WO2019134539A1 - Dihydropyrazolone and pyrimidine compound, preparation method and use therefor - Google Patents

Abstract

A dihydropyrazolone and pyrimidine compound, and a preparation method and a use therefor. Specifically, the present application relates to a compound of formula I, a pharmaceutical composition and a pharmaceutical formulation comprising said compound, a preparation method for said compound, and a use of said compound in the preparation of drugs for the prevention or treatment of Wee1 protein-related diseases.

Description

Dihydropyrazolone pyrimidine compound and preparation method and use thereof Technical field

The present application relates to a dihydropyrazolone pyrimidine compound, a process for its preparation and its use.

Background technique

The cell cycle is a highly regulated and controlled process that is tightly regulated by a complex network of proteins, metabolism, and microenvironment interactions, designed to allow cells to proliferate only to specific stimuli and appropriate conditions (S. Diaz-Moralli , M. Tarrado-Castellarnau, A. Miranda, M. Cascante, Pharmacology & Therapeutics, 2013, 138: 255-271). The standard cell cycle in turn undergoes G1 phase, S phase (DNA synthesis phase), G2 phase, and M phase (cell division phase). There are several periodic block checkpoints during G1-S conversion, S phase, G2-M conversion, etc., to maintain genome integrity and provide time to repair damaged DNA before entering mitosis. In response to DNA damage, cell cycle checkpoints can be activated during G1/S phase, S phase, and G2/M phase, and induce cell cycle arrest until repair is completed; if repair is unsuccessful, cell senescence or cells will be driven Apoptosis (R. Visconti, MRDella, D. Grieco, Journal of Experimental & Clinical Cancer Research, 2016, 35: 153).

Wee1 protein kinase is a member of the serine/threonine protein kinase family and is a key component of the G2/M checkpoint of the cell cycle, playing a key role in cell division (CJMatheson, DSBackos, P. Reigan, Trends in Pharmacological Sciences, 2016, 37: 872). Entry into the mitosis from the G2/M checkpoint depends on the phosphorylation state of CDK1 (also known as CDC2) and the binding state of cyclin B. Before mitosis, Wee1 phosphorylates the Tyr15 site of CDK1, and then the myelin transcription factor (MYT1) phosphorylates the Thr14 site of CDK1, maintains the inactive state of CDK1, and inhibits cells from entering M phase. Therefore, Wee1 is a negative regulator of cells from the G2 phase to the M phase.

Thus, inhibition of Wee1 kinase activity to remove the function of the G2/M checkpoint is a promising strategy to drive tumor cells into unplanned mitosis, thereby undergoing mitotic disorders leading to cell death. This cell is forced into mitosis without completing DNA replication and is highly toxic to cells, representing a novel mechanism for inducing tumor cell death. Therefore, Wee1 inhibitor has a good application prospect as a drug. Patent WO2007126128 reports that certain compounds exhibit the activity of Wee1 protein kinase inhibitors.

In order to meet better tumor treatment effects, safer or less side-effect drug requirements, the present application will provide a novel structure of Wee1 protein kinase inhibitor, and found that compounds of such structure exhibit excellent Wee1 protein inhibition and drugs. Duration of effectiveness.

Summary of the invention

An aspect of the present application provides a dihydropyrazolone pyrimidine compound having a Wee1 protein kinase inhibitory activity, or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a polymorph, a solvent a substance, metabolite or prodrug having the structure of formula I below:

among them,

X is selected from CH and N;

R ^{1 is} selected from the group consisting of hydroxy-C _1-6 alkyl-, C _1-6 alkoxy-C _1-6 alkyl- and cyano-C _3-6 cycloalkyl-;

R ^{2 is} selected from a 7-10 membered fused heterocyclic group, and the 7-10 membered fused heterocyclic group is optionally substituted by one or more C _1-6 alkyl groups, and each C _1-6 alkyl group may be the same or different; Or R ^{2 is} selected from piperidinyl, the piperidinyl is optionally substituted by one or more -NR ⁵ R ⁶ ; R ⁵ , R ⁶ are each independently selected from C _1-6 alkyl;

R ^{3 is} selected from the group consisting of C _1-6 alkyl and C _2-6 alkenyl;

R ^{4 is} selected from the group consisting of hydrogen and halogen.

In a partially preferred embodiment, the compound has the structure of formula I-1 below:

Wherein X, R ¹ , R ² , R ³ and R ⁴ are as defined in formula I.

In a partially preferred embodiment, the compound has the structure of formula I-2 below:

Wherein R ¹ and R ² are as defined in formula I.

Another aspect of the present application provides a method of preparing a compound of formula (I):

Wherein Hal ¹ is halogen (for example F, Cl or Br); Hal ² is halogen (for example Cl, Br or I), a boronic acid group or a boronic acid ester group; R ¹ , R ² , R ³ , R ⁴ The meaning of X and X is the same as that of the above formula I.

In some embodiments, the present application also provides a method of preparing a compound of Formula 1-2:

Wherein Hal ¹ is halogen (for example F, Cl or Br); Hal ² is halogen (for example Cl, Br or I), a boronic acid group or a boronic acid ester group; PG is a protecting group, which may be selected from a benzyl group, P-methoxybenzyl, tert-butoxycarbonyl, benzyloxycarbonyl, preferably tert-butoxycarbonyl; R ¹ and R ² are as defined above for the general formula I-2.

Another aspect of the present application provides a pharmaceutical composition comprising the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite thereof or Prodrug, and one or more pharmaceutically acceptable carriers.

Another aspect of the present application provides a method of preparing the pharmaceutical composition, which comprises the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph thereof The solvate, metabolite or prodrug is combined with one or more pharmaceutically acceptable carriers.

Another aspect of the present application provides a pharmaceutical formulation comprising the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or A drug, or the pharmaceutical composition.

Another aspect of the application provides the compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, the pharmaceutical composition or The pharmaceutical preparation is used for the preparation of a disease for preventing or treating Wee1 protein kinase, and preferably the disease is cancer.

Another aspect of the application provides the compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, or the pharmaceutical composition, It is used for the treatment of Wee1 protein kinase-related diseases, preferably the disease is cancer.

Another aspect of the present application provides a method of preventing or treating a Wee1 protein kinase-associated disease, the method comprising administering to an individual in need thereof an effective amount of the compound or a pharmaceutically acceptable salt thereof, a stereoisomer, and a mutual An isomer, polymorph, solvate, metabolite or prodrug, or the pharmaceutical composition; preferably, the disease is cancer.

definition

Unless otherwise defined below, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques that are generally understood in the art, including those that are obvious to those skilled in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the invention.

The terms "including", "comprising", "having", "containing", or "comprising", as used herein, and other variants thereof, are inclusive or open, and not Exclude other unlisted elements or method steps.

As used herein, the term "alkyl" is defined as a straight or branched saturated aliphatic hydrocarbon group. In some embodiments, an alkyl group has from 1 to 12, such as from 1 to 6 carbon atoms. For example, as used herein, the term " _C1-6 alkyl" refers to a straight or branched chain group having from 1 to 6 carbon atoms (eg, methyl, ethyl, n-propyl, isopropyl, N-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl or n-hexyl), which is optionally substituted by one or more (such as 1 to 3) suitable substituents such as halogen (at this time) This group is referred to as "haloalkyl" such as CF ₃ , C ₂ F ₅ , CHF ₂ , CH ₂ F, CH ₂ CF ₃ , CH ₂ Cl or -CH ₂ CH ₂ CF _{3 ,} and the like.

As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated, non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (eg, a monocyclic ring such as cyclopropyl, cyclobutyl, cyclopentyl). , cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, or bicyclic, including spiro, fused or bridging systems (such as bicyclo [1.1.1] pentyl, bicyclo [2.2.1] heptyl, bicyclic [3.2.1] Octyl or bicyclo [5.2.0] anthracenyl, decalinyl, etc.), which is optionally substituted by one or more (such as 1 to 3) suitable substituents. The group has 3 to 15, for example 3 to 6 carbon atoms. For example, the term "C _3-6 cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic ring having 3 to 6 ring-forming carbon atoms or a polycyclic (such as bicyclic) hydrocarbon ring (eg, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl), which is optionally substituted with one or more (such as 1 to 3) suitable substituents, such as A substituted cyclopropyl group.

The term "halo" or "halogen" group, as used herein, is defined to include fluoro, chloro, bromo or iodo.

As used herein, the term "alkoxy" refers to an alkyl group, as defined above, appended to the parent molecular moiety through an oxygen atom. Representative examples of _C1-6 alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, n-butoxy, isobutoxy, tert-butoxy, pentyloxy, hexyloxy, and the like. .

The term "heterocyclyl" as used herein refers to a saturated or partially unsaturated monocyclic or polycyclic group, for example having 2, 3, 4, 5, 6, 7, 8 or 9 carbons in the ring. An atom and one or more (eg, 1, 2, 3 or 4) heteroatoms selected from nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), such as but not limited to epoxy Ethyl, aziridine, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, tetrahydropyran Base, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, etc., correspondingly, the term "fused heterocyclyl" refers to each ring An adjacent atomic polycyclic heterocyclic group is shared with other rings in the system, wherein one or more of the rings may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system, one of which The plurality of ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen or S(O) _m (where m is an integer from 0 to 2), and the remaining ring atoms are carbon. It is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, it may be classified into a bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic group, preferably a bicyclic or tricyclic ring, more preferably a 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclic group.

The term "substituted" means that one or more (eg, 1, 2, 3 or 4) hydrogens on the designated atom are replaced by the selection of the indicated group, provided that the specified atom is not exceeded. The normal valence of the current case and the substitution form a stable compound. Combinations of substituents and/or variables are permissible only if such combinations form stable compounds.

If a substituent is described as "optionally substituted with", the substituent may be unsubstituted or (2) substituted. If the carbon of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the carbon (to the extent of any hydrogen present) may be independently and/or together independently The optional substituents selected are substituted. If the nitrogen of the substituent is described as being optionally substituted by one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent of any hydrogen present) may each be independently selected. Substitute substitution.

If a substituent is described as being "independently selected from" a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same or different from another (other) substituent.

As used herein, the term "one or more" means 1 or more than 1, such as 2, 3, 4, 5 or 10 under reasonable conditions.

Unless otherwise indicated, a point of attachment of a substituent, as used herein, may come from any suitable position of the substituent.

When a bond of a substituent is shown to pass through a bond connecting two atoms in the ring, such a substituent may be bonded to any of the ring-forming atoms in the substitutable ring.

The invention also includes all pharmaceutically acceptable isotopically-labeled compounds which are identical to the compounds of the invention, except that one or more atoms are of the same atomic number but the atomic mass or mass number differs from the atomic mass prevailing in nature. Or atomic substitution of mass. Examples of isotopes suitable for inclusion in the compounds of the invention include, but are not limited to, isotopes of hydrogen (e.g., ² H, ³ H); isotopes of carbon (e.g., ¹¹ C, ¹³ C, and ¹⁴ C); isotopes of chlorine (e.g. ³⁷ Cl); isotope of fluorine (eg ¹⁸ F); isotopes of iodine (eg ¹²³ I and ¹²⁵ I); isotopes of nitrogen (eg ¹³ N and ¹⁵ N); isotopes of oxygen (eg ¹⁵ O, ¹⁷ O and ¹⁸ O) ); an isotope of phosphorus (eg, ³² P); and an isotope of sulfur (eg, ³⁵ S). Certain isotopically-labeled compounds of the invention (e.g., those incorporating radioisotopes) are useful in drug and/or substrate tissue distribution studies (e.g., assays). The radioisotope ruthenium (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) are particularly useful for this purpose because of their ease of incorporation and ease of detection. Substitution with positron emitting isotopes (eg, ¹¹ C, ¹⁸ F, ¹⁵ O, and ¹³ N) can be used to examine substrate receptor occupancy in positron emission tomography (PET) studies. Isotopically labeled compounds of the invention can be prepared by replacing the previously employed non-labeled reagents with suitable isotopically labeled reagents by methods analogous to those described in the accompanying routes and/or examples and preparations. The pharmaceutically acceptable solvates of the present invention include those in which the crystallization solvent can be substituted with an isotope, for example, D ₂ O, acetone-d ₆ or DMSO-d ₆ .

The term "stereoisomer" denotes an isomer formed by at least one asymmetric center. In a compound having one or more (eg, 1, 2, 3, or 4) asymmetric centers, which can produce a racemic mixture, a single enantiomer, a mixture of diastereomers, and Separate diastereomers. Specific individual molecules can also exist as geometric isomers (cis/trans). Similarly, the compounds of the invention may exist as mixtures (often referred to as tautomers) of two or more different forms in a rapidly balanced structure. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers Wait. For example, the dihydropyrimidinyl group may exist in equilibrium in the following tautomeric forms in solution. It is to be understood that the scope of the present application covers all such ratios in any ratio (eg, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99). %) isomer or a mixture thereof.

Solid lines can be used in this article

Solid wedge

Virtual wedge

The carbon-carbon bond of the compound is depicted. The use of solid lines to delineate linkages bonded to an asymmetric carbon atom is intended to include all possible stereoisomers at the carbon atom (eg, specific enantiomers, racemic mixtures, etc.). The use of a solid or virtual wedge to characterize a bond to an asymmetric carbon atom is intended to indicate the presence of the stereoisomers shown. When present in a racemic mixture, solid and virtual wedges are used to define relative stereochemistry rather than absolute stereochemistry. Unless otherwise indicated, the compounds are intended to be stereoisomers (including cis and trans isomers, optical isomers (eg, R and S enantiomers), diastereomers, geometry Forms of isomers, rotamers, conformers, atropisomers, and mixtures thereof exist. The compounds may exhibit more than one type of isomerism and consist of a mixture thereof (e.g., a racemic mixture and a diastereomeric pair).

The present application encompasses all possible crystalline forms or polymorphs of the compounds, which may be a single polymorph or a mixture of more than one polymorph in any ratio.

It will also be understood that certain compounds of the present application may exist in free form for treatment or, where appropriate, in the form of their pharmaceutically acceptable derivatives. In the present application, pharmaceutically acceptable derivatives include, but are not limited to, pharmaceutically acceptable salts, solvates, metabolites or prodrugs which, after administration to a patient in need thereof, can be directly or indirectly A compound of the invention or a metabolite or residue thereof is provided. Thus, when reference is made herein to "a compound of the present application" or "the compound", it is also intended to encompass the various derivative forms described above for the compound.

The pharmaceutically acceptable salts of the compounds of the present application include the acid addition salts and base addition salts thereof.

Suitable acid addition salts are formed from acids which form pharmaceutically acceptable salts. Examples include acetate, adipate, aspartate, benzoate, bicarbonate/carbonate, hydrogen sulfate/sulfate, fumarate, glucoheptonate, gluconate , glucuronate, hexafluorophosphate, hydrobromide/bromide, hydroiodide/iodide, maleate, malonate, methyl sulfate, naphthoate ( Naphthylate), nicotinate, nitrate, orotate, oxalate, palmitate and other similar salts.

Suitable base addition salts are formed from bases which form pharmaceutically acceptable salts. Examples include aluminum salts, arginine salts, choline salts, diethylamine salts, lysine salts, magnesium salts, meglumine salts, potassium salts, sodium salts, tromethamine salts, and other similar salts.

For a review of suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, 2002). Methods for preparing pharmaceutically acceptable salts of the compounds of the present application are known to those skilled in the art.

The compounds of the present application may exist in the form of a solvate, preferably a hydrate, wherein the compound of the present application contains a polar solvent as a structural element of the crystal lattice of the compound, especially such as water, methanol or ethanol. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric ratios.

The application also includes metabolites of the compounds, ie, substances formed in vivo upon administration of the compounds. Such products may be produced, for example, by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic hydrolysis, and the like of the administered compound. Accordingly, the application includes metabolites of the compounds, including compounds made by contacting the compound with a mammal for a time sufficient to produce its metabolites.

The present application further encompasses prodrugs of the compounds which are certain derivatives of the compounds which may themselves have less or no pharmacological activity, may be cleavable by, for example, hydrolytic hydrolysis when administered to or in the body. Conversion to the compound with the desired activity. Typically such prodrugs will be functional group derivatives of the compounds which are readily converted in vivo to the desired therapeutically active compound. Additional information on the use of prodrugs can be found in "Pro-drugs as Novel Delivery Systems", Volume 14, ACS Symposium Series (T. Higuchi and V. Stella) and "Bioreversible Carriers in Drug Design," Pergamon Press, 1987 ( Edited by EBRoche, American Pharmaceutical Association). The prodrug can be replaced, for example, by using certain parts known to those skilled in the art as "pro-moiety" (for example as described in "Design of Prodrugs", H. Bundgaard (Elsevier, 1985)" Prepared by the appropriate functional groups present in the compounds of the present application.

The present application also encompasses such compounds containing a protecting group. In any process in which the compound is prepared, it may be necessary and/or desirable to protect sensitive groups or reactive groups on any of the molecules of interest, thereby forming a chemically protected form of the compound. This can be achieved by conventional protecting groups such as those described in Protective Groups in Organic Chemistry, ed. JFW McOmie, Plenum Press, 1973; and TW Greene & P. GM Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 1991. Protecting groups, which are incorporated herein by reference. The protecting group can be removed at a suitable subsequent stage using methods known in the art.

The term "about" means within ±10% of the stated value, preferably within ±5%, more preferably within ±2%.

Compound

It is an object of the present application to provide a compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, having the formula The structure of I:

among them,

X is selected from CH and N;

R ^{1 is} selected from the group consisting of hydroxy-C _1-6 alkyl-, C _1-6 alkoxy-C _1-6 alkyl- and cyano-C _3-6 cycloalkyl-;

R ^{2 is} selected from a 7-10 membered fused heterocyclic group, and the 7-10 membered fused heterocyclic group is optionally substituted by one or more C _1-6 alkyl groups, and each C _1-6 alkyl group may be the same or different; Or R ^{2 is} selected from piperidinyl, the piperidinyl is optionally substituted by one or more -NR ⁵ R ⁶ ; R ⁵ , R ⁶ are each independently selected from C _1-6 alkyl;

R ^{3 is} selected from the group consisting of C _1-6 alkyl and C _2-6 alkenyl;

R ^{4 is} selected from the group consisting of hydrogen and halogen.

In a preferred embodiment, the compound has the structure shown in Formula I-1.

Wherein X, R ¹ , R ² , R ³ and R ⁴ are as defined in formula I.

In some embodiments, X in Formula I or Formula I-1 is CH.

In some embodiments, X in Formula I or Formula I-1 is N.

In embodiments, portions, of Formula I or Formula I-1 wherein R ¹ is selected from hydroxy -C _1-4 alkyl -, C _1-3 alkoxy -C _1-4 alkyl - cyano and -C _{3- 6} cycloalkyl-. In some preferred embodiments, R ^{1 is} selected from the group consisting of 2-hydroxypropan-2-yl, 2-methoxypropan-2-yl, and 1-cyanocycloprop-1-yl.

In some embodiments, R ² in Formula I or Formula I-1 is selected from a 7-10 membered bicyclic fused heterocyclyl optionally substituted by one or more C _1-6 An alkyl group (e.g., C _1-4 alkyl, C _1-2 alkyl) is substituted. In some embodiments, R ^{2 is} selected from an 8-membered fused heterocyclyl, which is substituted with a C _1-6 alkyl (eg, C _1-4 alkyl, C _1-2 alkyl) . In some preferred embodiments, R ^{2 is} selected from the group consisting of an 8-membered bicyclic fused heterocyclyl group, which is a C _1-6 alkyl group (eg, C _1-4 alkyl, C _1-2 Alkyl) substitution. In some preferred embodiments, R ^{2 is} selected from

In some preferred embodiments, R ² is 5-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-yl.

In some embodiments, R ² in Formula I or Formula I-1 is piperidinyl, the piperidinyl is substituted by one or more —NR ⁵ R ⁶ , and R ⁵ and R ⁶ are each independently selected from C _{1 -4} alkyl. In some preferred embodiments, R ² is piperidinyl, the piperidinyl is substituted by one or more -NR ⁵ R ⁶ , and R ⁵ and R ⁶ are each independently selected from C _1-2 alkyl. In some preferred embodiments, R ² is

R ⁵ and R ⁶ are each independently selected from a C _1-6 alkyl group (e.g., a C _1-4 alkyl group, a C _1-2 alkyl group). In some preferred embodiments, R ² is 4-dimethylaminopiperidin-1-yl.

In some embodiments, R ^{3 of} Formula I or Formula I-1 is selected from the group consisting of C _1-4 alkyl and C _2-4 alkenyl. In some preferred embodiments, R ^{3 is} selected from the group consisting of allyl and isopropyl.

In some embodiments, R ^{4 of} Formula I or Formula I-1 is selected from the group consisting of hydrogen, fluorine, chlorine, and bromine. In some embodiments, R ^{4 is} selected from the group consisting of hydrogen and fluorine. In some embodiments, R ⁴ is hydrogen. In some embodiments, R ⁴ is fluoro.

In some embodiments, the compound has the structure of Formula I-2,

Wherein R ¹ and R ² are as defined in formula I.

In some embodiments, R ^{1 is} selected from the group consisting of hydroxy-C _1-6 alkyl-, C _1-6 alkoxy-C _1-6 alkyl-, and cyano-C _3-6 cycloalkyl-.

In some embodiments, R ^{2 is} selected from 7-10 membered fused heterocyclyl, and the 7-10 membered fused heterocyclyl is optionally substituted by one or more C _1-6 alkyl, each C _1-6 alkane The groups may be the same or different; or R ^{2 is} selected from the group consisting of piperidinyl, which is substituted by one or more -NR ⁵ R ⁶ , and R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl.

In some embodiments, R ^{1 is} selected from the group consisting of hydroxy-C _1-4 alkyl-, C _1-3 alkoxy-C _1-4 alkyl-, and cyano-C _3-6 cycloalkyl-; preferably R ^{1 is} selected from the group consisting of 2-hydroxyprop-2-yl, 2-methoxyprop-2-yl and 1-cyanocycloprop-1-yl.

In some embodiments, R ^{2 is} selected from an 8-membered fused heterocyclyl, and the 8-membered fused heterocyclyl is substituted with a C _1-6 alkyl;

Preferably, R ^{2 is} selected from an 8-membered bicyclic fused heterocyclic group substituted by a C _1-6 alkyl group (eg, C _1-4 alkyl, C _1-2 alkyl);

Preferably, R ^{2 is} selected from

More preferably, R ² is 5-methylhexahydropyrrolo[3,4-c]pyrrole-2(1H)-yl.

In some embodiments, R ^{2 is} selected from

R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl (e.g., C _1-4 alkyl, C _1-2 alkyl); preferably, R ² is 4-dimethylaminopiperidin-1-yl .

In some embodiments, the compound has the structure of Formula I-2 above,

Wherein R ^{1 is} selected from the group consisting of hydroxy-C _1-6 alkyl-, C _1-6 alkoxy-C _1-6 alkyl- and cyano-C _3-6 cycloalkyl-;

R ^{2 is} selected from a 7-10 membered fused heterocyclic group, and the 7-10 membered fused heterocyclic group is optionally substituted by one or more C _1-6 alkyl groups, and each C _1-6 alkyl group may be the same or different; Or R ^{2 is} selected from the group consisting of piperidinyl, which is substituted by one or more -NR ⁵ R ⁶ , and R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl.

In some embodiments, the compound has the structure of Formula I-2 above,

Wherein R ^{1 is} selected from the group consisting of hydroxy-C _1-4 alkyl-, C _1-3 alkoxy-C _1-4 alkyl- and cyano-C _3-6 cycloalkyl-; preferably, R ^{1 is} selected From 2-hydroxypropan-2-yl, 2-methoxypropan-2-yl and 1-cyanocycloprop-1-yl;

R ^{2 is} selected from an 8-membered fused heterocyclic group substituted by a C _1-6 alkyl group; preferably, R ^{2 is} selected from

Preferably, R ² is 5-methylhexahydropyrrole [3,4-c]pyrrole-2(1H)-yl;

Alternatively, R ^{2 is} selected from

R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl; preferably, R ² is 4-dimethylaminopiperidin-1-yl.

In some embodiments, the compound has the structure:

Preparation

Another object of the present application is to provide a process for the preparation of a compound of formula (I):

Wherein Hal ¹ is halogen (for example F, Cl or Br); Hal ² is halogen (for example Cl, Br or I), a boronic acid group or a boronic acid ester group; R ¹ , R ² , R ³ , R ⁴ The meaning of X and X is the same as that of the above formula I.

(1) reacting compound IN-1 with a hydrazine compound to obtain compound IN-2;

In some embodiments: Compound IN-1 can be reacted with an alkyl hydrazine compound to provide compound IN-2, preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, dimethylacetamide, dioxane, and any combination thereof, preferably tetrahydrofuran. The reaction is preferably carried out in the presence of a suitable organic base. The organic base may be selected from the group consisting of triethylamine, pyridine, 4-dimethylaminopyridine, diisopropylethylamine, preferably diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably 50-70 °C. The reaction is preferably carried out for a suitable period of time, for example 6-16 hours.

In other embodiments, the compound IN-1 may also be first reacted with hydrazine hydrate, and then reacted with an aldehyde compound to obtain the compound IN-2. The reaction with hydrazine hydrate is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of ethanol, methanol, tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, dimethylacetamide, dioxane, and any combination thereof, preferably ethanol. The reaction is preferably carried out at a suitable temperature, preferably from 0 to 25 °C. The reaction is preferably carried out for a suitable period of time, for example 1-6 hours. The reduction reaction with the aldehyde compound is preferably carried out in a suitable organic solvent and in the presence of a reducing agent. The solvent may be selected from the group consisting of methanol, ethanol, dichloromethane, chloroform, acetonitrile, preferably methanol. The reducing agent may be selected from sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride, etc., preferably sodium cyanoborohydride. The reaction temperature is usually preferably from 0 to 25 ° C, and the reaction time is usually preferably from 1 to 3 hours.

(2) cyclizing compound IN-2 to give compound IN-3;

The cyclization reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of methanol, ethanol, tetrahydrofuran, dioxane, and any combination thereof, preferably ethanol. The base used for the cyclization may be selected from inorganic bases such as sodium hydroxide, potassium hydroxide, potassium carbonate, etc., preferably sodium hydroxide. The cyclization reaction temperature is usually preferably room temperature (20 to 30 ° C), and the cyclization reaction time is usually preferably from 0.5 to 2 hours.

(3) reacting compound IN-3 with compound IN-4 to give compound IN-5;

Compound IN-3 was coupled with compound IN-4 to give compound IN-5. The coupling reaction is preferably carried out in the presence of a metal catalyst and a base. Preferably, the metal catalyst is a copper salt catalyst, preferably cuprous iodide, copper acetate or the like. The base is an inorganic base such as potassium carbonate, cesium carbonate, sodium carbonate, 2,2-bipyridine or potassium phosphate, preferably potassium carbonate. The ligand for the coupling reaction is selected from the group consisting of trans-N,N'-dimethyl-1,2-cyclohexanediamine, N,N'-dimethylethylenediamine, preferably trans-N,N '-Dimethyl-1,2-cyclohexanediamine. The solvent for the coupling reaction is selected from the group consisting of benzene, toluene, dioxane, dimethylformamide, ethylene glycol dimethyl ether, and the like, or a mixed solvent thereof, preferably dioxane and dimethylformamide. The coupling reaction temperature is usually preferably from 80 to 110 °C. The reaction time is usually preferably from 4 to 6 hours.

(4) oxidizing compound IN-5 to give compound IN-6;

The oxidation reaction can be preferably carried out in a suitable organic solvent and in the presence of an oxidizing agent. The solvent may be selected from the group consisting of benzene, toluene, dichloromethane, chloroform, acetonitrile, dimethylformamide and the like, preferably acetonitrile. The oxidizing agent may be selected from the group consisting of m-chloroperoxybenzoic acid, potassium peroxymonosulfate complex salt, and the like, preferably a potassium peroxymonosulfate complex salt. The reaction temperature is usually preferably room temperature (20 to 30 ° C), and the reaction time is usually preferably from 0.5 to 3 hours.

(5) reacting compound IN-6 with compound IN-7 to give a compound of formula I;

The reaction is preferably carried out in an inert solvent including toluene, benzene, chloroform, dioxane, dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide or the like or a mixed solvent thereof, preferably toluene. The reaction is preferably carried out in the presence of a suitable organic base which may be selected from the group consisting of triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like, preferably diisopropylethylamine. The reaction temperature is usually preferably from 60 to 120 ° C, and the reaction time is usually preferably from 4 to 12 hours.

A further object of the present application is to provide a process for the preparation of a compound of formula 1-2:

Wherein Hal ¹ is halogen (for example F, Cl or Br); Hal ² is halogen (for example Cl, Br or I), a boronic acid group or a boronic acid ester group; PG is a protecting group, which may be selected from a benzyl group, P-methoxybenzyl, tert-butoxycarbonyl, benzyloxycarbonyl, preferably tert-butoxycarbonyl; R ¹ and R ² are as defined above for the general formula I-2.

(1) reacting compound IN-1 with compound IN-8 to give compound IN-9;

The reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of tetrahydrofuran, dichloromethane, chloroform, dimethylformamide, dimethylacetamide, dioxane, and any combination thereof, preferably tetrahydrofuran. The reaction is preferably carried out in the presence of a suitable organic base. The organic base may be selected from the group consisting of triethylamine, pyridine, 4-dimethylaminopyridine, diisopropylethylamine, preferably diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from 50 to 70 °C. The reaction is preferably carried out for a suitable period of time, for example 6-16 hours.

(2) removing the protecting group PG from the compound IN-9 to obtain the compound IN-10;

The reaction for removing the protecting group PG can be applied to a hydrolysis reaction method of a carboxylic acid ester well known in the field of organic chemistry, in which the protecting group PG is a tert-butoxycarbonyl group, the reaction preferably passes through an ester in the presence of an acid. The acid hydrolysis reaction proceeds. The acid may be a mineral acid or a suitable organic acid including, but not limited to, hydrochloric acid, sulfuric acid, formic acid, trifluoroacetic acid; the reaction solvent may be selected from dioxane, dichloromethane, ethyl acetate or a mixed solvent thereof. The acid can also be used as a reaction solvent, preferably trifluoroacetic acid. The reaction temperature is usually preferably from 40 to 70 °C. The reaction time is usually preferably from 1 to 3 hours.

(3) cyclizing the compound IN-10 to give the compound IN-11;

The cyclization reaction is preferably carried out in a suitable organic solvent. The organic solvent may be selected from the group consisting of methanol, ethanol, tetrahydrofuran, dioxane, and any combination thereof, preferably ethanol. The base used in the cyclization reaction may be selected from inorganic bases such as sodium hydroxide, potassium hydroxide, potassium carbonate, etc., preferably sodium hydroxide. The cyclization reaction temperature is usually preferably room temperature (20 to 30 ° C), and the cyclization reaction time is usually preferably from 0.5 to 2 hours.

(4) reacting compound IN-11 with compound IN-12 to give compound IN-13;

Compound IN-11 was coupled with compound IN-12 to give compound IN-13. The coupling reaction is preferably carried out in the presence of a metal catalyst and a base. The metal catalyst is a copper salt catalyst, preferably cuprous iodide, copper acetate or the like. The base is an inorganic base such as potassium carbonate, cesium carbonate, sodium carbonate, 2,2-bipyridine or potassium phosphate, preferably potassium carbonate. The ligand for the coupling reaction is selected from the group consisting of trans-N,N'-dimethyl-1,2-cyclohexanediamine, N,N'-dimethylethylenediamine, preferably trans-N,N '-Dimethyl-1,2-cyclohexanediamine. The solvent for the coupling reaction is selected from the group consisting of benzene, toluene, dioxane, dimethylformamide, ethylene glycol dimethyl ether, and the like, or a mixed solvent thereof, preferably dioxane and dimethylformamide. The coupling reaction temperature is preferably from 80 to 110 °C. The reaction time is preferably 4 to 6 hours.

(5) oxidizing compound IN-13 to give compound IN-14;

The oxidation reaction can be preferably carried out in a suitable organic solvent and in the presence of an oxidizing agent. The solvent may be selected from the group consisting of benzene, toluene, dichloromethane, chloroform, acetonitrile, dimethylformamide and the like, preferably acetonitrile. The oxidizing agent may be selected from the group consisting of m-chloroperoxybenzoic acid, potassium peroxymonosulfate complex salt, and the like, preferably a potassium peroxymonosulfate complex salt. The reaction temperature is usually preferably room temperature (20 to 30 ° C), and the reaction time is usually preferably from 0.5 to 3 hours.

(6) reacting compound IN-14 with compound IN-15 to give a compound of formula 1-2;

The reaction is preferably carried out in an inert solvent selected from the group consisting of toluene, benzene, chloroform, dioxane, dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, and the like, or a mixed solvent thereof, preferably Toluene. The reaction is preferably carried out in the presence of a suitable organic base which may be selected from the group consisting of triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like, preferably diisopropylethylamine. The reaction temperature is preferably from 60 to 120 ° C, and the reaction time is usually preferably from 4 to 12 hours.

In the preparation of compounds of formula I or formula I-2, it may be desirable to protect the distal functional groups of the intermediate (e.g., hydroxyl or amino groups). The need for such protection can vary with the nature of the remote functional groups and the conditions of the method of preparation. For an overview of protecting groups and their use, see T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1991.

Pharmaceutical composition and pharmaceutical preparation

Another object of the present application is to provide a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of the compound or a pharmaceutically acceptable salt thereof, a stereoisomer, a tautomer, a polymorph, a solvent a compound, metabolite or prodrug, and one or more pharmaceutically acceptable carriers.

By "pharmaceutically acceptable carrier" herein is meant a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting humans and/or others within the scope of sound medical judgment. Animal tissue without excessive toxicity, irritation, allergic reactions or other problems or complications corresponding to a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers for use in the pharmaceutical compositions include, but are not limited to, sterile liquids such as water and oils, including those oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, Sesame oil and so on. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. It is also possible to use physiological saline and an aqueous solution of glucose and glycerin as a liquid carrier, particularly for injection. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene glycol, water, Ethanol and the like. The composition may also contain minor amounts of wetting agents, emulsifying agents or pH buffering agents as needed. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutically acceptable carriers are as described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical composition can act systemically and/or locally. For this purpose, they may be administered in a suitable route, for example by injection (for example intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, including instillation) or transdermal administration; or by oral, buccal, or oral administration. Nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

For these routes of administration, the pharmaceutical compositions can be administered in a suitable dosage form. The dosage forms include, but are not limited to, tablets, capsules, troches, hard candy, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions. Injectable solutions, elixirs, syrups, and the like.

The amount or amount of the compound in the pharmaceutical composition may be from about 0.01 mg to about 1000 mg, suitably from 0.1 to 500 mg, preferably from 0.5 to 300 mg, more preferably from 1 to 150 mg.

Another object of the present application is to provide a method of preparing the pharmaceutical composition, which comprises the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph thereof The solvate, metabolite or prodrug is combined with one or more pharmaceutically acceptable carriers.

Another object of the present application is to provide a pharmaceutical preparation comprising the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite thereof or Prodrug, or a mixture thereof, or the pharmaceutical composition.

Treatment and use

Another object of the present application is to provide a compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, or a combination thereof Use of the medicament for the preparation of a medicament for preventing or treating a Wee1 protein kinase-related disease. In some embodiments, the Wee1 protein kinase associated disease is cancer.

Another object of the present application is to provide a compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, or a combination thereof For use in the prevention or treatment of Wee1 protein kinase-associated diseases. In some embodiments, the Wee1 protein kinase associated disease is cancer.

Another object of the present application is to provide a method for preventing or treating a Wee1 protein kinase-related disease, which comprises administering an effective amount of the compound or a pharmaceutically acceptable salt thereof, a stereoisomer, and each other to an individual in need thereof An isomer, polymorph, solvate, metabolite or prodrug, or the pharmaceutical composition. In some embodiments, the Wee1 protein associated disease is cancer.

In some embodiments, the cancer includes, but is not limited to, head and neck cancer, ovarian cancer, colorectal cancer, bladder cancer, breast cancer, non-small cell lung cancer, and endometrial cancer.

The term "effective amount" as used herein refers to an amount of a compound that, to a certain extent, relieves one or more symptoms of the condition being treated after administration.

The dosing regimen can be adjusted to provide the optimal desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgent need for treatment. It is noted that the dose value can vary with the type and severity of the condition to be alleviated and can include single or multiple doses. It is to be further understood that for any particular individual, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering the composition or the composition of the supervised composition.

The amount of the compound administered will depend on the individual being treated, the severity of the condition or condition, the rate of administration, the handling of the compound, and the judgment of the prescribing physician. Generally, an effective dose will be from about 0.0001 to about 50 mg per kg body weight per day, for example from about 0.01 to about 10 mg/kg/day (single or divided doses). For a 70 kg person, this would add up to about 0.007 mg/day to about 3500 mg/day, such as from about 0.7 mg/day to about 700 mg/day. In some cases, a dose level that is not higher than the lower limit of the aforementioned range may be sufficient, while in other cases, a larger dose may still be employed without causing any harmful side effects, provided that the larger The dose is divided into several smaller doses to be administered throughout the day.

The term "treating" as used herein, unless otherwise indicated, means reversing, alleviating, inhibiting the progression of a condition or condition to which such a term applies or the progression of one or more symptoms of such a condition or condition, or preventing such A condition or condition or one or more symptoms of such condition or condition.

"Individual" as used herein includes human or non-human animals. Exemplary human individuals include a human individual (referred to as a patient) or a normal individual having a disease, such as the disease described herein. As used herein, "non-human animal" includes all vertebrates, such as non-mammals (eg, birds, amphibians, reptiles) and mammals, such as non-human primates, domestic animals, and/or domesticated animals (eg, sheep, dogs, Cats, cows, pigs, etc.).

Detailed ways

Example

In order to make the objects and technical solutions of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. Further, specific experimental methods not mentioned in the following examples were carried out in accordance with a conventional experimental method.

The abbreviations in this article have the following meanings:

abbreviation	meaning
TLC	Thin layer chromatography
LC-MS	Liquid chromatography-mass spectrometry
DIPEA	N,N-diisopropylethylamine
CD 3 OD	Deuterated methanol
CDCl 3	Deuterated chloroform
DMSO-d 6	Hexafluoro dimethyl sulfoxide
TMS	Tetramethylsilane
NMR	Nuclear magnetic resonance
MS	Mass spectrometry
s	Single peak (singlet)
d	Doublet
t	Triplet
q	Quadruple (quartet)
Dd	Double doublet
m	Multiplet
J	Coupling constant
Hz	hertz

The structure of the compound described in the following examples was confirmed by ¹ H-NMR or MS. ^{The 1} H-NMR measuring instrument was measured using a Bruker 400 MHz nuclear magnetic resonance apparatus, and the solvent was CD ₃ OD, CDCl ₃ or DMSO-d ₆ , and the internal standard substance was TMS. The total δ value was expressed by ppm. The mass spectrometer (MS) assay instrument used an Agilent (ESI) mass spectrometer, model Agilent 6120B.

Thin layer chromatography silica gel plates (TLC) were prepared using an aluminum plate (20 x 20 cm) manufactured by Merck, and separated by thin layer chromatography using GF 254 (0.4 to 0.5 mm).

The reaction was monitored by thin layer chromatography (TLC) or LC-MS. The developing solvent system used was: dichloromethane and methanol system, n-hexane and ethyl acetate system, petroleum ether and ethyl acetate system, solvent volume ratio. The adjustment is carried out depending on the polarity of the compound or by adding triethylamine or the like.

Microwave reaction

Initiator + (400 W, RT ~ 300 ° C) microwave reactor.

Column chromatography generally uses 200 to 300 mesh silica gel as a carrier. The system of the eluent includes: a dichloromethane and methanol system, a petroleum ether and an ethyl acetate system, and the volume ratio of the solvent is adjusted depending on the polarity of the compound, and may also be adjusted by adding a small amount of triethylamine.

Preparation of high performance liquid chromatography, instrument model: Agilent 1260, column: Waters XBridge Prep C ₁₈ OBD (19 mm × 150 mm × 5.0 μm); column temperature: 25 ° C; flow rate: 20.0 mL / min; detection wavelength: 214 nm; Elution gradient: (0 min: 10% A, 90% B; 16.0 min: 90% A, 10% B); mobile phase A: 100% acetonitrile; mobile phase B: 0.05% aqueous ammonium bicarbonate solution.

The reaction temperature of the examples is room temperature (20 ° C to 30 ° C) unless otherwise specified.

The reagents used in the present application were purchased from Acros Organics, Aldrich Chemical Company, Shanghai Tebo Chemical Technology Co., Ltd., and the like.

Preparation of intermediates:

Intermediate Preparation Example 1: Preparation of 2-allyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

First step: Preparation of ethyl 4-(2-allyl-2-(tert-butoxycarbonyl)indenyl)-2-(methylthio)pyrimidine-5-carboxylate

Ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (20 g, 85.8 mmol), tert-butyl 1-allylhydrazine carbonate (15 g, 85.8 mmol) and DIPEA (28 g, 215 mmol) (200 mL) was stirred under reflux with heating for 18 hours. The reaction mixture was cooled, and the solvent was evaporated, evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,

MS m/z (ESI): 369.2 [M+H] ⁺ .

Second step: Preparation of ethyl 4-(2-allylmethyl)-2-(methylthio)pyrimidine-5-carboxylate

Add to ethyl 4-(2-allyl-2-(tert-butoxycarbonyl)indenyl)-2-(methylthio)pyrimidine-5-carboxylate (31.7 g, 85.8 mmol) in an ice bath Trifluoroacetic acid (60 mL) was stirred at room temperature for 1 hour, and then heated at 70 ° C for 1 hour. The title compound was obtained from the title compound (23 g, yield: 100%).

MS m/z (ESI): 269.1 [M+H] ⁺ .

Step 3: Preparation of 2-allyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

Ethyl 4-(2-allylhydrazino)-2-(methylthio)pyrimidine-5-carboxylate (10 g, 38.2 mmol) was dissolved in ethanol (70 mL). The title compound (16 g, yield: 83%) was obtained.

MS m/z (ESI): 2221. [M+H] ⁺ .

Intermediate Preparation Example 2: Preparation of 2-(6-bromopyridin-2-yl)propan-2-ol

Methyl 6-bromopyridine-2-carboxylate (5 g, 23.1 mmol) was dissolved in diethyl ether (100 mL). Methyl magnesium iodide (17 mL, 50.8 mmol) was added under nitrogen, and stirred at room temperature for 0.5 hr. The organic layer was combined and washed with a saturated aqueous solution of sodium hydrogen carbonate and brine and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate gave the title compound (5 g, yield: 100%).

MS m/z (ESI): 216.0 [M+H] ⁺ .

Intermediate Preparation Example 3: 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylsulfinyl)-1H-pyrazolo[ Preparation of 3,4-d]pyrimidine-3(2H)-one

First step: 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylthio)-1H-pyrazolo[3,4- Preparation of d]pyrimidine-3(2H)-one

2-allyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (2 g, 9.0 mmol), 2-(6-bromopyridine-2 -yl)propan-2-ol (2.5 g, 11.7 mmol), cuprous iodide (1.7 g, 9.0 mmol), potassium carbonate (1.7 g, 12.6 mmol) and trans-N,N'-dimethyl- 1,2-cyclohexanediamine (1.4g, 9.9mmol) was added to dioxane (35mL), reacted at 95 ° C for 4 hours, cooled to room temperature, then added with aqueous ammonia, extracted with ethyl acetate three times, organic phase combined and concentrated The residue was subjected to silica gel column chromatography (eluent: EtOAc (EtOAc)

MS m/z (ESI): 358.1 [M+H] ⁺ .

The second step: 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylsulfinyl)-1H-pyrazolo[3, Preparation of 4-d]pyrimidine-3(2H)-one

2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylsulfinyl)-1H-pyrazolo[3,4-d] Pyrimidine-3(2H)-one (200mg, 0.56mmol) was dissolved in acetonitrile (5mL) and water (5mL), then added potassium hydrogen sulfate monohydrate (282mg, 1.68mmol), stirred at room temperature for 2 hours, add water, use The mixture was extracted with EtOAc (EtOAc)EtOAc.

MS m/z (ESI): 374.1 [M+H] ⁺ .

Intermediate Preparation Example 4: Preparation of 1-(6-bromopyridin-2-yl)cyclopropylcarbonitrile

First step: Preparation of 2-(6-bromopyridin-2-yl)acetonitrile

Acetonitrile (1.5 mL, 28.7 mmol) was dissolved in tetrahydrofuran (20 mL), n-butyllithium (11.2 mL, 27.9 mmol) was added dropwise at -78 ° C, stirred for 30 min, and 2,6-dibromopyridine (2 g, 8.4 mmol) was added. A solution of tetrahydrofuran (30 mL) was stirred for 45 minutes, the reaction was gradually warmed to room temperature, then extracted with water and ethyl acetate. The organic phase was concentrated and purified to silica gel column chromatography (eluent: petroleum ether / ethyl acetate Ester = 3/1) gave the title compound (1.28 g, yield: 77%).

MS m/z (ESI): 197.0 [M+H] ⁺ .

Second step: Preparation of 1-(6-bromopyridin-2-yl)cyclopropylcarbonitrile

2-(6-Bromopyridin-2-yl)acetonitrile (500 mg, 2.54 mmol), 1,2-dibromoethane (716 mg, 3.81 mmol), tetrabutylammonium hydrogen sulfate (164 mg, 0.51 mmol) dissolved in toluene (5 mL), a 50% aqueous sodium hydroxide solution (2.5 mL) was added, and the mixture was stirred at room temperature for 1 hour. Water and ethyl acetate were added to the reaction mixture, and the organic phase was combined, washed successively with water and saturated brine, and the residue obtained from the organic phase was concentrated. The title compound (300 mg, yield: 53%) was obtained.

MS m/z (ESI): 223.0 [M+H] ⁺ .

Intermediate Preparation Example 5: Preparation of 2-bromo-6-(2-methoxypropan-2-yl)pyridine

2-(6-Bromopyridin-2-yl)propan-2-ol (500 mg, 2.3 mmol) and iodomethane (980 mg, 6.9 mmol) were dissolved in tetrahydrofuran (15 mL). The mixture was stirred at rt EtOAc EtOAc.

MS m / z (ESI): 230.0 [M + H] +.

Intermediate Preparation Example 6 Preparation of 4-(5-Methylhexahydropyrrole[3,4-c]pyrrole-2(1H)-yl)-phenylamine

First step: Preparation of 5-(4-nitrophenyl)hexahydropyrrole[3,4-c]pyrrole-2(1H)-tert-butyl carbonate

1-Fluoro-4-nitrobenzene (312.4 mg, 2.2 mmol) was dissolved in acetonitrile (10 mL), followed by hexahydropyrrole[3,4-c]pyrrole-2(1H)-tert-butyl carbonate (500 mg) 2.4 mmol) and DIPEA (455.8 mg, 3.5 mmol) were stirred under reflux for 5 hours. The reaction mixture was cooled, filtered, dried and evaporated.

MS m/z (ESI): 334.2 [M+H] ⁺ .

Second step: Preparation of 2-(4-nitrophenyl)hexahydropyrrole [3,4-c]pyrrole hydrochloride

5-(4-Nitrophenyl)hexahydropyrrole[3,4-c]pyrrole-2(1H)-tert-butyl carbonate (620 mg, 1.9 mmol) dissolved in 4N hydrochloric acid in 1,4 dioxane The solution (20 mL) was stirred at room temperature for 2 hr.

MS m/z (ESI): 2321. [M+H] ⁺ .

The third step: preparation of 2-methyl-5-(4-nitrophenyl)hexahydropyrrole [3,4-c]pyrrole

2-(4-Nitrophenyl)hexahydropyrrole [3,4-c]pyrrole hydrochloride (560 mg, 2.4 mmol) and 40% aqueous formaldehyde (2.6 g, 36.0 mmol) were dissolved in dichloromethane (20 mL) After adding sodium borohydride hydride (1.5 g, 7.2 mmol), the mixture was stirred at room temperature for 1 hour, water was added, and the mixture was combined with EtOAc. The title compound of this step (600 mg, yield: 100%).

MS m / z (ESI): 248.1 [M + H] +.

Fourth step: Preparation of 4-(5-methylhexahydropyrrole[3,4-c]pyrrole-2(1H)-yl)-aniline

2-Methyl-5-(4-nitrophenyl)hexahydropyrrole [3,4-c]pyrrole (600 mg, 2.4 mmol) was dissolved in methanol (10 mL), and then lanthanide and hydrazine hydrate (606.6). After the mixture was stirred at room temperature for 2 hours, the reaction mixture was filtered over Celite. The filtrate was concentrated to give the title compound.

MS m/z (ESI): 218.2 [M+H] ⁺ .

Intermediate Preparation Example 7: Preparation of 2-isopropyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

First step: Preparation of ethyl 4-mercapto-2-(methylthio)pyrimidine-5-carboxylate

Ethyl 4-chloro-2-(methylthio)pyrimidine-5-carboxylate (5 g, 21.5 mmol) was dissolved in ethanol (150 mL), cooled to 0 ° C, and hydrated hydrazine (3.2 g). After stirring for 1 hour, the reaction mixture was evaporated to dryness mjjjjjjjjl ).

MS m/z (ESI): 229.1 [M+H] ⁺ .

Second step: Preparation of ethyl 2-(methylthio)-4-(2-(propan-2-ylidene)indenyl)pyrimidine-5-carboxylate

Ethyl 4-mercapto-2-(methylthio)pyrimidine-5-carboxylate (4.3 g, 18.8 mmol) was placed in a 150 mL single-necked flask, acetone (100 mL) was added, and the mixture was heated to 70 ° C overnight, and the reaction mixture was concentrated. The title compound of this step (5.1 g, yield: 100%) was obtained.

MS m/z (ESI): 269.1 [M+H] ⁺ .

Third step: Preparation of ethyl 4-(2-isopropylindolyl)-2-(methylthio)pyrimidine-5-carboxylate

Ethyl 2-(methylthio)-4-(2-(propan-2-ylidene)pyrimidin-5-carboxylate (5.1 g, 18.8 mmol) was dissolved in methanol (100 mL) 0 ° C, sodium cyanoborohydride (1.8 g, 28.2 mmol) and concentrated hydrochloric acid (0.1 mL) were added, and the mixture was stirred at 0 ° C for 2 hours, then a saturated aqueous solution of sodium hydrogencarbonate (50 mL) was added and the mixture was stirred for 10 min. The ethyl ester was extracted three times, and the combined organic layer was evaporated.

MS m/z (ESI): 2721. [M+H] ⁺ .

Step 4: Preparation of 2-isopropyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

Ethyl 4-(2-isopropylindolyl)-2-(methylthio)pyrimidine-5-carboxylate (4.9 g, 18.1 mmol) was dissolved in methanol (400 mL). After stirring at room temperature overnight, the reaction liquid was evaporated to dryness under reduced pressure of methanol, and the aqueous layer was extracted twice with ethyl acetate. The aqueous phase was adjusted to pH 2 with 5N diluted hydrochloric acid, and extracted with ethyl acetate 5 times. The sodium was dried, filtered, and evaporated to dryness crystals

MS m/z (ESI): 225.1 [M+H] ⁺ .

Intermediate Preparation Example 8 Preparation of 1-(4-Amino-2-fluorophenyl)-N,N-dimethylpiperidin-4-amine

First step: Preparation of 1-(4-nitro-2-fluorophenyl)-N,N-dimethylpiperidin-4-amine

1,2-Difluoro-4-nitrobenzene (1 g, 6.28 mmol), N,N-dimethylpiperidin-4-amine (0.96 g, 7.55 mmol) and potassium carbonate (1.72 g, 12.5 mmol) After adding acetonitrile (10 mL), the mixture was reacted at 50 ° C for 16 hours, and the reaction mixture was cooled, poured into water, filtered, and dried to give the title compound (1.2 g, yield: 71.4%).

MS m/z (ESI): 268.2 [M+H] ⁺ .

Second step: Preparation of 1-(4-amino-2-fluorophenyl)-N,N-dimethylpiperidin-4-amine

1-(4-Nitro-2-fluorophenyl)-N,N-dimethylpiperidin-4-amine (200 mg, 0.75 mmol) was dissolved in methanol (5 mL), and a catalytic amount of Raney nickel was added. The hydrazine hydrate (1 mL) was added dropwise, and the mixture was stirred at room temperature for 2 hr.

MS m/z (ESI): 238.2 [M+H] ⁺ .

Compound preparation examples:

Example 1: 2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(4-dimethylaminopiperidin-1-yl) Preparation of phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 1)

2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylsulfinyl)-1H-pyrazolo[3,4-d Pyrimidine-3(2H)-one (58 mg, 0.16 mmol) was dissolved in toluene (5 mL) and (4-dimethylaminopiperidin-1-yl)phenylamine (68 mg, 0.32 mmol) and DIPEA (82.6 mg, The reaction mixture was heated to 80 ° C for 4 hr.

MS m/z (ESI): 529.3 [M+H] ⁺

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 8.84 (s, 1H), 7.78 (t, J = 8.0 Hz, 1H), 7.74 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.4) Hz, 2H), 7.36 (d, J = 7.6 Hz, 1H), 6.92 (d, J = 8.8 Hz, 2H), 5.73-5.67 (m, 1H), 5.05 (dd, J = 10.0, 1.2 Hz, 1H) ), 4.94 (dd, J = 17.2, 1.2 Hz, 1H), 4.74 (d, J = 6.0 Hz, 2H), 3.74 (d, J = 12.4 Hz, 2H), 2.83 - 2.71 (m, 5H), 2.56 (s, 7H), 2.09 (d, J = 12.0 Hz, 2H), 1.83-1.80 (m, 2H), 1.59 (s, 6H).

Example 2: 2-allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-((4-(5-methylhexahydropyrrole[3,4] -c]Preparation of pyrrole-2(1H)-yl)-phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 2)

2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(methylsulfinyl)-1H-pyrazolo[3,4-d Pyrimidine-3(2H)-one (50 mg, 0.13 mmol) was dissolved in toluene (5 mL), followed by 4-(5-methylhexahydropyrrole[3,4-c]pyrrole-2(1H)-yl) An aniline (58 mg, 0.27 mmol) and DIPEA (67.1 mg, 0.52 mmol), EtOAc (EtOAc) 14%).

MS m/z (ESI): 527.3 [M+H] ⁺

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 8.83 (s, 1H), 7.87 (t, J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 7.6 Hz, 2H), 7.35 (d, J = 7.2 Hz, 1H), 6.72 (d, J = 8.8 Hz, 2H), 5.70 (d, J = 6.8 Hz, 1H), 5.35 (t, J = 4.8 Hz, 1H), 5.05 (d, J = 9.2 Hz, 1H), 4.94 (d, J = 18.0 Hz, 1H), 4.73 (d, J = 6.4 Hz, 2H), 3.75 (s, 3H), 3.44 (d, J = 10.0 Hz, 2H), 3.35 (s, 2H), 3.17 (s, 2H), 2.79 (s, 5H), 1.59 (s, 6H).

Example 3: 2-allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-((4-(4-dimethylaminopiperidine-1) -Phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 3)

First step: 2-allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-methylthio-1H-pyrazolo[3,4- Preparation of d]pyrimidine-3(2H)-one

2-Allyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (280 mg, 1.26 mmol), 2-bromo-6-(1) -Cyanocyclopropan-1-yl)pyridine (366 mg, 1.64 mmol), cuprous iodide (239 mg, 1.26 mmol), potassium carbonate (243 mg, 1.76 mmol) and trans-N,N'-dimethyl- 1,2-cyclohexanediamine (197 mg, 1.39 mmol) was added to dioxane (8 mL), and the mixture was reacted at 95 ° C for 4 hours. After cooling to room temperature, aqueous ammonia was added, and ethyl acetate was extracted three times. The residue was subjected to silica gel column chromatography (jjjjjjjjjjj

The second step: 2-allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-methylsulfinyl-1H-pyrazolo[3, Preparation of 4-d]pyrimidine-3(2H)-one

2-Allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-methylthio-1H-pyrazolo[3,4-d]pyrimidine -3(2H)-one (289mg, 0.8mmol) was dissolved in acetonitrile (5mL) and water (5mL), then added potassium hydrogensulfate complex salt (400mg, 2.4mmol), stirred at room temperature for 2 hours, added water, with acetic acid The ethyl ester was extracted three times, and the organic layer was evaporated, evaporated, evaporated, evaporated

The third step: 2-allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-((4-(4-dimethylaminopiperidine-1) Of phenyl)amino)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

2-Allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-methylsulfinyl-1H-pyrazolo[3,4-d Pyrimidine-3(2H)-one (62 mg, 0.16 mmol) was dissolved in toluene (5 mL), then (4-dimethylaminopiperidin-1-yl)phenylamine (72 mg, 0.33 mmol) and DIPEA (84 mg, 0.65) The reaction mixture was heated to 80 ° C for 4 hours, and the reaction mixture was evaporated.

MS m/z (ESI): 527.3 [M+H] ⁺

^{1 H-NMR (400MHz, CDCl} 3) δ: 7.95 (s, 1H), 7.35 (t, J = 15.2Hz, 1H), 7.08 (dd, J = 15.2,3.2Hz, 1H), 7.03-7.00 (m , 1H), 6.99-6.97 (m, 1H), 6.78-6.76 (m, 1H), 6.75-6.73 (m, 1H), 6.25 (dd, J = 14.8, 3.2 Hz, 1H), 5.96-5.79 (m ,1H),5.24-5.16(m,1H), 5.16-5.12(m,1H),4.95(s,1H),3.56-3.46(m,2H),3.44-3.32(m,2H),3.16-3.05 (m, 2H), 2.26 (s, 6H), 1.95-1.82 (m, 2H), 1.74-1.57 (m, 5H), 1.51-1.39 (m, 2H).

Example 4: 2-allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-((4-(5-methylhexahydropyrrole[3] , 4-c]pyrrole-2(1H)-yl)-phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 4)

2-Allyl-1-(6-(1-cyanocycloprop-1-yl)pyridin-2-yl)-6-(methylsulfinyl)-1H-pyrazolo[3,4 -d]pyrimidine-3(2H)-one (80 mg, 0.21 mmol) was dissolved in toluene (5 mL) and then 4-(5-methylhexahydropyrrole[3,4-c]pyrrole-2(1H)- The aniline (91.4 mg, 0.42 mmol) and DIPEA (108.5 mg, 0.84 mmol), mp. Yield: 28.6%).

MS m/z (ESI): 534.6 [M+H] ⁺

^{1 H-NMR (400MHz, CDCl} 3) δ: 7.95 (s, 1H), 7.35 (t, J = 7.2Hz, 1H), 7.08 (dd, J = 7.2,1.6Hz, 1H), 7.00 (d, J = 7.2 Hz, 2H), 6.76 (d, J = 7.2 Hz, 2H), 6.25 (dd, J = 7.2, 1.6 Hz, 1H), 5.92-5.82 (m, 1H), 5.20-5.14 (m, 2H) , 4.89 (s, 1H), 4.89 (s, 1H), 3.67-3.64 (m, 2H), 3.51 (d, J = 6.0 Hz, 1H), 3.47 (d, J = 6.4 Hz, 1H), 2.96- 2.85 (m, 4H), 2.18 (s, 3H), 2.14-2.08 (m, 2H), 1.84-1.77 (m, 2H), 1.74-1.67 (m, 2H), 1.48-1.42 (m, 2H).

Example 5: 2-allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-((4-(5-methylhexahydropyrrole[3] , 4-c]pyrrole-2(1H)-yl)-phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 5)

First step: 2-allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-methylthio-1H-pyrazolo[3,4- Preparation of d]pyrimidine-3(2H)-one

2-Allyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (140 mg, 0.63 mmol), 2-bromo-6-(2 -methoxypropan-2-yl)pyridine (188.6 mg, 0.82 mmol), cuprous iodide (119.5 mg, 0.63 mmol), potassium carbonate (121.5 mg, 0.88 mmol) and trans-N,N'-di Methyl-1,2-cyclohexanediamine (98.5 mg, 0.70 mmol) was added to dioxane (8 mL), and reacted at 95 ° C for 4 hours. After cooling to room temperature, ammonia water was added and ethyl acetate was extracted three times. The phases were combined and concentrated. EtOAcjjjjjjjjj

The second step: 2-allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-methylsulfinyl-1H-pyrazolo[3, Preparation of 4-d]pyrimidine-3(2H)-one

2-Allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-methylthio-1H-pyrazolo[3,4-d]pyrimidine -3(2H)-one (150 mg, 0.4 mmol) was dissolved in acetonitrile (5 mL) and water (5 mL). The organic layer was extracted with EtOAc (3 mL).

The third step: 2-allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-((4-(5-methylhexahydropyrrole[3] Of 4-(4-c]pyrrole-2(1H)-yl)-phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

2-Allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-methylsulfinyl-1H-pyrazolo[3,4-d Pyrimidine-3(2H)-one (75 mg, 0.19 mmol) was dissolved in toluene (5 mL), followed by 4-(5-methylhexahydropyrrole[3,4-c]pyrrole-2(1H)-yl) The aniline (84 mg, 0.39 mmol) and DIPEA (100 mg, 0.77 mmol) were reacted to 80 ° C for 4 hours, and the reaction mixture was concentrated to give the title compound (34 mg, yield: 33%).

MS m/z (ESI): 541.7 [M+H] ⁺

¹ H-NMR (400MHz, CDCl ₃ ) δ: 8.81 (s, 1H), 7.87-7.75 (m, 2H), 7.50-7.36 (m, 3H), 6.68 (d, J = 8.8 Hz, 2H), 5.72 -5.61(m,1H),4.99(d,J=9.6Hz,1H),4.92-4.80(m,3H),3.34-3.21(m,8H),3.13(s,2H),2.85(s,4H ), 2.53 (s, 3H), 1.56 (s, 6H).

Example 6: 2-allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-((4-(4-dimethylaminopiperidine-1) -Phenyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 6)

2-Allyl-1-(6-(2-methoxypropan-2-yl)pyridin-2-yl)-6-methylsulfinyl-1H-pyrazolo[3,4-d Pyrimidine-3(2H)-one (75 mg, 0.19 mmol) was dissolved in toluene (5 mL), then (4-dimethylaminopiperidin-1-yl)phenylamine (85 mg, 0.39 mmol) and DIPEA (100 mg, 0.77) The reaction mixture was heated to 80 ° C for 4 hr.

MS m/z (ESI): 543.3 [M+H] ⁺

^{1 H-NMR (400MHz, CDCl} 3) δ: 8.83 (s, 1H), 7.85 (t, J = 7.6Hz, 1H), 7.77 (d, J = 7.6Hz, 1H), 7.48 (d, J = 8.0 Hz, 3H), 6.93 (d, J = 9.2 Hz, 2H), 5.72-5.61 (m, 1H), 4.99 (d, J = 9.6 Hz, 1H), 4.91-4.82 (m, 3H), 3.73 (d , J = 12.4 Hz, 2H), 3.22 (s, 3H), 2.79-2.72 (m, 4H), 2.54 (s, 6H), 2.08 (d, J = 12.0 Hz, 2H), 1.86-1.74 (m, 2H), 1.56 (s, 6H).

Example 7: 6-((4-(4-(Dimethylamino)piperidin-1-yl)phenyl)amino)-1-(6-(2-hydroxypropan-2-yl)pyridine-2- Preparation of 2-acetyl-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 7)

First step: 1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1H-pyrazolo[3,4- Preparation of d]pyrimidine-3(2H)-one

2-Isopropyl-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (300 mg, 1.34 mmol) and 2-hydroxypropyl-6- Bromopyridine (347 mg, 1.61 mmol) was dissolved in 1,4-dioxane (50 mL), cuprous iodide (255 mg, 1.34 mmol), potassium carbonate (277 mg, 2.01 mmol) and trans-N, N' - dimethyl-1,2-cyclohexanediamine (285 mg, 2.01 mmol), added, warmed to 95 ° C, stirred overnight, the reaction solution was cooled to room temperature, filtered, the filtrate was concentrated, and the residue was applied to silica gel column chromatography Purification (eluent: petroleum ether / ethyl acetate = 3/1 to 1/1) gave the title compound (350 mg, yield: 72%).

MS m/z (ESI): 360.1 [M+H] ⁺ .

Second step: 6-((4-(4-(Dimethylamino)piperidin-1-yl)phenyl)amino)-1-(6-(2-hydroxypropan-2-yl)pyridine-2- Of 2-acetyl-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

1-(6-(2-Hydroxypropyl-2-yl)pyridin-2-yl)-2-isopropyl-6-(methylthio)-1H-pyrazolo[3,4-d] Pyrimidine-3(2H)-one (180 mg, 0.50 mmol) was dissolved in toluene (12 mL), and m-chloroperoxybenzoic acid (104 mg, 0.60 mmol) was added and stirred at room temperature for two hours, then diisopropylethylamine (258 mg, 2.00mmol) and (4-dimethylaminopiperidin-1-yl)aniline (220mg, 1.00mmol), after adding, warmed to 80 ° C and stirred overnight, the reaction liquid was evaporated to dryness under reduced pressure, and the residue was used for preparative high performance liquid chromatography Purification to give the title compound (165 mg, yield: 62.1%).

MS m/z (ESI): 531.3 [M+H] ⁺ .

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 10.12 (s, 1H), 8.75 (s, 1H), 8.07 (t, J = 7.2Hz, 1H), 7.71 (d, J = 8.0Hz, 1H ), 7.65 (d, J = 7.6 Hz, 1H), 7.54 (s, 2H), 6.90 (d, J = 9.2 Hz, 2H), 5.33 (s, 1H), 4.18-4.11 (m, 1H), 3.64 (d, J = 12.4 Hz, 2H), 2.64 - 2.58 (m, 2H), 2.19 (s, 6H), 2.18 - 2.14 (m, 1H), 1.84-1.81 (m, 2H), 1.52-1.48 (m , 2H), 1.44 (s, 6H), 1.37 (s, 3H), 1.36 (s, 3H).

Example 8: 2-allyl-6-((4-(4-(dimethylamino)piperidin-1-yl)-3-fluorophenyl)amino)-1-(6-(2-hydroxyl) Propane-2-yl)pyridin-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 8)

2-Allyl-1-(6-(2-hydroxypropan-2-yl)pyridin-2-yl)-6-(sulfinyl)-1H-pyrazolo[3,4-d]pyrimidine -3(2H)-one (100 mg, 0.27 mmol) and 1-(4-amino-2-fluorophenyl)-N,N-dimethylpiperidin-4-amine (80 mg, 0.32 mmol) dissolved in 1 , 4-dioxane (5mL), trifluoroacetic acid (0.5mL) was added dropwise, heated to 95 ° C for 2 hours, cooled to room temperature, the reaction solution was poured into water, ethyl acetate extracted impurities, water phase After the pH was adjusted to 8-9 with potassium carbonate, ethyl acetate was evaporated, EtOAcjjjjjjjjjjjj : 32.8%).

MS m/z (ESI): 547.3 [M+H] ⁺ .

^{1 H-NMR (400MHz, DMSO} -d 6) δ: 8.88 (s, 1H), 8.02 (t, J = 8.0Hz, 1H), 7.76-7.71 (m, 2H), 7.64 (d, J = 8.0Hz , 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.37-7.34 (m, 1H), 7.03-6.98 (t, J = 5.2 Hz, 1H), 5.70-5.63 (m, 1H), 5.35 ( s, 1H), 5.01-4.98 (m, 1H), 4.84-4.80 (m, 1H), 4.68 (d, J = 5.2 Hz, 1H), 2.65-2.59 (t, J = 10.8 Hz, 2H), 2.21. (s, 6H), 1.84 (d, J = 10.8 Hz, 2H), 1.57-1.52 (m, 2H), 1.44 (s, 6H).

Example 9: 2-allyl-6-((4-(4-(dimethylamino)piperidin-1-yl)phenyl)amino)-1-(3-(2-hydroxypropane-2-) Preparation of phenyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one (Compound 9)

First step: Preparation of 3-(1-hydroxy-1-methylethyl)bromobenzene

Methyl 3-bromobenzoate (1 g, 4.65 mmol) was added to diethyl ether (20 mL) under EtOAc. EtOAc (EtOAc m. After that, it was naturally raised to room temperature for 12 hours. The reaction mixture was poured into a saturated aqueous solution of EtOAc. EtOAc.

MS m / z (ESI): 245.0 [M + H] +.

Second step: Preparation of 3-(1-hydroxy-1-methylethyl)phenylboronic acid

3-(1-Hydroxy-1-methylethyl)bromobenzene (500 mg, 2.32 mmol) was added to tetrahydrofuran (10 mL) under nitrogen, and n-hexane solution of n-butyllithium (3 mL) was slowly added dropwise at -60 °C. , 2mol / L, 6mmol), stirred at -60 ° C for 1 hour, dropwise addition of trimethyl borate (480 mg, 4.62 mmol), reaction at room temperature for 12 hours, slowly adding water (20 mL), ethyl acetate extraction, aqueous phase with 1N hydrochloric acid The pH was adjusted to 2-3, and ethyl acetate was evaporated. EtOAcjjjjjjjjjjj

MS m/z (ESI): 181.0 [M+H] ⁺ .

The third step: 2-allyl-1-(3-(2-hydroxypropan-2-yl)phenyl)-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidine- Preparation of 3(2H)-one

2-Allyl-6-(methylthio)-1,2-dihydro-3H-pyrazolo[3,4-d]pyrimidin-3-one (200 mg, 0.56 mmol), copper acetate (160 mg) , 0.8 mmol) and 3-(1-hydroxy-1-methylethyl)phenylboronic acid (200 mg, 1.12 mmol) were sequentially added to chloroform (3 mL), and pyridine (0.4 mL) was added dropwise to react at room temperature for 48 hours. Add 30% ammonia water, stir for 30 minutes, extract with ethyl acetate, and extract the organic phase, dried over anhydrous sodium sulfate. Yield 15%).

MS m/z (ESI): 357.1 [M+H] ⁺ .

Fourth step: 2-allyl-1-(3-(2-hydroxypropan-2-yl)phenyl)-6-(methylsulfinyl)-1H pyrazolo[3,4-d] Preparation of pyrimidine-3(2H)-one

2-Allyl-1-(3-(2-hydroxypropan-2-yl)phenyl)-6-(methylthio)-1Hpyrazolo[3,4-d]pyrimidine-3 (2H The ketone (30 mg, 0.084 mmol) was added to a mixed solvent of acetonitrile (2 mL) and water (2 mL), and potassium hydrogen persulfate complex salt (34 mg, 0.2 mmol) was added, and the mixture was stirred at room temperature for 2 hours, and the reaction solution was diluted with 10 mL of water. The mixture was extracted with ethyl acetate. EtOAc evaporated.

MS m / z (ESI): 373.1 [M + H] +.

Step 5: 2-allyl-6-((4-(4-(dimethylamino)piperidin-1-yl)phenyl)amino)-1-(3-(2-hydroxypropane-2-) Of phenyl)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-3(2H)-one

2-Allyl-1-(3-(2-hydroxypropan-2-yl)phenyl)-6-(methylsulfinyl)-1H-pyrazolo[3,4-d]pyrimidine-3 (2H)-ketone (20 mg, 0.053 mmol) and (4-dimethylaminopiperidin-1-yl)phenylamine (13 mg, 0.059 mmol) were added to 1,4-dioxane (10 mL). Acetic acid (0.1 mL), the temperature was raised to 90 ° C for 4 hours, the reaction was cooled to room temperature, the reaction solution was poured into water, the impurities were extracted with ethyl acetate, and the aqueous phase was adjusted to pH 8-9 with potassium carbonate. The organic phase was combined, dried over anhydrous sodium sulfate

MS m/z (ESI): 528.3 [M+H] ⁺ .

^{1 H-NMR (400MHz, CDCl} 3) δ: 8.80 (s, 1H), 7.55-7.48 (m, 5H), 7.29-7.27 (m, 1H), 6.85 (d, J = 8.8Hz, 2H), 5.70 -5.62 (m, 1H), 5.18 (s, 1H), 5.08 (d, J = 9.2 Hz, 1H), 4.27 (br, 2H), 3.61 (d, J = 12.0 Hz, 2H), 2.59 (d, J = 8.0 Hz, 2H), 2.20 (s, 6H), 2.20-2.15 (m, 3H), 2.02-1.97 (m, 2H), 1.83-1.80 (m, 2H), 1.46 (s, 6H).

Pharmacological test

Experimental Example 1: Inhibition of Wee1 Kinase:

To test the inhibitory effect of the compound on Wee1 kinase in vitro, the experimental steps are as follows:

1) After pre-incubating Wee1 protein (Carna Biosciences) with different concentrations of the test compound for 15 minutes at room temperature, the substrate (Poly (Lys, Tyr), Sigma) and 10ATP were added according to the kit instructions to start the reaction, and the reaction was carried out at 30 ° C. minute.

2) Add an equal volume of ADP-Glo reagent (Promega) to the reaction system and incubate for 40 minutes at room temperature to terminate the reaction.

3) Add the kinase assay reagent and incubate for 30 minutes at room temperature. The product self-luminous signal is detected. The vehicle control group (DMSO) was used as the negative control, the Buffer reaction buffer group (with no enzyme and compound) was used as the blank control, and the half-inhibitory concentration (IC ₅₀ ) of the compound was calculated from the concentration of the remaining activity from the nearest 50%. The results are shown in Table 1. Shown as follows:

Table 1 Compounds on Wee1 Activity Inhibition Data

As can be seen from the data in Table 1, the compound has a significant inhibitory effect on Wee1 kinase.

Experimental Example 2: Study on the distribution of rat pharmacokinetics (PK)

The Wee1 inhibitor AZD1775 and the test compound were administered to the male SD rats at the clinical trial stage by intravenous (IV) and gavage (PO), respectively, and the pharmacokinetic characteristics of the test compounds were examined. The doses of IV and PO were 1 mg/kg and 5 mg/kg, respectively, and the medium of IV was 5% DMSO: 5% Solutol: 90% physiological saline, and the solvent of PO was 0.5% MC. Blood was collected at different time points after IV and PO administration. The blood was anticoagulated with EDTA-K ₂ and centrifuged to obtain a plasma sample. Plasma samples were processed by precipitation protein and analyzed by LC-MS/MS.

The pharmacokinetic parameters were calculated using WinNonlin 6.3 software using a non-compartmental model. The results are shown in Tables 2 and 3.

Table 2. Pharmacokinetic parameters of compounds in rats

The data in Table 2 shows that the bioavailability (F) of Compound 2 of the present application in mice is 49.7%, which is superior to AZD1775, indicating that the compound of the present application has good drug exposure and bioavailability in rats. Suitable for oral administration.

Table 3. Comparison of drug half-life of compounds in rats

Analyte	Compound 1	Compound 2	AZD1775
Route of administration	Stomach	Stomach	Stomach
gender	male	male	male
Dose (mg/kg)	5	5	5
T 1/2 (h)	4.37	3.79	1.73

The data in Table 3 shows that T _1/2 of Compound 1 and Compound 2 of the present application administered by a dose of 5 mg/kg, T _1/2 was 4.37 hours and 3.79 hours, respectively, indicating that the half-life of the compound of the present application is higher than that of the compound AZD1775. More than 1 time, the effect of the drug is longer, and has a significant advantage. Moreover, the compound of the present application can be administered once a day, which can reduce the number of administrations and improve patient compliance.

Experimental Example 3: Study on the distribution of pharmacokinetics (PK) in mice

The Wee1 inhibitor AZD1775 and the test compound were administered to female Balb/c mice at the clinical trial stage by intravenous (IV) and intragastric (PO), respectively, and the pharmacokinetic characteristics of the test compounds were examined. The doses of IV and PO were 1 mg/kg and 10 mg/kg, respectively, and the medium of IV was 5% DMSO: 5% Solutol: 90% physiological saline, and the solvent of PO was 0.5% MC. Blood was collected at different time points after IV and PO administration. The blood was anticoagulated with EDTA-K ₂ and centrifuged to obtain a plasma sample. Plasma samples were processed by precipitation protein and analyzed by LC-MS/MS.

The pharmacokinetic parameters were calculated using WinNonlin 6.3 software using a non-compartmental model. The results are shown in Tables 4 and 5.

Table 4. Pharmacokinetic parameters of compounds in mice

The data in Table 4 shows that when administered orally at 10 mg/kg, the AUC _last of Compound 1 of the present application in mice was 1770 h*ng/mL, which was superior to the AUC _last of AZD1775 at the same dose, indicating that the compound of the present application is The mice have good drug exposure and are suitable for oral administration.

Table 5. Comparison of drug half-life of compounds in mice

The data in Table 5 shows that the compound 1 of the present application has a T _1/2 of 1.15 hours and 2.90 hours in the intravenous and intragastric administration modes, respectively, indicating that the half-life of the compound of the present application is more than doubled than that of the compound AZD1775. Longer duration and significant advantages. Moreover, the compound of the present application can be administered once a day, which can reduce the number of administrations and improve patient compliance.

Claims (16)

1. a compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, having the structure of formula I below:

   

   among them,

   X is selected from CH and N;

   R ^{1 is} selected from the group consisting of hydroxy-C _1-6 alkyl-, C _1-6 alkoxy-C _1-6 alkyl- and cyano-C _3-6 cycloalkyl-;

   R ^{2 is} selected from a 7-10 membered fused heterocyclic group, which is optionally substituted by one or more C _1-6 alkyl groups; or R ² is piperidinyl, said piperidine The group is optionally substituted by one or more -NR ⁵ R ⁶ ; R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl;

   R ^{3 is} selected from the group consisting of C _1-6 alkyl and C _2-6 alkenyl;

   R ^{4 is} selected from the group consisting of hydrogen and halogen.
2. A compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

   Wherein R ^{1 is} selected from the group consisting of hydroxy-C _1-4 alkyl-, C _1-3 alkoxy-C _1-4 alkyl- and cyano-C _3-6 cycloalkyl-;

   Preferably, R ^{1 is} selected from the group consisting of 2-hydroxypropan-2-yl, 2-methoxypropan-2-yl and 1-cyanocycloprop-1-yl.
3. A compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

   Wherein R ^{2 is} selected from a 7-10 membered bicyclic fused heterocyclic group, and the 7-10 membered bicyclic fused heterocyclic group is optionally substituted by one or more C _1-6 alkyl groups;

   Preferably, R ^{2 is} selected from an 8-membered fused heterocyclic group substituted by a C _1-6 alkyl group;

   Preferably, R ^{2 is} selected from an 8-membered bicyclic fused heterocyclic group, and the 8-membered bicyclic fused heterocyclic group is substituted by a C _1-6 alkyl group;

   Preferably, R ^{2 is} selected from

   

   Preferably, R ² is 5-methyl-octahydro-pyrrolo[3,4-c]pyrrol-2-yl.
4. A compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

   Wherein R ² is piperidinyl, the piperidinyl group is substituted by one or more -NR ⁵ R ⁶ , and R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl;

   Preferably, R ² is piperidinyl, the piperidinyl group is substituted by one or more -NR ⁵ R ⁶ , and R ⁵ and R ⁶ are each independently selected from C _1-4 alkyl;

   Preferably, R ² is

   

   R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl;

   Preferably, R ² is 4-dimethylaminopiperidin-1-yl.
5. A compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

   Wherein R ^{3 is} selected from the group consisting of C _1-4 alkyl and C _2-4 alkenyl;

   Preferably, R ^{3 is} selected from the group consisting of allyl and isopropyl.
6. A compound according to claim 1 or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

   Wherein R ^{4 is} selected from the group consisting of hydrogen and fluorine.
7. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, wherein The compound has the structure shown in Formula I-1.

   

   Wherein X, R ¹ , R ² , R ³ and R ^{4 are} as defined in any one of claims 1 to 6.
8. The compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, wherein The compound has the structure shown in Formula I-2.

   

   Wherein R ¹ and R ^{2 are} as defined in any one of claims 1 to 6.
9. A compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

   Wherein R ^{1 is} selected from the group consisting of 2-hydroxyprop-2-yl, 2-methoxyprop-2-yl and 1-cyanocycloprop-1-yl;

   R ^{2 is} selected from an 8-membered fused heterocyclic group substituted by a C _1-6 alkyl group; preferably, R ² is 5-methylhexahydropyrrole [3,4-c]pyrrole -2(1H)-yl; or R ^{2 is} selected from

   

   R ⁵ and R ⁶ are each independently selected from C _1-6 alkyl; preferably, R ² is 4-dimethylaminopiperidin-1-yl.
10. The compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, The compound has the following structure:

    
11. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate thereof , a metabolite or prodrug, and one or more pharmaceutically acceptable carriers.
12. A pharmaceutical preparation comprising a compound according to any one of claims 1 to 10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, Or the pharmaceutical composition of claim 11; preferably, it is administered orally, intravenously, intraarterially, subcutaneously, intraperitoneally, intramuscularly or transdermally.
13. The compound of any one of claims 1 to 10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, the drug of claim 11. Use of the composition or the pharmaceutical preparation of claim 12 for the preparation of a medicament for preventing or treating a Wee1 protein-related disease; preferably, the Wee1 protein-related disease is cancer.
14. The use according to claim 13, wherein the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, colorectal cancer, bladder cancer, breast cancer, non-small cell lung cancer, and endometrial cancer.
15. A method of preparing a compound of any of claims 1-10, comprising the steps of:

    

    Wherein Hal ¹ is halogen (for example F, Cl or Br); Hal ^{2 is} selected from halogen (for example Cl, Br or I), a boronic acid group or a boronic acid ester group; R ¹ , R ² , R ³ , R ⁴ And X as defined in any one of claims 1-10;

    The reaction conditions of each step are as follows:

    (1) reacting compound IN-1 with a hydrazine compound to obtain compound IN-2;

    (2) cyclizing compound IN-2 to give compound IN-3;

    (3) reacting compound IN-3 with compound IN-4 to give compound IN-5;

    (4) oxidizing compound IN-5 to give compound IN-6;

    (5) Compound IN-6 is reacted with compound IN-7 to give a compound of formula I.
16. A method of preparing the compound of claim 8 comprising the steps of:

    

    Wherein Hal ¹ is halogen (eg F, Cl or Br); Hal ² is halogen (eg Cl, Br or I), a boronic acid group or a boronic acid ester group; PG is a protecting group selected from benzyl, para Oxybenzyl, tert-butoxycarbonyl, benzyloxycarbonyl; R ¹ and R ^{2 are} as defined in claim 8;

    The reaction conditions of each step are as follows:

    (1) reacting compound IN-1 with compound IN-8 to give compound IN-9;

    (2) removing the protecting group PG from the compound IN-9 to obtain the compound IN-10;

    (3) cyclizing the compound IN-10 to give the compound IN-11;

    (4) reacting compound IN-11 with compound IN-12 to give compound IN-13;

    (5) oxidizing compound IN-13 to give compound IN-14;

    (6) Compound IN-14 is reacted with compound IN-15 to give a compound of formula 1-2.