WO2023192629A1

WO2023192629A1 - Induction of mecp2 expression by dna methyl transferase inhibitors

Info

Publication number: WO2023192629A1
Application number: PCT/US2023/017183
Authority: WO
Inventors: Brian RASH; Pavan Ramkumar; Ryan Thomas JONES; Gaia SKIBINSKI; Sharath Hegde; Saul Kato; Brad Savall
Original assignee: Herophilus, Inc.
Priority date: 2022-04-01
Filing date: 2023-03-31
Publication date: 2023-10-05

Abstract

Provided herein are methods for the induction of MECP2 expression and/or reactivation via (S)-2-((3,5-dicyano-6-(dimethylamino)-4-ethylpyridin-2-yl)thio)-2-phenylacetamide or salts thereof, and compounds of Formula (I) or (I-A) or salts or prodrugs thereof.

Description

INDUCTION OF MECP2 EXPRESSION BY DNA METHYL TRANSFERASE INHIBITORS

CROSS-REFERENCE

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/326,562 filed on April 1, 2022, and U.S. Provisional Patent Application No. 63/348,866 filed on June 3, 2022, the entire contents of each of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Rett syndrome (RTT) is a progressive neurological disorder that affects 1 in 10,000-15,000 female births. Loss of function mutations in methyl-CpG-binding protein 2 (MeCP2, locus located on the X-chromosome) is the major cause of RTT. Random X chromosome inactivation causes cells to express either mutant or wild-type MeCP2, leading to a mosaic pattern of mutant MeCP2 protein expression throughout the body. Mouse models of RTT have shown that an increase of 5 to 10% in wild-type MeCP2 is sufficient to improve RTT phenotypes and delay premature death. Thus, new methods that increase MeCP2 protein levels are needed to treat RTT.

SUMMARY OF THE INVENTION

[0003] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease which comprise: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X- chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome. In some embodiments, the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation. In some embodiments, the MeCP2 Exon 2 region contains no genetic mutations. In some embodiments, the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409. In some embodiments, the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Alport syndrome, X-linked Charcot-Mari e- tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy. In some embodiments, the X-chromosome-linked disease is Rett Syndrome. In some embodiments, administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces wild-type MeCP2 in human neural tissue.

[0004] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease which further comprises the DNA methyltransferase inhibitor activating an inactive X-chromosome in the following in vitro assay: (a) reprogramming X-chromosome linked disease fibroblasts into induced pluripotent stem cells; (b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyltransferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and (d) detecting MeCP2 level in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyltransferase inhibitor. In some embodiments, the X-chromosome linked disease fibroblasts are Rett Syndrome fibroblasts.

[0005] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease in a subject in need thereof, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor is a compound of Formula (I):

wherein:

X¹ and X² are independently selected from: hydrogen, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, O C_1-6 alkyl, OR^e, SH, and SR^a; and cycloalkyl and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

Y is selected from -S-, -NH-, -NR^Z-, -O-, -S(O)-, and -S(O)2-;

R¹ is selected from:

NH2, -NHR^a, -NR^bR^c, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, O C_1-6 alkyl, OR^e, SH, and SR^a; and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

R² is selected from: hydrogen; and aryl and heteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

R³ is selected from: hydrogen, C_1-6 alkyl, R^e, -COOR^a, -CONHR^a, and -CONR^bR^c; and cycloalkyl and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

R⁴ is selected from: hydrogen, C_1-6 alkyl, R^e, -COOR^a, -CONHR^a, and -CONR^bR^c; and cycloalkyl and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

R⁵ is selected from:

-NH₂, -NHR^a, -NR^bR^c, -OC i-6 alkyl, -OR^e, -SH, and -SR^a; and aryl, -Oaryl, and -Oheteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d; wherein: each R^a is independently selected from C_1-6 alkyl and R^e; and aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d; each R^b and R^c are independently selected from hydrogen, C_1-6 alkyl, and R^e; and aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d, or

R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, -CN, oxo, -OH, -0P(0)(0H)2, -COOH, -CONH₂, -NO₂, -NH₂, -NH(Ci-₄alkyl), -N(H)R^e, -N(Ci-4alkyl)₂, -NR^eR^e, - SO2NH2, -S(O)₂CH₂CH₃, -S(O)₂CH₂CH₂CH₃, -S(O)₂CH₃, -S(O)₂phenyl, and C1.4 alkoxy optionally substituted with 1 to 6 substituents independently selected from fluoro, oxo, -OH, -COOH, -NH₂, and -CN; and aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d; each R^d is independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, C(O)H, -C(O)R^ZZ, -OC(O)H, - CO(O)R^ZZ, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO₂NH₂, - S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, -N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, - C(O)NR^XR^X, -COOH, -COOR^X, oxo, -OH, -NH₂, -NHR^X, -NR^XR^X, -NO₂, - CN, -NHC(O)NH₂, -NHC(O)NHR^X, -NHC(O)NR^XR^X, and Ci-₄ alkoxy optionally substituted with 1 to 4 times by fluoro; and aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and -Oaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^x; and

-C(O)aryl, C(O)heteroaryl, -OC(O)aryl, and -OC(O)heteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^zz; each R^eis independently selected from: C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-₆ alkyl, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, - S(O)₂R^X, OXO, -OH, -NH₂, -NHR^XX, -NR^XR^X, -NHC(=N)NH₂, -COOH, - C00R^x, -C(0)NH₂, -C(0)NHR^X, -C(0)NR^XR^X, SO₂NH₂, -S(0)₂NHR^X, - S(0)₂NR^XR^X, -NHS(0)₂H, -NHS(0)₂R^X, -NHC(0)NHR^XP, - NHC(0)NR^xpR^xp, -N0₂, and -CN; and

Ci -6 alkoxy optionally substituted with 1 to 6 substituents independently selected from fluoro, oxo, -OH, -COOH, -NH₂, and -CN; and aryl, -Oaryl, heteroaryl, -Oheteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^x; each R^x is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OH, -COOH, -NH₂ and -CN; each RN is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OR", NR"R^XV -COOH, and -CN; each R^xy is independently selected from: hydrogen, aryl, Ci-s alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OH, C1.5 alkoxy optionally substituted with 1 to 6 substituents independently selected from: fluoro and -COOH; each R^xp is independently selected from: heteroaryl, cyclcoalkyl, heterocycloalkyl, C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: -COOH, -NH2 and -CN; each R^z is independently selected from: C_1-6 alkyl, R^e, cyclcoalkyl optionally substituted with 1 to 4 substituents independently selected from R^d, and heterocyclcoalkyl optionally substituted with 1 to 4 substituents independently selected from R^d. each R^zz is independently selected from C_1-6 alkyl, and R^e. provided that: at least one of R², R³ and R⁴ is hydrogen, R², R³ and R⁴ are not all hydrogen, X¹ and X² are not both hydrogen; or a pharmaceutically acceptable salt or prodrug thereof.

[0006] In some embodiments, X¹ and X² are each independently selected from: H, -CN, fluoro, chloro, bromo, iodo, and methyl. In some embodiments, X¹ and X² are each -CN.

[0007] In some embodiments, Y is -S-.

[0008] In some embodiments, R¹ is C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, CM alkyloxy, -OH, -COOH, -NH2, - NH(CI-4 alkyl), -N(CI-4 alkyl)2 and -CN. In some embodiments, R¹ is ethyl.

[0009] In some embodiments, R² is phenyl optionally substituted with one or more substituents independently selected from: fluoro, chloro, -CH3, -CF3, -C(O)phenyl, pyrrolidinyl, -P(O)(CH3)2, -C(O)NH2, - S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH3)2, -CH₂C(O)NH₂, -NH-cyclopropyl, -N(CH₃)- cyclobutyl, -NH-oxetanyl, -N(R^ZZ)-S(O)2R^X, -NH(Ci-5alkyl), -N(Ci-5alkyl)2, S(O)₂CH₂CH3, -S(O)2CH₂CH₂CH3, -S(O)₂CH3, -SO2NH2, and -S(O)₂phenyl; and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, C1.4 alkyloxy, -OH, -COOH, -NHz, -NH(C 1.4 alkyl), -N(CI-4 alkyl)₂, -NHCOCH2NH2 and -CN.

[0010] In some embodiments, R² is selected from phenyl, -PI1CH3, -PI1CF3, -PhN(CH3)- S(O)₂CH₃ and -PhCH2NHCOCH₂NH2.

[0011] In some embodiments, R³ is hydrogen.

[0012] In some embodiments, R⁴ is selected from hydrogen and -CONH2.

[0013] In some embodiments, R⁵ is selected from -NHz, -NHR^a, and -NR^bR^c . [0014] The method of claim 11, wherein R⁵ is selected from -NH2,

[0015] In some embodiments, each R^a is independently selected from C_1-6 alkyl and R^e.

[0016] In some embodiments, each R^b and R^c is independently selected from hydrogen, C_1-6 alkyl, and R^e. In some embodiments, R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: C_1-6 alkyl, R^e, and -NH2.

[0017] In some embodiments, each R^d is independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, C(O)H, -C(O)R^ZZ, -OC(O)H, -CO(O)R^ZZ, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO2NH2, -S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, -N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, -COOH, -COOR^X, oxo, -OH, -NH₂, -NHR^X, -NR^XR^X, -NO2, -CN, -NHC(O)NH₂, -NHC(O)NHR^X, -NHC(O)NR^XR^X, and Ci-4 alkoxy optionally substituted with 1 to 4 times by fluoro. In some embodiments, each R^d is independently selected from: R^e and -N(R^ZZ)S(O)2R^X

[0018] In some embodiments, each R^eis independently selected from: C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, -SH, -SR^X, - S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, OXO, -OH, -NH₂, -NHR^XX, -NR^XR^X, -NHC(=N)NH₂, -COOH, - COOR^X, -C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, SO2NH2, -S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, - NHS(O)2R^X, -NHC(O)NHR^xp, -NHC(O)NR^xpR^xp, -NO₂, and -CN. In some embodiments, each R^e is independently selected from: C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, oxo, -OH, and -NHR^XX.

[0019] In some embodiments, each R^x is independently selected from: C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OH, -COOH, -NH2 and -CN. In some embodiments, wherein each R^xx is independently selected from C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: oxo, NR^xyR^xy, -COOH, and -CN.

[0020] In some embodiments, each R^xy is hydrogen.

[0021] In some embodiments, the DNA methyltransferase inhibitor is a compound of Formula (II):

or a pharmaceutically acceptable salt or prodrug thereof.

[0022] In some embodiments, the DNA methyltransferase inhibitor is a compound of Formula

(IIA):

or a pharmaceutically acceptable salt or prodrug thereof.

[0023] In some embodiments, R² is phenyl.

[0024] In some embodiments, R⁶ and R⁷ are each C_1-6 alkyl.

[0025] In some embodiments, R⁶ and R⁷ are each methyl.

[0026] In some embodiments, the DNA methyltransferase is compound A ((S)-2-((3,5-dicyano- 6-(dimethylamino)-4-ethylpyridin-2-yl)thio)-2-phenylacetamide) or a salt thereof, wherein compound A has the following structure:

[0027] In some embodiments, the DNA methyltransferase is compound B (2-((3,5-dicyano-6- (dimethylamino)-4-ethylpyridin-2-yl)thio)-2 -phenyl acetamide) or a salt thereof, wherein compound A has the following structure:

[0028] In some embodiments, the DNA methyltransferase inhibitor is a compound of Formula (I):

wherein:

X¹ and X² are independently selected from: -CN, methyl, fluoro, chloro, bromo and iodo;

Y is selected from -S-, -NR⁵-, -O- , -S(O)-, and -S(O)2-;

R¹ is selected from:

-NH(CI-4 alkyl), -N(Ci-4alkyl)2, and -S-Ci-4 alkyl; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, Ci-4 alkyloxy, -OH, -COOH, -NH2, -NH(C 1.4 alkyl), - N(Ci-4 alkyl)2 and -CN;

C1.4 alkyloxy optionally substituted with 1 to 4 times by fluoro; aryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; heteroaryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; cycloalkyl optionally substituted with from 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with from 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; R² is selected from:

Ci-4alkyl, thienyl, piperidinyl; phenyl optionally substituted with 1 or 2 substituents independently selected from: fluoro, chloro, -CH3, -CF3, -C(O)phenyl, pyrrolidinyl, -P(O)(CH3)2, -C(O)NH₂, - S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH3)2 and -CH₂C(O)NH₂; and pyridine optionally substituted with 1 or 2 substituents independently selected from fluoro, -CH3, -CF3, and -OCH3;

R⁶ and R⁷ are independently selected from: hydrogen; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: phenyl, morpholino, triazolyl, imidazolyl, pyrrolidinyl, -0C(0)NH2, - OCH₂CH₂NH₂, -ONHC(NH₂)NH₂, -NHCH₂C(CH₃)3, -NHOCH3, -NHOH, - NHCH₂CH2F, -N(CH3)CH₂CH₂OCH₃, -N(CH₂CH₃)₂, -NCH(CH₂OH)₂, - N(CH₂CH₂OH)₂, -NHCH₂CH₂OH, -NHCH₂CH₂NH₂, -N(CH₃)C(CH₃)₂CH₂OH, - NHCH₂CH₃, -NHCH₂CH₂OCH₃, -N(CH₃)CH₂CH₂OH, -NHC(O)C(O)NH₂, - N(CH₃)CH₂CH₂CH₂OH, -N(CH3)CH₂CH(OH)CH₂OH, -N(CH₃)CH₂CH₂NH₂, oxo, -NHCH₂C(CH₃)₂CH₂OH, -OH, -NH₂, -NHCH3, -NHCH₂CH₂CH₂OH, - N(CH₃)₂, — N(CH₃)CH₂CH₃, -NHOC(CH₃)₂NH₂, -N(CH₃)CH₂(cyclopropyl), - NHCH₂(cyclopropyl), -NH(oxetanyl), -NCH₂CH₂(triazole), piperazinyl, piperidinyl, pyrazolyl, azepinyl, azetidinyl, methoxy, and cyclopropylamino, wherein said phenyl, morpholino, triazolyl, imidazolyl, azepinyl, azetidinyl, pyrrolidinyl, piperazinyl, piperidinyl, oxetanyl, cyclopropyl, and pyrazolyl are optionally substituted with from 1 to 4 substituents independently selected from: methyl, fluoro, -NH₂, -N(CH3)₂, hydroxymethyl, oxo, -OH, and -CH₂NH₂; cycloalkyl optionally substituted with one to five substituents independently selected from: fluoro, chloro, -OH, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro and chloro; heterocycloalkyl optionally substituted with from 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, aryl, -CN, oxo, -OH, -C00H, -N0₂, -NH₂, SO₂NH₂; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, -OH, -NH₂ and -CN, and

Ci-4 alkoxy optionally substituted with 1 to 4 substituents independently selected from fluoro, oxo, -OH, -C00H, -NH₂, and -CN; or R⁶ and R⁷ are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, -CN, oxo, -OH, -OP(O)(OH)2, -COOH, -CONH2, -NO2, - NH2, aryl, cycloalkyl, -O-oxetanyl, -ONHC(NH)NH2, -NH-cyclopropyl, -N(CH3)- cyclobutyl, -NH-oxetanyl, -N(Ci-₅alkyl)₂, -S(O)₂CH₂CH3, -S(O)₂CH₂CH₂CH3, - S(O)₂CH₃, -SO₂NH₂, -S(O)₂phenyl, -NH(Ci-₅alkyl); C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, C1.4 alkoxy, oxo, phenyl, cycloalkyl, heterocycloalkyl, methylheterocycloalkyl-, -OH, -NH₂, aminoheterocycloalkyl-, -N(Ci-s alkyl)₂, -CN, - N(CI-4 alkyl)(CH₂OCH₃), -NH(CI-5 alkyl), and -NH(CI-4 alkyl) substituted by one or two substituents independently selected from oxo, NH₂, and -OH; heterocycloalkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, -C_1-6alkylOH, -C_1-6 alkylNH₂, oxo, -OH, - NH₂ and -CN;

C1.4 alkoxy optionally substituted with 1 to 4 substituents independently selected from: fluoro, oxo, -OH, -COOH, -NH₂, and -CN;

-NH(CI-6 alkyl) substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, oxo, phenyl, cycloalkyl, aminoCi-4alkoxy, heterocycloalkyl, methylheterocycloalkyl-, -OH, -NH₂, -NH(CI-4 alkyl), -N(CI-4 alkyl)₂, and -CN; benzoyl, benzylamino, -propylpyrrolidinyl, -methylcyclopropyl, cyclobutylamino, piperidinyl, imidazolyl, morpholinyl, morpholinylmethyl, methylpiperazinylmethyl, methylpiperazinyl, pyrrolidinyl, pyrrolidinylmethyl, (methoxypyridinylmethyl)amino, methylpyrrolidinyl, difluoropyrrolidinyl, dimethylpyrrolidinyl, (methylcyclopropylmethyl)amino, hydroxymethylpyrrolidinyl, fluoropyrrolidinyl, fluorophenylmethylamino, piperazinlymethyl, oxazolidinyl, (methyloxetanmethyl)amino, (methylcyclobutylmethyl)amino, oxoimidazolidinyl, and 2-hydroxyethylpiperidinyl; and R⁸ is selected from: hydrogen; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -CN, hydoxy, amino, mercapto and -O-C_1-6 alkyl; cycloalkyl optionally substituted with 1 to 4 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -CN, hydoxy, amino, mercapto and -O-C_1-6 alkyl; and heterocycloalkyl optionally substituted with 1 to 4 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -CN, hydoxy, amino, mercapto and -O-C_1-6 alkyl; provided that:

X¹ and X² are not both hydrogen, and

R³ and R⁴ are not both hydrogen; or a pharmaceutically acceptable salt or prodrug thereof.

[0029] In some aspects, the DNA methyltransferase inhibitor is a compound of Formula (II-

or a pharmaceutically acceptable salt or prodrug thereof.

[0030] In some aspects, X¹ and X² are each -CN. In some embodiments, Y is -S-. In some embodiments, R¹ is C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, Ci-4 alkyloxy, -OH, -COOH, -NH2, -NH(CI-4 alkyl), -N(CM alkyl)₂ and -CN. In some embodiments, R¹ is ethyl. In some embodiments, R² is phenyl optionally substituted with 1 or 2 substituents independently selected from: fluoro, chloro, -CH3, -CF3, - C(O)phenyl, pyrrolidinyl, -P(O)(CH₃)₂, -C(O)NH₂, -S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH₃)₂ and - CH₂C(O)NH₂. In some embodiments, R² is phenyl. In some embodiments, R⁶ and R⁷ are each C_1-6 alkyl. In some embodiments, R⁶ and R⁷ are each methyl.

[0031] In some embodiments, the DNA methyltransferase inhibitor is compound A:

or a pharmaceutically acceptable salt or prodrug thereof.

[0032] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, the method comprising: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X-chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome. In some embodiments, methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more. In some embodiments, compound A or a salt thereof is administered daily, every other day or once weekly. In some embodiments, the subject has Rett Syndrome.

[0033] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease, comprises administering compound A or a salt thereof to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer. In some embodiments, methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more. In some embodiments, compound A or a salt thereof is administered daily, every other day or once weekly. In some embodiments, the subject has Rett Syndrome.

[0034] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more. In some embodiments, methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more. In some embodiments, compound A or salt thereof is administered daily, every other day or once weekly. In some embodiments, the subject has Rett Syndrome.

[0035] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more. In some embodiments, methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more. In some embodiments, compound A or salt thereof is administered daily, every other day or once weekly. In some embodiments, the subject has Rett Syndrome.

[0036] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more. In some embodiments, methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more. In some embodiments, compound A or a salt thereof is administered daily, every other day or once weekly. In some embodiments, the subject has Rett Syndrome.

[0037] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more. In some embodiments, DNA methylation patterns in the subject’s blood is decreased by at least 5% for two weeks or more. In some embodiments, compound A or a salt thereof is administered daily, every other day or once weekly. In some embodiments, the subject has Rett Syndrome.

[0038] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease in a subject, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor selectively inhibits DMNT1 over at least one of DMNT3A and DMNT3B. In some aspects, the present disclosure provides a method of treating Rett Syndrome in a subject, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor selectively inhibits DMNT1 over at least one of DMNT3A and DMNT3B.

[0039] In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1 over DMNT3A. In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1 over DMNT3B.

[0040] In some aspects, the present disclosure provides a method of treating an X chromosome-linked disease, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor is a compound of Formula

(II):

[0041] In some embodiments, the DNA methyltransferase inhibitor is selected from:

[0042] In some embodiments, the method further comprises identifying the subject as having an X-chromosome genetic mutation prior to the administering of the DNA methyltransferase inhibitor to the subject.

[0043] In some aspects, the present disclosure provides a method of treating an X chromosome-linked disease, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor is a compound of Formula (II):

wherein:

Y is selected from -S-, -NR⁸-, -O-, -S(O)-, and -S(O)2-;

R¹ is selected from:

C1.4 alkyloxy optionally substituted with 1 to 4 times by fluoro; aryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; heteroaryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; cycloalkyl optionally substituted with from 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with from 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN;

R² is selected from:

Ci-4alkyl, thienyl, piperidinyl; phenyl optionally substituted with 1 or 2 substituents independently selected from: fluoro, chloro, -CH3, -CF3, -C(O)phenyl, pyrrolidinyl, -P(O)(CH3)2, -C(0)NH2, - S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH3)2 and -CH₂C(O)NH₂; and pyridine optionally substituted with 1 or 2 substituents independently selected from fluoro, -CH3, -CF3, and -OCH3;

R⁶ and R⁷ are independently selected from: hydrogen, C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: phenyl, morpholino, triazolyl, imidazolyl, pyrrolidinyl, -0C(0)NH2, - OCH2CH2NH2, -ONHC(NH₂)NH₂, -NHCH₂C(CH₃)3, -NHOCH3, -NH0H, - NHCH2CH2F, -N(CH3)CH₂CH₂OCH3, -N(CH₂CH₃)2, -NCH(CH₂OH)₂, - N(CH₂CH₂OH)₂, -NHCH₂CH₂OH, -NHCH2CH2NH2, -N(CH₃)C(CH3)₂CH₂OH, - NHCH2CH3, -NHCH₂CH₂OCH3, -N(CH3)CH2CH₂OH, -NHC(0)C(0)NH₂, - N(CH3)CH₂CH₂CH₂OH, -N(CH3)CH₂CH(OH)CH₂OH, -N(CH3)CH₂CH₂NH₂, oxo, -NHCH₂C(CH3)₂CH₂OH, -OH, -NH₂, -NHCH3, -NHCH₂CH₂CH₂OH, - N(CH₃)₂, — N(CH₃)CH₂CH3, -NHOC(CH₃)₂NH2, -N(CH₃)CH₂(cyclopropyl), - NHCH2(cyclopropyl), -NH(oxetanyl), -NCH2CH2(triazole), piperazinyl, piperidinyl, pyrazolyl, azepinyl, azetidinyl, methoxy, and cyclopropylamino, wherein said phenyl, morpholino, triazolyl, imidazolyl, azepinyl, azetidinyl, pyrrolidinyl, piperazinyl, piperidinyl, oxetanyl, cyclopropyl, and pyrazolyl are optionally substituted with from 1 to 4 substituents independently selected from: methyl, fluoro, -NH2, -N(CH3)₂, hydroxymethyl, oxo, -OH, and -CH2NH2; cycloalkyl optionally substituted with one to five substituents independently selected from: fluoro, chloro, -OH, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro and chloro; heterocycloalkyl optionally substituted with from 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, aryl, -CN, oxo, -OH, -COOH, -NO2, -NH2, SO2NH2; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN, and

Ci -4 alkoxy optionally substituted with 1 to 4 substituents independently selected from fluoro, oxo, -OH, -COOH, -NH2, and -CN; or

R⁶ and R⁷ are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, -CN, oxo, -OH, -0P(0)(0H)2, -COOH, -CONH2, -NO2, - NH2, aryl, cycloalkyl, -O-oxetanyl, -0NHC(NH)NH2, -NH-cyclopropyl, -N(CH3)- cyclobutyl, -NH-oxetanyl, -N(Ci-5alkyl)2, -S(O)2CH2CH3, -S(O)2CH2CH2CH3, - S(O)₂CH₃, -SO2NH2, -S(O)₂phenyl, -NH(Ci-₅alkyl); C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, C1.4 alkoxy, oxo, phenyl, cycloalkyl, heterocycloalkyl, methylheterocycloalkyl-, -OH, -NH2, aminoheterocycloalkyl-, -N(CI-5 alkyl)2, -CN, - N(Ci-4 alkyl)(CH2OCH3), -NH(CI-5 alkyl), and -NH(CI-4 alkyl) substituted by one or two substituents independently selected from oxo, NH2, and -OH; heterocycloalkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, -C_1-6alkylOH, -C_1-6 alkyfNdL, oxo, -OH, - NH₂ and -CN;

C1.4 alkoxy optionally substituted with 1 to 4 substituents independently selected from: fluoro, oxo, -OH, -COOH, -NH2, and -CN;

-NH(CI-6 alkyl) substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, oxo, phenyl, cycloalkyl, aminoCi.4alkoxy, heterocycloalkyl, methylheterocycloalkyl-, -OH, -NH2, -NH(Ci-4 alkyl), -N(CI-4 alkyl)2, and -CN; benzoyl, benzylamino, -propylpyrrolidinyl, -methylcyclopropyl, cyclobutylamino, piperidinyl, imidazolyl, morpholinyl, morpholinylmethyl, methylpiperazinylmethyl, methylpiperazinyl, pyrrolidinyl, pyrrolidinylmethyl, (methoxypyridinylmethyl)amino, methylpyrrolidinyl, difluoropyrrolidinyl, dimethylpyrrolidinyl, (methylcyclopropylmethyl)amino, hydroxymethylpyrrolidinyl, fluoropyrrolidinyl, fluorophenylmethylamino, piperazinlymethyl, oxazolidinyl, (methyloxetanmethyl)amino, (methylcyclobutylmethyl)amino, oxoimidazolidinyl, and 2-hydroxyethylpiperidinyl; and R⁸ is selected from: hydrogen; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -CN, hydoxy, amino, mercapto and -O-C_1-6 alkyl; cycloalkyl optionally substituted with 1 to 4 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -CN, hydoxy, amino, mercapto and -O-C_1-6 alkyl; and heterocycloalkyl optionally substituted with 1 to 4 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -CN, hydoxy, amino, mercapto and -O-C_1-6 alkyl; provided that:

X¹ and X² are not both hydrogen, and

R⁶ and R⁷ are not both hydrogen; or a pharmaceutically acceptable salt or prodrug thereof.

[0044] In some aspects, the DNA methyltransferase inhibitor is a compound of Formula (II- A):

or a pharmaceutically acceptable salt or prodrug thereof.

[0045] In some aspects, X¹ and X² are each -CN. In some embodiments, Y is selected from -S- and -O-. In some embodiments, Y is -S-. In some embodiments, R¹ is C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, Ci-4 alkyloxy, -OH, -COOH, -NH2, -NH(C 1.4 alkyl), -N(CI-4 alkyl)2 and -CN In some embodiments, R¹ is ethyl. In some embodiments, R² is phenyl optionally substituted with 1 or 2 substituents independently selected from: fluoro, chloro, -CH3, -CF3, -C(O)phenyl, pyrrolidinyl, -P(O)(CH3)2, - C(O)NH₂, -S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH3)2 and -CH₂C(O)NH₂. In some embodiments, R² is phenyl. In some embodiments, R⁶ and R⁷ are each C_1-6 alkyl. In some embodiments, R⁶ and R⁷ are each methyl.

[0046] In some embodiments, the DNA methyltransferase inhibitor is compound A:

or a pharmaceutically acceptable salt or prodrug thereof.

[0047] In some embodiments, the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Alport syndrome, X-linked Charcot-Mari e-tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy. In some embodiments, the X-chromosome-linked disease is Rett Syndrome. In some embodiments, the administration of a compound or a salt of Formula (I) or (I- A) induces wild-type MeCP2 expression. In some embodiments, the administration of a compound or a salt of Formula (I) or (I- A) induces wildtype MeCP2 expression in human neural tissue.

INCORPORATION BY REFERENCE

[0048] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0050] FIG. 1A-C illustrates a wild type MeCP2 protein expression assay design.

[0051] FIG. 2 illustrates compound A induced MeCP2 protein expression as quantified by MeCP2 reactivation at day 14.

[0052] FIG. 3 illustrates compound A induced MeCP2 protein expression after week 1 and comparison with post withdrawal expression at week 1, 2 and 4 as quantified by MeCP2 reactivation.

[0053] FIG. 4 illustrates comparison of compounds A, S and T induced MeCP2 protein expression as quantified by MeCP2 reactivation at day 14. [0054] FIG. 5 illustrates comparison of compounds A, S and I induced MeCP2 protein expression as quantified by MeCP2 reactivation at day 14 with 1 pM.

[0055] FIG. 6 illustrates comparison of compounds U, V and I induced MeCP2 protein expression as quantified by MeCP2 reactivation at day 14.

[0056] FIG. 7 illustrates the lack of cytotoxicity of compound A as quantified by % survival.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0057] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0058] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0059] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0060] The term “C_x-y” when used in conjunction with a chemical moiety, such as alkyl, is meant to include groups that contain from x to y carbons in the chain. For example, the term “C_1-6alkyl” refers to saturated hydrocarbon groups, including straight-chain alkyl and branched-chain alkyl groups that contain from 1 to 6 carbons. For example - C_1-6 alkyl- may be selected from methyl, ethyl, propyl, butyl, pentyl, and hexyl, any one of which is optionally substituted.

[0061] "Alkyl" as used herein refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, and preferably having from one to fifteen carbon atoms (i.e., C1-C15 alkyl). In certain embodiments, an alkyl comprises one to thirteen carbon atoms (i.e., C1-C13 alkyl). In certain embodiments, an alkyl comprises one to eight carbon atoms (i.e., C1-C8 alkyl). In other embodiments, an alkyl comprises one to five carbon atoms (i.e., C1-C5 alkyl). In other embodiments, an alkyl comprises one to four carbon atoms (i.e., C1-C4 alkyl). In other embodiments, an alkyl comprises one to three carbon atoms (i.e., C1-C3 alkyl). In other embodiments, an alkyl comprises one to two carbon atoms (i.e., C1-C2 alkyl). In other embodiments, an alkyl comprises one carbon atom (i.e., Ci alkyl). In other embodiments, an alkyl comprises five to fifteen carbon atoms (i.e., C5-C15 alkyl). In other embodiments, an alkyl comprises five to eight carbon atoms (i.e., C5-C15 alkyl). In other embodiments, an alkyl comprises two to five carbon atoms (i.e., C2-C5 alkyl). In other embodiments, an alkyl comprises three to five carbon atoms (i.e., C3-C5 alkyl). In certain embodiments, the alkyl group is selected from methyl, ethyl, 1 -propyl (n-propyl), 1 -methylethyl (/.w-propyl), 1 -butyl (n-propy)l, 1 -methylpropyl (sec-butyl), 2- methylpropyl (iso-butyl), 1,1 -dimethylethyl (tert-butyl), 1-pentyl (//-pentyl). The alkyl is attached to the rest of the molecule by a single bond.

[0062] “Halo" or "halogen" as used herein refers to halogen substituents such as bromo, chloro, fluoro and iodo substituents.

[0063] "Haloalkyl" as used herein refers to an alkyl radical, as defined above, that is substituted by one or more halogen radicals, for example, trifluoromethyl, dichloromethyl, bromomethyl, 2,2,2- trifluoroethyl, l-fluoromethyl-2-fluoroethyl, and the like. Examples of halogen substituted alkanes (“haloalkanes”) include halomethane (e.g., chloromethane, bromomethane, fluoromethane, iodomethane), di-and trihalomethane (e.g., trichloromethane, tribromomethane, trifluoromethane, triiodomethane), 1-haloethane, 2-haloethane, 1,2-dihaloethane, and any other suitable combinations of alkanes (or substituted alkanes) and halogens. When an alkyl group is substituted with more than one halogen radicals, each halogen may be independently selected, for example 1 -chloro, 2- bromoethane.

[0064] “Carbocycle” as used herein refers to a saturated, unsaturated or aromatic ring in which each atom of the ring is carbon. Carbocycle may include 3- to 10-membered monocyclic rings, 6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic carbocycle may be selected from saturated, unsaturated, and aromatic rings. In some embodiments, the carbocycle is an aryl. In some embodiments, the carbocycle is a cycloalkyl. In some embodiments, the carbocycle is a cycloalkenyl. In an exemplary embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated, unsaturated and aromatic bicyclic rings, as valence permits, are included in the definition of carbocyclic. Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl, adamantyl, phenyl, indanyl, and naphthyl. Carbocycle may be optionally substituted by one or more substituents such as those substituents described herein. Bicyclic carbocycles may be fused, bridged or spiro-ring systems. [0065] “Carbocycle-alkyl” refers to a radical alkylene bound to a carbocyclic group. An exemplary carbocycle-alkyl includes benzyl, cyclopropyl-methyl and phenethyl.

[0066] The term “heterocycle” as used herein refers to a saturated, unsaturated, non-aromatic or aromatic ring comprising one or more heteroatoms. Exemplary heteroatoms include N, O, Si, P, B, and S atoms. Heterocycles include 3- to 10-membered monocyclic rings and 6- to 12-membered bicyclic rings. Each ring of a bicyclic heterocycle may be selected from saturated, unsaturated, and aromatic rings. In some embodiments, the heterocycle comprises at least one heteroatom selected from oxygen, nitrogen, sulfur, or any combination thereof. In some embodiments, the heterocycle comprises at least one heteroatom selected from oxygen, nitrogen, or any combination thereof. In some embodiments, the heterocycle comprises at least one heteroatom selected from oxygen, sulfur, or any combination thereof. In some embodiments, the heterocycle comprises at least one heteroatom selected from nitrogen, sulfur, or any combination thereof. The heterocycle may be attached to the rest of the molecule through any atom of the heterocycle, valence permitting, such as a carbon or nitrogen atom of the heterocycle. In some embodiments, the heterocycle is a heteroaryl. In some embodiments, the heterocycle is a heterocycloalkyl. Exemplary heterocycles include pyrrolidinyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, piperidinyl, pyridinyl, pyrimidinyl, pyridazinyl, pyrazinyl, thiophenyl, oxazolyl, thiazolyl, morpholinyl, indazolyl, indolyl, and quinolinyl. Heterocycle may be optionally substituted by one or more substituents such as those substituents described herein Bicyclic heterocycles may be fused, bridged or spiro-ring systems. In an exemplary embodiment, a heterocycle, e.g., pyridyl, may be fused to a saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, or cyclohexene. Heterocycle may be optionally substituted by one or more substituents such as those substituents described herein.

[0067] The term “heteroaryl” refers to a radical derived from a 3- to 12-membered aromatic ring radical that comprises one to eleven carbon atoms and at least one heteroatom wherein each heteroatom may be selected from N, O, and S. In some embodiments, the heteroaryl comprises at least one heteroatom selected from oxygen, nitrogen, sulfur, or any combination thereof. In some embodiments, the heteroaryl comprises at least one heteroatom selected from oxygen, nitrogen, or any combination thereof. In some embodiments, the heteroaryl comprises at least one heteroatom selected from oxygen, sulfur, or any combination thereof. In some embodiments, the heteroaryl comprises at least one heteroatom selected from nitrogen, sulfur, or any combination thereof. As used herein, the heteroaryl ring may be selected from monocyclic or bicyclic and fused or bridged ring systems rings wherein at least one of the rings in the ring system is aromatic, i.e., it contains a cyclic, delocalized (4n+2) 7t-electron system in accordance with the Hiickel theory. The heteroatom(s) in the heteroaryl radical may be optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaryl may be attached to the rest of the molecule through any atom of the heteroaryl, valence permitting, such as a carbon or nitrogen atom of the heteroaryl. Heteroaryl includes aromatic single ring structures, preferably 5- to 6-membered rings, whose ring structures include at least one heteroatom, preferably one to four heteroatoms, more preferably one or two heteroatoms. Heteroaryl groups include, for example, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine, and the like. Heteroaryl may be optionally substituted by one or more substituents such as those substituents described herein. Heteroaryl also includes polycyclic ring systems having two or more rings in which two or more atoms are common to two adjoining rings wherein at least one of the rings is heteroaromatic, e.g., the other rings can be aromatic or non-aromatic carbocyclic, or heterocyclic. Heteroaryl may be optionally substituted by one or more substituents such as those substituents described herein.

[0068] "Heterocycloalkyl" refers to a stable 3- to 12-membered non-aromatic ring radical that comprises two to twelve carbon atoms and at least one heteroatom wherein each heteroatom may be selected from N, O, Si, P, B, and S atoms. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from oxygen, nitrogen, sulfur, or any combination thereof. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from oxygen, nitrogen, or any combination thereof. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from oxygen, sulfur, or any combination thereof. In some embodiments, the heterocycloalkyl comprises at least one heteroatom selected from nitrogen, sulfur, or any combination thereof. The heterocycloalkyl may be selected from monocyclic or bicyclic, and fused or bridged ring systems. The heteroatoms in the heterocycloalkyl radical are optionally oxidized. One or more nitrogen atoms, if present, are optionally quatemized. The heterocycloalkyl radical is partially or fully saturated. The heterocycloalkyl is attached to the rest of the molecule through any atom of the heterocycloalkyl, valence permitting, such as any carbon or nitrogen atoms of the heterocycloalkyl. Examples of heterocycloalkyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo-thiomorpholinyl. Heterocycloalkyl may be optionally substituted by one or more substituents such as those substituents described herein

[0069] "Aryl" as used herein refers to a radical derived from an aromatic monocyclic or aromatic multicyclic hydrocarbon ring system by removing a hydrogen atom from a ring carbon atom. The aromatic monocyclic or aromatic multicyclic hydrocarbon ring system contains only hydrogen and carbon and from five to eighteen carbon atoms, where at least one of the rings in the ring system is aromatic, i.e., it contains a cyclic, delocalized (4n+2) π-electron system in accordance with the Htickel theory. The ring system from which aryl groups are derived include, but are not limited to, groups such as benzene, fluorene, indane, indene, tetralin and naphthalene.

[0070] "Aryl-alkyl" as used herein refers to radical alkylene bound to an aryl ring, e g., benzyl, phenethyl, and phenpropyl.

[0071] “Substituted” refers to moieties having substituents replacing a hydrogen on one or more carbons or substitutable heteroatoms, e.g., an NH or NHz of a compound. It will be understood that “substitution” or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, i.e., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. In certain embodiments, substituted refers to moieties having substituents replacing two hydrogen atoms on the same carbon atom, such as substituting the two hydrogen atoms on a single carbon with an oxo, imino or thioxo group. As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of organic compounds. The permissible substituents can be one or more and the same or different for appropriate organic compounds.

[0072] A pharmaceutically acceptable salt also refers to any salt which may form in vivo as a result of administration of an acid, another salt, or a prodrug which is converted into an acid or salt. A salt comprises one or more ionic forms of the compound, such as a conjugate acid or base, associated with one or more corresponding counterions. Salts can form from or incorporate one or more deprotonated acidic groups (e.g. carboxylic acids), one or more protonated basic groups (e.g. amines ), or both (e.g. zwitterions).

[0073] The term “salt” or “pharmaceutically acceptable salt” refers to salts derived from a variety of organic and inorganic counter ions well known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, /?-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like. Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, specifically such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.

[0074] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0075] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0076] The term “in vivo” is used to describe an event that takes place in a subject’s body.

[0077] The term “ex vivo” is used to describe an event that takes place outside of a subject’s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “in vitro” assay.

[0078] The term “in vitro” is used to describe an event that takes place in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0079] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0080] The terms “subject,” “individual,” or “patient” are often used interchangeably herein. A “subject” can be a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a human. The subject may be diagnosed or suspected of being at high risk for a disease. In various embodiments, the subject can be a human (e g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain embodiments, the subject may not be under the care or prescription of a physician or other health worker. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease.

[0081] As used herein, the phrase "a subject in need thereof refers to a subject, as described infra, that suffers from, or is at risk for, a pathology to be prophylactically or therapeutically treated with a compound or salt described herein.

[0082] The terms “administer”, “administered”, “administers” and “administering” are defined as providing a composition to a subject via a route known in the art, including but not limited to intravenous, intraarterial, intracranial, intracerebral, intrathecal, intracerebroventricular, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, or intraperitoneal routes of administration. In certain embodiments, oral routes of administering a composition can be used. The terms “administer”, “administered”, “administers” and “administering” a compound should be understood to mean providing a compound of the invention or a prodrug of a compound of the invention to the individual in need.

[0083] As used herein, the term “intracerebroventricular injection” (ICV) refers to the administration of an injection, containing a substance, directly into the cerebrospinal fluid ICV helps bypass the blood-brain barrier allowing for higher concentrations of a substance to reach the brain of a subject. In some embodiments, the substance is a DNA methyltransferase inhibitor as disclosed herein. In some embodiments, the substance is a compound or salt of Formula (I) or (I-a), as disclosed herein.

[0084] As used herein, the terms “treatment” or “treating” are used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit may refer to eradication or amelioration of symptoms or of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease may undergo treatment, even though a diagnosis of this disease may not have been made.

[0085] The term “DNA methyltransferase inhibitors” are defined as molecules or compositions that inhibit the catalyzed transfer of a methyl group to DNA. Such molecules or compositions can be synthetically produced, naturally derived, or semi -synthetically produced. Such molecules and compositions can directly or indirectly inhibit the catalyzed transfer of a methyl group to DNA.

[0086] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0087] It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0088] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0089] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

Methods of treating X-chromosome-linked diseases

[0090] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease which comprise: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X- chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.

[0091] In some embodiments, the genetic mutation outside of the MeCP2 Exon 2 region is at least one of a missense, non-sense, frameshift, insertion, deletion or a duplication mutation, or any combination thereof. In some embodiments, the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation. In some embodiments, the genetic mutation is a missense mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a non-sense mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a frameshift mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is an insertion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a deletion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a duplication mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation outside of SEQ. ID NO. 1 region is at least one of a missense mutation, non-sense mutation, frameshift, insertion, deletion or duplication mutation, or any combination thereof. In some embodiments, the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or duplication mutation. In some embodiments, the genetic mutation is a missense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a non-sense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a frameshift mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is an insertion mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a deletion mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a duplication mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the MeCP2 Exon 2 region contains no genetic mutations. In some embodiments, the SEQ. ID NO. 1 region contains no genetic mutations. In some embodiments, the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409. In some embodiments, the SEQ. ID NO. 1 region contains mutations selected from: reference SNP rs267608409.

[0092] In some embodiments, the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie- tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy. In some embodiments, the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie-tooth disease, X-linked dominant porphyria, and Vitamin D resistant rickets. In some embodiments, the X-chromosome-linked disease is selected from fragile x syndrome and Rett syndrome. In some embodiments, the X-chromosome-linked disease is Rett syndrome.

[0093] In some embodiments, administering the DNA methyltransferase inhibitor induces wildtype MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression in human neural tissue.

[0094] In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1. Such selectivity is beneficial because it maintains efficacy in X-chromosome-linked diseases while avoiding unwanted side effects associated with off-target inhibitory activity (e.g., inhibition of other DMNT enzymes). In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1 over other DMNT enzymes. In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1 over at least one of DMNT3A and DMNT3B. In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1 over DMNT3A. In some embodiments, the DNA methyltransferase inhibitor selectively inhibits DMNT1 over DMNT3B. [0095] For example, in some embodiments, the DNA methyltransferase inhibitor is selective (e.g., at least 1.5-fold selective, at least 2-fold selective, at least 5-fold selective, at least 10-fold selective, at least 15-fold selective, at least 20-fold selective, at least 50-fold selective, at least 100- fold selective, at least 200-fold, at least 500-fold selective, at least 1000-fold selective, etc.) for the inhibition of DMNT1 over at least one of DMNT3A and DMNT3B. In some embodiments, the DNA methyltransferase inhibitor is selective (e.g., at least 1.5-fold selective, at least 2-fold selective, at least 5-fold selective, at least 10-fold selective, at least 15-fold selective, at least 20-fold selective, at least 50-fold selective, at least 100-fold selective, at least 200-fold, at least 500-fold selective, at least 1000-fold selective, etc.) for the inhibition of DMNT1 over DMNT3A. In some embodiments, the DNA methyltransferase inhibitor is selective (e.g., at least 1.5-fold selective, at least 2-fold selective, at least 5-fold selective, at least 10-fold selective, at least 15-fold selective, at least 20-fold selective, at least 50-fold selective, at least 100-fold selective, at least 200-fold, at least 500-fold selective, at least 1000-fold selective, etc.) for the inhibition ofDMNTl over DMNT3B.

[0096] In some embodiments, the DNA methyltransferase inhibitor is selective (e.g., between 1.5-fold and 500-fold selective, between 2-fold and 1000-fold selective, between 5-fold and 500-fold selective, between 10-fold and 500-fold selective, between 20-fold and 500-fold selective, between 50-fold and 500-fold selective, between 100-fold and 500-fold selective, between 200-fold and 500- fold selective, between 100-fold and 1000-fold selective, between 200-fold and 1000-fold selective, between 500-fold and 1000-fold selective, etc.) for the inhibition ofDMNTl over at least one of DMNT3A and DMNT3B. In some embodiments, the DNA methyltransferase inhibitor is selective (e g., between 1.5-fold and 500-fold selective, between 2-fold and 1000-fold selective, between 5- fold and 500-fold selective, between 10-fold and 500-fold selective, between 20-fold and 500-fold selective, between 50-fold and 500-fold selective, between 100-fold and 500-fold selective, between 200-fold and 500-fold selective, between 100-fold and 1000-fold selective, between 200-fold and 1000-fold selective, between 500-fold and 1000-fold selective, etc.) for the inhibition ofDMNTl over DMNT3A. In some embodiments, the DNA methyltransferase inhibitor is selective (e.g., between 1.5-fold and 500-fold selective, between 2-fold and 1000-fold selective, between 5-fold and 500-fold selective, between 10-fold and 500-fold selective, between 20-fold and 500-fold selective, between 50-fold and 500-fold selective, between 100-fold and 500-fold selective, between 200-fold and 500-fold selective, between 100-fold and 1000-fold selective, between 200-fold and 1000-fold selective, between 500-fold and 1000-fold selective, etc.) for the inhibition of DMNT1 over DMNT3B.

[0097] In some embodiments, the DNA methyltransferase inhibitor is a compound or salt of Formula (I). In some embodiments, the compound or salt of Formula (I) selectively inhibits DMNT1. In some embodiments, the compound or salt of Formula (I) selectively inhibits DMNT1 over other DMNT enzymes. In some embodiments, the compound or salt of Formula (I) selectively inhibits DMNT1 over at least one of DMNT3A and DMNT3B. In some embodiments, the compound or salt of Formula (I) selectively inhibits DMNT1 over DMNT3A. In some embodiments, the compound or salt of Formula (I) selectively inhibits DMNT1 over DMNT3B.

[0098] For example, in some embodiments, the compound or salt of Formula (I) is selective (e.g., at least 1.5-fold selective, at least 2-fold selective, at least 5-fold selective, at least 10-fold selective, at least 15-fold selective, at least 20-fold selective, at least 50-fold selective, at least 100- fold selective, at least 200-fold, at least 500-fold selective, at least 1000-fold selective, etc.) for the inhibition of DMNT1 over at least one of DMNT3A and DMNT3B. In some embodiments, the compound or salt of Formula (I) is selective (e.g., at least 1.5-fold selective, at least 2-fold selective, at least 5-fold selective, at least 10-fold selective, at least 15-fold selective, at least 20-fold selective, at least 50-fold selective, at least 100-fold selective, at least 200-fold, at least 500-fold selective, at least 1000-fold selective, etc.) for the inhibition of DMNT1 over DMNT3A. In some embodiments, the compound or salt of Formula (I) is selective (e g., at least 1.5-fold selective, at least 2-fold selective, at least 5-fold selective, at least 10-fold selective, at least 15-fold selective, at least 20-fold selective, at least 50-fold selective, at least 100-fold selective, at least 200-fold, at least 500-fold selective, at least 1000-fold selective, etc.) for the inhibition ofDMNTl over DMNT3B.

[0099] In some embodiments, the compound or salt of Formula (I) is selective (e.g., between 1.5-fold and 500-fold selective, between 2-fold and 1000-fold selective, between 5-fold and 500-fold selective, between 10-fold and 500-fold selective, between 20-fold and 500-fold selective, between 50-fold and 500-fold selective, between 100-fold and 500-fold selective, between 200-fold and 500- fold selective, between 100-fold and 1000-fold selective, between 200-fold and 1000-fold selective, between 500-fold and 1000-fold selective, etc.) for the inhibition ofDMNTl over at least one of DMNT3A and DMNT3B. In some embodiments, the compound or salt of Formula (I) is selective (e.g., between 1.5-fold and 500-fold selective, between 2-fold and 1000-fold selective, between 5- fold and 500-fold selective, between 10-fold and 500-fold selective, between 20-fold and 500-fold selective, between 50-fold and 500-fold selective, between 100-fold and 500-fold selective, between 200-fold and 500-fold selective, between 100-fold and 1000-fold selective, between 200-fold and 1000-fold selective, between 500-fold and 1000-fold selective, etc.) for the inhibition ofDMNTl over DMNT3A. In some embodiments, the compound or salt of Formula (I) is selective (e.g., between 1.5-fold and 500-fold selective, between 2-fold and 1000-fold selective, between 5-fold and 500-fold selective, between 10-fold and 500-fold selective, between 20-fold and 500-fold selective, between 50-fold and 500-fold selective, between 100-fold and 500-fold selective, between 200-fold and 500-fold selective, between 100-fold and 1000-fold selective, between 200-fold and 1000-fold selective, between 500-fold and 1000-fold selective, etc.) for the inhibition of DMNT1 over DMNT3B.

[0100] In some embodiments, the DNA methyltransferase inhibitor is a compound of Formula (I): wherein:

X¹ and X² are independently selected from: hydrogen, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, SH, and SR^a; and cycloalkyl and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

Y is selected from -S-, -NH-, -NR^Z-, -O-, -S(O)-, and -S(O)2-;

R¹ is selected from:

NH2, -NHR^a, -NR^bR^c, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, SH, and SR^a; and cycloalkyl, heterocycloalkyl, aryl, and heteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d;

R⁵ is selected from:

R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, -CN, oxo, -OH, -0P(0)(0H)2, -COOH, -CONH₂, -NO2, -NH₂, -NH(Ci-₄alkyl), -N(H)R^e, -N(Ci-₄alkyl)₂, -NR^eR^e, - SO2NH2, -S(O)₂CH₂CH3, -S(O)2CH₂CH₂CH₃, -S(O)₂CH₃, -S(O)₂phenyl, and Ci-₄ alkoxy optionally substituted with 1 to 6 substituents independently selected from fluoro, oxo, -OH, -COOH, -NH2, and -CN; and aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d; each R^d is independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, C(O)H, -C(O)R^ZZ, -OC(O)H, - CO(O)R^ZZ, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO2NH2, - S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, -N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, - C(O)NR^XR^X, -COOH, -COOR^X, oxo, -OH, -NH₂, -NHR^X, -NR^XR^X, -NO₂, - CN, -NHC(O)NH₂, -NHC(O)NHR^X, -NHC(O)NR^XR^X, and Ci-₄ alkoxy optionally substituted with 1 to 4 times by fluoro; and aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and -Oaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^x; and

-C(O)aryl, C(O)heteroaryl, -OC(O)aryl, and -OC(O)heteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^zz; each R^eis independently selected from: C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-₆ alkyl, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, - S(O)₂R^X, OXO, -OH, -NH₂, -NHR^XX, -NR^XR^X, -NHC(=N)NH₂, -C00H, - C00R^x, -C(0)NH₂, -C(0)NHR^X, -C(0)NR^XR^X, SO₂NH₂, -S(0)₂NHR^X, - S(0)₂NR^XR^X, -NHS(0)₂H, -NHS(0)₂R^X, -NHC(O)NHR^xp, - NHC(0)NR^xpR^xp, -N0₂, and -CN; and C_1-6 alkoxy optionally substituted with 1 to 6 substituents independently selected from fluoro, oxo, -OH, -C00H, -NH₂, and -CN; and aryl, -Oaryl, heteroaryl, -Oheteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^x; each R^x is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OH, -C00H, -NH₂ and -CN; each R™ is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OR", NR"R" -C00H, and -CN; each R" is independently selected from: hydrogen, aryl, C1.5 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, -OH, C1.5 alkoxy optionally substituted with 1 to 6 substituents independently selected from: fluoro and -C00H; each R^xp is independently selected from: heteroaryl, cyclcoalkyl, heterocycloalkyl, C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: -C00H, -NH₂ and -CN; each R^z is independently selected from: C_1-6 alkyl, R^e, cyclcoalkyl optionally substituted with 1 to 4 substituents independently selected from R^d, and heterocyclcoalkyl optionally substituted with 1 to 4 substituents independently selected from R^d. each R^zz is independently selected from C_1-6 alkyl, and R^e. provided that: at least one of R², R³ and R⁴ is hydrogen, R², R³ and R⁴ are not all hydrogen, X¹ and X² are not both hydrogen; or a pharmaceutically acceptable salt or prodrug thereof.

[0101] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), X¹ and X² are independently selected from: hydrogen, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, SH, and SR^a. In some embodiments, X¹ and X² are independently selected from: hydrogen, -CN, fluoro, chloro, bromo, C_1-6 alkyl, R^e, OC_1-6 alkyl, and OR^e. In some embodiments, X¹ and X² are independently selected from: hydrogen, -CN, fluoro, chloro, bromo, Ci- 6 alkyl, and R^e. In some embodiments, X¹ and X² are independently selected from: -CN, fluoro, chloro, bromo, C_1-6 alkyl and C_1-6 haloalkyl. In some embodiments, X¹ and X² are independently selected from: -CN, fluoro, chloro, and bromo. In some embodiments, X¹ and X² are independently selected from: -CN, fluoro, and chloro. In some embodiments, X¹ and X² are independently selected from: -CN.

[0102] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), Y is selected from -S-, -NH-, -NR^Z-, and -O-. In some embodiments, Y is selected from -S-, - O-, -S(O)-, and -S(O)2. In some embodiments, Y is selected from -S-, -NH-, -NR^Z-, -S(O)-, and - S(O)₂. In some embodiments, Y is selected from -S-, -S(O)-, and -S(O)2. In some embodiments, Y is selected from -S-, -NH-, -O-, -S(O)-, and -S(O)2. In some embodiments, Y is selected from -S-, - NH-, -NR^Z-, -O-, and -S(O)2. In some embodiments, Y is selected from -S-, -NH-, -O-, and -S(O)2. In some embodiments, Y is selected from -S-, -NH-, and -O-. In some embodiments, Y is selected from -S-, and -O-. In some embodiments, Y is selected from -S-.

[0103] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), R¹ is selected from: NFb, -NHR^a, -NR^bR^c, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, SH, SR^a, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl. In some embodiments, R¹ is selected from: NH2, -NHR^a, -NR^bR^c, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, SH, SR^a, cycloalkyl, and heterocycloalkyl. In some embodiments, R¹ is selected from: NH2, -NHR^a, -NR^bR^c, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, and OR^e. In some embodiments, R¹ is selected from: -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, and OR^e. In some embodiments, R¹ is selected from: NH2, -NHR^a, -NR^bR^c, -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, and cycloalkyl. In some embodiments, R¹ is selected from: - CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, and cycloalkyl. In some embodiments, In some embodiments, R¹ is selected from: -CN, fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, OC_1-6 alkyl, OR^e, and cyclopropyl. In some embodiments, R¹ is selected from: chloro, bromo, C_1-6 alkyl, R^e, OC_1-6 alkyl, and OR^e. In some embodiments, R¹ is selected from: C_1-6 alkyl, R^e, OC_1-6 alkyl, and OR^e. In some embodiments, R¹ is selected from: C_1-6 alkyl and R^e. In some embodiments, R¹ is selected from: C_1-6 alkyl.

[0104] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), R² is selected from aryl and heteroaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d In some embodiments, R² is selected from aryl optionally substituted with 1 to 4 substituents independently selected from R^d. In some embodiments, R² is selected from phenyl optionally substituted with 1 to 4 substituents independently selected from R^d. In some embodiments, R² is selected from phenyl optionally substituted with 1 to 2 substituents independently selected from R^d.

[0105] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), R² is phenyl optionally substituted with one or more substituents independently selected from: fluoro, chloro, -CH3, -CF3, -C(O)phenyl, pyrrolidinyl, -P(O)(CH₃)2, -C(O)NH₂, - S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH₃)₂, -CH₂C(O)NH₂, -NH-cyclopropyl, -N(CH₃)- cyclobutyl, -NH-oxetanyl, -N(R^ZZ)-S(O)₂R^X, -NH(Ci-5alkyl), -N(Ci-5alkyl)₂, S(O)₂CH₂CH₃, -S(O)₂CH₂CH₂CH₃, -S(O)₂CH₃, -SO₂NH₂, and -S(O)₂phenyl; and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, CM alkyloxy, -OH, -COOH, -NH₂, -NH(C 1.4 alkyl), -N(CI-4 alkyl)₂, -NHCOCH₂NH₂ and -CN.

[0106] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), R² is phenyl optionally substituted with one or more substituents independently selected from: fluoro, chloro, -CH₃, -CF3, -P(O)(CH₃)₂, -C(O)NH₂, -S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH₃)₂, - CH₂C(O)NH₂, -N(R^ZZ)-S(O)₂R^X, -NH(Ci.₅alkyl), -N(Ci-₅alkyl)₂, S(O)₂CH₂CH₃, -S(O)₂CH₂CH₂CH₃, -S(O)₂CH₃, -SO₂NH₂, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, C1.4 alkyloxy, -OH, -COOH, -NH₂, - NHCOCH₂NH₂ and -CN. In some embodiments, R² is phenyl optionally substituted with one or more substituents independently selected from: fluoro, chloro, -CH₃, -CF₃, -OCH₂CH₂N(CH₃)₂, - CH₂C(O)NH₂, -N(R^ZZ)-S(O)₂R^X, S(O)₂CH₂CH₃, -S(O)₂CH₂CH₂CH₃, -S(O)₂CH₃, -SO₂NH₂, and Ci-₆ alkyl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, -NH₂, -NHCOCH₂NH₂ and -CN. In some embodiments, R² is phenyl optionally substituted with one or more substituents independently selected from: fluoro, chloro, -CH₃, -CF₃, N(R^ZZ)-S(O)2R^X, -SO2NH2, and C_1-6 alkyl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, oxo, -NH2, -NHCOCH2NH2 and -CN. In some embodiments, R² is phenyl optionally substituted with one or more substituents independently selected from-CF3, N(R^ZZ)-S(O)2R^X, and C_1-6 alkyl optionally substituted with 1 to 4 substituents independently selected from: oxo, -NH2, and -NHCOCH2NH2. In some embodiments, R² is selected from phenyl, -PhCH₃, -PhCF₃, -PhN(CH₃)-S(O)₂CH₃ and -PhCItNHCOCIfcNJfe.

[0107] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), R³ is selected from: hydrogen, C_1-6 alkyl, R^e, -COOR^a, -CONHR^a, and -CONR^bR^c. In some embodiments, R³ is selected from: hydrogen, C_1-6 alkyl, and R^e. In some embodiments, R³ is selected from: hydrogen and C_1-6 alkyl. In some embodiments, R³ is hydrogen.

[0108] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), R⁴ is selected from: hydrogen, C_1-6 alkyl, R^e, -COOR^a, -CONHR^a, and -CONR^bR^c. In some embodiments, R⁴ is selected from: hydrogen, C_1-6 alkyl, R^e, -CONHR^a, and -CONR^bR^c. In some embodiments, R⁴ is selected from: hydrogen, C_1-6 alkyl, -CONHR^a, and -CONR^bR^c. In some embodiments, R⁴ is selected from: hydrogen, C_1-6 alkyl, R^e, and -CONR^bR^c. In some embodiments, R⁴ is selected from: hydrogen, -CONHR^a, and -CONR^bR^c. In some embodiments, R⁴ is selected from: hydrogen and -CONR^bR^c. In some embodiments, R⁴ is -CONR^bR^c. In some embodiments, R⁴ is hydrogen.

[0109] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), R⁵ is selected from: -NH2, -NHR^a, -NR^bR^c, -OC_1-6 alkyl, -OR^e, -SH, and -SR^a. In some embodiments, R⁵ is selected from: -NH2, -NHR^a, -NR^bR^c. -OC_1-6 alkyl, and -OR^e. In some embodiments, R⁵ is selected from: -NH2, -NHR^a, -NR^bR^c, and -OC_1-6 alkyl. In some embodiments, R⁵ is selected from: -NHR^a, -NR^bR^c, and -OC_1-6 alkyl. In some embodiments, R⁵ is selected from: - NHR^a, and -NR^bR^c. In some embodiments, R⁵ is selected from: -NR^bR^c. In some embodiments, R⁵ is selected from -NH2, and

[0110] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), each R^a is independently selected from C_1-6 alkyl and R^e. In some embodiments, each R^a is independently selected from aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d. In some embodiments, each R^a is independently selected from C_1-6 alkyl. [oni] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), each R^b and R^c are independently selected from hydrogen, C_1-6 alkyl, and R^e; and aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d. In some embodiments, each R^b and R^c are independently selected from hydrogen, C_1-6 alkyl, and R^e.

[0112] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, -CN, oxo, -OH, -OP(O)(OH)2, -COOH, -CONH2, - NO2, -NH2, -NH(Ci-4alkyl), -N(H)R^e, -N(Ci-4alkyl)2, and -NR^eR^e. In some embodiments, R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, -CN, oxo, -NH2, -NH(Ci-4alkyl), -N(H)R^e, -N(Ci-4alkyl)2, and -NR^eR^e. In some embodiments, R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, C_1-6 alkyl, R^e, oxo, -NH2, -NH(Ci-4alkyl), -N(H)R^e, -N(Ci-4alkyl)2, and -NR^eR^e In some embodiments, R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: C_1-6 alkyl, R^e, -NH2, -NH(Ci-4alkyl), -N(H)R^e, -N(Ci-4alkyl)2, and -NR^eR^e. In some embodiments, R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: C_1-6 alkyl, R^e, -NH2, and -NH(Ci-4alkyl). In some embodiments, R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: C_1-6 alkyl, R^e, and -NH2.

[0113] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), each R^d is independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, C(O)H, - C(O)R^ZZ, -OC(O)H, -CO(O)R^ZZ, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO2NH2, - S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, - N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, -COOH, -COOR^X, oxo, -OH, -NH₂, -NHR^X, - i-₆ , , , - , - , - , - , - , - ₂ , - ₂ , - SO2NH2, -S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, - NHC(O)R^X, -N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, -NH₂, -NHR^X, -NR^, and Ci-₄ alkoxy optionally substituted with 1 to 4 times by fluoro. In some embodiments, each R^d is independently selected from: Cue alkyl, R^e, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO₂NH₂, - S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, - N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, -NH₂, -NHR^X, -NR’TO, and Ci-₄ alkoxy optionally substituted with 1 to 4 times by fluoro. In some embodiments, each R^d is independently selected from: Ci-₆ alkyl, R^e, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO₂NH₂, -S(O)₂NHR^X, - S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, -N(Me)C(O)R^x, - C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, -NH₂, -NHR^X, and -NR^XR. In some embodiments, each R^d is independently selected from: Cue alkyl, R^e, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO₂NH₂, - S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, and -N(R^ZZ)S(O)₂R^X. In some embodiments, each R^d is independently selected from: Cue alkyl, R^e, -SO₂NH₂, -S(O)₂NHR^X, -S(O)₂NR^XR^X, - NHS(0)2H, -NHS(O)₂R^X, and -N(R^ZZ)S(O)₂R^X. In some embodiments, each R^d is independently selected from: Ci-e alkyl, R^e, -NHS(O)₂H, -NHS(O)₂R^X, and -N(R^ZZ)S(O)₂R^X. In some embodiments, each R^d is independently selected from: R^e, and -N(R^ZZ)S(O)₂R^X.

[0114] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II- A), each R^eis independently selected from: Ci-e alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-e alkyl, -SH, -SR^X, -S(O)H, -S(O)R^X, - S(O)₂H, -S(O)₂R^X, OXO, -OH, -NH₂, -NHR™, -NR^, -NHC(=N)NH₂, -COOH, -COOR^X, -C(0)NH₂, -C(O)NHR^X, -C(0)NR^xR^x, SO₂NH₂, -S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, - NHC(0)NHR^xp, -NHC(0)NR^xpR^xp, -N0₂, and -CN. In some embodiments, each R^eis independently selected from: C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-e alkyl, oxo, -OH, -NH₂, -NHR™, -NR^XR^X, -NHC(=N)NH₂, -COOH, - COOR^X, -C(O)NH₂, -C(0)NHR^X, -C(O)NR^XR^X, -N0₂, and -CN. In some embodiments, Cue alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, oxo, -OH, -NH₂, -NHR^XX, -NR^XR^X, -NHC(-N)NH₂, -COOH, -COOR^X, -C(O)NH₂, - C(O)NHR^X, and -C(O)NR^XR^X. In some embodiments, Ci-e alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, Ci-e alkyl, oxo, -OH, -NH₂, -NHR^XX, - NR^XR^X, -C(0)NH₂, -C(0)NHR^X, and -C(O)NR^XR^X. In some embodiments, C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, C_1-6 alkyl, oxo, -OH, -NH₂, -NHR^XX, - NR^XR^X, -C(0)NH₂, -C(O)NHR^X, and -C(O)NR^XR^X. In some embodiments, C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, C_1-6 alkyl, oxo, -OH, -NH2, -NHR^XX, and - NR^XR^X In some embodiments, C_1-6 alkyl substituted with 1 to 4 substituents independently selected from: fluoro, C_1-6 alkyl, oxo, -OH, -NH2, -NHR^XX, and -NR^XR^X. In some embodiments, C_1-6 alkyl substituted with 1 to 4 substituents independently selected from: fluoro, oxo, -OH, -NH2, -NHR^XX, and -NR^XR^X. In some embodiments, C_1-6 alkyl substituted with 1 to 4 substituents independently selected from: fluoro, oxo, -OH, -NHR^XX, and -NR^xR^x. In some embodiments, C_1-6 alkyl substituted with 1 to 4 substituents independently selected from: fluoro, oxo, -OH, and -NHR™.

[0115] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), each R^x is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, - OH, -COOH, -NH2 and -CN. In some embodiments, each R^x is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, and oxo. In some embodiments, each R^x is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and Ci-e alkyl. In some embodiments, each R^x is independently selected from: C_1-6 alkyl.

[0116] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), each R^xx is independently selected from: aryl, heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, - OR^xy, NR’HV⁵' -COOH, and -CN. In some embodiments, each R^xx is independently selected from: C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, - OR^XV, NR^XyR^Xy -COOH, and -CN. In some embodiments, each R^xx is independently selected from: C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, - OR^xy, and NR^xyR^xy. In some embodiments, each R^xx is independently selected from: C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, and NR^xyR^xy. In some embodiments, each R^xx is independently selected from: C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: oxo and NR^xyR^xy.

[0117] In some embodiments, for the compound, salt, or prodrug of Formula (I), (I-A), (II), or (II-A), each R^xy is independently selected from: hydrogen, aryl, and C1.5 alkyl. In some embodiments, each R^xy is independently selected from: hydrogen and C1.5 alkyl. In some embodiments, each R^xv is hydrogen. In some embodiments, R’’’ is independently selected from: heteroaryl, cyclcoalkyl, heterocycloalkyl, and C_1-6 alkyl. In some embodiments, R^xp is independently selected from C_1-6 alkyl. In some embodiments, each R^z is independently selected from: C_1-6 alkyl, R^e, and cyclcoalkyl. In some embodiments, each R^z is independently selected from: C_1-6 alkyl and R^e. In some embodiments, each R^z is independently selected from: C_1-6 alkyl. In some embodiments, each R^zz is independently selected from C_1-6 alkyl. [0118] In some embodiments, for the compound, salt, or prodrug of Formula (I) or (I-A), R⁵ is NR⁶R⁷. In some embodiments, R⁵ is NR⁶R⁷, R³ is hydrogen, and R⁴ is hydrogen. In some embodiments, for the compound, salt, or prodrug of Formula (I) or (I-A), R⁵ is NR⁶R⁷, R³ is hydrogen R⁴ is hydrogen, and Y is selected from -S-, -NH-, -NR⁸-, -O-, -S(O)-, and -S(O)2-. In some embodiments, R⁵ is NR⁶R⁷, R³ is hydrogen R⁴ is hydrogen, and Y is selected from -S-, -NH-, -NR⁸-, and -O-. In some embodiments, R⁵ is NR⁶R⁷, R³ is hydrogen R⁴ is hydrogen, and Y is selected from -S-.

[0119] In some embodiments, the DNA methyltransferase inhibitor is a compound of Formula (II):

wherein:

Y is selected from -S-, -NR⁸-, -O-, -S(O)-, and -S(O)2-;

R¹ is selected from:

-NH(CI-4 alkyl), -N(Ci-4alkyl)2, and -S-Ci-4 alkyl; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, Ci-4 alkyloxy, -OH, -COOH, -NH2, -NH(C 1-4 alkyl), - N(Ci-4 alkyl)2 and -CN;

C1.4 alkyloxy optionally substituted with 1 to 4 times by fluoro; aryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; heteroaryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; cycloalkyl optionally substituted with from 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with from 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN; R² is selected from:

R⁶ and R⁷ are independently selected from: hydrogen; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: phenyl, morpholino, triazolyl, imidazolyl, pyrrolidinyl, -OC(O)NH2, - OCH₂CH₂NH₂, -ONHC(NH₂)NH₂, -NHCH₂C(CH₃)3, -NHOCH3, -NHOH, - NHCH₂CH2F, -N(CH3)CH₂CH₂OCH₃, -N(CH₂CH₃)₂, -NCH(CH₂OH)₂, - N(CH₂CH₂OH)₂, -NHCH₂CH₂OH, -NHCH₂CH₂NH₂, -N(CH₃)C(CH₃)₂CH₂OH, - NHCH₂CH₃, -NHCH₂CH₂OCH₃, -N(CH₃)CH₂CH₂OH, -NHC(O)C(O)NH₂, - N(CH₃)CH₂CH₂CH₂OH, -N(CH3)CH₂CH(OH)CH₂OH, -N(CH₃)CH₂CH₂NH₂, oxo, -NHCH₂C(CH₃)₂CH₂OH, -OH, -NH₂, -NHCH3, -NHCH₂CH₂CH₂OH, - N(CH₃)₂, — N(CH₃)CH₂CH₃, -NHOC(CH₃)₂NH₂, -N(CH₃)CH₂(cyclopropyl), - NHCH₂(cyclopropyl), -NH(oxetanyl), -NCH₂CH₂(triazole), piperazinyl, piperidinyl, pyrazolyl, azepinyl, azetidinyl, methoxy, and cyclopropylamino, wherein said phenyl, morpholino, triazolyl, imidazolyl, azepinyl, azetidinyl, pyrrolidinyl, piperazinyl, piperidinyl, oxetanyl, cyclopropyl, and pyrazolyl are optionally substituted with from 1 to 4 substituents independently selected from: methyl, fluoro, -NH₂, -N(CH3)₂, hydroxymethyl, oxo, -OH, and -CH₂NH₂; cycloalkyl optionally substituted with one to five substituents independently selected from: fluoro, chloro, -OH, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro and chloro; heterocycloalkyl optionally substituted with from 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, aryl, -CN, oxo, -OH, -COOH, -N0₂, -NH₂, SO₂NH₂; C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, -OH, -NH₂ and -CN, and

Ci-4 alkoxy optionally substituted with 1 to 4 substituents independently selected from fluoro, oxo, -OH, -COOH, -NH₂, and -CN; or R⁶ and R⁷ are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, -CN, oxo, -OH, -0P(0)(0H)2, -COOH, -CONH2, -NO2, - NH2, aryl, cycloalkyl, -O-oxetanyl, -0NHC(NH)NH2, -NH-cyclopropyl, -N(CH3)- cyclobutyl, -NH-oxetanyl, -N(Ci-₅alkyl)₂, -S(O)₂CH₂CH3, -S(O)₂CH₂CH₂CH3, - S(O)₂CH₃, -SO₂NH₂, -S(O)₂phenyl, -NH(Ci-₅alkyl); C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, C1.4 alkoxy, oxo, phenyl, cycloalkyl, heterocycloalkyl, methylheterocycloalkyl-, -OH, -NH₂, aminoheterocycloalkyl-, -N(Ci-s alkyl)₂, -CN, - N(CI-4 alkyl)(CH₂OCH₃), -NH(CI-5 alkyl), and -NH(CI-4 alkyl) substituted by one or two substituents independently selected from oxo, NH₂, and -OH; heterocycloalkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, -C_1-6alkylOH, -C_1-6 alkylNH₂, oxo, -OH, - NH₂ and -CN;

X¹ and X² are not both hydrogen, and

[0120] In some embodiments, the DNA methyltransferase inhibitor is a compound of Formula (I- A):

or a pharmaceutically acceptable salt or prodrug thereof.

[0121] In some embodiments for the compound, salt, or prodrug of Formula (I), (I- A), (II), or (II- A), X¹ and X² are each -CN.

[0122] In some embodiments, Y is selected from -S- and -O-. In some embodiments, Y is -S-. In some embodiments, Y is -O-.

[0123] In some embodiments, R¹ is C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, CM alkyloxy, -OH, -COOH, -NH2, - NH(CI-4 alkyl), -N(CI-4 alkyl)2 and -CN. In some embodiments, R¹ is ethyl.

[0124] In some embodiments, R² is phenyl optionally substituted with 1 or 2 substituents independently selected from: fluoro, chloro, -CH3, -CF3, -C(O)phenyl, pyrrolidinyl, -P(O)(CH3)2, - C(O)NH₂, -S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH3)2 and -CH₂C(O)NH₂. In some embodiments, R² is phenyl.

[0125] In some embodiments for the compound, salt, or prodrug of Formula (I), (I- A), (II), or (II-A), R² is aryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, CM alkoxy, -CN, oxo, -OH, -NO2, -NHz, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN. In some embodiments for the compound, salt, or prodrug of Formula (I) or (I-A), R² is heteroaryl optionally substituted with 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH2 and -CN. In some embodiments for the compound, salt, or prodrug of Formula (I) or (I-A), R² is cycloalkyl optionally substituted with from 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1-4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with from 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, - NH2 and -CN. In some embodiments for the compound, salt, or prodrug of Formula (I) or (I-A), R² is heterocycle optionally substituted with from 1 to 4 substituents independently selected from: fluoro, chloro, bromo, iodo, C1.4 alkoxy, -CN, oxo, -OH, -NO2, -NH2, and C_1-6 alkyl optionally substituted with from 1 to 9 substituents independently selected from fluoro, chloro, bromo, iodo, oxo, -OH, -NH₂ and -CN;

[0126] In some embodiments, R⁶ and R⁷ are each C_1-6 alkyl. In some embodiments, R³ and R⁴ are each methyl.

[0127] In some embodiments, the DNA methyltransferase inhibitor is compound A:

or a pharmaceutically acceptable salt or prodrug thereof.

[0128] In some embodiments, the DNA methyltransferase inhibitor is compound B:

or a pharmaceutically acceptable salt or prodrug thereof.

[0129] Throughout the present disclosure, reference will be made to the use of compound A or salts thereof in the treatment of diseases and the induction of MeCP2 expression. However, other DNA methyltransferases inhibitors disclosed herein can be used in such treatment and induction, including for example compound B or salts thereof, and compounds C-V or salts thereof, as well as all compounds or salts thereof encompassed by Formula (I) and Formula (I-A).

[0130] In some embodiments, administering compound A or a salt thereof induces wild-type MeCP2 expression. In some embodiments, administering compound A or a salt thereof induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering compound A or a salt thereof induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering compound A or a salt thereof induces the reactivation of wild-type MeCP2 expression in human neural tissue.

[0131] In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering a compound or salt of Formula (I) or (I- A) induces the reactivation of wild-type MeCP2 expression in human neural tissue.

[0132] In some embodiments, the DNA methyl transferase inhibitor is selected from Compounds A-V, listed in Table 1 :

Table 1 - Selected DNA Methyl Transferase Inhibitors

[0133] In some embodiments, the DNA methyltransferase inhibitor is disclosed in U.S. Patent No. US 10,975,056, which is incorporated by reference herein in its entirety.

[0134] In some embodiments, the method of treating an X-chromosome-linked disease comprises a DNA methyltransferase inhibitor, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome in the following in vitro assay: (a) reprogramming X- chromosome linked disease fibroblasts into induced pluripotent stem cells; (b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyl transferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and (d) detecting MeCP2 level in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyltransferase inhibitor. In some embodiments, the X-chromosome-linked disease fibroblasts are Rett Syndrome fibroblasts. In some embodiments, the DNA methyltransferase inhibitor is compound A or a salt thereof. In some embodiments, the DNA methyltransferase inhibitor is compound B or a salt thereof. In some embodiments, the DNA methyltransferase inhibitor is a compound or salt of Formula (I) or (I-A). In some embodiments, the DNA methyltransferase inhibitor is 5’-0-triethylsilyl 5-aza-2'-deoxycytidine.

[0135] In some embodiments, the method of treating an X-chromosome-linked disease comprises a DNA methyltransferase inhibitor, wherein the DNA methyltransferase inhibitor induces wild-type MeCP2 in the following in vitro assay: (a) reprogramming X-chromosome linked disease fibroblasts into induced pluripotent stem cells; (b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyltransferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and (d) detecting MeCP2 level in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyl transferase inhibitor. In some embodiments, the X-chromosome-linked disease fibroblasts are Rett Syndrome fibroblasts. In some embodiments, the DNA methyltransferase inhibitor is compound A or a salt thereof. In some embodiments, the DNA methyltransferase inhibitor is compound B or a salt thereof. In some embodiments, the DNA methyltransferase inhibitor is a compound or salt of Formula (I) or (I-A). In some embodiments, the DNA methyltransferase inhibitor is compound B or a salt thereof. In some embodiments, the DNA methyltransferase is selected from compounds C-V or salts thereof. In some embodiments, the DNA methyltransferase inhibitor is 5 ’ -O-triethylsilyl 5- aza-2'-deoxy cytidine.

[0136] In some embodiments, the assay provides an increased level of MeCP2 expression compared to the level of MeCP2 expression prior to the administration of the DNA methyltransferase inhibitor. In some embodiments, the level of expression is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the level of expression is increased by 5% to about 50%.

[0137] In some embodiments, the DNA methyltransferase inhibitor is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, the DNA methyltransferase inhibitor is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering DNA methyltransferase inhibitor to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.

[0138] In some embodiments, a compound or salt of Formula (I) or (I-A) is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, a compound or salt of Formula (I) or (I- A) is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering a compound or salt of Formula (I) or (I-A) to a subject in need thereof over a period of two weeks or more, wherein the subj ect does not have cancer.

[0139] In some embodiments, compound A or a salt thereof is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, compound A or a salt thereof is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering compound A or a salt thereof to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.

[0140] In some embodiments, compound B or a salt thereof is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, compound B or a salt thereof is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering compound B or a salt thereof to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.

[0141] In some embodiments, the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie- tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy. In some embodiments, the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Rett syndrome, Alport syndrome, X-linked Charcot-Marie-tooth disease, X-linked dominant porphyria, and Vitamin D resistant rickets. In some embodiments, the X-chromosome-linked disease is selected from fragile x syndrome and Rett syndrome. In some embodiments, the X-chromosome-linked disease is Rett syndrome.

[0142] In some embodiments, administering the DNA methyltransferase inhibitor induces wildtype MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering the DNA methyltransferase inhibitor induces the reactivation of wild-type MeCP2 expression in human neural tissue.

[0143] In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces the reactivation of wild-type MeCP2 expression. In some embodiments, administering a compound or salt of Formula (I) or (I-A) induces wild-type MeCP2 expression in human neural tissue. In some embodiments, administering a compound or salt of Formula (I) or (I- A) induces the reactivation of wild-type MeCP2 expression in human neural tissue.

[0144] In some embodiments, a compound or salt of Formula (I) or (I-A) is administered to a subject in need thereof for a period to induce MeCP2 expression. In some embodiments, the subject in need thereof does not have cancer. In some embodiments, a compound or salt of Formula (I) or (I- A) is administered to a subject in need thereof for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some embodiments, the method of treating an X-chromosome-linked disease, comprises administering a compound or salt of Formula (I) or (I- A) to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.

[0145] In some embodiments, a compound or salt of Formula (I) or (I-A) can be formulated for administration as an injection. Non-limiting examples of formulations for injection can include a sterile suspension, solution or emulsion in oily or aqueous vehicles. Suitable oily vehicles can include, but are not limited to, lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. The suspension can also contain suitable stabilizers. Injections can be formulated for bolus injection or continuous infusion. Alternatively, the compositions can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, a compound or salt of Formula (I) or (I-A) can be formulated for administration as a intracerebroventricular injection (ICV). In some embodiments, the administering is by injection. In some embodiments, the injection is directly into the cerebrospinal fluid of the subject. In some embodiments, the injection bypasses the blood-brain barrier. In some embodiments the administering is by intracerebroventricular injection (ICV).

[0146] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease which comprise: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X- chromosome in a subject; and administering a DNA methyltransferase inhibitor as disclosed herein to the subject by injection, wherein the DNA methyltransferase inhibitor activates an inactive X- chromosome.

[0147] In some aspects, the present disclosure provides a method of treating an X-chromosome- linked disease which comprise: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X- chromosome in a subject; and administering a DNA methyltransferase inhibitor as disclosed herein to the subject by intracerebroventricular injection (ICV), wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.

Methods of Reactivating Inactive X-Chromosomes

[0148] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, the method comprising: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X-chromosome in a subject; and administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome. In some embodiments, the genetic mutation outside of the MeCP2 Exon 2 region is at least one of a missense, non-sense, frameshift, insertion, deletion or a duplication mutation, or any combination thereof. In some embodiments, the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation. In some embodiments, the genetic mutation is a missense mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a nonsense mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a frameshift mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is an insertion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a deletion mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation is a duplication mutation outside of the MeCP2 Exon 2 region. In some embodiments, the genetic mutation outside of SEQ. ID NO. 1 region is at least one of a missense mutation, non-sense mutation, frameshift, insertion, deletion or a duplication mutation, or any combination thereof. In some embodiments, the genetic mutation is a missense, non-sense, frameshift, insertion, deletion or a duplication mutation. In some embodiments, the genetic mutation is a missense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a non-sense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a frameshift mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is an insertion mutation outside of the SEQ ID NO. 1 region. In some embodiments, the genetic mutation is a deletion mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the genetic mutation is a nonsense mutation outside of the SEQ. ID NO. 1 region. In some embodiments, the MeCP2 Exon 2 region contains no genetic mutations. In some embodiments, the SEQ. ID NO. 1 region contains no genetic mutations. In some embodiments, the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409. In some embodiments, the SEQ. ID NO. 1 region contains mutations selected from: reference SNP rs267608409.

[0149] In some embodiments, the method of reactivating an inactive X-chromosome, comprises administering a DNA methyltransferase inhibitor to a subject in need thereof in an amount sufficient to increase at least one biomarker selected from: KCC2, brain-derived neurotrophic factor, oxidative stress biomarkers, and DNA methylation patterns for at least 1 week or more. In some embodiments, the biomarker is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the biomarker is increased by 5% to about 50%. In some embodiments, the biomarker is decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the biomarkers is decreased by 5% to about 50%. In some embodiments, the biomarker is increased for a period of at least 7 days, at least 10 days, at least 14 days, at least 20 days, at least 30 days, at least 90 days, at least 200 days, at least 350 days or at least 500 days. In some cases, the subject in need thereof is administered compound A or a salt thereof in an amount sufficient to increase at least one biomarker selected from: KCC2, and brain-derived neurotrophic factor, for at least 1 week or more. In some cases, the subject in need thereof is administered compound A or a salt thereof in an amount sufficient to decrease at least one biomarker selected from: oxidative stress biomarkers and DNA methylation patterns, for at least 1 week or more. In some cases, the subject in need thereof is administered a compound or salt of Formula (I), or (I-A) in an amount sufficient to decrease at least one biomarker selected from: oxidative stress biomarkers and DNA methylation patterns, for at least 1 week or more.

[0150] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more. In some embodiments, the cerebrospinal fluid levels of KCC2 is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the cerebrospinal fluid levels of KCC2 is increased by 5% to about 50%. KCC2 levels can be measured using any number of methods known to those skilled in the art.

[0151] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more. In some embodiments, the cerebrospinal fluid levels of brain- derived neurotrophic factor is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the brain-derived neurotrophic factor is increased by 5% to about 50%. Brain-derived neurotrophic factor levels can be measured using any number of methods known to those skilled in the art.

[0152] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more. In some embodiments, the oxidative stress biomarkers are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the oxidative stress biomarkers are decreased by 5% to about 50%. Oxidative stress biomarkers levels can be measured using any number of methods known to those skilled in the art.

[0153] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering compound A or a salt thereof to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more. In some embodiments, DNA methylation patterns are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, DNA methylation patterns are decreased by 5% to about 50%. DNA methylation patterns levels can be measured using any number of methods known to those skilled in the art. In some embodiments, DNA methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more. In some embodiments, the subject has Rett Syndrome. In some embodiments, compound A or a salt thereof is administered daily, every other day or once weekly. [0154] In some embodiments, the method comprises, chronically administering compound A or a salt thereof daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering compound A or a salt thereof daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering compound A or a salt thereof at least daily, every other day, every third day, once a week, or once a month.

[0155] In some embodiments, the method comprises chronically administering compound A or a salt thereof at least one time a week, two times a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, or more. In some embodiments, chronically administrating compound A or a salt thereof the compound over the course of at least about 7 days, 10 days, 14 days, 21 days, 30 days, 60 days, 90 days, or more.

[0156] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound B or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more. In some embodiments, the cerebrospinal fluid levels of KCC2 is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the cerebrospinal fluid levels of KCC2 is increased by 5% to about 50%. KCC2 levels can be measured using any number of methods known to those skilled in the art.

[0157] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound B or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more. In some embodiments, the cerebrospinal fluid levels of brain- derived neurotrophic factor is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the brain-derived neurotrophic factor is increased by 5% to about 50%. Brain-derived neurotrophic factor levels can be measured using any number of methods known to those skilled in the art. [0158] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering compound B or a salt thereof to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more. In some embodiments, the oxidative stress biomarkers are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the oxidative stress biomarkers are decreased by 5% to about 50%. Oxidative stress biomarkers levels can be measured using any number of methods known to those skilled in the art.

[0159] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering compound B or a salt thereof to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more. In some embodiments, DNA methylation patterns are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, DNA methylation patterns are decreased by 5% to about 50%. DNA methylation patterns levels can be measured using any number of methods known to those skilled in the art. In some embodiments, DNA methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more. In some embodiments, the subject has Rett Syndrome. In some embodiments, compound B or a salt thereof is administered daily, every other day or once weekly. [0160] In some embodiments, the method comprises, chronically administering compound B or a salt thereof daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering compound B or a salt thereof daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering compound B or a salt thereof at least daily, every other day, every third day, once a week, or once a month.

[0161] In some embodiments, the method comprises chronically administering compound B or a salt thereof at least one time a week, two times a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, or more. In some embodiments, chronically administrating compound B or a salt thereof the compound over the course of at least about 7 days, 10 days, 14 days, 21 days, 30 days, 60 days, 90 days, or more.

[0162] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering a compound or salt of Formula (I) or (I- A) to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more. In some embodiments, the cerebrospinal fluid levels of KCC2 is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the cerebrospinal fluid levels of KCC2 is increased by 5% to about 50%. KCC2 levels can be measured using any number of methods known to those skilled in the art. [0163] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering a compound or salt of Formula (I) or (I- A) to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more. In some embodiments, the cerebrospinal fluid levels of brain-derived neurotrophic factor is increased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the brain- derived neurotrophic factor is increased by 5% to about 50%. Brain-derived neurotrophic factor levels can be measured using any number of methods known to those skilled in the art.

[0164] In some aspects, the present disclosure provides a method of reactivating an inactive X- chromosomes, comprising administering a compound or salt Formula (I) or (I-A) to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more. In some embodiments, the oxidative stress biomarkers are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the oxidative stress biomarkers are decreased by 5% to about 50%. Oxidative stress biomarkers levels can be measured using any number of methods known to those skilled in the art. [0165] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, comprising administering a compound or salt of Formula (I) or (I-A) to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more. In some embodiments, DNA methylation patterns are decreased by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, DNA methylation patterns are decreased by 5% to about 50%. DNA methylation patterns levels can be measured using any number of methods known to those skilled in the art. In some embodiments, DNA methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more. In some embodiments, the subject has Rett Syndrome. In some embodiments, a compound or salt of Formula (I) or (I-A) is administered daily, every other day or once weekly.

[0166] In some embodiments, the method comprises, chronically administering a compound or salt of Formula (I) or (I-A) daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering a compound or salt of Formula (I) or (I-A) daily, every other day, every third day, once a week, or once a month. In some embodiments, the method comprises, chronically administering a compound or salt of Formula (I) or (I-A) at least daily, every other day, every third day, once a week, or once a month. [0167] In some embodiments, the method comprises chronically administering a compound or salt of Formula (I) or (I- A) at least one time a week, two times a week, three times a week, four times a week, five times a week, six times a week, seven times a week, eight times a week, nine times a week, ten times a week, or more. In some embodiments, chronically administrating a compound or salt of Formula (I) or (I- A) over the course of at least about 7 days, 10 days, 14 days, 21 days, 30 days, 60 days, 90 days, or more.

[0168] In some embodiments, a compound or salt of Formula (I) or (I-A) can be formulated for administration as an injection. Non-limiting examples of formulations for injection can include a sterile suspension, solution or emulsion in oily or aqueous vehicles. Suitable oily vehicles can include, but are not limited to, lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid esters, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension. The suspension can also contain suitable stabilizers. Injections can be formulated for bolus injection or continuous infusion. Alternatively, the compositions can be lyophilized or in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. In some embodiments, a compound or salt of Formula (I) or (I-A) can be formulated for administration as a intracerebroventricular injection (ICV). In some embodiments, the administering is by injection. In some embodiments, the injection bypasses the blood-brain barrier. In some embodiments, the injection is directly into the cerebrospinal fluid of the subject. In some embodiments the administering is by intracerebroventricular injection (ICV).

[0169] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, the method comprising: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X-chromosome in a subject; and administering a DNA methyltransferase inhibitor as described herein to the subject by injection, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome.

[0170] In some aspects, the present disclosure provides a method of reactivating inactive X- chromosomes, the method comprising: identifying a subject with an X-chromosome genetic mutation, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on the X-chromosome in a subject; and administering a DNA methyltransferase inhibitor as described herein to the subject by intracerebroventricular injection (ICV), wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome. EXAMPLES

[0171] The following examples are included to further describe some embodiments of the present disclosure and should not be used to limit the scope of the disclosure.

Example 1: Wild-type MeCP2 protein expression assay design

[0172] FIG. 1 A-C shows a wild type MeCP2 protein expression assay design. FIG. 1 A shows isogenic control patient derived organoid (PDO) models are generated from Rett patient iPSC lines in which the wild type MeCP2 gene (WT MeCP2) on the active X chromosome (Xa) is expressed. The mutated MeCP2 gene is on the inactive X chromosome (Xi) and not expressed. Immunolabeling with a C-terminal targeting MeCP2 antibody results in detectable MeCP2 expression in cells. FIG. IB shows PDO models of Rett syndrome generated from iPSC lines expressing mutated MeCP2 protein with premature truncation on the Xa and wild type MeCP2 on Xi. C-terminal targeting MeCP2 immunolabeling results in no detectable MeCP2 protein. FIG. 1C shows a drug compound inducing re-activation of wild type MeCP2 from the inactivated X chromosome (Xi*) that results in measurable C-terminal immunolabeling enabling quantification of percentage MeCP2 reactivation.

General Procedures

Stem cell lines

[0173] Patient fibroblasts, with mutation R270X, were obtained from Rett Syndrome Research Trust. Patient fibroblasts were reprogrammed into induced pluripotent stem cells (iPSCs) using nonintegrating Sendai Virus (Cytotune 2.0). Single clones were selected and expanded. The clones were validated for their expression of either the mutant MeCP2 270X allele, or the wildtype MeCP2 270R allele. The iPSCs were maintained in StemFlex medium on matrigel-coated plates.

Organoid production and dissociation

[0174] iPSCs were seeded at 10,000 cells/well in ultra-low attachment 96 well plates and differentiated into forebrain organoids using a dual SMAD inhibition and WNT inhibition protocol. Following neural induction, organoids were cultured in neuron culture media and fed every day. [0175] Once the organoids were 30-40 days old, they were incubated in TrypLE/DNasel at 37 °C for 30 mins. The organoids were then dissociated into single cells and plated at 25,000 cells/well onto matrigel-coated 96 well Cellvis plates. The cells were cultured in neuron culture media and fed every other day. Compound addition

[0176] Compounds (Decitabine, and Compounds A, I, S, T, U and V) were synthesized as described in US 10,975,056, the entire contents of which are incorporated herein by reference. Compounds were resuspended at up to 50 mM in DMSO, aliquoted and stored in -20 C. Compounds were diluted to desired working concentration in neuron culture medium while maintaining strict 0.1% DMSO. Compounds were added to dissociated cells every other day, using an 80% medium change. Control wild type and mutant wells were treated with 0.1% DMSO.

Immunofluorescence staining

[0177] Immunofluorescence staining was performed after 1-week or 2-weeks of compound addition. Dissociated cells were fixed with 2% paraformaldehyde and 4% sucrose in phosphate buffered saline (PBS) at room temperature (RT) for 10 minutes. The cells were permeabilized with 0.1% Triton X-100 at RT for 10 minutes, and then blocked with 5% donkey serum, 2% bovine serum albumin in lX PBS-0.1% Triton-X-100 (PBST) at RT for 1 hour. Primary antibodies for MeCP2 (C-terminus specific; 1:200, Rabbit, CST) and MAP2 (1 :1000, chicken, Abeam) were incubated at 4 °C overnight. Secondary antibodies (1 : 1000, donkey anti-rabbit 647 and donkey antichicken 488) were incubated at RT for 2 hours. DAPI was added at 1 : 1000 at RT for 5 minutes. A montage grid of 16 fluorescence images (each representing a non-overlapping field of view; FOV) was captured for each well using the GE InCell Analyzer. Additionally, brightfield light microscopy images were also acquired for each field of view.

Imaging and analysis

[0178] Digital images were analyzed with proprietary analysis pipelines built on top of open source nuclear segmentation software leveraging deep learning.

Preprocessing and segmentation

[0179] First, multiple z-plane images were projected onto a single flattened image for each channel and each FOV. Next, DNA-stained image channels (DAPI channels) were used to segment nuclei using cellpose (cellpose.org). Identified segments were post-hoc artifact-corrected for potential debris, background staining or imaging artifacts using thresholds on DAPI intensity and size, as well as brightfield image intensity. After rejecting spurious segments, the number of D PI positive nuclei were counted in each image.

[0180] Therapeutic efficacy and toxicity quantification metrics were computed for each image using DAPI positive nuclei as follows.

Efficacy metric

[0181] The MeCP2 reactivation rate was computed by first applying the DAPI segmented nuclei as a mask on the MeCP2 channel. Second, thresholding the MeCP2 channel intensity with suitably derived fixed intensity thresholds. Third, counting the number of MeCP2 positive cells and normalizing by DAPI positive cells expressed as a percent.

[0182] FIG. 2 shows compound A induced MeCP2 protein expression in a dose-dependent manner following 2 week treatment, with 10 pM inducing the greatest MeCP2 protein expression by MeCP2 reactivation quantification. FIG. 2 shows, from left to right, MeCP2 reactivation rates (normalized to positive control) for: Positive control, Negative control, 1 pM Compound A, 3 pM Compound A, and 10 pM Compound A. Positive control data (far left) were generated using Rett isogenic control brain organoids expressing wild type MeCP2. Negative control data (middle left) were generated using Rett brain organoids expressing mutated MeCP2 and either untreated or treated with DMSO vehicle. Reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells. Each dot represents median reactivation rate computed across all fluorescence microscopy images from within a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging. FIG. 2 shows dose dependent MeCP2 reactivation by Compound A (compared to the negative control) across the entire concentration range tested (1 pM to 10 pM).

[0183] FIG. 3 shows that both a 0.3 pM dose of Decitabine and a 10 pM dose of compound A induced MeCP2 protein expression following 7 days of treatment, and MeCP2 protein expression remained elevated up to 28 days after cessation of treatment (7 day, 14 day, and 28 day withdrawal). Further, Compound A inducing the greatest MeCP2 protein expression after 7 to 28 days following cessation of treatment (FIG. 3, right). The long-lasting effects of the compounds disclosed herein are particularly desired for local delivery to the brain, as the long-term efficacy of these compounds reduces the frequency of dosing to necessary to achieve sustained MeCP2 reactivation. Positive control data generated using Rett isogenic control brain organoids expressing wild type MeCP2. Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated, or treated with DMSO vehicle. MeCP2 reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells. Each dot represents median reactivation rate computed across all fluorescence microscopy images from within a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.

[0184] FIG. 4 shows that compounds A and S induced MeCP2 protein expression following 14 day treatment to a greater extent than Compound T, with Compound A at 10 pM and Compound S at 10 pM inducing the greatest MeCP2 protein expression by MeCP2 reactivation quantification. Positive control data generated using Rett isogenic control brain organoids expressing wild type MeCP2. Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated, or treated with DMSO vehicle. MeCP2 reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells. Each dot represents median reactivation rate computed across all fluorescence microscopy images from within a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.

[0185] FIG. 5 shows that compounds A, S and I induced MeCP2 protein expression following 14 day treatment, with compound I at 1 pM inducing the greatest MeCP2 protein expression by MeCP2 reactivation quantification. Positive control data generated using Rett isogenic control brain organoids expressing wild type MeCP2. Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated, or treated with DMSO vehicle. MeCP2 reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells. Each dot represents median reactivation rate computed across all fluorescence microscopy images from within a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.

[0186] FIG. 6 shows that compounds U, V and I induced MeCP2 protein expression following 14 day treatment, with compound U at 0.1 pM inducing MeCP2 protein expression at the lowest dose. Negative control data generated using Rett brain organoids expressing mutated MeCP2 treated with DMSO vehicle. MeCP2 reactivation rate quantified as the number of MeCP2 positive cells per 100 DAPI positive cells. Each dot represents median reactivation rate computed across all fluorescence microscopy images from within a well plated with 2-D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.

Survival metric

[0187] Survival was quantified based on estimates of percent cell survival in the treatment group relative to a negative control group (DMSO vehicle treated samples). Percent survival corresponds to the ratio of overall background-removed DAPI channel intensity from treatment and control groups, expressed as a percent.

[0188] FIG. 7 shows the quantified cytotoxicity of compound A treatment for 1 week and 2 weeks using percent survival relative to untreated or DMSO vehicle treated control using DAPI staining. No cytotoxicity at any efficacious dose was observed following 2 week treatment. Negative control data generated using Rett brain organoids expressing mutated MeCP2 and either untreated or treated with DMSO vehicle. Percent survival quantified as percent of background-removed DAPI intensity in treatment groups relative to negative control group. Each dot represents median reactivation rate computed across all single fluorescence microscopy images of a well plated with 2- D dissociated PDOs, then compound treated, fixed and immunolabeled prior to imaging.

[0189] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0190] It is to be understood that this disclosure is not limited to particular compositions and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

SEQUENCES

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A method of treating an X-chromosome-linked disease in a subject in need thereof, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor is a compound of Formula (I):

wherein:

Y is selected from -S-, -NH-, -NR^Z-, -O-, -S(O)-, and -S(O)2-;

R¹ is selected from:

R⁴ is selected from: hydrogen, C_1-6 alkyl, R^e, -COOR^a, -CONHR^a, and -CONR^bR^c; and cycloalkyl and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d; R⁵ is selected from:

R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, -CN, oxo, -OH, -0P(0)(0H)2, -COOH, -CONH₂, -NO2, -NH₂, -NH(Ci.₄alkyl), -N(H)R^e, -N(Ci-4alkyl)₂, -NR^eR^e, - SO2NH2, -S(O)₂CH₂CH₃, -S(O)2CH₂CH₂CH₃, -S(O)₂CH₃, -S(O)₂phenyl, and C1.4 alkoxy optionally substituted with 1 to 6 substituents independently selected from fluoro, oxo, -OH, -COOH, -NH2, and -CN; and aryl, heteroaryl, cycloalkyl, and heterocycloalkyl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^d; each R^d is independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, R^e, C(O)H, -C(O)R^ZZ, -OC(O)H, - CO(O)R^ZZ, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO₂NH₂, - S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, -N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, - C(O)NR^XR^X, -COOH, -COOR^X, oxo, -OH, -NH2, -NHR^X, -NR^XR^X, -NO₂, - CN, -NHC(O)NH₂, -NHC(O)NHR^X, -NHC(O)NR^XR^X, and Ci-₄ alkoxy optionally substituted with 1 to 4 times by fluoro; and aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and -Oaryl, each of which is optionally substituted with 1 to 4 substituents independently selected from R^x; and

2. The method of claim 1, wherein X¹ and X² are each independently selected from: H, -CN, fluoro, chloro, bromo, iodo, and methyl.

3. The method of any one of claims 1 to 2, wherein X¹ and X² are each -CN.

4. The method of any one of claims 1 to 3, wherein Y is -S-.

5. The method of any one of claims 1 to 4, wherein R¹ is C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, Ci-4 alkyloxy, -OH, -COOH, -NH₂, -NH(C I-4 alkyl), -N(CI-₄ alkyl)₂ and -CN.

6. The method of claim 5, wherein R¹ is ethyl.

7. The method of any one of claims 1 to 6, wherein R² is phenyl optionally substituted with one or more substituents independently selected from: fluoro, chloro, -CH₃, -CF₃, -C(O)phenyl, pyrrolidinyl, -P(O)(CH₃)₂, -C(O)NH₂, -

S(O)₂N(H)(CH₃), -OCH₂CH₂N(CH₃)₂, -CH₂C(O)NH₂, -NH-cyclopropyl, -N(CH₃)- cyclobutyl, -NH-oxetanyl, -N(R^ZZ)-S(O)₂R^X, -NH(Ci-5alkyl), -N(Ci-5alkyl)₂, S(O)₂CH₂CH₃, -S(O)₂CH₂CH₂CH₃, -S(O)₂CH₃, -SO₂NH₂, and -S(O)₂phenyl; and C_1-6 alkyl optionally substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, oxo, CM alkyloxy, -OH, -COOH, -NH₂, -NH(Ci-4 alkyl), -N(CI-4 alkyl)₂, -NHCOCH₂NH₂ and -CN.

8. The method of claim 7, wherein R² is selected from phenyl, -PhCH₃, -PhCF₃, -PhN(CH₃)- S(O)₂CH₃ and -PhCH₂NHCOCH₂NH₂.

9. The method of any one of claims 1 to 8, wherein R³ is hydrogen.

10. The method of any one of claims 1 to 9, wherein R⁴ is selected from hydrogen and -CONH₂.

11. The method of any one of claims 1 to 10, wherein R⁵ is selected from -NH₂, -NHR^a, and -

NR^bR^c .

12. The method of claim 11, wherein R⁵ is selected from -NH2,

13. The method of any one of claims 1 to 12, wherein each R^a is independently selected from Ci- 6 alkyl and R^e.

14. The method of any one of claims 1 to 13, wherein each R^b and R^c is independently selected from hydrogen, C_1-6 alkyl, and R^e.

15. The method of any one of claims 1 to 13, wherein R^b and R^c are taken together with the nitrogen to which they are attached, and optionally from 1 to 3 additional heteroatoms independently selected from O, N, and S, to form a heterocycloalkyl, which is optionally substituted with 1 to 5 substituents independently selected from: C_1-6 alkyl, R^e, and -NH2.

16. The method of any one of claims 1 to 15, wherein each R^d is independently selected from: fluoro, chloro, bromo, iodo, Ci-₆ alkyl, R^e, C(O)H, -C(O)R^ZZ, -OC(O)H, -CO(O)R^ZZ, -SH, -SR^X, - S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, -SO₂NH₂, -S(O)₂NHR^X, -S(O)₂NR^XR^X, -NHS(O)₂H, - NHS(0)2R^X, -N(R^ZZ)S(O)₂R^X, NHC(O)H, -NHC(O)R^X, -N(Me)C(O)R^x, -C(O)NH₂, -C(O)NHR^X, - C(O)NR^XR^X, -COOH, -COOR^X, oxo, -OH, -NH₂, -NHR^X, -NR^XR^X, -NO₂, -CN, -NHC(O)NH₂, - NHC(O)NHR^X, -NHC(O)NR^XR^X, and C1-4 alkoxy optionally substituted with 1 to 4 times by fluoro.

17. The method of claim 16, wherein each R^d is independently selected from: R^e and - N(R^ZZ)S(O)₂R^X.

18. The method of any one of claims 1 to 17, wherein each R^eis independently selected from: C1- 6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, chloro, bromo, iodo, C_1-6 alkyl, -SH, -SR^X, -S(O)H, -S(O)R^X, -S(O)₂H, -S(O)₂R^X, oxo, -OH, -NH₂, -NHR™, -NR^XR^X, - NHC(=N)NH₂, -COOH, -COOR^X, -C(O)NH₂, -C(O)NHR^X, -C(O)NR^XR^X, SO₂NH₂, -S(O)₂NHR^X, - S(O)₂NR^XR^X, -NHS(O)₂H, -NHS(O)₂R^X, -NHC(O)NHR^xp, -NHC(O)NR^xpR^xp, -NO2, and -CN.

19. The method of claim 18, wherein each R^eis independently selected from: C_1-6 alkyl substituted with 1 to 9 substituents independently selected from: fluoro, oxo, -OH, and -NHR^XX.

20. The method of any one of claims 1 to 19, wherein each R^x is independently selected from: C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: fluoro, oxo, - OH, -COOH, -NH₂ and -CN

21. The method of any one of claims 1 to 19, wherein each R™ is independently selected from C_1-6 alkyl optionally substituted with 1 to 6 substituents independently selected from: oxo, NR^x'R^xy, - COOH, and -CN

22. The method of any one of claims 1 to 21, wherein each R^xy- is hydrogen.

23. The method of any one of claims 1 to 8 and 13 to 22, wherein the DNA methyltransferase inhibitor is a compound of Formula (II):

or a pharmaceutically acceptable salt or prodrug thereof.

24. The method of claim 23, wherein the DNA methyltransferase inhibitor is a compound of

Formula (IIA):

or a pharmaceutically acceptable salt or prodrug thereof.

25. The method of claim 23 or claim 24, wherein R² is phenyl.

26. The method of any one of claims 23 to 25, wherein R⁶ and R⁷ are each C_1-6 alkyl.

27. The method of claim 26, wherein R⁶ and R⁷ are each methyl.

28. The method of claim 1, wherein the DNA methyltransferase inhibitor is selected from:

29. The method of any one of claims 1 to 28, wherein the method further comprises identifying the subject as having an X-chromosome genetic mutation prior to the administering of the DNA methyltransferase inhibitor to the subject.

30. The method of claim 29, wherein the X-chromosome genetic mutation is outside of the MeCP2 Exon 2 region on an X-chromosome of the subject.

31. The method of any one of claims 1 to 30, wherein the genetic mutation is a missense, nonsense, frameshift, insertion, deletion or a duplication mutation.

32. The method of claim 30 or claim 31, wherein the MeCP2 Exon 2 region contains no genetic mutations.

33. The method of claim 30 or claim 31, wherein the MeCP2 Exon 2 region contains mutations selected from: reference SNP rs267608409.

34. The method of any one of claims 1 to 33, wherein the X-chromosome-linked disease is selected from CDKL5 deficiency disorder, fragile x syndrome, Alport syndrome, X-linked Charcot- Marie-tooth disease, X-linked dominant porphyria, Vitamin D resistant rickets, Incontinentia pigmenti, CLCN4-related disorder, and facioscapulohumeral muscular dystrophy.

35. The method of claim 34, wherein the X-chromosome-linked disease is Rett Syndrome.

36. The method of any one of claims 1 to 35, wherein administering the DNA methyltransferase inhibitor induces wild-type MeCP2 expression.

37. The method of any one of claims 1 to 36, wherein the DNA methyltransferase inhibitor activates an inactive X-chromosome in the following in vitro assay:

(a) reprogramming X-chromosome linked disease fibroblasts into induced pluripotent stem cells;

(b) differentiating the induced pluripotent stem cells into brain organoids; (c) contacting said brain organoids in a vessel with a test DNA methyltransferase inhibitor, wherein the test DNA methyltransferase inhibitor is administered every other day for two weeks; and

(d) detecting MeCP2 levels in said vessel following step (c) and comparing the MeCP2 level to a MeCP2 level prior to said contacting in step (c), wherein when the MeCP2 levels are elevated by at least 1% and the test DNA methyltransferase inhibitor is a DNA methyltransferase inhibitor.

38. The method of claim 37, wherein the X-chromosome linked disease fibroblasts are Rett Syndrome fibroblasts.

39. A method of treating an X-chromosome-linked disease, comprising administering compound of Formula (I) or a salt thereof to a subject in need thereof over a period of two weeks or more, wherein the subject does not have cancer.

40. A method of reactivating an inactive X-chromosomes, comprising administering compound of Formula (I) or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of KCC2 by at least 10% for two weeks or more.

41. A method of reactivating an inactive X-chromosomes, comprising administering compound of Formula (I) or a salt thereof to a subject in need thereof in an amount sufficient to increase cerebrospinal fluid levels of brain-derived neurotrophic factor by at least 10% for two weeks or more.

42. A method of reactivating an inactive X-chromosomes, comprising administering compound of Formula (I) or a salt thereof to a subject in need thereof in an amount sufficient to reduce oxidative stress biomarkers by at least 10% for two weeks or more.

43. A method of reactivating inactive X-chromosomes, comprising administering compound of Formula (I) or a salt thereof to a subject in need thereof in an amount sufficient to reduce DNA methylation patterns in the subject’s blood by at least 1% for two weeks or more.

44. The method of claim 43, wherein DNA methylation patterns in the subject’s blood are decreased by at least 5% for two weeks or more.

45. The method of any one of claims 39 to 44, wherein the compound of formula (I) or salt thereof is administered daily, every other day or once weekly.

46. The method of any one of claims 39 to 45, wherein the subject has Rett Syndrome.

47. A method of treating an X-chromosome-linked disease in a subject, the method comprising: administering a DNA methyltransferase inhibitor to the subject, wherein the DNA methyltransferase inhibitor selectively inhibits DMNT1 over at least one of DMNT3A and DMNT3B.

48. The method of any one of claims 1 to 47, wherein the administering is by injection.

49. The method of claim 48, wherein the injection is directly into the cerebrospinal fluid of the subject.

50. The method of claim 48 or claim 49, wherein the administering is by intracerebroventricular injection (ICV).