the protease is proteinase K.

[0016] In some embodiments, the contact step is performed for about 5 minutes to about 120 minutes. In some embodiments, the contact step is performed for about 20 minutes to about 40 minutes. In some embodiments, the contact step is performed at a temperature of about 20°C to about 100°C. In some embodiments, the contact step is performed at a temperature of about 50°C to about 85°C.

[0017] This method involves contacting a sample with a solution of the compound of formula (I) or a salt thereof, the solution further comprising a buffer. In some embodiments, the solution comprises a buffer selected from tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate-buffered saline, glycine, and citrate.

[0018] In some embodiments, the method involves contacting a sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution obtained when the sample is contacted with the solution of the compound of formula (I) has a pH of about 4.0 to about 8.5. In some embodiments, the method involves contacting a sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution obtained when the sample is contacted with the solution of the compound of formula (I) has a pH of about 4.5 to about 6.5.

[0019] In some embodiments, the method involves contacting a sample with a solution of the compound of formula (I) or a salt thereof, the solution containing the compound of formula (I) at a concentration of about 1 mM to about 100 mM. In some embodiments, the method involves contacting a sample with a solution of the compound of formula (I) or a salt thereof, the solution containing the compound of formula (I) at a concentration of about 5 mM to about 50 mM.

[0020] In some embodiments, the method further includes extracting one or more components from the sample after the contact step. In some embodiments, the one or more components are selected from nucleic acids and proteins. In some embodiments, the one or more components are nucleic acids, and the method further includes a step of detecting and / or amplifying one or more nucleic acids. In some embodiments, the one or more components are proteins, and the method further includes a step of detecting one or more proteins. In some embodiments, the detection step includes colorimetric analysis, fluorescence spectroscopy, ultraviolet-visible spectroscopy, electrophoresis, immunoassay, or mass spectrometry.

[0021] In some embodiments, the method further includes a step selected from pigment binding, absorption, and enzymatic digestion.

[0022] In another aspect, this specification discloses a composition, Formaldehyde-crosslinked biological samples, and Compound of formula (I), [ka] or a salt thereof During the ceremony, (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (ii)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (iii)R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0023] In some embodiments, R 1 H is R 2 R is selected from C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, 3 -XR 4 And in the formula, X is -C(O)- and R 4The alkyl groups are selected from C1-C6 alkyl, aryl, and heteroaryl groups, where each alkyl group is independently unsubstituted or substituted with one substituent selected from hydroxy and carboxyl groups, and each aryl and heteroaryl group is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxyl groups.

[0024] In some embodiments, R 1 H is R 2 R is a C1-C6 alkyl group that is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl, 3 -XR 4 And in the formula, X is -C(O)-, and R 4 This is either an unsubstituted phenyl compound or a phenyl compound substituted with one or two substituents independently selected from methoxy, hydroxy, and halo.

[0025] In some embodiments, R 1 H is R 2 R is selected from aryl-C1~C4-alkyl and heteroaryl-C1~C4-alkyl, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. 3 H is H.

[0026] In some embodiments, R 1 and R 2 Together with the nitrogen atom to which they are bonded, they form a saturated 4-6 membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo, R 3 H is H.

[0027] In some embodiments, R1 H is R 2 and R 3 These atoms, together with the atoms to which they are bonded, form a saturated 4- to 7-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo.

[0028] In some embodiments, the compound of formula (I) is [ka] [ka] And selected from those salts.

[0029] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0030] In some embodiments, the compound of formula (I) is in the form of a salt. In some embodiments, the compound of formula (I) is in the form of a hydrochloride salt.

[0031] In some embodiments, the composition further comprises a buffer. In some embodiments, the buffer is selected from tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate-buffered saline, glycine, and citric acid.

[0032] In some embodiments, the composition comprises at least two different compounds of formula (I) or salts thereof.

[0033] In some embodiments, the sample is a formalin-fixed, paraffin-embedded tissue sample.

[0034] In another embodiment, this specification discloses a kit, which is: (A) Compound of formula (I), [ka] or a salt thereof (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (ii)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5selected from, wherein Y is selected from a bond, -C(O)-, and -SO2-, R 5 is selected from C1-C6 alkyl, aryl, and heteroaryl, R 3 is H, each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester, each alkyl is unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl, and optionally substituted 3-6 member heterocyclyl, or (iii)R 1 and R 2 together with the nitrogen atom to which they are attached form an optionally substituted 4- to 8-membered ring, R 3 is H, or (iv)R 1 is H, R 2 and R 3 together with the atom to which they are attached form an optionally substituted 4- to 8-membered ring, a compound of formula (I) or a salt thereof, (B) Instructions for use for crosslinking a formaldehyde-crosslinked biological sample by contacting it with a compound of formula (I) or a salt thereof, and.

[0035] In some embodiments, R 1 is H, R 2 is selected from C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, R 3 is -X-R 4 wherein X is -C(O)- and R 4is selected from C1-C6-alkyl, aryl, and heteroaryl, each alkyl being independently unsubstituted or substituted with one substituent selected from hydroxy and carboxy, and each aryl and heteroaryl being independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy.

[0036] In some embodiments, R 1 is H, R 2 is C1-C6 alkyl which is unsubstituted or substituted with one substituent selected from hydroxy and carboxy, and R 3 is -X-R 4 wherein X is -C(O)- and R 4 is unsubstituted phenyl or phenyl substituted with one or two substituents independently selected from methoxy, hydroxy, and halo.

[0037] In some embodiments, R 1 is H, R 2 is selected from aryl-C1-C4-alkyl and heteroaryl-C1-C4-alkyl, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy, and R 3 is H.

[0038] In some embodiments, R 1 and R 2 together with the nitrogen atom to which they are attached form a saturated 4- to 6-membered ring which is unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4-alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo, and R 3 is H.

[0039] In some embodiments, R1 H is R 2 and R 3 These atoms, together with the atoms to which they are bonded, form a saturated 4- to 7-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo.

[0040] In some embodiments, the compound of formula (I) is [ka] [ka] And selected from those salts.

[0041] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0042] In some embodiments, the compound of formula (I) is in the form of a salt. In some embodiments, the compound of formula (I) is in the form of a hydrochloride salt.

[0043] In some embodiments, the composition comprises at least two different compounds of formula (I) or salts thereof.

[0044] In some embodiments, the formaldehyde-crosslinked biological sample is a formalin-fixed, paraffin-embedded tissue sample.

[0045] In another embodiment, as specified herein, the sample is an effective amount of at least two different compounds of formula (I), [ka] A method for decrosslinking a formaldehyde-crosslinked biological sample is disclosed, which includes contact with a salt thereof. During the ceremony, (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (ii)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (iii)R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0046] In another aspect, this specification discloses a composition, Formaldehyde-crosslinked biological samples, and Compounds of at least two different formulas (I), [ka] or a salt thereof During the ceremony, (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (ii)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (iii)R 1and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0047] In another embodiment, this specification discloses a kit, which is: (A) Compounds of at least two different formulas (I), [ka] or a salt thereof (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (ii)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (iii)R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3These are compounds of formula (I) or salts thereof, which, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings, (B) Instructions for use for decrosslinking a formaldehyde-crosslinked biological sample by contacting it with a compound of formula (I) or a salt thereof. [Brief explanation of the drawing]

[0048] [Figure 1] This shows exemplary crosslinks formed when tissue is fixed using formalin, including protein-protein crosslinks and protein-DNA crosslinks. [Figure 2] An exemplary workflow used to test decrosslinking catalyst compounds is shown, along with positive and negative controls. [Figure 3] The DNA yield and quality purified from healthy FFPE colon tissue using the decrosslinking catalyst compounds and positive and negative controls of this disclosure are shown, as measured by the ProNex® DNA QC assay (75 bp, 150 bp, and 300 bp qPCR amplicons). [Figure 4] Figure 3 shows the volume and quality of DNA sequencing libraries prepared using Illumina's AmpliSeq for Cancer HotSpot Panel v2 from the purified DNA. A shows the molecular weight of the library determined by electrophoresis using Agilent TapeStation. B shows the quantification of the electrophoretic library. C shows the volume of the library determined by qPCR using the ProNex® NGS Library Quantification Kit. [Figure 5] Figure 3 shows the agreement in the number of single nucleotide variants with the purified DNA, as determined by sequencing using Illumina MiSeq. A shows the total number of mutant alleles with a frequency greater than 1%. B shows the mutant allele frequencies between DNA purified by rapid decrosslinking catalyst, positive control, and sequencing control gDNA. [Figure 6]This shows the amplifiable DNA yield from FFPE rapidly decrosslinked at 80°C for 10, 20, and 30 minutes using a catalytic compound. [Figure 7] This paper demonstrates the determination of the optimal pH for rapid decrosslinking reactions using catalytic compounds. [Figure 8] This shows the amplifiable DNA yield from FFPE rapidly decrosslinked with 20 mM, 10 mM, and 5 mM catalytic HCl salt compounds across various pH lysis buffers. [Figure 9A] This shows the yield of purified DNA from a rapid decrosslinking reaction with Cpd 1. DNA was purified from 13 human FFPE tissues and evaluated using an amplification assay. [Figure 9B] This shows the yield of purified DNA from a rapid decrosslinking reaction with Cpd 1. DNA was purified from 13 human FFPE tissues and evaluated using a dsDNA dye-binding assay. [Figure 9C] This shows the yield of purified DNA from a rapid decrosslinking reaction with Cpd 1. DNA was purified from 13 human FFPE tissues and evaluated using UV-Vis absorption. [Figure 10] This document describes the workflow for extracting proteins from FFPE tissue and the analytical techniques used. [Figure 11] The soluble and insoluble FFPE protein yields are shown as a function of decrosslinking incubation time at 90°C. A shows the protein yield determined by SDS-PAGE silver staining. B shows the soluble protein yield determined by BCA assay. [Figure 12] This shows the soluble protein yield from rapidly decrosslinked FFPE lysates, measured by SDS-PAGE silver staining. [Figure 13A] This shows the soluble protein yield from FFPE lysates rapidly decrosslinked with a catalyst over a wide pH range, as determined by SDS-PAGE silver staining. [Figure 13B] This shows the soluble protein yield from FFPE lysates rapidly decrosslinked with a catalyst over a wide pH range, as determined by SDS-PAGE silver staining. [Figure 13C] This shows the soluble protein yield from FFPE lysates rapidly decrosslinked with a catalyst over a wide pH range, as determined by SDS-PAGE silver staining. [Figure 13D] This shows the soluble protein yield from FFPE lysates rapidly decrosslinked with a catalyst over a wide pH range, as determined by SDS-PAGE silver staining. [Figure 13E] This shows the soluble protein yield from FFPE lysates rapidly decrosslinked with a catalyst over a wide pH range, as determined by SDS-PAGE silver staining. [Figure 14] This shows the soluble protein yield from FFPE lysates rapidly decrosslinked at various catalyst concentrations while maintaining a constant pH, as determined by SDS-PAGE silver staining. [Figure 15A] The results of determining the soluble protein yield from rapidly decrosslinked FFPE lysates at three separate time points ranging from 10 minutes to 8 hours at three different incubation temperatures (65°C, 80°C, and 95°C) using SDS-PAGE silver staining are shown. [Figure 15B] The results of determining the soluble protein yield from rapidly decrosslinked FFPE lysates at three separate time points ranging from 10 minutes to 8 hours at three different incubation temperatures (65°C, 80°C, and 95°C) using SDS-PAGE silver staining are shown. [Figure 15C] The results of determining the soluble protein yield from rapidly decrosslinked FFPE lysates at three separate time points ranging from 10 minutes to 8 hours at three different incubation temperatures (65°C, 80°C, and 95°C) using SDS-PAGE silver staining are shown. [Figure 16] This shows histone H3 protein from rapidly decrosslinked FFPE lysates, as determined by Western blotting. [Figure 17A] The volcano plot of rapidly decrosslinked FFPE protein compared to a low pH control group, as determined by LC-MS / MS, is shown. [Figure 17B]The volcano plot of rapidly decrosslinked FFPE protein compared to a low pH control group, as determined by LC-MS / MS, is shown. [Figure 17C] The volcano plot of rapidly decrosslinked FFPE protein compared to a low pH control group, as determined by LC-MS / MS, is shown. [Figure 18] Figures 17A-17C show the abundance of selected human proteins concentrated by rapid decrosslinking extraction. [Modes for carrying out the invention]

[0049] This specification provides compositions, methods, and kits for decrosslinking formaldehyde-crosslinked biological samples, such as FFPE tissue samples. In some embodiments, samples can be decrosslinked in significantly less time than currently used. For example, in some embodiments, FFPE samples can be efficiently decrosslinked in as little as 30 minutes without compromising sample quality.

[0050] definition Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have meanings generally understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and the chemistry and hybridization of proteins and nucleic acids as described herein are well known and commonly used in the art. The meaning and scope of terms should be clear, but in the event of potential ambiguity, the definitions provided herein shall take precedence over dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0051] As used herein and in the appended claims, the singular forms "a," "an," and "the" also include the plural forms unless the context clearly indicates otherwise.

[0052] As used herein, the term "and / or" includes any and all combinations of the listed items, including any one of the listed items individually. For example, "A, B, and / or C" includes A, B, C, AB, AC, BC, and ABC, each of which is considered to be individually described by the statement "A, B, and / or C".

[0053] In the descriptions of numerical ranges in this specification, each intervening number of the same precision is explicitly assumed. For example, for the range 6 to 9, in addition to 6 and 9, the numbers 7 and 8 are intended, and for the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitly intended.

[0054] The definitions of specific functional groups and chemical terms are described in more detail below. For the purposes of this disclosure, chemical elements are as defined in Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Specified according to the Ed. (endpaper), specific functional groups are generally defined as described therein. Furthermore, for general principles of organic chemistry, as well as specific functional groups and reactivity, see Sorrell, Organic Chemistry, 2 nd edition,University Science Books,Sausalito,2006;Smith,March's Advanced Organic Chemistry:Reactions,Mechanism,and Structure,7 th Edition,John Wiley & Sons,Inc.,New York,2013;Larock,Comprehensive Organic Transformations,3 rdEdition, John Wiley & Sons, Inc., New York, 2018; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd This information is contained in Edition, Cambridge University Press, Cambridge, 1987, and its entirety is incorporated herein by reference.

[0055] As used herein, the term "alkyl" refers to a radical of a linear or branched saturated hydrocarbon chain. An alkyl chain is, for example, a chain with 1 to 24 carbon atoms (C1-C1). 24 Alkyl), 1 to 16 carbon atoms (C1-C 16 Alkyl), 1 to 14 carbon atoms (C1-C 14 Alkyl), 1 to 12 carbon atoms (C1-C 12 Alkyl), 1 to 10 carbon atoms (C1-C 10 Alkyl compounds may include 1-8 carbon atoms (C1-C8 alkyl), 1-6 carbon atoms (C1-C6 alkyl), 1-4 carbon atoms (C1-C4 alkyl), 1-3 carbon atoms (C1-C3 alkyl), or 1-2 carbon atoms (C1-C2 alkyl). Typical examples of alkyl compounds include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, and n-dodecyl.

[0056] As used herein, the term “alkenyl” refers to a radical of a straight or branched hydrocarbon chain containing at least one carbon-carbon double bond and no triple bond. The double bond(s) can be located at any position(s) in the hydrocarbon chain. An alkenyl chain, for example, contains 2 to 24 carbon atoms (C2-C2). 24 Alkenyl), 2-16 carbon atoms (C2-C 16Alkenyl), 2-14 carbon atoms (C2-C 14 Alkenyl), 2-12 carbon atoms (C2-C 12 Alkenyl), 2 to 10 carbon atoms (C2-C 10 Alkenyls may include alkenyls with 2-8 carbon atoms (C2-C8 alkenyls), 2-6 carbon atoms (C2-C6 alkenyls), 2-4 carbon atoms (C2-C4 alkenyls), 2-3 carbon atoms (C2-C3 alkenyls), or 2 carbon atoms (C2 alkenyls). Typical examples of alkenyls, though not limited to them, include ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, 2-methyl-2-propenyl, 3-butenyl, pentenyl, pentadinyl, hexenyl, heptenyl, octenyl, and octatrienyl.

[0057] As used herein, the term "alkynyl" means a radical of a linear or branched hydrocarbon chain containing at least one carbon-carbon triple bond. An alkynyl chain is, for example, a chain containing 2 to 24 carbon atoms (C2-C2). 24 Alkynyl), 2-16 carbon atoms (C2-C 16 Alkynyl), 2-14 carbon atoms (C2-C 14 Alkynyl), 2-12 carbon atoms (C2-C 12 Alkynyl), 2 to 10 carbon atoms (C2-C 10 The hydrocarbon chain may contain alkynyl groups, 2-8 carbon atoms (C2-C8 alkynyl), 2-6 carbon atoms (C2-C6 alkynyl), 2-4 carbon atoms (C2-C4 alkynyl), 2-3 carbon atoms (C2-C3 alkynyl), or 2 carbon atoms (C2 alkynyl). Triple bonds (or more) can be located at any position (or more) in the hydrocarbon chain. Representative examples of alkynyls include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, and 2-butynyl.

[0058] As used herein, the term "alkoxy" refers to an alkyl group as defined herein, which is attached to the parent molecule via an oxygen atom. Typical examples of alkoxys include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, and tert-butoxy.

[0059] As used herein, the term "amino" refers to the group -NR x R y It refers to, and in the formula, R x and R y The group is selected from hydrogen and alkyl (e.g., C1-C4 alkyl). In this specification, the -NH (alkyl) group may be called "alkylamino," and the -N (alkyl)2 group may be called "dialkylamino."

[0060] As used herein, the term “aryl” refers to a monocyclic, bicyclic, or tricyclic 4n+2 aromatic ring system having 6 to 14 ring carbon atoms and 0 heteroatoms (e.g., having 6, 10, or 14 π electrons shared in a cyclic arrangement) (“C6~C 14 In some embodiments, the aryl group has six ring carbon atoms ("C6 aryl", i.e., phenyl). In some embodiments, the aryl group has ten ring carbon atoms ("C6 aryl"). 10 "Aryl" (for example, naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C"). 14 "Aryl" (e.g., anthracenyl and phenantrenyl).

[0061] As used herein, the term "carboxy" refers to the -COOH group.

[0062] As used herein, the term "carboxyalkyl" refers to an alkyl group, as defined herein, in which at least one hydrogen atom is replaced by a carboxyl group. Typical examples of carboxyalkyls include, but are not limited to, carboxymethyl, 2-carboxyethyl, and 3-carboxypropyl.

[0063] As used herein, the term "cycloalkyl" refers to a radical of a saturated carbocyclic ring system containing 3 to 10 carbon atoms and 0 heteroatoms. Cycloalkyls may be monocyclic, bicyclic, bridging, condensed, or spirocyclic. Typical examples of cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, adamantyl, bicyclo[2.2.1]heptanyl, bicyclo[3.2.1]octanyl, and bicyclo[5.2.0]nonanyl.

[0064] As used herein, the term “ester” refers to the group-COOR, where R is an alkyl group as defined herein (e.g., C1-C6, C1-C4, or C1-C3 alkyl group).

[0065] As used herein, the terms "halogen" or "halo" refer to F, Cl, Br, or I.

[0066] As used herein, the term “haloalkyl” refers to an alkyl group as defined herein in which at least one hydrogen atom (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 hydrogen atoms) is replaced by a halogen. In some embodiments, each hydrogen atom of the alkyl group is replaced by a halogen (“perhaloalkyl”). Typical examples of haloalkyls include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2-fluoroethyl, 2,2,2-trifluoroethyl, and 3,3,3-trifluoropropyl.

[0067] As used herein, the term “heteroaryl” refers to a monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in the ring arrangement) having a ring carbon atom and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5 to 10-membered ring heteroaryl”). In heteroaryl groups containing one or more nitrogen atoms, the bond site may be a carbon atom or a nitrogen atom, as long as the valence allows. A heteroaryl bicyclic ring system may contain one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems in which the heteroaryl ring defined above is fused with one or more aryl groups, where the bond site is in either the aryl or heteroaryl ring, and in such examples, the number of ring members indicates the number of ring members in the fused (aryl / heteroaryl) ring system. In the case of a bicyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl), the bond may be on either ring, i.e., the ring containing a heteroatom (e.g., 2-indolyl) or the ring not containing a heteroatom (e.g., 5-indolyl). Exemplary five-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary five-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary five-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary five-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary six-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Exemplary six-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary six-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetradinyl, respectively.Examples of seven-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Examples of 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranil, benzoisofuranil, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolidinyl, and prinyl. Examples of 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthylidinyl, pteridinyl, quinolinyl, isoquinolinyl, sinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl.

[0068] As used herein, the term “heterocyclyl” refers to a 3- to 10-membered non-aromatic cyclic radical having a ring carbon atom and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3- to 10-membered heterocyclyl”). In heterocyclyl groups containing one or more nitrogen atoms, the bond sites can be carbon atoms or nitrogen atoms, as long as the valence allows. Heterocyclyl groups can be monocyclic (“monocyclic heterocyclyl”), or condensed, bridging, or spirocyclic systems (e.g., bicyclic systems (“bicyclic heterocyclyl”)), and can be saturated or partially unsaturated. A heterocyclyl bicyclic system may contain one or more heteroatoms in one or both rings. "Heterocyclyl" includes ring systems in which the heterocyclyl ring defined above is fused with one or more cycloalkyl groups, with the bond site located on either the cycloalkyl ring or the heterocyclyl ring, or ring systems in which the heterocyclyl ring defined above is fused with one or more aryl or heteroaryl groups, with the bond site located on the heterocyclyl ring, in which case the ring member number continues to indicate the ring member number in the heterocyclyl ring system. A heterocyclyl group may be described, for example, as a 3- to 7-membered ring heterocyclyl, and the term "membered ring" refers to the ring atoms other than hydrogen within that part, namely carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Examples of 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azildinyl, oxyranyl, and thiorenyl. Examples of 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Examples of five-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranil, dihydrofuranil, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinil, dihydropyrrolyl, and pyrrolyl-2,5-dione. Examples of five-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanil, oxasulfuranil, disulfuranil, and oxazolidine-2-one.Examples of five-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Examples of six-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl (e.g., 2,2,6,6-tetramethylpiperidinyl), tetrahydropyranyl, dihydropyridinyl, pyridinonyl (e.g., 1-methylpyridine-2-onyl), and thianyl. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, pyridadinonyl (2-methylpyridazine-3-onyl), pyrimidinonyl (e.g., 1-methylpyrimidine-2-onyl, 3-methylpyrimidine-4-onyl), dithianyl, and dioxanyl. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinyl. Examples of seven-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Examples of eight-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azokanyl, oxekanyl, and thiokanyl. Examples of five-membered heterocyclyl groups condensed on a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocyclyl rings) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, and benzoxazolinonyl. Examples of five-membered heterocyclyl groups condensed on a heterocyclyl ring (also referred to herein as 5,5-bicyclic heterocyclyl rings) include, but are not limited to, octahydropyrrolopyrrolyl (e.g., octahydropyrrolo[3,4-c]pyrrolyl). Examples of six-membered heterocyclyl groups (also called 4,6-membered heterocyclyl rings) condensed on a heterocyclyl ring include, but are not limited to, diazaspirononanyl (e.g., 2,7-diazaspirononanyl).Examples of six-membered heterocyclyl groups condensed to an aryl ring (also referred to herein as 6,6-bicyclic heterocyclyl rings) include, but are not limited to, tetrahydroquinolinyl and tetrahydroisoquinolinyl. Examples of six-membered heterocyclyl groups condensed to a cycloalkyl ring (also referred to herein as 6,7-bicyclic heterocyclyl rings) include, but are not limited to, azabicyclooctanyl (e.g., (1,5)-8-azabicyclo[3.2.1]octanyl). Examples of six-membered heterocyclyl groups condensed to a cycloalkyl ring (also referred to herein as 6,8-bicyclic heterocyclyl rings) include, but are not limited to, azabicyclononanyl (e.g., 9-azabicyclo[3.3.1]nonanyl).

[0069] As used herein, the terms "hydroxy" or "hydroxyl" refer to the -OH group.

[0070] As used herein, the term "hydroxyalkyl" refers to an alkyl group as defined herein, in which at least one hydrogen atom is replaced by a hydroxyl group. Typical examples of hydroxyalkyls include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 3-hydroxypropyl.

[0071] As used herein, the term "oxo" refers to the =O group.

[0072] Where a group or part can be substituted, the term “substituted” indicates that one or more hydrogens on the group indicated by the “substituted” expression (e.g., 1, 2, 3, 4, 5, or 6; in some embodiments, 1, 2, or 3; in other embodiments, 1 or 2) can be replaced with one of the listed options of the indicated group or a suitable substituent known to those skilled in the art (e.g., one or more of the groups listed below), provided that the hydrogens do not exceed the normal valence of the specified atom. Substituents include, but are not limited to, alkyl, alkenyl, alkynyl, alkoxy, acyl, amino, amide, amidino, aryl, azide, carbamoyl, carboxy, carboxyalkyl, cyano, cycloalkyl, cycloalkenyl, ester, guanidino, halo, haloalkyl, haloalkoxy, heteroalkyl, heteroaryl, heterocyclyl, hydroxy, hydroxyalkyl, hydrazino, imino, oxo, nitro, phosphate, phosphonate, sulfonic acid, thiol, thion, or combinations thereof.

[0073] When used herein, the chemical structure is: [ka] The notation indicates a point where one part is bonded to another part (for example, a point where a substituent is bonded to the rest of the compound).

[0074] For the compounds described herein, the groups and substituents can be selected according to the allowable valencies of the atoms and substituents, and as a result, stable compounds that do not spontaneously undergo transformations such as rearrangement, cyclization, or elimination can be obtained through selection and substitution.

[0075] Compounds and compositions This specification discloses compositions, methods, systems, and kits for decrosslinking formaldehyde-crosslinked biological samples, such as FFPE samples. The compositions, methods, systems, and kits described below in further detail contain compounds of formula (I), [ka] or a salt thereof During the ceremony, (i)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (ii)R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, alkoxy, amino, amide, carboxy, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. or (iii)R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0076] In some embodiments, the compound of formula (I) or a salt thereof, R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 The elements are selected from H, C1-C6 alkyl, hydroxy-C1-C4-alkyl, carboxy-C1-C4-alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxyl, alkoxy, amino, amide, carboxyl, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines.

[0077] In some embodiments, R 1 H is H. In some embodiments, R 2 R is selected from C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, where C1-C6 alkyl is unsubstituted or substituted with one substituent selected from hydroxy and carboxy, and aryl or heteroarylaryl and heteroaryl are independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. In some embodiments, R 2 C is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl. 1 ~C 4 It is alkyl. In some embodiments, R 3 -XR 4 And X is -C(O)- and R 4 R is selected from C1-C6 alkyl, aryl, and heteroaryl, where C1-C6 alkyl is unsubstituted or substituted with one substituent selected from hydroxy and carboxy, and aryl or heteroarylaryl and heteroaryl are independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. In some embodiments, R 3 -XR 4And in the formula, X is -C(O)- and R 4 is phenyl, which is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl.

[0078] In some embodiments, R 1 H is R 2 R is selected from C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, 3 -XR 4 And in the formula, X is -C(O)- and R 4 R is selected from C1-C6 alkyl, aryl, and heteroaryl groups, where each alkyl group is independently unsubstituted or substituted with one substituent selected from hydroxy and carboxy, and each aryl and heteroaryl group is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. In some embodiments, R 1 H is R 2 R is a C1-C6 alkyl group that is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl, 3 -XR 4 And in the formula, X is -C(O)-, and R 4 is phenyl, which is either unsubstituted or substituted with one substituent selected from methoxy, hydroxy, and halo.

[0079] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0080] In some embodiments, the compound of formula (I) or a salt thereof, R 1 These are selected from H, C1-C6 alkyl, and carboxy-C1-C4 alkyl. R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1-C4 alkyl, C1-C4 alkoxy, amino, amide, carboxy, and ester. Each alkyl group is either unsubstituted or substituted with one or two substituents independently selected from halo, hydroxyl, alkoxy, amino, amide, carboxyl, ester, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines.

[0081] In some embodiments, R 1 H is H. In this embodiment, R 2 These include C1-C6 alkyl, aryl-C1-C4-alkyl, heteroaryl-C1-C4-alkyl, and -YR 5 Selected from, where Y is selected from the bonds, -C(O)- and -SO2-, R 5 R is selected from C1-C6 alkyl, aryl, and heteroaryl groups. In some embodiments, R 2 R is an aryl-C1~C4-alkyl or heteroaryl-C1~C4-alkyl, where each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, C1~C4 alkyl, C1~C4 alkoxy, amino, amide, carboxy, and ester. In some embodiments, R 2R is an aryl-C1~C4-alkyl or heteroaryl-C1~C4-alkyl, where each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. In some embodiments, R 2 R is an aryl-C1~C4-alkyl (e.g., benzyl), where the aryl is either unsubstituted or substituted with one or two substituents independently selected from halo (e.g., chloro), hydroxy, and methoxy. In some embodiments, R 2 R is a heteroaryl-C1~C4-alkyl, where the heteroaryl is a monocyclic heteroaryl having one or two heteroatoms independently selected from N, O, and S. In some embodiments, R 2 A is a -CH2-heteroaryl, where the heteroaryl is a monocyclic five-membered or six-membered heteroaryl having one heteroatom selected from N, O, and S.

[0082] In some embodiments, R 1 H is R 2 R is selected from aryl-C1~C4-alkyl and heteroaryl-C1~C4-alkyl, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy. 3 H is H. In some embodiments, R 1 H is R 2 R is an aryl-C1~C4-alkyl (e.g., benzyl), where the aryl is either unsubstituted or substituted with one or two substituents independently selected from halo (e.g., chloro), hydroxy, and methoxy. 3 H is H.

[0083] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0084] In some embodiments, the compound of formula (I) or a salt thereof, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is H.

[0085] In some embodiments, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form a saturated 4- to 8-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo. In some embodiments, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form a saturated 4- to 6-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo. In some embodiments, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form a saturated 5- to 6-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, -CH2OH, carboxy, -CH2COOH, and oxo. In some embodiments, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form a pyrrolidine, piperidine, or morpholine ring, each of which is either unsubstituted or substituted with one or two substituents independently selected from hydroxyl, -CH2OH, carboxyl, -CH2COOH, and oxo.

[0086] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0087] In some embodiments, the compound of formula (I) or a salt thereof, R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0088] In some embodiments, R 2 and R 3 These, together with the atoms to which they are bonded, form a saturated 4- to 7-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo. In some embodiments, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form a saturated 5- to 6-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxyl, -CH2OH, carboxyl, -CH2COOH, and oxo.

[0089] In some embodiments, R 2 and R 3 These, together with the atoms to which they are bonded, form a saturated 4- to 8-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo. In some embodiments, R 2 and R 3These, together with the atoms to which they are bonded, form a saturated 4- to 7-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1-C4 alkyl, C1-C4 alkoxy, carboxy, carboxy-C1-C4 alkyl, and oxo. In some embodiments, R 2 and R 3 Together with the atoms to which they are bonded, they form a saturated 4- to 7-membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, -CH2OH, -CH2CH2OH, methoxy, carboxy, and oxo. In some embodiments, R 2 and R 3 These, together with the atoms to which they are bonded, form a 1,2-oxazetidine ring, isoxazolidine ring, 1,2-oxadinane ring, or 1,2-oxazepane ring, each of which is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C1~C4 alkyl, C1~C4 alkoxy, carboxy, carboxy-C1~C4 alkyl, and oxo. In some embodiments, R 2 and R 3 Together with the atoms to which they are bonded, they form a 1,2-oxazetidine ring, isoxazolidine ring, 1,2-oxadinane ring, or 1,2-oxazepane ring, each of which is either unsubstituted or substituted with one substituent selected from hydroxy, -CH2OH, -CH2CH2OH, methoxy, carboxy, and oxo.

[0090] In some embodiments, the compound of formula (I) is [ka] And selected from those salts.

[0091] In some embodiments, the compound of formula (I) is [ka] [ka] And selected from those salts.

[0092] In some embodiments, this compound [ka] And selected from those salts.

[0093] In some embodiments, the compound of formula (I) is in the form of a salt. In some embodiments, the compound of formula (I) is in the form of an acid addition salt. Acid addition salts can be formed from inorganic or organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Organic acids from which salts can be derived include, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, toluenesulfonic acid, and sulfosalicylic acid. In some embodiments, the compound of formula (I) is in the form of a hydrochloride salt.

[0094] Unless otherwise specified, the structures shown herein include the geometric (or conformational) forms of the structures, such as the R and S configurations of each chiral center, the Z and E double bond isomers, and the Z and E conformational isomers. Therefore, single stereoisomers of the disclosed compounds, as well as enantiomers, diastereomers, and geometric (or conformational) mixtures, are within the scope of this disclosure. Unless otherwise specified, all tautomers of the compounds described herein are within the scope of this disclosure.

[0095] Therefore, unless otherwise specified, the structures shown herein include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds in which hydrogen is replaced with deuterium or tritium, or carbon 13 C or 14Compounds having the disclosed structure with carbon substituted with carbon rich in C are within the scope of the present disclosure.

[0096] The compounds of formula (I) are useful for the de-crosslinking of formaldehyde-crosslinked biological samples such as FFPE tissue samples. Thus, in some embodiments, there is disclosed herein a composition comprising a formaldehyde-crosslinked biological sample (e.g., an FFPE sample) and a compound of formula (I) described herein. In some embodiments, this composition comprises two or more different compounds of formula (I) (i.e., this composition comprises a mixture of two or more different compounds of formula (I)).

[0097] In some embodiments, the composition comprising the compound(s) of formula (I) is in the form of a solution such as an aqueous solution. In some embodiments, this composition further comprises a buffer. For example, in some embodiments, the buffer is selected from tris(hydroxymethyl)aminomethane (tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate buffered saline, glycine, and citric acid. Without being limited by theory, when used in the de-crosslinking methods described herein, certain buffers may help isolate / eliminate formaldehyde from the de-crosslinked FFPE within the sample.

[0098] method There is disclosed herein a method for de-crosslinking a formaldehyde-crosslinked biological sample comprising contacting the sample with an effective amount of a compound of formula (I) or a salt thereof. The disclosed method can use any compound of formula (I) described herein, such as those specifically exemplified above, or any combination of at least two compounds of formula (I) or salts thereof (i.e., at least two different compounds of formula (I) or salts thereof).

[0099] In embodiments where two or more different compounds of formula (I) are used, they can be added to the sample simultaneously (e.g., in the same composition or simultaneously from separate compositions) or sequentially.

[0100] In some embodiments, the sample is a formalin-fixed paraffin-embedded tissue sample. In some embodiments where the sample is an FFPE sample, the method further comprises the step of deparaffinizing the sample prior to contacting the sample with a compound of formula (I) (or a salt thereof). The deparaffinization can be carried out according to standard methods. A common deparaffinization protocol includes incubating the sample with mineral oil at about 80 °C for 1 - 3 minutes and optionally repeating the mineral oil treatment one or more times to complete the paraffin removal process. Other methods of deparaffinizing the sample, such as treatment with xylene, can also be used. In some embodiments, the FFPE sample is not deparaffinized prior to subsequent processing steps.

[0101] This method may further comprise the step of treating the sample with a protease prior to contacting the sample with a compound of formula (I) (or a salt thereof). When detecting nucleic acids, protease treatment can remove contaminating proteins such as DNase and RNase. In some embodiments, the protease is selected from proteinase K, trypsin, LysC, proalanylase, and pepsin. In some embodiments, the protease is proteinase K. The protease treatment can be carried out at an appropriate time and appropriate temperature, for example, at about 50 - 60 °C for about 30 minutes to about 18 hours.

[0102] The step of contacting the sample with the compound of formula (I) (or a salt thereof) can be carried out for a sufficient amount of time to decrosslink the sample. In some embodiments, the contact step is carried out for about 5 minutes to about 120 minutes, or about 20 minutes to about 40 minutes. In some embodiments, the contact step is carried out for about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes, or about 120 minutes.

[0103] The step of contacting the sample with the compound of formula (I) (or a salt thereof) can be carried out at a temperature suitable for decrosslinking the sample. In some embodiments, the contact step is carried out at a temperature of about 20°C to about 100°C, or about 50°C to about 80°C. In some embodiments, the contact step is carried out at ambient temperature (i.e., room temperature). In some embodiments, the contact step is carried out at a temperature of about 20°C, about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, or about 95°C.

[0104] The step of contacting the sample with the compound of formula (I) (or a salt thereof) can be carried out by contacting the sample with a solution of the compound of formula (I) (or a salt thereof), for example, an aqueous solution containing the compound of formula (I) (or a salt thereof). In such embodiments, the resulting solution has a pH of about 4 to about 8.5, or about 4.5 to about 6.5, when in contact with the sample. In some embodiments, the aqueous solution has a pH of about 4.0, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5.

[0105] In some embodiments, the aqueous solution contains the compound of formula (I) (or a salt thereof) at a concentration of about 1 mM to about 120 mM, or about 5 mM to about 100 mM. In some embodiments, the aqueous solution contains the compound of formula (I) (or a salt thereof) at concentrations of about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, About 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37mM, about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, about 51mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 6 1mM, about 62mM, about 63mM, about 64mM, about 65mM, about 66mM, about 67mM, about 68mM, about 69mM, about 70mM, about 71mM, about 72mM, about 73mM, about 74mM, about 75mM, about 76mM, about 77 mM, about 78mM, about 79mM, about 80mM, about 81mM, about 82mM, about 83mM, about 84mM, about 85mM, about 86mM, about 87mM, about 88mM, about 89mM, about 90mM, about 91mM, about 92mM, about 93m It contains M at concentrations of approximately 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, 100 mM, 101 mM, 102 mM, 103 mM, 104 mM, 105 mM, 106 mM, 107 mM, 108 mM, 109 mM, 110 mM, 111 mM, 112 mM, 113 mM, 114 mM, 115 mM, 116 mM, 117 mM, 118 mM, 119 mM, or 120 mM. In embodiments in which two or more compounds of formula (I) are used, each may be present individually at any of the concentrations shown.

[0106] This method can efficiently decrosslink crosslinked biological samples, such as FFPE tissue samples, without sacrificing the quality of the biological substances contained within the sample, such as nucleic acids. In some embodiments, the method further includes a step of extracting one or more components from the sample after the contact step. In some embodiments, the one or more components are selected from nucleic acids and proteins. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the one or more components are proteins.

[0107] These methods may further include methods for detecting one or more components after extraction from a sample. For example, in embodiments in which nucleic acids are extracted from a sample, the nucleic acids can be detected, for example, by nucleotide sequencing or sequence-specific hybridization. In some embodiments, the method further includes a step of amplifying one or more nucleic acids from the sample. Amplification at any point in time can be used, including polymerase chain reaction (PCR), quantitative PCR, real-time PCR, hot-start PCR, single-cell PCR, nested PCR, in-situ colony PCR, digital PCR (dPCR), Droplet Digital® PCR (ddPCR), emulsion PCR, ligase chain reaction (LCR), transcription-based amplification systems (TAS), nucleic acid sequence-based amplification (NASBA), strand-displacement amplification (SDA), rolling circle amplification (RCA), and hyperbranched RCA (HRCA). In some embodiments, the nucleic acid amplification reaction is a multiple nucleic acid amplification reaction. In some embodiments, the sequence analysis includes fragment analysis and / or Sanger sequencing analysis, or next-generation sequencing (NGS) analysis.

[0108] Other characterization methods include dye binding, absorption, and enzymatic digestion. For example, dye binding assays use fluorescent dyes specific to the nucleic acid and a fluorometer for quantification. Absorption assays are spectrophotometric assays that utilize the natural light absorption properties of nucleic acids to determine concentration and purity. Enzymatic digestion includes applications of enzymatically digesting FFPE nucleic acids to improve quality or as an intermediate step for further experiments. For example, uracil DNA glycosylase can improve the quality of FFPE DNA by removing uracil nucleic acid bases resulting from cytosine deamination, while S1 nuclease can improve the quality of FFPE DNA by digesting single-stranded DNA, increasing the proportion of double-stranded DNA, and reducing artifacts caused by single-stranded DNA templates.

[0109] In some embodiments, once a protein is extracted from a sample, it can be detected in various ways. In some embodiments, the protein can be detected by mass spectrometry. Suitable mass spectrometry methods include electrospray ionization and matrix-assisted laser desorption / ionization (MALDI) (e.g., MALDI time-of-flight (MALDI-TOF)). In some embodiments, the detection step includes a colorimetric assay (e.g., BCA assay or Bradford assay), fluorescence spectroscopy (e.g., by using dye binding such as a fluorescent peptide assay or by detecting fluorescence from tryptophan residues in the protein), ultraviolet-visible spectroscopy (e.g., absorbance at 280 nm), electrophoresis (e.g., SDS-PAGE, which may be followed by staining such as silver staining), or immunoassay (e.g., Western blotting or immunohistochemistry).

[0110] Systems and kits Furthermore, this specification also discloses systems and kits for decrosslinking formaldehyde-crosslinked biological samples. The systems and kits include a compound of formula (I) or a salt thereof, for example, any compound of formula (I) described herein (including those specifically illustrated above). In some embodiments, the system or kit includes one or more compounds of formula (I) (or salts thereof), for example, a combination of two or more compounds of formula (I) (or salts thereof).

[0111] In some embodiments, the system or kit includes the compound alone or dissolved in a solvent such as water, DMSO, or a buffer. If the compound is provided in the absence of a solvent, the system or kit may further include a solvent capable of dissolving the compound. The system or kit may include one or more reagents used to perform a decrosslinking reaction and / or a downstream assay such as a detection method described herein. The system or kit may further include instructions for use, such as instructions for performing a decrosslinking reaction of a crosslinked biological sample or instructions for performing a downstream assay such as a detection method.

[0112] In some embodiments, the system or kit further comprises other compounds used in the manner disclosed herein. For example, in some embodiments, the system or kit further comprises a protease such as proteinase K. In some embodiments, the system or kit further comprises a compound for carrying out a deparaffinization step such as mineral oil or xylene. [Examples]

[0113] Example 1 Screening of decrosslinked compounds [ka] Candidate decrosslinking catalyst compounds (compounds 1-5) were screened by adding them to FFPE tissue lysates after proteinase K (ProK) digestion (56°C, 30 minutes), followed by decrosslinking at 80°C. (Compound 6 was tested individually, and the results are shown below.) A decrosslinking treatment was performed at 80°C for 4 hours as a positive control, and at 80°C for 30 minutes as a negative control. Candidate decrosslinking compounds were placed in lysates buffered at 20 mM or 100 mM at pH 7.4, 8.4, or 9.6 and incubated with the sample at 80°C for 30 minutes. Decrosslinked nucleic acids were purified using the Maxwell® RSC DNA FFPE kit (Promega catalog number AS1450) in a Maxwell® RSC 48 instrument (Promega catalog number AS8500) according to the manufacturer's protocol. After nucleic acid extraction, DNA recovery was measured by qPCR analysis using primers specific to RNase P ("R") and telomerase reverse transcriptase ("T"), yielding amplicons of 102 bp and 164 bp, respectively. Screening was performed using artificial FFPE cell pellets obtained from various tissue types and immortalized cell lines. The results are shown in Tables 1 and 2, respectively, showing DNA yield (ng) (top) and relative DNA yield standardized against negative control (bottom). As can be seen, the decrosslinking compounds disclosed herein show similar recovery rates in 30 minutes compared to 4 hours of incubation without the decrosslinking compounds. [Table 1] [Table 2]

[0114] The quality of DNA depolymerized by these lead candidates was further evaluated by testing FFPE tissues collected from human donors using next-generation sequencing (NGS). Healthy colon FFPE donor tissues were subjected to rapid pretreatment using compounds 1, 3, and 5 (Cpd.1, Cpd.3, and Cpd.5, respectively), and depolymerization was carried out at 80 °C for 30 minutes, along with a positive control (4 hours at 80 °C) and a negative control (30 minutes at 80 °C). Subsequently, the DNA was purified using a Maxwell® RSC 48 instrument (Promega Corp). The amount of DNA and the fragment size were evaluated using Promega's ProNex® DNA QC assay, which amplifies qPCR amplicons of 75 bp, 150 bp, and 300 bp. The amount of amplifiable DNA and the fragment size from samples rapidly depolymerized in the reactions of Cpd1, Cpd5, and Cpd3 were equivalent to those of the positive control samples (Figure 3). The average of three purification replicates was plotted with error bars representing the standard deviation.

[0115] NGS libraries were constructed using Illumina's AmpliSeq for Cancer HotSpot Panel v2. Figure 4A shows the molecular weight of the libraries determined by electrophoresis using an Agilent TapeStation. Figure 4B shows the quantification of the electrophoresed libraries. Figure 4C shows the amount of the libraries determined by qPCR using the ProNex® NGS Library Quantification Kit. G304A is high-quality human gDNA (Promega catalog number G3041) pooled from multiple donors. HD803 is formalin-damaged human gDNA pooled from multiple immortalized cell lines for use as a multiplex reference standard (Horizon, HD803). The NGS libraries from the rapid depolymerization reactions were of the expected size and were present in sufficient amounts for sequencing.

[0116] The library was sequenced using Illumina MiSeq with 150 paired-end cycles. Sequence Analysis Viewer and FastQC were used to evaluate sequencing performance. Error rate, %Q30, cluster density, cluster %PF, and Phas / Prephas (%) were all within expected ranges (data not shown). Bioinformatics analysis was performed using Illumina's BaseSpace DNA Amplicon app. Single nucleotide variants (SNVs) and insertion / deletion (Indels) with variant allele frequencies (VAFs) greater than 1% were identified. The total number was similar between the positive control and rapid decrosslinking catalytic purification (Figure 5A). The VAFs of mutations identified in the positive control were found to be in high agreement with those of catalytic purification (Figure 5B). This indicates that rapidly decrosslinked DNA maintains high sequence quality, allowing for VAF calls near the detection limit using NGS.

[0117] Example 2 Changes in decrosslinking over time Time-course experiments were performed to determine the minimum decrosslinking time required for complete decrosslinking with the catalytic compound (Figure 6). Cell pellets of artificial FFPE (Amsbio LLC) were decrosslinked at 80°C for 10, 20, and 30 minutes at Cpd1–5. Amplifiable DNA yield was evaluated using a PrimeTime qPCR assay targeting the 102 bp amplicon of the RPPH1 gene (IDT). The minimum decrosslinking times ranged from 10 minutes (Cpd4), 20 minutes (Cpd1, Cpd2, Cpd3), and 30 minutes (Cpd5). DNA yield was compared to a positive control (positive) decrosslinked at 80°C for 4 hours without the catalyst.

[0118] Example 3 pH optimization To determine the optimal pH for rapid decrosslinking, cell pellets of artificial FFPE (Amsbio LLC) were decrosslinked at 80°C for 30 minutes with 20 mM Cpd1–5 HCl salts in various pH lysis buffers (Figure 7A). Amplifiable DNA yield was evaluated using a PrimeTime qPCR assay targeting the 164 bp amplicon of the TERT gene (IDT). DNA yield was compared to a positive control (positive) decrosslinked at 80°C for 4 hours in pH 8 lysis buffer without catalyst, and a negative control (simulated) decrosslinked at 80°C for 30 minutes in various pH lysis buffers without catalyst. To determine the working pH of the lysis buffer during rapid decrosslinking, 20 mM Cpd HCl salt was added to lysis buffer pH 7.75 (optimal values ​​are shown in Figure 7A). The optimal pH was 4.80–5.90 (Figure 7B). Experiments were performed using catalytic free bases to confirm that the pH requirements could be achieved by the buffer (Figure 7C). After proteinase K digestion, the pH of the lysate was lowered with a predetermined amount of HCl to create lysis buffers at various pH levels. 20 mM free bases Cpd1 and Cpd3 were added to the lysate, and decrosslinking was performed at 80°C for 30 minutes. The yield of amplifiable DNA was evaluated using a PrimeTime qPCR assay targeting the 164 bp amplicon of the TERT gene (IDT). DNA yield peaked in buffers with a pH of 4.5–6.0.

[0119] Example 4 Optimization of catalyst concentration The optimal concentrations of the decrosslinking catalyst compound were determined by adding 20 mM, 10 mM, and 5 mM Cpd HCl 1–5 and decrosslinking at 80°C for 30 minutes (Figure 8). Various pH lysis buffers were used to reduce the amount of HCl added (derived from Cpd HCl salts). The yield of amplifiable DNA was evaluated using a PrimeTime qPCR assay targeting the 102 bp amplicon of the RPPH1 gene (IDT). DNA yield was compared to a positive control (positive) decrosslinked with pH 8 lysis buffer at 80°C for 4 hours without the catalyst. The yield of amplifiable DNA was found to be concentration-dependent.

[0120] Example 5 Purification of catalytically decrosslinked FFPE DNA from various human tissues, as well as evaluation by amplification, dye binding, and absorption.

[0121] The catalyst was tested in 13 types of human FFPE tissue (Figures 9A-9C: bladder, stomach, kidney, skin, lung, esophagus, pancreas, prostate, uterus, gallbladder, liver, colon, and breast). 30 mM compound 1 (Cpd1) was added to each FFPE lysate and decrosslinked at 80°C for 30 minutes (Cpd1), and compared to untreated simulated compound (positive) after decrosslinking at 80°C for 4 hours. 1-3 donors were used per tissue, and 2-4 purification replicates were performed per donor. DNA yield was evaluated by RT-qPCR using Promega ProNex® DNA QC Assay (catalog number NG1002) (Figure 9A), fluorescent dsDNA dye using Promega QuantiFluor® dsDNA System (catalog number E2671) (Figure 9B), and absorption using ThermoFisher NanoDrop (Figure 9C).

[0122] Example 6 Overview of the FFPE protein extraction workflow As outlined in Figure 10, a workflow for extracting proteins from FFPE was developed. Briefly, FFPE blocks from the human colon and human lung were microtome-cut to 20 μm. The sections were deparaffinized by washing with 100% xylene followed by 100% ethanol. The deparaffinized FFPE tissue was resuspended in a lysis buffer containing 50 mM HEPES (pH 8.0), 50 mM NaCl, 2 mM MgCl2, 2% SDS, and 1% glycerol. The resulting lysate was sonicated for a total of 10 minutes using 10-second on / 10-second off pulses, and the microtip probe received a total of approximately 10,000 J. Benzoase was added at a concentration of 100 units / ml, and the lysate was incubated at 37°C for 30 minutes. The lysates were subjected to decrosslinking catalyst or simulated treatment (H2O or DMSO), followed by decrosslinking incubation at various temperatures (65°C to 95°C) and times (10 minutes to 8 hours). The resulting crude protein extracts were analyzed by Pierce BCA protein assay (ThermoFisher), SDS-PAGE silver staining, or Western blotting. Selected crude protein extracts were further reduced and alkylated with 10 mM TCEP and 40 mM chloroacetamide and incubated at 37°C for 30 minutes. Proteins were purified using the SP3 workflow (Hughes et al. Nat Protoc. 2019;14(1):68-85). Briefly, 250 μg of MagSil beads and 80% acetonitrile were added to each protein sample. The proteins were bound, washed with 70% ethanol, and then washed with 100% acetonitrile. The proteins were resuspended in 30 μl of proteolytic buffer (200 mM HEPES (pH 8.0), trypsin / Lys-C 1:25 E / S ratio) and incubated at 37°C for 18 hours. The digested peptides were quantified using the Pierce Quantitative Fluorometric Peptide Assay (ThermoScientific).Next, the normalized peptide amounts were subjected to LC-MS / MS using a 1-hour gradient (0–36% B (80% acetonitrile)) with DIA-MS detection on an Exploris 240 Orbitrap mass spectrometer (ThermoScientific).

[0123] Example 7 FFPE proteins require heat decrosslinking incubation. In this example, the role of decrosslinking incubation in FFPE protein extraction was characterized for the first time. FFPE was subjected to decrosslinking incubation times ranging from 0 to 120 minutes at a temperature of 90°C and tested three times. The resulting lysates were centrifuged to separate the soluble protein fraction from the pelletized insoluble protein fraction. The soluble and insoluble protein fractions were analyzed using SDS-PAGE and subsequently stained with silver (Figure 11A). With increasing decrosslinking time, the yield of mobile protein increased from both the soluble and insoluble fractions. The role of denaturants in decrosslinking was also tested using 20 mM DTT, but no effect on mobile protein was observed (Figure 11A). Soluble protein was further quantified using the Pierce BCA assay (Figure 11B). Yields ranged from less than 5 μg / ml without decrosslinking incubation to 120 μg / ml with decrosslinking incubation at 90°C for 120 minutes.

[0124] Example 8 Rapid decrosslinking of FFPE proteins by catalytic compounds In this example, the catalytic effect on FFPE protein yield was then tested. Cpd1 and Cpd3 were incubated with FFPE lysates at various concentrations (0–70 mM) and pH (5.3–7.6), and decrosslinked at 90°C for 30 minutes (Table 3). The soluble protein fraction was analyzed using SDS-PAGE, followed by silver staining (Figure 12). To benchmark protein yield, lysates without the catalyst were extracted in parallel and either decrosslinked without (0') or decrosslinked at 90°C for 120 minutes (120'). Increasing the concentration of Cpd1 and Cpd3 and lowering the pH of the lysate increased the yield of mobile FFPE protein. [Table 3]

[0125] Example 9 The role of pH in FFPE protein decrosslinking In this example, the role of pH in the rapid FFPE protein decrosslinking reaction was tested. Cpd1, Cpd3, Cpd5, and Cpd6 were added to the lysate at a concentration of 70 mM in the pH range of 4.0–10.0, followed by decrosslinking at 90°C for 30 minutes. To control the effect of pH on decrosslinking, a catalyst-free treatment (using simulated addition of H2O or DMSO) was also added in the pH range of 4.0–10.0, followed by decrosslinking at 90°C for 30 minutes. To benchmark protein yield, catalyst-free lysates were extracted in parallel and decrosslinked at 90°C for 120 minutes (positive) or 90°C for 30 minutes (negative). Soluble protein fractions were analyzed using SDS-PAGE, followed by silver staining (Figures 13A–13E). Lowering the pH of the lysate alone was sufficient to increase the yield of mobile proteins, but the addition of the catalyst increased the yield compared to positive and negative controls. The catalyst also induced the appearance of higher molecular weight protein bands in a pH-dependent manner.

[0126] Example 10 The role of catalyst concentration in FFPE protein decrosslinking. In this example, the role of catalyst concentration in the rapid FFPE protein decrosslinking reaction was tested. While maintaining the pH of the lysate at 5.4–5.6, Cpd1, Cpd3, Cpd5, and Cpd6 were added to the lysate at concentrations of 0–120 mM, followed by decrosslinking at 90°C for 30 minutes. To benchmark protein yield, lysates decrosslinked without catalyst at 90°C for 120 minutes (positive) were also extracted in parallel. Soluble protein fractions were analyzed using SDS-PAGE, followed by silver staining (Figures 14A–14B). As the catalyst concentration increased, the yield of mobile proteins increased, and a band of higher molecular weight proteins appeared.

[0127] Example 11 The role of time and temperature in FFPE protein decrosslinking In this example, the time and temperature elements of decrosslinking incubation were then investigated. 70 mM Cpd1, Cpd3, Cpd5, and Cpd6 were tested at temperatures of 65°C, 80°C, and 95°C at three separate time points ranging from 10 minutes to 8 hours. For control, uncatalyzed extraction (simulation) was also performed in parallel. The soluble protein fraction was analyzed using SDS-PAGE, followed by silver staining (Figures 15A-15C). As expected, decrosslinking at higher temperatures and longer incubation times increased the yield of mobile proteins. The addition of catalysts, particularly Cpd1 and Cpd5, resulted in higher protein yields compared to the simulation at all temperatures and time points tested.

[0128] Example 12 Western blot evaluation of catalytic decrosslinked FFPE proteins Soluble protein extracts were tested by Western blotting using a primary histone H3 antibody (Figure 16). A 17 kDa histone H3 protein band was detected in the extracts of Cpd1, Cpd3, and Cpd5. The histone H3 protein band was also detected in uncatalyzed extracts decrosslinked at 90°C for 120 minutes (positive), 90°C for 30 minutes (negative), or at pH 5.5 at 90°C for 30 minutes (low pH).

[0129] Example 13 LC-MS / MS analysis of rapidly decrosslinked FFPE proteins The FFPE lysates were rapidly decrosslinked using six extraction repeats under each condition with 70 mM catalyst at pH approximately 5.5 (Cpd1, Cpd3, Cpd5) and a low pH approximately 5.5 control. The total protein extracts were then SP3 purified, desalted, proteolytic, and subjected to LC-MS / MS as described in Example 9. The protein identity and abundance of each catalyst were plotted on the low pH control and evaluated using volcano plots (Figures 17A-17C). A significant increase in protein was observed in the catalyst-treated samples compared to the control (Log2 change factor, p=0.05).

[0130] Example 14 Enrichment of human FFPE protein using rapid decrosslinking chemical reaction This example shows specific human proteins that were detected more abundantly after catalytic treatment compared to a low-pH control sample (Figure 18). These values ​​are calculated from the peak area (expressed as log2 abundance) of MS2 chromatography. Significant increases in protein peak area have been observed with the use of various catalysts.

Claims

1. The sample contains an effective amount of compound (I) of formula (I). 【Chemistry 1】 A method for decrosslinking a formaldehyde-crosslinked biological sample, comprising contacting it with a salt thereof, (i) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 is selected from H, C 1 ~C 6 alkyl, hydroxy-C 1 ~C 4 -alkyl, carboxy-C 1 ~C 4 -alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -alkyl, and heteroaryl-C 1 ~C 4 -alkyl, and is selected from R 3 is, -X-R 4 In the formula, X is -C(O)- and -SO 2 - Selected from, R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (ii) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Y-R 5 Selected from, where Y is the bond, -C(O)-, and -SO 2 - Selected from, R 5 C 1 ~C 6 Selected from alkyl, aryl, and heteroaryl, R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (iii) R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 The method wherein these atoms, together with the atoms to which they are bonded, form a optionally substituted 4- to 8-membered ring.

2. R 1 H is, R 2 C 1 ~C 6 Alkyl, -CH 2 -aryl and -CH 2 - Selected from heteroaryls, R 3 is, -X-R 4 In the formula, X is -C(O)-, and R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each alkyl group is independently either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl groups. The method according to claim 1, wherein each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy.

3. R 1 H is, R 2 C is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl. 1 ~C 6 It is alkyl, R 3 is, -X-R 4 And in the formula, X is -C(O)-, and R 4 The method according to claim 1 or 2, wherein is an unsubstituted phenyl or a phenyl substituted with one or two substituents independently selected from methoxy, hydroxy, and halo.

4. R 1 H is, R 2 is aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxyl groups. R 3 The method according to claim 1, wherein H is

5. R 1 and R 2 together with the nitrogen atom to which they are attached form a saturated 4- to 6-membered ring which is unsubstituted or substituted with one or two substituents independently selected from hydroxy, hydroxy-C 1 to C 4 alkyl, C 1 to C 4 -alkoxy, carboxy, carboxy-C 1 to C 4 alkyl, and oxo, R 3 The method according to claim 1, wherein H is...

6. R 1 H is, R 2 and R 3 together with the atoms to which they are attached form an unsubstituted or substituted saturated 4- to 7-membered ring with one or two substituents independently selected from hydroxy, hydroxy-C 1 to C 4 alkyl, C 1 to C 4 -alkoxy, carboxy, carboxy-C 1 to C 4 alkyl, and oxo, the method according to claim 1.

7. The compound of formula (I) is 【Chemistry 2-1】 【Chemistry 2-2】 The method according to claim 1, as well as a selection from those salts.

8. The compound of formula (I) is 【Transformation 3】 The method according to claim 1, as well as a selection from those salts.

9. The method according to any one of claims 1 to 8, wherein the compound of formula (I) is in the form of a salt.

10. The method according to any one of claims 1 to 9, wherein the compound of formula (I) is in the form of a hydrochloride salt.

11. The method according to any one of claims 1 to 10, comprising contacting the sample with an effective amount of at least two different compounds of formula (I) or salts thereof.

12. The method according to claim 11, wherein at least two different compounds of formula (I) or salts thereof are added to the sample simultaneously.

13. The method according to claim 11, wherein at least two different compounds of formula (I) or salts thereof are added sequentially to the sample.

14. The method according to any one of claims 1 to 13, wherein the sample is a formalin-fixed paraffin-embedded tissue sample.

15. The method according to claim 14, further comprising the step of deparaffinizing the sample before contacting the sample with the compound of formula (I) or a salt thereof.

16. The method according to any one of claims 1 to 15, further comprising the step of contacting the sample with a protease before contacting the sample with the compound of formula (I) or a salt thereof.

17. The method according to claim 16, wherein the protease is proteinase K.

18. The method according to any one of claims 1 to 17, wherein the contact step is performed for about 5 minutes to about 120 minutes.

19. The method according to claim 18, wherein the contact step is performed for about 20 minutes to about 40 minutes.

20. The method according to any one of claims 1 to 19, wherein the contact step is performed at a temperature of about 20°C to about 100°C.

21. The method according to any one of claims 1 to 20, wherein the contact step is performed at a temperature of about 50°C to about 85°C.

22. The method according to any one of claims 1 to 21, comprising contacting the sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution further comprises a buffer.

23. The method according to claim 22, wherein the solution comprises a buffer selected from tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate-buffered saline, glycine, and citrate.

24. The method according to any one of claims 1 to 23, comprising contacting the sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution obtained when the sample is contacted with the solution of the compound of formula (I) has a pH of about 4.0 to about 8.

5.

25. The method according to any one of claims 1 to 24, comprising contacting the sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution obtained when the sample is contacted with the solution of the compound of formula (I) has a pH of about 4.5 to about 6.

5.

26. The method according to any one of claims 1 to 25, comprising contacting the sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution contains the compound of formula (I) at a concentration of about 1 mM to about 120 mM.

27. The method according to any one of claims 1 to 26, comprising contacting the sample with a solution of the compound of formula (I) or a salt thereof, wherein the solution contains the compound of formula (I) at a concentration of about 5 mM to about 100 mM.

28. The method according to any one of claims 1 to 27, further comprising extracting one or more components from the sample after the contact step.

29. The method according to claim 28, wherein the one or more components are selected from nucleic acids and proteins.

30. The method according to claim 29, wherein the one or more components are nucleic acids, and the method further comprises the step of detecting and / or amplifying one or more nucleic acids.

31. The method according to claim 30, further comprising a step selected from pigment binding, absorption, and enzymatic digestion.

32. The method according to claim 29, wherein the one or more components are proteins, and the method further comprises the step of detecting one or more proteins.

33. The method according to claim 32, wherein the detection step includes colorimetric analysis, fluorescence spectroscopy, ultraviolet-visible spectroscopy, electrophoresis, immunoassay, or mass spectrometry.

34. A composition, Formaldehyde-crosslinked biological samples, and Compound of formula (I), 【Chemistry 4】 or a salt thereof During the ceremony, (i) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, hydroxy-C 1 ~C 4 -Alkyl, carboxy-C 1 ~C 4 -Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 is, -X-R 4 In the formula, X is -C(O)- and -SO 2 - Selected from, R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (ii) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Y-R 5 Selected from, where Y is the bond, -C(O)-, and -SO 2 - Selected from, R 5 C 1 ~C 6 Selected from alkyl, aryl, and heteroaryl, R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (iii) R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 The composition wherein these atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

35. R 1 H is, R 2 C 1 ~C 6 Alkyl, -CH 2 -aryl and -CH 2 - Selected from heteroaryls, R 3 is, -X-R 4 In the formula, X is -C(O)-, and R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each alkyl group is independently either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl groups. The composition according to claim 34, wherein each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy.

36. R 1 H is, R 2 C is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl. 1 ~C 6 It is alkyl, R 3 is, -X-R 4 And in the formula, X is -C(O)-, and R 4 The composition according to claim 34 or 35, wherein is an unsubstituted phenyl or a phenyl substituted with one or two substituents independently selected from methoxy, hydroxy, and halo.

37. R 1 H is, R 2 is aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxyl groups. R 3 The composition according to claim 34, wherein H is present.

38. R 1 and R 2 These, together with the nitrogen atom to which they are bonded, are either unsubstituted or hydroxy, hydroxy-C 1 ~C 4 Alkyl, C 1 ~C 4 -Alkoxy, Carboxy, Carboxy-C 1 ~C 4 A saturated 4-6 membered ring is formed by substituting one or two substituents independently selected from alkyl and oxo groups. R 3 The composition according to claim 34, wherein H is present.

39. R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, are either unsubstituted or hydroxy, hydroxy-C 1 ~C 4 Alkyl, C 1 ~C 4 -Alkoxy, Carboxy, Carboxy-C 1 ~C 4 The composition according to claim 34, which forms a saturated 4- to 7-membered ring substituted with one or two substituents independently selected from alkyl and oxo groups.

40. The compound of formula (I) is 【Chemistry 5-1】 【Chemistry 5-2】 The composition according to claim 34, further selected from those salts.

41. The compound of formula (I) is 【Transformation 6】 The composition according to claim 34, further selected from those salts.

42. The composition according to any one of claims 34 to 41, wherein the compound of formula (I) is in the form of a salt.

43. The composition according to any one of claims 34 to 42, wherein the compound of formula (I) is in the form of a hydrochloride salt.

44. The composition according to any one of claims 34 to 43, further comprising a buffer solution.

45. The composition according to claim 44, wherein the buffer solution is selected from tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate-buffered saline, glycine, and citric acid.

46. The composition according to any one of claims 34 to 45, comprising at least two different compounds of formula (I) or a salt thereof.

47. The composition according to any one of claims 34 to 46, wherein the sample is a formalin-fixed paraffin-embedded tissue sample.

48. It's a kit, (A) Compound of formula (I), 【Transformation 7】 or a salt thereof (i) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, hydroxy-C 1 ~C 4 -Alkyl, carboxy-C 1 ~C 4 -Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 is, -X-R 4 In the formula, X is -C(O)- and -SO 2 - Selected from, R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (ii) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Y-R 5 Selected from, where Y is the bond, -C(O)-, and -SO 2 - Selected from, R 5 C 1 ~C 6 Selected from alkyl, aryl, and heteroaryl, R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (iii) R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 The compounds of formula (I) or salts thereof, which, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings, (B) Instructions for use for decrosslinking a formaldehyde-crosslinked biological sample by contacting it with a compound of formula (I) or a salt thereof, the kit comprising:

49. R 1 H is, R 2 C 1 ~C 6 Alkyl, -CH 2 -aryl and -CH 2 - Selected from heteroaryls, R 3 is, -X-R 4 In the formula, X is -C(O)-, and R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each alkyl group is independently either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl groups. The kit according to claim 48, wherein each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxy.

50. R 1 H is, R 2 C is either unsubstituted or substituted with one substituent selected from hydroxyl and carboxyl. 1 ~C 6 It is alkyl, R 3 is, -X-R 4 And in the formula, X is -C(O)-, and R 4 The kit according to claim 48 or 49, wherein is an unsubstituted phenyl or a phenyl substituted with one or two substituents independently selected from methoxy, hydroxy, and halo.

51. R 1 H is, R 2 is aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, methoxy, amino, and carboxyl groups. R 3 The kit according to claim 48, wherein H is present.

52. R 1 and R 2 These, together with the nitrogen atom to which they are bonded, are either unsubstituted or hydroxy, hydroxy-C 1 ~C 4 Alkyl, C 1 ~C 4 -Alkoxy, Carboxy, Carboxy-C 1 ~C 4 A saturated 4-6 membered ring is formed by substituting one or two substituents independently selected from alkyl and oxo groups. R 3 The kit according to claim 48, wherein H is present.

53. R 1 H is, R 2 and R 3 These atoms, together with the atoms to which they are bonded, are either unsubstituted or hydroxy, hydroxy-C 1 ~C 4 Alkyl, C 1 ~C 4 -Alkoxy, Carboxy, Carboxy-C 1 ~C 4 The kit according to claim 48, which forms a saturated 4- to 7-membered ring substituted with one or two substituents independently selected from alkyl and oxo groups.

54. The compound of formula (I) is 【Chemistry 8-1】 【Chemistry 8-2】 The kit according to claim 48, further comprising salts selected from those salts.

55. The compound of formula (I) is 【Chemistry 9】 The kit according to claim 48, further comprising salts selected from those salts.

56. The kit according to any one of claims 48 to 55, wherein the compound of formula (I) is in the form of a salt.

57. The kit according to any one of claims 48 to 56, wherein the compound of formula (I) is in the form of a hydrochloride salt.

58. The kit according to any one of claims 48 to 57, wherein the composition comprises at least two different compounds of formula (I) or salts thereof.

59. The kit according to any one of claims 48 to 58, wherein the formaldehyde-crosslinked biological sample is a formalin-fixed paraffin-embedded tissue sample.

60. A method for decrosslinking a formaldehyde-crosslinked biological sample, wherein the sample is subjected to an effective amount of at least two different compounds of formula (I). 【Chemistry 10】 or including contact with a salt thereof, (B)R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, hydroxy-C 1 ~C 4 -Alkyl, carboxy-C 1 ~C 4 -Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 is, -X-R 4 In the formula, X is -C(O)- and -SO 2 - Selected from, R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (ii) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Y-R 5 Selected from, where Y is the bond, -C(O)-, and -SO 2 - Selected from, R 5 C 1 ~C 6 Selected from alkyl, aryl, and heteroaryl, R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (iii) R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 The method wherein these atoms, together with the atoms to which they are bonded, form a optionally substituted 4- to 8-membered ring.

61. A composition, Formaldehyde-crosslinked biological samples, and Compounds of at least two different formulas (I), 【Chemistry 11】 or a salt thereof During the ceremony, (B)R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, hydroxy-C 1 ~C 4 -Alkyl, carboxy-C 1 ~C 4 -Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 is, -X-R 4 In the formula, X is -C(O)- and -SO 2 - Selected from, R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (ii) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Y-R 5 Selected from, where Y is the bond, -C(O)-, and -SO 2 - Selected from, R 5 C 1 ~C 6 Selected from alkyl, aryl, and heteroaryl, R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (iii) R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 The composition wherein these atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

62. It's a kit, (A) At least two different compounds of formula (I), 【Chemistry 12】 or a salt thereof (B)R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, hydroxy-C 1 ~C 4 -Alkyl, carboxy-C 1 ~C 4 -Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 is, -X-R 4 In the formula, X is -C(O)- and -SO 2 - Selected from, R 4 C 1 ~C 6 - Selected from alkyl, aryl, and heteroaryl, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (ii) R 1 H, C 1 ~C 6 Alkyl and carboxy-C 1 ~C 4 - Selected from alkyl groups, R 2 C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Y-R 5 Selected from, where Y is the bond, -C(O)-, and -SO 2 - Selected from, R 5 C 1 ~C 6 Selected from alkyl, aryl, and heteroaryl, R 3 H is, Each aryl and heteroaryl is independently either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkyl, C 1 ~C 4 Substituting with one or two substituents independently selected from alkoxy, amino, amide, carboxy, and ester, Each alkyl group is either unsubstituted or optionally substituted with halo, hydroxy, alkoxy, amino, amide, carboxy, or ester C. 3 ~C 6 It is substituted with one or two substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. or (iii) R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. R 3 H is, or (iv)R 1 H is, R 2 and R 3 The compounds of formula (I) or salts thereof, which, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings, (B) Instructions for use for decrosslinking a formaldehyde-crosslinked biological sample by contacting it with the compound of formula (I) or a salt thereof, The kit includes the above.

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