Method and composition for decrosslinking biological samples

Abstract

This specification provides methods, compositions, and kits 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 / 500,184, filed on 4 May 2023, which is incorporated herein by reference in its entirety.

[0002] This specification provides methods, compositions, and kits 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. Before nucleic acids or other biomolecules can be extracted and used in downstream assays, these crosslinks must be reversed, which requires considerable pretreatment time. Typically, after deparaffinization and short-term proteinase K digestion, the tissue is decrosslinked for an extended period (several hours to several days) at high temperatures (60-90°C), with the industry standard being 4 hours at 80°C. While this decrosslinking step is necessary, it can lead to extraction failure and reduced sample quality, posing significant challenges for clinical and research applications. [Overview of the project]

[0004] In one embodiment, this specification discloses a method for decrosslinking a formaldehyde-crosslinked biological sample, comprising the step of simultaneously contacting the sample with a protease and a decrosslinking catalyst, wherein the decrosslinking catalyst is a compound of formula (I), formula (II), or formula (III). The compound of formula (I) is [ka] or a salt thereof, During the ceremony, R 1 is selected from H and C1-C6 alkyl, R 2 is selected from H, C1-C6 alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl, R 3 is selected from H, C1-C6 alkyl, and -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 more substituents independently selected from halo, C1-C4 alkyl, hydroxy, C1-C4 alkoxy, -NR a1 R b1 、-C(O)NR c1 R d1 、-COOR e1 、and -SO2NR f1 R g1 ; each alkyl is unsubstituted or substituted with one or more substituents independently selected from halo, hydroxy, C1-C4 alkoxy, thiol, C1-C4 alkylthio, -NR a1 R b1 、-C(O)NR c1 R d1 、-COOR e1 、-SO2NR f1 R g1 、-ONR h1 R i1 、-NR j1 OR k1 、-PO3H2, -SO3H, optionally substituted C3-C6 cycloalkyl, and optionally substituted 3-6 member heterocyclyl; wherein R a1 、R b1 、R c1 、R d1 、R e1 、R f1 、R g1 、R h1 、R i1 、R j1, and R k1 Each of these is independently selected from H, C1-C4 alkyl, C1-C4 hydroxyalkyl, and C1-C4 carboxyalkyl. In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine, optionally substitute to form 4-8 membered rings. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4-8 membered ring with optional substitutions. The compound of formula (II) is A-NR x R y (II) or a salt thereof, in the formula, A is H, C1~C 6- Alkyl, aryl, heteroaryl, C3-C 6- Cycloalkyl, aryl-C1~C4-alkyl, heteroaryl-C1~C4-alkyl, and -QR 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO2-, -SO2NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C1~C 6- Alkyl, aryl, heteroaryl, and C3-C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 Each is independently selected from H and C1-C6 alkyl groups. R x and R y Each is independently selected from H and C1-C6 alkyl, or R x and R yThese, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, -P(O)(OH)2, -B(OH)2, --COOR g2 ,-CONR h2 R i2 -SO2NR j2 R k2 , and -SO2OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group may be unsubstituted, or a halo, C1-C4 alkoxy, hydroxy, thiol, C1-C4 alkylthio, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 member heterocyclyl, or -COOR. g2 -PO3H2, -SO2OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is BX(III) or a salt thereof, in the formula, B is aryl, heteroaryl, C1-C6 alkyl, aryl-C1-C4-alkyl-, aryl-C2-C4-alkoxy-, heteroaryl-C1-C4-alkyl-, and heteroarylC2-C4-alkoxy-. X is selected from -COOH, -PO3H2, -B(OH)2, and -SO3H. Each aryl and heteroaryl is independently unsubstituted or substituted with one or more substituents independently selected from halo, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 hydroxyalkyl, -NR a3 R b3 、-COOR c3 、-SO3R d3 、-PO3H2, and -B(OH)2, Each alkyl is unsubstituted or substituted with one or more substituents independently selected from halo, C1-C4 alkoxy, hydroxy, thiol, C1-C4 alkylthio, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 member heterocyclyl, -COOH, -PO3H2 and -SO3H, wherein R a3 、R b3 、R c3 、and R d3 are each independently selected from H, -CH3, and -CH2CH3.

[0005] In some embodiments, the crosslinking catalyst is a compound of formula (I) or a salt thereof.

[0006] In some embodiments, R 1 is H, R 2 is selected from H, C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, R 3 is selected from H, C1-C6 alkyl, -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 and C1-C4 alkoxy, and each alkyl is independently unsubstituted or substituted with hydroxy, C1-C4 alkoxy, -NR a1 R b1 、-COOR e1 、-ONR h1 Ri1 , and -NR j1 Ure k1 It is substituted with one substituent selected from, where R a1 , R b1 , R e1 , R h1 , R i1 , R j1 , and R k1 Each of these is independently selected from H and -CH3.

[0007] 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.

[0008] 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.

[0009] In some embodiments, the compound of formula (I) is [ka] [ka] and selected from those salts.

[0010] In some embodiments, the decrosslinking catalyst is a compound of formula (II) or a salt thereof.

[0011] In some embodiments, R x H is R y A is selected from H and -CH3. In some embodiments, A is phenyl, monocyclic heteroaryl, and C5-C 6- Selected from cycloalkyl groups, each is either unsubstituted or substituted with one or two substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, -P(O)(OH)2, -B(OH)2, and -COOH groups.

[0012] In some embodiments, A is C1~C 6- Alkyl and -QR 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO2-, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C1~C 6- Alkyl, phenyl, or monocyclic heteroaryl, where each alkyl is either unsubstituted or hydroxy, -P(O)(OH) 2、 -COOR g2 , and -NR m2 R n2 It is substituted with one or two substituents independently selected from, where R a2 , R b2 , R d2 、 R e2 , R f2 、 R g2 、 R m2 , and R n2 These are H.

[0013] In some embodiments, R x and R yThese, together with the nitrogen atoms to which they are bonded, form a six-membered ring selected from morpholine, thiomorpholine, selenomorpholine, and piperazine, each of which is either unsubstituted or substituted with one or two oxo groups, and A is -CH3, -CH2CH3, -CH2COOH, -CH2CH2OH, and -QR 5 Selected from, Q is -NR a2 -or-NR b2 CO-, and in the formula, R a2 and R b2 H and R 5 This is either H or -CH3.

[0014] In some embodiments, the compound of formula (II) is [ka] and selected from those salts.

[0015] In some embodiments, the decrosslinking catalyst is a compound of formula (III) or a salt thereof.

[0016] In some embodiments, B is an aryl or heteroaryl, each of which is either unsubstituted or substituted with one or two substituents independently selected from halo, methyl, methoxy, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H.

[0017] In some embodiments, B is a monocyclic heteroaryl having one heteroatom selected from phenyl or N, O, and S, each of which is either unsubstituted or substituted with one substituent selected from methyl, methoxy, fluoro, chloro, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H.

[0018] In some embodiments, the compound of formula (III) is [ka] and selected from those salts.

[0019] In some embodiments, the decrosslinking catalyst is [ka] and selected from those salts.

[0020] In some embodiments, the decrosslinking catalyst is in the form of a salt. In some embodiments, the decrosslinking catalyst is in the form of a hydrochloride salt.

[0021] In some embodiments, the method involves contacting a sample with an effective amount of at least two different decrosslinking catalysts or salts thereof. In some embodiments, at least two different decrosslinking catalysts or salts thereof are added to the sample simultaneously. In some embodiments, at least two different decrosslinking catalysts or salts thereof are added to the sample sequentially.

[0022] 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 it with a protease and a decrosslinking catalyst. In some embodiments, the protease is proteinase K.

[0023] 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.

[0024] In some embodiments, the process involves contacting the sample with a solution containing the decrosslinking catalyst and the protease. In some embodiments, after contacting the sample with the solution, the resulting solution has a pH of about 4.0 to about 8.5. In some embodiments, after contacting the sample with the solution, the resulting solution has a pH of about 4.5 to about 6.5.

[0025] In some embodiments, the solution contains a decrosslinking catalyst at a concentration of about 1 mM to about 100 mM. In some embodiments, the solution contains a decrosslinking catalyst at a concentration of about 10 mM to about 50 mM.

[0026] In some embodiments, the protease and / or decrosslinking catalyst is added to the buffer solution. In some embodiments, the buffer solution includes tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate-buffered saline, glycine, or citrate.

[0027] In some embodiments, the method involves contacting a sample with a first solution containing a protease and a second solution containing a decrosslinking catalyst. In some embodiments, the pH of the solution obtained after the contact step is about 4.0 to about 8.5. In some embodiments, the pH of the solution obtained after the contact step is about 4.5 to about 6.5. In some embodiments, the second solution contains a decrosslinking catalyst at a concentration of about 1 mM to about 100 mM. In some embodiments, the second solution contains a decrosslinking catalyst at a concentration of about 5 mM to about 50 mM.

[0028] 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 the step of detecting and / or amplifying one or more nucleic acids. In some embodiments, the method further includes a step selected from dye binding, absorption, and enzymatic digestion.

[0029] In another embodiment, this specification discloses a composition comprising a protease and a decrosslinking catalyst, wherein the decrosslinking catalyst is a compound of formula (I), formula (II), or formula (III). The compound of formula (I) is [ka] or a salt thereof, During the ceremony, R 1 It is selected from H and C1-C6 alkyl groups. R 2 The group is selected from H, C1-C6 alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 These are H, C1-C6 alkyl, and -XR 4 Selected from, where X is selected from -C(O)- and -SO2-, R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently unsubstituted or halo, C1-C4 alkyl, hydroxy, C1-C4 alkoxy, -NR a1 R b1 -C(O)NR c1 R d1 ,-COOR e1 , and -SO2NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or contains halo, hydroxy, C1-C4 alkoxy, thiol, C1-C4 alkylthio, or -NR. a1 R b1 -C(O)NR c1 R d1 ,-COOR e1 -SO2NR f1 R g1 ,-ONR h1 R i1 , -NR j1 Ure k1 It is substituted with one or more substituents independently selected from -PO3H2, -SO3H, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 Each of these is independently selected from H, C1-C4 alkyl, C1-C4 hydroxyalkyl, and C1-C4 carboxyalkyl. In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine, optionally substitute to form 4-8 membered rings. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4-8 membered ring with optional substitutions. The compound of formula (II) is A-NR x R y (II) or a salt thereof, in the formula, A is H, C1~C 6- Alkyl, aryl, heteroaryl, C3-C 6- Cycloalkyl, aryl-C1~C4-alkyl, heteroaryl-C1~C4-alkyl, and -QR 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO2-, -SO2NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C1~C 6- Alkyl, aryl, heteroaryl, and C3-C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 Each is independently selected from H and C1-C6 alkyl groups. R x and R y Each is independently selected from H and C1-C6 alkyl, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, -P(O)(OH)2, -B(OH)2, --COOR g2 ,-CONR h2 R i2 -SO2NR j2 R k2 , and -SO2OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group may be unsubstituted, or a halo, C1-C4 alkoxy, hydroxy, thiol, C1-C4 alkylthio, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 member heterocyclyl, or -COOR. g2 -PO3H2, -SO2OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, Rg2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is BX(III) or a salt thereof, in the formula, B is aryl, heteroaryl, C1-C6 alkyl, aryl-C1-C4-alkyl-, aryl-C2-C4-alkoxy-, heteroaryl-C1-C4-alkyl-, and heteroarylC2-C4-alkoxy-. X is selected from -COOH, -PO3H2, -B(OH)2, and -SO3H. Each aryl and heteroaryl is independently unsubstituted or halo, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 hydroxyalkyl, -NR a3 R b3 ,-COOR c3 , -SO3R d3 It is substituted with one or more substituents independently selected from -PO3H2 and -B(OH)2, Each alkyl group is either unsubstituted or substituted with one or more substituents independently selected from halo, C1-C4 alkoxy, hydroxy, thiol, C1-C4 alkylthio, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 membered heterocyclyl, -COOH, -PO3H2, and -SO3H. In the formula, R a3 , R b3 , R c3 , and R d3 Each of these is independently selected from H, -CH3, and -CH2CH3.

[0030] In some embodiments, the decrosslinking catalyst is a compound of formula (I) or a salt thereof.

[0031] In some embodiments, R 1H is R 2 R is selected from H, C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, 3 H, C1-C6 alkyl, -XR 4 Selected from, where X is selected from -C(O)- and -SO2-, R 4 The alkyl groups are selected from C1-C6 alkyl, aryl, and heteroaryl groups, and each aryl and heteroaryl group is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, and C1-C4 alkoxy groups, and each alkyl group is independently unsubstituted or substituted with hydroxy, C1-C4 alkoxy, -NR groups. a1 R b1 ,-COOR e1 ,-ONR h1 R i1 , and -NR j1 Ure k1 It is substituted with one substituent selected from, where R a1 , R b1 , R e1 , R h1 , R i1 , R j1 , and R k1 Each of these is independently selected from H and -CH3.

[0032] 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.

[0033] In some embodiments, R 1 H is R 2 and R 3These 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.

[0034] In some embodiments, the compound of formula (I) is [ka] [ka] and selected from those salts.

[0035] In some embodiments, the decrosslinking catalyst is a compound of formula (II) or a salt thereof. In some embodiments, R x H is R y A is selected from H and -CH3. In some embodiments, A is phenyl, monocyclic heteroaryl, and C5-C 6- Selected from cycloalkyl groups, each of which is either unsubstituted or substituted with one or two substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, -P(O)(OH)2, -B(OH)2, and -COOH. In some embodiments, A is C1-C 6- Alkyl and -QR 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO2-, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C1~C 6- Alkyl, phenyl, or monocyclic heteroaryl, where each alkyl is either unsubstituted or hydroxy, -P(O)(OH) 2、 -COOR g2 , and -NR m2 R n2It is substituted with one or two substituents independently selected from, where R a2 , R b2 , R d2 、 R e2 , R f2 、 R g2 、 R m2 , and R n2 These are H.

[0036] In some embodiments, R x and R y These, together with the nitrogen atoms to which they are bonded, form a six-membered ring selected from morpholine, thiomorpholine, selenomorpholine, and piperazine, each of which is either unsubstituted or substituted with one or two oxo groups, and A is -CH3, -CH2CH3, -CH2COOH, -CH2CH2OH, and -QR 5 Selected from, Q is -NR a2 -or-NR b2 CO-, and in the formula, R a2 and R b2 H and R 5 This is either H or -CH3.

[0037] In some embodiments, the compound of formula (II) is [ka] and selected from those salts.

[0038] In some embodiments, the de-crosslinking catalyst is a compound of formula (III), or a salt thereof. In some embodiments, B is aryl or heteroaryl, each of which is unsubstituted or substituted with one or two substituents independently selected from halo, methyl, methoxy, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H. In some embodiments, B is phenyl or monocyclic heteroaryl having one heteroatom selected from N, O, and S, each of which is unsubstituted or substituted with one substituent selected from methyl, methoxy, fluoro, chloro, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H.

[0039] In some embodiments, the compound of formula (III) is

Chemical formula

[0040] In some embodiments, the de-crosslinking catalyst is

Chemical formula

[0041] In some embodiments, the de-crosslinking catalyst is in the form of a salt. In some embodiments, the de-crosslinking catalyst is in the form of a hydrochloride.

[0042] In some embodiments, the protease is proteinase K.

[0043] In some embodiments, the composition comprises a solution of the depolymerization catalyst and the protease. In some embodiments, when the solution of the depolymerization catalyst and the protease is added to a crosslinked biological sample, the solution has a pH of from about 4.0 to about 8.5. In some embodiments, when the solution of the depolymerization catalyst and the protease is added to a crosslinked biological sample, the solution has a pH of from about 4.5 to about 6.5. In some embodiments, the solution comprises a depolymerization catalyst at a concentration of from about 1 mM to about 100 mM. In some embodiments, the solution comprises a depolymerization catalyst at a concentration of from about 10 mM to about 50 mM.

[0044] In some embodiments, the composition comprises at least two different depolymerization catalysts, or salts thereof.

[0045] In some embodiments, the composition further comprises a buffer. In some embodiments, the buffer is tris(hydroxymethyl)aminomethane.

[0046] In some embodiments, the composition further comprises a formaldehyde-crosslinked biological sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded tissue sample.

[0047] In another aspect, there is disclosed herein a kit for depolymerizing a formaldehyde-crosslinked biological sample, the kit comprising: (i) a depolymerization catalyst which is a compound of formula (I), formula (II), or formula (III), and (ii) instructions for depolymerizing a formaldehyde-crosslinked biological sample that instruct the user to contact the sample with the depolymerization catalyst and the protease simultaneously, The compound of formula (I) is

Chemical formula

[0048] In some embodiments, the kit further comprises a protease. In some embodiments, the protease is proteinase K.

[0049] In some embodiments, the kit comprises at least two different decrosslinking catalysts, or salts thereof.

[0050] In some embodiments, the kit further comprises at least one component selected from a buffer, a salt, and packaging material.

[0051] In another embodiment, the Specified herein discloses a method for decrosslinking a formaldehyde-bridged biological sample, comprising the step of simultaneously contacting the sample with a protease and at least two different decrosslinking catalysts, each decrosslinking catalyst being a compound of formula (I), formula (II), or formula (III) as defined herein.

[0052] In another aspect, the present disclosure provides a composition comprising a protease and at least two different depolymerization catalysts, each depolymerization catalyst being a compound of formula (I), formula (II), or formula (III) as defined herein.

[0053] In another aspect, the present disclosure provides a kit for depolymerizing a formaldehyde-crosslinked biological sample, the kit comprising: (i) at least two different depolymerization catalysts, each depolymerization catalyst being a compound of formula (I), formula (II), or formula (III) as defined herein; and (ii) instructions for depolymerizing a formaldehyde-crosslinked biological sample that instruct the user to contact the sample with the depolymerization catalysts and protease simultaneously.

[0054] In another aspect, the present disclosure provides a kit for depolymerizing a formaldehyde-crosslinked biological sample, the kit comprising: (i) at least two different depolymerization catalysts, each depolymerization catalyst being a compound of formula (I), formula (II), or formula (III) as defined herein; and (ii) instructions for depolymerizing a formaldehyde-crosslinked biological sample that instruct the user to contact the sample simultaneously with a first solution comprising protease and a second solution comprising depolymerization catalyst. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] [Figure 1] Exemplary crosslinks formed when using formalin to fix tissue, including protein-protein crosslinks and protein-DNA crosslinks, are shown. [Figure 2] A current DNA extraction protocol is shown compared to the workflow according to the method described herein. [Figure 3] An exemplary workflow used to screen depolymerization catalyst compounds is shown, together with positive and negative controls. [Figure 4]This shows the DNA yield and quality purified from healthy FFPE colon tissue using decrosslinking catalyst compounds and positive and negative controls, as measured by ProNex® DNA QC assays (75bp, 150bp, and 300bp qPCR amplicons). [Figure 5] Figures A-C show the quantity and quality of DNA sequencing libraries prepared using Illumina's AmpliSeq for Cancer HotSpot Panel v2 from the purified DNA in Figure 4. Figure 5A shows the molecular weight of the library determined by electrophoresis using Agilent TapeStation. Figure 5B shows the quantification of the electrophoretic library. Figure 5C shows the quantity of the library determined by qPCR using the ProNex® NGS Library Quantification Kit. [Figure 6] Figures A and B show the agreement in the number of single nucleotide variants with the DNA purified from Figure 5, as determined by sequencing using Illumina MiSeq. Figure 6A shows the total number of mutant alleles with a frequency greater than 1%. Figure 6B shows the mutant allele frequencies between DNA purified by rapid decrosslinking catalyst, positive control, and sequencing control gDNA. [Figure 7] This shows the amplifiable DNA yield from FFPE rapidly decrosslinked at 80°C for 10, 20, and 30 minutes using a catalytic compound. [Figure 8] Figures A through C show the optimal pH for rapid decrosslinking reactions using catalyst compounds. [Figure 9] 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 10] This document describes a screening method for determining whether a decrosslinking catalyst compound is compatible with proteinase K digestion (including positive and negative controls). [Figure 11] Figure 7 shows the total yield (ng) of amplified DNA determined by the duplex qPCR assay from the workflow described. [Figure 12]Figures A and B show the effect of protease K (ProK) digestion time on DNA yield. Figure 12A shows a schematic diagram of the experimental design. Figure 12B shows the total yield (ng) of amplified DNA determined by duplex qPCR assays and ProNex® QC qPCR assays (75 bp, 150 bp, and 300 bp amplicons). [Figure 13] Figures A and B show the FFPE DNA yield from a single-step ProK digestion and decrosslinking incubation. Figure 13A shows a schematic diagram of the experimental design. Figure 13B shows the total yield (ng) of amplifiable DNA determined by duplex qPCR assay. [Figure 14A] Figures A–C show the yield of purified DNA from the rapid decrosslinking reaction with Cpd 1. DNA was purified from 13 human FFPE tissues and evaluated using amplification assays (Figure 14A), dsDNA dye-binding assays (Figure 14B), and UV-Vis absorption (Figure 14C). [Figure 14B] Figures A–C show the yield of purified DNA from the rapid decrosslinking reaction with Cpd 1. DNA was purified from 13 human FFPE tissues and evaluated using amplification assays (Figure 14A), dsDNA dye-binding assays (Figure 14B), and UV-Vis absorption (Figure 14C). [Figure 14C] Figures A–C show the yield of purified DNA from the rapid decrosslinking reaction with Cpd 1. DNA was purified from 13 human FFPE tissues and evaluated using amplification assays (Figure 14A), dsDNA dye-binding assays (Figure 14B), and UV-Vis absorption (Figure 14C). [Modes for carrying out the invention]

[0056] This specification provides compositions, methods, and kits for decrosslinking formaldehyde-crosslinked biological samples, such as FFPE tissue samples. In some embodiments, both decrosslinking and protease digestion can be performed in a single step, allowing samples to be decrosslinked in significantly less time than currently used methods. For example, in some embodiments, FFPE samples can be efficiently decrosslinked in as little as 30 minutes without compromising sample quality.

[0057] 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.

[0058] As used herein, in the appended claims, the singular forms "a," "an," and "the" also include the plural forms unless the context clearly indicates otherwise.

[0059] 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".

[0060] 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.

[0061] 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 rd Edition, 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 the entire contents of each of these are incorporated herein by reference.

[0062] 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.

[0063] 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 16 Alkenyl), 2-14 carbon atoms (C2-C 14 Alkenyl), 2-12 carbon atoms (C2-C 12 Alkenyl), 2 to 10 carbon atoms (C2-C 10Alkenyls 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.

[0064] As used herein, the term "alkynyl" means a linear or branched hydrocarbon chain radical 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.

[0065] 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.

[0066] As used herein, the term "alkylthio" refers to a molecule in which an alkyl group is added to the parent molecule via a sulfur atom, as defined herein. Typical examples of alkoxys include, but are not limited to, methylthio, ethylthio, and propylthio.

[0067] 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."

[0068] 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).

[0069] As used herein, the term "carboxy" refers to the -COOH group.

[0070] 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.

[0071] As used herein, the term "cycloalkyl" refers to a saturated carbocyclic radical 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.

[0072] 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).

[0073] As used herein, the terms "halogen" or "halo" refer to F, Cl, Br, or I.

[0074] As used herein, the term “haloalkyl” refers to an alkyl group 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, as defined herein. 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.

[0075] 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 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. The bond site of a bicyclic heteroaryl group in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.) may be on either ring, i.e., a ring containing a heteroatom (e.g., 2-indolyl) or a ring without a heteroatom (e.g., 5-indolyl). Examples of five-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Examples of five-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Examples of five-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Examples of five-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Examples of six-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Examples of six-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl.Examples of 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.

[0076] As used herein, the term “heterocyclyl” refers to a radical of a 3- to 10-membered non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, selenium, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the bond site may be a carbon or nitrogen atom, 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 ring 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).

[0077] As used herein, the terms "hydroxy" or "hydroxyl" refer to the -OH group.

[0078] As used herein, the term "hydroxyalkyl" refers to an alkyl group in which at least one hydrogen atom is replaced by a hydroxyl group, as defined herein. Typical examples of hydroxyalkyls include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 3-hydroxypropyl.

[0079] As used herein, the term "oxo" refers to the =O group.

[0080] As used herein, the term "thiol" refers to the -SH group. 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 by a selection of the listed indicated groups 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.

[0081] As used herein, the term “simultaneous” means, with respect to multiple steps, that the steps are performed simultaneously or sequentially without any removal or washing steps in between. For example, when a sample comes into contact with two compounds A and B simultaneously, the sample comes into contact with compounds A and B simultaneously (e.g., with a single composition containing both compounds A and B), or the sample comes into contact with compound A first, followed by compound B without removing compound A or washing the sample, or the sample comes into contact with compound B first, followed by compound A without removing compound B or washing the sample.

[0082] Where used herein, the chemical structure is indicated as follows: [ka] This represents a point where one part is bonded to another part (for example, a substituent bonded to the rest of the compound).

[0083] Where a divalent substituent is specified by a conventional chemical formula written from left to right, such notation shall also include substituents resulting from writing the structure from right to left. For example, where a divalent group is indicated as -CH2O-, such notation shall also include -OCH2-, and similarly, -OC(O)NH- shall also include -NHC(O)O-.

[0084] 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.

[0085] Decrosslinking catalyst This specification discloses compositions, methods, and kits for decrosslinking formaldehyde-crosslinked biological samples, such as FFPE samples. The compositions, methods, and kits described below in further detail include a decrosslinking catalyst which is a compound of formula (I), a compound of formula (II), or a compound of formula (III).

[0086] In some embodiments, the decrosslinking catalyst is a compound of formula (I). [ka] or a salt thereof, During the ceremony, R 1 It is selected from H and C1-C6 alkyl groups. R 2 The group is selected from H, C1-C6 alkyl, aryl, heteroaryl, aryl-C1-C4-alkyl, and heteroaryl-C1-C4-alkyl. R 3 These are H, C1-C6 alkyl, and -XR 4 Selected from, where X is selected from -C(O)- and -SO2-, R 4 These are selected from C1-C6 alkyl, aryl, and heteroaryl groups. Each aryl and heteroaryl is independently unsubstituted or halo, C1-C4 alkyl, hydroxy, C1-C4 alkoxy, -NR a1 R b1 -C(O)NR c1 R d1 ,-COOR e1 , and -SO2NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or contains halo, hydroxy, C1-C4 alkoxy, thiol, C1-C4 alkylthio, or -NR. a1 R b1 -C(O)NR c1 R d1 ,-COOR e1 -SO2NR f1 R g1 ,-ONR h1 R i1 , -NR j1 Ure k1 It is substituted with one or more substituents independently selected from -PO3H2, -SO3H, optionally substituted C3-C6 cycloalkyl groups, and optionally substituted 3-6 membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 Each of these is independently selected from H, C1-C4 alkyl, C1-C4 hydroxyalkyl, and C1-C4 carboxyalkyl. In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine, optionally substitute to form 4-8 membered rings. In the formula, R 2 and R 3 These atoms, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings.

[0087] In some embodiments, R 1 H is R 2 R is selected from H, C1-C6 alkyl, -CH2-aryl, and -CH2-heteroaryl, 3 H, C1-C6 alkyl, -XR 4 Selected from, where X is selected from -C(O)- and -SO2-, R 4 The alkyl groups are selected from C1-C6 alkyl, aryl, and heteroaryl groups, and each aryl and heteroaryl group is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, and C1-C4 alkoxy groups, and each alkyl group is independently unsubstituted or substituted with hydroxy, C1-C4 alkoxy, -NR groups. a1 R b1 ,-COOR e1 ,-ONR h1 R i1 , and -NR j1 Ure k1 It is substituted with one substituent selected from, where R a1 , R b1 , R e1 , R h1 , R i1 , R j1 , and R k1 Each of these is independently selected from H and -CH3.

[0088] In some embodiments, R 1 H is R 2 is selected from H and C1-C6 alkyl groups, R 3 The alkyl group is selected from H and C1-C6 alkyl groups, and each alkyl group is independently unsubstituted or hydroxyl-NR a1 R b1 ,-COOR e1 ,-ONR h1 R i1 , and -NR j1 Ure k1 It is substituted with one substituent selected from, where R a1 , R b1 , R e1 , Rh1 , R i1 , R j1 , and R k1 Each is independently selected from H and -CH3. In some embodiments, R 1 H is R 2 R is selected from H and C1-C2 alkyl groups. 3 The alkyl group is selected from H and C1-C2 alkyl groups, and each alkyl group is independently unsubstituted or hydroxyl-NR a1 R b1 ,-COOR e1 ,-ONR h1 R i1 , and -NR j1 Ure k1 It is substituted with one substituent selected from, where R a1 , R b1 , R e1 , R h1 , R i1 , R j1 , and R k1 Each is independently selected from H and -CH3. In some embodiments, R 1 H is R 2 R is selected from H, -CH3, -CH2CH3, -CH2CH2OH, and -CH2CH2NHOH. 3 is selected from H, -CH3, -CH2CH3, -CH2CH2OH, -CH2CH2NH2, -CH2CH2ONH2, -CH2CH2ONHCH3, and -CH2COOH. In some embodiments, the compound of formula (I) is [ka] and selected from those salts.

[0089] In some embodiments, R 1 H is R 2 is selected from H and C1-C6 alkyl groups, R 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4The alkyl groups are selected from C1-C6 alkyl and aryl groups, and each alkyl group is independently unsubstituted or hydroxy-COOR. e1 It is substituted with one or two substituents independently selected from, where R e1 R is selected from H and CH3, and each aryl and heteroaryl is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, and C1-C4 alkoxy. In some embodiments, R 1 H is R 2 R is selected from H and C1-C4 alkyl groups, where the alkyl group is either unsubstituted or substituted with one substituent selected from -OH and -COOH groups. 3 -XR 4 In the formula, X is selected from -C(O)- and -SO2-, and R 4 R is selected from -CH3 and phenyl, where phenyl is either unsubstituted or substituted with one substituent selected from methoxy, hydroxy, and halo. In some embodiments, R 1 H is R 2 R is selected from H, -CH3, -CH2CH2CH2CH3, -CH2COOH, and -CH2CH2OH. 2 The compound is selected from -COCH3, -COPh, -CO(4-methoxyphenyl), -SO2CH3, and -SO2Ph. In some embodiments, the compound of formula (I) is [ka] and selected from those salts.

[0090] 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 independently substituted with one or two substituents selected from halo, hydroxy, and C1~C4 alkoxy, 3H is H. In some embodiments, R 1 H is R 2 R is selected from -CH2-aryl and -CH2-heteroaryl, each of which is independently unsubstituted or substituted with one or two substituents independently selected from halo, hydroxy, and C1-C4 alkoxy, 3 H is H. In some embodiments, R 1 H is R 2 R is -CH2-phenyl and -CH2-heteroaryl, where the heteroaryl is a monocyclic heteroaryl (e.g., pyridyl, furanyl, or thiophenyl) having one heteroatom selected from N, O, and S, and the phenyl or heteroaryl is unsubstituted or substituted with one or two substituents independently selected from chloro, hydroxy, and methoxy, 3 is H. In some embodiments, the compound of formula (I) is [ka] and selected from those salts.

[0091] In some embodiments, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring, R 3 H is H. In some embodiments, R 1 and R 2 Together with the nitrogen atom to which they are bonded, they form a saturated 4-8 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, R 3 H is H. In some embodiments, R 1 and R 2Together 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. In some embodiments, R 1 and R 2 Together with the nitrogen atom to which they are bonded, they form a saturated 5-6 membered ring that is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, -CH2OH, carboxy, -CH2COOH, and oxo, R 3 H is H. 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, where each ring is either unsubstituted or substituted with one or two substituents independently selected from hydroxy, -CH2OH, carboxy, -CH2COOH, and oxo, R 3 is H. In some embodiments, the compound of formula (I) is [ka] and selected from those salts.

[0092] In some embodiments, R 1 H is R 2 and R 3 These, together with the atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. In some embodiments, R 1 H is 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, R1 H is 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 1 H is 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 1 H is R 2 and R 3 These, together with the atoms to which they are bonded, form a 1,2-oxazetidine, isoxazolidine, 1,2-oxazinane, or 1,2-oxazepane ring, where each ring 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 H is R 2 and R 3 These, together with the atoms to which they are bonded, form a 1,2-oxazetidine, isoxazolidine, 1,2-oxazinane, or 1,2-oxazepane ring, where each ring is either unsubstituted or substituted with one substituent selected from hydroxy, -CH2OH, -CH2CH2OH, methoxy, carboxy, and oxo. In some embodiments, the compound of formula (I) is [ka] and selected from those salts.

[0093] In some embodiments, the compound of formula (I) is [ka] [ka] and selected from those salts.

[0094] In some embodiments, the decrosslinking catalyst is a compound of formula (II). A-NR x R y (II) or a salt thereof, in the formula, A is H, C1~C 6- Alkyl, aryl, heteroaryl, C3-C 6- Cycloalkyl, aryl-C1~C4-alkyl, heteroaryl-C1~C4-alkyl, and -QR 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO2-, -SO2NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C1~C 6- Alkyl, aryl, heteroaryl, and C3-C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 Each is independently selected from H and C1-C6 alkyl groups. R x and R y Each is independently selected from H and C1-C6 alkyl, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, C1-C4 hydroxyalkyl, -P(O)(OH)2, -B(OH)2, -COOR g2 ,-CONR h2 R i2 -SO2NR j2 R k2 , and -SO2OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group may be unsubstituted, or a halo, C1-C4 alkoxy, hydroxy, thiol, C1-C4 alkylthio, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 member heterocyclyl, or -COOR. g2 -PO3H2, -SO2OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each of these is independently selected from H and methyl.

[0095] In some embodiments, R x H is R y is selected from H and -CH3. In some embodiments, R x and R y These are H.

[0096] In some embodiments, A is phenyl, monocyclic heteroaryl, and C5-C 6-Selected from cycloalkyl groups, each is either unsubstituted or substituted with one or two substituents independently selected from C1-C4 alkyl, C1-C4 alkoxy, -P(O)(OH)2, -B(OH)2, and -COOH. In some embodiments, A is selected from phenyl and cyclohexyl groups, each is either unsubstituted or substituted with one or two substituents independently selected from methyl, methoxy, -P(O)(OH)2, -B(OH)2, and -COOH. In some embodiments, the compound of formula (II) is [ka] and selected from those salts.

[0097] In some embodiments, A is C1~C 6- Alkyl and -QR 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO2-, and -NR d2 COCONR e2 NR f2 Selected from, R 5 H, C1~C 6- Alkyl, phenyl, or monocyclic heteroaryl, where each alkyl is either unsubstituted or hydroxy, -P(O)(OH) 2、 -COOR g2 , and -NR m2 R n2 It is substituted with one or two substituents independently selected from R a2 , R b2 , R d2 、 R e2 , R f2 、 R g2 、 R m2 , and R n2Each of these is H. In some embodiments, A is selected from -CH2CH2P(O)(OH)2, -CH2CH(OH)CH2OH, -NHCH3, -NHC(O)CH3, -NHC(O)CH2OH, -NHC(O)CH2CH2CH(COOH)NH2, -NC(O)C(O)NHNH2, NHC(O)-pyridyl, and -SO2-phenyl. In some embodiments, the compound of formula (II) is [ka] and selected from those salts.

[0098] In some embodiments, R x and R y Together with the nitrogen atoms to which they are bonded, they form optionally substituted 4- to 8-membered rings. In some embodiments, R x and R y These, together with the nitrogen atoms to which they are bonded, form a six-membered ring selected from morpholine, thiomorpholine, selenomorpholine, and piperazine, each of which is either unsubstituted or substituted with one or two oxo groups, and A is -CH3, -CH2CH3, -CH2COOH, -CH2CH2OH, and -QR 5 Selected from, Q is -NR a2 -or-NR b2 CO-, and in the formula, R a2 and R b2 H and R 5 is H or -CH3. In some embodiments, the compound of formula (II) is [ka] and selected from those salts.

[0099] In some embodiments, the compound of formula (II) is [ka] and selected from those salts.

[0100] In some embodiments, the decrosslinking catalyst is a compound of formula (III). BX(III) or a salt thereof, in the formula, B is aryl, heteroaryl, C1-C6 alkyl, aryl-C1-C4-alkyl-, aryl-C2-C4-alkoxy-, heteroaryl-C1-C4-alkyl-, and heteroarylC2-C4-alkoxy-. X is selected from -COOH, -PO3H2, -B(OH)2, and -SO3H. Each aryl and heteroaryl is independently unsubstituted or halo, C1-C4 alkyl, C1-C4 alkoxy, hydroxy, C1-C4 hydroxyalkyl, -NR a3 R b3 ,-COOR c3 , -SO3R d3 It is substituted with one or more substituents independently selected from -PO3H2 and -B(OH)2, Each alkyl group is either unsubstituted or substituted with one or more substituents independently selected from halo, C1-C4 alkoxy, hydroxy, thiol, C1-C4 alkylthio, optionally substituted C3-C6 cycloalkyl, optionally substituted 3-6 membered heterocyclyl, -COOH, -PO3H2, and -SO3H. In the formula, R a3 , R b3 , R c3 , and R d3 Each of these is independently selected from H, -CH3, and -CH2CH3.

[0101] In some embodiments, B is an aryl or heteroaryl, each of which is unsubstituted or substituted with one or two substituents independently selected from halo, methyl, methoxy, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H. In some embodiments, B is a monocyclic heteroaryl having one heteroatom selected from phenyl or N, O, and S, each of which is unsubstituted or substituted with one substituent selected from methyl, methoxy, fluoro, chloro, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H. In some embodiments, B is selected from phenyl, pyridyl, furanyl, and thiophenyl, each of which is unsubstituted or substituted with one substituent selected from methyl, methoxy, fluoro, chloro, -COOH, and -PO3H2, and X is selected from -COOH, -PO3H2, and -SO3H. In some embodiments, the compound of formula (III) is [ka] and selected from those salts.

[0102] In some embodiments, the decrosslinking catalyst is [ka] and selected from those salts.

[0103] In some embodiments, the decrosslinking catalyst is [ka] and selected from those salts.

[0104] In some embodiments, the decrosslinking catalyst (e.g., a compound of formula (I), (II), or (III)) is in the form of a salt. In some embodiments, the decrosslinking catalyst is in the form of an acid addition salt. The acid addition salt can be formed from an inorganic or organic acid. Inorganic acids that can derive a salt include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. Organic acids that can derive a salt 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, sulfosalicylic acid, etc. In some embodiments, the decrosslinking catalyst is in the form of a hydrochloride salt.

[0105] 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.

[0106] 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 14 Compounds having the disclosed structure, substituted with carbon-rich carbon atoms, are within the scope of this disclosure.

[0107] Method and composition The decrosslinking catalysts disclosed herein (e.g., compounds of formulas (I), (II), and (III)) are useful for decrosslinking formaldehyde-crosslinked biological samples in a one-step process in which the sample is simultaneously contacted with the decrosslinking catalyst and a protease. Accordingly, a method for decrosslinking a formaldehyde-crosslinked biological sample is disclosed herein, comprising the step of simultaneously contacting the sample with a protease and a decrosslinking catalyst, wherein the decrosslinking catalyst is a compound of formula (I), formula (II), or formula (III) (described in detail above).

[0108] 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 includes the step of deparaffinizing the sample before contacting it with a decrosslinking catalyst. Deparaffinization can be carried out according to standard methods. A common deparaffinization protocol involves incubating the sample with mineral oil at approximately 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 for deparaffinizing the sample, such as treatment with xylene, can also be used. In some embodiments, FFPE samples are not deparaffinized before the subsequent processing steps.

[0109] Decrosslinking catalysts help remove formaldehyde crosslinks from a sample and release nucleic acids from the sample. Protease treatment can remove contaminating proteins such as DNase and RNase. In some embodiments, the protease is selected from proteinase K, trypsin, LysC, proalanase, and pepsin. In some embodiments, the protease is proteinase K.

[0110] The step of contacting the sample with the decrosslinking catalyst and protease can be carried out for a sufficient amount of time to decrosslink the sample and cleave the proteins in 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.

[0111] The step of contacting the sample with the decrosslinking catalyst and protease can be performed at a temperature suitable for decrosslinking the sample and cleaving proteins in the sample. In some embodiments, the contact step is performed 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 performed at ambient temperature (i.e., room temperature). In some embodiments, the contact step is performed 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.

[0112] The step of contacting the sample with the decrosslinking catalyst and protease can be carried out by contacting the sample with a single solution containing both the decrosslinking catalyst and protease, for example, an aqueous solution containing the decrosslinking catalyst and protease. In other embodiments, the step of contacting the sample with the decrosslinking catalyst and protease can be carried out by contacting the sample with a first solution containing the protease (e.g., a first aqueous solution containing the protease) and a second solution containing the decrosslinking catalyst (e.g., a second aqueous solution containing the decrosslinking catalyst). Unless a washing step is performed between the continuous contact steps, the sample can be contacted with the first solution and the second solution simultaneously or continuously. In some embodiments, the sample comes into contact with the first solution and the second solution in any order within approximately 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, or 30 minutes, without a washing step in between. In some embodiments, the sample is brought into contact with a first solution containing a protease, followed by contact with a second solution containing a decrosslinking catalyst, with an interval of approximately 30 seconds to 30 minutes between the contact steps, during which no washing step is performed.

[0113] The pH of the solution after contacting the sample with the solution (e.g., an aqueous solution containing a decrosslinking catalyst and protease, the first solution, and / or the second solution) may be approximately 4 to approximately 8.5, or approximately 4.5 to approximately 6.5, or approximately 4.5 to approximately 6.0. In some embodiments, the pH of the solution obtained after the contact step (i.e., the step of contacting the crosslinked biological sample with the solution) may be approximately 4.0, approximately 4.2, approximately 4.3, approximately 4.4, approximately 4.5, approximately 4.6, approximately 4.7, approximately 4.8, approximately 4.9, approximately 5.0, approximately 5.1, approximately 5.2, approximately 5.3, approximately 5.4, approximately 5.5, approximately 5.6, or approximately 5. 7, approximately 5.8, approximately 5.9, approximately 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, or approximately 8.5.

[0114] In some embodiments, the aqueous solution or the first solution contains a decrosslinking catalyst at a concentration of about 1 mM to about 100 mM, or about 5 mM to about 50 mM. In some embodiments, the aqueous solution contains the decrosslinking catalyst 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 M, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37m M, 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 50m M, about 51mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61mM, about 62mM, about 63m M, 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 76m The catalysts are present in concentrations of approximately 77 mM, 78 mM, 79 mM, 80 mM, 81 mM, 82 mM, 83 mM, 84 mM, 85 mM, 86 mM, 87 mM, 88 mM, 89 mM, 90 mM, 91 mM, 92 mM, 93 mM, 94 mM, 95 mM, 96 mM, 97 mM, 98 mM, 99 mM, or 100 mM. In embodiments where multiple decrosslinking catalysts are used, each catalyst may be present individually at any of the indicated concentrations.

[0115] In some embodiments, the method involves contacting a sample with a protease and two or more decrosslinking catalysts described herein, for example, two different decrosslinking catalysts, for example, two different compounds of formula (I), formula (II), or formula (III), or salts thereof. In some embodiments, the method involves contacting a sample with two different decrosslinking catalysts, each being a different compound of formula (I) or a salt thereof. In some embodiments, the method involves contacting a sample with two different decrosslinking catalysts, each being a different compound of formula (II) or a salt thereof. In some embodiments, the method involves contacting a sample with two different decrosslinking catalysts, each being a different compound of formula (III) or a salt thereof. In some embodiments, the method involves contacting a sample with two different decrosslinking catalysts, one being a compound of formula (I) or a salt thereof and the other being a compound of formula (II) or a salt thereof. In some embodiments, the method involves contacting a sample with two different decrosslinking catalysts, one being a compound of formula (I) or a salt thereof and the other being a compound of formula (III) or a salt thereof. In some embodiments, the method involves contacting a sample with two different decrosslinking catalysts, one of which is a compound of formula (II) or a salt thereof, and the other being a compound of formula (III) or a salt thereof.

[0116] In some embodiments, the solution (e.g., an aqueous solution containing a decrosslinking catalyst and a protease, the first solution, and / or the second solution) 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 citrate. While not limited to theory, certain buffers may help to sequester / eliminate formaldehyde from decrosslinked FFPE in the sample.

[0117] This method can efficiently decrosslink crosslinked biological samples and degrade proteins in samples such as FFPE tissue samples without sacrificing the quality of the nucleic acids contained in the sample. 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 nucleic acids. In some embodiments, the nucleic acids are DNA. In some embodiments, the nucleic acids are RNA. In some embodiments, the one or more components are proteins.

[0118] These methods may further include methods for detecting one or more components after extraction from a sample. For example, if 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 time point 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.

[0119] 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.

[0120] Furthermore, compositions comprising a protease and a decrosslinking catalyst (e.g., a decrosslinking catalyst of formula (I), (II), or (III), or a salt thereof) are also disclosed herein. In some embodiments, the protease is selected from proteinase K, trypsin, LysC, proalanase, and pepsin. In some embodiments, the protease is proteinase K.

[0121] In some embodiments, the composition is a solution comprising a protease and a decrosslinking catalyst. In some embodiments, when the solution is added to a crosslinked biological sample, the solution has a pH of about 4 to about 8.5. In some embodiments, when the solution is added to a crosslinked biological sample, the solution has a pH of about 4.5 to about 6.5, or about 4.5 to about 6.0. In some embodiments, when the solution is added to a crosslinked biological sample, the solution has a pH of about 4.0, about 4.1, 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 It has a pH of 6.0, approximately 6.1, approximately 6.2, approximately 6.3, approximately 6.4, approximately 6.5, approximately 6.6, approximately 6.7, approximately 6.8, approximately 6.9, approximately 7.0, approximately 7.1, approximately 7.2, approximately 7.3, approximately 7.4, approximately 7.5, approximately 7.6, approximately 7.7, approximately 7.8, approximately 7.9, approximately 8.0, approximately 8.1, approximately 8.2, approximately 8.3, approximately 8.4, or approximately 8.5.

[0122] In some embodiments, the solution contains a decrosslinking catalyst at concentrations of about 1 mM to about 100 mM, or about 5 mM to about 50 mM. In some embodiments, the solution contains a decrosslinking catalyst 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 M, about 25mM, about 26mM, about 27mM, about 28mM, about 29mM, about 30mM, about 31mM, about 32mM, about 33mM, about 34mM, about 35mM, about 36mM, about 37m M, 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 50m M, about 51mM, about 52mM, about 53mM, about 54mM, about 55mM, about 56mM, about 57mM, about 58mM, about 59mM, about 60mM, about 61mM, about 62mM, about 63m M, 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 76m It contains M, approximately 77mM, approximately 78mM, approximately 79mM, approximately 80mM, approximately 81mM, approximately 82mM, approximately 83mM, approximately 84mM, approximately 85mM, approximately 86mM, approximately 87mM, approximately 88mM, approximately 89mM, approximately 90mM, approximately 91mM, approximately 92mM, approximately 93mM, approximately 94mM, approximately 95mM, approximately 96mM, approximately 97mM, approximately 98mM, approximately 99mM, or approximately 100mM.

[0123] In some embodiments, the composition comprises two different decrosslinking catalysts, for example, two different compounds of formula (I), formula (II), or formula (III), or salts thereof. In some embodiments, the composition comprises two different decrosslinking catalysts, each being a different compound of formula (I) or a salt thereof. In some embodiments, the composition comprises two different decrosslinking catalysts, each being a different compound of formula (II) or a salt thereof. In some embodiments, the composition comprises two different decrosslinking catalysts, each being a different compound of formula (III) or a salt thereof. In some embodiments, the composition comprises two different decrosslinking catalysts, one being a compound of formula (I) or a salt thereof, and the other being a compound of formula (II) or a salt thereof. In some embodiments, the composition comprises two different decrosslinking catalysts, one being a compound of formula (I) or a salt thereof, and the other being a compound of formula (III) or a salt thereof. In some embodiments, the composition comprises two different decrosslinking catalysts, one being a compound of formula (II) or a salt thereof, and the other being a compound of formula (III) or a salt thereof. In embodiments in which the composition comprises two or more decrosslinking catalysts, each can be present individually at any of the concentrations shown above.

[0124] In some embodiments, the composition further comprises a formaldehyde-crosslinked biological sample, such as a formalin-fixed paraffin-embedded tissue sample.

[0125] Systems and kits Furthermore, this specification also discloses systems and kits for decrosslinking formaldehyde-crosslinked biological samples. The systems and kits include a decrosslinking catalyst, such as any of the compounds (or salts thereof) of formula (I), (II), or (III) described herein (specifically including those exemplified above), and instructions for decrosslinking formaldehyde-crosslinked biological samples, instructing the user to contact the sample with the decrosslinking catalyst and protease simultaneously. In some embodiments, the system or kit includes one or more decrosslinking catalysts disclosed herein (e.g., two different compounds of formula (I), two different compounds of formula (II), two different compounds of formula (III), a compound of formula (I) and a compound of formula (II), a compound of formula (I) and a compound of formula (III), or a compound of formula (II) and a compound of formula (III), or a salt thereof).

[0126] 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 the detection method described herein.

[0127] 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 protease 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]

[0128] Example 1 Screening of decrosslinked compounds [ka] Candidate decrosslinking catalyst compounds, compounds 1-5, were screened according to the general workflow shown in Figure 3 (compound 6 was tested individually). The compounds were added to FFPE tissue lysates after proteinase K (ProK) digestion (56°C for 30 minutes) and subsequently decrosslinked at 80°C. 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 according to the manufacturer's protocol using the Maxwell® RSC DNA FFPE kit (Promega catalog number AS1450) and the Maxwell RSC 48 instrument (Promega catalog number AS8500). After nucleic acid extraction, DNA recovery was determined 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]

[0129] The quality of DNA decrosslinked by these leading candidates was further evaluated by testing FFPE tissue collected from human donors using next-generation sequencing (NGS). Healthy colon FFPE donor tissue underwent rapid pretreatment with compounds 1, 3, and 5 (compound 1, compound 3, and compound 5, respectively), and decrosslinked for 30 minutes at 80°C, along with a positive control (4 hours at 80°C) and a negative control (30 minutes at 80°C). The DNA was then purified using a Maxwell® RSC 48 instrument (Promega Corp). The amount of DNA and fragment size were evaluated using Promega's ProNex® DNA QC assay to amplify 75 bp, 150 bp, and 300 bp qPCR amplicons. The amount of amplified DNA and fragment size from samples rapidly decrosslinked in reactions Cpd 1, Cpd 5, and Cpd 3 were comparable to those from the positive control samples (Figure 4). The mean of three purification replicates is plotted along with error bars representing the standard deviation.

[0130] The NGS library was constructed using Illumina's AmpliSeq for Cancer HotSpot Panel v2. Figure 5A shows the molecular weight of the library determined by electrophoresis using Agilent TapeStation. Figure 5B shows the quantification of the electrophoresed library. Figure 5C shows the volume of the library 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 (Horizon, HD803) pooled from multiple immortalized cell lines for use as a multiplex reference standard. The NGS library from the rapid decrosslinking reaction was of expected size and sufficient volume for sequencing.

[0131] 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 6A). The VAFs of mutations identified in the positive control were found to be in high agreement with those of catalytic purification (Figure 6B). This indicates that rapidly decrosslinked DNA maintains high sequence quality and allows for VAF calling near the detection limit using NGS.

[0132] 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 7). Cell pellets of artificial FFPE (Amsbio LLC) were decrosslinked at 80°C for 10, 20, and 30 minutes at Cpd 1–5. 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 (Cpd 4), 20 minutes (Cpd 1, Cpd 2, Cpd 3), and 30 minutes (Cpd 5). DNA yield was compared to a positive control (Pos) decrosslinked at 80°C for 4 hours without the catalyst.

[0133] 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 Cpd 1–5 HCl salts in various pH lysis buffers (Figure 8A). 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 (Pos) decrosslinked at 80°C for 4 hours in pH 8 lysis buffer without catalyst, and a negative control (mock) decrosslinked at 80°C for 30 minutes in various pH lysis buffers without catalyst. To determine the working pH of the lysate during rapid decrosslinking, 20 mM Cpd HCl salt was added to lysis buffer pH 7.75 (optimal values ​​are shown in Figure 8A). The optimal pH ranged from 4.80 to 5.90 (Figure 8B). Experiments were performed using catalytic free bases to confirm that the pH requirements could be achieved by the buffers (Figure 8C). After proteinase K digestion, the pH of the lysate was lowered with a predetermined amount of HCl to create various pH lysis buffers. 20 mM free bases Cpd 1 and Cpd 3 were added to the lysate, and decrosslinking was performed at 80°C for 30 minutes. Amplifiable DNA yield 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.

[0134] 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 9). Various pH lysis buffers were used to reduce the amount of HCl added (derived from Cpd HCl salts). Amplified DNA yield 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 (Pos) decrosslinked with pH 8 lysis buffer at 80°C for 4 hours without the catalyst. Amplified DNA yield was found to be concentration-dependent.

[0135] Example 5 Protease compatibility screening After candidate decrosslinking compounds were identified, secondary screening was performed to confirm the compatibility of the compounds with ProK. A general screening protocol, including positive and negative controls, is shown in Figure 10. The decrosslinking catalyst was included in the lysis buffer master mix and added before the ProK digestion incubation. The effect on DNA yield was compared to samples in which the decrosslinking compound was added after ProK digestion, a positive control (decrosslinking for 4 hours without the decrosslinking catalyst), and a negative control (decrosslinking for 30 minutes without the decrosslinking catalyst). The data are shown in Figure 11. The results regarding termination differed depending on the nucleophile. Compound 3, added before ProK digestion, and to a lesser extent, compound 1, showed an increase in DNA yield compared to the negative control. This indicates that the ProK digestion and nucleophilic activity of compounds 1 and 3 did not adversely affect each other.

[0136] Example 6 Evaluation of the incubation period ProK is a potent and stable enzyme, but it rapidly loses its activity at temperatures above 60°C. To evaluate the effect of incubation time on ProK digestion, samples were digested for 0 minutes, 5 minutes, 15 minutes, and 30 minutes, then decrosslinked with compound 1 at 80°C for 30 minutes (see Figure 12A). The total yield (ng) of amplifiable DNA was then evaluated by duplex qPCR assay and ProNEX® QC qPCR assay (75 bp, 150 bp, and 300 bp amplicons).

[0137] The results are shown in Figure 12B. Incubation at 56°C for 5 minutes yielded high levels of amplifiable DNA. Interestingly, incubation for 0 minutes (i.e., directly at 80°C) also produced high levels of DNA compared to the control without ProK. This suggests that ProK digestion may have occurred while the lysis buffer was being mixed with the tissue (approximately 3 minutes at room temperature) and while the lysate was being heated to 80°C for decrosslinking (approximately 5 minutes at 21°C–80°C).

[0138] Example 7 Single-step decrosslinking After identifying decrosslinking compounds compatible with ProK (Figure 11) and enabling rapid ProK digestion (Figure 12B), a single-step assay was used to determine if it was sufficient for high FFPE DNA yield (see Figure 13A). Deparaffinized FFPE cells were given MC118 lysis buffer (Promega Corp.), ProK solution, and a master mix of 20 mM compound 1 or compound 3. Lysates were incubated at 80°C for 30 minutes, and DNA was purified using a Maxwell® RSC instrument. Amplified DNA was evaluated using qPCR (Figure 13B). Both compounds 1 and 3 yielded DNA levels similar to a standard two-step protocol with a positive control. Therefore, these compounds enable a one-step incubation for FFPE tissue pretreatment and subsequent nucleic acid purification.

[0139] Example 8 Catalytically decrosslinked FFPE DNA was purified from various human tissues and evaluated by amplification, dye binding, and absorption. The catalyst was tested in 13 different human FFPE tissues (Figures 14A-14C: 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 simulated compound treatment (positive) decrosslinked at 80°C for 4 hours. One to three donors were used per tissue, and two to four purification replicates were performed per donor. DNA yield was evaluated by RT-qPCR using Promega ProNex® DNA QC Assay (catalog no. NG1002) (Figure 14A), fluorescent dsDNA dye using Promega QuantiFluor® dsDNA System (catalog no. E2671) (Figure 14B), and absorption using ThermoFisher NanoDrop (Figure 14C).

Claims

1. A method for decrosslinking a formaldehyde-crosslinked biological sample, comprising the step of simultaneously contacting the sample with a protease and a decrosslinking catalyst, wherein the decrosslinking catalyst is a compound of formula (I), formula (II), or formula (III), The compound of formula (I) is 【Chemistry 1】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 is selected from H, C 1 ~C 6 alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -alkyl, and heteroaryl-C 1 ~C 4 -alkyl, and is selected from R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 selected from R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 、R b2 、R c2 、R d2 、R e2 、and R f2 are each independently selected from H and C 1 to C 6 alkyl, R x and R y H and C are independent of each other. 1 ~C 6 Selected from alkyl groups, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is either unsubstituted or halo, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, hydroxy, C 1 ~C 4 Hydroxyalkyl, -NR a3 R b3 , -COOR c3 , -SO 3 R d3 , -PO 3 H 2 , and -B(OH) 2 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The method selected from the above.

2. The method according to claim 1, wherein the decrosslinking catalyst is a compound of formula (I) or a salt thereof.

3. R 1 H is, R 2 H, C 1 ~C 6 Alkyl, -CH 2 -aryl and -CH 2 - Selected from heteroaryls, R 3 H, C 1 ~C 6 Alkyl, -X-R 4 Selected from, where 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 unsubstituted or halo, hydroxy, and C. 1 ~C 4 It is substituted with one or two substituents independently selected from the alkoxy, Each alkyl group is independently either unsubstituted or hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -COOR e1 , -ONR h1 R i1 , and -NR j1 OR k1 It is substituted with one substituent selected from the following: In the formula, R a1 , R b1 , R e1 , R h1 , R i1 , R j1 , and R k1 These are H and -CH, respectively, independently. 3 The method according to claim 2, selected from the following.

4. 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 method according to claim 2, wherein H is

5. 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 method according to claim 2, wherein a saturated 4- to 7-membered ring is formed by substituting one or two substituents independently selected from alkyl and oxo groups.

6. The compound of formula (I) is 【Chemistry 2-1】 【Chemistry 2-2】 The method according to claim 2, selected from and salts thereof.

7. The method according to claim 1, wherein the decrosslinking catalyst is a compound of formula (II) or a salt thereof.

8. R x H is R y H and -CH 3 The method according to claim 7, selected from the following.

9. A is phenyl, monocyclic heteroaryl, and C 5 ~C 6- Selected from cycloalkyl groups, each of which is either unsubstituted or C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, -P(O)(OH) 2 , -B(OH) 2 The method according to claim 8, wherein the molecule is substituted with one or two substituents independently selected from -COOH.

10. A is C 1 -C 6- Alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- They are alkyl, phenyl, or monocyclic heteroaryl compounds. Each alkyl group is either unsubstituted or hydroxy, -P(O)(OH) 2、 - COOR g2 , and -NR m2 R n2 It is substituted with one or two substituents independently selected from the original molecule. In the formula, R a2 , R b2 , R d2 、 R e2 , R f2 、 R g2 、 R m2 , and R n2 The method according to claim 8, wherein each of them is H.

11. R x and R y These, together with the nitrogen atoms to which they are bonded, form a six-membered ring selected from morpholine, thiomorpholine, selenomorpholine, and piperazine, each of which is either unsubstituted or substituted with one or two oxo groups. A is H, -CH 3 ien-CH 2 CH 3 ien-CH 2 COOH, -CH 2 CH 2 OH, and -Q-R 5 Selected from, Q is -NR a2 - or - NR b2 CO-, and in the formula, R a2 and R b2 These are H, R 5 is H or -CH 3 The method according to claim 7.

12. The compound of formula (II) is 【Transformation 3】 The method according to claim 7, wherein a selection is made from salts thereof.

13. The method according to claim 1, wherein the decrosslinking catalyst is a compound of formula (III) or a salt thereof.

14. B is an aryl or heteroaryl compound, which is either unsubstituted or contains halo, methyl, methoxy, -COOH, and -PO 3 H 2 It is substituted with one or two substituents independently selected from the original molecule. X is -COOH, -PO 3 H 2 , and -SO 3 The method according to claim 13, selected from H.

15. B is a monocyclic heteroaryl having one heteroatom selected from phenyl or N, O, and S, each of which is either unsubstituted or methyl, methoxy, fluoro, chloro, -COOH, and -PO 3 H 2 It is substituted with one substituent selected from the following: X is -COOH, -PO 3 H 2 , and -SO 3 The method according to claim 13, selected from H.

16. The compound of formula (III) is 【Chemistry 4】 The method according to claim 13, selected from and salts thereof.

17. The aforementioned decrosslinking catalyst is 【Transformation 5】 The method according to claim 1, wherein a selection is made from salts thereof.

18. The method according to any one of claims 1 to 17, wherein the decrosslinking catalyst is in the form of a salt.

19. The method according to any one of claims 1 to 18, wherein the decrosslinking catalyst is in the form of a hydrochloride salt.

20. The method according to any one of claims 1 to 19, wherein the method comprises contacting the sample with an effective amount of at least two different decrosslinking catalysts or salts thereof.

21. The method according to claim 20, wherein at least two different decrosslinking catalysts or salts thereof are added simultaneously to the sample.

22. The method according to claim 20, wherein the at least two different decrosslinking catalysts or salts thereof are added sequentially to the sample.

23. The method according to any one of claims 1 to 22, wherein the sample is a formalin-fixed paraffin-embedded tissue sample.

24. The method according to claim 23, further comprising the step of deparaffinizing the sample before contacting the sample with a protease and a decrosslinking catalyst.

25. The method according to any one of claims 1 to 24, wherein the protease is proteinase K.

26. The method according to any one of claims 1 to 25, wherein the contact step is performed for about 5 minutes to about 120 minutes.

27. The method according to claim 26, wherein the contact step is performed for about 20 minutes to about 40 minutes.

28. The method according to any one of claims 1 to 27, wherein the contact step is performed at a temperature of about 20°C to about 100°C.

29. The method according to any one of claims 1 to 28, wherein the contact step is performed at a temperature of about 50°C to about 85°C.

30. The method according to any one of claims 1 to 29, comprising contacting the sample with a solution containing the decrosslinking catalyst and the protease.

31. The method according to claim 30, wherein after contacting the sample with the solution, the resulting solution has a pH of about 4.0 to about 8.

5.

32. The method according to claim 31, wherein after contacting the sample with the solution, the resulting solution has a pH of about 4.5 to about 6.

5.

33. The method according to any one of claims 30 to 32, wherein the solution contains the decrosslinking catalyst at a concentration of about 1 mM to about 100 mM.

34. The method according to claim 33, wherein the solution contains the decrosslinking catalyst at a concentration of about 10 mM to about 50 mM.

35. The method according to any one of claims 1 to 34, wherein the protease and / or decrosslinking catalyst is added to the buffer solution.

36. The method according to claim 35, wherein the buffer solution comprises tris(hydroxymethyl)aminomethane (Tris), 2-(N-morpholino)ethanesulfonic acid (MES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), phosphate-buffered saline, glycine, or citrate.

37. The method according to any one of claims 1 to 29, comprising contacting the sample with a first solution containing the protease and a second solution containing the decrosslinking catalyst.

38. The method according to claim 37, wherein the pH of the solution obtained after the contact step is about 4.0 to about 8.

5.

39. The method according to claim 38, wherein the pH of the solution obtained after the contact step is about 4.5 to about 6.

5.

40. The method according to any one of claims 37 to 39, wherein the second solution contains the decrosslinking catalyst at a concentration of about 1 mM to about 100 mM.

41. The method according to claim 40, wherein the second solution contains the decrosslinking catalyst at a concentration of about 5 mM to about 50 mM.

42. The method according to any one of claims 1 to 41, further comprising extracting one or more components from the sample after the contact step.

43. The method according to claim 42, wherein the one or more components are selected from nucleic acids and proteins.

44. The method according to claim 43, 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.

45. The method according to claim 44, further comprising a step selected from pigment binding, absorption, and enzymatic digestion.

46. A composition comprising a protease and a decrosslinking catalyst, wherein the decrosslinking catalyst is a compound of formula (I), formula (II), or formula (III), The compound of formula (I) is 【Transformation 6】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 These are H and C, respectively, independently. 1 ~C 6 Selected from alkyl groups, R x and R y H and C are independent of each other. 1 ~C 6 Selected from alkyl groups, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is either unsubstituted or halo, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, hydroxy, C 1 ~C 4 Hydroxyalkyl, -NR a3 R b3 , -COOR c3 , -SO 3 R d3 , -PO 3 H 2 , and -B(OH) 2 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The composition selected from the above.

47. The composition according to claim 46, wherein the decrosslinking catalyst is a compound of formula (I) or a salt thereof.

48. R 1 H is, R 2 H, C 1 ~C 6 Alkyl, -CH 2 -aryl and -CH 2 - Selected from heteroaryls, R 3 H, C 1 ~C 6 Alkyl, -X-R 4 Selected from, where 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 unsubstituted or halo, hydroxy, and C. 1 ~C 4 It is substituted with one or two substituents independently selected from the alkoxy, Each alkyl group is independently either unsubstituted or hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -COOR e1 , -ONR h1 R i1 , and -NR j1 OR k1 It is substituted with one substituent selected from the following: In the formula, R a1 , R b1 , R e1 , R h1 , R i1 , R j1 , and R k1 These are H and -CH, respectively, independently. 3 A composition according to claim 47, selected from the following.

49. 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 47, wherein is H.

50. 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 47, which forms a saturated 4- to 7-membered ring substituted with one or two substituents independently selected from alkyl and oxo groups.

51. The compound of formula (I) is 【Chemistry 7-1】 【Chemistry 7-2】 The composition according to claim 47, selected from and salts thereof.

52. The composition according to claim 46, wherein the decrosslinking catalyst is a compound of formula (II) or a salt thereof.

53. R x H is R y H and -CH 3 A composition according to claim 52, selected from the following.

54. A is phenyl, monocyclic heteroaryl, and C 5 ~C 6- Selected from cycloalkyl groups, each of which is either unsubstituted or C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, -P(O)(OH) 2 , -B(OH) 2 The composition according to claim 53, wherein the substituents are substituted with one or two substituents independently selected from -COOH.

55. A is C 1 -C 6- Alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- They are alkyl, phenyl, or monocyclic heteroaryl compounds. Each alkyl group is either unsubstituted or hydroxy, -P(O)(OH) 2、 - COOR g2 , and -NR m2 R n2 It is substituted with one or two substituents independently selected from the original molecule. In the formula, R a2 , R b2 , R d2 、 R e2 , R f2 、 R g2 、 R m2 , and R n2 The composition according to claim 53, wherein each of the elements is H.

56. R x and R y These, together with the nitrogen atoms to which they are bonded, form a six-membered ring selected from morpholine, thiomorpholine, selenomorpholine, and piperazine, each of which is either unsubstituted or substituted with one or two oxo groups. A is H, -CH 3 ien-CH 2 CH 3 ien-CH 2 COOH, -CH 2 CH 2 OH, and -Q-R 5 Selected from, Q is -NR a2 - or - NR b2 CO-, and in the formula, R a2 and R b2 These are H, R 5 is H or -CH 3 The composition according to claim 52.

57. The compound of formula (II) is 【Transformation 8】 The composition according to claim 52, selected from and salts thereof.

58. The composition according to claim 46, wherein the decrosslinking catalyst is a compound of formula (III) or a salt thereof.

59. B is an aryl or heteroaryl compound, each of which is either unsubstituted or contains halo, methyl, methoxy, -COOH, and -PO 3 H 2 It is substituted with one or two substituents independently selected from the original molecule. X is -COOH, -PO 3 H 2 , and -SO 3 A composition according to claim 58, selected from H.

60. B is a monocyclic heteroaryl having one heteroatom selected from phenyl or N, O, and S, each of which is either unsubstituted or methyl, methoxy, fluoro, chloro, -COOH, and -PO 3 H 2 It is substituted with one substituent selected from the following: X is -COOH, -PO 3 H 2 , and -SO 3 A composition according to claim 58, selected from H.

61. The compound of formula (III) is 【Chemistry 9】 The composition according to claim 58, selected from and salts thereof.

62. The aforementioned decrosslinking catalyst is 【Chemistry 10】 The composition according to claim 46, selected from and salts thereof.

63. The composition according to any one of claims 46 to 62, wherein the decrosslinking catalyst is in the form of a salt.

64. The composition according to any one of claims 46 to 63, wherein the decrosslinking catalyst is in the form of a hydrochloride salt.

65. The composition according to any one of claims 46 to 64, wherein the protease is proteinase K.

66. The composition according to any one of claims 46 to 65, comprising a solution of the decrosslinking catalyst and the protease.

67. The composition according to claim 66, wherein when the solution of the decrosslinking catalyst and the protease is added to a crosslinked biological sample, the solution has a pH of about 4.0 to about 8.

5.

68. The composition according to claim 67, wherein when the solution of the decrosslinking catalyst and the protease is added to a crosslinked biological sample, the solution has a pH of about 4.5 to about 6.

5.

69. The composition according to any one of claims 66 to 68, wherein the solution contains the decrosslinking catalyst at a concentration of about 1 mM to about 100 mM.

70. The composition according to claim 69, wherein the solution contains the decrosslinking catalyst at a concentration of about 10 mM to about 50 mM.

71. The composition according to any one of claims 46 to 70, comprising at least two different decrosslinking catalysts or salts thereof.

72. The composition according to any one of claims 46 to 71, further comprising a buffer solution.

73. The composition according to claim 72, wherein the buffer solution is tris(hydroxymethyl)aminomethane.

74. The composition according to any one of claims 46 to 73, further comprising a formaldehyde-crosslinked biological sample.

75. The composition according to claim 74, wherein the sample is a formalin-fixed paraffin-embedded tissue sample.

76. A kit for decrosslinking a formaldehyde-crosslinked biological sample, the kit comprising (i) a decrosslinking catalyst which is a compound of formula (I), formula (II), or formula (III), and (ii) instructions for decrosslinking a formaldehyde-crosslinked biological sample which instruct the user to contact the sample with the decrosslinking catalyst and the protease simultaneously. The compound of formula (I) is 【Chemistry 11】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 These are H and C, respectively, independently. 1 ~C 6 Selected from alkyl groups, R x and R y H and C are independent of each other. 1 ~C 6 Selected from alkyl groups, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is either unsubstituted or halo, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, hydroxy, C 1 ~C 4 Hydroxyalkyl, -NR a3 R b3 , -COOR c3 , -SO 3 R d3 , -PO 3 H 2 , and -B(OH) 2 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The kit selected from the above.

77. The kit according to claim 76, further comprising a protease.

78. The kit according to claim 77, wherein the protease is proteinase K.

79. The kit according to any one of claims 76 to 78, wherein the kit comprises at least two different decrosslinking catalysts or salts thereof.

80. The kit according to any one of claims 76 to 79, further comprising at least one component selected from a buffer, a salt, and packaging material.

81. A method for decrosslinking a formaldehyde-crosslinked biological sample, comprising the step of simultaneously contacting the sample with a protease and at least two different decrosslinking catalysts, wherein each decrosslinking catalyst is a compound of formula (I), formula (II), or formula (III), The compound of formula (I) is 【Chemistry 12】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 、R b2 、R c2 、R d2 、R e2 、and R f2 are each independently selected from H and C 1 to C 6 alkyl, R x and R y are each independently selected from H and C 1 ~C 6 alkyl, or R x and R y together with the nitrogen atom to which they are attached form an optionally substituted 4- to 8-membered ring, Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is independently unsubstituted or substituted with one or more substituents independently selected from halo, C 1 ~C 4 alkyl, C 1 ~C 4 alkoxy, hydroxy, C 1 ~C 4 hydroxyalkyl, -NR a3 R b3 -, -COOR c3 -, -SO 3 R d3 -, -PO 3 H 2 -, and -B(OH) 2 and is substituted with one or more substituents independently selected from Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The method selected from the above.

82. A composition comprising a protease and at least two different decrosslinking catalysts, each decrosslinking catalyst being a compound of formula (I), formula (II), or formula (III), The compound of formula (I) is 【Chemistry 13】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 These are H and C, respectively, independently. 1 ~C 6 Selected from alkyl groups, R x and R y H and C are independent of each other. 1 ~C 6 Selected from alkyl groups, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is either unsubstituted or halo, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, hydroxy, C 1 ~C 4 Hydroxyalkyl, -NR a3 R b3 , -COOR c3 , -SO 3 R d3 , -PO 3 H 2 , and -B(OH) 2 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The composition selected from the above.

83. A kit for decrosslinking a formaldehyde-crosslinked biological sample, the kit comprising: (i) at least two different decrosslinking catalysts, each of which is a compound of formula (I), formula (II), or formula (III); and (ii) instructions for decrosslinking a formaldehyde-crosslinked biological sample, which instruct the user to contact the sample with the decrosslinking catalyst and the protease simultaneously. The compound of formula (I) is 【Chemistry 14】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 These are H and C, respectively, independently. 1 ~C 6 Selected from alkyl groups, R x and R y H and C are independent of each other. 1 ~C 6 Selected from alkyl groups, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is either unsubstituted or halo, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, hydroxy, C 1 ~C 4 Hydroxyalkyl, -NR a3 R b3 , -COOR c3 , -SO 3 R d3 , -PO 3 H 2 , and -B(OH) 2 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The kit selected from the above.

84. A kit for decrosslinking a formaldehyde-crosslinked biological sample, the kit comprising: (i) at least two different decrosslinking catalysts, each being a compound of formula (I), formula (II), or formula (III); and (ii) instructions for decrosslinking a formaldehyde-crosslinked biological sample, instructing the user to contact the sample with a first solution containing the protease and a second solution containing the decrosslinking catalyst. The compound of formula (I) is 【Chemistry 15】 or a salt thereof, During the ceremony, R 1 H and C 1 ~C 6 Selected from alkyl groups, R 2 H, C 1 ~C 6 Alkyl, aryl, heteroaryl, aryl-C 1 ~C 4 -Alkyl and heteroaryl-C 1 ~C 4 - Selected from alkyl groups, R 3 H, C 1 ~C 6 Alkyl and -X-R 4 Selected from, where 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 either unsubstituted or halo, C 1 ~C 4 Alkyl, hydroxy, C 1 ~C 4 Alkoxy, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , and -SO 2 NR f1 R g1 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, hydroxy, or C. 1 ~C 4 Alkoxy, thiol, C 1 ~C 4 Alkylthio, -NR a1 R b1 , -C(O)NR c1 R d1 , -COOR e1 , -SO 2 NR f1 R g1 , -ONR h1 R i1 , -NR j1 OR k1 , -PO 3 H 2 , -SO 3 H, replaced by C of any choice 3 ~C 6 It is substituted with one or more substituents independently selected from cycloalkyl groups and optionally substituted 3- to 6-membered heterocyclines. In the formula, R a1 , R b1 , R c1 , R d1 , R e1 , R f1 , R g1 , R h1 , R i1 , R j1 , and R k1 These are H and C, which are independent of each other. 1 ~C 4 Alkyl, C 1 ~C 4 Hydroxyalkyl, and C 1 ~C 4 Selected from carboxyalkyl groups, In the formula, R 1 and R 2 These, together with the nitrogen atoms to which they are bonded, optionally combine together, optionally substitute to form a 4- to 8-membered ring. In the formula, R 2 and R 3 These, together with the atoms to which they are bonded, form a 4- to 8-membered ring with optional substitutions. The compound of formula (II) is _?|| x ( y (=) or a salt thereof, in the formula, A is H, C 1 ~C 6- Alkyl, aryl, heteroaryl, C 3 ~C 6- Cycloalkyl, aryl-C 1 ~C 4 -Alkyl, heteroaryl-C 1 ~C 4 -alkyl and -Q-R 5 Selected from, Q is -NR a2 -, -NR b2 CO-, -SO 2 -, -SO 2 NR c2 -, and -NR d2 COCONR e2 NR f2 - Selected from, R 5 H, C 1 ~C 6- Alkyl, aryl, heteroaryl, and C 3 -C 6- Selected from cycloalkyl groups, R a2 , R b2 , R c2 , R d2 , R e2 , and R f2 These are H and C, respectively, independently. 1 ~C 6 Selected from alkyl groups, R x and R y H and C are independent of each other. 1 ~C 6 Selected from alkyl groups, or R x and R y These, together with the nitrogen atoms to which they are bonded, form optionally substituted 4- to 8-membered rings. Each aryl or heteroaryl is either unsubstituted or halo, hydroxyl, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, C 1 ~C 4 Hydroxyalkyl, -P(O)(OH) 2 , -B(OH) 2 , --COOR g2 , -CONR h2 R i2 , -SO 2 NR j2 R k2 , and -SO 2 OR l2 It is independently selected and replaced, Each alkyl or cycloalkyl group is either unsubstituted or halo-C. 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOR g2 , -PO 3 H 2 , -SO 2 OR l2 , and -NR m2 R n2 It is substituted with one or more substituents independently selected from, R g2 , R h2 , R i2 , R j2 , R k2 , R l2 , R m2 , and R n2 Each is independently selected from H and methyl, The compound of formula (III) is B-X (III) or a salt thereof, in the formula, B is aryl, heteroaryl, C 1 ~C 6 Alkyl, aryl-C 1 ~C 4 -Alkyl-, Aryl-C 2 ~C 4 -alkoxy-, heteroaryl-C 1 ~C 4 -Alkyl- and heteroaryl C 2 ~C 4 -alkoxy-, X is -COOH, -PO 3 H 2 , -B(OH) 2 , and -SO 3 Selected from H, Each aryl and heteroaryl is either unsubstituted or halo, C 1 ~C 4 Alkyl, C 1 ~C 4 Alkoxy, hydroxy, C 1 ~C 4 Hydroxyalkyl, -NR a3 R b3 , -COOR c3 , -SO 3 R d3 , -PO 3 H 2 , and -B(OH) 2 It is substituted with one or more substituents independently selected from, Each alkyl group is either unsubstituted or halo, C 1 ~C 4 Alkoxy, hydroxy, thiol, C 1 ~C 4 Alkylthio, optionally substituted C 3 ~C 6 Cycloalkyl, optionally substituted 3- to 6-membered heterocyclyl, -COOH, -PO 3 H 2 and -SO 3 It is substituted with one or more substituents independently selected from H, In the formula, R a3 , R b3 , R c3 , and R d3 These are H and -CH, respectively, independently. 3 , and -CH 2 CH 3 The kit selected from the above.

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