Purine compound and use thereof

Abstract

Provided are a purine compound and the use thereof. The purine compound has a structure as represented by formula I, and the definition of each substituent is as described in the description and the claims. By means of specifically targeting ZMYND8 and inhibiting the activity thereof, the compound inhibits the transdifferentiation of prostate cancer to neuroendocrine tumors and the growth of prostate cancer, and can also inhibit the growth and progression of small cell lung cancer.

Description

A class of purine compounds and their applications Technical Field

[0001] The present invention belongs to the field of medicinal chemistry, and in particular relates to a class of purine compounds and applications thereof. Background Art

[0002] Prostate cancer and lung cancer, both adenocarcinomas, can transdifferentiate into poorly differentiated neuroendocrine (NE) tumors, namely neuroendocrine prostate cancer (NEPC) and small cell lung cancer (SCLC), after relapse or progression following targeted therapy. Despite their distinct tissue origins, NEPC and SCLC share highly similar genomic and transcriptomic alterations, displaying common features such as high expression of neuroendocrine markers and the acquisition of numerous genetic or epigenetic modifications. A key event in the development of neuroendocrine tumors is the nearly universal loss of the tumor suppressors RB1 and TP53. SCLC tumors can be divided into four molecular subtypes based on the expression of dominant transcription factors: ASCL1, NEUROD1, POU2F3, and YAP1, with ASCL1 being the most prevalent. In NEPC, ASCL1 and NEUROD1 subtypes have been described, but subtype analysis has not been routinely performed. This process of cell differentiation toward a neuroendocrine phenotype, termed neuroendocrine transdifferentiation, is an important mechanism of resistance to endocrine therapy and is clearly associated with poor clinical prognosis.

[0003] Disruptions in epigenetic regulation, primarily marked by covalent chromatin modification, can lock some important genes or entire gene networks into a heritable transcriptional aberration, playing a crucial role in the development and progression of tumors. Histone modifying enzymes and effector proteins often contain enzymatic or histone recognition domains, potentially enabling drug targeting. Therefore, genes associated with epigenetic modification have become popular targets in the field of tumor treatment. For example, the histone deacetylase inhibitors vorinostat and romidepsin have been approved by the FDA for the treatment of cutaneous T-cell lymphoma.

[0004] The histone modification recognition factor ZMYND8 contains a plant homeodomain (PHD), a bromodomain (BRD), a Pro-Trp-Trp-Pro domain (PWWP), and a MYND domain containing a zinc finger structure. The PHD, BRD, and PWWP domains are responsible for recognizing histone modifications such as H3K4me1 and H3K14ac, while MYND is mainly responsible for protein-protein interactions. ZMYND8 can recognize and bind to enhancers, and together with the transcription factor FOXM1, specifically promote the expression of neuroendocrine-related transcription factors, thereby promoting the formation and development of neuroendocrine tumors. Therefore, targeted inhibition of ZMYND8 may have the effect of selectively intervening in the growth and progression of neuroendocrine tumors, and may have the effect of treating NEPC and SCLC. However, there are currently no small molecule inhibitory drugs specifically targeting ZMYND8 on the market, and the field urgently hopes to discover highly selective small molecule inhibitors of ZMYND8.

[0005] Summary of the Invention

[0006] The present invention aims to provide a purine compound and its application. The compound can effectively inhibit ZMYND8 activity without obvious cytotoxicity to other tissue cells, thereby significantly inhibiting tumor growth without significant side effects.

[0007] In a first aspect of the present invention, there is provided a compound represented by general formula (I), a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug thereof,

[0008] in, Indicates a double bond or a single bond;

[0009] X is WR4, wherein W is selected from N, O, and S;

[0010] R1 is selected from the group consisting of: H, SF3, SF5, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-12 cycloalkyl, substituted or unsubstituted 5-8 membered heterocyclyl, substituted or unsubstituted C6-14 aryl, substituted or unsubstituted 5-8 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the group consisting of: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3 , SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclic group, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl, or any two substituents are linked to form a 5-8 membered aliphatic ring or 5-8 membered aromatic ring containing or not containing heteroatoms;

[0011] R2 is selected from: substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-12 cycloalkyl, substituted or unsubstituted 5-8 membered heterocyclyl, substituted or unsubstituted C6-14 aryl, substituted or unsubstituted 5-10 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3 -8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl, or any two substituents are linked to the attached carbon to form a 5-8 membered aliphatic ring or 5-8 membered aromatic ring containing or not containing heteroatoms;

[0012] R3 is none or selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-8 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C6-14 aryl, 5-8 membered heteroaryl;

[0013] R4 is none or selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-8 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following groups: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl.

[0014] In another preferred embodiment, R1 is selected from: H, SF3, SF5, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 5-6 membered heterocyclyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted 5-6 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the group consisting of D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF 3. SF5, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl, or any two substituents linked to form a 5-8 membered aliphatic ring or 5-8 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

[0015] In another preferred embodiment, R1 is selected from: H, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 5-6 membered heterocyclyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-6 cycloalkyl, cyano, nitro, C1-6 alkoxy, 5-6 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-4 alkyl-(C=O)-, C1-4 alkyl-(O=S=O)-, C1-4 alkyl-S-, C6-10 aryl, 5-6 membered heteroaryl, or any two substituents are connected to form a 5-6 membered aliphatic ring or 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

[0016] In another preferred embodiment, R2 is selected from: substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 5-6 membered heterocyclyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted 5-6 membered heteroaryl; wherein the substitution refers to substitution with one or more substituents selected from the group consisting of D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3 -6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-10 aryl, 5-8 membered heteroaryl, or any two substituents are linked to the attached carbon to form a 5-8 membered aliphatic ring or 5-8 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

[0017] In another preferred embodiment, R2 is selected from: substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-6 cycloalkyl, substituted or unsubstituted 5-6 membered heterocyclyl, substituted or unsubstituted C6-10 aryl, substituted or unsubstituted 5-6 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-6 cycloalkyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-4 alkyl-(C=O)-, C1-4 alkyl-(O=S=O)-, C1-4 alkyl-S-, C6-10 aryl, 5-8 membered heteroaryl, or any two substituents are connected to the connected carbon to form a 5-6 membered aliphatic ring or 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

[0018] In another preferred embodiment, R3 is none or selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-6 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-4 alkyl, SF3, SF5, C3-6 cycloalkyl, C2-4 alkenyl, C2-4 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-4 haloalkyl, C1-4 alkylamino, hydroxymethyl, C1-4 alkyl-(C=O)-, C6-10 aryl, 5-8 membered heteroaryl.

[0019] In another preferred embodiment, R3 is none or selected from: hydrogen, C1-6 alkyl, C3-6 cycloalkyl.

[0020] In another preferred embodiment, R4 is none or selected from: hydrogen, C1-6 alkyl, C3-6 cycloalkyl.

[0021] In another preferred embodiment, the compound has a structure shown in Formula II:

[0022] Wherein, X is O or S; R3 is selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, optionally substituted or unsubstituted C3-8 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following groups: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-8 cycloalkyl, SF3, SF5, C2-6 alkenyl, C2-6 alkynyl, cyano, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-10 aryl, 5-8 membered heteroaryl; the definitions of R1 and R2 are as described above.

[0023] In another preferred embodiment, the compound has a structure shown in Formula III:

[0024] Wherein, R3 is selected from: hydrogen, optionally substituted or unsubstituted alkyl, optionally substituted or unsubstituted cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following groups: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-8 cycloalkyl, SF3, SF5; the definitions of R1 and R2 are as described above.

[0025] In another preferred embodiment, R1 is selected from: H, C3-C8 cycloalkyl,

[0026] in, Indicates a double bond or a single bond;

[0027] W1 is selected from CRx or N; wherein Rx is H, D, halogen, nitro, hydroxy, cyano, C1-6 alkoxy, C1-6 alkyl, C1-6 alkyl-S-, C1-6 alkylamino, amino, C1-6 haloalkyl, C6-10 aryl, C1-6 alkyl-(O=S=O)-;

[0028] R 1a and R 1b Each is independently selected from H, D, halogen, hydroxy, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-14 aryl, 5-8 membered heteroaryl; or R 1a and R 1bConnected to the attached carbon to form a 5-6 membered aliphatic ring or a 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms;

[0029] Y is selected from NRy, O or S; Ry is H, C1-6 alkyl or C3-8 cycloalkyl;

[0030] R 1c Selected from H, D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-14 aryl, 5-8 membered heteroaryl.

[0031] In another preferred embodiment, R1 is selected from: H, C3-C6 cycloalkyl,

[0032] in, Indicates a double bond or a single bond;

[0033] W1 is selected from CRx or N; wherein Rx is H, D, halogen, nitro, hydroxy, cyano, C1-4 alkoxy, C1-4 alkyl, C1-4 alkyl-S-, C1-4 alkylamino, amino, C1-4 haloalkyl, C6-10 aryl, C1-4 alkyl-(O=S=O)-;

[0034] R 1a and R 1b Each is independently selected from H, D, halogen, hydroxy, amino, carboxyl, C1-4 alkyl, C3-6 cycloalkyl, cyano, nitro, C1-4 alkoxy, 5-6 membered heterocyclyl, C1-4 haloalkyl, C1-4 alkylamino, hydroxymethyl, C1-4 alkyl-(C=O)-, C1-4 alkyl-(O=S=O)-, C1-4 alkyl-S-, C6-10 aryl, 5-6 membered heteroaryl; or R 1a and R 1b It is connected to the carbon to form a 5-6 membered aliphatic ring or a 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

[0035] In another preferred embodiment, Y is selected from NRy, O or S; Ry is H, C1-4 alkyl or C3-6 cycloalkyl.

[0036] In another preferred embodiment, R 1cSelected from H, D, halogen, hydroxyl, amino, carboxyl, C1-4 alkyl, SF3, SF5, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-6 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-10 aryl, 5-6 membered heteroaryl.

[0037] In another preferred embodiment, R2 is C1-4 alkyl or

[0038] wherein W2 is selected from CRz or N; Rz is halogen, H, D, cyano, C1-4 alkyl, C1-4 alkoxy, phenyl, hydroxy, amino, carboxyl, nitro, C3-6 cycloalkyl, C1-4 haloalkyl, or C1-6 alkylamino;

[0039] R 2a and R 2b Each is independently selected from H, D, halogen, hydroxy, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-10 aryl, 5-8 membered heteroaryl; or R 2a and R 2b It is connected to the carbon to form a 5-6 membered aliphatic ring or a 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

[0040] In another preferred embodiment, R2 is C1-4 alkyl or

[0041] wherein W2 is selected from CRz or N; Rz is halogen, H, D, cyano, C1-4 alkyl, C1-4 alkoxy, nitro, hydroxy, amino, carboxyl, C3-6 cycloalkyl, C1-4 haloalkyl, or C1-6 alkylamino;

[0042] R 2a and R 2b Each is independently selected from H, D, halogen, hydroxy, amino, carboxyl, C1-4 alkyl, SF3, SF5, C3-6 cycloalkyl, C2-4 alkenyl, C2-4 alkynyl, cyano, nitro, C1-4 alkoxy, 5-6 membered heterocyclyl, C1-4 haloalkyl, C1-4 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, phenyl, 5-6 membered heteroaryl;

[0043] or R 2a and R2b Connected to the attached carbon to form a benzene ring or a 5-6 membered aliphatic ring containing 1, 2 or 3 oxygen atoms.

[0044] In another preferred embodiment, R2 is C1-4 alkyl, A substituted or unsubstituted benzene ring or naphthalene ring; the substitution means that the hydrogen of the benzene ring is replaced by 1, 2 or 3 substituents selected from the following groups: halogen, D, cyano, C1-4 alkyl, C1-4 alkoxy, phenyl, nitro, hydroxy, amino, carboxyl, C3-6 cycloalkyl, C1-4 haloalkyl, C1-6 alkylamino.

[0045] In another preferred embodiment, R3 is none, H, C1-4 alkyl or C3-C6 cycloalkyl.

[0046] In another preferred embodiment, the compound is selected from the following group: Compound 1-Compound 63.

[0047] The second aspect of the present invention provides a pharmaceutical composition comprising:

[0048] The compound of the first aspect, a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug thereof; and a pharmaceutically acceptable carrier.

[0049] The third aspect of the present invention provides the compound described in the first aspect, its pharmaceutically acceptable salt, or its enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug or the use of the pharmaceutical composition of the third aspect for preparing a ZMYND8 small molecule inhibitor; or for preparing a drug for treating neuroendocrine tumors.

[0050] In another preferred embodiment, the neuroendocrine tumor is selected from the group consisting of neuroendocrine prostate cancer and small cell lung cancer.

[0051] The advantages of the present invention are:

[0052] 1. The small molecule compounds of the present invention (such as iZMYND8-34) specifically inhibit neuroendocrine prostate cancer and small cell lung cancer.

[0053] 2. The small molecule compound iZMYND8-34 had no obvious toxic side effects on mice at a dosage concentration of 50 mg / kg.

[0054] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equal, or similar purpose. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] FIG1 shows that overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy.

[0056] FIG2 shows that ZMYND8 knockout in neuroendocrine prostate cancer organoids, cell lines, and small cell lung cancer cells results in reduced tumor growth.

[0057] FIG3 shows that iZMYND8-34 directly binds to ZMYND8 and inhibits the interaction of ZMYND8 with H3K4me1-H3K14ac.

[0058] FIG4 shows that iZMYND8-34 has no obvious toxic effect on the body.

[0059] FIG5 shows that iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer.

[0060] FIG6 shows that iZMYND8-34 specifically inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer. DETAILED DESCRIPTION

[0061] After extensive and in-depth research, the inventors of this application have developed a class of purine compounds that effectively inhibit neuroendocrine transdifferentiation in adenocarcinomas, thereby preventing further malignant progression. Specifically, they specifically target the histone-associated molecule ZMYND8 and inhibit its activity, thereby specifically inhibiting the progression of neuroendocrine tumors. Based on this, the present invention was completed.

[0062] the term

[0063] In the present invention, the halogen is F, Cl, Br or I.

[0064] In the present invention, unless otherwise specified, the terms used have the general meanings commonly known to those skilled in the art.

[0065] In the present invention, the term "C 1-6 ” means having 1, 2, 3, 4, 5 or 6 carbon atoms, “C 3-8 ” means having 3, 4, 5, 6, 7 or 8 carbon atoms, and so on. “5-8 membered” means having 5, 6, 7 or 8 ring atoms, and so on. 1-4 means 1, 2, 3 or 4; 1-6 means 1, 2, 3, 4, 5 or 6, and so on.

[0066] In the present invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon moiety. For example, the term "C1-C6 alkyl" refers to a straight or branched alkyl group having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl and hexyl, etc.; preferably ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0067] In the present invention, the term "alkoxy" refers to -O-(alkyl), where alkyl is as defined above. For example, the term "C1-C6 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy, and butoxy.

[0068] In the present invention, the term "alkenyl" refers to a straight chain or branched hydrocarbon moiety containing at least one double bond. For example, the term "C2-C6 alkenyl" refers to a straight chain or branched alkenyl group containing one double bond having 2 to 6 carbon atoms, including but not limited to ethenyl, propenyl, butenyl, isobutenyl, pentenyl and hexenyl.

[0069] In the present invention, the term "alkynyl" refers to a straight or branched chain alkynyl group containing a triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl.

[0070] In the present invention, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon moiety. For example, the term "C3-C8 cycloalkyl" refers to a cyclic alkyl group having 3 to 8 carbon atoms in the ring, including but not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc. The term "C3-C6 cycloalkyl" has a similar meaning.

[0071] In the present invention, the term "aryl" refers to a hydrocarbon moiety containing one or more aromatic rings. For example, the term "C6-C 14 "Aryl" refers to an aromatic ring group having 6 to 14 carbon atoms and no heteroatoms in the ring, such as phenyl, naphthyl, etc. The term "C6-C12 aryl" has a similar meaning. Examples of aryl include, but are not limited to, phenyl (Ph), naphthyl, pyrenyl, anthracenyl, and phenanthrenyl.

[0072] In the present invention, the term "heterocyclyl" refers to a saturated or unsaturated, non-aromatic cyclic group containing at least one (such as 1, 2, 3 or 4) ring heteroatom (such as N, O or S), such as tetrahydropyridyl, pyrrolinyl, dihydropyridyl, dihydrofuranyl, dihydrothiophenyl, morpholinyl.

[0073] In the present invention, the term "heteroaryl" refers to an aromatic cyclic group containing at least one (such as 1, 2, 3 or 4) ring heteroatom (such as N, O or S), such as furyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, quinolyl, isoquinolyl, indolyl, pyrimidinyl, pyranyl.

[0074] In the present invention, alkylamino refers to monoalkylamino (NH(alkyl)) or dialkylamino (N(alkyl)(alkyl)). For example, C1-6 alkylamino refers to NH(C1-6 alkyl), N(C1-6 alkyl)(C1-6 alkyl), including NHCH3, NHCH2CH3, N(CH3)2, N(CH2CH3)2, N(CH3)(CH2CH3), etc.

[0075] In the present invention, the heteroatom refers to N, O, S or P.

[0076] Unless otherwise specified, the alkyl, alkoxy, cycloalkyl, heteroaryl, heterocyclyl and aryl groups described herein are substituted and unsubstituted groups. Possible substituents on alkyl, alkoxy, cycloalkyl, heterocyclyl and aryl groups include, but are not limited to, hydroxy, amino, nitro, nitrile, halogen, C1-C6 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocycloalkyl, C1-C20 heterocycloalkenyl, C1-C6 alkoxy, C6-C10 aryl, heteroaryl, heteroaryloxy, C1-C10 alkylamino, C1-C20 dialkylamino, C6- C10 arylamino, diC6-C10 arylamino, C1-C10 alkylsulfamoyl, C6-C10 arylsulfamoyl, C1-C10 alkylimino, C1-C10 alkylsulfoimino, C6-C10 arylsulfoimino, mercapto, C1-C10 alkylthio, C1-C10 alkylsulfonyl, C6-C10 arylsulfonyl, acylamino, aminoacyl, aminothioacyl, guanidino, ureido, cyano, acyl, thioacyl, acyloxy, carboxyl, and carboxylate groups. On the other hand, cycloalkyl, heterocycloalkyl, heterocycloalkenyl, aryl, and heteroaryl groups may be fused to each other.

[0077] In the present invention, the substitution is mono- or poly-substituted, and the poly-substituted is di-, tri-, tetra-, or penta-substituted. The di-substituted refers to having two substituents, and so on.

[0078] Pharmaceutically acceptable salts of the present invention can be salts formed by positively charged groups on anions and compounds of formula I. Suitable anions are chloride, bromide, iodide, sulfate, nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate, acetate, malate, toluenesulfonate, tartrate, fumarate, glutamate, glucuronide, lactate, glutarate or maleate. Similarly, salts can be formed by negatively charged groups on cations and compounds of formula I. Suitable cations include sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, such as tetramethylammonium ion.

[0079] In another preferred embodiment, "pharmaceutically acceptable salts" refer to salts formed by the compound of formula I with an acid selected from the group consisting of hydrofluoric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, oxalic acid, sulfuric acid, nitric acid, methanesulfonic acid, aminosulfonic acid, salicylic acid, trifluoromethanesulfonic acid, naphthalenesulfonic acid, maleic acid, citric acid, acetic acid, lactic acid, tartaric acid, succinic acid, oxalic acid, pyruvic acid, malic acid, glutamic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalenedisulfonic acid, malonic acid, fumaric acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonic acid, 2-acetoxybenzoic acid and isethionic acid; or sodium salts, potassium salts, calcium salts, aluminum salts or ammonium salts formed by the compound of formula I with an inorganic base; or methylamine salts, ethylamine salts or ethanolamine salts formed by the compound of general formula I with an organic base.

[0080] Pharmaceutical composition

[0081] The present invention also provides a pharmaceutical composition comprising an active ingredient within a safe and effective amount and a pharmaceutically acceptable carrier.

[0082] The "active ingredient" of the present invention refers to the compound of formula I of the present invention.

[0083] The active ingredient and pharmaceutical composition of the present invention are used to prepare a small molecule inhibitor of ZMYND8 and a drug for treating tumors selected from the group consisting of neuroendocrine prostate cancer and small cell lung cancer.

[0084] A "safe and effective amount" refers to an amount of the active ingredient sufficient to significantly improve the condition without causing serious side effects. Typically, a pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably 10-200 mg per dose. Preferably, "one dose" is one tablet.

[0085] "Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the active ingredients of the present invention and with each other without significantly reducing the efficacy of the active ingredients. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0086] There is no particular limitation on the administration of the active ingredient or pharmaceutical composition of the present invention. Representative administration methods include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous) and the like.

[0087] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.

[0088] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active ingredient, the liquid dosage form can comprise the inert diluents conventionally employed in this area, such as water or other solvents, solubilizing agents and emulsifying agents, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethyl formamide and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances etc. In addition to these inert diluents, the composition can also comprise adjuvants, such as wetting agents, emulsifying agents and suspending agents, sweeteners, flavorings and spices.

[0089] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0090] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0091] The compounds of the present invention can be administered alone or in combination with other therapeutic drugs (such as anti-tumor drugs).

[0092] When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is administered to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage will also take into account factors such as the route of administration and the patient's health condition, all of which are within the skill of a skilled physician.

[0093] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions (such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989)) or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0094] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those familiar to those skilled in the art. Furthermore, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0095] preparation

[0096] Route 1:

[0097] Route 2:

[0098] Among them, the synthesis of compounds 1-56 refers to route 1, and the synthesis of compounds 57-63 refers to route 2.

[0099] The preparation of representative compounds is as follows. Other compounds can be prepared by replacing the starting materials and using similar methods with reference to the above routes.

[0100] Preparation Example 1: Synthesis of Compound 1

[0101] Step 1: Synthesis of intermediate 1b:

[0102] To a 100ml eggplant-shaped flask, diaminomaleonitrile (0.540g, 5.00mmol) and compound 1a (0.856g, 5.25mmol) were added, followed by anhydrous tetrahydrofuran (15ml). The mixture was stirred at room temperature for 24 hours. After the reaction, the solvent was dried, and the solid was washed with glacial ethanol (10ml). The solid was filtered, and the filter cake was washed with glacial ethanol (10ml) and then glacial ether (10ml). The filter cake was dried under vacuum at 60°C for two hours to obtain 1.200g of a brownish-gray solid product (88.6% yield).

[0103] Step 2: Synthesis of compound 1:

[0104] To a 100ml eggplant-shaped flask, compound 1b (0.500g, 1.85mmol) and 3-ethoxysalicylaldehyde (0.628g, 3.78mmol) were added, along with methanol (15ml) and triethylamine (0.196g, 1.94mmol). After stirring at room temperature for 1 hour, the reaction solution became clear. Stirring was continued at room temperature for 24 hours, during which time the product gradually precipitated. After the reaction, the mixture was filtered directly, and the filter cake was dissolved in dimethyl sulfoxide (5ml). Glacial ethanol (50ml) was added, resulting in the precipitation of a solid. This was filtered, and the filter cake was vacuum-dried at 60°C for two hours to yield 0.408g of a black solid product (50.8% yield).

[0105] Preparation Example 2: Synthesis of Compound 21

[0106] Step 1: Synthesis of intermediate 21b

[0107] Same synthesis as intermediate 1b.

[0108] Step 2: Synthesis of compound 21

[0109] To a 100ml eggplant-shaped flask, compound 21b (0.500g, 1.85mmol) and 4-nitrobenzaldehyde (0.571g, 3.78mmol) were added, along with methanol (15ml) and triethylamine (0.196g, 1.94mmol). After stirring at room temperature for 1 hour, the reaction solution became clear. Stirring was continued at room temperature for 24 hours, during which time the product gradually precipitated. After the reaction, the mixture was filtered directly, and the filter cake was dissolved in dimethyl sulfoxide (5ml). Glacial ethanol (50ml) was added, resulting in the precipitation of a solid. The filter cake was then filtered and dried under vacuum at 60°C for two hours to yield 0.676g of a tan solid product (87.2% yield).

[0110] Preparation Example 3: Synthesis of Compound 61

[0111] Step 1: Synthesis of intermediate 61b

[0112] Synthesis of the same intermediate 1b

[0113] Step 2: Synthesis of intermediate 61c

[0114] Same synthesis as compound 21

[0115] Step 3: Synthesis of intermediate 61d

[0116] Compound 61c (0.100 g, 0.238 mmol) and phosphorus oxychloride (3 ml) were added to a 50 ml eggplant-shaped flask and stirred at 100°C for 12 hours. After the reaction, the reaction solution was added dropwise to ice water (50 ml) with stirring. The mixture was stirred for 30 minutes after the addition was complete. Ethyl acetate (20 ml x 3) was added for extraction. The organic phases were separated, combined, washed with water (20 ml x 3), and concentrated under reduced pressure. Column chromatography (ethyl acetate:petroleum ether = 1:1) afforded 0.055 g of a yellow solid, with a yield of 57.5%.

[0117] Step 4: Synthesis of intermediate 61e

[0118] To a 50 ml eggplant-shaped flask, compound 61d (0.110 g, 0.274 mmol) and cesium carbonate (0.178 mg, 0.547 mmol) were added, along with N,N-dimethylformamide (3 ml) and iodomethane (0.058 g, 0.410 mmol). The mixture was stirred at room temperature for 12 hours. After completion of the reaction, water (10 ml) was added, and the mixture was extracted with ethyl acetate (20 ml x 3). The organic phases were separated, combined, washed with water (20 ml x 3), and concentrated under reduced pressure. Column chromatography (ethyl acetate:petroleum ether = 1:3) afforded 0.084 g of a yellow solid, yielding 73.7%.

[0119] Step 5: Synthesis of Compound 61

[0120] Compound 61e (0.050 g, 0.120 mmol) and cesium carbonate (0.118 mg, 0.361 mmol) were added to a 50 ml eggplant-shaped flask. Dimethyl sulfoxide (3 ml) was then added, followed by 100 μl of 30% hydrogen peroxide in an ice bath. The mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was filtered and the filter cake was dried under vacuum at 60°C for 2 hours to obtain 0.039 g of a yellow solid product in a yield of 75.1%.

[0121] Preparation Example 4: Synthesis of Compound 63

[0122] Step 1: Synthesis of intermediate 63b

[0123] To a 100 ml eggplant-shaped flask, diaminomaleonitrile (0.540 g, 5.00 mmol) and compound 63a (0.719 g, 5.25 mmol) were added, followed by anhydrous tetrahydrofuran (15 ml) and stirred at room temperature for 24 hours. After the reaction, the solvent was dried, and the solid was washed with glacial ethanol (10 ml) and filtered. The filter cake was then washed with glacial ethanol (10 ml) and glacial ether (10 ml). The filter cake was dried under vacuum at 60°C for two hours to obtain 1.087 g of a brown solid product (88.7% yield).

[0124] Step 2: Synthesis of intermediate 63c

[0125] To a 100ml eggplant-shaped flask, compound 63b (0.500g, 2.04mmol) and 4-nitrobenzaldehyde (0.632g, 4.18mmol) were added, along with methanol (15ml) and triethylamine (0.216g, 2.14mmol). After stirring at room temperature for 1 hour, the reaction solution became clear. Stirring was continued at room temperature for 24 hours, during which time the product gradually precipitated. After the reaction, the mixture was filtered directly, and the filter cake was dissolved in dimethyl sulfoxide (5ml). Glacial ethanol (50ml) was added, resulting in the precipitation of a solid. The filter cake was then filtered and dried under vacuum at 60°C for two hours to yield 0.544g of a tan solid product in a 67.7% yield.

[0126] Step 3: Synthesis of intermediate 63d

[0127] Compound 63c (0.100 g, 0.254 mmol) and phosphorus oxychloride (3 ml) were added to a 50 ml eggplant-shaped flask and stirred at 100°C for 12 hours. After the reaction, the reaction solution was added dropwise to ice water (50 ml) with stirring. The mixture was stirred for 30 minutes after the addition was complete. Ethyl acetate (20 ml x 3) was added for extraction. The organic phases were separated, combined, washed with water (20 ml x 3), and concentrated under reduced pressure. Column chromatography (ethyl acetate:petroleum ether = 1:1) afforded 0.092 g of a yellow solid, with a yield of 96.3%.

[0128] Step 4: Synthesis of intermediate 63e

[0129] To a 50 ml eggplant-shaped flask, compound 63d (0.100 g, 0.266 mmol) and cesium carbonate (0.173 mg, 0.532 mmol) were added, along with N,N-dimethylformamide (3 ml) and iodomethane (0.057 g, 0.399 mmol). The mixture was stirred at room temperature for 12 hours. After the reaction, water (10 ml) was added, and the mixture was extracted with ethyl acetate (20 ml x 3). The organic phases were separated, combined, washed with water (20 ml x 3), and concentrated under reduced pressure. Purification by column chromatography (ethyl acetate:petroleum ether = 1:3) afforded 0.093 g of a yellow solid, yielding 89.6%.

[0130] Step 5: Synthesis of compound 63

[0131] Compound 63e (0.090 g, 0.231 mmol) and cesium carbonate (0.226 mg, 0.692 mmol) were added to a 50 ml eggplant-shaped flask. Dimethyl sulfoxide (3 ml) was then added, followed by 100 μl of 30% hydrogen peroxide in an ice bath. The mixture was stirred at room temperature for 1 hour. After the reaction, the mixture was filtered and the filter cake was dried under vacuum at 60°C for 2 hours to obtain 0.082 g of a yellow solid product in an 87.0% yield.

[0132] Table 1 Structure and characterization of the compounds in the examples

[0133] Experimental Example 1 Overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy

[0134] 1. In vitro, overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy

[0135] Experimental Methods: ZMYND8-OE overexpressing plasmids were transfected into androgen-sensitive prostate cancer cell lines (LNCaP, VCaP, and MycCaP cells) to construct stable overexpressing lines as experimental groups (ZMYND8-OE). A control group (CTRL) was also transfected with a blank vector. After stable lines were established, equal numbers of cells were plated in 96-well plates, with 3-6 replicates per group. Enzalutamide (ENZ) was then used to simulate androgen deprivation therapy (MDT) treatment. During culture, viable cell counts were assessed using MTS reagent (100:1 volume ratio) in the culture medium. After a 2-hour incubation, the OD value at 490 nm was measured using a microplate reader. The OD value reflects the viable cell count. Day 0 was defined as the time cells first attached to the culture medium. Thereafter, viable cell counts were measured every 2-3 days. Changes in cell number were recorded for 5-7 days to assess changes in drug resistance.

[0136] Experimental results: As shown in Figures 1A to C, compared with the control, the ZMYND8-overexpressing cell line grew faster during androgen deprivation therapy and exhibited stronger androgen deprivation resistance. This result indicates that ZMYND8 overexpression promotes androgen deprivation resistance in androgen-sensitive prostate cancer cell lines.

[0137] 2. In vivo experiments, ZMYND8 overexpression in androgen-sensitive prostate cancer cell lines promoted resistance to androgen deprivation therapy

[0138] Experimental methods: ZMYND8-overexpressing stable cells were constructed using androgen-sensitive prostate cancer cell lines 22RV1 and MycCaP as the experimental group (ZMYND8-OE), and empty vector-constructed stable cells were used as the control group (CTRL). The constructed cells were injected subcutaneously into 8-10 week-old nude mice in equal amounts. Seven days after injection, castration and enzalutamide (ENZ) were co-treated (30 mg / kg / time, 3 times / week) to simulate androgen deprivation therapy. Thereafter, the size of the subcutaneous tumor was measured with a vernier caliper every 7 days, and the size of the tumor was calculated based on the 0.5×(length×width) value. 2 Tumor volume was calculated using the formula ( ). Changes in tumor size were recorded, and differences in tumor growth were statistically calculated.

[0139] Experimental Results: As shown in Figure 1D and E, ZMYND8-overexpressing cell lines injected subcutaneously into nude mice grew faster during androgen deprivation therapy compared to the control group and were less sensitive to androgen deprivation therapy. These results indicate that in vivo, overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy.

[0140] Experimental Example 2: ZMYND8 knockout in neuroendocrine prostate cancer organoids, cell lines, and small cell lung cancer cells leads to reduced tumor growth

[0141] 1. Knockout of ZMYND8 in neuroendocrine prostate cancer organoids inhibits tumor growth

[0142] Experimental methods: Tumor-derived primary neuroendocrine tumor organoids (ST88) and mouse prostate neuroendocrine tumor primary cells (selected from Probasin Cre / + ;Pten flox / flox ;Tp53 flox / flox ;Rb1 flox / floxIn a triple-knockout mouse model (Pten; Tp53; Rb1 TKO), Zmynd8 knockdown (shZmynd8-1, shZmynd8-2) or knockout (ZMYND8-sgRNA1, ZMYND8-sgRNA2) stable transgenic lines were constructed as experimental groups, and an empty vector-constructed stable transgenic line was transferred into the corresponding model as a control group (EV, CTRL-sgRNA). Protein immunoblotting was first used to verify the changes in protein levels to ensure that the expression of Zmynd8 in the control group in the organoid model had been reduced. By taking pictures under a microscope and measuring and comparing the changes in organoid size and formation rate using ImageJ, the regulatory role of ZMYND8 on the growth of neuroendocrine prostate cancer organoids was clarified.

[0143] Experimental results: As shown in Figure 2 AD, knocking down or knocking out ZMYND8 in primary mouse prostate cancer cells (Pten; Tp53; Rb1 TKO) and primary human neuroendocrine prostate cancer cells ST88 significantly inhibited the organoid formation ability of neuroendocrine cells.

[0144] 2. Knockout of ZMYND8 in neuroendocrine prostate cell lines and small cell lung cancer cell lines inhibits tumor growth

[0145] Experimental Methods: ZMYND8 knockout plasmids were transfected into the neuroendocrine prostate cancer cell lines NE1.3 and NCI-H660, and the small cell lung cancer cell line H82 to create stable knockout lines (ZMYND8-KO1 and ZMYND8-KO2). A control group (CTRL) was transfected with a blank vector. Once stable lines were established, equal numbers of cells from each group were plated in 96-well plates, with 3-6 replicates per group. During culture, MTS reagent was added to the culture medium at a 100:1 ratio by volume. After a 2-hour incubation, the OD value at 490 nm was measured using a microplate reader. The OD value reflects the number of viable cells. Day 0 was defined as the time cells first attached to the plate. Thereafter, the viable cell count was measured every 2-3 days. Changes in cell number were recorded continuously for 5-7 days to analyze changes in cell growth.

[0146] Experimental results: As shown in Figure 2E, knockout of ZMYND8 can significantly inhibit tumor growth in both neuroendocrine prostate cancer cell lines and small cell lung cancer cell lines.

[0147] 3. Knockout of ZMYND8 in androgen-sensitive cell lines and non-small cell lung cancer cell lines does not affect cell growth

[0148] Experimental Methods: ZMYND8 knockout plasmids were transfected into the androgen-sensitive cell lines LNCaP and LAPC4, as well as the non-small cell lung cancer cell line A549, to construct stable knockout lines (ZMYND8-KO1 and ZMYND8-KO2) for experimental groups. A control group (CTRL) was transfected with a blank vector. Once stable lines were constructed, equal numbers of cells from each group were plated in 96-well plates, with 3-6 replicates per group. During culture, MTS reagent was added to the culture medium at a 100:1 ratio by volume. After a 2-hour incubation, the OD value at 490 nm was measured using a microplate reader. The OD value reflects the number of viable cells. Day 0 was defined as the time cells first attached to the culture medium. Thereafter, the viable cell count was measured every 2-3 days. Changes in cell number were recorded continuously for 5-7 days to analyze changes in cell growth.

[0149] Experimental results: As shown in Figure 2F, knockout of ZMYND8 did not affect tumor growth in androgen-sensitive cell lines and non-small cell lung cancer cell lines.

[0150] These results indicate that ZMYND8 knockout in neuroendocrine prostate cancer organoids, cell lines, and small cell lung cancer cells leads to reduced tumor growth, and that the function of ZMYND8 is specific in neuroendocrine tumors and small cell lung cancer.

[0151] Experimental Example 3 iZMYND8-34 directly binds to ZMYND8 and inhibits the interaction between ZMYND8 and H3K4me1-H3K14ac

[0152] 1. Surface Plasmon Resonance (SPR) Analysis of the Binding of iZMYND8-34 to ZMYND8 Protein

[0153] Experimental method: The SPR experiment was performed on Biacore 8K (GE Healthcare) using HBS (10mM HEPES, pH 7.4, 150mM NaCl) as the buffer. ZMYND8 protein (aa103-426) was covalently coupled to the CM5 chip using a standard amino coupling procedure in the presence of 10mM sodium acetate (pH 4.0). iZMYND8-34 was diluted to 5, 1.2, 0.8, 0.6, and 0.4 μM with HBS buffer. During the analysis, the sample was injected at a flow rate of 30 μL / min for 120 seconds, then dissociated for 180 seconds, and the response signal was recorded over time during the process. After the experiment, the equilibrium dissociation constant (K) of iZMYND8-34 and ZMYND8 protein was calculated using the steady-state analysis module in the Biacore 8K evaluation software (GE Healthcare). Dvalue).

[0154] Experimental results: As shown in Figure 3A, SPR experiments showed that the small molecule compound iZMYND8-34 directly bound to the ZMYND8 protein, and its equilibrium dissociation constant (K D value) was 2.38 μM.

[0155] 2. Homogeneous Time Resolved Fluorescence (HTRF) Analysis of iZMYND8-34 Disruption of the Interaction between ZMYND8 Protein and H3K4me1-H3K14ac

[0156] Experimental method: GST-ZMYND8 protein (aa103-426) and Biotin-labeled polypeptide (ART{Lys(Me)}QTARKSTGG{Lys-Ac}APRKQL) were diluted to 40 nM and 2 μM respectively with HTRF buffer (20 mM HEPES, pH 7.4, 10 mM NaCl, 0.01% BSA). The experiment was performed in a white 384-well plate (6007290, PerkinElmer). 1 μL of compound and 9 μL of GST-ZMYND8 protein were added to the plate (1 μL of DMSO and 9 μL of HTRF buffer were added to the control group) and incubated at room temperature for 15 minutes. Then, 5 μL of biotin-labeled substrate peptide was added and incubated at room temperature for 15 minutes. Finally, 5 μL of anti-GST-Tb (Cisbio, 61GSTTLB) and anti-streptavidin-XL665 (Cisbio, 610SAXLA) mixture was added. After incubation at room temperature for 30 minutes, the HTRF signal value was detected at 620 nm and 665 nm using a TECAN SPARK microplate reader.

[0157] Experimental results: As shown in Figure 3B, HTRF experiments showed that the small molecule compound iZMYND8-34 can inhibit the binding of ZMYND8 protein to H3K4me1-H3K14ac in a concentration-dependent manner, with an IC 50 The value is 0.66μM.

[0158] Experimental Example 4 Toxicity Analysis of Small Molecule Compound iZMYND8-34

[0159] Experimental Methods: 8-10 week-old WT mice were intraperitoneally administered iZMYND8-34 or a DMSO control at a dose of 50 mg / kg once daily for 14 days. Body weights were measured daily. On day 14, blood was collected and serum levels of alanine aminotransferase (ALT, reflecting liver toxicity) and alkaline phosphatase (ALP, reflecting kidney toxicity) were analyzed by ELISA.

[0160] Experimental Results: As shown in Figure 4A, treatment with iZMYND8-34 at a concentration of 50 mg / kg did not significantly change the body weight of mice. As shown in Figures 4B and 4C, treatment with iZMYND8-34 at a concentration of 50 mg / kg did not significantly increase the levels of ALT and ALP in the mouse serum. These results indicate that iZMYND8-34 treatment did not cause significant toxic effects in mice.

[0161] Experimental Example 5 iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer

[0162] 1. Inhibitory effect of iZMYND8-34 on neuroendocrine prostate cancer PDX

[0163] Experimental method: 8-10 week old NSG mice were subcutaneously inoculated with neuroendocrine prostate cancer PDX LTL-545 and LTL-651. When the tumors grew to a measurable size (approximately 80 mm 3 ), the experimental group was given iZMYND8-34 intraperitoneally at a dose of 50 mg / kg daily, and the control group was injected with an equal volume of PBS. The tumor size was measured every 5-7 days, and the tumor growth curve was drawn.

[0164] Experimental results: As shown in Figure 5A, iZMYND8-34 significantly inhibited the growth of various tumors, indicating that the ZMYND8 inhibitor can inhibit the growth of neuroendocrine prostate cancer PDX. This indicates that iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer PDX.

[0165] 2. iZMYND8-34 inhibits tumor formation in genetically engineered mice with neuroendocrine prostate cancer

[0166] Experimental Methods: 6-week-old triple-knockout neuroendocrine prostate cancer genetic mice were used to Cre / + ;Pten flox / flox ;Tp53 flox / flox ;Rb1 flox / flox iZMYND8-34 was administered intraperitoneally daily at a dose of 50 mg / kg for 4-6 weeks. The prostates of the mice were harvested and stained with H&E to analyze changes in the malignancy of prostate cancer. Immunohistochemical staining for the neuroendocrine marker protein Syp was also performed, and differences in marker protein expression were analyzed.

[0167] Experimental results: As shown in Figure 5B, in the genetic mouse model, the malignancy of the tumor was weakened after treatment with iZMYND8-34, and the expression level of the neuroendocrine prostate cancer-specific expression molecule Syp was reduced, indicating that iZMYND8-34 inhibits tumor formation in genetic mice with neuroendocrine prostate cancer.

[0168] 3. iZMYND8-34 inhibits the formation of small cell lung cancer xenografts

[0169] Experimental method: Small cell lung cancer cells H82 or H209 were injected subcutaneously into 8-week-old nude mice, with 10 cells per tumor. 6 After injection, the experimental group was given iZMYND8-34 at a concentration of 50 mg / kg for intraperitoneal administration, while the control group was injected with an equal volume of PBS. The size of the subcutaneous tumor was measured with a vernier caliper every 7 days. 2 ) formula to calculate the tumor volume.

[0170] Experimental results: As shown in Figure 5C, after treatment with iZMYND8-34, the tumorigenicity of small cell lung cancer cells was weakened and tumor growth was significantly slowed, indicating that iZMYND8-34 inhibits the formation and growth of small cell lung cancer.

[0171] The above experiments show that the small molecule inhibitor iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer.

[0172] Example 6 iZMYND8-34 specifically inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer

[0173] 1. iZMYND8-34 has no inhibitory effect on androgen-sensitive prostate cancer and non-small cell lung cancer cell lines

[0174] Experimental Methods: The effect of iZMYND8-34 on cell growth was assessed using the androgen-sensitive prostate cancer cell line LNCaP and the non-small cell lung cancer cell line A549. The experimental group received 1 μM iZMYND8-34 in the culture medium, while the control group received an equal volume of DMSO. Equal numbers of cells were plated in 96-well plates, with 3-6 replicates per group. During the culture period, MTS reagent was added to the culture medium at a 100:1 ratio. After a 2-hour incubation, the OD value at 490 nm was measured using a microplate reader. The OD value reflects the number of viable cells. Day 0 was defined as the time when cells first attached to the culture medium. Thereafter, the viable cell count was measured every 2-3 days. Changes in cell number were recorded continuously for 5-7 days to analyze changes in cell growth.

[0175] Experimental results: As shown in Figure 6A and B, iZMYND8-34 did not affect the growth of the androgen-sensitive prostate cancer cell line LNCaP and the non-small cell lung cancer cell line A549, indicating that iZMYND8-34 has no inhibitory effect on androgen-sensitive prostate cancer and non-small cell lung cancer cell lines.

[0176] 2. Inhibitory effect of iZMYND8-34 on neuroendocrine prostate cancer and small cell lung cancer

[0177] Experimental Methods: The effects of iZMYND8-34 on cell growth were assessed using neuroendocrine prostate cancer cell lines NE1.3 and NCI-H660, and small cell lung cancer cell lines H82 and H209. Experimental groups were treated with 1 μM iZMYND8-34 in the culture medium, while controls were treated with an equal volume of DMSO. Equal numbers of cells were plated in 96-well plates, with 3-6 replicates per group. During culture, viable cell counts were assessed using MTS reagent (100:1 volume ratio). After a 2-hour incubation, the OD at 490 nm was measured using a microplate reader to measure viable cell counts. Day 0 was defined as the time cells first attached to the culture medium. Thereafter, viable cell counts were assessed every 2-3 days. Changes in cell number were recorded continuously for 5-7 days to assess cell growth.

[0178] Experimental results: As shown in Figure 6C and D, iZMYND8-34 significantly inhibited the growth of neuroendocrine prostate cancer cell lines NE1.3 and NCI-H660 and small cell lung cancer cell lines H82 and H209.

[0179] The above experiments show that iZMYND8-34 has a specific inhibitory effect on the growth of neuroendocrine prostate cancer and small cell lung cancer cells.

[0180] Experimental Example 7 Homogeneous Time Resolved Fluorescence (HTRF) Analysis of Compounds Disrupting the Binding of ZMYND8 Protein to Its Peptide H3K4me1-H3K14ac

[0181] GST-ZMYND8 protein (aa103-426) and Biotin-tagged polypeptide (ART{Lys(Me)}QTARKSTGG{Lys-Ac}APRKQL) were diluted to 40 nM and 2 μM with HTRF buffer (20 mM HEPES, pH 7.4, 10 mM NaCl, 0.01% BSA), respectively. The experiment was performed in a white 384-well plate (6007290, PerkinElmer). 1 μL of compound and 9 μL of GST-ZMYND8 protein were added to the plate (the control group was treated with 1 μL of DMSO and 9 μL of HTRF buffer). The plate was incubated at room temperature for 15 minutes. Then, 5 μL of biotin-labeled substrate peptide was added and incubated at room temperature for 15 minutes. Finally, 5 μL of a mixture of anti-GST-Tb (Cisbio, 61GSTTLB) and anti-streptavidin-XL665 (Cisbio, 610SAXLA) was added. After incubation at room temperature for 30 minutes, the HTRF signal values ​​were measured at 620 nm and 665 nm using a TECAN SPARK microplate reader. The compound inhibition rate was calculated as follows: Inhibition rate = (1-(compound group - control group) / (DMSO group - control group))*100%, IC 50 The values ​​were determined by curve fitting with variable slope (four parameters) nonlinear regression in Graphpad Prism (8.0.1). The results are shown in Table 2.

[0182] Table 2 IC values ​​of compounds inhibiting the binding of ZMYND8 protein to H3K4me1-H3K14ac 50 value

[0183] A stands for IC 50 value less than or equal to 10 μM;

[0184] B stands for IC 50 values ​​greater than 10 μM and less than 50 μM;

[0185] C stands for IC 50 The value was greater than 50 μM.

[0186] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A compound represented by general formula (I), a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug thereof, in, Indicates a double bond or a single bond; X is WR4, wherein W is selected from N, O, S; R1 is selected from: H, SF3, SF5, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-12 cycloalkyl, substituted or unsubstituted 5-8 membered heterocyclyl, substituted or unsubstituted C6-14 aryl, substituted or unsubstituted 5-8 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3 , SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl, or any two substituents are connected to form a 5-8 membered aliphatic ring or 5-8 membered aromatic ring containing or not containing heteroatoms; R2 is selected from: substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C2-6 alkenyl, substituted or unsubstituted C2-6 alkynyl, substituted or unsubstituted C3-12 cycloalkyl, substituted or unsubstituted 5-8 membered heterocyclyl, substituted or unsubstituted C6-14 aryl, substituted or unsubstituted 5-10 membered heteroaryl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3 -8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl, or any two substituents are connected to the connected carbon to form a 5-8 membered aliphatic ring or 5-8 membered aromatic ring containing or not containing heteroatoms; R3 is none or selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-8 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C6-14 aryl, 5-8 membered heteroaryl; R4 is none or selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C3-8 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1- 6 alkoxy, 5-8 membered heterocyclic group, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, C1-6 alkyl-(C=O)-, C1-6 alkyl-(O=S=O)-, C1-6 alkyl-S-, C6-14 aryl, 5-8 membered heteroaryl.

2. The compound according to claim 1, having the structure shown in Formula II: in, X is O or S; R3 is selected from: hydrogen, substituted or unsubstituted C1-6 alkyl, arbitrarily substituted or unsubstituted C3-8 cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-8 cycloalkyl, SF3, SF5, C2-6 alkenyl, C2-6 alkynyl, cyano, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-10 aryl, 5-8 membered heteroaryl; The definitions of R1 and R2 are as described in claim 1.

3. The compound according to claim 1, having the structure shown in formula III: in, R3 is selected from: hydrogen, any substituted or unsubstituted alkyl, any substituted or unsubstituted cycloalkyl; wherein the substitution refers to substitution by one or more substituents selected from the group consisting of: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-8 cycloalkyl, SF3, SF5; The definitions of R1 and R2 are as described in claim 1.

4. The compound according to claim 1, wherein R1 is selected from the group consisting of: H, C3-C8 cycloalkyl, in, Indicates a double bond or a single bond; W1 is selected from CRx or N; wherein Rx is H, D, halogen, nitro, hydroxyl, cyano, C1-6 alkoxy, C1-6 alkyl, C1-6 alkyl-S-, C1-6 alkylamino, amino, C1-6 haloalkyl, C6-10 aryl, C1-6 alkyl-(O=S=O)-; R 1a and R 1b Each is independently selected from H, D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-14 aryl, 5-8 membered heteroaryl; or R 1a and R 1b Connected to the attached carbon to form a 5-6 membered aliphatic ring or a 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms; Y is selected from NRy, O or S; Ry is H, C1-6 alkyl or C3-8 cycloalkyl; R 1c Selected from H, D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-8 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-14 aryl, 5-8 membered heteroaryl.

5. The compound according to claim 1, wherein R2 is C1-4 alkyl or in, W2 is selected from CRz or N; Rz is halogen, H, D, cyano, C1-4 alkyl, C1-4 alkoxy, phenyl, hydroxy, amino, carboxyl, nitro, C3-6 cycloalkyl, C1-4 haloalkyl, C1-6 alkylamino; R 2a and R 2b Each is independently selected from H, D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, SF3, SF5, C3-6 cycloalkyl, C2-6 alkenyl, C2-6 alkynyl, cyano, nitro, C1-6 alkoxy, 5-8 membered heterocyclyl, C1-6 haloalkyl, C1-6 alkylamino, hydroxymethyl, CH3-(C=O)-, CH3-(O=S=O)-, CH3-S-, C6-10 aryl, 5-8 membered heteroaryl; or R 2a and R 2b It is connected to the carbon to form a 5-6 membered aliphatic ring or a 5-6 membered aromatic ring containing 1, 2 or 3 heteroatoms or no heteroatoms.

6. The compound of claim 1, wherein R3 is none, H, C1-4 alkyl or C3-C6 cycloalkyl.

7. The compound according to claim 1, characterized in that The compound is selected from the group consisting of:

8. Provide a pharmaceutical composition comprising: A compound according to claim 1, a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug thereof; and A pharmaceutically acceptable carrier.

9. Use of the compound according to claim 1, its pharmaceutically acceptable salt, or its enantiomer, diastereomer, tautomer, solvate, polymorph or prodrug or the pharmaceutical composition according to claim 8, characterized in that, Used to prepare ZMYND8 small molecule inhibitors; or used to prepare drugs for treating neuroendocrine tumors.

10. The use according to claim 9, characterized in that The neuroendocrine tumor is selected from the group consisting of neuroendocrine prostate cancer and small cell lung cancer.

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