HK40008902B - Cdk4/6 inhibitor - Google Patents

Description

CDK4/6 inhibitors

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application CN201611170508.1 filed on December 16, 2016 and Chinese patent application CN 201710787583.0 filed on September 4, 2017, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to a series of compounds serving as CDK4/6 inhibitors, and specifically discloses compounds represented by formula (I), pharmaceutically acceptable salts thereof or isomers thereof, pharmaceutical compositions containing the same, and their use in preparing drugs for treating cancer.

Background Art

The cell cycle is the continuous, dynamic process that a normally dividing cell undergoes from the end of one mitosis to the end of the next. The mammalian cell cycle consists of four phases: G1 (pre-DNA synthesis), S (DNA synthesis), G2 (late DNA synthesis), and M (mitosis). The M phase is followed by cytokinesis, resulting in the formation of two daughter cells. Although the newly formed cells re-enter the cell cycle after cell cycle division, at a certain point in late G1 (called the restriction point or R point), cell cycle regulatory mechanisms determine the cell's ultimate fate: continued cell cycle cycling or exiting active proliferation and entering a quiescent (G0) state. Cell cycle regulation is primarily influenced by a series of serine/threonine kinases, also known as cyclin-dependent kinases (CDKs). These kinases regulate the cell cycle by binding to their corresponding regulatory subunits, cyclins. To date, at least 10 cyclin-dependent kinases (CDKs) and 15 cyclins have been identified. They can form the following pairing complexes: CDK1 pairs with cyclin A or B; CDK2 pairs with cyclin A or E; CDK3 pairs with an unknown cyclin; CDK4 pairs with cyclin D (1-3); CDK5 pairs with cyclin D or p35Nck5A; CDK6 pairs with cyclin D; CDK7 pairs with cyclin H; CDK8 pairs with cyclin C; CDK9 pairs with cyclin T.

Abnormal cell proliferation and dysregulation of the normal cell cycle are common hallmarks of all cancer types. For this reason, inhibitors of key cell cycle regulators have become attractive novel anti-tumor targets. During the early G1 phase of the cell cycle, a complex formed by CDK4/6 and cyclin D is activated by extracellular growth factors. This activated complex phosphorylates the retinoblastoma protein (RB), releasing the transcription factor E2F to which it binds in its unphosphorylated state. E2F activation further promotes transcriptional activation, driving the cell cycle past the R point and progression from G1 to S phase. After crossing the R point, other cyclins are sequentially activated, regulating the entire cell cycle. For example, cyclin E binds to CDK2 to control entry into S phase; cyclin A binds to CDK2 to control S phase progression; then, during G2, cyclin A binds to CDK1; and finally, cyclin B binds to CDK1 to control entry into mitosis. The complex formed by CDK4/6 and cyclin D is a key "master switch" in cell cycle regulation. Inhibiting CDK4/6 and preventing it from forming the Cyclin D-CDK4/6 complex can block the progression of the cell cycle from the G1 phase to the S phase, thereby inhibiting tumor proliferation. Therefore, CDK4/6 has become an important anti-cancer target.

In recent years, several small molecule CDK4/6 inhibitors have entered clinical trials for the treatment of cancer, either as monotherapy or in combination. Based on interim data from the Phase II PALOMA-1 clinical trial, the FDA approved palbociclib in February 2015 for use in combination with letrozole as a first-line treatment for postmenopausal women with ER-positive/HER2-negative metastatic breast cancer. Palbociclib is also currently undergoing Phase III clinical trials for the treatment of non-small cell lung cancer. Furthermore, based on results from the Phase III MONALEESA-2 trial, the US FDA granted Breakthrough Therapy designation to the CDK4/6 inhibitor ribociclib (LEE-011) in August 2016 for use in combination with letrozole as a first-line treatment for advanced or metastatic hormone receptor-positive/HER2-negative breast cancer. Eli Lilly and Company's CDK4/6 inhibitor abemaciclib (LY2835219) is also in the Phase III MONARCH 2 clinical trial, with final results expected in the first half of 2017. In addition to breast cancer treatment, these small molecule heterocyclic compounds can also be used clinically to treat a variety of other cancers. These patents include WO2014128588, WO2012018540, WO2012129344, WO2011101409, WO2011130232, WO2010075074, WO2009126584, WO2008032157, WO2005094830, WO2005117980, and WO2003062236.

Although much effort has been made to develop CDK4/6 inhibitors for the treatment of cancer and other diseases, only one drug targeting this target (Palbociclib) has been marketed so far, and its indication is only ER-positive/HER2-negative postmenopausal metastatic breast cancer. Although clinical research on CDK4/6 inhibitors for lung cancer has progressed to Phase III clinical trials, no drugs have yet been marketed. Therefore, there is still an urgent need to develop novel, safer and more effective CDK4/6 inhibitors that can treat multiple cancers (including lung cancer). On the other hand, although Palbociclib has been approved for marketing, literature has reported that its brain penetration is poor, making it difficult for it to cross the blood-brain barrier and unable to treat cancer with brain metastases.

Summary of the Invention

In one aspect, the present invention provides a compound represented by formula (I), a pharmaceutically acceptable salt thereof or an isomer thereof,

in,

_R1 is selected from H, or C1-3 alkyl, C1-3 heteroalkyl, which are optionally substituted by 1 _{, 2 or} 3 R, _R2 is independently selected from H, OH, CN, halogen, or _C1-5 alkyl, _{C1-5 heteroalkyl} , C3-6 cycloalkyl, _3-6 membered heterocycloalkyl, which are optionally substituted by 1, 2 or 3 R;

Ring A is a 4- to 11-membered heterocycloalkyl group;

Ring B is selected from: C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, optionally substituted with 1, 2 or 3 R;

R is selected from halogen, OH, CN, NH ₂ , NO ₂ , or selected from: C _1-3 alkyl, C _1-3 heteroalkyl, optionally substituted with 1, 2 or 3 R′;

R′ is selected from the group consisting of: F, Cl, Br, I, OH, CN, NH ₂ ;

The "hetero" of the C _1-3 heteroalkyl, C _1-5 heteroalkyl, 3-6 membered heterocycloalkyl, 4-11 membered heterocycloalkyl, and 5-6 membered heteroaryl is independently selected from the group consisting of: N, -O-, -S-, -NH-, -(C=O)-, -(S=O)-, and -(S=O) ₂ -;

In any of the above cases, the number of heteroatoms or heteroatom groups is independently selected from 1, 2 or 3.

In some embodiments of the present invention, R is selected from F, _Cl , Br, OH, CN, _NH2 , _CH3 , _CH3CH2 , _CH3O , _CF3 , _CHF2 , _CH2F , and other variables are as defined in the present invention.

In some embodiments of the present invention, R ₁ is selected from H, or CH ₃ , CH ₃ CH ₂ , CH ₃ (C═O)—, optionally substituted with 1, 2 or 3 R groups, and R and other variables are as defined herein.

In some embodiments of the present invention, R ₁ is selected from CH ₃ , CHF ₂ , CH ₃ (C═O)—, and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring B is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, and phenyl, optionally substituted with 1, 2, or 3 R groups, wherein R and other variables are as defined herein.

In some embodiments of the present invention, the ring B is selected from: cyclopentyl, cyclohexyl, phenyl, and other variables are as defined in the present invention.

In some embodiments of the present invention, R ₂ is independently selected from H, OH, CN, F, Cl, or CH ₃ , oxetanyl, piperazinyl, morpholinyl, optionally substituted with 1, 2 or 3 R, and R and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is independently selected from H, or selected from CH 3 optionally substituted with 1, 2 or 3 R: CH ₃ , R and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is independently selected from: H, CH ₃ , and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is a 5- to 9-membered heterocycloalkyl group, and other variables are as defined herein.

In some embodiments of the present invention, the structural unit is selected from:

_R2 and other variables are as defined herein.

In some embodiments of the present invention, the above structural unit is selected from: R ₂ and other variables are as defined in the present invention.

In some embodiments of the present invention, the above structural units are selected from: other variables are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer is selected from:

wherein R ₂ , R and ring A are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer is selected from:

wherein R ₁ and R ₂ are as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer is selected from:

wherein R ₂ is as defined in the present invention.

In some embodiments of the present invention, _R1 is selected from H, or selected from C1-3 alkyl, _C1-3 heteroalkyl, which are optionally substituted by 1 _{, 2} or 3 R; _R2 is independently selected from H, OH, CN, halogen, or selected from _C1-5 alkyl, _C1-5 heteroalkyl, _{C3-6 cycloalkyl, 3-6} membered heterocycloalkyl, which are optionally substituted by 1, 2 or 3 R;

Ring A is selected from a 4- to 11-membered heterocyclic group;

Ring B is selected from: C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, optionally substituted with 1, 2 or 3 R;

R is selected from halogen, OH, CN, NH ₂ , or selected from: C _1-3 alkyl, C _1-3 heteroalkyl, optionally substituted with 1, 2 or 3 R';

R' is selected from the group consisting of: F, Cl, Br, I, OH, CN, NH ₂ ;

The "hetero" of the C _1-3 heteroalkyl, C _1-5 heteroalkyl, 3-6 membered heterocycloalkyl, 4-11 membered heterocyclyl, and 5-6 membered heteroaryl is independently selected from the group consisting of: N, -O-, =O, -S-, -NH-, -(C=O)-, -(S=O)-, and -(S=O) ₂ -;

In any of the above cases, the number of heteroatoms or heteroatom groups is independently selected from 1, 2 or 3.

_In some embodiments of the present invention, R is selected from F, Cl, Br, OH, CN, _NH2 , _CH3 , _CH3CH2 , _CH3O , _CF3 , _CHF2 , _CH2F , and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₁ is selected from H, or selected from CH ₃ , CH ₃ CH ₂ , and CH ₃ (C═O) optionally substituted with 1, 2, or 3 Rs.

In some embodiments of the present invention, R ₁ is selected from CH ₃ , CHF ₂ , and CH ₃ (C═O), and R and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring B is selected from the group consisting of cyclobutyl, cyclopentyl, cyclohexyl, and phenyl, optionally substituted with 1, 2, or 3 R groups, wherein R and other variables are as defined herein.

In some embodiments of the present invention, the ring B is selected from: cyclopentyl, cyclohexyl, phenyl, and other variables are as defined in the present invention.

In some embodiments of the present invention, R ₂ is selected from H, OH, CN, F, Cl, or CH ₃ , oxetanyl, piperazinyl, morpholinyl, optionally substituted with 1, 2 or 3 R, and R and other variables are as defined in the present invention.

In some embodiments of the present invention, R ₂ is selected from H, or is selected from CH 3 optionally substituted with 1, 2 or 3 R: CH ₃ , R and other variables are as defined in the present invention.

In some embodiments of the present invention, the above R ₂ is selected from: H, CH ₃ ,

In some embodiments of the present invention, the ring A is selected from: m is independently selected from 0, 1 or 2; X is independently selected from CH ₂ , NH or O; Y is independently selected from CH or N, and other variables are as defined in the present invention.

In some embodiments of the present invention, the ring A is selected from: and other variables are as defined in the present invention.

In some embodiments of the present invention, the above structural unit is selected from: R ₂ and other variables are as defined in the present invention.

In some embodiments of the present invention, the above structural units are selected from: other variables are as defined in the present invention.

In some embodiments of the present invention, the compound is selected from:

Wherein, R ₂ and Ring A are as defined in the present invention.

In some embodiments of the present invention, the above compound is selected from

wherein R ₂ is as defined in the present invention.

In some embodiments of the present invention, the above-mentioned compound, its pharmaceutically acceptable salt or its isomer is selected from:

Some other solutions of the present invention are obtained by arbitrarily combining the above variables.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of any one of the above-mentioned compounds, pharmaceutically acceptable salts or isomers thereof, and a pharmaceutically acceptable carrier.

The present invention also provides the use of the above-mentioned compound, its pharmaceutically acceptable salt or isomer or the above-mentioned pharmaceutical composition in the preparation of a drug for treating cancer.

Definition and Description

Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and/or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to salts of the compounds of the present invention, prepared by reacting the compounds of the present invention with relatively nontoxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts, or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of acid in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, bisulfate, hydroiodic acid, phosphorous acid, and the like; and organic acid salts such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; and salts of amino acids such as arginine, and organic acids such as glucuronic acid (see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functional groups and can be converted into either base or acid addition salts.

Preferably, the neutral form of the compound is regenerated by contacting the salt with a base or acid in a conventional manner and isolating the parent compound. The parent form of the compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents.

As used herein, "pharmaceutically acceptable salts" are derivatives of the compounds of the present invention wherein the parent compound is modified by acid or base salt formation. Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of bases such as amines, alkali metal or organic salts of acids such as carboxylic acids, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound, such as salts formed with non-toxic inorganic or organic acids. Conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from the group consisting of 2-acetoxybenzoic acid, 2-hydroxyethanesulfonic acid, acetic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, bicarbonate, carbonic acid, citric acid, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptose, gluconic acid, glutamic acid, glycolic acid, hydrobromic acid, hydrochloric acid, hydroiodide, hydroxy, hydroxynaphthalene, isethionic acid, lactic acid, lactose, dodecylsulfonic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, nitric acid, oxalic acid, pamoic acid, pantothenic acid, phenylacetic acid, phosphoric acid, polygalacturonic acid, propionic acid, salicylic acid, stearic acid, acetic acid, succinic acid, sulfamic acid, p-aminobenzenesulfonic acid, sulfuric acid, tannin, tartaric acid, and p-toluenesulfonic acid.

The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in water or an organic solvent, or a mixture of the two. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.

In addition to the form of salts, the compounds provided by the present invention also exist in prodrug form. The prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to be converted into the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

Certain compounds of the present invention may exist in unsolvated forms as well as solvated forms, including hydrates. In general, the solvated forms are equivalent to the unsolvated forms and are encompassed within the scope of the present invention.

Certain compounds of the present invention may have asymmetric carbon atoms (optical centers) or double bonds. Racemates, diastereomers, geometric isomers and individual isomers are all within the scope of the present invention.

Unless otherwise indicated, the absolute configuration of a stereocenter is represented by a solid wedge and a dashed wedge bond, the relative configuration of a stereocenter is represented by a solid straight bond and a dashed straight bond, and a solid wedge or a dashed wedge bond or a solid straight bond and a dashed straight bond are represented by a wavy line.

When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, are intended to include both E and Z geometric isomers. Likewise, all tautomeric forms are encompassed within the scope of the present invention.

The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis- and trans-isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are encompassed within the scope of the present invention.

Unless otherwise indicated, the term "enantiomer" or "optical isomer" refers to stereoisomers that are mirror images of one another.

Unless otherwise indicated, the term "cis-trans isomers" or "geometric isomers" arises from the inability to rotate freely about double bonds or single bonds forming ring carbon atoms.

Unless otherwise indicated, the term "diastereomer" refers to stereoisomers that have two or more chiral centers and that are not mirror images of each other.

Unless otherwise indicated, "(D)" or "(+)" indicates dextrorotatory, "(L)" or "(-)" indicates levorotatory, and "(DL)" or "(±)" indicates racemic.

The compounds of the present invention may exist in specific forms. Unless otherwise indicated, the term "tautomer" or "tautomeric form" means that at room temperature, different functional group isomers are in dynamic equilibrium and can quickly convert to each other. If tautomerism is possible (such as in solution), the chemical equilibrium of the tautomers can be achieved. For example, proton tautomers (proton tautomers) (also known as prototropic tautomers) include interconversions carried out by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence isomers (valence tautomers) include interconversions carried out by the reorganization of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the two tautomers of pentane-2,4-dione and 4-hydroxypent-3-ene-2-one.

Unless otherwise indicated, the terms "enriched in one isomer", "isomerically enriched", "enriched in one enantiomer" or "enantiomerically enriched" mean that the content of one isomer or enantiomer is less than 100%, and the content of that isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise indicated, the term "isomer excess" or "enantiomeric excess" refers to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the content of the other isomer or enantiomer is 10%, the isomer or enantiomeric excess (ee value) is 80%.

Optically active (R)- and (S)-isomers, as well as D and L isomers, can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a diastereomeric salt is formed with an appropriate optically active acid or base, and then the diastereoisomers are resolved by conventional methods known in the art, and then the pure enantiomer is recovered. In addition, the separation of enantiomers and diastereomers is typically accomplished by using chromatography, which employs a chiral stationary phase and is optionally combined with a chemical derivatization method (e.g., carbamate formation from an amine).

The compounds of the present invention may contain unnatural proportions of atomic isotopes on one or more atoms comprising the compound. For example, the compound may be labeled with a radioactive isotope, such as tritium ( ³ H), iodine-125 ( ¹²⁵ I), or carbon-14 ( ¹⁴ C). As another example, deuterated drugs may be formed by replacing hydrogen with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared to non-deuterated drugs, deuterated drugs have advantages such as reduced toxic side effects, increased drug stability, enhanced therapeutic efficacy, and prolonged drug biological half-life. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of the present invention.

The term "pharmaceutically acceptable carrier" refers to any formulation or carrier medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical medicine. For additional information on carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For the oral dosage forms of the present invention, an "effective amount" of an active substance in the composition means the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may be substituted or unsubstituted, and unless otherwise specified, the type and number of substituents can be any on the basis of chemical achievable.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 0-2 Rs, the group may be optionally substituted with up to two Rs, with each occurrence of R being an independent choice. Furthermore, combinations of substituents and/or their variants are permissible only if such combinations result in stable compounds.

When the number of a linking group is 0, such as -(CRR)0-, it means that the linking group is a single bond.

When one of the variables is selected from a single bond, it means that the two groups it connects are directly connected. For example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When a substituent is vacant, it means that the substituent does not exist. For example, when X is vacant in A-X, it means that the structure is actually A.

When a substituent's bond crosses two atoms on a ring, the substituent may be bonded to any atom on the ring. When a substituent is listed without specifying the atom through which it is bonded to a compound included but not specifically mentioned in the general chemical formula, the substituent may be bonded to any atom therein. Combinations of substituents and/or their variants are permitted only if such combinations result in stable compounds. For example, a structural unit may indicate that substitution may occur at any position on a cyclohexyl group or a cyclohexadiene group.

When the linking group is listed without specifying its connection direction, its connection direction is arbitrary. For example, when the linking group L is -M-W-, -M-W- can connect ring A and ring B in the same direction as the reading order from left to right, or it can connect ring A and ring B in the opposite direction as the reading order from left to right.

Unless otherwise specified, the term "hetero" means a heteroatom or heteroatom group (i.e., a group containing heteroatoms), including atoms other than carbon (C) and hydrogen (H), and groups containing these heteroatoms, for example, including oxygen (O), nitrogen (N), sulfur (S), silicon (Si), germanium (Ge), aluminum (Al), boron (B), -O-, -S-, ＝O, ＝S, -C(＝O)O-, -C(＝O)-, -C(＝S)-, -S(＝O), -S(＝O) _2- , and optionally substituted -C(＝O)N(H)-, -N(H)-, -C(＝NH)-, -S(＝O) _2N (H)-, or -S(＝O)N(H)-.

Unless otherwise specified, "ring" means a substituted or unsubstituted cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, aryl or heteroaryl group. The so-called ring includes a monocyclic, linked, spirocyclic, fused or bridged ring. The number of atoms in the ring is usually defined as the number of ring members, for example, "5- to 7-membered ring" means 5 to 7 atoms arranged around. Unless otherwise specified, the ring optionally contains 1 to 3 heteroatoms. Therefore, "5- to 7-membered ring" includes, for example, phenyl, pyridine and piperidinyl; on the other hand, the term "5- to 7-membered heterocycloalkyl ring" includes pyridinyl and piperidinyl, but does not include phenyl. The term "ring" also includes a ring system containing at least one ring, each of which "rings" independently meets the above definition.

Unless otherwise specified, the term "heterocycle" or "heterocyclyl" means a stable monocyclic, bicyclic, or tricyclic ring containing a heteroatom or heteroatom group, which may be saturated, partially unsaturated, or unsaturated (aromatic), and comprises carbon atoms and 1, 2, 3, or 4 ring heteroatoms independently selected from N, O, and S, wherein any of the above heterocycles may be fused to a benzene ring to form a bicyclic ring. The nitrogen and sulfur heteroatoms may optionally be oxidized (i.e., NO and S(O)p, where p is 1 or 2). The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or other substituents as defined herein). The heterocycle may be attached to any heteroatom or carbon atom pendant to form a stable structure. The heterocycles described herein may be substituted at the carbon or nitrogen positions if the resulting compound is stable. Nitrogen atoms in the heterocycle may be quaternized. Preferably, when the total number of S and O atoms in the heterocycle exceeds 1, the heteroatoms are not adjacent to each other. Another preferred embodiment is that the total number of S and O atoms in the heterocyclic ring does not exceed 1. As used herein, the term "aromatic heterocyclic group" or "heteroaryl" refers to a stable 5-, 6-, or 7-membered monocyclic or bicyclic or 7-, 8-, 9-, or 10-membered bicyclic heterocyclic aromatic ring comprising carbon atoms and 1, 2, 3, or 4 ring heteroatoms independently selected from N, O, and S. The nitrogen atom may be substituted or unsubstituted (i.e., N or NR, where R is H or another substituent as defined herein). The nitrogen and sulfur heteroatoms may be optionally oxidized (i.e., NO and S(O)p, where p is 1 or 2). Notably, the total number of S and O atoms in the aromatic heterocyclic ring does not exceed 1. Bridged rings are also included in the definition of heterocycle. A bridged ring is formed when one or more atoms (i.e., C, O, N, or S) link two non-adjacent carbon atoms or nitrogen atoms. Preferred bridged rings include, but are not limited to, one carbon atom, two carbon atoms, one nitrogen atom, two nitrogen atoms, and a carbon-nitrogen group. Notably, a bridge always converts a monocyclic ring into a tricyclic ring. In bridged rings, substituents on the ring may also be present on the bridge.

Examples of heterocyclic compounds include, but are not limited to, acridinyl, azcinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzothiazolyl, benzotriazolyl, benzotetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromene, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3-b]tetrahydro furanyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolene, indolinyl, indolizinyl, indolyl, 3H-indolyl, isobenzofuranyl, isoindolyl, isoindolinyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, Oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazine, phenothiazine, benzoxanthinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridoxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl , tetrahydrofuranyl, tetrahydroisoquinolyl, tetrahydroquinolyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, isothiazolylthienyl, thienoxazolyl, thienothiazolyl, thienoimidazolyl, thienyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl. Also included are fused ring and spirocyclic compounds.

Unless otherwise specified, the term "hydrocarbyl" or its subordinate concepts (such as alkyl, alkenyl, alkynyl, aryl, etc.) by itself or as part of another substituent means a straight-chain, branched or cyclic hydrocarbon radical or combinations thereof, which can be fully saturated (such as alkyl), mono- or polyunsaturated (such as alkenyl, alkynyl, aryl), can be monosubstituted or polysubstituted, can be monovalent (such as methyl), divalent (such as methylene) or polyvalent (such as methine), can include divalent or polyvalent radicals, have the specified number of carbon atoms (such as C1-C12 means 1 to 12 carbons, _C1-12 is selected from _C1 , _C2 , _C3 , _C4 , _C5 , _C6 , _C7 , _C8 , _C9 , _C10 , _C11 and _C12 ; _C3-12 is selected from _C3 , _C4 , _C5 , _C6 , _C7 , C8 _, , C ₉ , C ₁₀ , C ₁₁ and C _12. ). "Hydrocarbon" includes but is not limited to aliphatic and aromatic hydrocarbon groups, the aliphatic hydrocarbon group includes chain and ring, specifically but not limited to alkyl, alkenyl, alkynyl, the aromatic hydrocarbon group includes but is not limited to 6-12 membered aromatic hydrocarbon groups, such as benzene, naphthalene, etc. In some embodiments, the term "hydrocarbon" represents a linear or branched atomic group or a combination thereof, which can be fully saturated, mono- or polyunsaturated, and can include divalent and polyvalent atomic groups. Examples of saturated hydrocarbon atomic groups include but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, sec-butyl, isobutyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, and homologues or isomers of atomic groups such as n-pentyl, n-hexyl, n-heptyl, and n-octyl. Unsaturated hydrocarbon groups have one or more double or triple bonds, examples of which include, but are not limited to, ethenyl, 2-propenyl, butenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and higher homologs and isomers.

Unless otherwise specified, the term "heteroalkyl" or its subordinate concepts (such as heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl, etc.), by itself or in combination with another term, refers to a stable linear, branched, or cyclic hydrocarbon radical, or combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom. In some embodiments, the term "heteroalkyl" by itself or in combination with another term refers to a stable linear, branched, or cyclic hydrocarbon radical, or combination thereof, consisting of a certain number of carbon atoms and at least one heteroatom. In a typical embodiment, the heteroatom is selected from B, O, N, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. The heteroatom or heteroatom group can be located at any interior position of the heteroalkyl group, including the position at which the hydrocarbon group is attached to the rest of the molecule, but the terms "alkoxy,""alkylamino," and "alkylthio" (or thioalkoxy) are conventional expressions and refer to those alkyl groups that are attached to the rest of the molecule through an oxygen atom, an amino group, or a sulfur atom, respectively. Examples include, but are not limited to, _-CH2 - _CH2 -O- _CH3 , _-CH2 - _CH2 -NH- _CH3 , _{-CH2 -CH2} -N( _CH3 ) _-CH3 , -CH2 _- S- _CH2 - _CH3 , -CH2 _{- CH2} , -S(O) _-CH3 , _-CH2- CH2 _- S(O) _{2- CH3} , -CH=CH-O- _CH3 , _-CH2 -CH=N- _OCH3 , and -CH=CH-N( _CH3 ) _-CH3 . Up to two heteroatoms may be consecutive, for example, _-CH2- NH- _OCH3 .

Unless otherwise specified, the terms "cycloalkyl," "heterocycloalkyl," or their sub-groups (e.g., aryl, heteroaryl, cycloalkyl, heterocycloalkyl, cycloalkenyl, heterocycloalkenyl, cycloalkynyl, heterocycloalkynyl, etc.), by themselves or in combination with other terms, refer to a cyclized "alkyl" or "heteroalkyl," respectively. Furthermore, in the case of heteroalkyl or heterocycloalkyl (e.g., heteroalkyl, heterocycloalkyl), a heteroatom may occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Non-limiting examples of heterocyclic groups include 1-(1,2,5,6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuranindol-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, and 2-piperazinyl.

Unless otherwise specified, the term "alkyl" is used to refer to a straight or branched saturated hydrocarbon group, which may be monosubstituted (e.g., -CH ₂ F) or polysubstituted (e.g., -CF ₃ ), and may be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, s-butyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.

Unless otherwise specified, "alkenyl" refers to an alkyl group having one or more carbon-carbon double bonds at any position of the chain, which may be monosubstituted or polysubstituted and may be monovalent, divalent, or polyvalent. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, piperyl, hexadienyl, and the like.

Unless otherwise specified, "alkynyl" refers to an alkyl group having one or more carbon-carbon triple bonds at any position of the chain, which may be monosubstituted or polysubstituted and may be monovalent, divalent, or polyvalent. Examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, and the like.

Unless otherwise specified, cycloalkyl includes any stable cyclic or polycyclic hydrocarbon radical, any carbon atom of which is saturated, which may be monosubstituted or polysubstituted, and which may be monovalent, divalent, or polyvalent. Examples of such cycloalkyl radicals include, but are not limited to, cyclopropyl, norbornyl, [2.2.2]bicyclooctane, [4.4.0]bicyclodecane, and the like.

Unless otherwise specified, cycloalkenyl includes any stable cyclic or polycyclic hydrocarbon radical containing one or more unsaturated carbon-carbon double bonds at any position of the ring, which may be monosubstituted or polysubstituted and may be monovalent, divalent or polyvalent. Examples of such cycloalkenyl radicals include, but are not limited to, cyclopentenyl, cyclohexenyl, and the like.

Unless otherwise specified, cycloalkynyl groups include any stable cyclic or polycyclic hydrocarbon group containing one or more carbon-carbon triple bonds at any position of the ring, which may be monosubstituted or polysubstituted, and may be monovalent, divalent, or polyvalent.

Unless otherwise specified, the term "halo" or "halogen" by itself or as part of another substituent represents a fluorine, chlorine, bromine or iodine atom. In addition, the term "haloalkyl" is intended to include monohaloalkyl and polyhaloalkyl. For example, the term "halo (C1-C4) alkyl" is intended to include but is not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl and 3-bromopropyl, etc. Unless otherwise specified, examples of haloalkyl include but are not limited to trifluoromethyl, trichloromethyl, pentafluoroethyl, and pentachloroethyl.

"Alkoxy" represents an alkyl group as described above with a specific number of carbon atoms connected through an oxygen bridge. Unless otherwise specified, _C1-6 alkoxy includes _C1 , _C2 , _C3 , _C4 , _C5 and _C6 alkoxy. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and S-pentoxy.

Unless otherwise specified, the term "aryl" refers to a polyunsaturated aromatic hydrocarbon substituent that can be monosubstituted or polysubstituted, can be monovalent, divalent or polyvalent, can be monocyclic or polycyclic (e.g., 1 to 3 rings; at least one of which is aromatic), which are fused together or covalently linked. The term "heteroaryl" refers to an aryl group (or ring) containing one to four heteroatoms. In an exemplary embodiment, the heteroatoms are selected from B, N, O and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom is optionally quaternized. The heteroaryl group can be attached to the rest of the molecule through the heteroatom. Non-limiting examples of aryl or heteroaryl groups include phenyl, naphthyl, biphenyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, oxazolyl, phenyl-oxazolyl, isoxazolyl, thiazolyl, furanyl, thienyl, pyridyl, pyrimidinyl, benzothiazolyl, purinyl, benzimidazolyl, indolyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl phenyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl. Substituents for any of the above aryl and heteroaryl ring systems are selected from the group consisting of acceptable substituents described below.

Unless otherwise specified, aryl when used in conjunction with other terms (e.g., aryloxy, arylthio, arylalkyl) includes aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is intended to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl, etc.), including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom, such as phenoxymethyl, 2-pyridyloxymethyl-3-(1-naphthyloxy)propyl, etc.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (e.g., an affine substitution reaction). For example, representative leaving groups include trifluoromethanesulfonate; chloro, bromo, iodo; sulfonate groups such as methanesulfonate, toluenesulfonate, p-bromobenzenesulfonate, p-toluenesulfonate, etc.; acyloxy groups such as acetoxy and trifluoroacetoxy, etc.

The term "protecting group" includes, but is not limited to, an "amino protecting group," a "hydroxy protecting group," or a "thiol protecting group." The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the amino nitrogen position. Representative amino protecting groups include, but are not limited to, formyl; acyl, such as alkanoyl (e.g., acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butyloxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), trityl (Tr), 1,1-bis-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like. The term "hydroxy protecting group" refers to a protecting group suitable for preventing side reactions at the hydroxyl group. Representative hydroxy protecting groups include, but are not limited to, alkyl groups such as methyl, ethyl and tert-butyl; acyl groups such as alkanoyl (e.g., acetyl); arylmethyl groups such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm) and diphenylmethyl (diphenylmethyl, DPM); silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), and the like.

The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

The compounds were named manually or by software, and commercially available compounds used the supplier's catalog names.

The present invention uses the following abbreviations: MeCN represents acetonitrile; DCM represents dichloromethane; THF represents tetrahydrofuran; AcOH represents acetic acid; TFA represents trifluoroacetic acid; DMF represents N,N-dimethylformamide; _H2O represents water; Boc represents tert-butyloxycarbonyl, Bn represents benzyl, both of which are amine protecting groups; DIPEA represents diisopropylethylamine; _MnO2 represents manganese dioxide; DIBAL-H represents diisobutylaluminum hydride; NaH represents sodium hydride; MeMgBr represents methylmagnesium bromide; LiHMDS represents lithium hexamethyldisilazide; _Pd2 (dba) ₃ represents tris(dibenzylideneacetone)dipalladium; Pd(dppf) _Cl2 represents [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium; Pd(OAc) ₂ represents palladium acetate; Pd( _PPh3 ) ₄ represents triphenylphosphinepalladium; Pd( _PPh3 ) _{2Cl 2} represents bis(triphenylphosphine)palladium dichloride; PO represents oral administration; Xphos represents 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbiphenyl; BINAP represents (±)-2,2′-bis-(diphenylphosphino)-1,1′-binaphthyl; Xphos-Pd-G1 represents chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-aminoethylphenyl)]palladium(II); Xphos-PD-G ₂ represents chloro(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II); Xphos-Pd-G3 represents methanesulfonic acid(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2′-amino-1,1′-biphenyl)]palladium(II); NIS represents N-iododibutyrimide; NBS represents N-bromodibutyrimide; _Br2 represents liquid bromine; _NH2OH ·HCl represents hydroxylamine hydrochloride; NaOAc represents sodium acetate; _Cs2CO3 represents cesium _carbonate ; _OsO4 represents osmium tetroxide; _NaIO4 represents sodium periodate; DAST represents diethylaminosulfur trifluoride; PO represents oral administration; QD represents once a day.

Technical Effects

The compounds of the present invention have significant inhibitory activity against CDK4 and CDK6 kinases. They also have significant proliferation inhibition activity against H358 lung cancer cells. Certain compounds of the present invention have higher NCI-H358 cell proliferation inhibition activity than the reference compound, Palbociclib.

Compared to the reference compounds Palbociclib and LY2835219, the compounds of the present invention have higher permeability and are less likely to be affected by efflux transporters in vivo. This improved permeability allows the compounds of the present invention to be more widely distributed in tissues (such as the lungs), resulting in improved anti-tumor efficacy in vivo. Furthermore, this improved permeability makes it possible for the compounds of the present invention to penetrate the blood-brain barrier, achieving the goal of treating cancers with brain metastases (including lung cancer).

The compounds of this invention exhibit higher kinetic solubility than Palbociclib. Kinetic solubility can help us better understand in vitro and in vivo biological test data. Furthermore, the compounds of this invention exhibit good liver microsomal stability in humans, rats, and mice, and exhibit low clearance rates. In subcutaneous implantation of the HCT-116 colorectal cancer model, the compounds of this invention resulted in less weight loss in the animals, demonstrating their improved safety.

The compounds of the present invention demonstrated significant anti-tumor activity in a human-derived tumor tissue xenograft (PDX) model derived from a LU-01-0393 lung cancer patient. Although certain compounds of the present invention were comparable to the reference compound Palbociclib in inhibiting tumor volume growth, their dosage was only half that of the reference compound. This indicates that, at the same dose, the compounds of the present invention exhibit superior anti-tumor activity. From a dosing perspective, this can reduce the patient's medication dosage and improve compliance. Furthermore, in a subcutaneous implantation NCI-H358 non-small cell lung cancer model, under the same dosage conditions, the body weight of animals receiving the compounds of the present invention not only did not significantly decrease, but also showed a trend of gradual increase, suggesting that the compounds of the present invention have comparable or superior safety compared to existing technologies. Overall, the compounds of the present invention have better drug development prospects than existing technologies.

DETAILED DESCRIPTION

The present invention is described in detail below by way of examples, but is not intended to limit the present invention in any way. While the present invention has been described in detail herein, and specific embodiments thereof have been disclosed, it will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the present invention.

The compounds of the present invention can be prepared through a series of synthetic steps, wherein R ₁ , R ₂ , Ring A and Ring B are the same as defined above.

Reaction Scheme 1: Preparation of the compound represented by formula (I)

When the heterocyclic aromatic amine (B) lacks an N-Boc or N-Bn protecting group, in the reaction shown in Reaction Scheme 1, 2-chloro-1,6-naphthyridin-2-one (A) reacts with the heterocyclic aromatic amine (B) to produce the compound represented by Formula (I). This reaction requires a suitable catalyst (e.g., palladium acetate), a suitable ligand (e.g., Xphos), a suitable base (e.g., cesium carbonate), and a suitable solvent (e.g., 1,4-dioxane). According to Reaction Scheme 1, this reaction is preferably carried out at an elevated temperature.

When the heterocyclic aromatic amine (B) has an N-Boc or N-Bn protecting group, in the following reaction shown in Reaction Scheme 1, the compound represented by Formula (I) can still be prepared by reacting 2-chloro-1,6-naphthyridin-2-one (A) with the heterocyclic aromatic amine (B), but the Boc group must be removed under strong acid conditions (such as trifluoroacetic acid), and the Bn group must be removed under reducing conditions (such as wet palladium carbon/ammonium formate). Finally, the deprotected intermediate undergoes a reductive amination reaction under reducing conditions (such as sodium cyanoborohydride) or a nucleophilic substitution reaction under alkaline conditions (such as potassium carbonate) to obtain the compound represented by Formula (I).

Reaction Scheme 2: Preparation of 2-chloro-1,6-naphthyridin-2-one (A)

When _R1 is an acetyl group, in the reaction shown in Reaction Scheme 2, 2-chloro-3-bromo-1,6-naphthyridin-2-one (C) and a tin reagent (D) undergo a coupling reaction to produce compound (E). This reaction requires a suitable catalyst (e.g., Pd( _PPh3 ) ₄ ) and a suitable solvent (e.g., toluene). According to Reaction Scheme 2, this reaction is preferably carried out at an elevated temperature. Compound (E) is then deprotected under strongly acidic conditions (e.g., trifluoroacetic acid) to produce 2-chloro-1,6-naphthyridin-2-one (A).

When R ₁ is difluoromethyl, in the following reaction shown in Reaction Scheme 2, 2-chloro-3-bromo-1,6-naphthyridin-2-one (C) undergoes a coupling reaction with a vinyl boron reagent (F) to produce compound (G). This reaction requires a suitable catalyst (e.g., Pd(PPh ₃ ) ₂ Cl ₂ ), a suitable base (e.g., cesium carbonate), and a suitable solvent (e.g., 1,4-dioxane/water). According to Reaction Scheme 2, this reaction is preferably carried out at an elevated temperature. In the presence of an oxidizing agent, compound (G) undergoes an oxidation reaction to produce compound (H). This reaction requires a suitable oxidizing agent (e.g., sodium periodate). Compound (H) then reacts with a fluorinating agent (I) to produce 2-chloro-1,6-naphthyridin-2-one (A). This reaction requires a suitable fluorinating agent (e.g., DAST).

Reaction Scheme 3: Preparation of 2-chloro-3-bromo-1,6-naphthyridin-2-one (C)

In the reaction shown in Reaction Scheme 3, 4,6-dichloronicotinate (J) reacts with a primary amine to produce Compound (K). This reaction requires a suitable base (e.g., triethylamine) and a suitable solvent (e.g., acetonitrile). Compound (K) then undergoes a reduction reaction to produce Compound (L). This reaction requires a suitable reducing agent (e.g., DIBAL-H) and a suitable solvent (e.g., anhydrous tetrahydrofuran). Compound (M) can be prepared by oxidation of Compound (L), which requires a suitable oxidizing agent (e.g., activated manganese dioxide). Compound (M) undergoes a nucleophilic addition reaction with methylmagnesium bromide to produce Compound (N). This reaction requires a suitable solvent (e.g., anhydrous tetrahydrofuran). According to Reaction Scheme 3, this reaction is preferably carried out at low temperatures. Compound (N) undergoes an oxidation reaction to produce Compound (O), which requires a suitable oxidizing agent (e.g., activated manganese dioxide). Compound (Q) can be prepared by condensation and cyclization of compound (O) with a cyclizing agent (P). This reaction requires a suitable cyclizing agent (e.g., triethyl phosphoacetate, ethyl acetate), a suitable base (e.g., sodium hydride, LiHMDS), and a suitable solvent (e.g., tetrahydrofuran). According to Reaction Scheme 3, this reaction is preferably carried out at an elevated temperature. Compound (Q) is then halogenated to obtain compound (C). The halogenating agent may be Br ₂ , NBS, or NIS. This reaction requires a suitable solvent (e.g., N,N-dimethylformamide, acetonitrile).

Reaction Scheme 4: Preparation of Heterocyclic Aromatic Amines (B)

In the reaction shown in Reaction Scheme 4, heterocyclic aromatic amine (B) can be prepared by the following two methods: 1) A bromine atom on 2,5-dibromopyrazine (R) is first coupled with a borate ester compound (S) under palladium catalysis to produce compound (T). Compound (T) is first reacted with diphenylmethylimine (U) under palladium catalysis, and then reacted with hydroxylamine hydrochloride under alkaline conditions to produce compound (V). Finally, compound (V) undergoes double bond reduction to produce heterocyclic aromatic amine (B). 2) A bromine atom on 2,5-dibromopyrazine (R) is first replaced with a commercially available or synthetic amine (W) to produce compound (X). Compound (X) can be prepared from heterocyclic aromatic amine (B) by two methods: ① Compound (X) is first reacted with diphenylmethylimine (U) under palladium catalysis, and then reacted with hydroxylamine hydrochloride under alkaline conditions to produce heterocyclic aromatic amine (B); ② Compound (X) is reacted with LiHMDS under palladium catalysis to produce heterocyclic aromatic amine (B).

Plan A

Synthesis of intermediate A and intermediate B:

Step 1:

To a solution of ethyl 4,6-dichloronicotinate (Compound 1) (10.00 g, 45.44 mmol, 1.00 equiv) in acetonitrile (100.00 ml) were added N,N-diisopropylethylamine (17.62 g, 136.32 mmol, 3.00 equiv) and cyclopentylamine (3.87 g, 45.44 mmol, 1.00 equiv). The reaction mixture was stirred at 25°C for 16 hours. TLC indicated that the starting material remained. The reaction mixture was then heated to 50°C and stirred for 8 hours. TLC (petroleum ether:ethyl acetate = 10:1) indicated that the reaction was complete. The mixture was concentrated, and the resulting crude product was dissolved in ethyl acetate (100 ml), washed with saturated brine (50 ml x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 10:1) to give the target compound (Compound 2) (9.50 g, 35.35 mmol, yield: 77.80%). ¹ HNMR (400MHz, CDCl ₃ )δ8.67 (s, 1H), 8.20 (d, J=4.8Hz, 1H), 6.58 (s, 1H), 4.35 (q, J=7.2Hz, 2H), 3.88-3.80 (m, 1H), 2.12-2.04 (m, 2H ), 1.82-1.75 (m, 2H), 1.74-1.67 (m, 2H), 1.63-1.57 (m, 2H), 1.40 (t, J=7.2Hz, 3H); LCMS (ESI) m/z: 269.0 (M+1).

Step 2:

To a solution of ethyl 6-chloro-4-(cyclopentylamino)nicotinate (Compound 2) (9.50 g, 35.35 mmol, 1.00 equiv) in tetrahydrofuran (100.00 mL) at -30°C under nitrogen was added DIBAL-H (1 M, 70.70 mL, 2.00 equiv). After the addition was complete, the reaction mixture was warmed to 25°C and stirred for 16 hours. TLC (petroleum ether:ethyl acetate = 5:1) indicated the reaction was complete. The mixture was cooled to 0°C, quenched with saturated aqueous sodium sulfate (50 mL), and then extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated to yield the title compound (Compound 3) (7.50 g, 33.08 mmol, yield: 93.59%). ¹ HNMR (400MHz, CDCl ₃ ) δ7.71 (s, 1H), 6.51 (s, 1H), 5.57 (d, J = 5.2Hz, 1H), 4.60 (s, 2H), 3.86-3.77 (m, 1H), 2.12-2.03 (m, 2H), 1.82-1.62 (m, 4H), 1.60-1.50 (m, 2H).

Step 3:

To a solution of (6-chloro-4-(cyclopentylamino)-pyridin-3-yl)methanol (Compound 3) (7.50 g, 33.08 mmol, 1.00 equiv) in dichloromethane (80.00 mL) was added activated manganese dioxide (28.76 g, 330.80 mmol, 10.00 equiv). The reaction mixture was stirred at 25°C for 16 hours. TLC (petroleum ether:ethyl acetate = 5:1) indicated the reaction was complete. The reaction mixture was filtered, the filter cake washed with dichloromethane (50 mL), and the filtrate concentrated to afford the title compound (Compound 4) (7.00 g, 31.15 mmol, yield: 94.18%). ¹ H NMR (300MHz, CDCl ₃ ) δ 9.75 (s, 1H), 8.57 (d, J=6.8Hz, 1H), 8.20 (s, 1H), 6.53 (s, 1H), 3.85-3.73 (m, 1H), 2.05-1.94 (m, 2H), 1.78-1.48 (m, 6H).

Step 4:

To a solution of 6-chloro-4-(cyclopentylamino)nicotinaldehyde (Compound 4) (7.0 g, 31.15 mmol, 1.00 equiv) in tetrahydrofuran (70.00 mL) was slowly added dropwise methylmagnesium bromide (3 M, 25.96 mL, 2.50 equiv) at -10°C under nitrogen. After the addition was complete, the reaction mixture was stirred at this temperature for 1 hour. TLC (petroleum ether:ethyl acetate = 5:1) indicated the reaction was complete. The reaction mixture was quenched with saturated aqueous ammonium chloride (30 mL) and extracted with ethyl acetate (50 mL x 3). The combined organic phases were washed with saturated brine (80 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to yield the title compound (Compound 5) (6.70 g, 27.83 mmol, yield: 89.35%). ¹ H NMR (300MHz, CDCl ₃ )δ7.50 (s, 1H), 6.37 (s, 1H), 6.01 (d, J = 6.4Hz, 1H), 4.76 (q, J = 6.4Hz, 1H), 3. 75-3.64 (m, 1H), 1.97-1.90 (m, 2H), 1.75-1.50 (m, 6H), 1.46 (d, J=6.6Hz, 3H).

Step 5:

To a solution of 1-(6-chloro-4-(cyclopentylamino)pyridin-3-yl)ethanol (Compound 5) (6.70 g, 27.83 mmol, 1.00 equiv) in dichloromethane (70.00 ml) was added activated manganese dioxide (24.20 g, 278.30 mmol, 10.00 equiv), and the reaction mixture was stirred at 25°C for 16 hours. TLC indicated that the starting material remained, and the reaction mixture was then heated to 50°C and stirred for 8 hours. TLC (petroleum ether:ethyl acetate = 5:1) indicated that the reaction was complete. The reaction mixture was cooled to 20°C, filtered, and the filter cake was washed with dichloromethane (50 ml). The filtrate was concentrated to give the title compound (Compound 6) (6.00 g, 25.14 mmol, yield: 90.32%). ¹ H NMR (300MHz, CDCl ₃ )δ9.22 (s, 1H), 8.59 (s, 1H), 6.60 (s, 1H), 3.90-3.79 (m, 1H), 2.58 (s, 3H), 2.14-2.00 (m, 2H), 1.87-1.67 (m, 4H), 1.63-1.53 (m, 2H).

Step 6:

To a solution of triethyl phosphoacetate (14.65 g, 65.36 mmol, 2.60 eq) in tetrahydrofuran (60.00 ml) at 0°C under nitrogen was added sodium hydride (2.61 g, 65.36 mmol, 2.60 eq, 60% purity) portionwise. The reaction mixture was stirred at this temperature for 20 minutes, followed by the addition of 1-(6-chloro-4-(cyclopentylamino)pyridin-3-yl)ethanone (Compound 6) (6.00 g, 25.14 mmol, 1.00 eq). After the addition was complete, the reaction mixture was heated to 70°C and stirred for 16 hours. TLC (petroleum ether:ethyl acetate = 5:1) indicated the reaction was complete. The reaction mixture was cooled to 25°C, quenched with saturated aqueous ammonium chloride (20 ml), and extracted with ethyl acetate (30 ml x 3). The combined organic phases were washed with saturated brine (50 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 50:1 to 20:1) to give the title compound (Compound 7) (5.00 g, 3.19 mmol, yield: 75.70%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.68 (s, 1H), 7.35 (s, 1H), 6.54 (s, 1H), 5.49 (q, J = 9.2 Hz, 1H), 2.50 (s, 3H), 2.24-2.00 (m, 6H), 1.84-1.76 (m, 2H); LCMS (ESI) m/z: 263.0 (M+1).

Step 7:

To a solution of 7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Compound 7) (1.00 g, 3.81 mmol, 1.00 equiv) in acetic acid (20.00 ml) were added sodium acetate (1.25 g, 15.22 mmol, 4.00 equiv) and liquid bromine (1.22 g, 7.61 mmol, 2.00 equiv) in that order. The reaction mixture was heated to 70°C and stirred for 20 hours. The reaction mixture was concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford the title compound (Intermediate A) (1.10 g, 3.22 mmol, yield: 84.51%). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.80 (s, 1H), 7.40 (s, 1H), 5.40-5.30 (m, 1H), 2.73 (s, 3H), 2.29-2.12 (m, 4H), 2.07-1.98 (m, 2H), 1.81-1.75 (m, 2H).

Step 8:

Under nitrogen, tributyl(1-ethoxyvinyl)tin (580.01 mg, 1.61 mmol, 1.10 equiv) and Pd(PPh 3 ) 4 (168.71 mg, 146.00 μmol, 0.10 equiv) were added to a solution of 3-bromo-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2 _- one (Compound ₈ ) (500.00 mg, 1.46 mmol, 1.00 equiv) in toluene (5.00 mL). The reaction mixture was heated to 110°C and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to obtain the title compound (Compound 9) (400.00 mg, 1.20 mmol, yield: 82.32%). ¹ H NMR (300MHz, CD ₃ OD) δ 8.84 (s, 1H), 7.72 (s, 1H), 5.41-5.30 (m, 1H), 4.57 (d, J = 2.4Hz, 1H), 4.15 (d, J = 2.4Hz, 1H), 3.94 (q, J = 7.2 Hz, 2H), 2.56 (s, 3H), 2.24-2.05 (m, 6H), 1.84-1.78 (m, 2H), 1.35 (t, J=6.8Hz, 3H); LCMS (ESI) m/z: 333.1 (M+1).

Step 9:

To a solution of 7-chloro-1-cyclopentyl-3-(1-ethoxyvinyl)-4-methyl-1,6-naphthyridin-2-one (Compound 9) (400.00 mg, 1.20 mmol, 1.00 equiv) in dichloromethane (5.00 mL) was added trifluoroacetic acid (3.00 mL), and the reaction mixture was stirred at 25°C for 1 hour. TLC (petroleum ether:ethyl acetate = 5:1) and LCMS both indicated completion of the reaction. The reaction solution was concentrated, followed by addition of water (5 mL) and extraction with ethyl acetate (10 mL x 3). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated, and the resulting crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 10:1) to afford the target compound (Intermediate B) (300.00 mg, 984.35 μmol, yield: 81.90%). LCMS (ESI) m/z: 305.2 (M+1).

Example 1

Step 1:

To a solution of 2,5-dibromopyrazine (10.00 g, 42.04 mmol, 1.00 equiv) in 1-methylpyrrolidin-2-one (100.00 ml) were added tert-butyl piperazine-1-carboxylate (7.83 g, 42.04 mmol, 1.00 equiv) and potassium carbonate (8.72 g, 63.06 mmol, 1.50 equiv). The mixture was heated to 100°C and stirred for 18 hours. TLC (petroleum ether:ethyl acetate = 10:1) indicated the reaction was complete. The reaction mixture was diluted with water (200 ml) and extracted with ethyl acetate (200 ml x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 5:1) to obtain the title compound (11.00 g, 32.05 mmol, yield: 76.24%). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.15 (d, J=1.38Hz, 1H), 7.87 (d, J=1.38Hz, 1H), 3.56 (m, 8H), 1.49 (s, 9H).

Step 2:

Under nitrogen, to a solution of tert-butyl 4-(5-bromopyrazin-2-yl)piperazine-1-carboxylate (10.00 g, 29.14 mmol, 1.00 equiv) and tri-tert-butylphosphonium tetrafluoroborate (2.54 g, 8.74 mmol, 0.30 equiv) in toluene (100.00 mL) were added LiHMDS (1 M, 60.00 mL, 2.06 equiv) and _Pd2 (dba) ₃ (2.60 g, 2.84 mmol, 0.10 equiv). The reaction mixture was heated to 65°C and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was quenched with water (50 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic phases were concentrated and the resulting crude product was purified by preparative HPLC (basic) to afford the title compound (5.00 g, 17.90 mmol, yield: 61.43%). LCMS (ESI) m/z: 280.1 (M+1).

Step 3:

To a solution of 3-acetyl-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Intermediate B) (100.00 mg, 328.12 μmol, 1.00 eq), tert-butyl 4-(5-aminopyrazin-2-yl)piperazine-1-carboxylate (137.48 mg, 492.17 μmol, 1.50 eq), and potassium tert-butoxide (110.45 mg, 984.35 μmol, 3.00 eq) in tetrahydrofuran (2.00 ml) was added Xphos-Pd-G2 (25.82 mg, 32.81 μmol, 0.10 eq). The reaction mixture was heated to 80° C. and stirred for 16 hours. TLC (petroleum ether:ethyl acetate=1:1) indicated complete reaction of the starting material. The reaction solution was cooled to room temperature and concentrated. The crude product was purified by preparative TLC (petroleum ether:ethyl acetate=1:1) to obtain the title compound (40.00 mg, 73.04 μmol, yield: 22.26%).

Step 4:

To a solution of tert-butyl 4-(5-((3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1,2-dihydro-1,6-naphthalen-7-yl)amino)pyrazin-2-yl)piperazine-1-carboxylate (60.00 mg, 109.56 μmol, 1.00 equiv) in dichloromethane (1.00 mL) was added trifluoroacetic acid (0.5 mL), and the mixture was stirred for 0.5 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the resulting crude product was purified by preparative HPLC (hydrochloric acid) to give the hydrochloride salt of the title compound (22.78 mg, 50.90 μmol, yield: 46.46%). ¹ H NMR (400MHz, CD ₃ OD)δ8.78(s,1H),8.27(s,1H),8.19(s,1H),7.30(s,1H),5.44-5.32(m,1H),3.93-3.88(m,4H),3.44-3.38(m,4H ), 2.51 (s, 3H), 2.40 (s, 3H), 2.31-2.16 (m, 4H), 2.08 (d, J=8.0Hz, 2H), 1.82 (m, 2H); LCMS (ESI) m/z: 448.1 (M+1).

Example 2

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (150.00 mg, 335.17 μmol, 1.00 equiv) in dichloroethane (2.00 mL) at 25°C were added acetaldehyde solution (553.66 mg, 5.03 mmol, 700.83 μL, 15.00 equiv) and sodium acetate borohydride (213.11 mg, 1.01 mmol, 3.00 equiv). The mixture was stirred for 1 hour. LCMS indicated the detection of approximately 26% of the title compound. The mixture was concentrated, and the resulting crude product was purified by preparative HPLC (basic) to yield the title compound (14.35 mg, 27.71 μmol, 8.27% yield, 91.83% purity). ¹ H NMR (400MHz, CDCl ₃ )δ8.65 (s, 1H), 8.17 (d, J = 1.3Hz, 1H), 7.89-7.75 (m, 2H), 7.65 (s, 1H), 5.73 (qum, J = 9.3Hz, 1H), 3.60-3.48(m, 4H), 2.65-2.58(m, 4H), 2.55(s, 3H), 2.49(q, J=7.3Hz, 2H), 2.40(s, 3H), 2.29(br dd, J＝12.4, 7.3Hz, 2H), 2.13(br dd, J=7.9, 5.5Hz, 2H), 2.02-1.95 (m, 2H), 1.82-1.73 (m, 2H), 1.15 (t, J=7.2Hz, 3H); LCMS (ESI) m/z: 492.3 (M+1).

Example 3

The synthesis of Example 3 was carried out with reference to Example 2. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.65 (s, 1H), 8.17 (d, J=1.3 Hz, 1H), 7.87-7.74 (m, 2H), 7.46 (s, 1H), 5.74 (quin, J=9.3 Hz, 1H), 3.63-3.43 (m, 4H), 2.77 (td, J=13.0, 6.5 Hz, 1H), 2.73-2.66 (m, 4H), 2.56 (s, 3H), 2.41 (s, 3H), 2.35-2.24 (m, 2H), 2.19-2.07 (m, 2H), 2.03-1.95 (m, 2H), 1.77 (br d, J=4.8Hz, 2H), 1.11 (d, J=6.5Hz, 6H); LCMS (ESI) m/z: 490.2 (M+1).

Example 4

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (150.00 mg, 335.17 μmol, 1.00 eq) in methanol (2.00 mL) at 25° C. was added (1-ethoxycyclopropyloxy)trimethylsilane (292.12 mg, 1.68 mmol, 335.77 μL, 5.00 eq) and sodium cyanoborocyanide (63.19 mg, 1.01 mmol, 3.00 eq). The mixture was stirred at 25° C. for 1 hour. LCMS showed that the starting material was not completely consumed. The reaction mixture was heated to 60° C. and stirred for 18 hours. LCMS showed that the target compound was detected. The reaction mixture was concentrated and the resulting crude product was purified by preparative HPLC (basic) to afford the title compound (31.98 mg, 65.17 μmol, yield: 19.44%, purity: 99.37%). ¹ H NMR (400MHz, CDCl ₃ )δ8.66 (s, 1H), 8.17 (d, J=1.4Hz, 1H), 7.84-7.77 (m, 2H), 7.44 (s, 1H), 5.74 (quin, J=9.3Hz, 1H), 3.54-3.44 (m, 4H ), 2.83-2.73(m, 4H), 2.56(s, 3H), 2.41(s, 3H), 2.35-2.24(m, 2H), 2.19-2.08(m, 2H), 2.04-1.93(m, 2H), 1.78(br dd, J=10.3, 5.5Hz, 2H), 0.56-0.45 (m, 4H); LCMS (ESI) m/z: 488.3 (M+1).

Example 5

The synthesis of Example 5 was carried out in accordance with Example 2. ¹ H NMR (400 MHz, CDCl ₃ )δ8.65 (s, 1H), 8.17 (d, J=1.1Hz, 1H), 7.88-7.73 (m, 2H), 7.65 (s, 1H), 5.73 (quin, J=9.3Hz, 1H), 3.57-3.48 (m, 4H), 2.79 (quin, J=7.8Hz, 1H), 2.5 5(s, 3H), 2.51-2.44(m, 4H), 2.40(s, 3H), 2.35-2.23(m, 2H), 2.18-2.04( m, 4H), 2.02-1.86 (m, 6H), 1.83-1.70 (m, 2H); LCMS (ESI) m/z: 502.3 (M+1).

Example 6

The synthesis of Example 6 was carried out in accordance with Example 2. ¹ H NMR (400 MHz, CDCl ₃ )δ8.66 (s, 1H), 8.19 (d, J=1.1Hz, 1H), 7.82 (s, 2H), 7.55 (s, 1H), 5.74 (quin, J=9.3Hz, 1H), 4.70 (td, J=19.4, 6.4Hz, 4H), 3.68-3.47 (m, 5H), 2.56 (s, 3H), 2.53-2.45 (m, 4H), 2.41 (s, 3H), 2.36-2.23 (m, 2H), 2.19-2.08 (m , 2H), 2.02-1.94 (m, 2H), 1.81-1.75 (m, 2H); LCMS (ESI) m/z: 504.2 (M+1).

Example 7

The synthesis of Example 7 was carried out in accordance with Example 2. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.65 (s, 1H), 8.17 (d, J=1.4 Hz, 1H), 7.85-7.75 (m, 2H), 7.70-7.58 (m, 1H), 5.73 (t, J=9.3 Hz, 1H), 3.61-3.45 (m, 4H), 2.70-2.62 (m, 4H), 2.61-2.48 (m, 4H), 2.40(s, 3H), 2.34-2.22(m, 2H), 2.17-2.06(m, 2H), 1.98-1.87(m, 4H), 1.81-1 .68 (m, 4H), 1.64-1.53 (m, 2H), 1.51-1.41 (m, 2H); LCMS (ESI) m/z: 516.3 (M+1).

Example 8

Step 1:

To a solution of tert-butyl (2R)-2-methylpiperazine-1-carboxylate (841.94 mg, 4.20 mmol, 1.00 equiv) in 1-methylpyrrolidin-2-one (10.00 mL) were added 2,5-dibromopyrazine (1.00 g, 4.20 mmol, 1.00 equiv) and potassium carbonate (871.51 mg, 6.30 mmol, 1.50 equiv). The mixture was heated to 100°C and stirred for 18 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to room temperature and diluted with water (100 mL), followed by extraction with ethyl acetate (50 mL x 3). The combined organic phases were washed sequentially with water (50 mL x 3) and brine (50 mL), then concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford the title compound (900.00 mg, 2.52 mmol, yield: 59.98%). ¹ H NMR (400MHz, CDCl ₃ ) δ8.12 (d, J=1.4Hz, 1H), 7.84 (d, J=1.5Hz, 1H), 4.35 (br s, 1H), 4.09-4.02 (m, 1H), 3.96 (td, J=13.2, 2.0Hz, 2H), 3.32-3.21 (m, 2H), 3.05 (dt, J=11.9, 3.8Hz, 1H), 1.49 (s, 9H), 1.19 (d, J=6.7Hz, 3H).

Step 2:

To a solution of (2R)-4-(5-bromopyrazin-2-yl)-2-methyl-piperazine-1-carboxylic acid tert-butyl ester (900.00 mg, 2.52 mmol, 1.00 equiv) in 1,4-dioxane (10.00 mL) under nitrogen was added diphenylmethylimine (502.37 mg, 2.77 mmol, 465.16 μL, 1.10 equiv), cesium carbonate (1.64 g, 5.04 mmol, 2.00 equiv), Pd(OAc) ₂ (56.56 mg, 252.00 μmol, 0.10 equiv), and BINAP (313.73 mg, 504.00 μmol, 0.20 equiv). The mixture was heated to 100° C. and stirred for 18 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to room temperature, diluted with dichloromethane (10 ml) and filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 40: 1 to 10: 1) to obtain the title compound (850.00 mg, 1.86 mmol, yield: 73.72%). ¹ H NMR (400MHz, CDCl ₃ )δ7.82-7.77(m, 3H), 7.55(d, J=1.4Hz, 1H), 7.51-7.47(m, 1H), 7.44-7.38(m, 2H), 7.36-7.30(m, 3H), 7.21-7.15(m, 2H), 4.32(br s, 1H), 4.01-3.83 (m, 3H), 3.26-3.13 (m, 2H), 2.93 (dt, J=12.0, 3.7Hz, 1H), 1.49 (s, 9H), 1.18 (d, J=6.8Hz, 3H).

Step 3:

To a solution of (2R)-4-(5-(benzhydrylideneamino)pyrazin-2-yl)-2-methyl-piperazine-1-carboxylic acid tert-butyl ester (850.00 mg, 1.86 mmol, 1.00 equiv) in methanol (10.00 ml) were added sodium acetate (183.09 mg, 2.23 mmol) and hydroxylamine hydrochloride (232.65 mg, 3.35 mmol, 1.80 equiv), and the mixture was stirred at 20°C for 30 minutes. LCMS indicated the reaction was complete. The reaction mixture was concentrated, and the resulting crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 3:1) to afford the title compound (160.00 mg, 545.40 μmol, yield: 29.32%). ¹ H NMR (400MHz, CDCl ₃ ) δ7.69 (d, J=1.3Hz, 1H), 7.64 (d, J=1.3Hz, 1H), 4.36 (br s, 1H), 4.14-4.05 (m, 2H), 3.96 (br d, J=13.4Hz, 1H), 3.86 (br d, J＝12.2Hz, 1H), 3.73(br d, J=12.3Hz, 1H), 3.24 (dt, J=12.7, 3.5Hz, 1H), 3.00 (dd, J=12.4, 4.0Hz, 1H), 2.79 (dt, J=12.0, 3.6Hz, 1H), 1.49 (s, 9H), 1.25 (d, J=7.0Hz, 3H).

Step 4:

Under nitrogen, to a solution of (2R)-4-(5-aminopyrazin-2-yl)-2-methyl-piperazine-1-carboxylic acid tert-butyl ester (160.00 mg, 545.40 μmol, 1.00 equiv) and 3-acetyl-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Intermediate B) (166.22 mg, 545.40 μmol, 1.00 equiv) in tetrahydrofuran (2.00 ml) were added Xphos-Pd-G3 (46.17 mg, 54.54 μmol, 0.10 equiv) and potassium tert-butoxide (122.40 mg, 1.09 mmol, 2.00 equiv). The mixture was heated to 70° C. and stirred for 18 hours. LCMS showed that the reaction was complete. The reaction mixture was cooled to room temperature, diluted with dichloromethane (5 ml), filtered, and the filtrate was concentrated. The crude product was purified by preparative TLC (petroleum ether: ethyl acetate = 1:1) to give the title compound (200.00 mg, 313.35 μmol, yield: 57.45%, purity: 88%). LCMS (ESI) m/z: 562.2 (M+1).

Step 5:

To a solution of (2R)-4-(5-((3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1,6-naphthyridin-7-yl)amino)pyrazin-2-yl-2-methyl-piperazine-1-carboxylic acid tert-butyl ester (200.00 mg, 313.35 μmol, 1.00 equiv) in dichloromethane (2.00 mL) was added trifluoroacetic acid (1.00 mL) at 20°C, and the mixture was stirred for 15 minutes. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness, and the crude product was purified by preparative HPLC (hydrochloric acid) to give the hydrochloride salt of the title compound (66.91 mg, 123.94 μmol, yield: 39.55%, purity: 99%). ¹ H NMR (400 MHz, CD ₃ OD) δ8.76 (s, 1H), 8.24 (d, J = 1.1Hz, 1H), 8.20 (s, 1H), 7.27 (s, 1H), 5.36 (quin, J = 8.7Hz , 1H), 4.50-4.39 (m, 2H), 3.56-3.45 (m, 2H), 3.31-3.21 (m, 2H), 3.10 (dd, J=14.1, 10.8Hz 3H), 2.49 (s, 3H), 2.38 (s, 3H), 2.29-2.12 (m, 4H), 2.10-1.99 (m, 2H), 1.87-1.72 (m, 2H), 1.45 (d, J=6.6 Hz, 3H); LCMS (ESI) m/z: 462.1 (M+1); SFC (AD-3S_4_40_3ML column: Chiralpak AD-3 100×4.6 mm ID, 3 μm; mobile phase A: 40% isopropanol (containing 0.05% diethylamine); mobile phase B: CO ₂ ; flow rate: 3 mL/min; wavelength: 280 nm): RT=2.834 min.

Example 9

The starting material of Example 9 is tert-butyl (2S)-2-methylpiperazine-1-carboxylate, and its synthesis method is similar to Example 8. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.76 (s, 1H), 8.24 (d, J = 1.3 Hz, 1H), 8.20 (d, J = 1.3 Hz, 1H), 7.26 (s, 1H), 5.37 (quin, J = 8.7 Hz, 1H), 4.52-4.38 (m, 2H), 3.58-3.44 (m, 2H), 3.36-3.31 (m, 2H), 3.09 (dd, J = 14.1, 10 77 (s, 3H), 2.49 (s, 3H), 2.36 (s, 3H), 2.73 (m, 4H), 2.11 (m, 2H), 1.99 (m, 2H), 1.94 (m, 2H), 1.96 (m, 3H), 1.96 (m, 4H); LCMS (ESI) m/z: 462.1 (M+1); SFC (AD-3S_4_40_3ML column: Chiralpak AD-3 100×4.6 mm ID, 3 μm; mobile phase A: 40% isopropanol (containing 0.05% diethylamine); mobile phase B: CO ₂ ; flow rate: 3 mL/min; wavelength: 280 nm): RT=3.284 min.

Example 10

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[[5-[(3S)-3-methylpiperazin-1-yl]pyrazin-2-yl]amino]-1,6-naphthyridin-2-one (200.00 mg, 433.31 μmol, 1.00 equiv) in methanol (20.00 mL) was added formaldehyde solution (351.68 mg, 4.33 mmol, 322.64 μL, 10.00 equiv), acetic acid (500.00 μL), and wet palladium on carbon (100.00 mg). The reaction flask was purged three times with nitrogen and then hydrogen. Maintaining hydrogen pressure (50 Psi), the reaction solution was heated to 50°C and stirred for 3 hours. LCMS indicated the reaction was complete. The reaction solution was filtered and the filtrate was concentrated. The crude product was purified by preparative HPLC (basic) to give the title compound (42.66 mg, 88.31 μmol, yield: 20.38%, purity: 98.45%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.65 (s, 1H), 8.16 (s, 1H), 7.81-7.79 (m, 3H), 5.71 (quin, J = 9.3 Hz, 1H), 3.98-3.93 (m, 2H), 3.07 (dt, J = 11.4, 3.0 Hz, 1H), 2.92 (td, J = 11.4, 3.2 Hz, 1H), 2.70-2.6 (m, 1H), 2.56 (s , 3H), 2.39 (s, 3H), 2.38-2.37 (m, 1H), 2.35 (s, 3H), 2.31-2.20 (m, 3H), 2.17-2.04 (m, 3H), 2.01-1.93 (m, 2H), 1.84-1.71 (m, 2H), 1.17 (d, J=5.6Hz, 3H); LCMS (ESI) m/z: 476.3 (M+1).

Example 11

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[[5-[(3S)-3-methylpiperazin-1-yl]pyrazin-2-yl]amino]-1,6-naphthyridin-2-one (100.00 mg, 216.66 μmol, 1.00 equiv) in acetonitrile (10.00 mL) were added potassium carbonate (149.72 mg, 1.08 mmol, 5.00 equiv) and 2-iodopropane (736.60 mg, 4.33 mmol, 433.29 μL, 20.00 equiv). The reaction mixture was heated to 80°C and stirred for 18 hours. LCMS indicated approximately 13.5% of the starting material remained, and approximately 75.5% of the desired compound was produced. The reaction mixture was cooled to 20°C, filtered, and the filtrate concentrated. The crude product was purified by preparative HPLC (basic) to give the title compound (9.00 mg, 17.87 μmol, yield: 8.25%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.65 (s, 1H), 8.16 (d, J = 1.4 Hz, 1H), 7.81 (d, J = 1.3 Hz, 1H), 7.77 (s, 1H), 7.40 (s, 1H), 5.74 (quin, J = 9.3 Hz, 1H), 3.96-3.89 (m, 2H), 3.33 (spt, J = 6.5 Hz, 1H), 3.06 (dt, J = 11.4, 3.0 Hz, 1H), 2.92 (td, J = 11.4, 3.2 Hz, 1H), 2. .85-2.70(m,2H),2.56(s,3H),2.49-2.47(m,1H),2.41(s,3H),2.35-2.24(m,2H),2.19-2.07(m,2H),2.03-1. 93 (m, 2H), 1.84-1.75 (m, 2H), 1.17 (dd, J=6.2, 4.2Hz, 6H), 0.93 (d, J=6.4Hz, 3H); LCMS (ESI) m/z: 504.3 (M+1).

Example 12

The synthesis of Example 12 was carried out by referring to Example 1 to obtain the hydrochloride salt of the target compound. ¹ HNMR (400 MHz, CD ₃ OD) δ 8.77 (s, 1H), 8.26 (d, J = 1.2Hz, 1H), 8.24 (d, J = 1.2Hz, 1H), 7.27 (s, 1H), 5.44-5.35(m, 1H), 4.58-4.54(m, 2H), 3.55-5.45(m, 2H), 3.00-2.9 3(m, 2H), 2.51(s, 3H), 2.40(s, 3H), 2.30-2.15(m, 4H), 2.10-2.05(m, 2H ), 1.84-1.78 (m, 2H), 1.46 (d, J=7.2Hz, 6H); LCMS (ESI) m/z: 476.3 (M+1).

Example 13

The synthesis of Example 13 was carried out by referring to Example 1 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.76 (s, 1H), 8.23 (d, J = 8.9 Hz, 2H), 7.26 (s, 1H), 5.43-5.29 (m, 1H), 3.99-3.83 (m, 2H), 3.76 (s, 2H), 3.50-3.39 (m, 2H), 2.53-2.46 (m, 3H), 2.39 (s, 3H), 2.29-2.13 (m, 4H), 2.11-2.01 (m, 2H), 1.82 (br d, J = 5.9 Hz, 2H), 1.51 (s, 6H); LCMS (ESI) m/z: 476.2 (M+1).

Example 14

To a solution of 3-acetyl-1-cyclopentyl-7-[[5-(3,3-dimethylpiperazin-1-yl)pyrazin-2-yl]amino]-4-methyl-1,6-naphthyridin-2-one (100.00 mg, 186.31 μmol, 1.00 equiv), formaldehyde solution (27.97 mg, 931.55 μmol, 25.66 μL, 5.00 equiv), and acetic acid (44.75 mg, 745.24 μmol, 42.62 μL, 4.00 equiv) in dichloroethane (1.00 mL) was added sodium acetate borohydride (98.72 mg, 465.77 μmol, 2.50 equiv). The reaction was heated to 60°C and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction was concentrated, and saturated aqueous sodium bicarbonate solution was added dropwise to the resulting residue to adjust the pH to 9. The resulting aqueous phase was extracted with dichloromethane (5 mL x 2). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by preparative HPLC (basic) to afford the title compound (31.00 mg, 61.56 μmol, yield: 33.04%, purity: 97.229%). ¹ H NMR (400MHz, CDCl ₃ )δ8.64(s, 1H), 8.14(s, 1H), 7.80(s, 1H), 7.73(s, 1H), 7.53(s, 1H), 5.84-5.59(m, 1H), 3.62-3. 44(m, 2H), 3.27(s, 2H), 2.68(brt, J=5.0Hz, 2H), 2.55(s, 3H), 2.39(s, 3H), 2.29(s, 3H), 2.11(br s, 2H), 1.97 (brd, J=5.4Hz, 2H), 1.78 (br d, J=5.8Hz, 4H), 1.10 (s, 6H); LCMS (ESI) m/z: 490.2 (M+1).

Example 15

The synthesis of Example 15 was carried out with reference to Example 11. ¹ H NMR (400MHz, CDCl ₃ )δ8.64 (s, 1H), 8.12 (d, J = 1.3Hz, 1H), 7.79 (d, J = 1.3Hz, 1H), 7.70 (s, 1H), 7.44(s, 1H), 5.86-5.63(m, 1H), 3.49(brs, 2H), 3.39-3.28(m, 1H), 3.23(br s, 2H), 2.82-2.69 (m, 2H), 2.55 (s, 3H), 2.39 (s, 3H), 2.34-2.20 (m, 2H), 2.17-2.05 (m, 2H), 2.03-1.91 (m, 2H), 1.76 (br dd, J=10.4, 5.6Hz, 2H), 1.17 (s, 6H), 1.03 (br d, J=6.4Hz, 6H); LCMS (ESI) m/z: 518.3 (M+1).

Example 16

The synthesis of Example 16 was carried out according to Example 1 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400MHz, CD ₃ OD) δ 8.75 (s, 1H), 8.20 (s, 1H), 8.02 (s, 1H), 7.27 (s, 1H), 5.45-5.20 (m, 1H), 4.15-3.97 (m, 2H), 3.81 (brt, J=5.8Hz, 2H), 3.44 (br d, J＝4.4Hz, 2H), 3.39-3.33(m, 2H), 2.48(s, 3H), 2.37(s, 3H), 2.31-2.13(m, 6H), 2.05(br d, J＝8.8Hz, 2H), 1.79(br s, 2H); LCMS (ESI) m/z: 462.2 (M+1).

Example 17

The synthesis of Example 17 was carried out by referring to Example 1 to obtain the hydrochloride of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.74 (s, 1H), 8.21 (s, 1H), 7.90 (s, 1H), 7.26 (s, 1H), 5.35 (t, J=8.3 Hz, 1H), 5.04 (br. s., 1H), 4.61 (s, 1H), 3.86-3.77 (m, 1H), 3.77-3.69 (m, 1H), 3.4 9-3.39 (m, 2H), 2.49 (s, 3H), 2.38 (s, 3H), 2.32 (d, J=11.2Hz, 2H), 2.25-2.10 (m, 5H), 2.05 (d, J=9.3Hz, 2H), 1.80 (br.s., 2H); LCMS (ESI) m/z: 460.3 (M+1).

Example 18

The synthesis of Example 18 was carried out by referring to Example 11 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400MHz, CD ₃ OD) δ 8.74 (d, J=7.9Hz, 1H), 8.20 (d, J=4.1Hz, 1H), 7.94-7.87 (m, 1H), 7.26 (d, J=4.6Hz, 1H), 5.44-5.27 (m, 1H), 5.03 (br s, 1H), 4.82-4.72(m, 1H), 4.00-3.88(m, 1H), 3.85-3.67(m, 2H), 3.65-3.38(m , 2H), 2.49(s, 3H), 2.38(s, 3H), 2.36-2.25(m, 2H), 2.25-2.11(m, 4H), 2.05(br d, J=8.9Hz, 2H), 1.80 (br s, 2H), 1.51 (d, J=6.3Hz, 2H), 1.46 (br d, J=5.8Hz, 1H), 1.43-1.35 (m, 1H), 1.39 (br d, J=6.3Hz, 2H); LCMS (ESI) m/z: 502.2 (M+1).

Example 19

The synthesis of Example 19 was carried out with reference to Examples 1 and 2 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.77 (s, 1H), 8.25 (s, 1H), 8.16-8.03 (m, 1H), 7.30 (s, 1H), 5.44-5.26 (m, 1H), 4.31 (br d, J=13.2 Hz, 2H), 4.21-4.06 (m, 2H), 3.70-3.35 (m, 2H), 2.93 (s, 3H), 2.55-2.45 (m, 3H), 2.38 (s, 3H), 2.37-1.92 (m, 10H), 1.80 (br s, 2H); LCMS (ESI) m/z: 488.1 (M+1).

Example 20

The synthesis of Example 20 was carried out by referring to Example 11 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400MHz, CD ₃ OD) δ8.76 (s, 1H), 8.31-8.20 (m, 1H), 8.14-8.05 (m, 1H), 7.27 (s, 1H), 5.37 (br t, J=8.5Hz, 1H), 4.54-4.35(m, 2H), 4.34-4.04(m, 2H), 3.67-3.45(m, 2H), 3.37(t d, J＝12.6, 6.4Hz, 1H), 2.54-2.45(m, 3H), 2.38(s, 3H), 2.34-2.26(m, 2H), 2.23(br d, J＝10.8Hz, 2H), 2.18(br d, J=7.5Hz, 2H), 2.14-2.08 (m, 2H), 2.07-1.98 (m, 2H), 1.86-1.73 (m, 1H), 1.80 (br s, 1H), 1.51 (d, J=6.4Hz, 6H); LCMS (ESI) m/z: 516.2 (M+1).

Example 21

The synthesis of Example 21 was carried out with reference to Example 1 to obtain the trifluoroacetate salt of the title compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.71 (s, 1H), 8.24 (s, 1H), 8.07 (br s, 1H), 7.36 (s, 1H), 5.38 (quin, J = 8.7 Hz, 1H), 4.92-4.47 (m, 2H), 4.20-3.58 (m, 5H), 3.31-3.01 (m, 2H), 2.50 (s, 3H), 2.37 (s, 3H), 2.32-2.13 (m, 6H), 2.09-2.01 (m, 2H), 1.81 (br d, J = 5.9 Hz, 2H); LCMS (ESI) m/z: 488.2 (M+1).

Example 22

Step 1:

To a solution of 2,5-dibromopyrazine (1.00 g, 4.20 mmol, 1.00 equiv) in 1-methylpyrrolidone (10.00 ml) was added 1-benzyl-1,7-diazaspiro[4,4]nonane (908.55 mg, 4.20 mmol, 1.00 equiv) and potassium carbonate (870.72 mg, 6.30 mmol, 1.50 equiv). The reaction mixture was heated to 100°C and stirred for 16 hours. LCMS showed that the reaction was complete. Water (15 ml) was added to the reaction mixture and extracted with ethyl acetate (30 ml x 3). The combined organic phases were washed with water (20 ml x 4), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 10:1) to give the title compound (952.00 mg, 2.37 mmol, yield: 56.47%, purity: 93%). ¹ H NMR (400MHz, CDCl ₃ )δ8.04 (d, J=1.2Hz, 1H), 7.63-7.51 (m, 1H), 7.29-7.20 (m, 4H), 7.18-7.13 (m, 1H), 3.68-3.53 (m, 3H), 3.52-3.32 (m, 2H), 3.29-3.18 (m, 1H), 2.72-2.55 (m, 2H), 2.28-2.14 (m, 1H), 1.94-1.68 (m, 5H); LCMS (ESI) m/z: 375.0 (M+1).

Step 2:

Under nitrogen, to a solution of 1-benzyl-7-(5-bromopyrazin-2-yl)-diazaspiro[4,4]nonane (800.00 mg, 2.14 mmol, 1.00 equiv) in toluene (10.00 mL) were added LiHMDS (1 M, 5.36 mL, 2.50 equiv), Pd ₂ (dba) ₃ (196.25 mg, 214.31 μmol, 0.10 equiv), and tri-tert-butylphosphine tetrafluoroborate (186.53 mg, 642.93 μmol, 0.30 equiv). The reaction mixture was heated to 65°C and stirred for 16 hours. TLC (petroleum ether:ethyl acetate = 10:1) indicated the reaction was complete. The reaction solution was concentrated, and the resulting residue was diluted with ethyl acetate (10 mL). Saturated aqueous potassium fluoride (10 mL) was added to the organic phase, and the resulting mixture was stirred at 30°C for 16 hours. The mixed solution was extracted with ethyl acetate (10 mL x 3), and the organic phase was concentrated. The resulting crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to dichloromethane:methanol = 20:1) to afford the title compound (700.00 mg, crude product). LCMS (ESI) m/z: 310.3 (M+1).

Step 3:

To a solution of 5-(1-benzyl-1,7-diazaspiro[4,4]nonan-7-yl)pyrazin-2-amine (400.00 mg, 1.29 mmol, 1.00 equiv) in tetrahydrofuran (5.00 ml) was added 3-acetyl-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Intermediate B) (472.80 mg, 1.55 mmol, 1.20 equiv) and potassium tert-butoxide (435.19 mg, 3.88 mmol, 3.00 equiv) and Xphos-Pd-G3 (191.01 mg, 258.56 μmol, 0.20 equiv). The reaction mixture was heated to 70° C. and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the resulting crude product was purified by preparative TLC (petroleum ether:ethyl acetate=3:1) to give the title compound (110.00 mg, crude product). LCMS (ESI) m/z: 578.2 (M+1).

Step 4:

To a mixed solution of 3-acetyl-7-[[5-(1-benzyl-1,7-diazaspiro[4,4]nonan-7-yl)pyrazin-2-yl]amino]-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (5.00 mg, 8.65 μmol, 1.00 equiv) in tetrahydrofuran (2.00 ml) and methanol (2.00 ml) was added palladium on carbon (5.00 mg). Maintaining hydrogen pressure (15 psi), the reaction mixture was stirred at 30°C for 32 hours. LCMS indicated the reaction was complete. The reaction solution was filtered and the filtrate was concentrated. The crude product was purified by preparative HPLC (hydrochloric acid) to give the hydrochloride salt of the title compound (550.00 μg, 1.05 μmol, yield: 12.13%, purity: 93%). ¹ H NMR (400 MHz, CD ₃ OD)δ8.74(s,1H),8.28-8.14(m,1H),7.93-7.84(m,1H),7.31-7.14(m,1H),5.48- 5.37(m, 1H), 4.08-4.06(m, 1H), 4.17-4.01(m, 1H), 3.93-3.77(m, 2H), 3.73-3.63( m, 1H), 3.55-3.50 (m, 1H), 2.96-2.88 (m, 1H), 2.51 (s, 5H), 2.43-2.36 (m, 3H), 2.3 1-2.13 (m, 8H), 2.11-2.01 (m, 2H), 1.89-1.78 (m, 2H); LCMS (ESI) m/z: 488.2 (M+1).

Example 23

The synthesis of Example 23 was carried out in accordance with Example 1. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.63 (s, 1H), 8.14 (d, J = 1.1 Hz, 1H), 7.63 (s, 1H), 7.58 (d, J = 1.1 Hz, 1H), 7.46 (s, 1H), 5.84-5.59 (m, 1H), 3.77 (dd, J = 9.8, 7.2 Hz, 1H), 3.72-3.63 (m, 1H), 3.46 (dt, J = 9.9, 6.9 Hz, 1H), 3.37- 3.25(m, 1H), 2.92-2.79(m, 1H), 2.59-2.51(m, 3H), 2.39(s, 3H), 2.34(s, 6H), 2.30-2.25(m, 2H ), 2.08 (brd, J=4.9Hz, 2H), 2.00-1.95 (m, 4H), 1.77-1.74 (m, 2H); LCMS (ESI) m/z: 476.2 (M+1).

Example 24

The synthesis of Example 24 was carried out with reference to Example 8 to obtain the hydrochloride of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.75 (s, 1H), 8.20 (s, 1H), 8.10 (br s, 1H), 7.27 (s, 1H), 5.40-5.25 (m, 1H), 4.52 (br d, J=12.3 Hz, 2H), 3.56 (br t, J=11.7 Hz, 1H), 3.01 (br t, J=12.5 Hz, 2H), 2.92 (s, 6H), 2.50 (s, 3H), 2.38 (s, 3H), 2.29-2.13 (m, 6H), 2.07 (br d, J=8.8 Hz, 2H), 1.80 (br d, J=7.8Hz, 4H); LCMS (ESI) m/z: 490.2 (M+1).

Example 25

The synthesis of Example 25 was carried out by referring to Example 1 to obtain the hydrochloride of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.75 (s, 1H), 8.19 (d, J = 1.0 Hz, 1H), 8.01 (s, 1H), 7.24 (s, 1H), 5.37 (quin, J = 8.7 Hz, 1H), 4.06 (td, J = 14.7, 4.8 Hz, 1H), 3.93-3.84 (m, 1H), 3.75-3.59 (m, 2 H), 3.51-3.40 (m, 1H), 2.85 (d, J=1.9Hz, 6H), 2.51 (s, 3H), 2.39 (s, 4H), 2.31-2 .13 (m, 6H), 2.12-1.97 (m, 3H), 1.91-1.71 (m, 4H); LCMS (ESI) m/z: 504.3 (M+1).

Example 26

Step 1:

To a solution of benzyl 4-piperidone-1-carboxylate (10.00 g, 42.87 mmol, 8.55 mL, 1.00 equiv), tert-butyl piperazine-1-carboxylate (9.58 g, 51.44 mmol, 1.20 equiv), and acetic acid (2.57 g, 42.87 mmol, 2.45 mL, 1.00 equiv) in methanol (100.00 mL) was added sodium acetate borohydride (22.71 g, 107.18 mmol, 2.50 equiv) at 25°C. The reaction mixture was stirred for 20 hours. LCMS indicated the formation of the title compound. The reaction mixture was concentrated, and the resulting residue was diluted with ethyl acetate. The resulting organic phase was washed sequentially with water (100 mL) and saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 10:1 to 1:1) to give the title compound (14.00 g, 27.65 mmol, yield: 64.50%, purity: 79.7%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.43-7.31 (m, 5H), 5.16-5.12 (m, 2H), 4.36-4.16 (m, 2H), 4.02-3.81 (m, 1H), 3.49-3.40 (m, 4H), 3.23-3.09 (m, 1H), 2.81 (br s, 2H), 2.59-2.45 (m, 4H), 1.81 (br d, J = 10.5 Hz, 2H), 1.58-1.39 (m, 11H).

Step 2:

To a solution of tert-butyl 4-(1-benzyloxycarbonyl-4-piperidinyl)piperazine-1-carboxylate (9.00 g, 22.30 mmol, 1.00 equiv) in tetrahydrofuran (90.00 ml) at 25°C was added palladium on carbon (1.00 g). The suspension was degassed under vacuum and purged with hydrogen several times. Maintaining hydrogen pressure (15 psi), the mixture was stirred at 25°C for 16 hours. LCMS showed that the starting material was completely consumed and the MS of the target compound was detected. The reaction mixture was filtered and the filtrate was concentrated to give the target compound (5.25 g, 19.49 mmol, yield: 87.40%). LCMS (ESI) m/z: 270.1 (M+1).

Step 3:

To a mixed solution of tert-butyl 4-(4-piperidinyl)piperazine-1-carboxylate (2.27 g, 8.41 mmol, 1.00 equiv) and 2,5-dibromopyrazine (2.00 g, 8.41 mmol, 1.00 equiv) in dimethyl sulfoxide (30.00 ml) and water (6.00 ml) was added potassium carbonate (1.28 g, 9.25 mmol, 1.10 equiv). The reaction mixture was heated to 90°C and stirred for 16 hours. LCMS showed complete consumption of the starting material, and MS of the title compound was detected. The reaction mixture was cooled to 25°C, water (30 ml) was added, and the resulting mixture was stirred for 30 minutes and then filtered. The filter cake was dissolved in dichloromethane (30 ml) and washed sequentially with water (20 ml x 2) and saturated brine (20 ml x 2). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to give the title compound (1.92 g, 4.41 mmol, yield: 52.41%, purity: 97.88%), which was used directly in the next reaction without purification. ¹ H NMR (400MHz, CDCl ₃ ) δ8.10 (d, J=1.4Hz, 1H), 7.87 (d, J=1.4Hz, 1H), 4.28 (br d, J＝13.3Hz, 2H), 3.52-3.33(m, 4H), 2.98-2.80(m, 2H), 2.60-2.42(m, 4H), 1.92(br d, J＝12.3Hz, 2H), 1.78(br s, 1H), 1.59-1.50 (m, 2H), 1.46 (s, 9H); LCMS (ESI) m/z: 426.0 (M+1).

Step 4:

Under nitrogen, to a solution of tert-butyl 4-[1-(5-bromopyrazin-2-yl)-4-piperidinyl]piperazine-1-carboxylate (1.50 g, 3.44 mmol, 1.00 equiv) and tri-tert-butylphosphine tetrafluoroborate (299.73 mg, 1.03 mmol, 0.30 equiv) in toluene (15.00 mL) were added LiHMDS (1 M, 7.09 mL, 2.06 equiv) and _Pd2 (dba) ₃ (315.34 mg, 344.36 μmol, 0.10 equiv). The reaction mixture was heated to 65°C and stirred for 16 hours. LCMS indicated that most of the starting material had been consumed, and the target compound was detected. The reaction mixture was quenched with saturated aqueous ammonium chloride (10 mL) and extracted with ethyl acetate (20 mL). The organic phase was adjusted to pH 2 with 10% aqueous citric acid solution, followed by separation. The aqueous phase was adjusted to pH 9 with 10% sodium hydroxide solution and filtered. The filter cake was vacuum-dried to yield the title compound (672.00 mg, 1.85 mmol, yield: 53.72%, purity: 100%), which was used directly in the next reaction without purification. LCMS (ESI) m/z: 363.1 (M+1).

Step 5:

Under nitrogen, to a solution of tert-butyl 4-[1-(5-aminopyrazin-2-yl)-4-piperidinyl]piperazine-1-carboxylate (300.00 mg, 827.65 μmol, 1.00 equiv), 3-acetyl-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Intermediate B) (252.24 mg, 827.65 μmol, 1.00 equiv), and potassium tert-butoxide (278.61 mg, 2.48 mmol, 3.00 equiv) in tetrahydrofuran (20.00 ml) was added Xphos-Pd-G3 (35.03 mg, 41.38 μmol, 0.05 equiv). The reaction mixture was heated to 80° C. and stirred for 16 hours. LCMS indicated that most of the starting material had been consumed, and MS of the target compound was detected. The reaction mixture was concentrated, and the resulting residue was dissolved in ethyl acetate (15 mL) and washed with water (10 mL x 2). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by preparative TLC (dichloromethane:methanol = 10:1) to yield the title compound (63.00 mg, 82.87 μmol, yield: 10.01%, purity: 82.976%). LCMS (ESI) m/z: 631.3 (M+1).

Step 6:

To a solution of tert-butyl 4-[1-[5-[(3-acetyl-1-cyclohexyl-4-methyl-2-oxo-1,6-naphthyridin-7-yl)amino]pyrazin-2-yl]-4-piperidin]piperazine-1-carboxylate (63.00 mg, 82.87 μmol, 1.00 equiv) in dichloromethane (3.00 mL) at 25°C was added trifluoroacetic acid (1.54 g, 13.51 mmol, 1.00 mL, 162.98 equiv), and the reaction mixture was stirred for 30 minutes. LCMS indicated complete consumption of the starting material and the MS of the target compound was detected. The reaction mixture was concentrated, and the crude product was purified by preparative HPLC (hydrochloric acid) to provide the hydrochloride salt of the target compound (13.00 mg, 19.34 μmol, yield: 23.34%, purity: 95.231%). ¹ HNMR (400MHz, CD ₃ OD) δ 8.73 (s, 1H), 8.20 (s, 1H), 8.13 (s, 1H), 7.24 (s, 1H), 5.35 (quin, J=8.5Hz, 1H), 4.56 (br d, J＝13.1Hz, 2H), 4.05-3.43(m, 9H), 3.03(br t, J＝12.3Hz, 2H), 2.49(s, 3H), 2.38(s, 3H), 2.34(br s, 2H), 2.29-2.12(m, 4H), 2.11-1.99(m, 2H), 1.96-1.84(m, 2H), 1.80(br d, J=5.6Hz, 2H); LCMS (ESI) m/z: 531.3 (M+1).

Example 27

The synthesis of Example 27 was carried out with reference to Example 26 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.75 (s, 1H), 8.22 (s, 1H), 8.13 (s, 1H), 7.26 (s, 1H), 5.45-5.31 (m, 1H), 4.56 (br d, J = 13.4 Hz, 2H), 4.12 (br d, J = 10.1 Hz, 2H), 3.89 (br t, J = 12.0 Hz, 2H), 3.58 (br d, J = 12.2 Hz, 3H), 3.30-3.20 (m, 2H), 3.02 (br t, J = 12.6 Hz, 2H), 2.51 (s, 3H), 2.39 (s, 3H), 2.33 (br d, J=10.1Hz, 2H), 2.15-2.03 (m, 3H), 2.26-2.00 (m, 3H), 1.90-1.74 (m, 4H); LCMS (ESI) m/z: 532.3 (M+1).

Example 28

Step 1:

Under nitrogen, to a mixed solution of 2,5-dibromopyrazine (2.00 g, 8.41 mmol, 1.00 equiv) in 1,4-dioxane (20.00 ml) and water (2.00 ml) were added tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxoboran-2-yl)-3,6-dihydro-2H-piperidine-1-carboxylate (2.60 g, 8.41 mmol, 1.00 equiv), potassium phosphate (3.57 g, 16.82 mmol, 2.00 equiv), and Pd(dppf) _Cl₂ (307.60 mg, 420.50 μmol, 0.05 equiv). The reaction mixture was heated to 100° C. and stirred for 16 hours. TLC (petroleum ether:ethyl acetate = 20:1) indicated the reaction was complete. The reaction solution was concentrated and the crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1 to 10:1) to give the title compound (1.60 g, crude product).

Step 2:

Under nitrogen, to a solution of tert-butyl 4-(5-bromopyrazin-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (1.50 g, 4.41 mmol, 1.00 equiv) in dioxane (20.00 mL) were added benzhydryl imine (879.15 mg, 4.85 mmol, 814.03 μL, 1.10 equiv), cesium carbonate (2.87 g, 8.82 mmol, 2.00 equiv), Pd(OAc) _₂ (99.01 mg, 441.00 μmol, 0.10 equiv), and BINAP (274.60 mg, 441.00 μmol, 0.10 equiv). The reaction was heated to 100° C. and stirred for 18 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to 20°C, diluted with dichloromethane (20 mL), filtered, and the filtrate was concentrated. The crude product was purified by preparative HPLC (basic) to give the title compound (1.40 g, 3.14 mmol, yield: 71.12%, purity: 98.7%). ¹ H NMR (400MHz, CDCl ₃ ) δ8.36 (d, J=1.1Hz, 1H), 7.88 (d, J=1.1Hz, 1H), 7.82 (br d, J=6.7Hz, 2H), 7.58-7.39 (m, 4H), 7.30 (br d, J=7.1Hz, 2H), 7.17 (br d, J=6.2Hz, 2H), 6.55 (br s, 1H), 4.11 (br d, J=2.4Hz, 2H), 3.62 (brt, J=5.5Hz, 2H), 2.57 (br s, 2H), 1.48 (s, 9H).

Step 3:

To a solution of tert-butyl 4-[5-(benzylideneamino)pyrazin-2-yl]-3,6-dihydro-2H-pyridine-1-carboxylate (500.00 mg, 1.12 mmol, 1.00 equiv) in methanol (10.00 ml) was added sodium acetate (110.27 mg, 1.34 mmol, 1.20 equiv) and hydroxylamine hydrochloride (140.12 mg, 2.02 mmol, 1.80 equiv) at 25°C and stirred for 30 minutes. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified by preparative TLC (petroleum ether:ethyl acetate = 1:1) to give the title compound (190.00 mg, 687.58 μmol, yield: 61.38%). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.10 (d, J=1.2Hz, 1H), 7.95 (d, J=1.3Hz, 1H), 6.42 (br s, 1H), 4.59 (br s, 2H), 4.11 (br d, J=2.7Hz, 2H), 3.64 (br t, J=5.6Hz, 2H), 2.58 (br s, 2H), 1.49 (s, 9H).

Step 4:

To a solution of tert-butyl 4-(5-aminopyrazin-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate (180.00 mg, 651.40 μmol, 1.00 equiv) in methanol (3.00 mL) was added wet palladium on carbon (100.00 mg). The reaction mixture was stirred at 25°C for 1 hour while maintaining a hydrogen pressure (15 psi). LCMS indicated approximately 60% starting material remained. The reaction mixture was stirred under these conditions for an additional 18 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered and the filtrate was concentrated to afford the title compound (160.00 mg, 574.82 μmol, yield: 88.24%). 1H NMR (400MHz, CDCl ₃ ) δ7.94 (d, J=1.4Hz, 1H), 7.87 (d, J=1.4Hz, 1H), 4.46 (br s, 2H), 4.25 (br s, 2H), 2.82 (br t, J=10.5Hz, 2H), 2.78-2.69 (m, 1H), 1.85 (br d, J=13.2Hz, 2H), 1.69 (dd, J=12.7, 4.1Hz, 2H), 1.48 (s, 9H).

Step 5:

Under nitrogen, to a solution of tert-butyl 4-(5-aminopyrazin-2-yl)piperidine-1-carboxylate (150.00 mg, 538.89 μmol, 1.00 equiv) in tetrahydrofuran (3.00 ml) were added 3-acetyl-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Intermediate B) (164.24 mg, 538.89 μmol, 1.00 equiv), potassium tert-butoxide (120.94 mg, 1.08 mmol, 2.00 equiv) and Xphos-Pd-G3 (45.61 mg, 53.89 μmol, 0.10 equiv). The reaction mixture was heated to 70° C. and stirred for 18 hours. LCMS showed that the reaction was complete. The reaction mixture was cooled to 25° C. and diluted with dichloromethane (10 ml), filtered, and the filtrate concentrated. The crude product was purified by preparative TLC (petroleum ether: ethyl acetate = 1:2) to give the title compound (92.00 mg, 168.29 μmol, yield: 31.23%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.70 (s, 1H), 8.40 (d, J = 1.4 Hz, 1H), 8.27 (s, 1H), 8.29-8.23 (m, 1H), 8.09 (d, J = 1.3 Hz, 1H), 7.65 (s, 1H), 5.82 (quin, J = 9.4 Hz, 1H), 4.29 (br s, 2H), 2.87 (ddd, J=11.8, 8.4, 3.8Hz, 3H), 2.57 (s, 3H), 2.43 (s, 3H), 2.39-2.28 (m, 2H), 2.28-2.1 6(m, 2H), 2.08-1.99(m, 2H), 1.95-1.88(m, 2H), 1.87-1.71(m, 4H), 1.49(s, 8H), 1.51-1.47(m, 1H).

Step 6:

To a solution of tert-butyl 4-[5-[(3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1,6-naphthyridin-7-yl)amino]piperazin-2-yl]piperidine-1-carboxylate (87.00 mg, 159.15 μmol, 1.00 equiv) in dichloromethane (1.00 mL) was added trifluoroacetic acid (500.00 μL). The reaction was stirred at 30°C for 0.5 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the crude product was purified by preparative HPLC (hydrochloric acid) to afford the hydrochloride salt of the title compound (37.58 mg, 70.61 μmol, yield: 44.36%, purity: 97.599%). ¹ H NMR (400MHz, CD ₃ OD) δ 8.86 (s, 1H), 8.62 (d, J = 0.8Hz, 1H), 8.43 (s, 1H), 7.47 (s, 1H), 5.40 (quin, J = 8.6Hz, 1H), 3.55 (br d, J=12.8Hz, 2H), 3.29-3.17 (m, 3H), 2.50 (s, 3H), 2.41 (s, 3H), 2.30-2.01 (m, 10H), 1.86-1.74 (m, 2H); LCMS (ESI) m/z: 447.2 (M+1).

Example 29

The synthesis of Example 29 was carried out by referring to Example 1 to obtain the trifluoroacetate salt of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.69 (s, 1H), 8.32 (d, J=1.1 Hz, 1H), 8.08 (d, J=1.5 Hz, 1H), 7.70 (br s, 1H), 3.91-3.71 (m, 4H), 4.88-4.87 (m, 1H), 3.42-3.35 (m, 4H), 2.67-2.51 (m, 2H), 2.48 (s, 3H), 2.37 (s, 3H), 2.04-1.93 (m, 2H), 1.80 (br d, J=11.8Hz, 3H), 1.54 (q, J=13.0Hz, 2H), 1.40 (brt, J=12.7Hz, 1H); LCMS (ESI) m/z: 462.3 (M+1).

Example 30

The synthesis of Example 30 was carried out with reference to Example 1 to obtain the hydrochloride salt of the target compound. ¹ H NMR (400 MHz, CD ₃ OD) δ 8.84 (s, 1H), 8.17 (d, J = 1.5 Hz, 1H), 8.05 (d, J = 1.5 Hz, 1H), 7.75-7.60 (m, 3H), 7.47-7.36 (m, 2H), 6.30 (s, 1H), 3.94-3.78 (m, 4H), 3.41-3.34 (m, 4H), 2.52 (s, 3H), 2.49 (s, 3H); LCMS (ESI) m/z: 456.1 (M+1).

Plan B

Example 31

Step 1:

Under nitrogen, to a mixed solution of 3-bromo-7-chloro-1-cyclopentyl-4-methyl-1,6-naphthyridin-2-one (Intermediate A) (3.00 g, 7.86 mmol, 1.00 equiv), potassium vinyl trifluoroborate (1.26 g, 9.43 mmol, 1.20 equiv), and cesium carbonate (5.12 g, 15.72 mmol, 2.00 equiv) in dioxane (30.00 ml) and water (6.00 ml) was added Pd( _PPh₃ ) _₂Cl₂ (275.76 _mg , 392.88 μmol, 0.05 equiv). The reaction mixture was heated to 110°C and stirred for 2 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to 25°C, water (20 ml) was added, and the mixture was concentrated to remove the dioxane. The aqueous phase was extracted with ethyl acetate (30 mL x 3). The combined organic phases were washed with saturated brine (30 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 30:1) to obtain the title compound (Compound 9) (1.00 g, 3.46 mmol, yield: 44.03%). ¹ H NMR (400MHz, CDCl ₃ )δ8.77 (s, 1H), 7.34 (s, 1H), 6.82-6.72 (m, 1H), 5.75 (s, 1H), 5.71 (dd, J=7.4, 1.9Hz, 1H), 5.41 (quin, J=8.9Hz, 1H), 2 .60 (s, 3H), 2.28-2.18 (m, 2H), 2.17-2.08 (m, 2H), 2.05-1.97 (m, 2H), 1.82-1.74 (m, 2H); LCMS (ESI) m/z: 251.1 (M+1).

Step 2:

To a solution of 7-chloro-1-cyclopentyl-4-methyl-3-vinyl-1,6-naphthyridin-2-one (Compound 9) (700.00 mg, 2.42 mmol, 1.00 equiv) in dioxane (8.00 mL) and water (2.00 mL) were added sodium periodate (1.56 g, 7.27 mmol, 402.97 μL, 3.00 equiv) and osmium tetroxide (24.65 mg, 96.96 μM, 5.03 μL, 0.04 equiv). The mixture was stirred at 30°C for 2 hours. TLC indicated the reaction was complete. The reaction mixture was diluted with water (10 mL), filtered, and the filtrate extracted with ethyl acetate (20 mL x 2). The combined organic phases were washed with saturated brine (20 mL x 2), dried over anhydrous sodium sulfate, filtered, and the filtrate concentrated. The crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to give the title compound (Compound 10) (560.00 mg, 1.93 mmol, yield: 79.62%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 10.46 (s, 1H), 8.87 (s, 1H), 7.28 (s, 1H), 5.33 (quin, J = 8.9 Hz, 1H), 2.77 (s, 3H), 2.15 (br dd, J = 12.1, 7.6 Hz, 2H), 2.05 (br dd, J = 8.4, 5.4 Hz, 2H), 1.99-1.93 (m, 2H), 1.76-1.69 (m, 2H).

Step 3:

To a solution of 7-chloro-1-cyclopentyl-1-4-methyl-2-oxo-1,6-naphthyridine-3-carbaldehyde (Compound 10) (560.00 mg, 1.93 mmol, 1.00 equiv) in dichloromethane (6.00 mL) was added DAST (1.56 g, 9.65 mmol, 1.27 mL, 5.00 equiv) at 25°C. The reaction mixture was stirred for 16 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated, and the resulting crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 20:1) to afford the title compound (Compound 11) (500.00 mg, 1.60 mmol, yield: 82.84%). ¹ H NMR (400MHz, CDCl ₃ )δ10.46 (s, 1H), 8.87 (s, 1H), 7.28 (s, 1H), 5.33 (quin, J=8.9Hz, 1H), 2.77 (s, 3 H), 2.20-2.11(m, 2H), 2.08-2.01(m, 2H), 1.99-1.92(m, 2H), 1.78-1.70(m, 2H).

Step 4:

Under nitrogen, to a solution of 7-chloro-1-cyclopentyl-3-(difluoromethyl)-4-methyl-1,6-naphthyridin-2-one (Compound 11) (100.00 mg, 319.75 μmol, 1.00 equiv) and tert-butyl 4-(5-aminopyrazin-2-yl)piperazine-1-carboxylate (107.18 mg, 383.71 μmol, 1.20 equiv) in dioxane (2.00 ml) were added cesium carbonate (208.36 mg, 639.51 μmol, 2.00 equiv), Xphos (15.24 mg, 31.98 μmol, 0.10 equiv), and Pd(OAc) ₂ (3.59 mg, 15.99 μmol, 0.05 equiv). The reaction mixture was heated to 100° C. and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to 30°C, filtered, and the filtrate was concentrated. The crude product was purified by preparative TLC (petroleum ether:ethyl acetate = 2:1) to yield the title compound (Compound 12) (16.70 mg, 30.06 μmol, yield: 9.40%). LCMS (ESI) m/z: 251.1 (M+1).

Step 5:

To a solution of tert-butyl 4-[5-[[1-cyclopentyl-3-(difluoromethyl)-4-methyl-2-oxo-1,6-naphthyridin-7-yl]amino]pyrazin-2-yl]piperazine-1-carboxylate (Compound 12) (20.00 mg, 36.00 μmol, 1.00 equiv) in dichloromethane (2.00 mL) was added trifluoroacetic acid (1.00 mL) at 30°C, and the reaction was stirred for 0.5 hours. LCMS indicated the reaction was complete. The reaction solution was concentrated under reduced pressure to remove dichloromethane and trifluoroacetic acid. The crude product was purified by preparative HPLC (hydrochloric acid) to afford the hydrochloride salt of the title compound (15.00 mg, 28.03 μmol, yield: 77.86%, purity: 98.74%). ¹ H NMR (400MHz, CD ₃ OD) δ8.86 (s, 1H), 8.28 (d, J=0.9Hz, 1H), 8.22 (s, 1H), 7.33-7.06 (m, 2H), 5.45-5.34 (m, 1H), 3. 96-3.88(m, 4H), 3.45-3.39(m, 4H), 2.70(s, 3H), 2.30-2.16(m, 4H), 2.12-2.02(m, 2H), 1.83(br d, J=5.5Hz, 2H); LCMS (ESI) m/z: 251.1 (M+1).

Example 32

To a solution of 1-cyclopentyl-3-(difluoromethyl)-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (177.00 mg, 388.58 μmol, 1.00 equiv) in dichloroethane (5.00 mL) were added acetone (112.84 mg, 1.94 mmol, 142.84 μL, 5.00 equiv), sodium acetate borohydride (205.89 mg, 971.45 μmol, 2.50 equiv), and acetic acid (46.67 mg, 777.16 μmol, 44.45 μL, 2.00 equiv). The reaction mixture was stirred at 30°C for 2 hours. LCMS indicated complete consumption of the starting material, and MS of the target compound was detected. The reaction solution was concentrated under reduced pressure, and the obtained crude product was purified by preparative HPLC (hydrochloric acid) to obtain the hydrochloride salt of the target compound (49.67 mg, 85.49 μmol, yield: 22.00%, purity: 98.19%). ¹ H NMR (400 MHz, CD ₃ OD) δ 8.86 (s, 1H), 8.27 (d, J = 1.1 Hz, 1H), 8.23 (d, J = 1.2 Hz, 1H), 7.34-7.07 (m, 1H), 7.24 (s, 1H), 5.45-5.33 (m, 1H), 4.58 (br d, J = 13.6 Hz, 2H), 3.68 (br d, J=13.1Hz, 2H), 3.72-3.66 (m, 1H), 3.43-3.35 (m, 2H), 3.32-3.22 (m, 2H), 2.70 (s, 3H), 2.32-2.1 3 (m, 4H), 2.12-2.00 (m, 2H), 1.88-1.75 (m, 2H), 1.46 (d, J=6.6Hz, 6H); LCMS (ESI) m/z: 498.0 (M+1).

Example 33

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (0.2 g, 446.89 μmol, 1 eq) in DMF (4 mL) was added 2-bromoethanol (72.60 mg, 580.96 μmol, 41.25 μL, 1.3 eq) and sodium carbonate (142.10 mg, 1.34 mmol, 3.0 eq). The reaction mixture was heated to 80°C and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered and purified by preparative HPLC (basic) to provide the title compound. ¹ H NMR (400MHz, DMSO-d ₆ ) δ9.95 (br s, 1H), 8.75 (s, 1H), 8.54 (s, 1H), 7.97 (s, 1H), 7.63 (s, 1H), 5.57 (quin, J=9.1Hz, 1H), 3.55 (t, J=6.2Hz, 2H), 3.48-3.41 (m, 4H), 2.57 -2.53 (m, 4H), 2.47-2.39 (m, 5H), 2.34 (s, 3H), 2.25-2.06 (m, 4H), 1.94-1.82 (m, 2H), 1.77-1.65 (m, 2H); LCMS (ESI) m/z: 492.4 (M+1).

Example 34

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (200 mg, 446.89 μmol, 1 eq), 2-chloro-N,N-dimethylethanamine (64.37 mg, 446.89 μmol, 1 eq, hydrochloride), and sodium carbonate (142.10 mg, 1.34 mmol, 3 eq) in DMF (5 mL) was added sodium iodide (13.40 mg, 89.38 μmol, 0.2 eq). The reaction mixture was heated to 80°C and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (hydrochloric acid) to provide the hydrochloride salt of the title compound. ¹ H NMR (400MHz, CD ₃ OD) δ8.75 (s, 1H), 8.27 (d, J=0.9Hz, 1H), 8.22 (s, 1H), 7.25 (s, 1H), 5.41 (quin, J=8.8Hz, 1H), 3.82-3.70 (m, 4H), 3.69- 3.35(m, 4H), 3.33-3.31(m, 4H), 3.06(s, 6H), 2.51(s, 3H), 2.40(s, 3H), 2.32-2.16(m, 4H), 2.13-2.03(m, 2H), 1.83(br d, J=5.9Hz, 2H); LCMS (ESI) m/z: 519.5 (M+1).

Example 35

Step 1:

To a solution of 3-acetyl-1-cyclopentyl-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (0.15 g, 335.17 μmol, 1 eq) in DMF (3 mL) were added tert-butyl N-(2-bromoethyl)carbamate (90.13 mg, 402.21 μmol, 1.2 eq) and sodium carbonate (106.57 mg, 1.01 mmol, 3.0 eq). The reaction mixture was heated to 80°C and stirred for 16 hours. LCMS indicated that the starting material had reacted and the desired product was detected. The reaction mixture was filtered to obtain a DMF solution of the desired compound, which was used directly in the next step without further purification. LCMS (ESI) m/z: 591.5 (M+1).

Step 2:

To a solution of tert-butyl N-[2-[4-[5-[(3-acetyl-1-cyclopentyl-4-methyl-2-oxo-1,6-naphthyridin-7-yl)amino]pyrazin-2-ylpiperazin-1-yl]ethyl]carboxylate (197.99 mg, 335.17 μmol, 1 eq) in DMF (3 mL) was added trifluoroacetic acid (1 mL), and the reaction mixture was stirred at 20°C for 15 hours. LCMS showed that the starting material was not completely converted, but a large amount of the desired product was produced. The reaction mixture was filtered, and the filtrate was purified by preparative HPLC (hydrochloric acid) to provide the hydrochloride salt of the desired compound. ¹ H NMR (400MHz, DMSO-d ₆ ) δ 11.52 (br s, 1H), 10.72 (br s, 1H), 8.78 (s, 1H), 8.57 (s, 1H), 8.46 (br s, 3H), 8.14 (d, J=1.0Hz, 1H), 7.67 (s, 1H), 5.53 (br t, J=8.9Hz, 1H), 4.34(br s, 2H), 3.49-3.30(m, 6H), 2.44(s, 3H), 2.33(s, 3H), 2.26-2.04(m, 4H), 1.90(br d, J=8.6Hz, 2H), 1.77-1.65 (m, 2H); LCMS (ESI) m/z: 491.4 (M+1).

Example 36

To a solution of O-ethylhydroxylamine hydrochloride (130.78 mg, 1.34 mmol, 6 eq) in pyridine (2 mL) was added 3-acetyl-1-cyclopentyl-4-methyl-7-[(5-piperazin-1-ylpyrazin-2-yl)amino]-1,6-naphthyridin-2-one (0.1 g, 223.45 μmol, 1 eq). The reaction mixture was heated to 70°C and stirred for 16 hours. LCMS indicated the reaction was complete. The reaction mixture was cooled to 20°C and concentrated to dryness. The resulting residue was purified by preparative HPLC (hydrochloric acid) to afford the hydrochloride salt of target compound 36a or 36b. ¹ H NMR (400MHz, CD ₃ OD) δ9.45 (br s, 2H), 8.74 (s, 1H), 8.53 (s, 1H), 8.09 (d, J=1.1Hz, 1H), 7.64 (s, 1H), 5.57-5.46 (m, 1H), 4.11 (br d, J=7.0Hz, 4H), 3.76-3.71 (m, 4H), 3.21 (br s, 4H), 2.37 (s, 3H), 2.23-2.09 (m, 4H), 2.00 (s, 3H), 1.89 (brd, J=9.0Hz, 2H), 1.70 (br s, 2H), 1.24 (t, J=7.0 Hz, 3H); LCMS (ESI) m/z: 491.3 (M+1); HPLC of the target compound: RT=2.088 min.

Pharmacology

The compounds described in this invention are CDK4/6 inhibitors. The following experimental results confirm that the compounds listed in this patent are indeed CDK4/6 inhibitors and have potential as anticancer drugs. The _IC50 value used herein refers to the concentration of an agent that produces 50% maximal inhibition.

Experimental Example 1: Enzyme Activity Test

Experimental Materials:

CDK4/cyclin D1 and CDK6/cyclin D1 were assayed using Life Science. ULight-labeled peptide substrates ULight-4E-BP1 and ULight-MBP were used (PerkinElmer). Europium-labeled anti-myelin basic protein antibodies and europium-labeled rabbit antibodies were used (PerkinElmer). Signals were detected using the Envision Multilabel Analyzer (PerkinElmer).

Experimental methods:

The compounds to be tested were diluted three-fold, comprising 10 concentration steps, with the final concentration range being 5 μM to 0.25 nM.

●CDK4/cyclin D1 enzyme reaction system

The standard Lance Ultra method was performed using a 10 μL enzyme reaction system containing 0.3 nM CDK4/cyclin D1 protein, 50 nM ULight-4E-BP1 peptide, and 350 μM ATP. Each reaction was dissolved in enzyme buffer consisting of 50 mM hydroxyethylpiperazine ethanesulfonic acid (pH 7.5), 1 mM EDTA, 10 mM magnesium chloride, 0.01% Brij-35, and 2 mM dithiothreitol. After initiation of the reaction, the OptiPlate 384-well plate was sealed with TopSeal-A and incubated at room temperature for 180 minutes.

CDK6/cyclin D1 enzyme reaction system

The standard Lance Ultra method was performed using a 10 μL enzyme reaction containing 0.8 nM CDK6/cyclin D1 protein, 50 nM ULight-4E-BP1 peptide, and 250 μM ATP. Each reaction was dissolved in enzyme buffer consisting of 50 mM hydroxyethylpiperazine ethanesulfonic acid (pH 7.5), 1 mM EDTA, 10 mM magnesium chloride, 0.01% Brij-35, and 2 mM dithiothreitol. After initiation of the reaction, the OptiPlate 384-well plate was sealed with TopSeal-A and incubated at room temperature for 180 minutes.

Prepare enzyme reaction stop buffer and dissolve EDTA in a 1x dilution of assay buffer. Terminate the reaction at room temperature for 5 minutes. Add 5 μL of the assay mix (prepared with europium-labeled anti-MYB antibody and europium-labeled rabbit antibody, respectively) to the CDK4/cyclin D1 and CDK6/cyclin D1 reactions. Incubate at room temperature for 60 minutes. Detect the reaction signal using time-resolved fluorescence resonance energy transfer (TRFE) using an Envision instrument.

Data Analysis:

The raw data were converted to inhibition rates using the equation (Max-Ratio)/(Max-Min)*100%, and the _IC50 values were obtained by four-parameter curve fitting (derived using the 205 model in XLFIT5, iDBS). Table 1 provides the inhibitory activities of the compounds of the present invention against CDK4/CDK6 kinases.

Experimental results: see Table 1.

Experimental conclusion:

The compounds of the present invention have significant inhibitory activity against CDK4 and CDK6 kinases.

Experimental Example 2: Cell Viability Test

Experimental Materials:

RPMI 1640 medium (Invitrogen-22400089), fetal bovine serum (Gibco-10099141), penicillin/streptomycin antibiotics (Hyclone-SV30010), and L-glutamine (Invitrogen-35050079). The NCI-H358 cell line was obtained from the WuXi AppTec Biological Cell Bank. Envision multi-label analyzer (PerkinElmer) was used.

Experimental methods:

1) Add 100 μL of phosphate buffered saline to the outer wells of a 384-well microplate. Add 40 μL of NCI-H358 cell suspension (containing 250 NCI-H358 cells) to each of the remaining wells. Then, place the cell plate in a CO2 incubator and culture overnight.

2) The test compound was diluted 3-fold using Echo, and each compound was diluted into 10 concentration gradients (from 25 μM to 1.27 nM) and 100 nL was added to the corresponding wells of the cell plate. The cell plate was then returned to the CO2 incubator and cultured for 7 days.

3) Add 20 μL of Promega CellTiter-Glo reagent to each well of the cell plate and shake at room temperature in the dark for 10 minutes to allow the luminescence signal to stabilize. Read the data using a PerkinElmer Envision Multilabel Analyzer.

Data Analysis:

The raw data were converted to inhibition rates using the equation (Max-Sample)/(Max-Min)*100%. _IC50 values were then calculated using a four-parameter curve fit (calculated using the log(inhibitor) vs. response - Variable slope fitting formula in GraphPad Prism). Table 1 provides the inhibitory activity of the compounds of the present invention against H358 cell proliferation.

Experimental results: see Table 1.

Experimental conclusion:

The compound of the present invention has better proliferation inhibition activity on NCI-H358 lung cancer cells than the reference compound Palbociclib.

Table 1

Test compound Palbociclib 5.5 1.3 314 Example 3 6.1 2.9 176 Example 9 9.1 3.4 195.6 Example 11 5.6 3.1 278 Example 14 7.1 2.4 197 Example 15 5.7 3.1 262 Example 19 7.0 3.7 214 Example 28 8.3 3.5 295 Example 33 8.62 4.66 153 Example 34 7.45 3.65 179 Example 35 5.06 2.97 189

Experimental Example 3: Caco-2 Cell Bidirectional Permeability Evaluation Experiment

Purpose of the experiment:

Caco-2 cells, a type of human colon cancer cell, are a widely used in vitro model for studying small intestinal absorption. The Caco-2 cell monolayer model has been widely used to evaluate passive and active transport processes during small intestinal absorption. This assay was used to determine the bidirectional permeability of the compounds of this invention and the reference compounds, palbociclib and LY2835219, across the Caco-2 cell model.

Experimental operation:

The standard experimental conditions are as follows:

- Test concentration: 2 μM (DMSO ≤ 1%);

- Replicate: n = 2;

- Direction: Bidirectional transport, including A→B (intracellular→extracellular) and B→A (extracellular→intracellular);

- Incubation time: single time point, 2 hours;

-Transport buffer: HBSS, pH 7.4;

- Incubation conditions: 37°C, 5% CO ₂ .

After incubation, sample solutions from the dosing and receiving wells were immediately mixed with cold acetonitrile containing an internal standard. The concentration of the test compound in all samples (including the initial dosing solution, the supernatant from the dosing well, and the receiving solution) was analyzed using LC/MS/MS. Parameters such as the apparent permeability coefficient and efflux ratio were also calculated.

Experimental results:

See Table 2. Table 2 lists the permeability coefficients of the compounds of the present invention and reference compounds Palbociclib and LY2835219 in Caco-2 monolayer cells.

Experimental conclusion:

Compared to the reference compounds Palbociclib and LY2835219, the compounds of the present invention have high permeability and are less likely to be affected by efflux transporters in vivo. This improved permeability allows the compounds of the present invention to be distributed more widely in tissues (such as the lungs), resulting in improved anti-tumor efficacy in vivo. Furthermore, this improved permeability makes it possible for the compounds of the present invention to penetrate the blood-brain barrier, achieving the goal of treating lung cancer with brain metastases.

Table 2

Experimental Example 4: Solubility Experiment

(1) Kinetic solubility experiment

Purpose of the experiment:

The kinetic solubility of the compounds was determined using conventional biological screening analytical conditions.

Detection principle:

Kinetic solubility is pH-dependent, and the pH of the solution is typically set at 7.4. This experiment employed shake-flask HPLC analysis. Each compound was prepared in DMSO to a 10 mM stock solution and diluted to a theoretical concentration of 200 μM in phosphate buffer (containing 2% DMSO). The solution was shaken and equilibrated at room temperature for 24 hours. The supernatant was then filtered and analyzed by HPLC-UV.

Experimental operation:

Kinetic Solubility Test Solution

Buffer (pH 7.4)

50 mM phosphate buffer, pH 7.4.

Preparation of standard solution:

A dilution solution was obtained by mixing 50% acetonitrile solution and 50% buffer solution.

10 mM (10 μL/compound) stock solution was added to 490 μL of diluent to prepare a 200 μM detection standard solution.

Dilute 200 μM UV detection standard solution with 10-fold or 200-fold diluent to obtain 20 μM or 1 μM UV standard solution.

UV standard solutions at 1, 20, and 200 μM were used as standard solutions for the kinetic solubility test.

method:

Sample preparation, shaking and filtration

Dissolve the compound in DMSO to prepare a 10 mM stock solution. A minimum of 100 μL of stock solution is required. Amiodarone hydrochloride, carbamazepine, and chloramphenicol are used as QCs for solubility testing.

Accurately measure 490 μL of dissolution medium (buffer) into 2 mL 96-well plates.

Add 10 μL of each stock solution of the test compound and QC to the dissolution medium (buffer) corresponding to the kinetic solubility solution at pH 7.4, containing 2% DMSO, and a theoretical maximum concentration of 200 μM for the test compound. Cap the vial. The theoretical maximum concentration of the test compound is 200 μM. If a higher theoretical maximum concentration is desired, increase the concentration of the stock solution.

Incubate on a shaker at 600 rpm at room temperature for 24 hours.

The samples were transferred to a 96-well filter plate and filtered.

The compound concentration in the filtrate was tested by HPLC-UV.

QC samples:

Data Analysis:

Inject three UV standards into the HPLC system, starting from low to high concentrations, followed by a test sample of the filtrate of the test compound. The test sample is injected into two parallel injections. Integrate the UV chromatographic peaks. Simulate the standard curve and calculate the kinetic solubility of the sample.

Experimental results:

See Table 3-1. Table 3-1 lists the kinetic solubility data of the compound of the present invention and the reference compound Palbociclib.

Experimental conclusion:

Compared to the reference compound Palbociclib, the compounds of the present invention have higher kinetic solubility.

Table 3-1

Test compound Kinetic solubility (pH=7.4, μM) Palbociclib 103 Example 3 171 Example 34 194

(2) Thermodynamic solubility experiment

Purpose of the experiment:

This experiment can accurately and reliably determine the thermodynamic solubility of compounds through filtration and HPLC methods.

Detection principle:

The thermodynamic solubility of compounds was determined using the shake flask method and HPLC. Compound solubility is a key property that influences compound drug screening and absorption in animals and humans. To determine compound solubility, a saturated solution of the compound must first be obtained, followed by quantitative analysis using HPLC.

Experimental operation:

Thermodynamic solubility solution

Buffer (pH 7.4)

50 mM phosphate buffer, pH 7.4

Preparation of standard solution:

Mix 50% acetonitrile solution and 50% buffer solution to obtain a dilution solution.

10 mM (10 μL/compound) stock solution was added to the diluent (490 μL/compound) and mixed to obtain a 200 μM UV detection standard solution.

Dilute the 200 μM UV detection standard solution with 10-fold or 100-fold dilution to obtain 20 μM or 2 μM UV standard solutions.

UV standard solutions of 2, 20, and 200 μM were used as standard samples for the thermodynamic solubility test.

method:

Sample preparation, shaking and filtration

Weigh at least 2 mg of sample powder into a Whatman miniuniprep vial. If the thermodynamic solubility of the sample is to be tested in multiple buffer solutions, a separate vial is required for each test.

Add 450 μL of buffer (pH 7.4) to each Whatman miniuniprep vial.

After adding the buffer, the piston cap of the Whatman miniuniprep with filter was installed and pressed above the liquid level so that the filter was in contact with the buffer solution during shaking.

Vortex the solubility sample for 2 minutes and record the solution behavior.

The mixture was shaken at 550 rpm at room temperature (about 22-25°C) for 24 hours.

Press the cap of the Whatman Miniunipreps filter bottle to the bottom to obtain the filtrate of the sample solubility solution. All sample vials should be checked for insoluble matter and leakage before and after filtration.

The sample dilution solution was obtained by diluting the buffer solution 50 times.

Inject three UV standards into the HPLC from low to high concentration, followed by the dilution and supernatant of the test compound. Inject the test sample twice.

Integrate UV chromatographic peaks, simulate standard curves, and calculate the thermodynamic solubility of samples.

QC samples:

Experimental results:

See Table 3-2. Table 3-2 lists the thermodynamic solubility data of the compounds of the present invention and the reference compound Palbociclib.

Experimental conclusion:

Compared with the reference compound Palbociclib, the compounds of the present invention have higher thermodynamic solubility.

Table 3-2

Test compound Thermodynamic solubility (pH = 7.4, μM) Palbociclib 65.3 Example 34 6420

Experimental Example 5: Metabolic stability study of rat, mouse and human liver microsomes

Purpose of the experiment:

This test is used to test the metabolic stability of the test substance in rat, mouse and human liver microsomes

Experimental operation:

1) The test compound concentration was 1 μM and incubated with liver microsomes with a protein concentration of 0.5 mg/mL in a 37°C water bath under the action of a reduced coenzyme II regeneration system.

2) Positive controls include: testosterone (3A4 substrate), propantheline (2D6 substrate), and diclofenac (2C9 substrate). The incubation conditions for the positive controls should be the same as those for the compound.

3) The reaction time points were: 0, 5, 10, 20, 30, and 60 minutes. The reaction was terminated at the corresponding time points using a stop solution containing an internal standard. The compound was also incubated with the microsomes for 60 minutes without the presence of a reduced coenzyme II regeneration system as a negative control.

4) Each time point is a single point (n=1).

5) Samples were analyzed by LC/MS/MS, and compound concentrations were displayed as the ratio of compound peak area to internal standard peak area (non-standard curve).

6) In the project report summary, the half-life and clearance rate will be calculated.

7) The following formula is used to calculate the clearance rate:

Note:

a) Microsomal protein in incubation: protein concentration during incubation

Liver weight to body weight ratio: rat, mouse and human parameters are 40g/kg, 88g/kg and 20g/kg respectively

The clearance in the whole liver was calculated using:

Note:

a) microsomes: microsomes;

b) liver: liver;

c)body weight: body weight;

Experimental results:

The experimental results are shown in Table 4.

Experimental conclusion:

The stability of the compound of the present invention in liver microsomes of humans, rats and mice is significantly better than that of the reference compounds LY2835219 and Palbociclib.

Table 4

Test substance Palbociclib 44.7/47.8/53.3 LY2835219 2.69/6.34/2.36 Example 34 43.1/＞145/40.2

Experimental Example 6: In vivo efficacy study (I)

In vivo efficacy studies were performed on BALB/c nude mice subcutaneously implanted with human-derived tumor xenografts (PDXs) derived from LU-01-0393 lung cancer patients.

Experimental operation:

BALB/c nude mice, female, 6-8 weeks, weighing approximately 17-21 grams, were kept in a special pathogen-free environment in single ventilated cages (5 mice per cage). All cages, bedding, and water were disinfected before use. All animals had free access to standard certified commercial laboratory diets. A total of 36 mice purchased from Beijing Vital River Laboratory Animal Co., LTD were used for the study. Each mouse was implanted subcutaneously with a tumor LU-01-0393FP4 slice (20-30 cubic millimeters) in the right back for tumor growth. Dosing was initiated when the average tumor volume reached approximately 150-200 cubic millimeters. The test compound was administered orally daily at the doses shown in Table 2. Tumor volume was measured twice a week with a two-dimensional caliper, and the volume was measured in cubic millimeters and calculated by the following formula: V= ^0.5axb2 , where a and b are the long and short diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the mean tumor volume increase in compound-treated animals by the mean tumor volume increase in untreated animals.

Experimental results: see Table 5.

Experimental conclusion:

The compounds of the present invention demonstrated significant antitumor activity in a human-derived tumor xenograft (PDX) model derived from a LU-01-0393 lung cancer patient. As shown in Table 5, 20 days after the start of the experiment, the tumor volume in the untreated group rapidly increased from an initial 144 cubic millimeters to 437 cubic millimeters. During the same period, the tumor volume in the group treated with Example 1 increased slowly from an initial 144 cubic millimeters to 212 cubic millimeters. This growth rate was similar to that of the group treated with the reference compound Palbociclib. However, the dose of Example 1 (60 mg/kg) was only half that of the reference compound Palbociclib (120 mg/kg). This indicates that the compounds of the present invention have superior antitumor activity to the reference compound.

Table 5

Experimental Example 7: In vivo efficacy study (II)

In vivo efficacy experiments were performed on BALB/c nude mice subcutaneously implanted with the NCI-H358 non-small cell lung cancer model.

Experimental operation:

The experimental animal information of Example 3 is as follows: BALB/c nude mice, female, 6-8 weeks, weighing approximately 17-20 grams, were kept in a special pathogen-free environment and in single ventilated cages (3-5 mice per cage). All cages, bedding and water were disinfected before use. All animals had free access to standard certified commercial laboratory diets. A total of 86 mice purchased from Beijing Vital River Laboratory Animal Co., LTD were used for the study. The experimental animal information of Example 34 is as follows: BALB/c nude mice, female, 6-8 weeks, weighing approximately 16-18 grams, were kept in a special pathogen-free environment and in single ventilated cages (4 mice per cage). All cages, bedding and water were disinfected before use. All animals had free access to standard certified commercial laboratory diets. A total of 56 mice purchased from Shanghai Lingchang Biotechnology Co., Ltd. were used for the study.

Each mouse was implanted subcutaneously with NCI-H358 tumor cells on the right back for tumor growth. Dosing was initiated when the average tumor volume reached approximately 100-200 cubic millimeters. The test compound was administered orally daily. The dosages for Example 3 and Example 34 are shown in Table 6-1 and Table 6-2, respectively. Tumor volume was measured twice weekly using a two-dimensional caliper. Volume was measured in cubic millimeters and calculated using the following formula: V = 0.5 a x ^{b 2} , where a and b are the major and minor diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the mean tumor volume increase in animals treated with the compound by the mean tumor volume increase in untreated animals, while compound safety was determined by weight change in animals treated with the compound.

Experimental results: See Table 6-1 and Table 6-2.

Experimental conclusion:

The compounds of the present invention exhibited significant antitumor activity in the NCI-H358 model of non-small cell lung cancer and had good safety. In addition, in this model, the antitumor effect of the compounds of the present invention showed a dose-dependent trend.

Table 6-1

Table 6-2

TGI: Tumor Growth Inhibition. TGI (%) = [(1 - (average tumor volume of a treatment group at the end of drug administration - average tumor volume of the treatment group at the start of drug administration)) / (average tumor volume of the solvent control group at the end of treatment - average tumor volume of the solvent control group at the start of treatment)] × 100%.

Experimental Example 8: In vivo efficacy study (III)

In vivo efficacy experiments were performed on BALB/c nude mice bearing the subcutaneous colorectal cancer HCT-116 model.

Experimental operation:

BALB/c nude mice, female, 6-8 weeks old, weighing approximately 18-22 grams, were maintained in a special pathogen-free environment in single ventilated cages (3-5 mice per cage). All cages, bedding, and water were disinfected before use. All animals had free access to a standard certified commercial laboratory diet. A total of 48 mice purchased from Shanghai Lingchang Laboratory Animal Co., Ltd. were used for the study. 0.2 mL of 5×106 HCT-116 cells were subcutaneously inoculated into the right back of each nude mouse. Group dosing began when the average tumor volume reached 132 ^mm3 . The test compound was orally administered daily at the dose shown in Table 5. Tumor volume was measured twice a week using a two-dimensional caliper, and the volume was measured in cubic millimeters and calculated using the following formula: V=0.5ax ^b2 , where a and b are the long and short diameters of the tumor, respectively. Antitumor efficacy was determined by dividing the average tumor volume increase in animals treated with the compound by the average tumor volume increase in untreated animals.

Experimental results: see Table 7.

Experimental conclusion:

The compound of the present invention exhibits good anti-tumor activity and high safety in the colorectal cancer HCT-116 model.

Table 7

Claims (14)

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, in, _R1 is selected from _CH3 (C＝O)-; _R2 is independently selected from H, OH, or selected from _{C1-5 alkyl groups that are optionally substituted with 1, 2} , or 3 Rs. Ring A is a 5- to 9-membered heterocyclic alkyl group, wherein the heteroatom is a nitrogen atom, and the number of heteroatoms is 1 or 2; Ring B is selected from _C3-6 cycloalkyl groups; R is selected from OH, _NH2 , _{CH3 and CH3CH2} . 2. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein ring B is selected from cyclobutyl, cyclopentyl, and cyclohexyl. 3. The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein ring B is selected from cyclopentyl and cyclohexyl. 4. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein _R2 is independently selected from H, OH, or selected from _CH3 , which is optionally substituted with 1, 2 or 3 Rs. 5. The compound according to claim 4 or a pharmaceutically acceptable salt thereof, wherein _R2 is independently selected from H, or selected from _CH3 groups optionally substituted with 1, 2 or 3 Rs. 6. The compound according to claim 5 or a pharmaceutically acceptable salt thereof, wherein _R2 is independently selected from: H, _CH3 , 7. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the structural units are selected from: 8. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein the structural units are selected from: 9. The compound according to claim 7 or 8, or a pharmaceutically acceptable salt thereof, wherein the structural unit Selected from: 10. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: Wherein, R ₂ is as defined in any of claims 1 and 4 to 6; R is as defined in claim 1; Ring A is as defined in any of claims 1 to 9. 11. The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is selected from: Wherein, R ₂ is as defined in any of claims 1 and 4 to 6. 12. The following compound or its pharmaceutically acceptable salt: 13. A pharmaceutical composition comprising a therapeutically effective amount of the compound according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. 14. The use of the compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, or the pharmaceutical composition according to claim 13, in the preparation of a medicament for treating cancer.