Purine compound and application thereof

Abstract

The invention discloses a purine compound and application thereof, the structure of the purine compound is as shown in formula I, and the definition of each substituent group is as shown in the specification and claims. The compound provided by the invention specifically targets ZMYND8 and inhibits the activity of ZMYND8, thereby inhibiting the transdifferentiation and growth of prostatic cancer adenoma to neuroendocrine tumor, and also inhibiting the growth and development of small cell lung cancer. # imgabs0 #

Description

Technical Field

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

[0002] Both prostate cancer and lung cancer are adenocarcinomas that can transdifferentiate into poorly differentiated neuroendocrine (NE) tumors, namely neuroendocrine prostate cancer (NEPC) and small cell lung cancer (SCLC), after relapse or progression of targeted therapy. Despite their different tissues of origin, NEPC and SCLC have highly similar genomic and transcriptomic alterations, showing common features of high expression of neuroendocrine marker molecules and acquisition of numerous genetic or epigenetic modifications. A key event in the development of neuroendocrine tumors is the almost universal loss of tumor suppressors RB1 and TP53. SCLC tumors can be divided into four molecular subtypes based on dominant transcription factor expression, ASCL1, NEUROD1, POU2F3, and YAP1, of which ASCL1 is the most prevalent subtype. In NEPC, ASCL1 and NEUROD1 subtypes have been reported, but subtype analysis has not been routinely performed. This process of cell differentiation to acquire a neuroendocrine phenotype is called neuroendocrine transdifferentiation, which is an important mechanism of endocrine therapy resistance and is clearly associated with poor clinical prognosis.

[0003] Disturbances in epigenetic regulation, mainly marked by covalent chromatin modification, can cause some important genes or entire gene networks to be locked in a heritable transcriptional abnormality state, which plays an important role in the occurrence and development of tumors. Histone modifying enzymes and effector proteins usually have enzymatic activity or histone recognition domains, which can be potentially targeted by drugs. Therefore, genes related to epigenetic modification have become hot 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, of which 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 is eager to discover highly selective small molecule inhibitors of ZMYND8. Summary of the invention

[0005] The purpose of the present invention is to provide a purine compound and its application. The compound can effectively inhibit the activity of ZMYND8 and has no obvious cytotoxicity to other tissue cells, thereby significantly inhibiting tumor growth without significant side effects.

[0006] 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,

[0007]

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

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

[0010] 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;

[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 connected to the connected 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 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.

[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 by 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 connected to the connected 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]

[0023] 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 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 above.

[0024] In another preferred embodiment, the compound has a structure shown in Formula III:

[0025]

[0026] Wherein, 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 following group: D, halogen, hydroxyl, amino, carboxyl, C1-6 alkyl, C3-8 cycloalkyl, SF3, SF5; R1 and R2 are defined as above.

[0027] In another preferred embodiment, R1 is selected from: H, C3-C8 cycloalkyl,

[0028] in, Indicates a double bond or a single bond;

[0029] 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)-;

[0030] R 1a and R 1bEach 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;

[0031] Y is selected from NRy, O or S; Ry is H, C1-6 alkyl or C3-8 cycloalkyl;

[0032] 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.

[0033] In another preferred embodiment, R1 is selected from: H, C3-C6 cycloalkyl,

[0034] in, Indicates a double bond or a single bond;

[0035] W1 is selected from CRx or N; wherein Rx is H, D, halogen, nitro, hydroxyl, 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)-;

[0036] R 1a and R 1b Each is independently selected from H, D, halogen, hydroxyl, 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.

[0037] In another preferred embodiment, Y is selected from NRy, O or S; Ry is H, C1-4 alkyl or C3-6 cycloalkyl.

[0038] In another preferred embodiment, R 1c Selected 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.

[0039] In another preferred embodiment, R2 is C1-4 alkyl or

[0040] Wherein, W2 is selected from CRz or N; Rz is halogen, H, D, cyano, C1-4 alkyl, C1-4 alkoxy, phenyl, hydroxyl, amino, carboxyl, nitro, C3-6 cycloalkyl, C1-4 haloalkyl, C1-6 alkylamino;

[0041] 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.

[0042] In another preferred embodiment, R2 is C1-4 alkyl or

[0043] Wherein, W2 is selected from CRz or N; Rz is halogen, H, D, cyano, C1-4 alkyl, C1-4 alkoxy, nitro, hydroxyl, amino, carboxyl, C3-6 cycloalkyl, C1-4 haloalkyl, C1-6 alkylamino;

[0044] R 2a and R 2bEach is independently selected from H, D, halogen, hydroxyl, 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;

[0045] or R 2a and R 2b Connected to the attached carbon to form a benzene ring or a 5-6 membered aliphatic ring containing 1, 2 or 3 oxygen atoms.

[0046] 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 group: halogen, D, cyano, C1-4 alkyl, C1-4 alkoxy, phenyl, nitro, hydroxyl, amino, carboxyl, C3-6 cycloalkyl, C1-4 haloalkyl, C1-6 alkylamino.

[0047] In another preferred embodiment, R3 is none, H, C1-4 alkyl or C3-C6 cycloalkyl.

[0048] In another preferred embodiment, the compound is selected from the following group: Compound 1-Compound 63.

[0049] The second aspect of the present invention provides a pharmaceutical composition comprising:

[0050] 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.

[0051] 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.

[0052] In another preferred embodiment, the neuroendocrine tumor is selected from the group consisting of neuroendocrine prostate cancer and small cell lung cancer.

[0053] The advantages of the present invention are:

[0054] 1. The small molecule compounds of the present invention (such as iZMYND8-34) specifically inhibit neuroendocrine prostate cancer and small cell lung cancer.

[0055] 2. The small molecule compound iZMYND8-34 had no obvious toxic side effects on mice at a dosage concentration of 50 mg / kg.

[0056] 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 specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. 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 described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy.

[0058] Figure 2 It was shown that ZMYND8 knockout in neuroendocrine prostate cancer organoids, cell lines, and small cell lung cancer cells resulted in reduced tumor growth.

[0059] Figure 3 It was shown that iZMYND8-34 directly binds to ZMYND8 and inhibits the interaction of ZMYND8 with H3K4me1-H3K14ac.

[0060] Figure 4 It shows that iZMYND8-34 has no obvious toxic effect on the body.

[0061] Figure 5 iZMYND8-34 was shown to inhibit the growth of neuroendocrine prostate cancer and small cell lung cancer.

[0062] Figure 6 iZMYND8-34 was shown to specifically inhibit the growth of neuroendocrine prostate cancer and small cell lung cancer. DETAILED DESCRIPTION

[0063] The inventors of the present application have conducted extensive and in-depth research and developed a class of purine compounds that can effectively inhibit the neuroendocrine transdifferentiation of adenocarcinoma and thus inhibit the further malignant outcome of anti-tumor. Specifically, they can specifically target the histone-related molecule ZMYND8 and inhibit its activity, thereby specifically inhibiting the development of neuroendocrine tumors. On this basis, the present invention was completed.

[0064] the term

[0065] In the present invention, the halogen is F, Cl, Br or I.

[0066] In the present invention, unless otherwise specified, the terms used have the general meanings well known to those skilled in the art.

[0067] 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.

[0068] 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.

[0069] In the present invention, the term "alkoxy" means -O-(alkyl), and the definition of alkyl is the same as above. For example, the term "C1-C6 alkoxy" refers to a straight or branched alkoxy group having 1 to 6 carbon atoms, including but not limited to methoxy, ethoxy, n-propoxy, isopropoxy and butoxy.

[0070] 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.

[0071] In the present invention, the term "alkynyl" refers to a straight or branched alkynyl group containing one triple bond, including but not limited to ethynyl, propynyl, butynyl, isobutynyl, pentynyl and hexynyl.

[0072] 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.

[0073] In the present invention, the term "aryl" refers to a hydrocarbon moiety containing one or more aromatic rings. 14 "Aryl" refers to an aromatic ring group having 6 to 14 carbon atoms without 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.

[0074] 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.

[0075] 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), for example furanyl, pyrrolyl, thienyl, oxazolyl, imidazolyl, thiazolyl, pyridyl, quinolyl, isoquinolyl, indolyl, pyrimidinyl, pyranyl.

[0076] 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.

[0077] In the present invention, the hetero atom refers to N, O, S or P.

[0078] 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 also be condensed with each other.

[0079] In the present invention, the substitution is mono- or poly-substitution, and the poly-substitution is di-, tri-, tetra- or penta-substitution. The di-substitution means having two substituents, and so on.

[0080] Pharmaceutically acceptable salts of the present invention can be salts formed by negatively charged groups on anions and compounds of formula I. Suitable negatively charged groups 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 ions, potassium ions, magnesium ions, calcium ions and ammonium ions, such as tetramethylammonium ions.

[0081] In another preferred embodiment, "pharmaceutically acceptable salt" refers to the salts formed by the compound of formula I with an acid selected from the following group: 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 the sodium salt, potassium salt, calcium salt, aluminum salt or ammonium salt formed by the compound of formula I with an inorganic base; or the methylamine salt, ethylamine salt or ethanolamine salt formed by the compound of general formula I with an organic base.

[0082] Pharmaceutical composition

[0083] The present invention also provides a pharmaceutical composition, which comprises an active ingredient within a safe and effective amount range and a pharmaceutically acceptable carrier.

[0084] The "active ingredient" described in the present invention refers to the compound of formula I described in the present invention.

[0085] 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.

[0086] "Safe and effective amount" means: the amount of active ingredient is sufficient to significantly improve the condition without causing serious side effects. Usually, the pharmaceutical composition contains 1-2000 mg of active ingredient per dose, more preferably, 10-200 mg of active ingredient per dose. Preferably, the "one dose" is one tablet.

[0087] "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 ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0088] 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), etc.

[0089] Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.

[0090] 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 may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide 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 may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and spices.

[0091] In addition to the active ingredients, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methanol and agar, or mixtures of these substances.

[0092] Compositions for parenteral injection may include 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.

[0093] The compounds of the present invention can be administered alone or in combination with other therapeutic drugs (such as anti-tumor drugs).

[0094] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1 to 2000 mg, preferably 20 to 500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the health status of the patient, which are all within the skill of a skilled physician.

[0095] 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 only and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not indicated in the following examples are usually performed under conventional conditions (such as the conditions described in Sambrook et al., Molecular Cloning: 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 weight parts.

[0096] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein can be applied to the methods of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0097] preparation

[0098] Route 1:

[0099]

[0100] Route 2:

[0101]

[0102]

[0103] Among them, the synthesis of compounds 1-56 refers to route 1, and the synthesis of compounds 57-63 refers to route 2.

[0104] 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.

[0105] Preparation Example 1: Synthesis of Compound 1

[0106]

[0107] Step 1: Synthesis of intermediate 1b:

[0108] Add diaminomaleonitrile (0.540 g, 5.00 mmol) and compound 1a (0.856 g, 5.25 mmol) to a 100 ml eggplant-shaped bottle, add anhydrous tetrahydrofuran (15 ml), and stir at room temperature for 24 hours. After the reaction is completed, spin dry the solvent, wash the solid with ice ethanol (10 ml), filter, and wash the filter cake with ice ethanol (10 ml) and ice ether (10 ml) respectively. The filter cake is vacuum dried at 60 ° C for two hours to obtain 1.200 g of brown-grey solid product with a yield of 88.6%.

[0109] Step 2: Synthesis of compound 1:

[0110] Compound 1b (0.500 g, 1.85 mmol) and 3-ethoxy salicylaldehyde (0.628 g, 3.78 mmol) were added to a 100 ml eggplant-shaped bottle, methanol (15 ml) was added, and triethylamine (0.196 g, 1.94 mmol) was added. After stirring at room temperature for 1 hour, the reaction liquid became clear. Stirring was continued at room temperature for 24 hours, and the product gradually precipitated. After the reaction was completed, it was directly filtered, the filter cake was dissolved with dimethyl sulfoxide (5 ml), and ice ethanol (50 ml) was added. Solid precipitated, filtered, and the filter cake was vacuum dried at 60°C for two hours to obtain 0.408 g of black solid product, with a yield of 50.8%.

[0111] Preparation Example 2: Synthesis of Compound 21

[0112]

[0113] Step 1: Synthesis of intermediate 21b

[0114] Same synthesis as intermediate 1b.

[0115] Step 2: Synthesis of compound 21

[0116] Compound 21b (0.500 g, 1.85 mmol) and 4-nitrobenzaldehyde (0.571 g, 3.78 mmol) were added to a 100 ml eggplant-shaped bottle, methanol (15 ml) was added, and triethylamine (0.196 g, 1.94 mmol) was added. After stirring at room temperature for 1 hour, the reaction solution became clear. Stirring was continued at room temperature for 24 hours, and the product gradually precipitated. After the reaction was completed, it was directly filtered, the filter cake was dissolved with dimethyl sulfoxide (5 ml), and ice ethanol (50 ml) was added. Solid precipitated, filtered, and the filter cake was vacuum dried at 60°C for two hours to obtain 0.676 g of a yellow-brown solid product with a yield of 87.2%.

[0117] Preparation Example 3: Synthesis of Compound 61

[0118]

[0119] Step 1: Synthesis of intermediate 61b

[0120] Synthesis of the same intermediate 1b

[0121] Step 2: Synthesis of intermediate 61c

[0122] Same synthesis as compound 21

[0123] Step 3: Synthesis of intermediate 61d

[0124] Compound 61c (0.100 g, 0.238 mmol) was added to a 50 ml eggplant-shaped bottle, and phosphorus oxychloride (3 ml) was added, and stirred at 100°C for 12 hours. After the reaction was completed, the reaction solution was added dropwise to ice water (50 ml) while stirring, and stirred for 30 minutes after the addition was complete. Ethyl acetate (20 ml × 3) was added for extraction, and the liquids were separated. The organic phases were combined, and the organic phases were washed with water (20 ml × 3) and concentrated under reduced pressure. After column chromatography (ethyl acetate: petroleum ether = 1:1), 0.055 g of yellow solid was obtained, with a yield of 57.5%.

[0125] Step 4: Synthesis of intermediate 61e

[0126] Compound 61d (0.110 g, 0.274 mmol) and cesium carbonate (0.178 mg, 0.547 mmol) were added to a 50 ml eggplant-shaped bottle, and N,N-dimethylformamide (3 ml) and iodomethane (0.058 g, 0.410 mmol) were added, and stirred at room temperature for 12 hours. After the reaction was completed, water (10 ml) was added, and ethyl acetate (20 ml × 3) was used for extraction, and the liquids were separated, and the organic phases were combined, and the organic phases were washed with water (20 ml × 3), and concentrated under reduced pressure. After column chromatography (ethyl acetate: petroleum ether = 1:3), 0.084 g of yellow solid was obtained, and the yield was 73.7%.

[0127] Step 5: Synthesis of compound 61

[0128] 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 bottle, followed by dimethyl sulfoxide (3 ml), 100 μl of 30% hydrogen peroxide under ice bath, and stirred at room temperature for 1 hour. After the reaction was completed, the mixture was directly 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 with a yield of 75.1%.

[0129] Preparation Example 4: Synthesis of Compound 63

[0130]

[0131] Step 1: Synthesis of intermediate 63b

[0132] Add diaminomaleonitrile (0.540 g, 5.00 mmol) and compound 63a (0.719 g, 5.25 mmol) to a 100 ml eggplant-shaped bottle, add anhydrous tetrahydrofuran (15 ml), and stir at room temperature for 24 hours. After the reaction is completed, spin dry the solvent, wash the solid with ice ethanol (10 ml), filter, and wash the filter cake with ice ethanol (10 ml) and ice ether (10 ml) respectively. The filter cake is vacuum dried at 60 ° C for two hours to obtain 1.087 g of brown solid product, with a yield of 88.7%.

[0133] Step 2: Synthesis of intermediate 63c

[0134] Compound 63b (0.500 g, 2.04 mmol) and 4-nitrobenzaldehyde (0.632 g, 4.18 mmol) were added to a 100 ml eggplant-shaped bottle, methanol (15 ml) was added, and triethylamine (0.216 g, 2.14 mmol) was added. After stirring at room temperature for 1 hour, the reaction solution became clear. Stirring was continued at room temperature for 24 hours, and the product gradually precipitated. After the reaction was completed, it was directly filtered, the filter cake was dissolved with dimethyl sulfoxide (5 ml), and ice ethanol (50 ml) was added. Solid precipitated, filtered, and the filter cake was vacuum dried at 60°C for two hours to obtain 0.544 g of a yellow-brown solid product with a yield of 67.7%.

[0135] Step 3: Synthesis of intermediate 63d

[0136] Compound 63c (0.100 g, 0.254 mmol) was added to a 50 ml eggplant-shaped bottle, and phosphorus oxychloride (3 ml) was added, and stirred at 100°C for 12 hours. After the reaction was completed, the reaction solution was added dropwise to ice water (50 ml) while stirring, and stirred for 30 minutes after the addition was complete. Ethyl acetate (20 ml × 3) was added for extraction, and the liquids were separated. The organic phases were combined, and the organic phases were washed with water (20 ml × 3) and concentrated under reduced pressure. After column chromatography (ethyl acetate: petroleum ether = 1:1), 0.092 g of yellow solid was obtained, with a yield of 96.3%.

[0137] Step 4: Synthesis of intermediate 63e

[0138] Compound 63d (0.100 g, 0.266 mmol) and cesium carbonate (0.173 mg, 0.532 mmol) were added to a 50 ml eggplant-shaped bottle, and N,N-dimethylformamide (3 ml) and iodomethane (0.057 g, 0.399 mmol) were added, and stirred at room temperature for 12 hours. After the reaction was completed, water (10 ml) was added, and ethyl acetate (20 ml × 3) was used for extraction, and the liquids were separated, and the organic phases were combined, and the organic phases were washed with water (20 ml × 3), and concentrated under reduced pressure. After column chromatography (ethyl acetate: petroleum ether = 1:3), 0.093 g of yellow solid was obtained, and the yield was 89.6%.

[0139] Step 5: Synthesis of compound 63

[0140] 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 bottle, followed by dimethyl sulfoxide (3 ml), 100 μl of 30% hydrogen peroxide under ice bath, and stirred at room temperature for 1 hour. After the reaction was completed, the mixture was directly 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 with a yield of 87.0%.

[0141] Table 1 Structure and characterization of compounds in the examples

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Experimental Example 1 Overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy

[0158] 1. In vitro, overexpression of ZMYND8 in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy

[0159] Experimental methods: In androgen-sensitive prostate cancer cell lines (LNCaP, VCaP and MycCaP cells), ZMYND8 overexpression plasmid was transferred to construct overexpression stable strains as experimental group (ZMYND8-OE); empty vector was transferred to construct stable strains as control group (CTRL). After the stable strains were constructed, the two types of cells were equally plated in 96-well plates, with 3-6 duplicate wells in each group. 1μM enzalutamide (ENZ) was used to simulate androgen deprivation therapy. During the culture process, MTS reagent was used to detect the number of viable cells, that is, MTS reagent was added to the culture medium at a volume ratio of 100:1. After incubation for 2 hours, the 490nm OD value was detected using an enzyme reader. The high and low OD values ​​reflected the number of viable cells. The time when the cells just attached to the wall was taken as day 0, and the number of viable cells was detected every 2-3 days thereafter, and the changes in the number of cells were continuously recorded within 5-7 days to count the changes in cell resistance.

[0160] Experimental results: Figure 1 As shown in Figures AC, compared with the control, the cell line with overexpression of ZMYND8 grew faster during androgen deprivation therapy and showed stronger androgen deprivation therapy resistance. This result shows that ZMYND8 overexpression promotes androgen deprivation therapy resistance in androgen-sensitive prostate cancer cell lines.

[0161] 2. In vivo experiments, ZMYND8 overexpression in androgen-sensitive prostate cancer cell lines promotes resistance to androgen deprivation therapy

[0162] Experimental methods: The androgen-sensitive prostate cancer cell lines 22RV1 and MycCaP were used to construct a stable ZMYND8-overexpressing cell as the experimental group (ZMYND8-OE), and an empty vector-constructed stable cell was transferred as the control group (CTRL). The constructed cells were injected subcutaneously into 8-10 week-old nude mice in equal amounts. Seven days after the 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 subcutaneous tumor was calculated based on 0.5×(length×width)=1. 2 ) formula to calculate tumor volume. Changes in tumor size were recorded, and differences in tumor growth were statistically calculated.

[0163] Experimental results: Figure 1 As shown in D and E, compared with the control group, the ZMYND8-overexpressing cell line grew faster during androgen deprivation therapy after being injected subcutaneously into nude mice, showing less sensitivity to androgen deprivation therapy. These results indicate that in vivo experiments, ZMYND8 overexpression in androgen-sensitive prostate cancer cell lines promotes androgen deprivation therapy resistance.

[0164] Experimental Example 2 ZMYND8 knockout in neuroendocrine prostate cancer organoids, cell lines and small cell lung cancer cells leads to reduced tumor growth

[0165] 1. Knockout of ZMYND8 in neuroendocrine prostate cancer organoids inhibits tumor growth

[0166] 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 / flox In the three-gene 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 the empty vector stable transgenic lines were transferred into the corresponding models as control groups (EV, CTRL-sgRNA). First, the protein level changes were verified by protein immunoblotting to ensure that the expression of Zmynd8 in the control group in the organoid model had been reduced. By taking pictures with a microscope and measuring and comparing the changes in organoid size and formation rate with ImageJ, the regulatory role of ZMYND8 on the growth of neuroendocrine prostate cancer organoids was clarified.

[0167] Experimental results: Figure 2 As shown in Figures 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.

[0168] 2. Knockout of ZMYND8 in neuroendocrine prostate cell lines and small cell lung cancer cell lines inhibits tumor growth

[0169] Experimental methods: In neuroendocrine prostate cancer cell lines NE1.3 and NCI-H660, and small cell lung cancer cell line H82, ZMYND8 knockout plasmids were transferred to construct knockout stable transfectants as experimental groups (ZMYND8-KO1, ZMYND8-KO2); empty vectors were transferred to control groups (CTRL). After stable transfectants were constructed, equal amounts of cells in each group were plated in 96-well plates, with 3-6 replicates per group. During the culture process, MTS reagent was used to detect the number of live cells, that is, MTS reagent was added to the culture medium at a volume ratio of 100:1. After incubation for 2 hours, the 490nm OD value was detected using an ELISA instrument. The high and low OD values ​​reflected the number of live cells. The time when the cells just attached to the wall was taken as day 0, and the number of live cells was detected every 2-3 days thereafter, and the changes in the number of cells were continuously recorded within 5-7 days to count the changes in cell growth.

[0170] Experimental results: Figure 2 As shown in E, knockout of ZMYND8 can significantly inhibit tumor growth in both neuroendocrine prostate cancer cell lines and small cell lung cancer cell lines.

[0171] 3. Knockout of ZMYND8 in androgen-sensitive cell lines and non-small cell lung cancer cell lines does not affect cell growth

[0172] Experimental methods: In the androgen-sensitive cell lines LNCaP and LAPC4, and the non-small cell lung cancer cell line A549, the ZMYND8 knockout plasmid was transferred to construct knockout stable strains as experimental groups (ZMYND8-KO1, ZMYND8-KO2); the empty vector was transferred as the control group (CTRL). After the stable strains were constructed, the cells of each group were plated in 96-well plates in equal amounts, and 3-6 duplicate wells were set for each group. During the culture process, the number of live cells was detected using MTS reagent, that is, MTS reagent was added to the culture medium at a volume ratio of 100:1. After incubation for 2 hours, the 490nm OD value was detected using an enzyme reader. The high and low OD values ​​reflected the number of live cells. The time when the cells just attached to the wall was taken as day 0, and the number of live cells was detected every 2-3 days thereafter, and the changes in the number of cells were continuously recorded within 5-7 days to count the changes in cell growth.

[0173] Experimental results: Figure 2 As shown in F, knockout of ZMYND8 did not affect tumor growth in androgen-sensitive cell lines and non-small cell lung cancer cell lines.

[0174] 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 the function of ZMYND8 in neuroendocrine tumors and small cell lung cancer is specific.

[0175] Experimental Example 3iZMYND8-34 directly binds to ZMYND8 and inhibits the interaction between ZMYND8 and H3K4me1-H3K14ac

[0176] 1. Surface Plasmon Resonance (SPR) analysis of iZMYND8-34 binding to ZMYND8 protein

[0177] Experimental method: The SPR experiment was performed on Biacore 8K (GE Healthcare) with HBS (10 mM HEPES, pH 7.4, 150 mM NaCl) as the buffer. ZMYND8 protein (aa103-426) was covalently coupled to the CM5 chip by a standard amino coupling procedure under the condition of 10 mM 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 steady-state analysis module in the Biacore 8K evaluation software (GE Healthcare) was used to calculate the equilibrium dissociation constant (K) between iZMYND8-34 and ZMYND8 protein. D value).

[0178] Experimental results: Figure 3 As shown in Figure 1, 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.

[0179] 2. Homogeneous Time Resolved Fluorescence (HTRF) analysis of iZMYND8-34 disrupting the interaction between ZMYND8 protein and H3K4me1-H3K14ac

[0180] Experimental method: GST-ZMYND8 protein (aa103~426) and Biotin-labeled polypeptide (ART{Lys(Me)}QTARKSTGG{Lys-Ac}APRKQL) were diluted to 40nM and 2μM respectively with HTRF buffer (20mM HEPES, pH7.4, 10mM NaCl, 0.01% BSA). The experiment was carried out 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), incubated at room temperature for 15 min, then 5 μL of Biotin-labeled substrate peptide was added, incubated at room temperature for 15 min, and 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 min, the HTRF signal value was detected at 620 nm and 665 nm using a TECAN SPARK microplate reader.

[0181] Experimental results: Figure 3 As shown in B, HTRF experiments showed that the small molecule compound iZMYND8-34 could inhibit the binding of ZMYND8 protein to H3K4me1-H3K14ac in a concentration-dependent manner, and its IC 50 The value is 0.66μM.

[0182] Experimental Example 4 Toxicity Analysis of Small Molecule Compound iZMYND8-34

[0183] Experimental method: 8-10 week old WT mice were intraperitoneally injected with iZMYND8-34 or DMSO control at a dose of 50 mg / kg once a day for 14 days, and the body weight was measured daily. On day 14, blood was collected and serum alanine aminotransferase (ALT, reflecting liver toxicity) and alkaline phosphatase (ALP, reflecting kidney toxicity) levels were analyzed by ELISA.

[0184] Experimental results: Figure 4 As shown in Figure A, when mice were treated with iZMYND8-34 at a concentration of 50 mg / kg, there was no significant change in the body weight of the mice. Figure 4 As shown in B and C, after iZMYND8-34 was treated at a concentration of 50 mg / kg, the levels of ALT and ALP in the serum of mice did not increase significantly. These results indicate that iZMYND8-34 treatment of mice does not cause significant toxic effects.

[0185] Experimental Example 5 iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer

[0186] 1. Inhibitory effect of iZMYND8-34 on neuroendocrine prostate cancer PDX

[0187] Experimental method: 8-10 week old NSG mice were subcutaneously inoculated with neuroendocrine prostate cancer PDX LTL-545 and LTL-651. When the tumor grew to a measurable size (about 80mm 3 ), the experimental group was given iZMYND8-34 intraperitoneal injection 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.

[0188] Experimental results: Figure 5 As shown in middle A, iZMYND8-34 significantly inhibited the growth of various tumors, indicating that ZMYND8 inhibitors can inhibit the growth of neuroendocrine prostate cancer PDX. This indicates that iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer PDX.

[0189] 2. iZMYND8-34 inhibits tumor formation in genetically derived mice with neuroendocrine prostate cancer

[0190] Experimental Methods: Probasin was used as a 6-week-old triple knockout neuroendocrine prostate cancer genetic mouse Cre / + ;Pten flox / flox ;Tp53 flox / flox ; Rb1 flox / flox , iZMYND8-34 was intraperitoneally injected at a dose of 50 mg / kg daily for 4-6 weeks, and the prostate of the mice was collected for H&E staining to count the changes in the malignancy of prostate cancer in the mice. At the same time, immunohistochemical staining of the neuroendocrine marker protein Syp was performed, and the differences in the expression of the marker protein were counted.

[0191] Experimental results: Figure 5 As shown in B, in the genetic mouse model, the tumor malignancy was weakened after iZMYND8-34 treatment, and the expression level of the neuroendocrine prostate cancer-specific expression molecule Syp was weakened, indicating that iZMYND8-34 inhibits tumor formation in genetic mice with neuroendocrine prostate cancer.

[0192] 3. iZMYND8-34 inhibits the formation of small cell lung cancer xenografts

[0193] Experimental method: Small cell lung cancer cells H82 or H209 were subcutaneously injected into 8-week-old nude mice, with 10 cells per tumor. 6After injection, the experimental group iZMYND8-34 was intraperitoneally administered at a concentration of 50 mg / kg, and the control group was injected with an equal volume of PBS. 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) 2 ) formula to calculate tumor volume.

[0194] Experimental results: Figure 5 As shown in middle C, after iZMYND8-34 treatment, the tumorigenicity of small cell lung cancer was weakened and the tumor growth was significantly slowed down, indicating that iZMYND8-34 inhibits the formation and growth of small cell lung cancer.

[0195] The above experiments show that the small molecule inhibitor iZMYND8-34 inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer.

[0196] Example 6 iZMYND8-34 specifically inhibits the growth of neuroendocrine prostate cancer and small cell lung cancer

[0197] 1. iZMYND8-34 has no inhibitory effect on androgen-sensitive prostate cancer and non-small cell lung cancer cell lines

[0198] Experimental methods: The androgen-sensitive prostate cancer cell line LNCaP and the non-small cell lung cancer cell line A549 were used to detect the effect of iZMYND8-34 on cell growth. The experimental group was treated with 1 μM iZMYND8-34 in the culture medium, and the control group was treated with an equal volume of DMSO. The cells in each group were equally plated in a 96-well plate, and 3-6 replicates were set for each group. During the culture process, the number of live cells was detected using MTS reagent, that is, MTS reagent was added to the culture medium at a volume ratio of 100:1. After incubation for 2 hours, the OD value at 490nm was detected using an enzyme reader. The high and low OD values ​​reflected the number of live cells. The time when the cells just attached to the wall was taken as day 0, and the number of live cells was detected every 2-3 days thereafter, and the changes in the number of cells were continuously recorded within 5-7 days, and the changes in cell growth were counted.

[0199] Experimental results: Figure 6 As shown in A and B, iZMYND8-34 did not affect the growth of androgen-sensitive prostate cancer cell line LNCaP and non-small cell lung cancer cell line A549, indicating that iZMYND8-34 had no inhibitory effect on androgen-sensitive prostate cancer and non-small cell lung cancer cell lines.

[0200] 2. Inhibitory effect of iZMYND8-34 on neuroendocrine prostate cancer and small cell lung cancer

[0201] Experimental methods: Neuroendocrine prostate cancer cell lines NE1.3, NCI-H660 and small cell lung cancer cell lines H82, H209 were used to detect the effect of iZMYND8-34 on cell growth. The experimental group was treated with 1μM iZMYND8-34 added to the culture medium, and the control was treated with an equal volume of DMSO. Each group of cells was plated in a 96-well plate in equal amounts, and 3-6 replicates were set for each group. During the culture process, the number of live cells was detected using MTS reagent, that is, MTS reagent was added at a volume ratio of 100:1. After incubation for 2 hours, the 490nm OD value was detected using an enzyme reader to reflect the number of live cells. The time when the cells just attached to the wall was taken as day 0, and the number of live cells was detected every 2-3 days thereafter, and the changes in the number of cells were continuously recorded within 5-7 days, and the changes in cell growth were counted.

[0202] Experimental results: Figure 6 As shown in C 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.

[0203] 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.

[0204] Experimental Example 7 Homogeneous Time Resolved Fluorescence (HTRF) Analysis of Compounds Disrupting the Binding of ZMYND8 Protein and Its Peptide H3K4me1-H3K14ac

[0205] 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 conducted 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), incubated at room temperature for 15 minutes, then 5 μL of Biotin-labeled substrate peptide was added, incubated at room temperature for 15 minutes, and finally 5 μL of Anti-GST-Tb (Cisbio, 61GSTTLB) and Anti-Streptavidin-XL665 (Cisbio, 610SAXLA) mixed solution was added. After incubation at room temperature for 30 minutes, the HTRF signal value was detected at 620nm and 665nm 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.

[0206] Table 2 IC of compounds inhibiting the binding of ZMYND8 protein to H3K4me1-H3K14ac 50 value

[0207] serial number <![CDATA[IC 50 (μM)]]> serial number <![CDATA[IC 50 (μM)]]> serial number <![CDATA[IC 50 (μM)]]> 1 A 22 A 43 C 2 A 23 A 44 B 3 A 24 A 45 A 4 A 25 A 46 A 5 A 26 C 47 A 6 A 27 C 48 B 7 A 28 C 49 A 8 A 29 C 50 A 9 A 30 C 51 B 10 B 31 A 52 A 11 B 32 A 53 A 12 B 33 A 54 A 13 A 34 A 55 A 14 A 35 A 56 A 15 B 36 A 57 A 16 A 37 A 58 B 17 B 38 A 59 B 18 C 39 A 60 A 19 A 40 A 61 A 20 A 41 A 62 A 21 A 42 A 63 A

[0208] A stands for IC 50 Value less than or equal to 10 μM;

[0209] B stands for IC 50 values ​​greater than 10 μM and less than 50 μM;

[0210] C stands for IC 50 The value was greater than 50 μM.

[0211] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

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 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.

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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