CN111315747A

CN111315747A - Dihydropyrazolone pyrimidine compound and preparation method and application thereof

Info

Publication number: CN111315747A
Application number: CN201880072261.0A
Authority: CN
Inventors: 刘金明; 何婷; 蔡家强; 王利春; 王晶翼
Original assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Current assignee: Sichuan Kelun Biotech Biopharmaceutical Co Ltd
Priority date: 2018-01-05
Filing date: 2018-12-21
Publication date: 2020-06-19
Anticipated expiration: 2038-12-21
Also published as: WO2019134539A1; CN111315747B

Abstract

The application relates to a dihydropyrazolone pyrimidine compound, a preparation method and application thereof. In particular, the application relates to a compound shown in a formula I, a pharmaceutical composition and a pharmaceutical preparation containing the compound, a preparation method of the compound and application of the compound in preparing a medicine for preventing or treating diseases related to Wee1 protein.

Description

Dihydropyrazolone pyrimidine compound and preparation method and application thereof

Technical Field

The application relates to a dihydropyrazolone pyrimidine compound, a preparation method and application thereof.

Background

The cell cycle is a highly regulated and controlled process, tightly regulated by a complex network formed by a series of protein, metabolic and microenvironment interactions, aimed at proliferating cells only in accordance with specific stimuli and appropriate conditions (S.Diaz-Moralli, M.Tarrago-Castellarnau, A.Miranda, M.Cascante, Pharmacology & Therapeutics,2013,138: 255-one 271). The standard cell cycle undergoes a G1 phase, an S phase (DNA synthesis phase), a G2 phase, and an M phase (cell division phase) in this order. There are several cycle-block checkpoints during G1-S transition, S phase, G2-M transition, etc., to maintain genome integrity and provide time for repair of damaged DNA prior to entry into mitosis. In response to DNA damage, cell cycle checkpoints can be activated at stages G1/S, S and G2/M and induce cell cycle arrest until repair is complete; if repair is unsuccessful, cell senescence or apoptosis will be driven (R.Visconti, M.R.Della, D.Grieco, Journal of Experimental & Clinical Cancer Research,2016,35: 153).

The Wee1 protein kinase is a member of the serine/threonine protein kinase family, is a key element of the G2/M checkpoint of the cell cycle, and plays a key role in the scheduling of cell division (c.j. matheson, d.s. backkos, p.reigan, Trends in pharmaceutical Sciences,2016,37: 872). Entry into mitosis from the G2/M checkpoint is dependent 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 myelin transcription factor (MYT1) phosphorylates the Thr14 site of CDK1, maintaining the inactive state of CDK1, inhibiting the cell from entering M phase. Wee1 is therefore a negative regulator of cell entry from G2 into M.

Thus, inhibiting Wee1 kinase activity to remove the function of the G2/M checkpoint is a potential strategy to drive tumor cells into an unplanned mitosis, thereby undergoing mitotic catastrophe leading to cell death. This way, in which cells are forced into mitosis without complete DNA replication, is highly toxic to cells and represents a novel mechanism for inducing tumor cell death. Therefore, the Wee1 inhibitor has good application prospect as a medicine. Patent WO2007126128 reports that certain compounds exhibit activity as Wee1 protein kinase inhibitors.

In order to meet the drug requirements of better tumor treatment effect, safer or low side effect, the application provides a novel structure Wee1 protein kinase inhibitor, and finds that the compound with the structure shows excellent Wee1 protein inhibition effect and drug effect duration.

Disclosure of Invention

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

wherein,

x is selected from CH and N;

R¹selected from hydroxy-C_1-6Alkyl radical-、C_1-6alkoxy-C_1-6Alkyl-and cyano-C_3-6Cycloalkyl-;

R²selected from 7-10 membered fused heterocyclyl, said 7-10 membered fused heterocyclyl being optionally substituted by one or more C_1-6Alkyl substituted, each C_1-6The alkyl groups may be the same or different; or R²Selected from piperidinyl, said piperidinyl being optionally substituted by one or more-NR⁵R⁶Substitution; r⁵、R⁶Each independently selected from C_1-6An alkyl group;

R³is selected from C_1-6Alkyl and C_2-6An alkenyl group;

R⁴selected from hydrogen and halogen.

In some preferred embodiments, the compounds have the following structure of formula I-1:

wherein, X, R¹、R²、R³And R⁴The definition is shown in the general formula I.

In some preferred embodiments, the compounds have the following structure of formula I-2:

wherein R is¹And R²The definition is shown in the general formula I.

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

in the formula, Hal¹Is halogen (e.g., F, Cl or Br); hal²Is halogen (e.g., Cl, Br, or I), a boronic acid group, or a boronic ester group; r¹、R²、R³、R⁴And X have the same meanings as in the above formula I.

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

in the formula, Hal¹Is halogen (e.g., F, Cl or Br); hal²Is halogen (e.g., Cl, Br, or I), a boronic acid group, or a boronic ester group; PG is a protecting group selected from benzyl, p-methoxybenzyl, tert-butoxycarbonyl, benzyloxycarbonyl, preferably tert-butoxycarbonyl; r¹And R²The definition of (a) is the same as that of the above 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, or prodrug thereof, and one or more pharmaceutically acceptable carriers.

Another aspect of the present application provides a method of making the pharmaceutical composition, the method comprising combining the compound, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite, or prodrug thereof, 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 prodrug thereof, or the pharmaceutical composition.

Another aspect of the present application provides a use of the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, the pharmaceutical composition or the pharmaceutical preparation for the manufacture of a medicament for the prevention or treatment of a Wee1 protein kinase-associated disease, preferably, the disease is cancer.

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

Another aspect of the present application provides a method for preventing or treating a Wee1 protein kinase related disease, the method comprising administering to a subject in need thereof an effective amount of the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, or the pharmaceutical composition; preferably, the disease is cancer.

Definition of

Unless defined otherwise below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. Reference to the techniques used herein is intended to refer to those techniques commonly understood in the art, including those variations of or alternatives to those techniques that would be apparent 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 present invention.

As used herein, the terms "comprises," "comprising," "has," "containing," or "involving," and other variations thereof herein, are inclusive or open-ended and do not exclude additional unrecited elements or method steps.

The term "alkyl" as used herein is defined as a straight or branched chain saturated aliphatic hydrocarbon group. In some embodiments, the alkyl group has 1 to 12, e.g., 1 to 6, carbon atoms. For example, as used herein, the term "C_1-6Alkyl "refers to a straight or branched chain group having 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, or n-hexyl), which is optionally substituted with one or more (such as 1 to 3) suitable substituents such as halo (when the group is referred to as" haloalkyl ", e.g., CF)₃、C₂F₅、CHF₂、CH₂F、CH₂CF₃、CH₂Cl or-CH₂CH₂CF₃Etc.).

As used herein, the term "cycloalkyl" refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., monocyclic, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, or bicyclic ring, including spiro, fused or bridged systems (such as bicyclo [ 1.1.1)]Pentyl, bicyclo [2.2.1]Heptyl, bicyclo [3.2.1]Octyl or bicyclo [5.2.0]Nonyl, decalinyl, etc.), optionally substituted with one or more (such as 1 to 3) suitable substituents. The cycloalkyl group has 3 to 15, for example 3 to 6, carbon atoms. For example, the term "C_3-6Cycloalkyl "refers to a saturated or partially unsaturated non-aromatic monocyclic or polycyclic (such as bicyclic) hydrocarbon ring (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl) having 3 to 6 ring carbon atoms, optionally substituted with one or more (such as 1 to 3) suitable substituents, for example, methyl-substituted cyclopropyl.

As used herein, the term "halo" or "halogen" group is defined to include fluorine, chlorine, bromine, or iodine.

The term "alkoxy," as used herein, means an alkyl group, as defined above, appended to the parent molecular moiety through an oxygen atom. C_1-6Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, n-butoxy, isobutoxy, t-butoxy, pentoxy, hexoxy, and the like.

As used herein, the term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic group, e.g., having 2, 3,4, 5, 6, 7, 8, or 9 carbon atoms in the ring and one or more (e.g., 1,2, 3, or 4) members selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer from 0 to 2) heteroatoms such as, but not limited to, oxiranyl, aziridinyl, azetidinyl, oxetanyl, tetrahydrofuranyl, pyrrolidinyl, pyrrolidinonyl, imidazolidinyl, pyrazolidinyl, tetrahydropyranyl, piperidinyl, morpholinyl, dithianylThe term "fused heterocyclyl" refers to polycyclic heterocyclyl groups in which each ring shares an adjacent pair of atoms with the other rings in the system, one or more of the rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system, wherein one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicyclic fused heterocyclic groups.

The term "substituted" means that one or more (e.g., 1,2, 3, or 4) hydrogens on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency at the present time is not exceeded and the substitution results in a stable compound. Combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

If a substituent is described as "optionally substituted with …", the substituent may be (1) unsubstituted or (2) substituted. If a carbon of a substituent is described as being optionally substituted with one or more of the list of substituents, one or more hydrogens on the carbon (to the extent of any hydrogens present) may be replaced individually and/or together with an independently selected optional substituent. If the nitrogen of a substituent is described as being optionally substituted with one or more of the list of substituents, then one or more hydrogens on the nitrogen (to the extent any hydrogen is present) may each be replaced with an independently selected optional substituent.

If a substituent is described as "independently selected from" a group of groups, each substituent is selected independently of the other. Thus, each substituent may be the same as 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 indicated, as used herein, the point of attachment of a substituent may be from any suitable position of the substituent.

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

The invention also includes all pharmaceutically acceptable isotopically-labeled compounds, which are identical to those of the present invention, except that one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number prevailing in nature. Examples of isotopes suitable for inclusion in compounds of the invention include, but are not limited to, isotopes of hydrogen (e.g. hydrogen)²H、³H) (ii) a Isotopes of carbon (e.g. of¹¹C、¹³C and¹⁴C) (ii) a Isotopes of chlorine (e.g. of chlorine)³⁷Cl); isotopes of fluorine (e.g. of fluorine)¹⁸F) (ii) a Isotopes of iodine (e.g. of iodine)¹²³I and¹²⁵I) (ii) a Isotopes of nitrogen (e.g. of¹³N and¹⁵n); isotopes of oxygen (e.g. of¹⁵O、¹⁷O and¹⁸o); isotopes of phosphorus (e.g. of phosphorus)³²P); and isotopes of sulfur (e.g. of³⁵S). Certain isotopically-labeled compounds of the present invention (e.g., those into which a radioisotope is incorporated) are useful in drug and/or substrate tissue distribution studies (e.g., assays). Radioisotope tritium (i.e. tritium³H) And carbon-14 (i.e.¹⁴C) Are particularly useful for this purpose because of their ease of incorporation and ease of detection. Using positron-emitting isotopes (e.g. of the type¹¹C、¹⁸F、¹⁵O and¹³n) can be used to examine substrate receptor occupancy in Positron Emission Tomography (PET) studies. Isotopically labeled compounds of the present invention can be prepared by processes analogous to those described in the accompanying schemes and/or in the examples and preparations by using an appropriate isotopically labeled reagent in place of the non-labeled reagent employed previously. Pharmaceutically acceptable solvates of the invention include those in which the crystallization solvent may be isotopically substituted, e.g., D₂O, acetone-d₆Or DMSO-d₆。

The term "stereoisomer" denotes an isomer formed as a result of at least one asymmetric center. In compounds having one or more (e.g., 1,2, 3, or 4) asymmetric centers, they can result in racemic mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Certain individual molecules may also exist as geometric isomers (cis/trans). Similarly, the compounds of the invention may exist as mixtures of two or more structurally different forms (commonly referred to as tautomers) in rapid equilibrium. Representative examples of tautomers include keto-enol tautomers, phenol-keto tautomers, nitroso-oxime tautomers, imine-enamine tautomers, and the like. For example, dihydropyrimidine groups may exist in solution in equilibrium with the following tautomeric forms. It is understood that the scope of this application encompasses all such isomers or mixtures thereof in any ratio (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%).

Solid lines may be used herein

Solid wedge shape

Or virtual wedge shape

Carbon-carbon bonds of the compounds are depicted. The use of a solid line to depict a bond to an asymmetric carbon atom is intended to indicate that all possible stereoisomers (e.g., particular enantiomers, racemic mixtures, etc.) at that carbon atom are included. The use of solid or dashed wedges to depict bonds to asymmetric carbon atoms is intended to indicate that the stereoisomers shown are present. When present in a racemic mixture, solid and dotted wedges are used to define the relative stereochemistry, not the absolute stereochemistry. Unless otherwise indicated, the compounds are describedIt is intended that the compounds may exist as stereoisomers, including cis and trans isomers, optical isomers (e.g., R and S enantiomers), diastereomers, geometric isomers, rotamers, conformers, atropisomers, and mixtures thereof. The compounds may exhibit more than one type of isomerization and consist of mixtures thereof (e.g., racemic mixtures and diastereomeric pairs).

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

It will also be appreciated that certain compounds of the present application may be present in free form for use in therapy or, where appropriate, in the form of a pharmaceutically acceptable derivative thereof. In the present application, pharmaceutically acceptable derivatives include, but are not limited to: pharmaceutically acceptable salts, solvates, metabolites or prodrugs thereof, which upon administration to a patient in need thereof are capable of providing, directly or indirectly, a compound of the present invention or a metabolite or residue thereof. 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 of the compound described above.

Pharmaceutically acceptable salts of the compounds of the present application include 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, bisulfate/sulfate, fumarate, glucoheptonate, gluconate, glucuronate, hexafluorophosphate, hydrobromide/bromide, hydroiodide/iodide, maleate, malonate, methylsulfate, naphthoate, 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 Stahl and Wermuth, "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" (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 be present in the form of solvates, preferably hydrates, wherein the compounds of the present application comprise as structural element of the crystal lattice of the compound a polar solvent, such as in particular water, methanol or ethanol. The amount of polar solvent, particularly water, may be present in stoichiometric or non-stoichiometric proportions.

The present application also includes metabolites of the compounds, i.e., substances formed in vivo upon administration of the compounds. Such products may result, for example, from oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic hydrolysis, etc. of the administered compound. Accordingly, the present application includes metabolites of the compounds, including compounds made by the method of contacting the compounds with a mammal for a time sufficient to produce a metabolite thereof.

The present application further includes prodrugs of the compounds which are certain derivatives of the compounds which may themselves have little or no pharmacological activity which may be converted to the compounds having the desired activity by, for example, hydrolytic cleavage when administered into or onto the body. Typically such prodrugs will be functional derivatives of the compounds which are readily convertible in vivo into the desired therapeutically active compound. Further 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(E.B.Roche editions, American Pharmaceutical Association). Such Prodrugs can be prepared, for example, by substituting certain moieties known to those skilled in the art as "pro-moieties" (e.g., "Design of drugs", h. bundgaard (Elsevier,1985) ") for the appropriate functional groups present in the compounds of the present application.

The application also encompasses such compounds containing protecting groups. In any process for the preparation of said compounds, it may be necessary and/or desirable to protect sensitive or reactive groups on any of the molecules concerned, thereby forming a chemically protected form of said compounds. This can be achieved by conventional protecting Groups, for example, as described in Protective Groups in Organic Chemistry, ed.j.f.w.mcomie, Plenum Press, 1973; and T.W.Greene & P.G.M.Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons,1991, which are incorporated herein by reference. The protecting group may be removed at a suitable subsequent stage using methods known in the art.

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

Compound (I)

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 structure of formula I:

wherein,

x is selected from CH and N;

R¹selected from hydroxy-C_1-6Alkyl-, C_1-6alkoxy-C_1-6Alkyl-and cyano-C_3-6Cycloalkyl-;

R³is selected from C_1-6Alkyl and C_2-6An alkenyl group;

R⁴selected from hydrogen and halogen.

In some preferred embodiments, the compounds have the structure shown in formula I-1,

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 some embodiments, R in formula I or formula I-1¹Selected from hydroxy-C_1-4Alkyl-, C_1-3alkoxy-C_1-4Alkyl-and cyano-C_3-6Cycloalkyl-. In some preferred embodiments, R¹Selected from the group consisting of 2-hydroxypropan-2-yl, 2-methoxyprop-2-yl and 1-cyanocyclopropyl-1-yl.

In some embodiments, R in formula I or formula I-1²Selected from 7-10 membered bicyclic fused heterocyclic group, said 7-10 membered bicyclic fused heterocyclic group optionally substituted with one or more C_1-6Alkyl (e.g. C)_1-4Alkyl radical, C_1-2Alkyl) substituted. In some embodiments, R²Selected from 8-membered fused heterocyclic groups, said 8-membered fused heterocyclic group being substituted by a C_1-6Alkyl (e.g. C)_1-4Alkyl radical, C_1-2Alkyl) substituted. In some preferred embodiments, R²Selected from 8-membered bicyclic fused heterocyclic group, said 8-membered bicyclic fused heterocyclic group being substituted by a C_1-6Alkyl (e.g. C)_1-4Alkyl radical, C_1-2Alkyl) substituted. In some preferred embodiments, R²Is selected from

In some preferred embodiments, R²Is 5-methyl hexahydro-pyrrolo [3,4-c]Pyrrol-2 (1H) -yl.

In some embodiments, R in formula I or formula I-1²Being piperidinyl, said piperidinyl being substituted by one or more-NR⁵R⁶Substituted, R⁵、R⁶Each independently selected from C_1-4An alkyl group. In some preferred embodiments, R²Being piperidinyl, said piperidinyl being substituted by one or more-NR⁵R⁶Substituted, R⁵、R⁶Each independently selected from C_1-2An alkyl group. In some preferred embodiments, R²Is composed of

R⁵、R⁶Each independently selected from C_1-6Alkyl (e.g. C)_1-4Alkyl radical, C_1-2Alkyl groups). In some preferred embodiments, R²Is 4-dimethylaminopiperidin-1-yl.

In some embodiments, R in formula I or formula I-1³Is selected from C_1-4Alkyl and C_2-4An alkenyl group. In some preferred embodiments, R³Selected from allyl and isopropyl.

In some embodiments, R in formula I or formula I-1⁴Selected from hydrogen, fluorine, chlorine and bromine. In some embodiments, R⁴Selected from hydrogen and fluorine. In some embodiments, R⁴Is hydrogen. In some embodiments, R⁴Is fluorine.

In some embodiments, the compounds have the structure shown in formula I-2,

wherein R is¹And R²The definition is defined in the general formula I.

In some embodiments, R¹Selected from hydroxy-C_1-6Alkyl-, C_1-6alkoxy-C_1-6Alkyl-and cyano-C_3-6Cycloalkyl-.

In some embodiments, R²Selected from 7-10 membered fused heterocyclyl, said 7-10 membered fused heterocyclyl being optionally substituted by one or more C_1-6Alkyl substituted, each C_1-6The alkyl groups may be the same or different; or R²Selected from piperidinyl, said piperidinyl being substituted by one or more-NR⁵R⁶Substituted, R⁵、R⁶Each independently selected from C_1-6An alkyl group.

In some embodiments, R¹Selected from hydroxy-C_1-4Alkyl-, C_1-3alkoxy-C_1-4Alkyl-and cyano-C_3-6Cycloalkyl-; preferably, R¹Selected from the group consisting of 2-hydroxypropan-2-yl, 2-methoxyprop-2-yl and 1-cyanocyclopropyl-1-yl.

In some embodiments, R²Selected from 8-membered fused heterocyclic groups, said 8-membered fused heterocyclic group being substituted by a C_1-6Alkyl substitution;

preferably, R²Selected from 8-membered bicyclic fused heterocyclic group, said 8-membered bicyclic fused heterocyclic group being substituted by a C_1-6Alkyl (e.g. C)_1-4Alkyl radical, C_1-2Alkyl) substitution;

preferably, R²Is selected from

More preferably, R²Is 5-methyl hexahydro-pyrrolo [3,4-c]Pyrrol-2 (1H) -yl.

In some embodiments, R²Is selected from

R⁵、R⁶Each independently selected from C_1-6Alkyl (e.g. C)_1-4Alkyl radical, C_1-2Alkyl groups); preferably, R²Is 4-dimethylaminopiperidin-1-yl.

In some embodiments, the compounds have the structure of formula I-2 above,

wherein R is¹Selected from hydroxy-C_1-6Alkyl-, C_1-6alkoxy-C_1-6Alkyl-and cyano-C_3-6Cycloalkyl-;

R²selected from 7-10 membered fused heterocyclyl, said 7-10 membered fused heterocyclyl being optionally substituted by one or more C_1-6Alkyl substituted, each C_1-6The alkyl groups may be the same or different; or R²Selected from piperidinyl, said piperidinyl being substituted by one or more-NR⁵R⁶Substituted, R⁵、R⁶Each independently selected from C_1-6An alkyl group.

In some embodiments, the compounds have the structure of formula I-2 above,

wherein R is¹Selected from hydroxy-C_1-4Alkyl-, C_1-3alkoxy-C_1-4Alkyl-and cyano-C_3-6Cycloalkyl-; preferably, R¹Selected from 2-hydroxyprop-2-yl, 2-methoxyprop-2-yl and 1-cyanocyclopropyl-1-yl;

R²selected from 8-membered fused heterocyclic groups, said 8-membered fused heterocyclic group being substituted by a C_1-6Alkyl substitution; preferably, R²Is selected from

Preferably, R²Is 5-methyl hexahydropyrrole [3,4-c]Pyrrol-2 (1H) -yl;

or, R²Is selected from

R⁵、R⁶Each independently selected from C_1-6An alkyl group; preferably, R²Is 4-dimethylaminopiperidin-1-yl.

In some embodiments, the compound has the following structure:

preparation method

It is another object of the present application to provide a process for preparing a compound of formula (I):

in the formula, Hal¹Is halogen (e.g., F, Cl or Br); hal²Is halogen (e.g., Cl, Br, or I), a boronic acid group, or a boronic ester group; r¹、R²、R³、R⁴And X have the same meaning as in formula I above.

(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 alkylhydrazine compound to give compound IN-2, preferably 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 can be selected from triethylamine, pyridine, 4-dimethylaminopyridine and diisopropylethylamine, and preferably is diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from 50 to 70 ℃. The reaction is preferably carried out for a suitable time, for example 6 to 16 hours.

In other embodiments: the compound IN-1 can also be reacted with hydrazine hydrate firstly and then subjected to reduction reaction with aldehyde compounds 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 ℃. The reaction is preferably carried out for a suitable time, for example 1 to 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 can be selected from methanol, ethanol, dichloromethane, chloroform, acetonitrile, preferably methanol. The reducing agent may be selected from sodium cyanoborohydride, sodium triacetoxyborohydride, sodium borohydride and the like, preferably sodium cyanoborohydride. The reaction temperature is preferably 0 to 25 ℃ in general, and the reaction time is preferably 1 to 3 hours in general.

(2) Cyclizing the compound IN-2 to obtain a compound IN-3;

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

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

the compound IN-3 is subjected to a coupling reaction with the compound IN-4 to obtain the 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, cupric acetate, and the like. The base is an inorganic base such as potassium carbonate, cesium carbonate, sodium carbonate, 2-bipyridine, potassium phosphate, preferably potassium carbonate. The ligand of the coupling reaction is selected from trans-N, N ' -dimethyl-1, 2-cyclohexanediamine and N, N ' -dimethylethylenediamine, preferably trans-N, N ' -dimethyl-1, 2-cyclohexanediamine. The solvent for the coupling reaction is selected from benzene, toluene, dioxane, dimethylformamide, ethylene glycol dimethyl ether, etc. or a mixed solvent thereof, and dioxane and dimethylformamide are preferred. The coupling reaction temperature is preferably from 80 to 110 ℃ in general. The reaction time is preferably 4 to 6 hours in general.

(4) Oxidizing the compound IN-5 to obtain a compound IN-6;

the oxidation reaction may preferably be carried out in a suitable organic solvent and in the presence of an oxidizing agent. The solvent may be selected from benzene, toluene, dichloromethane, chloroform, acetonitrile, dimethylformamide and the like, preferably acetonitrile. The oxidant can be selected from m-chloroperoxybenzoic acid, potassium peroxymonosulfate complex salt and the like, and potassium peroxymonosulfate complex salt is preferred. The reaction temperature is usually preferably room temperature (20 to 30 ℃ C.), and the reaction time is usually preferably 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, dimethylsulfoxide, etc., 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 triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like, preferably diisopropylethylamine. The reaction temperature is preferably 60 to 120 ℃ and the reaction time is preferably 4 to 12 hours.

It is a further object of the present application to provide a process for preparing a compound of 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 can be selected from triethylamine, pyridine, 4-dimethylaminopyridine and diisopropylethylamine, and preferably is diisopropylethylamine. The reaction is preferably carried out at a suitable temperature, preferably from 50 to 70 ℃. The reaction is preferably carried out for a suitable time, for example 6 to 16 hours.

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

this reaction for removing the protecting group PG can be applied to a hydrolysis reaction method of a carboxylic ester well known in the field of organic chemistry, and in an embodiment in which the protecting group PG is a t-butoxycarbonyl group, the reaction is preferably carried out by an acid hydrolysis reaction of an ester in the presence of an acid. The acid may be an inorganic acid or a suitable organic acid including, but not limited to, hydrochloric acid, sulfuric acid, formic acid, trifluoroacetic acid; the reaction solvent can be selected from dioxane, dichloromethane, ethyl acetate or their mixture, acid can also be used as reaction solvent, preferably trifluoroacetic acid. The reaction temperature is usually preferably 40 to 70 ℃. The reaction time is preferably 1 to 3 hours in general.

(3) Cyclizing compound IN-10 to give compound IN-11;

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

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

the compound IN-11 is subjected to a coupling reaction with the compound IN-12 to obtain the 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-bipyridine, potassium phosphate, preferably potassium carbonate. The ligand of the coupling reaction is selected from trans-N, N ' -dimethyl-1, 2-cyclohexanediamine and N, N ' -dimethylethylenediamine, preferably trans-N, N ' -dimethyl-1, 2-cyclohexanediamine. The solvent for the coupling reaction is selected from benzene, toluene, dioxane, dimethylformamide, ethylene glycol dimethyl ether, etc. or a mixed solvent thereof, and dioxane and dimethylformamide are preferred. The coupling reaction temperature is preferably 80 to 110 ℃. The reaction time is preferably 4 to 6 hours.

(5) Oxidizing the compound IN-13 to obtain a compound IN-14;

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

the reaction is preferably carried out in an inert solvent selected from toluene, benzene, chloroform, dioxane, dimethylformamide, N-methylpyrrolidone, dimethylsulfoxide, etc., 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 triethylamine, diisopropylethylamine, pyridine, 4-dimethylaminopyridine and the like, preferably diisopropylethylamine. The reaction temperature is preferably 60 to 120 ℃ and the reaction time is usually preferably 4 to 12 hours.

In preparing compounds of formula I or formula I-2, it may be desirable to protect remote functional groups (e.g., hydroxyl or amino groups) of the intermediate. The need for such protection may vary with the nature of the distal functional group and the conditions of the preparation method. For a summary 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

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

By "pharmaceutically acceptable carrier" herein is meant a diluent, adjuvant, excipient, or vehicle that is administered with the therapeutic agent and which is, within the scope of sound medical judgment, suitable for contact with the tissues of humans and/or other animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.

Pharmaceutically acceptable carriers that may be employed in the pharmaceutical compositions include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is an exemplary carrier when the pharmaceutical composition is administered intravenously. Physiological saline and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, maltose, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition may also optionally contain minor amounts of wetting agents, emulsifying agents, or pH buffering agents. Oral formulations may contain standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. Examples of suitable pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1990).

The pharmaceutical composition may act systemically and/or locally. For this purpose, they may be administered by a suitable route, for example by injection (e.g. intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular injection, including instillation) or transdermally; or by oral, buccal, nasal, transmucosal, topical, in the form of ophthalmic preparations or by inhalation.

For these routes of administration, the pharmaceutical composition may be administered in a suitable dosage form. Such dosage forms include, but are not limited to, tablets, capsules, lozenges, hard candies, powders, sprays, creams, ointments, suppositories, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like.

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

It is another object of the present application to provide a process for preparing the pharmaceutical composition, the process comprising combining the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof with one or more pharmaceutically acceptable carriers.

It is another object of the present application to provide a pharmaceutical formulation comprising said compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, or a mixture thereof, or said pharmaceutical composition.

Methods of treatment and uses

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

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

It is another object of the present application to provide a method for preventing or treating a Wee1 protein kinase-associated disease, comprising administering to a subject in need thereof an effective amount of the compound or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, or the pharmaceutical composition. In some embodiments, the Wee1 protein-related 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, when administered, will alleviate one or more symptoms of the condition being treated to some extent.

The dosing regimen may be adjusted to provide the best 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 exigencies of the therapeutic situation. It is noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single or multiple doses. It is further understood that for any particular individual, the specific dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering the composition or supervising the administration of the composition.

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

As used herein, unless otherwise specified, the term "treating" or "treatment" means reversing, alleviating, inhibiting the progression of, or preventing such a disorder or condition, or one or more symptoms of such a disorder or condition, to which such term applies.

As used herein, "individual" includes a human or non-human animal. Exemplary human individuals include human individuals (referred to as patients) having a disease (e.g., a disease described herein) or normal individuals. Herein, "non-human animal" includes all vertebrates, such as non-mammals (e.g., birds, amphibians, reptiles) and mammals, such as non-human primates, livestock, and/or domesticated animals (e.g., sheep, dogs, cats, cows, pigs, etc.).

Detailed Description

Examples

In order to make the objects and technical solutions of the present invention clearer, the present invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, specific experimental methods not mentioned in the following examples were carried out according to the usual experimental methods.

The abbreviations herein have the following meanings:

abbreviations	Means of
TLC	Thin layer chromatography
LC-MS	Liquid chromatography-mass spectrometry
DIPEA	N, N-diisopropylethylamine
CD ₃OD	Deuterated methanol
CDCl ₃	Deuterated chloroform
DMSO-d ₆	Hexahydro-deuterated dimethyl sulfoxide
TMS	Tetramethylsilane
NMR	Nuclear magnetic resonance
MS	Mass spectrometry
s	Single peak (singlet)
d	Double peak (doublet)
t	Triple peak (triplet)
q	Quartet (quartz)
dd	Double two peak (double)
m	Multiplet (multiplex)
J	Coupling constant
Hz	Hertz's scale

The following fruitThe structures of the compounds described in the examples are given by¹H-NMR or MS.¹H-NMR measurement apparatus Using Bruker 400MHz NMR spectrometer, CD was used as a measurement solvent₃OD、CDCl₃Or DMSO-d₆The internal standard substance is TMS, and all delta values are expressed in ppm. Mass Spectrometry (MS) was performed using an Agilent (ESI) mass spectrometer, model Agilent 6120B.

Thin layer chromatography silica gel plate (TLC) an aluminum plate (20X 20cm) from Merck was used, and GF 254 (0.4-0.5 mm) was used for separation and purification by thin layer chromatography.

The reaction was monitored by Thin Layer Chromatography (TLC) or LC-MS using a developing system of: dichloromethane and methanol system, n-hexane and ethyl acetate system, petroleum ether and ethyl acetate system, and volume ratio of solvent is regulated according to different polarities of the compounds or by adding triethylamine and the like.

Use of microwave reaction

Initiator + (400W, RT-300 ℃) microwave reactor.

The column chromatography generally uses 200-300 mesh silica gel as a carrier. The system of eluents comprises: the volume ratio of the solvent is adjusted according to different polarities of the compounds in a dichloromethane and methanol system and a petroleum ether and ethyl acetate system, and a small amount of triethylamine can also be added for adjustment.

Preparing a high performance liquid chromatograph, wherein the instrument model is as follows: agilent 1260, column: waters Xbridge Prep C₁₈OBD (19 mm. times.150 mm. times.5.0. mu.m); temperature of the chromatographic column: 25 ℃; 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.

Unless otherwise specified, the reaction temperatures of the examples are room temperature (20 ℃ C. to 30 ℃ C.).

Reagents used in this application are available from Acros Organics, Aldrich Chemical Company, Shanghai Teber Chemical science and technology, Inc., and the like.

Preparation of an intermediate:

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

The first step is as follows: preparation of ethyl 4- (2-allyl-2- (tert-butoxycarbonyl) hydrazino) -2- (methylthio) pyrimidine-5-carboxylate

Ethyl 4-chloro-2- (methylthio) pyrimidine-5-carboxylate (20g,85.8mmol), tert-butyl 1-allylhydrazinecarbonate (15g,85.8mmol) and DIPEA (28g,215mmol) were added to tetrahydrofuran (200mL) and stirred under reflux for 18 hours. The reaction mixture was cooled, the reaction solvent was distilled off under reduced pressure, ether (100mL) was added to the residue, the precipitated solid was filtered, and the filtrate was concentrated to give the title compound of this step (31.7g, yield: 100%).

MS m/z(ESI):369.2[M+H]⁺。

The second step is that: preparation of ethyl 4- (2-allylhydrazino) -2- (methylthio) pyrimidine-5-carboxylate

To ethyl 4- (2-allyl-2- (tert-butoxycarbonyl) hydrazino) -2- (methylthio) pyrimidine-5-carboxylate (31.7g,85.8mmol) was added trifluoroacetic acid (60mL) while cooling on ice, and the mixture was stirred at room temperature for 1 hour, heated at 70 ℃ for 1 hour, and the reaction mixture was concentrated to give the title compound of this step (23g, yield: 100%).

MS m/z(ESI):269.1[M+H]⁺。

The third step: preparation of 2-allyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

Ethyl 4- (2-allylhydrazino) -2- (methylthio) pyrimidine-5-carboxylate (10g,38.2mmol) was dissolved in ethanol (70mL), and 6N aqueous sodium hydroxide (140mL) was added under ice bath, stirred at room temperature for 15 minutes, adjusted to pH 1 with concentrated hydrochloric acid, and the ethanol was distilled off under reduced pressure, filtered, and the resulting solid was washed with ether to obtain the title compound (16 g, yield: 83%) after drying.

MS m/z(ESI):223.1[M+H]⁺。

Intermediate preparation example 2: preparation of 2- (6-bromopyridin-2-yl) propan-2-ol

Methyl 6-bromopyridine-2-carboxylate (5g,23.1mmol) was dissolved in ether (100mL), magnesium methyliodide (17mL,50.8mmol) was added under nitrogen protection, the mixture was stirred at room temperature for 0.5 hour, water and 2N hydrochloric acid were added to the reaction mixture, ethyl acetate was extracted 3 times, the organic layers were combined and washed with a saturated aqueous sodium bicarbonate solution and a saturated aqueous sodium chloride solution in this order, and dried over anhydrous sodium sulfate. Filtration and concentration of the filtrate gave the title compound (5g, yield: 100%).

MS m/z(ESI):216.0[M+H]⁺。

Intermediate preparation example 3: preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

The first step is as follows: preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-allyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (2g,9.0mmol), 2- (6-bromopyridin-2-yl) propan-2-ol (2.5g,11.7mmol), cuprous iodide (1.7g,9.0mmol), potassium carbonate (1.7g,12.6mmol) and trans-N, N' -dimethyl-1, 2-cyclohexanediamine (1.4g,9.9mmol) were added to dioxane (35mL) and reacted at 95 ℃ for 4 hours, after cooling to room temperature, aqueous ammonia was added, ethyl acetate was extracted 3 times, the organic phases were combined and concentrated, and the residue was subjected to silica gel column chromatography (eluent: petroleum ether/ethyl acetate 3/1) to give the title compound of this step (2.6g, yield: 81%).

MS m/z(ESI):358.1[M+H]⁺。

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

2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (methylsulfinyl) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (200mg,0.56mmol) was dissolved in acetonitrile (5mL) and water (5mL), potassium hydrogen peroxymonosulfate complex salt (282mg,1.68mmol) was added, stirred at room temperature for 2 hours, water was added, extracted 3 times with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound (200mg, yield: 96%).

MS m/z(ESI):374.1[M+H]⁺。

Intermediate preparation example 4: preparation of 1- (6-bromopyridin-2-yl) cyclopropylcarbonitrile

The first step is as follows: preparation of 2- (6-bromopyridin-2-yl) acetonitrile

Acetonitrile (1.5mL,28.7mmol) was dissolved in tetrahydrofuran (20mL), n-butyllithium (11.2mL,27.9mmol) was added dropwise at-78 ℃ and stirred for 30 minutes, a solution of 2, 6-dibromopyridine (2g,8.4mmol) in tetrahydrofuran (30mL) was added, stirring was continued for 45 minutes, the reaction was gradually raised to room temperature, water and ethyl acetate were added and the organic phase was concentrated and the resulting residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate ═ 3/1) to obtain the title compound of this step (1.28g, yield: 77%).

MS m/z(ESI):197.0[M+H]⁺。

The second step is that: preparation of 1- (6-bromopyridin-2-yl) cyclopropylcarbonitrile

2- (6-bromopyridin-2-yl) acetonitrile (500mg,2.54mmol), 1, 2-dibromoethane (716mg,3.81mmol), tetrabutylammonium hydrogensulfate (164mg,0.51mmol) were dissolved in toluene (5mL), 50% aqueous sodium hydroxide solution (2.5mL) was added, stirring was performed at room temperature for 1 hour, the reaction mixture was extracted with water and ethyl acetate, the organic phase was combined and washed with water and saturated brine in this order, and the residue obtained by concentrating the organic phase was purified by preparative thin-layer chromatography (eluent: petroleum ether/ethyl acetate: 1/1) to give the title compound (300mg, yield: 53%).

MS m/z(ESI):223.0[M+H]⁺。

Intermediate preparation example 5: preparation of 2-bromo-6- (2-methoxyprop-2-yl) pyridine

2- (6-Bromopyridin-2-yl) propan-2-ol (500mg,2.3mmol) and iodomethane (980mg,6.9mmol) were dissolved in tetrahydrofuran (15mL), sodium hydride (276mg,6.9mmol) was added, the mixture was stirred at room temperature overnight, the reaction solution was extracted with water and ethyl acetate, the organic phases were combined, washed successively with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound (377mg, 71%).

MS m/z(ESI):230.0[M+H]⁺。

Intermediate preparation example 6: preparation of 4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -phenylamine

The first step is as follows: preparation of 5- (4-nitrophenyl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carbonic acid tert-butyl ester

1-fluoro-4-nitrobenzene (312.4mg,2.2mmol) was dissolved in acetonitrile (10mL), followed by the addition of hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carbonic acid tert-butyl ester (500mg,2.4mmol) and DIPEA (455.8mg,3.5mmol) followed by heating under reflux and stirring for 5 hours. The reaction solution was cooled, filtered with suction, and dried to obtain the title compound (620mg, yield: 79.0%) of this step.

MS m/z(ESI):334.2[M+H]⁺。

The second step is that: preparation of 2- (4-nitrophenyl) hexahydropyrrolo [3,4-c ] pyrrole hydrochloride

5- (4-Nitrophenyl) hexahydropyrrolo [3,4-c ] pyrrole-2 (1H) -carbonic acid tert-butyl ester (620mg,1.9mmol) was dissolved in 4N hydrochloric acid in 1,4 dioxane (20mL), stirred at room temperature for 2 hours, and the reaction solution was concentrated to obtain the title compound of this step (560mg, yield: 100%).

MS m/z(ESI):234.1[M+H]⁺。

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

2- (4-Nitrophenyl) hexahydropyrrolo [3,4-c ] pyrrole hydrochloride (560mg,2.4mmol) and 40% aqueous formaldehyde (2.6g,36.0mmol) were dissolved in dichloromethane (20mL), and after addition of sodium borohydride acetate (1.5g,7.2mmol), stirring was carried out at room temperature for 1 hour, water was added, extraction was carried out 3 times with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the title compound (600mg, yield: 100%) of this step.

MS m/z(ESI):248.1[M+H]⁺。

The fourth step: preparation of 4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -phenylamine

2-methyl-5- (4-nitrophenyl) hexahydropyrrolo [3,4-c ] pyrrole (600mg,2.4mmol) was dissolved in methanol (10mL), raney nickel and hydrazine hydrate (606.6mg,12.1mmol) were sequentially added, the mixture was stirred at room temperature for 2 hours, the reaction solution was filtered through celite, methanol was distilled off under reduced pressure, water and dichloromethane were added and extracted 3 times, the organic layer was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound (342mg, yield: 65%).

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

The first step is as follows: preparation of ethyl 4-hydrazino-2- (methylthio) pyrimidine-5-carboxylate

Ethyl 4-chloro-2- (methylthio) pyrimidine-5-carboxylate (5g,21.5mmol) was dissolved in ethanol (150mL), cooled to 0 ℃ in an ice bath, hydrazine hydrate (3.2g) was added, the reaction mixture was stirred at 0 ℃ for 1 hour, then evaporated to dryness under reduced pressure, the residue was added to methyl t-butyl ether (100mL), stirred for 30 minutes and filtered, and the filter cake was dried under vacuum to give the title compound of this step (4.3g, yield: 88%).

MS m/z(ESI):229.1[M+H]⁺。

The second step is that: preparation of ethyl 2- (methylthio) -4- (2- (prop-2-ylidene) hydrazino) pyrimidine-5-carboxylate

Ethyl 4-hydrazino-2- (methylthio) pyrimidine-5-carboxylate (4.3g,18.8mmol) was placed in a 150mL single-necked flask, acetone (100mL) was added, the temperature was raised to 70 ℃ and stirred overnight, and the reaction solution was concentrated to give the title compound of this step (5.1g, yield: 100%).

MS m/z(ESI):269.1[M+H]⁺。

The third step: preparation of ethyl 4- (2-isopropylhydrazino) -2- (methylthio) pyrimidine-5-carboxylate

Ethyl 2- (methylthio) -4- (2- (prop-2-ylidene) hydrazino) pyrimidine-5-carboxylate (5.1g,18.8mmol) was dissolved in methanol (100mL), cooled to 0 ℃ in an ice bath, sodium cyanoborohydride (1.8g,28.2mmol) and concentrated hydrochloric acid (0.1mL) were added, and after stirring for 2 hours while maintaining 0 ℃, a saturated aqueous sodium bicarbonate solution (50mL) was added to the reaction solution, followed by stirring for 10 minutes, ethyl acetate extraction 3 times, merging of the organic phases, drying over anhydrous sodium sulfate, filtration, and evaporation of the filtrate under reduced pressure to dryness to obtain the title compound of this step (4.9g, yield: 96%).

MS m/z(ESI):271.1[M+H]⁺。

The fourth step: preparation of 2-isopropyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

Ethyl 4- (2-isopropylhydrazino) -2- (methylthio) pyrimidine-5-carboxylate (4.9g,18.1mmol) was dissolved in methanol (400mL), 5N aqueous sodium hydroxide solution (100mL) was added, stirring was carried out at room temperature overnight, the reaction solution was evaporated under reduced pressure to remove methanol, the remaining aqueous phase was extracted twice with ethyl acetate, the aqueous phase was adjusted to pH 2 with 5N diluted hydrochloric acid, extracted 5 times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated under reduced pressure to dryness to give the title compound (1.3g, yield: 31%).

MS m/z(ESI):225.1[M+H]⁺。

Intermediate preparation example 8: preparation of 1- (4-amino-2-fluorophenyl) -N, N-dimethylpiperidin-4-amine

The first step is as follows: preparation of 1- (4-nitro-2-fluorophenyl) -N, N-dimethylpiperidin-4-amine

1, 2-difluoro-4-nitrobenzene (1g,6.28mmol), N-dimethylpiperidin-4-amine (0.96g,7.55mmol) and potassium carbonate (1.72g,12.5mmol) were added to acetonitrile (10mL) and reacted at 50 ℃ for 16 hours, the reaction solution was cooled and poured into water, filtered and the solid was dried to obtain the title compound of this step (1.2g, yield: 71.4%).

MS m/z(ESI):268.2[M+H]⁺。

The second step is that: preparation of 1- (4-amino-2-fluorophenyl) -N, N-dimethylpiperidin-4-amine

1- (4-Nitro-2-fluorophenyl) -N, N-dimethylpiperidin-4-amine (200mg,0.75mmol) was dissolved in methanol (5mL), a catalytic amount of Raney nickel was added thereto, hydrazine hydrate (1mL) was added dropwise, the mixture was stirred at room temperature for 2 hours, and the mixture was filtered through a small pad of celite to obtain the title compound (170mg, yield: 96%) after the filtrate was concentrated.

MS m/z(ESI):238.2[M+H]⁺。

Compound preparation example:

example 1: preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- ((4- (4-dimethylaminopiperidin-1-yl) 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 ] pyrimidin-3 (2H) -one (58mg,0.16mmol) was dissolved in toluene (5mL), followed by addition of (4-dimethylaminopiperidin-1-yl) aniline (68mg,0.32mmol) and DIPEA (82.6mg,0.64mmol), heating to 80 ℃ for 4 hours, concentration of the reaction solution, and purification of the residue by preparative high performance liquid chromatography gave the title compound (29mg, yield: 34%).

MS m/z(ESI):529.3[M+H]⁺

¹H-NMR(400MHz,CDCl₃)δ:8.84(s,1H),7.87(t,J＝8.0Hz,1H),7.74(d,J＝8.0 Hz,1H),7.48(d,J＝8.4Hz,2H),7.36(d,J＝7.6Hz,1H),6.92(d,J＝8.8Hz,2H),5.73-5.67(m,1H),5.05(dd,J＝10.0,1.2Hz,1H),4.94(dd,J＝17.2,1.2Hz,1H),4.74(d,J＝6.0Hz,2H),3.74(d,J＝12.4Hz,2H),2.83-2.71(m,5H),2.56(s,7H),2.09(d,J＝12.0Hz,2H),1.83-1.80(m,2H),1.59(s,6H)。

Example 2: preparation of 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- ((4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-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 ] pyrimidin-3 (2H) -one (50mg,0.13mmol) was dissolved in toluene (5mL), and 4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -aniline (58mg,0.27mmol) and DIPEA (67.1mg,0.52mmol) were sequentially added, heated to 80 ℃ for 4 hours, and the reaction was concentrated, and the residue was purified by preparative high performance liquid chromatography to give the title compound (9mg, yield: 14%).

MS m/z(ESI):527.3[M+H]⁺

¹H-NMR(400MHz,CDCl₃)δ:8.83(s,1H),7.87(t,J＝8.0Hz,1H),7.73(d,J＝8.0Hz,1H),7.46(d,J＝7.6Hz,2H),7.35(d,J＝7.2Hz,1H),6.72(d,J＝8.8Hz,2H),5.70(d,J＝6.8Hz,1H),5.35(t,J＝4.8Hz,1H),5.05(d,J＝9.2Hz,1H),4.94(d,J＝18.0Hz,1H),4.73(d,J＝6.4Hz,2H),3.75(s,3H),3.44(d,J＝10.0Hz,2H),3.35(s,2H),3.17(s,2H),2.79(s,5H),1.59(s,6H)。

Example 3: preparation of 2-allyl-1- (6- (1-cyanocycloprop-1-yl) pyridin-2-yl) -6- ((4- (4-dimethylaminopiperidin-1-yl) phenyl) amino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (compound 3)

The first step is as follows: preparation of 2-allyl-1- (6- (1-cyanocycloprop-1-yl) pyridin-2-yl) -6-methylsulfanyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-allyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (280mg,1.26mmol), 2-bromo-6- (1-cyanocycloprop-1-yl) pyridine (366mg,1.64mmol), cuprous iodide (239mg,1.26mmol), potassium carbonate (243mg,1.76mmol) and trans-N, N' -dimethyl-1, 2-cyclohexanediamine (197mg,1.39mmol) were added to dioxane (8mL), reacted at 95 ℃ for 4 hours, cooled to room temperature, followed by addition of aqueous ammonia, extraction with ethyl acetate 3 times, organic phase combination and concentration, and the residue was chromatographed on a silica gel column (eluent: petroleum ether/ethyl acetate: 3/1) to give the title compound of this step (289mg, yield: 63%).

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

2-allyl-1- (6- (1-cyanocycloprop-1-yl) pyridin-2-yl) -6-methylsulfanyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (289mg,0.8mmol) was dissolved in acetonitrile (5mL) and water (5mL), potassium monopersulfate complex salt (400mg,2.4mmol) was added, stirred at room temperature for 2 hours, water was added, extracted 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound of this step (292mg, yield: 96%).

The third step: preparation of 2-allyl-1- (6- (1-cyanocycloprop-1-yl) pyridin-2-yl) -6- ((4- (4-dimethylaminopiperidin-1-yl) phenyl) 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 ] pyrimidin-3 (2H) -one (62mg,0.16mmol) was dissolved in toluene (5mL), followed by addition of (4-dimethylaminopiperidin-1-yl) aniline (72mg,0.33mmol) and DIPEA (84mg,0.65mmol), heating to 80 ℃ for 4 hours, concentration of the reaction solution, and purification of the residue by preparative high performance liquid chromatography gave the title compound (30mg, yield: 35%).

MS m/z(ESI):527.3[M+H]⁺

¹H-NMR(400MHz,CDCl₃)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＝14.8,3.2Hz,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: preparation of 2-allyl-1- (6- (1-cyanocycloprop-1-yl) pyridin-2-yl) -6- ((4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-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 ] pyrimidin-3 (2H) -one (80mg,0.21mmol) was dissolved in toluene (5mL), 4- (5-methylhexahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) -aniline (91.4mg,0.42mmol) and DIPEA (108.5mg,0.84mmol) were sequentially added, heated to 80 ℃ for 4 hours, the reaction solution was concentrated, and the residue was purified by preparative HPLC to give the title compound (32mg, yield: 28.6%).

MS m/z(ESI):534.6[M+H]⁺

¹H-NMR(400MHz,CDCl₃)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＝7.2Hz,2H),6.76(d,J＝7.2Hz,2H),6.25(dd,J＝7.2,1.6Hz,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＝6.0Hz,1H),3.47(d,J＝6.4Hz,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: preparation of 2-allyl-1- (6- (2-methoxypropan-2-yl) pyridin-2-yl) -6- ((4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -phenyl) amino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (Compound 5)

The first step is as follows: preparation of 2-allyl-1- (6- (2-methoxyprop-2-yl) pyridin-2-yl) -6-methylsulfanyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-allyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (140mg,0.63mmol), 2-bromo-6- (2-methoxyprop-2-yl) pyridine (188.6mg,0.82mmol), cuprous iodide (119.5mg,0.63mmol), potassium carbonate (121.5mg,0.88mmol) and trans-N, N' -dimethyl-1, 2-cyclohexanediamine (98.5mg,0.70mmol) were added to dioxane (8mL) to react at 95 ℃ for 4 hours, after cooling to room temperature, aqueous ammonia was added, ethyl acetate was extracted 3 times, the organic phase was combined and concentrated, and the residue was chromatographed on a silica gel column (eluent: petroleum ether/ethyl acetate: 3/1) to give the title compound of this step (166mg, yield: 71%).

The second step is that: preparation of 2-allyl-1- (6- (2-methoxyprop-2-yl) pyridin-2-yl) -6-methylsulfinyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-allyl-1- (6- (2-methoxyprop-2-yl) pyridin-2-yl) -6-methylsulfanyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (150mg,0.4mmol) was dissolved in acetonitrile (5mL) and water (5mL), potassium hydrogen peroxymonosulfate complex salt (203.7mg,1.2mmol) was added, stirred at room temperature for 2 hours, water was added, extraction was performed 3 times with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give the title compound of this step (152mg, yield: 98%).

The third step: preparation of 2-allyl-1- (6- (2-methoxyprop-2-yl) pyridin-2-yl) -6- ((4- (5-methylhexahydropyrrolo [3,4-c ] pyrrol-2 (1H) -yl) -phenyl) amino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-allyl-1- (6- (2-methoxyprop-2-yl) pyridin-2-yl) -6-methylsulfinyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (75mg,0.19mmol) was dissolved in toluene (5mL), 4- (5-methylhexahydropyrrole [3,4-c ] pyrrol-2 (1H) -yl) aniline (84mg,0.39mmol) and DIPEA (100mg,0.77mmol) were added in this order, the mixture was heated to 80 ℃ for 4 hours, and the reaction mixture was concentrated to prepare the title compound (34mg, yield: 33%) which was purified by high performance liquid chromatography.

MS m/z(ESI):541.7[M+H]⁺

¹H-NMR(400MHz,CDCl₃)δ:8.81(s,1H),7.87-7.75(m,2H),7.50-7.36(m,3H),6.68(d,J＝8.8Hz,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: preparation of 2-allyl-1- (6- (2-methoxypropan-2-yl) pyridin-2-yl) -6- ((4- (4-dimethylaminopiperidin-1-yl) phenyl) amino) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (compound 6)

2-allyl-1- (6- (2-methoxyprop-2-yl) pyridin-2-yl) -6-methylsulfinyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (75mg,0.19mmol) was dissolved in toluene (5mL), followed by addition of (4-dimethylaminopiperidin-1-yl) aniline (85mg,0.39mmol) and DIPEA (100mg,0.77mmol), heating to 80 ℃ for reaction at 4 hours, and concentration of the reaction mixture to afford the title compound (35mg, yield: 34%) by HPLC purification.

MS m/z(ESI):543.3[M+H]⁺

¹H-NMR(400MHz,CDCl₃)δ:8.83(s,1H),7.85(t,J＝7.6Hz,1H),7.77(d,J＝7.6Hz,1H),7.48(d,J＝8.0Hz,3H),6.93(d,J＝9.2Hz,2H),5.72-5.61(m,1H),4.99(d,J＝9.6Hz,1H),4.91-4.82(m,3H),3.73(d,J＝12.4Hz,2H),3.22(s,3H),2.79-2.72(m,4H),2.54(s,6H),2.08(d,J＝12.0Hz,2H),1.86-1.74(m,2H),1.56(s,6H)。

Example 7: preparation of 6- ((4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -2-isopropyl-1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (Compound 7)

The first step is as follows: preparation of 1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -2-isopropyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-isopropyl-6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (300mg,1.34mmol) and 2-hydroxypropyl-6-bromopyridine (347mg,1.61mmol) were dissolved in 1, 4-dioxane (50mL), cuprous iodide (255mg,1.34mmol), potassium carbonate (277mg,2.01mmol) and trans-N, N' -dimethyl-1, 2-cyclohexanediamine (285mg,2.01mmol) were added, and after completion, the temperature was raised to 95 ℃ and the mixture was stirred overnight, the reaction solution was cooled to room temperature, filtered, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate 3/1-1/1) to obtain the title compound (350mg, yield: 72%) of this step.

MS m/z(ESI):360.1[M+H]⁺。

The second step is that: preparation of 6- ((4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -2-isopropyl-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 ] pyrimidin-3 (2H) -one (180mg,0.50mmol) was dissolved in toluene (12mL), m-chloroperoxybenzoic acid (104mg,0.60mmol) was added, and after stirring at room temperature for two hours, diisopropylethylamine (258mg,2.00mmol) and (4-dimethylaminopiperidin-1-yl) aniline (220mg,1.00mmol) were added, and after completion of addition, the temperature was raised to 80 ℃ and stirring was continued overnight, the reaction solution was evaporated to dryness under reduced pressure, and the residue was purified by preparative high performance liquid chromatography to give the title compound (165mg, yield: 62.1%).

MS m/z(ESI):531.3[M+H]⁺。

¹H-NMR(400MHz,DMSO-d₆)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＝7.6Hz,1H),7.54(s,2H),6.90(d,J＝9.2Hz,2H),5.33(s,1H),4.18-4.11(m,1H),3.64(d,J＝12.4Hz,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-hydroxypropan-2-yl) pyridin-2-yl) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (compound 8)

Dissolving 2-allyl-1- (6- (2-hydroxypropan-2-yl) pyridin-2-yl) -6- (sulfinyl) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (100mg,0.27mmol) and 1- (4-amino-2-fluorophenyl) -N, N-dimethylpiperidin-4-amine (80mg,0.32mmol) in 1, 4-dioxane (5mL), dropwise adding trifluoroacetic acid (0.5mL), heating to 95 ℃ for reaction for 2 hours, cooling to room temperature, pouring the reaction solution into water, extracting impurities with ethyl acetate, adjusting the pH of the aqueous phase to 8-9 with potassium carbonate, extracting with ethyl acetate, combining the organic phases, drying over anhydrous sodium sulfate, filtration and concentration of the filtrate were carried out, and the residue was purified by preparative high performance liquid chromatography to give the title compound (48mg, yield: 32.8%).

MS m/z(ESI):547.3[M+H]⁺。

¹H-NMR(400MHz,DMSO-d₆)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＝8.0Hz,1H),7.37-7.34(m,1H),7.03-6.98(t,J＝5.2Hz,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＝5.2Hz,1H),2.65-2.59(t,J＝10.8Hz,2H),2.21(s,6H),1.84(d,J＝10.8Hz,2H),1.57-1.52(m,2H),1.44(s,6H)。

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

The first step is as follows: preparation of 3- (1-hydroxy-1-methylethyl) bromobenzene

Adding methyl 3-bromobenzoate (1g,4.65mmol) into diethyl ether (20mL) under the protection of nitrogen, cooling in an ice-water bath, dropwise adding a diethyl ether solution of methyl magnesium iodide (4mL,3mol/L,12mmol), and naturally heating to room temperature for reaction for 12 hours after dropwise adding. The reaction solution was slowly poured into a saturated aqueous ammonium chloride solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the title compound of this step (820mg, yield: 82%).

MS m/z(ESI):245.0[M+H]⁺。

The second step is that: preparation of 3- (1-hydroxy-1-methylethyl) phenylboronic acid

Under nitrogen protection, 3- (1-hydroxy-1-methylethyl) bromobenzene (500mg,2.32mmol) was added to tetrahydrofuran (10mL), a N-hexane solution of N-butyllithium (3mL,2mol/L,6mmol) was slowly added dropwise at-60 deg.C, stirring was carried out at-60 deg.C for 1 hour, trimethyl borate (480mg,4.62mmol) was added dropwise, reaction was carried out at room temperature for 12 hours, water (20mL) was slowly added dropwise, extraction was carried out with ethyl acetate, the aqueous phase was adjusted to pH 2-3 with 1N hydrochloric acid, extraction was carried out with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the title compound of this step (220mg, yield: 52.6%).

MS m/z(ESI):181.0[M+H]⁺。

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

2-allyl-6- (methylthio) -1, 2-dihydro-3H-pyrazolo [3,4-d ] pyrimidin-3-one (200mg,0.56mmol), copper acetate (160mg,0.8mmol) and 3- (1-hydroxy-1-methylethyl) phenylboronic acid (200mg,1.12mmol) were sequentially added to chloroform (3mL), pyridine (0.4mL) was added dropwise at room temperature for reaction for 48 hours, 30% aqueous ammonia was added to the reaction, stirring was carried out for 30 minutes, ethyl acetate extraction was carried out, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the residue was purified by silica gel thin chromatography to give the title compound of this step (30mg, yield 15%).

MS m/z(ESI):357.1[M+H]⁺。

The fourth step: preparation of 2-allyl-1- (3- (2-hydroxypropan-2-yl) phenyl) -6- (methylsulfinyl) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one

2-allyl-1- (3- (2-hydroxypropan-2-yl) phenyl) -6- (methylthio) -1H-pyrazolo [3,4-d ] pyrimidin-3 (2H) -one (30mg,0.084mmol) was added to a mixed solvent of acetonitrile (2mL) and water (2mL), oxone complex salt (34mg,0.2mmol) was added thereto, the mixture was stirred at room temperature for 2 hours, the reaction mixture was diluted with 10mL of water, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain the title compound (20mg, yield 63.6%) of this step

MS m/z(ESI):373.1[M+H]⁺。

The fifth step: preparation of 2-allyl-6- ((4- (4- (dimethylamino) piperidin-1-yl) phenyl) amino) -1- (3- (2-hydroxypropan-2-yl) 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 ] pyrimidin-3 (2H) -one (20mg,0.053mmol) and (4-dimethylaminopiperidin-1-yl) aniline (13mg,0.059mmol) were added to 1, 4-dioxane (10mL), trifluoroacetic acid (0.1mL) was added dropwise, the mixture was heated to 90 ℃ to react for 4 hours, the reaction was cooled to room temperature, the reaction solution was poured into water, ethyl acetate was used to extract impurities, the aqueous phase was adjusted to pH 8 to 9 with potassium carbonate, ethyl acetate was used to extract, the organic phase was combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the residue was purified by preparative high performance liquid chromatography to give the title compound (5mg, yield 18%).

MS m/z(ESI):528.3[M+H]⁺。

¹H-NMR(400MHz,CDCl₃)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＝9.2Hz,1H),4.27(br,2H),3.61(d,J＝12.0Hz,2H),2.59(d,J＝8.0Hz,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 testing

Experimental example 1: inhibition assay of Wee1 kinase:

to test the inhibitory effect of the compounds on the in vitro activity of Wee1 kinase, the experimental procedure was as follows:

1) after pre-incubation of Wee1 protein (Carna Biosciences) with different concentrations of test compounds for 15 min at room temperature, the reaction was initiated by adding substrate (Poly (Lys, Tyr), Sigma) and 10ATP according to the kit instructions and reacted for 90 min at 30 ℃.

2) An equal volume of ADP-Glo reagent (Promega) was added to the reaction system, and the reaction was terminated by incubation at room temperature for 40 minutes.

3) Kinase detection reagent was added and incubated for 30 minutes at room temperature. Detecting the self-luminescence signal of the product. The half maximal Inhibitory Concentration (IC) of the compound was calculated as the concentration of the remaining activity closest to 50% using the vehicle group (DMSO) as a negative control and the Buffer reaction Buffer group (containing no enzyme and compound) as a blank control₅₀) The results are shown in table 1:

table 1 data for inhibition of Wee1 Activity by Compounds

As can be seen from the data in Table 1, the compound has obvious inhibition effect on Wee1 kinase.

Experimental example 2: rat Pharmacokinetic (PK) profile study

Male SD rats were given the Wee1 inhibitor AZD1775 and the test compound at the clinical trial stage by Intravenous (IV) and intragastric (PO) administration, respectively, and the pharmacokinetic profile of the test compound was examined. The dosages of IV and PO were 1mg/kg and 5mg/kg, respectively, with IV vehicle being 5% DMSO, 5% Solutol, 90% physiological saline, and PO vehicle being 0.5% MC. Blood was collected at various time points after IV and PO administration. EDTA-K is adopted for blood₂Anticoagulated and centrifuged to obtain a plasma sample. Plasma samples were processed for precipitated protein and analyzed by LC-MS/MS.

Pharmacokinetic parameters were calculated using WinNonlin 6.3 software using a non-compartmental model, and the results are shown in tables 2 and 3.

TABLE 2 pharmacokinetic parameters of the Compounds in rats

The data in table 2 show that compound 2 of the present application has a bioavailability (F) in rats of 49.7% better than AZD1775, indicating that the compound of the present application has good drug exposure and bioavailability in rats and is suitable for oral administration.

TABLE 3 drug half-life comparison of Compounds in rats

Test object	Compound 1	Compound 2	AZD1775
Route of administration	Gavage stomach	Gavage stomach	Gavage stomach
Sex	Male sex	Male sex	Male sex
Dosage (mg/kg)	5	5	5
T _1/2(h)	4.37	3.79	1.73

The data in Table 3 show that Compound 1 and Compound 2, T of the present application are administered by gavage at a dose of 5mg/kg_1/2The half-life period of the compound is improved by more than 1 time compared with that of the AZD1775 compound, the effect time of the medicine is longer, and the medicine has obvious advantages. Moreover, the compound can be administrated 1 time per day, so that the administration frequency can be reduced, and the compliance of patients can be improved.

Experimental example 3: mouse Pharmacokinetic (PK) profile study

Female Balb/c mice were given the Wee1 inhibitor AZD1775 and test compound in clinical trials via Intravenous (IV) and intragastric (PO) administration, respectively, and the pharmacokinetic profile of the test compound was examined. The dosages of IV and PO were 1mg/kg and 10mg/kg, respectively, with IV vehicle being 5% DMSO, 5% Solutol, 90% physiological saline, and PO vehicle being 0.5% MC. Blood was collected at various time points after IV and PO administration. EDTA-K is adopted for blood₂Anticoagulated and centrifuged to obtain a plasma sample. Plasma samples were processed for precipitated protein and analyzed by LC-MS/MS.

Pharmacokinetic parameters were calculated using the WinNonlin 6.3 software using a non-compartmental model, and the results are shown in tables 4 and 5.

TABLE 4 pharmacokinetic parameters of Compounds in mice

The data in Table 4 show the AUC in mice for Compound 1 of the present application when administered 10mg/kg orally_last1770h ng/mL, better than the AUC of AZD1775 at the same dose_lastThe compounds of the present application are shown to have good drug exposure in mice and are suitable for oral administration.

TABLE 5 drug half-life comparison of Compounds in mice

The data in Table 5 show that Compound 1 of the present application, T, was administered intravenously and by gavage_1/2The half-life period of the compound is 1.15 hours and 2.90 hours respectively, which shows that the half-life period of the compound is improved by more than 1 time compared with that of the compound AZD1775, the effect action time is longer, and the compound has obvious advantages. Moreover, the compound can be administrated 1 time per day, so that the administration frequency can be reduced, and the compliance of patients can be improved.

Claims

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

wherein,

x is selected from CH and N;

R¹selected from hydroxy-C_1-6Alkyl-, C_1-6alkoxy-C_1-6Alkyl-and cyano-C_3-6Cycloalkyl-;

R²selected from 7-10 membered fused heterocyclyl, said 7-10 membered fused heterocyclyl being optionally substituted by one or more C_1-6Alkyl substitution; or R²Is piperidinyl, said piperidinyl group being optionally substituted by one or more-NR⁵R⁶Substitution; r⁵、R⁶Each independently selected from C_1-6An alkyl group;

R³is selected from C_1-6Alkyl and C_2-6An alkenyl group;

R⁴selected from hydrogen and halogen.
The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

wherein R is¹Selected from hydroxy-C_1-4Alkyl-, C_1-3alkoxy-C_1-4Alkyl-and cyano-C_3-6Cycloalkyl-;

preferably, R¹Selected from the group consisting of 2-hydroxypropan-2-yl, 2-methoxyprop-2-yl and 1-cyanocyclopropyl-1-yl.
The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

wherein R is²Selected from 7-10 membered bicyclic fused heterocyclic group, said 7-10 membered bicyclic fused heterocyclic group optionally substituted with one or more C_1-6Alkyl substitution;

preferably, R²Selected from 8-membered fused heterocyclic groups, said 8-membered fused heterocyclic group being substituted by a C_1-6Alkyl substitution;

preferably, R²Selected from 8-membered bicyclic fused heterocyclic group, said 8-membered bicyclic fused heterocyclic group being substituted by a C_1-6Alkyl substitution;

preferably, R²Is selected from

Preferably, R²Is 5-methyl-octahydro-pyrrolo [3,4-c]Pyrrol-2-yl.
The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

wherein R is²Being piperidinyl, said piperidinyl being substituted by one or more-NR⁵R⁶Substituted, R⁵、R⁶Each independently selected from C_1-6An alkyl group;

preferably, R²Being piperidinyl, said piperidinyl being substituted by one or more-NR⁵R⁶Substituted, R⁵、R⁶Each independently selected from C_1-4An alkyl group;

preferably, R²Is composed of
R⁵、R⁶Each independently selected from C_1-6An alkyl group;

preferably, R²Is 4-dimethylaminopiperidin-1-yl.
The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

wherein R is³Is selected from C_1-4Alkyl and C_2-4An alkenyl group;

preferably, R³Selected from allyl and isopropyl.
The compound according to claim 1, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof,

wherein R is⁴Selected from hydrogen and fluorine.
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, R¹、R²、R³And R⁴As defined in any one of claims 1 to 6.
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 a structure represented by formula I-2,

wherein R is¹And R²As defined in any one of claims 1 to 6.
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 is¹Selected from 2-hydroxyprop-2-yl, 2-methoxyprop-2-yl and 1-cyanocyclopropyl-1-yl;

R²selected from 8-membered fused heterocyclic groups, said 8-membered fused heterocyclic group being substituted by a C_1-6Alkyl substitution; preferably, R²Is 5-methyl hexahydropyrrole [3,4-c]Pyrrol-2 (1H) -yl; or R²Is selected from
R⁵、R⁶Each independently selected from C_1-6An alkyl group; preferably, R²Is 4-dimethylaminopiperidin-1-yl.
A compound according to any one of claims 1 to 9, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite or prodrug thereof, said compound having the structure:
a pharmaceutical composition comprising a prophylactically or therapeutically effective amount of a compound according to any one of claims 1-10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite, or prodrug thereof, and one or more pharmaceutically acceptable carriers.
A pharmaceutical formulation comprising a compound of any one of claims 1-10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite, or prodrug thereof, or a pharmaceutical composition of claim 11; preferably, it is administered by oral, intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular or transdermal routes.
Use of a compound of any one of claims 1-10, or a pharmaceutically acceptable salt, stereoisomer, tautomer, polymorph, solvate, metabolite, or prodrug thereof, a pharmaceutical composition of claim 11, or a pharmaceutical formulation of claim 12, for the manufacture of a medicament for the prevention or treatment of a disease associated with the Wee1 protein; preferably, the Wee1 protein-related disease is cancer.
The use of 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.
A process for preparing a compound according to any one of claims 1 to 10, comprising the steps of:

wherein Hal^lIs halogen (e.g., F, Cl or Br); hal²Selected from halogen (e.g., Cl, Br or I), boronic acid groups or boronic ester groups; r¹、R²、R³、R⁴And X is as defined in any one of claims 1 to 10;

the reaction conditions for each step were as follows:

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

(2) cyclizing the compound IN-2 to obtain a compound IN-3;

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

(4) oxidizing the compound IN-5 to obtain a compound IN-6;

(5) reacting compound IN-6 with compound IN-7 to obtain the compound of formula I.
A process for preparing the compound of claim 8, comprising the steps of:

wherein Hal¹Is halogen (e.g., F, Cl or Br); hal²Is halogen (e.g., Cl, Br, or I), a boronic acid group, or a boronic ester group; PG is a protecting group selected from benzyl, p-methoxybenzyl, tert-butoxycarbonyl, benzyloxycarbonyl; r¹And R²As defined in claim 8;

the reaction conditions for each step were 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 a compound IN-10;

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

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

(5) oxidizing the compound IN-13 to obtain a compound IN-14;

(6) reacting compound IN-14 with compound IN-15 to give the compound of formula I-2.