4-amino substituted pyridazinone compound, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a 4-amino substituted pyridazinone compound, and a preparation method therefor and the use thereof. The 4-amino substituted pyridazinone compound of the present invention has a structure as represented by formula I, wherein the definition of each substituent is as described in the description and claims. The compound of the present invention can be used in the preparation of anti-tumor drugs, particularly in the preparation of drugs for treating tumors such as melanoma.

Description

4-Amino-substituted pyridazinone compounds, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of pharmacology, specifically relating to a class of compounds with 4-amino-6-phenylpyridazine-3(2H)-one as the parent nucleus, their preparation methods, pharmaceutical compositions, and their applications in the preparation of antitumor drugs, especially for the treatment of melanoma. Background Technology

[0002] Melanoma is a malignant tumor that originates from melanocytes. It manifests as asymmetrical black or gray patches, nodules, or masses in the affected area. More than 280,000 new cases are diagnosed worldwide each year, and more than 60,000 people die from it (CA CANCER J CLIN 2018; 68:39).

[0003] Melanoma is a highly malignant tumor that primarily occurs in the skin, but can also occur in various locations or tissues such as mucous membranes (including visceral mucosa), the uvea of ​​the eye, and the pia mater. Early-stage malignant melanoma of the skin can metastasize via the lymphatic system and bloodstream, leading to an extremely poor prognosis; the five-year overall survival rate is less than 15% (Exon Publications (2017):161-174).

[0004] Early-stage melanoma is mainly treated with surgery, while for tumors that have metastasized deeply or spread to the lymph nodes, chemotherapy and radiotherapy are the main treatments. As a result, the demand for drugs to treat melanoma is constantly expanding, and new therapeutic drugs need to be developed. Summary of the Invention

[0005] The purpose of this invention is to provide a class of 4-amino-substituted pyridazinone compounds for the preparation of antitumor drugs, especially drugs for the treatment of melanoma.

[0006] In a first aspect, the present invention provides a compound of Formula I, a stereoisomer thereof, a prodrug, or a pharmaceutically acceptable salt thereof.

[0007] Where: R 0 Selected from H, -CF3, -OCF3, halogen, C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl;

[0008] R 1 Selected from -CF3, -OCF3, halogen, C1-C6 alkoxy, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl;

[0009] R 2 Selected from halogens, -CN, substituted or unsubstituted C1-C10 Alkyl, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic, wherein,

[0010] R a and R b Each is independently H, substituted or unsubstituted C1-C 10 Alkyl groups, substituted or unsubstituted C3-C6 cycloalkyl groups, substituted or unsubstituted 4-8 membered heterocyclic groups, or R a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, 4, or 5 heteroatoms.

[0011] R c C1-C, whether substituted or not 10 Alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic groups;

[0012] Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl.

[0013] The compounds shown in Formula I do not include

[0014] In another preferred embodiment, R 0 Selected from H, halogens, and C1-C4 alkoxy groups;

[0015] R 1 Selected from -CF3, -OCF3, halogen, C1-C4 alkoxy, C1-C4 alkyl, and C3-C6 cycloalkyl.

[0016] In another preferred embodiment, R 0 Selected from H, F, Cl, Br, methoxy, ethoxy, propoxy, and butoxy;

[0017] R 1 Selected from -CF3, -OCF3, F, Cl, Br, methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert-butoxy, methyl, ethyl, propyl, isopropyl, butyl, cyclopropyl, cyclobutyl, tert-butyl.

[0018] In another preferred embodiment, R 2Selected from -CN, C2-C6 alkyl, substituted C1-C6 alkyl, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-7 membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-7 membered heterocyclic, wherein,

[0019] R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted 4-8 membered heterocyclic group, or R. a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, or 4 heteroatoms.

[0020] R c The substituted C1-C6 alkyl, C3-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic groups;

[0021] Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl.

[0022] In another preferred embodiment, R 2 Selected from -CN, substituted C1-C6 alkyl groups, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-7 membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-7 membered heterocyclic, wherein,

[0023] R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted 4-8 membered heterocyclic group, or R. a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, or 4 heteroatoms.

[0024] R c The substituted C1-C6 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic groups;

[0025] Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl.

[0026] In another preferred embodiment, R 2 Selected from -CN, substituted C1-C6 alkyl, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-7-membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-7-membered heterocyclic, wherein the substitution independently refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: halogen, C3-C6 cycloalkyl, -OH;

[0027] R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, or a substituted or unsubstituted 4-6 membered heterocyclic group, wherein the substitution independently refers to substitution by 1, 2, 3, or 4 substituents selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, or

[0028] R a and R b Together with N, it forms a substituted or unsubstituted 4-8 membered heterocycle containing 1, 2, 3 or 4 heteroatoms, wherein the substitution independently refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: -OH, =O, C1-C4 alkyl, C1-C4 haloalkyl;

[0029] R c The substitution refers to a substituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, or a substituted or unsubstituted 4-8 membered heterocyclic group, wherein the substitution independently refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: -OH, C1-C4 alkyl, and C1-C4 haloalkyl.

[0030] In another preferred embodiment, the compound has the structure shown in Formula II:

[0031] R 2 R 1 The definition is as described above.

[0032] In another preferred embodiment, R 1Selected from -CF3, -OCF3, F, Cl, Br, methoxy, ethoxy, propoxy, butoxy, methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl.

[0033] In another preferred embodiment, R 1 Selected from -CF3, -OCF3, methoxy, ethoxy, propoxy, butoxy, cyclopropyl, and cyclobutyl.

[0034] In another preferred embodiment, R 2 Selected from cyano, C1-C6 haloalkyl, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic, wherein,

[0035] R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted 4-6 membered heterocyclic group, or R. a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, 4, or 5 heteroatoms.

[0036] R c It can be a substituted or unsubstituted C3-C6 cycloalkyl group or a substituted or unsubstituted 4-8 membered heterocyclic group;

[0037] Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl.

[0038] In some implementations, R2 is -N(R a (R) b ) or -O(R c ),in,

[0039] R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, or a substituted or unsubstituted 4-6 membered heterocyclic group, wherein the substitution independently refers to substitution by 1, 2, 3, or 4 substituents selected from the group consisting of: -OH, C1-C4 alkyl, C1-C4 haloalkyl, or

[0040] R aand R b Together with N, it forms a substituted or unsubstituted 4-8 membered heterocycle containing 1, 2, 3 or 4 heteroatoms, wherein the substitution independently refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: -OH, =O, C1-C4 alkyl, C1-C4 haloalkyl;

[0041] R c The substitution is a substituted or unsubstituted C3-C6 cycloalkyl group, or a substituted or unsubstituted 4-8 membered heterocyclic group, wherein the substitution independently refers to being substituted by 1, 2, 3 or 4 substituents selected from the group consisting of: -OH, C1-C4 alkyl, and C1-C4 haloalkyl.

[0042] In another preferred embodiment, the prodrug of the compound has a structure represented by general formula III, IV, or V:

[0043] In the formula, R 0 R 1 R 2 The definition is the same as before, and R2 or NH is further replaced by R. In another preferred embodiment, each R is independently an amino acid, phosphate or phosphate ester structure; in another preferred embodiment, each R is independently a valine, alanine or phosphate structure.

[0044] In another preferred embodiment, R is

[0045] In another preferred embodiment, the compound or its prodrug is selected from the group consisting of:

[0046] The compounds of the present invention may also exist in the form of solvates (including hydrates), polymorphs, isotope labels (e.g., deuterated compounds), tautomers, or prodrugs, and these forms are also included within the scope of protection of the present invention.

[0047] In another preferred embodiment, the compound or its prodrug is selected from the group consisting of:

[0048] A second aspect of the invention provides a pharmaceutical composition comprising a compound of formula I as described in the first aspect, a stereoisomer thereof, a prodrug, or a pharmaceutically acceptable salt thereof; and

[0049] Pharmaceutically acceptable carrier.

[0050] A third aspect of the invention provides the use of the compound of formula I as described in the first aspect, its stereoisomers, prodrugs, or pharmaceutically acceptable salts thereof, or the pharmaceutical compositions described in the second aspect, for:

[0051] (1) Preparation of PDE3 modulators / inhibitors;

[0052] (2) Prepare a drug for treating diseases that simultaneously express PDE3 and SLFN12; preferably, PDE3 and SLFN12 are overexpressed;

[0053] (3) Preparation of a drug that regulates the interaction between PDE3 and SLFN12; preferably, the drug is capable of enhancing and / or promoting the interaction between PDE3 and SLFN12; or

[0054] (4) Promotes the formation of a molecular glue of PDE3 and SLFN12 into a complex.

[0055] In another preferred embodiment, the disease that simultaneously expresses PDE3 and SLFN12 is a tumor.

[0056] In another preferred embodiment, the tumor is selected from: brain cancer, breast cancer, cervical cancer, AML, lung cancer, skin cancer, esophageal cancer, ovarian cancer, pancreatic cancer, prostate cancer, melanoma, liver cancer, glioma, and sarcoma.

[0057] In another preferred embodiment, the tumor is selected from melanoma, glioma, ovarian cancer, or lung cancer.

[0058] In another preferred embodiment, the tumor is selected from: melanoma, glioma, glioblastoma, and non-small cell lung cancer (lung adenocarcinoma).

[0059] This invention introduces a primary amino group at the 4-position of the pyridazinone core, which can effectively improve the physicochemical properties of the molecule, increase its water solubility, improve its metabolic properties, and reduce its potential toxicity. At the same time, the introduction of a polar amino fragment can increase potential hydrogen bonding and other interactions, thereby increasing the molecule's antitumor activity.

[0060] In vitro cell experiments showed that this series of compounds can effectively inhibit tumor cell growth, with an IC50 inhibitory activity of 100%. 50 All compounds have a molecular weight of less than 100 nM, with some compounds less than 10 nM, exhibiting potent antitumor activity. Furthermore, they showed significant inhibitory activity against multiple different tumor cell lines, indicating broad antitumor potential. The addition of a PDE3 inhibitor effectively reversed the activity of this series of compounds, resulting in the loss of their antitumor activity, suggesting a PDE3-related molecular gel mechanism.

[0061] Animal metabolism studies have shown that this series of compounds have good oral bioavailability in rats, achieving sufficient in vivo drug efficacy exposure. Furthermore, their plasma clearance rate is significantly higher than that of compound YHHU-258, which has no amino substitution at the 4-position, and can be cleared within 24 hours without any drug accumulation trend.

[0062] The prodrug compound in this invention can be effectively hydrolyzed in animals to release active molecules, achieving sufficient drug efficacy exposure and further producing biological effects.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] Figure 1 shows the inhibitory effect of compound I-25 on the proliferation of several different tumor lines.

[0065] Figure 2 shows that representative compounds lose their inhibitory activity against tumor cell proliferation after PDE3 "knockout".

[0066] Figure 3 shows the in vivo concentration curves of compound I-1 in rats after oral and intravenous administration (po: 20 mg / kg, iv: 2.5 mg / kg).

[0067] Figure 4 shows the in vivo concentration curves of compound YHHU-258 in rats after oral and intravenous administration (po: 20 mg / kg, iv: 20 mg / kg). Detailed Implementation

[0068] Through extensive and in-depth research, the inventors have developed a class of 4-amino-substituted pyridazinone compounds. These compounds exhibit strong inhibitory activity against the proliferation of multiple tumor cell lines, including melanoma cells, in vitro, demonstrating their broad anti-tumor application potential. Furthermore, they exhibit favorable metabolic properties in animals, showing no significant drug accumulation trend and reducing potential toxicity. Based on these findings, this invention was completed.

[0069] the term

[0070] In this invention, unless otherwise specifically stated, the meaning of substituents is defined as follows:

[0071] The halogen atoms refer to F, Cl, Br, and I.

[0072] In this invention, the terms "C1-C6" refer to having 1, 2, 3, 4, 5, or 6 carbon atoms, "C1-C8" refers to having 1, 2, 3, 4, 5, 6, 7, or 8 carbon atoms, and so on. "3-8" refers to having 3, 4, 5, 6, 7, or 8 ring atoms, and so on.

[0073] In this invention, the term "alkyl" refers to a saturated linear or branched hydrocarbon group. For example, the term C1-C6 straight-chain or branched alkyl refers to a straight-chain or branched alkyl group having 1 to 6 carbon atoms. Specific examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethylbutyl, etc.

[0074] In this invention, the term "alkoxy" refers to an -O-(alkyl) group. For example, the term C1-C4 straight-chain or branched alkoxy refers to a straight-chain or branched alkoxy having 1 to 4 carbon atoms, and specific examples include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, etc.

[0075] In this invention, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon group. For example, the term C3-C6 cycloalkyl refers to a cyclic alkyl group having 3 to 6 carbon atoms, and specific examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc.

[0076] In this invention, the term "heterocyclic group" refers to a saturated or unsaturated, non-aromatic cyclic group containing at least one (e.g., 1, 2, 3, or 4) cyclic heteroatoms (e.g., N, O, or S), such as tetrahydropyridyl, pyrrolinyl, dihydropyridyl, dihydrofuranyl, dihydrothiophenyl, or morpholinyl.

[0077] In this invention, the term "aryl" refers to a hydrocarbon group comprising one or more aromatic rings. For example, the term "C6-C..." 10 "Aryl" refers to an aromatic cyclic group having 6 to 10 carbon atoms on the ring, such as phenyl and naphthyl. Examples of aryl groups include, but are not limited to, phenyl (Ph), naphthyl, pyrene, anthracene, and phenanthryl.

[0078] In this invention, the term "heteroaryl" refers to an aromatic cyclic group containing at least one (e.g., 1, 2, 3, or 4) cyclic heteroatoms (e.g., N, O, or S). For example, a 5-7 membered heteroaryl refers to an aromatic ring having 5-7 atoms on the ring. Specific examples include pyrroleyl, furanyl, thiopheneyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, pyrimidinyl, pyridazinyl, etc.

[0079] Unless otherwise stated, the alkyl, alkoxy, cycloalkyl, heteroaryl, heterocyclic, and aryl groups mentioned herein are substituted and unsubstituted groups. Possible substituents on the above groups include, but are not limited to: hydroxyl, amino, nitro, nitrile, halogen, C1-C6 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C3-C20 cycloalkyl, C3-C20 cycloalkenyl, C1-C20 heterocyclic alkyl, C1-C20 heterocyclic alkenyl, C1-C6 alkoxy, C6-C10 aryl, heteroaryl, heteroaryloxy, C1-C10 alkylamino, C1-C20 dialkylamino, and C6-C10 arylamino. The groups include alkyl, di-C6-C10 arylamino, C1-C10 alkylaminosulfonyl, C6-C10 arylaminosulfonyl, C1-C10 alkylimino, C1-C10 alkylsulfonylimino, C6-C10 arylsulfonylimino, mercapto, C1-C10 alkylthio, C1-C10 alkylsulfonyl, C6-C10 arylsulfonyl, acylamino, aminoacyl, aminothioacyl, guanidine, urea, cyano, acyl, thioacyl, acyloxy, carboxyl, and carboxylic acid ester groups. On the other hand, cycloalkyl, heterocyclic, aryl, and heteroaryl groups can also be fused together.

[0080] In this invention, the substitution can be monosubstituted or polysubstituted, and the polysubstituted can be disubstituted, trisubstituted, tetrasubstituted, or pentasubstituted. Disubstituted means having two substituents, and so on.

[0081] The pharmaceutically acceptable salts described in this invention can be inorganic acid salts, such as hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, carbonate, etc.; or organic acid salts, such as formate, acetate, propionate, oxalate, malonate, succinate, fumarate, maleate, adipic acid, lactate, malate, citrate, tartrate, carbonate, picrate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, glutamate, dihydroxynaphthyl salt, etc.

[0082] The compounds of this invention may also contain prodrugs; therefore, these prodrugs are also included within the scope of protection of the compounds of this invention. A prodrug refers to a compound obtained by chemically modifying a drug, which is inactive or has low activity in vitro, but releases an active drug in vivo through enzymatic or non-enzymatic conversion to exert its pharmacological effect. The form of the prodrug in this invention is not particularly limited, as long as it releases the active drug in vivo through enzymatic or chemical action to exert the expected pharmacological effect; it can be a carrier prodrug or a biological prodrug.

[0083] The compounds of the present invention can exist in a non-solventized form and a solvated form (also called a solvate) containing a pharmaceutically acceptable solvent (such as water, ethanol, etc.). The compounds of the present invention include both solvated and non-solventized forms. The solvate is a complex formed by a compound of general formula I and a pharmaceutically acceptable solvent. Optionally, the pharmaceutically acceptable solvent includes water, ethanol, acetic acid, N,N-dimethylformamide, or dimethyl sulfoxide, etc.

[0084] The compounds of the present invention may also exist in different tautomer forms, all of which are included within the scope of the present invention. The terms "tautomer" or "tautomer form" refer to structural isomers with different energies that interconvert via low energy barriers.

[0085] This invention also covers isotopically labeled compounds of the invention, which are identical to those described herein except that one or more atoms are replaced by atoms with atomic masses or mass numbers different from those commonly found in nature. Examples of isotopes that may be incorporated into the compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as: 2-hydrogen, 3-hydrogen, 11-carbon, 13-carbon, 14-carbon, 13-nitrogen, 15-nitrogen, 15-oxygen, 17-oxygen, 18-oxygen, 31-phosphorus, 32-phosphorus, 35-sulfur, 18-fluorine, 123-iodine, 125-iodine, and 36-chlorine. The isotopically labeled compounds of the invention can generally be prepared by following methods similar to those disclosed in the schemes and / or the examples below, by replacing non-isotopically labeled reagents with isotopically labeled reagents.

[0086] "Pharmaceutically acceptable carriers" refer to one or more compatible solid or liquid fillers or gel substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredient of the invention without significantly reducing the efficacy of the active ingredient. 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0087] Molecular glues (MG) are a class of compounds or molecules that play a crucial role in binding and stabilizing protein-protein interactions in biological systems. These molecules act as "glue" by enhancing the affinity between proteins, ultimately influencing various cellular processes. MG compounds have attracted widespread attention in drug discovery, chemical biology, and basic research due to their potential to regulate protein interactions, thereby affecting various cellular pathways. They have opened up medical pathways previously considered "drug-free." Molecular glues (MG drugs) differ from traditional drugs in their mechanism of action, characterized by a drug targeting two (or more) targets. That is, the drug molecule simultaneously or sequentially binds to two different proteins, forming a ternary complex that induces protein-protein interaction (PPI), one of which is the target protein (POI), resulting in structural degradation and loss of function. The pharmacological effect exhibited by this type of protein interaction is induced by the molecular glue, but the molecules themselves do not participate in the degradation or inhibition process; their role is to induce the event. Currently, there is no rational or systematic method for discovering molecular glues.

[0088] Preparation method

[0089] This invention provides a method for preparing the compound described above, the main steps of which are as follows:

[0090] Route 1:

[0091] Route 2:

[0092] Route 3:

[0093] Including the following steps:

[0094] Route 1: Trichlorpyridazine undergoes a nucleophilic reaction with ammonia to obtain compounds 1-2, which are then hydrolyzed under reflux with glacial acetic acid as the solvent, followed by removal of the acetyl group with NaOH to obtain compounds 1-3. These compounds are then subjected to a Suzuki coupling reaction with arylboronic acid or arylboronic ester to obtain the target compound.

[0095] Route 2: Compound 2-1 or 2-3 is obtained from Route 1, and undergoes a nucleophilic reaction with an amine or alcohol to obtain compound 2-2; then undergoes a Suzuki coupling reaction with arylboronic acid or borate ester to obtain compound 2-4;

[0096] Route 3: Compound 3-1 undergoes a nucleophilic reaction to give compound 3-2; then it reacts with a borate ester to give compound 3-3.

[0097] X is a halogen or other group capable of coupling reaction, and R... k1 ~R k5 The corresponding substituent can be monosubstituted or polysubstituted; specifically, R k1 It can be H, halogen, -CF3, -OCF3, -CN, C1-C6 alkoxy, C1-C6 alkyl or cycloalkyl, 5-8 membered heterocyclic group, and can be monosubstituted or polysubstituted; R k2 and R k4 It is a halogen, -CF3, -OCF3, C1-C6 alkoxy, C1-C6 alkyl, or cycloalkyl; R k3 It is a C1-C10 substituted or unsubstituted group, which can be chain-like or cyclic, and can contain 1 to 5 heteroatoms; R k5 For substituted or unsubstituted 5-8 aryl or heteroaryl groups; R k6 It is a C1-C6 group containing 1 to 5 heteroatoms.

[0098] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise 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 as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0099] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0100] In the following examples, 1H NMR spectra were recorded using a Bruker AMX-400 NMR spectrometer. Chemical shifts δ are expressed in ppm. NMR calibrations were: δH 7.26 ppm (CDCl3), 2.50 ppm (DMSO-d6), 3.15 ppm (CD3OD). Unless otherwise specified, all reaction solvents were purified using conventional methods. All solvents were analytical grade reagents, purchased from Sinopharm Chemical Reagent Co., Ltd. Ultraviolet fluorescence was used for colorimetric analysis. Removal of organic solvents under reduced pressure was performed in a rotary evaporator.

[0101] Example 1: Synthesis of Compound I-1

[0102] Synthesis 1-2: 20 g of compound 1-1 was weighed and added to a reaction flask, along with 150 ml of ammonia solution (0.25-0.28 by mass) and 150 ml of 1,4-dioxane. The mixture was stirred overnight at 75°C. After the reaction was complete as monitored by TLC, most of the solvent was removed by vacuum distillation. The mixture was extracted with ethyl acetate (150 ml x 3), washed with saturated brine, separated from the organic phase, dried over anhydrous sodium sulfate, filtered, and concentrated to give 17 g of a pale yellow solid (94.9% yield), which was used in the next reaction.

[0103] 1 H NMR (400MHz, DMSO-d6) δ7.16 (s, 2H), 6.82 (s, 1H); ESI-MS: m / z=164[M+H] + .

[0104] Synthesis 1-3: 10g of compound 1-2 was weighed and added to a round-bottom flask. 150ml of glacial acetic acid was added, and the mixture was heated to reflux at 120℃. TLC monitoring showed no residual starting material. The glacial acetic acid solution was removed by vacuum distillation. Ethyl acetate and water were added for extraction (150ml x 3 of ethyl acetate, 100ml of water). The mixture was washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated to give 8g of a pale yellow solid. The 8g pale yellow solid was dissolved in a mixture of 50ml methanol and 50ml water. 10g of sodium hydroxide solid was added, and the mixture was heated to 75℃. After the reaction was complete, TLC monitoring showed that the methanol was removed by vacuum concentration. Ethyl acetate was added for extraction (100ml x 3), and the mixture was washed with saturated brine. The organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered and concentrated, and column chromatography was performed to give 6.3g of a white solid, with a yield of 70%.

[0105] 1 H NMR (400MHz, DMSO-d6) δ12.61(s,1H),6.81(s,2H),6.18(s,1H); ESI-MS: m / z=146[M+H] + .

[0106] Synthesis I-1: 100 mg of compounds 1-3 were weighed into a 50 mL round-bottom flask, and 188 mg of the corresponding arylphenylboronic acid, 55 mg of catalyst Pd(dppf)₂Cl₂, 288 mg of potassium phosphate, 10 mL of 1,4-dioxane, and 2 mL of H₂O were added. The mixture was completely purged with nitrogen and reacted at 80 °C for 24 hours. The reaction was monitored by TLC until completion. 50 mL of ethyl acetate and 20 mL of water were added for extraction, followed by washing with saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to give 128 mg of a white solid, with a yield of 65%.

[0107] 1H NMR (400MHz, DMSO-d6) δ12.83(s,1H),8.14(d,J=2.2Hz,1H),8.02(dd,J=8.4,2. 2Hz, 1H), 7.80 (d, J=8.4Hz, 1H), 6.80 (s, 1H), 6.57 (s, 2H); ESI-MS: m / z=290[M+H] + .

[0108] Example 2: Synthesis of Compound I-2

[0109] I-2 Synthesis: Compound 2-1 was obtained according to the synthesis method of Example 1.

[0110] 1 H NMR (400MHz, DMSO-d6) δ12.79(s,1H),8.30–7.97(m,2H),7.78–7.49(m,1H),6.78(s,1H),6.54(s,2H); ESI-MS: m / z=274[M+H] + .

[0111] 100 mg of compound 2-1 was weighed into a 25 ml sealed reaction tube, and 1 ml of liquid cyclopentanamine was added. The reaction was carried out at 150 °C for 48 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with 50 ml of ethyl acetate and 20 ml of water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was then filtered, concentrated, and separated by column chromatography to give 60 mg of a white solid, with a yield of 51%.

[0112] 1 H NMR(400MHz,Chloroform-d)δ7.94(s,1H),7.73(d,J=8.9Hz,1H),6.94(d,J=8.9Hz,1 H),6.70(s,1H),4.95(s,2H),3.42(s,4H),2.03–1.91(m,4H); ESI-MS:m / z=325[M+H] + .

[0113] Example 3: Synthesis of Compound I-3

[0114] Synthesis of I-3: 100 mg of compound I-1 was weighed into a 50 mL round-bottom flask, and 48 mg of 4-pyridinephenylboronic acid, 27 mg of Xphos Pd G2, 35 mg of X-phos, 90 mg of potassium carbonate, 10 mL of 1,4-dioxane, and 2 mL of H2O were added. The mixture was completely purged with nitrogen and reacted at 80 °C for 24 hours. The reaction was monitored by TLC until completion. 50 mL of ethyl acetate and 20 mL of water were added for extraction. The mixture was washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and separated by column chromatography to give 95 mg of a white solid, with a yield of 84%.

[0115] 1 H NMR (400MHz, DMSO-d6) δ12.84(s,1H),8.68(d,J=5.1Hz,2H),8.19(d,J=1.7Hz,1H),8.09(dd,J=8.0,1. 7Hz,1H),7.54(d,J=8.0Hz,1H),7.40(d,J=5.1Hz,2H),6.86(s,1H),6.57(s,2H); ESI-MS:m / z=333[M+H] + .

[0116] Example 4: Synthesis of Compound I-4

[0117] Synthesis of I-4-2: 100 mg of compound 2-1 was weighed into a 25 mL sealed reaction tube, and 100 mg of 1-4-1 and 2 mL of DMSO were added. The reaction was carried out at 150 °C for 48 hours. After the reaction was completed, the mixture was cooled to room temperature, extracted with 50 mL of ethyl acetate and 20 mL of water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was then filtered, concentrated, and separated by column chromatography to give 73 mg of a colorless oily liquid, with a yield of 50%.

[0118] 1 H NMR(400MHz,DMSO-d6)δ12.72(s,1H),7.95(d,J=8.3Hz,1H),7.58(m,2H),6.76(s,1H),6.4 9(s,2H),3.92(m,4H),2.96(t,J=5.4Hz,4H),1.75(t,J=5.4Hz,4H); ESI-MS:m / z=397[M+H] + .

[0119] Synthesis of I-4: 73 mg of compound 1-4-2 was weighed into a 50 mL reaction flask, and 15 mL of 1 N dilute hydrochloric acid solution and tetrahydrofuran were added. The mixture was reacted at 100 °C for 24 hours. The reaction was monitored by TLC until completion. The mixture was extracted with 50 mL of ethyl acetate and 20 mL of water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and then separated by column chromatography to give 58 mg of a white solid (90% yield).

[0120] 1 H NMR(400MHz,Chloroform-d)δ10.13(s,1H),8.04(s,1H),7.90(d,J=8.4Hz,1H),7.40(d,J=8.4Hz, 1H), 6.72 (s, 1H), 5.05 (s, 2H), 3.27 (t, J=5.7Hz, 4H), 2.63 (t, J=5.7Hz, 4H); ESI-MS: m / z=353[M+H] + .

[0121] Example 5: Synthesis of Compound I-5

[0122] Synthesis of I-5-1: 12 mg of cuprous iodide was weighed into a 100 mL reaction flask, purged with nitrogen, and 40 mL of anhydrous tetrahydrofuran was added. Then, 1.64 mL of cyclopropylmagnesium bromide (0.5 M in THF) was added, and the mixture was stirred for 30 minutes in an ice bath. 200 mg of 3-1 solution was slowly added dropwise, and the mixture was allowed to return to room temperature overnight. The reaction was monitored by TLC until completion. The reaction was quenched by adding saturated ammonium chloride solution in an ice bath. Volatile solvents were removed by vacuum distillation. The mixture was extracted with 50 mL of ethyl acetate and 20 mL of water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and then separated by column chromatography to obtain 70 mg of a colorless oily liquid, with a yield of 40%.

[0123] 1 H NMR(400MHz,Chloroform-d)δ7.75–7.72(m,2H),7.49–7.40(m,1H),2.67(d,J=6.9Hz,2H),1 .04–0.87(m,1H),0.68–0.46(m,2H),0.24(dt,J=6.0,4.6Hz,2H); ESI-MS:m / z=278.99[M+H] + .

[0124] Synthesis of I-5-2: 70 mg of compound I-5-1, 77 mg of pinacol diborate, 10 mg of Pd(dppf)₂Cl₂, and 39 mg of potassium acetate were weighed into a 100 mL reaction flask and completely purged with nitrogen. 10 mL of 1,4-dioxane and 1 mL of water were added, and the mixture was reacted at 85 °C for 12 hours. The reaction was monitored by TLC until completion. 50 mL of ethyl acetate and 20 mL of water were added for extraction. The mixture was washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and separated by column chromatography to obtain 57 mg of a colorless oily liquid, with a yield of 70%.

[0125] 1 H NMR(400MHz,Chloroform-d)δ7.85–7.75(m,2H),7.56(d,J=7.7Hz,1H),2.74(d,J=6.9Hz,2H),1. 35(s,9H),1.08–0.95(m,1H),0.60–0.47(m,2H),0.25(q,J=5.1Hz,2H).ESI-MS:m / z=327.17[M+H] + .

[0126] Synthesis of I-5: 100 mg of compounds 1-3 were weighed into a 50 mL round-bottom flask, and 200 mg of the corresponding arylphenylboronic acid, 55 mg of catalyst Pd(dppf)₂Cl₂, 288 mg of potassium phosphate, 10 mL of 1,4-dioxane, and 2 mL of H₂O were added. The mixture was completely purged with nitrogen and reacted at 80 °C for 24 hours. The reaction was monitored by TLC until completion. 50 mL of ethyl acetate and 20 mL of water were added for extraction, followed by washing with saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 128 mg of a white solid, with a yield of 60%.

[0127] 1 H NMR (400MHz, DMSO-d6) δ12.75(s,1H),8.01(d,J=1.8Hz,1H),7.95(dd,J=8.2,1.9Hz,1H),7.73(d,J=8.1Hz,1H),6.79(s,1H),6.51(s,2 H),2.70(d,J=6.8Hz,2H),1.03(dq,J=12.5,6.1,5.6Hz,1H),0.61–0.46(m,2H),0.28(dd,J=4.8,1.5Hz,2H).ESI-MS:m / z=310.11[M+H] + .

[0128] Examples 6-44: Compounds I-6 to I-14, I-31 to I-33, and I-42 to I-44 were synthesized using Route 1, with a synthetic method similar to that in Example 1; compounds I-15 to I-19 were synthesized using Route 2, with a synthetic method similar to that in Example 3; compounds I-20 to I-30, and I-34 to I-41 were synthesized using Route 2, with a synthetic method similar to that in Example 2; relevant compound data are as follows:

[0129] Examples 45-47 are prodrug compounds that can be effectively hydrolyzed in animals to release active molecules and exert biological functions.

[0130] Example 45: Synthesis of Compound I-45

[0131] Synthesis I-45-1: 200 mg of compounds 1-3 were weighed into a 50 mL round-bottom flask, and 237 mg of 4-methoxybenzyl bromide, 675 mg of cesium carbonate, and 15 mL of DMF were added. The reaction was stirred at room temperature for 12 hours. The reaction was monitored by TLC until complete. The mixture was extracted with 50 mL of ethyl acetate and 20 mL of water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and separated by column chromatography to give 294 mg of a white solid, with a yield of 80%.

[0132] 1 H NMR (400MHz, DMSO-d6) δ7.24(d,J=8.2Hz,2H),6.91–6.87(m,4H),6.20(s,1H),5.07(s,2H),3.72(d,J=1.5Hz,3H).ESI-MS:m / z=266.06[M+H] + .

[0133] Synthesis of I-45-2: 294 mg of compound I-45-1 was weighed into a 50 mL round-bottom flask, and 481 mg of (Boc)₂O, 13 mg of DMAP, 285 mg of DIPEA, and 15 mL of dichloromethane were added. The reaction was stirred at room temperature for 12 hours. The reaction was monitored by TLC until completion. The volatile solvent was removed by vacuum distillation. The mixture was extracted with 50 mL of ethyl acetate and 20 mL of water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and separated by column chromatography to give 282 mg of a white solid, with a yield of 70%.

[0134] 1H NMR(400MHz,Chloroform-d)δ7.38(d,J=8.7Hz,2H),7.07(s,1H),6.84(d,J=8.7Hz,2H),5.21(s,2H),3.78(s,3H),1.41(s,9H).ESI-MS:m / z=366.11[M+H] + .

[0135] Synthesis of I-45-3: Intermediate I-45-3 was synthesized in a similar manner to that in Example 5, as a white solid with a yield of 50%.

[0136] 1 H NMR(400MHz,Chloroform-d)δ7.88(s,1H),7.77(d,J=8.3Hz,1H),6.56(d,J=8.3Hz,1H),4.64(s,1H),4.24–4.10(m,1H),3. 77(s,1H),3.59–3.42(m,1H),2.95(dtd,J=9.6,6.8,3.3Hz,2H),1.86–1.77(m,2H),1.32(s,12H).ESI-MS:m / z=358.17[M+H] + .

[0137] Synthesis of I-45-4: 282 mg of compound I-45-2 was weighed into a 50 mL round-bottom flask, and 358 mg of I-45-3, 60 mg of catalyst Pd(dppf)₂Cl₂, 328 mg of potassium phosphate, 20 mL of 1,4-dioxane, and 2 mL of H₂O were added. The mixture was completely purged with nitrogen and reacted at 80 °C for 24 hours. The reaction was monitored by TLC until completion. 50 mL of ethyl acetate and 20 mL of water were added for extraction, followed by washing with saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to obtain 281 mg of a white solid, with a yield of 65%.

[0138] 1 H NMR(400MHz,DMSO-d6)δ8.53(s,1H),8.07–7.97(m,1H),7.85–7.75(m,2H),7.38–7.17(m,2H),6.98–6.73(m,3H),5.52(s,1H),5.25(s ,2H),5.09(s,1H),4.00–3.80(m,1H),3.56–3.40(m,1H),2.81–2.65(m,2H),1.88–1.80(m,2H),1.49(s,9H).ESI-MS: m / z=561.22[M+H] + .

[0139] Synthesis of I-45-5: 160 mg of compound I-45-4 was weighed into a 50 mL round-bottom flask, and 75 mg of BOC-L-valine, 88 mg of N,N'-dicyclohexylcarbodiimide, 3.5 mg of DMAP, and 20 mL of dichloromethane were added. The mixture was stirred at room temperature for 12 hours. The reaction was monitored by TLC until completion. The volatile solvent was removed by vacuum distillation, and the mixture was extracted with 50 mL of ethyl acetate and 20 mL of water. The extract was washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The mixture was filtered, concentrated, and separated by column chromatography to give 195 mg of a white solid, with a yield of 90%.

[0140] 1 H NMR(400MHz,Chloroform-d)δ8.12(s,1H),7.98–7.92(m,2H),7.85–7.77(m,1H),7.42(d,J=8.4Hz,2 H),6.85(d,J=8.5Hz,2H),6.62(d,J=8.7Hz,1H),5.32(s,2H),5.03–4.81(m,2H),4.62(d,J=5.7Hz,1 H),4.25–4.16(m,1H),3.70(q,J=7.0Hz,1H),3.15–2.88(m,2H),2.20–2.11(m,1H),2.02–1.94(m,2H ),1.52(s,9H),1.45(s,9H),0.98(d,J=6.8Hz,3H),0.90(d,J=6.9Hz,3H).ESI-MS:m / z=760.35[M+H] + .

[0141] Synthesis of I-45: 195 mg of compound I-45-5 was weighed into a 50 mL round-bottom flask, and 10 mL of trifluoroacetic acid was added. The reaction was stirred at 40 °C for 12 hours. The reaction was monitored by TLC until completion. The volatile solvent was removed by vacuum distillation, and the pH was adjusted to 9–10 with saturated sodium bicarbonate solution. Extraction was performed with 50 mL of ethyl acetate and 10 mL of water, followed by washing with saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to give 107 mg of a white solid (95% yield).

[0142] 1H NMR(400MHz,DMSO-d6)δ12.55(s,1H),7.84–7.69(m,2H),6.83(d,J=8.8Hz,1H) ,6.69(s,1H),6.38(s,2H),5.69(d,J=6.4Hz,1H),4.85–4.67(m,1H),3.79–3.61 (m,1H),3.10(d,J=5.3Hz,1H),2.94–2.76(m,2H),2.21–2.04(m,2H),1.94–1.61 (m,3H),0.88(d,J=6.8Hz,3H),0.83(d,J=6.8Hz,3H).ESI-MS:m / z=440.18[M+H] + .

[0143] Example 46: Synthesis of Compound I-46

[0144] Synthesis of I-46-1: 183 mg N-Boc-L-Alanine, 486.4 mg N,N,N′,N′-tetramethyl-O-(7-azabenzotriazol-1-yl)hexafluorophosphate, and 16 mg 4-dimethylaminopyridine were weighed into a reaction flask. 5 mL of DMF was added and the mixture was stirred at room temperature. 165 mg of DIPEA was added dropwise to the reaction system and the mixture was stirred at room temperature for 30 minutes. 200 mg of compound I-31 was added to the reaction system and the mixture was stirred at room temperature for 48 hours. The reaction was monitored by TLC until completion. 30 mL of ethyl acetate and 10 mL of water were added for extraction. The mixture was washed with saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by column chromatography to give 171 mg of a white solid, with a yield of 50%.

[0145] 1 H NMR (600MHz, DMSO-d6) δ13.46(s,1H),12.38(s,1H),8.53(s,1H),8.05(dd,J=8.8,2.3Hz,1H),7.96(d,J=2.3Hz,1H),7.66(d,J=8.7H z,1H),4.11(tt,J=6.0,2.9Hz,1H),3.93–3.87(m,1H),2.99(s,3H),0.93–0.82(m,2H),0.79–0.65(m,2H).ESI-MS:m / z=483.46[M+H] + .

[0146] Synthesis of I-46: 171 mg of compound I-46-1 was weighed and dissolved in 10 mL of dichloromethane. 1 mL of trifluoroacetic acid was added dropwise under ice bath conditions, and the mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC until completion, and the volatile solvent was removed by vacuum distillation. The pH was adjusted to alkaline by adding saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate and water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and separated by column chromatography to give 116 mg of a white solid, with a yield of 95%.

[0147] 1 H NMR (600MHz, DMSO-d6) δ13.42(s,1H),8.54(s,1H),8.04(dd,J=8.7,2.3Hz,1H),7.96(d,J=2.3Hz,1H),7.67(d,J=8.8Hz,1H),4.11( dd,J=6.0,3.1Hz,1H),3.54(q,J=7.0Hz,1H),1.27(d,J=7.0Hz,3H),0.91–0.82(m,2H),0.75–0.64(m,2H).ESI-MS:m / z=383.12[M+H] + .

[0148] Example 47: Synthesis of Compound I-47

[0149] Synthesis of I-47-1: 200 mg of compound I-31 was weighed into a reaction flask, 252 mg of cesium carbonate was added, and 5 mL of DMF was added. The mixture was stirred at 0 °C for 5 minutes. Under ice bath conditions, 250 mg of di-tert-butylchloromethyl phosphate was added dropwise to the reaction system, and the mixture was stirred at room temperature for 6 hours. The reaction was monitored by TLC until completion. The mixture was extracted with ethyl acetate and water, washed with saturated brine, and the organic phase was separated and dried over anhydrous sodium sulfate. The solution was filtered, concentrated, and separated by column chromatography to give 206 mg of a white solid, with a yield of 60%.

[0150] 1 H NMR(400MHz,Chloroform-d)δ8.02(s,2H),7.95(d,J=2.2Hz,1H),7.92(dd,J=8.6Hz,2.2Hz,1H),7.40( d,J=8.6Hz,1H),6.67(s,1H),5.93(d,J=7.3Hz,2H),3.95–3.77(m,1H),1.50(s,18H),0.87–0.81(m,4H) . ESI-MS: m / z = 534.19 [M+H] + .

[0151] Synthesis of I-47: 206 mg of compound I-47-1 was weighed into a reaction flask, and 5 mL of glacial acetic acid solution and 5 mL of ethanol solution were added. The mixture was stirred at room temperature for 4 hours. The reaction was monitored by TLC until completion. The volatile solvent was removed by vacuum distillation, and the mixture was purified by C-18 column chromatography to obtain 114 mg of a white solid.

[0152] 1 H NMR (600MHz, DMSO-d6) δ8.03(d,J=10.1Hz,1H),7.95(s,1H),7.62(d,J=8.7Hz,1H),6.75(s,1H),6. 67(s,2H),5.73(d,J=6.8Hz,2H),4.09(m,1H),0.87(m,2H),0.70(m,2H).ESI-MS:m / z=422.07[M+H] + .

[0153] Example 48: Pharmacological tests to assess the in vitro inhibitory activity of the compound against tumor cell proliferation.

[0154] Experimental protocol: Healthy CHL-1 cells were seeded in 96-well plates at a density of 1800 cells / well and incubated overnight at 37°C with 5% CO2. The test drug was added to the 96-well plates to achieve a maximum final concentration of 10 μM, and then serially diluted 5-fold to obtain 7 concentration points. The plates were incubated at 37°C with 5% CO2 for 72 h. The IC50 of the drug was determined using the SRB method. 50 .

[0155] Reagents and consumables: DMEM medium (Shanghai Peiyuan Biotechnology, #L110KJ), fetal bovine serum (Gibco, #10902096), CHL-1 cells (ATCC, #CRL3619), 96-well microplates (Thermo Fisher, #167425), SRB (Sigma-Aldrich, #S9012), DMSO (Sigma-Aldrich, #D4540).

[0156] Table 1 below shows the pharmacological results of the compounds of the present invention. The data represent the in vitro inhibitory activity of the compounds against the proliferation of melanoma CHL-1 cells. Among them, YHHU-258 is a compound disclosed in CN101537006B, with the following structure: The structure of the reference compound is

[0157] Table 1. Inhibitory activity of compounds against CHL-1 cell proliferation in vitro

[0158] As shown in Table 1, the experimental results indicate that this series of compounds exhibited strong inhibitory activity against the proliferation of CHL-1 melanoma cells in vitro, with an IC50 value of [missing information]. 50 The values ​​were all less than 100 nM, with some compounds less than 10 nM, indicating significant antitumor effects. Among these, the control compounds, compared to compounds I-1 to I-44, lacked substituents at the meta position, resulting in significantly reduced activity.

[0159] Furthermore, the inhibitory activity of compound I-25 against multiple different tumor cell lines (human lung adenocarcinoma NCI-H2122, human glioma H4, human glioblastoma DBTRG-05MG, human non-small cell lung cancer NCI-H1563, human melanoma CHL-1, and human non-small cell lung cancer NCI-H647) was further tested. The results are shown in Figure 1. The results indicate that the compound exhibits significant in vitro proliferation inhibitory activity against multiple tumor cell lines, including human lung cancer cell lines and glioma cell lines, demonstrating its broad anti-tumor potential.

[0160] Example 49: Pharmacological experimental mechanism verification that PDE3 enzyme activity inhibitors can reverse the drug efficacy of compounds.

[0161] Experimental protocol: Well-grown CHL-1 cells were seeded in 96-well plates at a density of 1800 cells / well and cultured overnight at 37°C with 5% CO2. The test drug was added to the 96-well plates at a concentration of 1 μM. The plates were then cultured at 37°C with 5% CO2 for 72 h, and the inhibitory rate on cell proliferation was measured. The effect of the 1 μM test compound was reversed in the 96-well plates using 1 μM Trequinsin or 10 μM Cilostamide, and the reversal efficiency was determined using the SRB method.

[0162] Reagents and consumables: DMEM medium (Shanghai Peiyuan Biotechnology, #L110KJ), fetal bovine serum (Gibco, #10902096), CHL-1 cells (ATCC, #CRL3619), 96-well microplates (Thermo Fisher, #167425), SRB (Sigma-Aldrich, #S9012), DMSO (Sigma-Aldrich, #D4540), Trequinsin (MCE, #HY-18740A), Cilostamide (Selleck, #S5806).

[0163] Detection:

[0164] The results are shown in Figure 2. The results indicate that the addition of the PDE3 inhibitors 1 μM Trequinsin or 10 μM Cilostamide effectively reversed the drug's efficacy, and it lost its inhibitory activity on tumor cell proliferation. This suggests that the series of compounds exert their biological effects by inducing the formation of a molecular glue effect of the complex between PDE3 and SLFN12.

[0165] Example 50: Pharmacokinetic Study of the Compound in Rats

[0166] Experimental protocol:

[0167] Compound I-1: Six male SD rats (weighing 200-220g) were fasted for 12 hours before administration and continued to fast for 2 hours after administration. The room temperature was controlled at 18-29℃, the humidity at 30%-70%, and the rats were exposed to light for 12 hours and protected from light for 12 hours. The oral dose of Compound I-1 was 20mg / kg in 0.5% CMC-Na suspension; the intravenous dose was 2.5mg / kg, with a solvent volume ratio of DMSO / EtOH / PEG300 / 0.9% NaCl = 5 / 5 / 40 / 50. Blood samples were collected at different time points (0.05h, 0.25h, 0.75h, 2.00h, 4.00h, 8.00h, and 24.0h) to detect the drug concentration in the blood samples and calculate the corresponding pharmacokinetic parameters.

[0168] Compound YHHU-258:

[0169] Four healthy male SD rats, weighing 200–250 g, were administered the drug via gavage. The dosage was 20 mg / kg, and the administration volume was 10 ml / kg. The rats were fasted for 12 hours prior to administration but had free access to water. At 0.25, 0.5, 1.0, 2.0, 3.0, 5.0, 7.0, 9.0, and 24 hours post-administration, 0.3 ml of venous blood was collected from the retroocular venous plexus of the rats, placed in heparinized tubes, centrifuged at 3500 rpm for 10 min, and the plasma was separated and stored at 20°C for analysis.

[0170] Four healthy male SD rats, weighing 200–250 g, were administered the drug intravenously. The dosage was 20 mg / kg, and the administration volume was 10 ml / kg. The rats were fasted for 12 hours prior to administration but had free access to water. At 5 min, 15 min, 0.5, 1.0, 2.0, 4.0, 6.0, 9.0, and 24 h post-administration, 0.3 ml of venous blood was collected from the retroocular venous plexus of the rats. The blood samples were placed in heparinized tubes, centrifuged at 3500 rpm for 10 min, and the plasma was separated and stored at 20 °C for analysis.

[0171] Table 2. Pharmacokinetics of Compound I-1 and YHHU-258 in rats

[0172] As shown in Figures 3 and 4, the pharmacokinetic results indicate that compound I-1 has good oral bioavailability in rats, at 82.7%, achieving sufficient in vivo drug efficacy exposure. Furthermore, its plasma clearance rate (CL) is significantly higher than that of compound YHHU-258, which has no amino substitution at position 4, and it can be cleared within 24 hours without any drug accumulation trend.

[0173] Example 51: Pharmacokinetic Study of Prodrug Compounds I-45 to I-47 in Rats

[0174] Six male SD rats (weighing 200-220g) were fasted for 12 hours before drug administration and continued to be fasted for 2 hours after administration. The room temperature was controlled at 18-29℃, and the humidity at 30%-70%. They were exposed to light for 12 hours and then protected from light for 12 hours. Prodrug compounds I-45-I-47 and active drugs I-25 and I-31 were orally administered. Blood samples were collected at different time points (0.05h, 0.25h, 0.75h, 2.00h, 4.00h, 8.00h, and 24.0h) to detect the drug concentrations of prodrug and active molecules in the blood samples, and the corresponding pharmacokinetic parameters were calculated. The results are shown in the table below.

[0175] Table 3. Pharmacokinetics of compounds I-45 and I-25 in rats

[0176] Table 4. Pharmacokinetics of compounds I-46 and I-31 in rats

[0177] Table 5. Pharmacokinetics of compounds I-47 and I-31 in rats

[0178] The pharmacokinetic results are shown in the table above. After oral administration, prodrug molecules I-45 to I-47 can be effectively hydrolyzed in rats to release active molecules I-25 or I-31. The drug exposure is significantly better than that of active molecules I-25 or I-31 alone, achieving sufficient drug efficacy exposure and thus further exerting biological effects.

[0179] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A compound represented by Formula I, a stereoisomer, a prodrug, or a pharmaceutically acceptable salt thereof, in: R 0 selected from H, -CF3, -OCF3, halogen, C1-C6alkoxy, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl; R 1 selected from -CF3, -OCF3, halogen, C1-C6alkoxy, substituted or unsubstituted C1-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl; R 2 Selected from halogens, -CN, substituted or unsubstituted C1-C 10 Alkyl, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclic, wherein, R a and R b Each is independently H, substituted or unsubstituted C1-C 10 Alkyl groups, substituted or unsubstituted C3-C6 cycloalkyl groups, substituted or unsubstituted 4-8 membered heterocyclic groups, or R a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, 4, or 5 heteroatoms. R c substituted or unsubstituted C1-C 10 alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclyl; Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl. Compounds of formula I do not include 2. The compound, stereoisomer, prodrug or pharmaceutically acceptable salt thereof of claim 1, wherein, R 0 selected from H, halogen, C1-C4alkoxy; R 1 selected from -CF3, -OCF3, halogen, C1-C4alkoxy, C1-C4alkyl, C3-C6cycloalkyl.

3. The compound, stereoisomer, prodrug or pharmaceutically acceptable salt thereof of claim 1, wherein R 2 Selected from -CN, C2-C6 alkyl, substituted C1-C6 alkyl, -N(R) a (R) b ), -O(R c ), substituted or unsubstituted C6-C 10 Aryl, substituted or unsubstituted 5-7 membered heteroaryl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted 4-7 membered heterocyclic, wherein, R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted 4-8 membered heterocyclic group, or R. a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, or 4 heteroatoms. R c substituted C1-C6alkyl, C3-C6alkyl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclyl; Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl.

4. The compound, stereoisomer, prodrug or pharmaceutically acceptable salt thereof of claim 1, wherein The compounds have a structure according to Formula II: R 2 , R 1 The definitions are as in claim 1.

5. The compound, stereoisomer, prodrug or pharmaceutically acceptable salt thereof of claim 4, wherein, R 1 selected from -CF3, -OCF3, F, Cl, Br, methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, cyclopropyl, cyclobutyl.

6. The compound, stereoisomer, prodrug or pharmaceutically acceptable salt thereof of claim 4, wherein R 2 selected from cyano, C1-C6haloalkyl, -N(R a )(R b ), -O(R c ), substituted or unsubstituted C6-C 10 aryl, substituted or unsubstituted 5-8 membered heteroaryl, substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclyl, wherein, R a and R b Each of the following is independently H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted 4-6 membered heterocyclic group, or R. a and R b Together with N, they form substituted or unsubstituted 4-8 membered heterocycles, said heterocycles containing 1, 2, 3, 4, or 5 heteroatoms. R c substituted or unsubstituted C3-C6cycloalkyl, substituted or unsubstituted 4-8 membered heterocyclyl; Each of the above substitutions refers independently to substitution by one or more substituents selected from the group consisting of: -OH, =O, -NH2, -NH(CH3), -N(CH3)2, halogen, C1-C4 alkyl, C1-C4 haloalkyl, and C3-C6 cycloalkyl.

7. The compound, stereoisomer, prodrug or pharmaceutically acceptable salt thereof of claim 1, wherein the compound or prodrug thereof is selected from the group consisting of:

8. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1-7, a stereoisomer thereof, a prodrug, or a pharmaceutically acceptable salt thereof; and Pharmaceutically acceptable carrier.

9. Use of the compound of Formula I as claimed in any one of claims 1-7, its stereoisomers, prodrugs, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of claim 8, for the purpose of: (1) Preparation of PDE modulators / inhibitors; (2) Prepare a drug for treating diseases that simultaneously express PDE3 and SLFN12; preferably, PDE3 and SLFN12 are overexpressed; (3) Preparation of a drug that regulates the interaction between PDE3 and SLFN12; preferably, the drug is capable of enhancing and / or promoting the interaction between PDE3 and SLFN12; or (4) Promotes the formation of a molecular glue of PDE3 and SLFN12 into a complex.

10. The use according to claim 9, characterized in that, The disease that simultaneously expresses PDE3 and SLFN12 is a tumor, preferably melanoma, glioma, ovarian cancer or lung cancer.

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