RU2839367C1 - Heterocyclic compound of pyrimidin-4(3h)-one, method for production thereof and pharmaceutical use thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a compound of general formula (II) or pharmaceutically acceptable salts, stereoisomers, stable isotopic derivatives and mixtures thereof, which can be used to treat and/or prevent diseases associated with abnormal SHP2 activity. In formula (II), ring A is selected from the group consisting of a phenyl ring and 6-member heteroaryl ring containing one heteroatom selected from N; ring B is selected from the group consisting of 5-member heteroaryl ring fused with ring A, where 5-member heteroaryl ring contains one or two heteroatoms selected from N; and wherein the phenyl ring and the heteroaryl ring are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, C_1-6 alkyl, -OR^a and -NR^aR^b; R¹ is selected from a group consisting of H, D, cyano, C_1-2 alkyl and -NR^aR^b; R² is selected from a group consisting of H and C_1-2 alkyl, where one or more hydrogen atoms of the alkyl are optionally substituted with one or more substitutes selected from the group consisting of D and fluorine; R^4a and R^4b each is independently selected from a group consisting of C_1-6 alkyl, where alkyl is optionally substituted with one or more substitutes selected from the group consisting of D or -NR^aR^b; R^4a and R^4b optionally together with the carbon atom to which the R^4a and R^4b, form C_3-7 carbocyclic ring or 4–7 membered heterocyclic ring containing one oxygen; R^a and R^b each independently represents H. Invention also relates to a compound of general formula (IV), in which X is selected from the group consisting of -O- and -CH₂-; R⁷ is selected from a group consisting of H, D and C_1-2 alkyl, where one or more hydrogen atoms of the alkyl are optionally substituted with one or more substitutes selected from the group consisting of D and fluorine; values A, B, R¹ and R² are defined above. Invention also relates to specific compounds, a pharmaceutical composition containing said compounds and use thereof to produce a medicinal agent.

EFFECT: treatment and/or prevention of diseases associated with abnormal activity of SHP2, and treatment and/or prevention of diseases associated with SHP2, wherein the diseases are cancer selected from leukaemia, Noonan syndrome, leopard syndrome, neuroblastoma, melanoma, lung cancer, breast cancer, oesophagus cancer, colon cancer, head and neck cancer and stomach cancer.

6 cl, 3 tbl, 19 ex

Description

AREA OF TECHNOLOGY

The present invention relates to a novel heterocyclic pyrimidin-4(3H)-one compound for inhibiting or regulating SHP2 or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof, a method for producing the compound or a pharmaceutically acceptable salt thereof, the use of the compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing the compound or a pharmaceutically acceptable salt thereof for producing a medicament for treating and/or preventing SHP2-mediated diseases, in particular cancer, and a method for administering the same.

LEVEL OF TECHNOLOGY

Src homology 2 domain-containing phosphatase 2 (SHP2) is a non-receptor protein tyrosine phosphatase encoded by the non-receptor protein tyrosine phosphatase type 11 (PTPN11) gene. SHP2 contains two Src homology (SH2) domains, one protein tyrosine phosphatase (PTP) domain, and one C-terminal tail. Under normal conditions, SHP2 adopts an autoinhibitory conformation and its N-SH2 binds to PTP, blocking the substrate channel of the catalytic site of PTP, thereby inhibiting PTP activity. When SH2 binds to a diphosphotyrosine peptide (such as IRS-1), the autoinhibitory SH2-PTP interaction is interrupted and the catalytic site of PTP is exposed, allowing SHP2 to be in an active state to catalyze tyrosine dephosphorylation.

SHP2 is widely expressed, which as an oncogene mediates the activation of various oncogenic cell signaling pathways, such as RAS-ERK, PI3K-AKT, and JAK-STAT pathways, and promotes the survival and proliferation of cancer cells. SHP2 can bind and dephosphorylate RAS, enhance RAS-RAF association, and activate downstream cell proliferation signals. SHP2 also mediates compensatory activation pathways after kinase inhibition, such as MEK, leading to drug resistance in tumor therapy (Ruess DA, et al., Nat. Med. 2018, 24, 954-960). Therefore, SHP2 activation is closely associated with the pathogenesis of various diseases, such as leukemia, melanoma, breast cancer, lung cancer, colon cancer, neuroblastoma, and hepatocellular carcinoma.

SHP2 also plays an important role in the PD-1 and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways, which not only inhibits T cell activation but also promotes T cell anergy (Li J, et al., Cancer Res. 2015, 75, 508-518).

Therefore, SHP2 has attracted much attention as an antitumor target. However, the high protein sequence homology of the PTP catalytic site and the high hydrophilicity of the PTP catalytic pocket result in poor selectivity, poor cell permeability, and low bioavailability of the SHP2 catalytic site. The discovery of the allosteric inhibitor SHP099 by Novartis (Chen Y, et al., Nature 2016, 535, 148-52) provides a new pipeline for the development of highly specific oral SHP2 inhibitors. At present, several patent applications for SHP2 inhibitors have been disclosed by many companies, including WO2015107495, WO2016203405, WO2018057884, WO2018013597 and WO2017211303. The present invention provides compounds having a structure represented by the general formula (I), and it has been found that compounds having such a structure exhibit an excellent effect of inhibiting SHP2 activity.

ESSENCE OF THE INVENTION

The present invention relates to a compound represented by the general formula (I) as an SHP2 inhibitor, or its prodrugs, stable isotopic derivatives, pharmaceutically acceptable salts and isomers, as well as mixtures thereof:

Where:

ring A is selected from the group consisting of phenyl and 6-membered heteroaryl, ring B is selected from the group consisting of a 5-membered heteroaryl ring fused to ring A, wherein phenyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7-membered heterocyclyl, 5-6-membered heteroaryl, -OR ^a , -NR ^a R ^b , -C(O)R ^a , -C(O)NR ^a R ^b , -S(O) ₂ R ^a , -S(O) ₂ NR ^a R ^b , -NR ^a S(O) ₂ R ^b and -P(O)(CH ₃ ) ₂ , wherein alkyl, cycloalkyl, heterocyclyl, and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7-membered heterocyclyl, -OR ^a , -NR ^a R ^b , -C(O)R ^a and -C(O)NR ^a R ^b ;

R ¹ is selected from the group consisting of H, D, cyano, C _1-2 alkyl, cyclopropyl, -OR ^a , -NR ^a R ^b and -C(O)NR ^a R ^b , wherein one or more hydrogen atoms of the alkyl and cyclopropyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ² is selected from the group consisting of H, C _1-2 alkyl and cyclopropyl, wherein one or more hydrogen atoms of the alkyl and cyclopropyl are optionally substituted with one or more substituents selected from the group consisting of D, fluorine and hydroxyl;

R ^4a and R ^4b are each independently selected from the group consisting of H, D, halogen, cyano, -OR ^a , -NR ^a R ^b , -C(O)R ^a , -C(O)NR ^a R ^b , C _1-6 alkyl, C _3-6 cycloalkyl, 4-7-membered heterocyclyl, and 5-6-membered heteroaryl, with the proviso that R ^4a and R ^4b cannot simultaneously be selected from the group consisting of cyano, -OR ^a and -NR ^a R ^b , wherein the alkyl, cycloalkyl, heterocyclyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7-membered heterocyclyl, 5-6-membered heteroaryl, -OR ^a , -NR ^a R ^b , -C(O)R ^a and -C(O)NR ^a R ^b ; R ^4a and R ^4b optionally together with the carbon atom to which R ^4a and R ^4b are attached form a C _3-7 carbocyclic ring or a 4-8-membered heterocyclic ring;

R ^5a , R ^5b , R ^6a and R ^6b are each independently selected from the group consisting of H, D, fluoro and methyl;

R ^a and R ^b are each independently selected from the group consisting of H, C _1-6 alkyl, C _3-6 cycloalkyl and 4-7 membered heterocyclyl, wherein alkyl, cycloalkyl and heterocyclyl are optionally substituted with one or more substituents selected from the group consisting of D, fluoro, cyano, oxo, hydroxyl, -OCH ₃ and -NH ₂ .

In an embodiment, the present invention relates to a compound represented by the above general formula (I), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof, which is a compound represented by the general formula (II), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof:

Where:

ring A is selected from the group consisting of phenyl and 6-membered heteroaryl, ring B is selected from the group consisting of a 5-membered heteroaryl ring fused to ring A, wherein phenyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7 membered heterocyclyl, 5-6 membered heteroaryl, -OR ^a , -NR ^a R ^b , -C(O)R ^a , -C(O)NR ^a R ^b , -S(O) ₂ R ^a , -S(O) ₂ NR ^a R ^b and -NR ^a S(O) ₂ R ^b , wherein alkyl, cycloalkyl, heterocyclyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7 membered heterocyclyl, -OR ^a , -NR ^a R ^b , -C(O)R ^a and -C(O)NR ^a R ^b ;

R ¹ is selected from the group consisting of H, D, cyano, C _1-2 alkyl, -OR ^a and -NR ^a R ^b , wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ² is selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ^4a and R ^4b are each independently selected from the group consisting of H, D, halogen, cyano, -OR ^a , -NR ^a R ^b , -C(O)NR ^a R ^b , C 6 alkyl, C 3_6 cycloalkyl, 4-7-membered heterocyclyl, and 5-6-membered heteroaryl, with the proviso that R ^4a and R ^4b cannot simultaneously be selected from the group consisting of cyano, -OR ^a and -NR ^a R ^b , wherein the alkyl, cycloalkyl, heterocyclyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7-membered heterocyclyl, 5-6-membered heteroaryl, -OR ^a , -NR ^a R ^b , -C(O)R ^a and -C(O)NR ^a R ^b ; R ^4a and R ^4b optionally together with the carbon atom to which R ^4a and R ^4b are attached form a C _3-7 carbocyclic ring or a 4-7-membered heterocyclic ring;

R ^a and R ^b are each independently selected from the group consisting of H, C _1-6 alkyl, C _3-6 cycloalkyl and 4-7 membered heterocyclyl, wherein alkyl, cycloalkyl and heterocyclyl are optionally substituted with one or more substituents selected from the group consisting of D, fluoro, cyano, oxo, hydroxyl, -OCH ₃ and -NH ₂ .

In another embodiment, the present invention relates to a compound represented by the above general formula (I), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof, which is a compound represented by the general formula (III), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof:

Where:

ring A is selected from the group consisting of phenyl and 6-membered heteroaryl, ring B is selected from the group consisting of a 5-membered heteroaryl ring fused to ring A, wherein phenyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-6 alkyl, C _3-6 cycloalkyl, 4-7-membered heterocyclyl, 5-6-membered heteroaryl, -OR ^a , -NR ^a R ^b , -C(O)R ^a and -C(O)NR ^a R ^b , wherein alkyl, cycloalkyl, heterocyclyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-2 alkyl, -OR ^a and -NR ^a R ^b ;

X is selected from the group consisting of -O- and -CR ^8a R ^8b -;

R ¹ is selected from the group consisting of H, D, cyano, C _1-2 alkyl and -NR ^a R ^b , wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ² is selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ^7a , R ^7b , R ^8a , R ^8b , R ^9a and R ^9b are each independently selected from the group consisting of H, D, halogen, cyano, C _1-2 alkyl and -OR ^c , wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D, fluoro and hydroxyl, with the proviso that R ^7a and R ^7b , R ^8a and R ^8b and R ^9a and R ^9b optionally cannot simultaneously be selected from the group consisting of -OR ^c ;

R ^a and R ^b are each independently selected from the group consisting of H, C _1-6 alkyl, C _3-6 cycloalkyl and 4-7 membered heterocyclyl, wherein alkyl, cycloalkyl and heterocyclyl are optionally substituted with one or more substituents selected from the group consisting of D, fluoro, cyano, oxo, hydroxyl, -OCH ₃ and -NH ₂ ;

R ^c is selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro.

In another embodiment, the present invention relates to a compound represented by the above general formula (I), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof, which is a compound represented by the general formula (IV), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof:

Where:

ring A is selected from the group consisting of phenyl and 6-membered heteroaryl, ring B is selected from the group consisting of a 5-membered heteroaryl ring fused to ring A, wherein phenyl and heteroaryl are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, oxo, C _1-2 alkyl, -OR ^a and -NR ^a R ^b , wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

X is selected from the group consisting of -O- and -CH ₂ -;

R ¹ is independently selected from the group consisting of H, D, C _1-2 alkyl and -NR ^a R ^b , wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ² is selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ⁷ is selected from the group consisting of H, D and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro;

R ^a and R ^b are each independently selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro.

The present invention further relates to a compound represented by the above general formula (I), wherein the compound is selected from the group consisting of:

or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof.

The compound of the present invention can effectively inhibit the activity of SHP2 with a preferable IC ₅₀ of less than 50 nM. The compound of the present invention has a significant inhibitory effect on the proliferation of NCI-H358 cells with a preferable IC ₅₀ of less than 1000 nM.

In another aspect, the present invention relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises a compound represented by general formula (I), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof, as well as pharmaceutically acceptable carriers and excipients.

The present invention further relates to a pharmaceutical composition, wherein the pharmaceutical composition comprises a compound represented by the general formula (I), or its pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof, and at least one additional drug, wherein the at least one additional drug includes, but is not limited to, chemotherapeutic agents, targeted drugs, DNA synthesis inhibitors, antibody-based drugs, antibody-drug conjugates, antitumor drugs and immunosuppressants.

The present invention further relates to the use of a compound represented by the general formula (I), or its pharmaceutically acceptable salts, prodrugs, stable isotope derivatives and isomers, as well as mixtures thereof, or a pharmaceutical composition for the preparation of a medicament, wherein the medicament is indicated for the treatment or prevention of SHP2-mediated diseases, wherein the diseases include, but are not limited to, leukemia, Noonan syndrome, leopard syndrome, neuroblastoma, lung cancer, breast cancer, colon cancer, esophageal cancer, gastric cancer and head and neck cancer.

According to the present invention, the medicament may be in any dosage form, including, but not limited to, tablets, capsules, solutions, lyophilized preparations and injectable preparations.

The pharmaceutical composition of the present invention can be administered in the form of a dosage unit containing a predetermined dose of the active pharmaceutical ingredient. Depending on the disease to be treated, the route of administration and the age, body weight and condition of the patient, such a unit can contain from 0.5 mg to 1 g, preferably from 1 mg to 700 mg and more preferably from 5 mg to 500 mg of the compound of the present invention. In addition, the pharmaceutical composition can be prepared by methods known in the pharmaceutical field, for example by mixing the active pharmaceutical ingredient with one or more excipients and/or adjuvants.

The pharmaceutical composition of the present invention is suitable for administration by any suitable route, including transoral (including oral or sublingual), transrectal, transnasal, topical (including oral, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) approaches.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise specified, the following terms in the description and claims have the following meanings.

"C _x-y " refers to a range of carbon atom numbers where x and y are both integers, for example, C _3-8 cycloalkyl means a cycloalkyl having 3-8 carbon atoms, namely 3, 4, 5, 6, 7 or 8 carbon atoms. Furthermore, it should be understood that "C _3-8 " further includes any subrange, for example, C _3-7 , C _3-6 , C _4-7 , C _4-6 and C _5-6 .

"Alkyl" refers to a saturated hydrocarbon group, straight or branched chain, having from 1 to 20 carbon atoms, such as 1 to 8, 1 to 6, or 1 to 4 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-amyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, and 2-ethylbutyl.

"Cycloalkyl or carbocyclic ring" refers to a saturated cyclic hydrocarbon substituent containing from 3 to 14 carbon atoms in the ring. Cycloalkyl may be a monocarbocyclic ring, typically containing from 3 to 8, from 3 to 7, or from 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl. Cycloalkyl may also be a fused bi- or tricarbocyclic ring, such as decahydronaphthyl, bicyclo[2.2.2]octane, and spiro[3.3]heptane.

"Heterocyclyl or heterocyclic ring" refers to a saturated or partially unsaturated mono- or polycyclic group having from 3 to 20 ring atoms, such as 3 to 14, 3 to 12, 3 to 10, 3 to 8, 3 to 6, or 5 to 6 ring atoms, wherein one or more are selected from nitrogen, oxygen, or S(O) _m (wherein m is an integer from 0 to 2), excluding -O-O-, -OS-, or -SS- in the ring structure, the remainder being carbon atoms. Preferably there are from 3 to 12, more preferably 3 to 10, even more preferably 4 to 7, even more preferably 4 to 6, and most preferably 5 to 6 ring atoms, wherein 1 to 4, more preferably 1 to 3, and most preferably 1 to 2 are heteroatoms. Non-limiting examples of a monoheterocyclic group include, but are not limited to, pyrrolidinyl, oxetanyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and azetidinyl. A polyheterocyclic group includes a fused, bridged, or spiropolyheterocycle, such as octahydrocyclopentadieno[c]pyrrole, octahydropyrrolo[1,2-a]pyrazine, 3,8-diazabicyclo[3.2.1]octane, 5-azaspiro[2.4]heptane, and 2-oxa-7-azaspiro[3.5]nonane.

"Aryl or aryl ring" refers to a monocyclic or fused polycyclic aromatic group having from 6 to 14 carbon atoms, preferably 6-10 membered, such as phenyl and naphthyl, and most preferably phenyl. The aromatic ring may be fused to a heteroaryl, heterocyclic or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring. Non-limiting examples include, but are not limited to:

"Heteroaryl or heteroaryl ring" refers to a heteroaromatic system having from 5 to 14 ring atoms, of which from 1 to 4 are selected from oxygen, sulfur and nitrogen heteroatoms. Heteroaryl is preferably 5-10 membered and more preferably 5- or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolinyl, isoquinolinyl, indolyl and isoindolyl. The heteroaryl ring can be fused to an aryl, heterocyclic or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring. Non-limiting examples include, but are not limited to:

"Halogen" refers to F, Cl, Br, or I.

"Cyano" refers to -CN.

"Oxo" refers to =O.

"Optionally" means that the event or environment described below may, but need not, occur, and includes instances in which the event or environment does or does not occur. For example, "a heterocyclic group optionally substituted with alkyl" means that alkyl may be present, but need not be present, and this description includes instances in which the heterocyclic group is substituted with alkyl and instances in which the heterocyclic group is not substituted with alkyl.

"Substituted" refers to one or more hydrogen atoms in the group, preferably 5 and more preferably 1 to 3, being independently replaced by the appropriate number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine possible or impossible substitution without much effort (either theoretically or experimentally). For example, amino or hydroxyl with a free hydrogen may be unstable when bonded to carbon atoms having unsaturated bonds (e.g., olefins). Substituents include, but are not limited to, halogen, cyano, nitro, oxo, _-SF5 , _C1-4 alkyl, _C3-7 cycloalkyl, 4-7-membered heterocyclyl, phenyl, and 5-6-membered heteroaryl.

"Isomer" refers to a compound that has the same molecular formula but different bonding properties, or their order, or a different spatial arrangement of its atoms. An isomer that has a different spatial arrangement of its atoms is called a "stereoisomer". Stereoisomers include optical isomer, geometric isomer, and conformer.

The compounds of the present invention may exist in the form of optical isomers. Optical isomers include enantiomers and diastereomers. An enantiomer is one of two stereoisomers that are mirror images of each other but are not superimposable. A racemic mixture or racemate is a mixture that has an equal number of left and right enantiomers of a chiral molecule. Diastereomers are stereoisomers that are not mirror images of each other and are not superimposable. When a compound is a single isomer and has a defined absolute configuration, it is called an "R" or "S" isomer according to the configuration of the substituents on the chiral carbon atom; when its absolute configuration is not defined, it is called a (+) or (-) isomer according to the measured optical rotation value. Methods for preparing and separating optical isomers are known in the art.

The compounds of the present invention may also exist in the form of geometric isomers resulting from the distribution of substituents around carbon-carbon double bonds, carbon-nitrogen double bonds, cycloalkyls, or heterocyclic groups. Substituents around carbon-carbon double bonds or carbon-nitrogen double bonds are designated as being in the Z- or E-configuration, and substituents around cycloalkyl or heterocyclic rings are designated as being in the cis- or trans-configuration.

The compounds of the present invention may further exhibit tautomerism, such as keto-enol tautomerism.

It should be noted that the present invention includes any tautomers or stereoisomers, as well as mixtures thereof, and is not limited to simply any tautomeric or stereoisomeric forms used in the nomenclature of compounds or chemical structural formula.

"Isotopes" include all isotopes of the atoms present in the compounds of the present invention. Isotopes include those atoms that have the same atomic numbers but different masses. Examples of isotopes suitable for inclusion in the compounds of the present invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as, but not limited to, ² H (D), ³ H, ¹³ C, ¹⁴ C, ¹⁵ N, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F and ³⁶ Cl, respectively. Isotopically labeled compounds of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by methods similar to those described in embodiments of the invention, using appropriate isotopically labeled reagents in place of non-isotopically labeled reagents. Such compounds have various potential uses, such as standards and reagents for determining biological activity. In the case of stable isotopes, such compounds can beneficially alter biological, pharmacological or pharmacokinetic properties. Deuterium (D) may be a preferred isotope in the present invention. For example, hydrogen in methyl, methylene or methine may be replaced by deuterium.

The compounds of the present invention can be administered in the form of a prodrug. "Prodrugs" refer to derivatives that can be converted into biologically active compounds under physiological conditions in vivo, such as oxidation, reduction and hydrolysis (any of which can occur in the absence or presence of an enzyme). Examples of prodrugs include compounds of the present invention in which the amino group is acylated, alkylated or phosphorylated, such as eicosanoyl amino, alanylamino and pivaloyloxymethylamino; or the hydroxyl is acylated, alkylated, phosphorylated or converted into a boronate salt, such as acetoxy, palmitoyloxy, pivaloyloxy, succinyloxy, fumaryloxy and alanyloxy; the carboxyl is esterified or amidated; the thiol forms a disulfide bridge with a carrier molecule, such as a peptide, which selectively delivers the drug to the target and/or the cytosol in the cell. Prodrugs can be prepared from the compounds of the present invention according to well-known methods.

"Pharmaceutical salts" or "pharmaceutically acceptable salts" refer to salts prepared from pharmaceutically acceptable bases or acids, including inorganic bases or acids and organic bases or acids. The present invention further includes pharmaceutically acceptable salts of the compounds of the present invention that contain one or more acidic or basic groups. Accordingly, the compounds of the present invention having acidic groups can exist in the form of a salt and are used according to the present invention, for example, in the form of alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of these salts can include sodium salts, potassium salts, calcium salts, magnesium salts or salts formed with ammonia or organic amines, for example, ethylamine, ethanolamine, triethanolamine or amino acids. The compounds of the present invention having basic groups can exist in the form of a salt and are used according to the present invention in the form of salts prepared with inorganic or organic acids. Examples of suitable acids may include hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, trimethylacetic acid, propanedioic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to those skilled in the art. Provided that the compounds of the present invention contain both acidic and basic groups in the molecule, the present invention further includes internal salts or betaine salts in addition to the mentioned salt forms. Each salt can be prepared by conventional methods known to those skilled in the art, for example, by mixing the compound with an organic or inorganic acid or base in a solvent or dispersing agent, or by anion or cation exchange with another salt.

"Pharmaceutical composition" refers to a composition containing one or more compounds described herein, or pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers thereof, as well as mixtures thereof and other components such as pharmaceutically acceptable carriers and excipients. The pharmaceutical composition is intended for easy administration into the body and better absorption of the active pharmaceutical ingredients to exhibit biological activity.

Therefore, when reference is made in this application to “compound(s),” “compound(s) of the present invention,” or “compound(s) of the present invention,” all forms of the compounds, such as their pharmaceutically acceptable salts, prodrugs, stable isotopic derivatives and isomers, as well as mixtures thereof, are included.

As used herein, the term “therapeutically effective dose” refers to a dose of a compound of the present invention that is capable of effectively inhibiting SHP2 function and/or treating or preventing diseases.

In this document, the term "patients" refers to mammals, in particular humans.

Methods of synthesis

The present invention further relates to a method for producing the compounds. The compounds represented by the general formula (I) of the present invention can be produced by the following typical methods and examples, but these methods and examples should not be construed as limiting the scope of the present invention in any way. The compounds of the present invention can be further synthesized by synthesis methods known to those skilled in the art or by using a combination of methods known in the art and the methods of the present invention. The products obtained in each reaction step can be obtained by separation methods known in the art, including but not limited to extraction, filtration, distillation, crystallization and chromatographic separation. The starting materials and chemical reagents used for the synthesis can be synthesized in a conventional manner according to the literature (queries in SciFinder) or purchased.

The heterocyclic compound represented by the general formula (I) of the present invention can be synthesized according to the route shown in Method A: 1) A1 and NH of the piperidine compound A2 undergo a substitution reaction by base catalysis or a condensation reaction in the presence of a condensing agent to obtain an intermediate compound A3; 2) A3 and sulfhydryl of 5,6-fused heteroaryl undergo a Buchwald-Hartwig cross-coupling reaction to form a product A4.

Method A:

The heterocyclic compounds represented by the general formula (I) of the present invention can also be synthesized according to the route shown in Method B: 1) A1 substitution reactions occur with (R)-2-methylpropane-2-sulfinamide undergoes reductive amination to give A5a; 3) A5a and the sulfhydryl from the 5,6-fused heteroaryl undergo a Buchwald-Hartwig cross-coupling reaction to give A6a; 4) A6a is deprotected in acidic medium to give product A4a.

Method B

Example

The starting materials of the present invention were synthesized according to methods known in the art or purchased from chemical companies such as ABCR GmbH&Co. KG, Acros Organics, Aldrich Chemical Company, Accela ChemBio Inc. and Beijing Ouhe Technology Co., Ltd.

The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). For NMR determination, a Bruker Ascend 400 MHz NMR spectrometer was used, the solvents were deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ) or deuterated methanol (CD ₃ OD), etc., the internal standard was tetramethylsilane (TMS), and the chemical shifts were given in units of 10 ^-6 (ppm). For MS determination, an Agilent SQD (ESI) mass spectrometer (Agilent 6120) was used.

For high-performance liquid chromatography (HPLC) determination, an Agilent 1260 DAD High Performance Liquid Chromatograph (Poroshell120 EC-C18 column, 50 × 3.0 mm, 2.7 μm) or a Waters Arc High Performance Liquid Chromatograph (Sunfire C18 column, 150 × 4.6 mm, 5 μm) was used.

Unless otherwise indicated in embodiments of the invention, reactions were carried out at room temperature (20 to 30°C).

Unless otherwise stated in the embodiments of the invention, the reactions were carried out in an argon or nitrogen atmosphere. An argon or nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen cylinder with a volume of about 1 liter.

Hydrogen atmosphere means that the reaction flask is connected to a hydrogen cylinder with a volume of about 1 liter after evacuation and filling with hydrogen (repeated three times).

A CEM Discover-SP Microwave Reactor was used in the microwave reaction.

The reaction processes in the examples were monitored either by an Agilent LC/MS system (1260/6120) or by thin layer chromatography (TLC) using silica gel plates with a thickness of 0.15 to 0.2 mm (GF254, Qingdao Haiyang Chemical Co., Ltd.).

The compounds were purified by column chromatography or TLC, where 200 to 300 mesh silica gel (Qingdao Haiyang Chemical Co., Ltd.) was used for column chromatography, and GF254 silica gel sheets (Qingdao Haiyang Chemical Co., Ltd.) with a thickness of 0.4 to 0.5 mm were used for TLC.

The eluent solvent systems for column chromatography or TLC typically included: a) dichloromethane-methanol system, b) petroleum ether-ethyl acetate system, or those systems shown in the examples. The volume ratio of the solvents was adjusted according to the different polarity of the compounds or further adjusted by adding a small amount of triethylamine or other acidic or basic reagents.

Compounds were alternatively purified using a Waters Automated MS Preparation System (MS detector: SQD2) with a reversed-phase column (XBridge-C18, 19×150 mm, 5 μm) with gradient elution at a flow rate of 20 mL/min in appropriate acetonitrile/water (containing 0.1% trifluoroacetic or formic acid or 0.05% aqueous ammonia) according to the polarity of the compounds. In some examples, 1 N dilute hydrochloric acid was added after purification using the automated preparation system, followed by removal of the solvent under reduced pressure to yield the hydrochloride salts.

The abbreviation DMF refers to N,N-dimethylformamide.

The abbreviation DIPEA stands for N,N-diisopropylethylamine.

The abbreviation DBU refers to 1,8-diazabicyclo[5.4.0]undec-7-ene.

The abbreviation NBS stands for N-bromosuccinimide.

The abbreviation NIS stands for N-iodosuccinimide.

The abbreviation XantPhos refers to 9,9-dimethyl-4,5-bis(diphenylphosphino)xanthene.

The abbreviation Pd ₂ (dba) ₃ refers to tris(dibenzylideneacetone) dipalladium.

Castros reagent is a benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate.

Example 1

(R)-5-((1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one hydrochloride

Stage 1

2-Chloro-5-iodopyrimidin-4(3H)-one (1b)

2,4-Dichloro-5-iodopyrimidine 1a (8.0 g, 29.1 mmol) was dissolved in tetrahydrofuran (THF) (100 mL) and sodium hydroxide solution (1 N, 45 mL) was added at 0 °C; the reaction mixture was warmed up to room temperature and stirred for 16 h. The pH was adjusted to acidic with citric acid and the reaction mixture was extracted with ethyl acetate (60 mL × 3). The organic phases were combined, washed with brine (30 mL × 3), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to give the desired product 1b (6.7 g, solid) in 91% yield.

MS m/z (ESI): 257 [M+1]

Stage 2

2-Chloro-5-iodo-3-methylpyrimidin-4(3H)-one (1c)

1b (5.6 g, 21.7 mmol) was dissolved in THF (100 mL), iodomethane (3.7 g, 26 mmol) and DIPEA (8.4 g, 65.1 mmol) were added, heated to 60 °C and stirred for 16 h. The resulting mixture was poured into water (120 mL) and extracted with ethyl acetate (80 mL × 3). The organic phases were combined, washed with brine (30 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 3/7) to give the desired product 1c (2.2 g, solid) in 37% yield.

MS m/z (ESI): 271 [M+1]

Stage 3

8-(5-Iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-one (1d)

To a solution of 1c (1.0 g, 3.65 mmol) in acetonitrile (25 mL) were added 8-azaspiro[4.5]decan-1-one (hydrochloride, 586 mg, 3.1 mmol) and potassium carbonate (1.51 g, 11 mmol) and the reaction mixture was heated to 80 °C and stirred for 16 h. After cooling to room temperature, the reaction mixture was poured into water (50 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 7/3) to give the desired product 1d (1.2 g, solid) in 85% yield.

MS m/z (ESI): 388 [M+1]

Stage 4

(R)-N-((R)-8-(5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (1e)

(R)-2-methylpropane-2-sulfinamide (750 mg, 6.2 mmol) and tetraethyl titanate (2.82 g, 12.4 mmol) were added to a solution of 1d (1.2 g, 3.1 mmol) in THF (30 mL), and the reaction mixture was heated to 90 °C and stirred for 16 h. After cooling to 0 °C, methanol (10 mL) and lithium borohydride in THF (2.0 M, 1.5 mL, 3 mmol) were added to the mixture and stirred for 1 h. The reaction was quenched with ammonium chloride solution and the resulting mixture was filtered. The filtrate was extracted with ethyl acetate (25 mL × 3); the organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 2/1) to give the desired product 1e (1.4 g, solid) in 93% yield.

MS m/z (ESI): 493 [M+1]

Stage 5

Ethyl 3-((1H-pyrrolo[2,3-b]pyridin-4-yl)thio)propionate (1g)

To a solution of 4-bromo-1H-pyrrolo[2,3-b]pyridine 1f (300 mg, 1.52 mmol) in dioxane (20 mL) were added ethyl 3-mercaptopropanoate (408 mg, 3.04 mmol), DIPEA (588 mg, 4.56 mmol), Pd ₂ (dba) ₃ (140 mg, 0.152 mmol) and XantPhos (132 mg, 0.228 mmol). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 7/3) to give the desired product 1g (200 mg, oil) in 53% yield.

MS m/z (ESI): 251 [M+1]

Stage 6

Sodium 1H-pyrrolo[2,3-6]pyridine-4-thiolate (1h)

Sodium methoxide in methanol (30%, 158 mg, 0.88 mmol) was added to a solution of 1g (200 mg, 0.8 mmol) in methanol (20 mL) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure to give the desired product 1h (100 mg, solid) in 73% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 151 [M+1]

Stage 7

(R)-N-((R)-8-(5-((1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (1i)

1h (55 mg, 0.32 mmol) was dissolved in dioxane (6 ml), 1e (82 mg, 0.16 mmol), DIPEA (128 mg, 0.99 mmol), _Pd2 (dba) ₃ (30 mg, 0.033 mmol) and XantPhos (29 mg, 0.049 mmol) were added. The reaction mixture was heated to 80 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 1i (30 mg, solid) in 37% yield.

MS m/z (ESI): 515 [M+1]

Stage 8

(R)-5-((1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one hydrochloride (1)

1i (30 mg, 0.058 mmol) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative reversed-phase high-performance liquid chromatography (preparative RP-HPLC) to give the desired product 1 (7.1 mg, solid) in 31% yield.

MS m/z (ESI): 411 [M+1]

1H NMR (400 MHz, DMSO-d ₆ ) δ 12.68 (s, 1H), 8.24 - 8.14 (m, 5H), 7.68 (s, 1H), 6.91 - 6.87 (m, 1H), 6.74 (s, 1H), 3.75 - 3.64 (m, 2H), 3.43 (s, 3H), 3.19 - 3.08 (m, 3H), 2.08 - 2.05 (m, 1H), 1.92 - 1.64 (m, 7H), 1.54 - 1.51 (m, 1H), 1.44 - 1.41 (m, 1H).

Example 2

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)thio)pyrimidin-4(3H)-one formate

Stage 1

Ethyl 3-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)thio)propionate (2b)

Pd ₂ (dba) ₃ (87 mg, 0.095 mmol) was added to a mixture of 4-bromo-1-methyl-1H-pyrrolo[2,3-b]pyridine 2a (200 mg, 0.95 mmol), ethyl 3-mercaptopropanoate (255 mg, 1.9 mmol), DIPEA (368 mg, 2.85 mmol), XantPhos (55 mg, 0.095 mmol) and dioxane (10 mL), and the reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature, water (10 mL) was added and extracted with ethyl acetate (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent, the solvent was removed from the filtrate under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 3/7) to give the target product 2b (240 mg, solid) in 96% yield.

MS m/z (ESI): 265 [M+1]

Stage 2

1-Methyl-1H-pyrrolo[2,3-b]pyridine-4-thiol (2c)

Potassium tert-butoxide (204 mg, 1.82 mmol) was added to a solution of compound 2b (240 mg, 0.91 mmol) in THF (5 mL), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was acidified with hydrochloric acid (6 N) to pH = 4 and extracted with ethyl acetate (20 mL). The organic phase was dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the solvent was removed from the filtrate under reduced pressure to give the desired product 2c (130 mg, solid) in 87% yield.

MS m/z (ESI): 165 [M+1]

Stage 3

(R)-2-methyl-N-((R)-8-(1-methyl-5-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (2d)

_Pd2 (dba) ₃ (20 mg, 0.022 mmol) was added to a mixture of 2c (50 mg, 0.24 mmol), 1e (110 mg, 0.22 mmol), DIPEA (85 mg, 0.66 mmol), XantPhos (13 mg, 0.022 mmol) and dioxane (5 mL), and the reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 2d (80 mg, solid) in 68% yield.

MS m/z (ESI): 529 [M+1]

Stage 4

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)thio)pyrimidin-4(3H)-one formate (2)

2d (80 mg, 0.14 mmol) was dissolved in methanol (8 mL), HCl solution in dioxane (4.0 M, 2 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 2 (21.7 mg, solid) in 34% yield.

MS m/z (ESI): 425 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.34 (s, 1H), 8.14 (s, 1H), 8.03 (d, J=5.1 Hz, 1H), 7.51 (d, J=3.5 Hz, 1H), 6.58 (d, J=5.1 Hz, 1H), 6.44 (d, J=3.5 Hz, 1H), 3.80 (s, 3H), 3.66-3.58 (m, 2H), 3.41 (s, 3H), 3.07 (t, J=11.5 Hz, 2H), 2.99 - 2.96 (m, 1H), 2.0 - 1.92 (m, 1H), 1.80-1.68 (m,4H), 1.60 - 1.53 (m, 3H), 1.40 (d, J=12.9 Hz, 1H), 1.33 (d, J=13.1 Hz, 1H).

Example 3

(R)-5-((1H-indazol-4-yl)thio)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one hydrochloride

Stage 1

Ethyl 3-((1H-indazol-4-yl)thio)propionate (3b)

To a solution of 4-bromo-1H-indazole 3a (500 mg, 2.53 mmol) in dioxane (20 mL) were added ethyl 3-mercaptopropanoate (680 mg, 5.06 mmol), DIPEA (979 mg, 7.59 mmol), Pd ₂ (dba) ₃ (231 mg, 0.253 mmol) and XantPhos (219 mg, 0.397 mmol). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 2.6/1) to give the desired product 3b (560 mg, solid) in 89% yield.

MS m/z (ESI): 251 [M+1]

Stage 2

1H-indazole-4-sodium thiolate (3c)

Sodium methoxide in methanol (30%, 443.5 mg, 2.46 mmol) was added to a solution of 3b (560 mg, 2.24 mmol) in methanol (20 mL) and the mixture was stirred at 30 °C for 1 h. The solvent was removed from the reaction mixture under reduced pressure to give the desired product 3c (200 mg, solid) in 51% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 151 [M+1]

Stage 3

(R)-N-((R)-8-(5-((1H-indazol-4-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (3d)

To a solution of 3c (70 mg, 0.41 mmol) in dioxane (6 mL) were added 1e (50 mg, 0.10 mmol), DIPEA (37.2 mg, 0.30 mmol), _Pd2 (dba) ₃ (9.1 mg, 0.01 mmol) and XantPhos (8.7 mg, 0.015 mmol). The reaction mixture was heated to 80 °C under nitrogen atmosphere and reacted for 4 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 3d (50 mg, solid) in 96% yield.

MS m/z (ESI): 515 [M+1]

Stage 4

(R)-5-((1H-indazol-4-yl)thio)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one hydrochloride (3)

3d (50 mg, 0.097 mmol) was dissolved in methanol (5 mL), HCl solution in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 3 (16 mg, solid) in 41% yield.

MS m/z (ESI): 411 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.14 (brs, 3H), 8.06 (s, 1H), 7.97 (s, 1H), 7.40 (d, J=8.4 Hz, 1H), 7.24 (dd, J=8.3, 7.2 Hz, 1H), 6.83 (d, J=6.8 Hz, 1H), 3.60 (d, J=13.5 Hz, 1H), 3.52 (d,J=13.3 Hz, 1H), 3.40 (s, 3H), 3.17 - 3.12 (m, 1H), 3.05 - 2.98 (m, 2H), 2.07 - 2.04 (m, 1H), 1.77- 1.62 (m, 7H), 1.49 (d, J=12.8 Hz, 1H), 1.39 (d, J=13.1 Hz, 1H).

Example 4

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-(pyrazolo[1,5-a]pyridin-4-ylthio)pyrimidin-4(3H)-one formate

Stage 1

Ethyl 4-bromopyrazolo[1,5-a]pyridine-3-carboxylate (4b)

To a solution of 3-bromopyridine 4a (5.0 g, 32.0 mmol) in acetonitrile (25 mL) was added o-(2,4-dinitrophenyl)hydroxylamine (6.37 g, 32.0 mmol), and the mixture was heated to 40 °C and stirred for 16 h. The solvent was removed from the reaction mixture under reduced pressure, and the residue was suspended in ether and dried to give a yellow solid (3.8 g).

The above product (2.0 g, 5.6 mmol) was dissolved in DMF (20 mL), potassium carbonate (12.1 g, 88.1 mmol) and ethyl propiolate (7.1 g, 52.8 mmol) were added, and the reaction mixture was stirred at room temperature for 20 h. The resulting mixture was poured into water (50 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 3/7) to give the objective product 4b (350 mg, solid) in 8% yield.

MS m/z (ESI): 269 [M+1]

Stage 2

4-Bromopyrazolo[1,5-a]pyridine (4c)

4b (350 mg, 1.30 mmol) was dissolved in hydrobromic acid solution (12 ml), heated to 100 °C and stirred for 6 h. The reaction mixture was cooled to room temperature, neutralized with sodium hydroxide solution (2 N) and extracted with ethyl acetate (20 ml × 3). The organic phases were combined, washed with brine (15 ml × 3), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/4) to give the desired product 4c (200 mg, solid) in 79% yield.

MS m/z (ESI): 197 [M+1]

Stage 3

Ethyl 3-(pyrazolo[1,5-a]pyridin-4-ylthio)propionate (4d)

Ethyl 3-mercaptopropanoate (680 mg, 5.06 mmol), DIPEA (394 mg, 3.06 mmol), _Pd2 (dba) ₃ (93.3 mg, 0.102 mmol) and XantPhos (88.4 mg, 0.153 mmol) were added to a solution of 4c (200 mg, 1.02 mmol) in dioxane (20 mL). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 3/7) to give the desired product 4d (240 mg, solid) in 94% yield.

MS m/z (ESI): 251 [M+1]

Stage 4

Sodium pyrazolo[1,5-a]pyridine-4-thiolate (4e)

Sodium methoxide in methanol (30%, 182 mg, 1.01 mmol) was added to a solution of 4d (230 mg, 0.92 mmol) in methanol (20 mL) and the reaction mixture was stirred at 30 °C for 4 h. The solvent was removed from the reaction mixture under reduced pressure to give the desired product 4e (150 mg, solid) in 95% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 151 [M+1]

Stage 5

(R)-2-methyl-N-((R)-8-(1-methyl-6-oxo-5-(pyrazolo[1,5-a]pyridin-4-ylthio)-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (4f)

1e (70 mg, 0.142 mmol), DIPEA (54.9 mg, 0.426 mmol), _Pd2 (dba) ₃ (12.9 mg, 0.0142 mmol) and XantPhos (12.3 mg, 0.0213 mmol) were added to 4e (85.3 mg, 0.495 mmol) in dioxane (6 mL). The reaction mixture was heated to 80 °C under nitrogen atmosphere and reacted for 4 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/13) to give the desired product 4f (40 mg, solid) in 55% yield.

MS m/z (ESI): 515 [M+1]

Stage 6

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-(pyrazolo[1,5-a]pyridin-4-ylthio)pyrimidin-4(3H)-one formate (4)

4f (40 mg, 0.078 mmol) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 4 (16 mg, solid) in 51% yield.

MS m/z (ESI): 411 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.56 (d, J=6.9 Hz, 1H), 8.39 (s, 1H), 8.09 (s, 1H), 8.03 (d, J=2.2 Hz, 1H), 6.89 (d, J=7.0 Hz, 1H), 6.80 (t, J=7.0 Hz, 1H), 6.63 (d, J=1.7 Hz, 1H), 3.64 - 3.53 (m, 2H), 3.39 (s, 3H), 3.05 - 2.97 (m, 3H), 1.98 - 1.95 (m, 1H), 1.80 - 1.67 (m, 4H), 1.64- 1.53 (m, 3H), 1.40 (d, J=13.0 Hz, 1H), 1.32 (d, J=13.3 Hz, 1H).

Example 5

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-(pyrazolo[1,5-a]pyridin-5-ylthio)pyrimidin-4(3H)-one hydrochloride

Stage 1

Ethyl 3-(pyrazolo[1,5-a]pyridin-5-ylthio)propionate (5b)

Ethyl 3-mercaptopropanoate (326 mg, 2.43 mmol), DIPEA (627 mg, 4.86 mmol), _Pd2 (dba) ₃ (148 mg, 0.162 mmol) and XantPhos (140 mg, 0.243 mmol) were added to a solution of 5-bromopyrazolo[1,5-a]pyridine 5a (320 mg, 1.62 mmol) in dioxane (20 mL). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 6 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 3/1) to give the target product 5b (400 mg, oil) in 97% yield.

MS m/z (ESI): 251 [M+1]

Stage 2

Pyrazolo[1,5-a]pyridine-5-thiol (5c)

Sodium methoxide in methanol (30%, 317 mg, 1.76 mmol) was added to a solution of 5b (400 mg, 1.6 mmol) in methanol (20 mL), and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was poured into water (100 mL), adjusted to slightly acidic pH with citric acid, and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 2/3) to give the desired product 5c (100 mg, oil) in 42% yield.

MS m/z (ESI): 151 [M+1]

Stage 3

(R)-2-methyl-N-((R)-8-(1-methyl-6-oxo-5(pyrazolo[1,5-a]pyridin-5-ylthio)-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)propane-2-sulfinamide (5d)

5c (30 mg, 0.20 mmol) was dissolved in dioxane (6 mL), and 1e (50 mg, 0.10 mmol), DIPEA (38.7 mg, 0.30 mmol), _Pd2 (dba) ₃ (9.0 mg, 0.010 mmol), and XantPhos (8.5 mg, 0.015 mmol) were added. The reaction mixture was heated to 80 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 5d (60 mg, solid, crude product).

MS m/z (ESI): 515 [M+1]

Stage 4

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-(pyrazolo[1,5-a]pyridin-5-ylthio)pyrimidin-4(3H)-one hydrochloride (5)

5d (60 mg, crude product) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 5 (14.1 mg, solid) in 34% yield in two steps.

MS m/z (ESI): 411 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.56 (d, J=7.3 Hz, 1H), 8.13 (s, 1H), 8.06 (brs, 3H), 7.95 (s, 1H), 7.94 (d, J=2.2 Hz, 2H), 7.33 (d, J=1.4 Hz, 1H), 6.68 (dd, J=7.3, 2.1 Hz, 1H), 6.44 (dd, J=2.2, 0.7 Hz, 1H), 3.66 (d, J=13.4 Hz, 1H), 3.58 (d, J=13.3 Hz, 1H), 3.41 (s, 3H), 3.21-3.14 (m, 1H), 3.10-3.02 (m, 2H), 2.07 - 2.03 (m, 1H), 1.87-1.58 (m, 7H), 1.50 (d, J=12.8 Hz, 1H), 1.41 (d, J=12.9 Hz, 1H).

Example 6

(R)-5-((1H-indazol-5-yl)thio)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one hydrochloride

Stage 1

Ethyl 3-((1H-indazol-5-yl)thio)propionate (6b)

_Pd2 (dba) ₃ (243 mg, 0.265 mmol) was added to a mixture of 5-bromo-1H-indazole 6a (520 mg, 2.65 mmol), ethyl 3-mercaptopropanoate (533 mg, 3.98 mmol), DIPEA (1.02 g, 7.95 mmol), XantPhos (153 mg, 0.265 mmol) and dioxane (10 mL), and then the reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/1) to give the objective product 6b (630 mg, solid) in 95% yield.

MS m/z (ESI): 251 [M+1]

Stage 2

1H-Indazole-5-thiol (6c)

To a solution of 6b (500 mg, 2.0 mmol) in THF (10 mL) was added potassium tert-butoxide (672 mg, 6.0 mmol) and the mixture was stirred at room temperature for 2 h. The reaction mixture was acidified with hydrochloric acid (6 N) to pH = 4 and extracted with dichloromethane (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent and the solvent was removed from the filtrate under reduced pressure to give the desired product 6c (300 mg, solid) in 100% yield.

MS m/z (ESI): 151 [M+1]

Stage 3

(R)-N-((R)-8-(5-((1H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (6d)

_Pd2 (dba) ₃ (18 mg, 0.02 mmol) was added to a mixture of 6c (60 mg, 0.4 mmol), 1e (100 mg, 0.2 mmol), DIPEA (77 mg, 0.6 mmol), XantPhos (12 mg, 0.02 mmol) and dioxane (5 mL), and then the reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/1) to give the objective product 6d (70 mg, solid) in 67% yield.

MS m/z (ESI): 515 [M+1]

Stage 4

(R)-5-((1H-indazol-5-yl)thio)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one hydrochloride (6)

6d (70 mg, 0.14 mmol) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 6 (30 mg, solid) in 54% yield.

MS m/z (ESI): 411 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.28 (s, 3H), 8.05 (s, 1H), 7.79 (s, 1H), 7.69 (s, 1H), 7.55 (d, J=8.7 Hz, 1H), 7.34 (dd, J=8.7, 1.6 Hz, 1H), 3.56 (d, J=13.2 Hz, 1H), 3.49 (d, J=13.5 Hz, 1H), 3.39 (s, 3H), 3.17 - 3.13 (m, 1H), 3.06 - 2.96 (m, 2H), 2.07 - 2.01 (m, 1H), 1.90 - 1.61 (m, 7H), 1.52 (d, J=13.1 Hz, 1H), 1.39 (d, J=13.0 Hz, 1H).

Example 7

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one hydrochloride

Stage 1

5-Bromo-4-chloro-2H-indazole (7b)

An aqueous solution (4 mL) of sodium nitrite (396 mg, 5.73 mmol) was added to 4-bromo-3-chloro-2-methylaniline 7a (1.0 g, 4.58 mmol) in acetic acid (20 mL) cooled in an ice bath, and the mixture was stirred at room temperature for 1 h. Most of the solvent was removed on a rotary evaporator, and the residue was suspended in water (30 mL) and filtered. The filter cake was washed with water (25 mL × 3) and air-dried to give the desired product 7b (780 mg, solid) in 74% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 231 [M+1]

Stage 2

5-Bromo-4-chloro-2-methyl-2H-indazole (7c)

Trimethyloxonium tetrafluoroborate (733 mg, 4.95 mmol) was added to 7b (760 mg, 3.3 mmol) in ethyl acetate (15 mL) cooled in an ice bath, and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with petroleum ether (30 mL), stirred for 10 min, and filtered. The filtrate was added to ethyl acetate (20 mL) and washed with sodium bicarbonate solution (30 mL × 3) and brine (20 mL × 3). The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1.4/1) to give the objective product 7c (460 mg, solid) in 57% yield.

MS m/z (ESI): 245 [M+1]

Stage 3

Ethyl 3-((4-chloro-2-methyl-2H-indazol-5-yl)thio)propionate (7d)

Ethyl 3-mercaptopropanoate (483 mg, 3.6 mmol), DIPEA (697 mg, 5.4 mmol), _Pd2 (dba) ₃ (165 mg, 0.18 mmol) and XantPhos (156 mg, 0.27 mmol) were added to a solution of 7c (440 mg, 1.8 mmol) in dioxane (20 mL). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 16 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1.7/1) to give the desired product 7d (300 mg, solid) in 59% yield.

MS m/z (ESI): 299 [M+1]

Stage 4

Sodium 4-Chloro-2-methyl-2H-indazole-5-thiolate (7e)

Sodium methoxide in methanol (30%, 93 mg, 0.52 mmol) was added to a solution of 7d (140 mg, 0.47 mmol) in methanol (20 mL) and stirred at 30 °C for 32 h. The solvent was removed from the reaction mixture under reduced pressure to give the desired product 7e (100 mg, solid) in 96% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 199 [M+1]

Stage 5

(R)-N-((R)-8-(5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (7f)

7e (80 mg, 0.36 mmol) was dissolved in dioxane (8 mL), and 1e (100 mg, 0.20 mmol), DIPEA (77 mg, 0.60 mmol), _Pd2 (dba) ₃ (18 mg, 0.020 mmol), and XantPhos (18 mg, 0.030 mmol) were added. The reaction mixture was heated to 90 °C under nitrogen atmosphere and reacted for 2 h. After cooling to room temperature, the reaction mixture was poured into a mixed solvent of ethyl acetate (15 mL) and petroleum ether (15 mL) and filtered. The filter cake was dried to give the desired product 7f (61 mg, solid) in 54% yield.

MS m/z (ESI): 563 [M+1]

Stage 6

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one hydrochloride (7)

7f (61 mg, 0.11 mmol) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 7 (16 mg, solid) in 31% yield.

MS m/z (ESI): 459 [M+l]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (s, 1H), 8.12 (brs, 3H), 7.90 (s, 1H), 7.51 (dd, J=9.0, 0.7 Hz, 1H), 6.99 (d, J=9.0 Hz, 1H), 4.17 (s, 3H), 3.60 (d, J=13.3 Hz, 1H), 3.53 (d, J=13.3 Hz, 1H), 3.40 (s, 3H), 3.16 - 3.13 (m, 1H), 3.06 - 2.98 (m, 2H), 2.07 - 2.04 (m, 1H), 1.87 - 1.60 (m, 7H), 1.49 (d, J=13.0 Hz, 1H), 1.39 (d, J=13.1 Hz, 1H).

Example 8

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate

Stage 1

tert-Butyl [[(2,4,6-trimethylphenyl)sulfonyl]oxy]carbamate (8b) 2,4,6-Trimethylbenzenesulfonyl chloride 8a (2.0 g, 9.15 mmol) and tert-butyl hydroxycarbamate (1.22 g, 9.15 mmol) were dissolved in tert-butyl methyl ether (30 mL) and cooled to 0 °C, triethylamine (1.4 mL, 10.1 mmol) was added dropwise and the reaction mixture was warmed to room temperature and stirred for 4 h. The reaction mixture was washed with water, the organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/5) to give the target product 8b (2.5 g, solid) in 87% yield.

MS m/z (ESI): 338 [M+23], 216 [M+1-100]

Stage 2

O-(mesitylsulfonyl)hydroxylamine (8c)

8b (2.5 g, 7.93 mmol) was added portionwise into trifluoroacetic acid (20 mL) at 0 °C and the reaction mixture was stirred at 0 °C for 2 h. Ice water was added slowly and the mixture was stirred for 15 min. After filtration, the filter cake was washed with water until the filtrate was neutral. The filter cake was dried to give the desired product 8c (1.5 g, solid) in 88% yield.

MS m/z (ESI): 216 [M+1]

Stage 3

Ethyl 5-bromo-4-chloropyrazolo[1,5-a]pyridine-3-carboxylate (8e)

4-Bromo-3-chloropyridine 8d (1.4 g, 7.28 mmol) was dissolved in 25 mL of acetonitrile, 8 c (1.6 g, 7.28 mmol) was added and the reaction mixture was stirred overnight at 40 °C. After filtration, the filter cake was dried to give a yellow solid (1.7 g).

The above product (1.6 g, 3.92 mmol) and ethyl propiolate (0.42 g, 4.32 mmol) were dissolved in DMF (15 mL), potassium carbonate (1.1 g, 7.84 mmol) was added and stirred at room temperature for 3 h. The reaction was quenched with water and the reaction mixture was extracted with ethyl acetate (100 mL × 3). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/2) to give the desired product 8e (120 mg, solid) in 5.8% yield.

MS m/z (ESI): 303, 305 [M+1]

Stage 4

5-Bromo-4-chloropyrazolo[1,5-a]pyridine (8f)

8e (110 mg, 0.36 mmol) was dissolved in hydrobromic acid solution (3 ml), heated to 100 °C and stirred for 3 h. The reaction mixture was cooled to room temperature, neutralized with sodium hydroxide solution (2 M) and extracted with dichloromethane (30 ml × 3). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 3/7) to give the desired product 8f (65 mg, solid) in 77% yield.

MS m/z (ESI): 231, 233 [M+1]

Stage 5

Ethyl 3-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)propionate (8g)

Ethyl 3-mercaptopropanoate (38 mg, 0.29 mmol), DIPEA (67 mg, 0.52 mmol), _Pd2 (dba) ₃ (24 mg, 0.026 mmol) and XantPhos (30 mg, 0.052 mmol) were added to a solution of 8f (60 mg, 0.26 mmol) in dioxane (5 mL). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 3 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/28) to give the desired product 8g (60 mg, solid) in 81% yield.

MS m/z (ESI): 285 [M+1]

Stage 6

Sodium 4-Chloropyrazolo[1,5-a]pyridine-5-thiolate (8h)

Sodium methoxide in methanol (30%, 45.5 mg, 0.25 mmol) was added to a solution of 8g (60 mg, 0.21 mmol) in methanol (3 mL), and the mixture was stirred at 30 °C for 4 h. The solvent was removed from the reaction mixture under reduced pressure to give the desired product 8h (40 mg, solid) in 92% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 185 [M+1]

Stage 7

(R)-N-((R)-8-(5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (8i)

8h (40 mg, 0.19 mmol) was dissolved in dioxane (5 mL), and 1e (95 mg, 0.19 mmol), DIPEA (49 mg, 0.38 mmol), _Pd2 (dba) ₃ (17 mg, 0.02 mmol), and XantPhos (22 mg, 0.04 mmol) were added. The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 4 h. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/28) to give the desired product 8i (32 mg, oil) in 30% yield.

MS m/z (ESI): 549 [M+1]

Stage 8

(R)-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate (8)

8i (32 mg, 0.06 mmol) was dissolved in methanol (2 mL), HCl in dioxane (4.0 M, 1 mL) was added and stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 8 (13 mg, solid) in 47% yield.

MS m/z (ESI): 445 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.54 (d, J=7.3 Hz, 1H), 8.35 (s, 1H), 8.19 (s, 1H), 8.06 (d, J=2.2 Hz, 1H), 6.60 (d, J=1.8 Hz, 1H), 6.48 (d, J=7.3 Hz, 1H), 3.69-3.60 (m, 2H), 3.41 (s, 3H), 3.09 (t, J=11.7 Hz, 2H), 3.02-2.99 (m, 1H), 2.00-1.91 (m, 1H), 1.76-1.50 (m, 7H), 1.42 (d,J=13.2 Hz, 1H), 1.35 (d, J=12.9 Hz, 1H).

Example 9

(R)-5-((2-(1-amino-8-azaspiro[4.5]decan-8-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-4-chloro-2-methyl-2H-indazole-3-carbonitrile

Stage 1

5-Bromo-4-chloro-2-methyl-2H-indazole-3-carbaldehyde (9a)

7c (800 mg, 3.28 mmol) was dissolved in THF (20 mL), cooled to -78 °C, lithium diisopropylamide in THF (2 M, 2.95 mL, 5.9 mmol) was added, stirred for 90 min, warmed up to 0 °C and stirring was continued for 30 min. Then the reaction mixture was cooled to -78 °C, DMF (0.76 mL) was added dropwise, stirred for 0.5 h, warmed up to room temperature and stirring was continued for 2 h. The reaction was quenched with saturated ammonium chloride solution and the mixture was extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, filtered to remove the drying agent and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=3/7) to give the desired product 9a (520 mg, solid) in 60% yield.

MS m/z (ESI): 273 [M+1]

Stage 2

5-Bromo-4-chloro-2-methyl-2H-indazole-3-carboxaldoxime (9a) 9a (490 mg, 1.8 mmol) was dissolved in isopropanol (8 mL), methanol (8 mL) and water (8 mL), hydroxylamine hydrochloride (497 mg, 7.2 mmol) and sodium carbonate (763 mg, 7.2 mmol) were added, heated to 50 °C and stirred for 16 h. The resulting mixture was poured into water (60 mL) and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=3/7) to give the desired product 9b (470 mg, solid) in 92% yield.

MS m/z (ESI): 288 [M+1]

Stage 3

5-Bromo-4-chloro-2-methyl-2H-indazole-3-carbonitrile (9c)

9b (470 mg, 1.63 mmol) was dissolved in acetonitrile (30 mL), copper acetate (147 mg, 0.81 mmol) was added, heated to 85 °C and stirred overnight. The reaction mixture was removed under reduced pressure, the residue was poured into water (50 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with brine (25 mL × 3), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure to give the desired product 9c (390 mg, solid) in 89% yield.

MS m/z (ESI): 270 [M+1]

Stage 4

Ethyl 3-((4-chloro-3-cyano-2-methyl-2H-indazol-5-yl)thio)propionate (9d) Ethyl 3-mercaptopropanoate (385 mg, 2.8 mmol), DIPEA (542 mg, 4.2 mmol), Pd ₂ (dba) ₃ (128 mg, 0.14 mmol) and XantPhos (121 mg, 0.21 mmol) were added to a solution of 9c (380 mg, 1.4 mmol) in dioxane (5 mL). The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 16 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/1) to give the desired product 9d (350 mg, solid) in 77% yield.

MS m/z (ESI): 324 [M+1]

Stage 5

Sodium 4-Chloro-3-cyano-2-methyl-2H-indazole-5-thiolate (9e)

Sodium methoxide in methanol (30%, 130 mg, 0.72 mmol) was added to a solution of 9d (195 mg, 0.60 mmol) in methanol (20 mL) and stirred at 30 °C for 16 h. The solvent was removed from the reaction mixture under reduced pressure to give the desired product 9e (220 mg, solid, crude product). The product was used directly in the next step without purification.

MS m/z (ESI): 224 [M+1]

Stage 6

(R)-N-((R)-8-(5-((4-chloro-3-cyano-2-methyl-2H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (9f)

9e (89.2 mg, 0.36 mmol) was dissolved in dioxane (6 mL), and 1e (120 mg, 0.24 mmol), DIPEA (92.8 mg, 0.72 mmol), _Pd2 (dba) ₃ (21.9 mg, 0.024 mmol), and XantPhos (20.8 mg, 0.036 mmol) were added. The reaction mixture was heated to 80 °C under nitrogen atmosphere and reacted for 16 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/12) to give the desired product 9f (90 mg, solid) in 63% yield in two steps.

MS m/z (ESI): 588 [M+1]

Stage 7

(R)-5-((2-(1-amino-8-azaspiro[4.5]decan-8-yl)-1-methyl-6-oxo-1,6-dihydropyrimidin-5-yl)thio)-4-chloro-2-methyl-2H-indazole-3-carbonitrile formate (9)

9f (90 mg, 0.15 mmol) was dissolved in methanol (10 mL), HCl in dioxane (4.0 M, 10 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 9 (14 mg, solid) in 19% yield.

MS m/z (ESI): 484 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.14 (s, 1H), 7.79 (brs, 3H), 7.75 (d, J=9.0 Hz, 1H), 7.05 (d, J=9.1 Hz, 1H), 4.34 (s, 3H), 3.69 (d, J=14.1 Hz, 1H), 3.61 (d,J=14 Hz, 1H), 3.41 (s, 3H), 3.24-3.16 (m, 1H), 3.13-3.06 (m, 2H), 2.08-2.04 (m, 1H), 1.80-1.64 (m, 7H), 1.49-1.41 (m, 2H).

Example 10

(S)-2-(4-amino-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one

Stage 1

2-((tert-Butyldimethylsilyl)oxy)ethan-1-ol (10b)

tert-Butyldimethylsilyl chloride (50 g, 334 mmol) was added dropwise to a mixture of ethylene glycol 10a (124 g, 2 mol), imidazole (34 g, 500 mmol) and dichloromethane (500 mL) at 0 °C, and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (300 mL) and dichloromethane (600 mL) and separated. The aqueous phase was extracted with dichloromethane (300 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/5) to give the desired product 10b (35 g, oil) in 10% yield.

MS m/z (ESI): 177 [M+1]

Stage 2

2-((tert-Butyldimethylsilyl)oxy)acetaldehyde (10c)

Oxalyl chloride (27.6 g, 218.9 mmol) was added dropwise into dichloromethane (500 mL) at -30 °C and the mixture was cooled to -78 °C, to which dimethyl sulfoxide (21.7 g, 278.6 mmol) was added dropwise. The reaction mixture was stirred at -78 °C for 30 min, a solution of 10b (35 g, 199 mmol) in dichloromethane (100 mL) was slowly added and stirred for 1 h. Triethylamine (100.5 g, 995 mmol) was added dropwise and the mixture was stirred at -78 °C for 30 min and then at room temperature overnight. The reaction mixture was washed successively with water (300 mL), dilute hydrochloric acid (1N, 400 mL × 2), saturated sodium bicarbonate solution (400 mL) and saturated brine (400 mL). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the desired product 10c (34 g, oil) in 98% yield.

^1H NMR (400 MHz, CDCl ₃ ) δ 9.60 (s, 1H), 4.11 (s, 2H), 0.82 (s, 9H), 0.00 (s, 6H).

Stage 3

1-tert-Butyl 4-ethyl-4-(2-((tert-butyldimethylsilyl)oxy)-1-hydroxyethyl)piperidine-1,4-dicarboxylate (10 d)

1-tert-Butyl 4-ethylpiperidine-1,4-dicarboxylate (50 g, 197 mmol) in THF (100 mL) was added dropwise to a solution of lithium diisopropylamide in THF (2 M, 148 mL, 296 mmol) and THF (400 mL) at -10 °C and the reaction mixture was stirred at 0 °C for 30 min. 10C (34 g, 197 mmol) was added and the reaction mixture was stirred at 0 °C for 1 h and at room temperature for 1 h. A mixture of saturated sodium bicarbonate solution and water (400 mL, 1/4 v/v) was added followed by ethyl acetate (200 mL) and the resulting mixture was separated. The aqueous phase was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 2/3) to give the desired product 10d (75 g, oil) in 88% yield.

MS m/z (ESI): 332 [M+1-100]

Stage 4

tert-Butyl 4-(2-((tert-butyldimethylsilyl)oxy)-1-hydroxyethyl)-4-(hydroxymethyl)piperidine-1-carboxylate (10e)

Lithium borohydride in THF (2 M, 130 mL, 261 mmol) was added to 10d (75 g, 174 mmol) in THF (600 mL) and the mixture was stirred at room temperature for 2 h. After cooling to 0 °C, the reaction was quenched with saturated sodium bicarbonate solution and water (150 mL, 1/2 v/v), the reaction mixture was diluted with ethyl acetate (200 mL) and filtered. The filtrate was separated and the aqueous phase was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the desired product 10e (55 g, oil) in 81% yield.

MS m/z (ESI): 290 [M+1-100]

Stage 5

tert-Butyl 4-(1,2-dihydroxyethyl)-4-(hydroxymethyl)piperidine-1-carboxylate (10f)

Tetrabutylammonium fluoride in THF (1 M, 193 mL, 193 mmol) was added to 10e (55 g, 128.5 mmol) in THF (400 mL) and the mixture was stirred at room temperature for 2 h. The reaction mixture was diluted with saturated sodium bicarbonate solution and water (100 mL, 1/2 v/v), ethyl acetate (200 mL) was added and the two phases were separated. The aqueous phase was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/4 to 1/0) to give the desired product 10f (23 g, oil) in 65% yield.

MS m/z (ESI): 176 [M+1-100]

Stage 6

tert-Butyl 4-hydroxy-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (10g)

Sodium hydride (60%, 11.7 g, 292.6 mmol) was suspended in THF (100 mL), cooled to 0 °C, and 10f (23 g, 83.6 mmol) in THF (100 mL) and p-toluenesulfonyl chloride (16 g, 83.6 mmol) in THF (200 mL) were added dropwise. The reaction mixture was stirred at 0 °C for 2 h. Saturated ammonium chloride solution (50 mL) was slowly added, and the reaction mixture was stirred vigorously until gas evolution ceased. Saturated ammonium chloride solution (100 mL) and brine (100 mL) were added, and the resulting mixture was extracted with ethyl acetate (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 0/1 to 1/0) to give the desired product 10h (8.5 g, oil) in 39% yield.

MS m/z (ESI): 158 [M+1-100]

Stage 7

tert-Butyl 4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (10h) A reaction mixture of 10g (8.5 g, 32.9 mmol), Dess-Martin periodinane (27.4 g, 64.56 mmol) and dichloromethane (200 mL) was stirred at 0 °C for 2 h. Saturated sodium bicarbonate solution and saturated sodium thiosulfate solution (100 mL, 1/1 v/v) were added and the resulting mixture was stirred vigorously. The two phases were separated and the aqueous phase was extracted with dichloromethane (200 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=2/3) to give the target product 10h (6.2 g, oil) in 74% yield.

MS m/z (ESI): 156 [M+1-100]

Stage 8

2-Oxa-8-azaspiro[4.5]decan-4-one (10i)

HCl in dioxane (4 M, 2 mL) was added to a solution of 10h (500 mg, 1.96 mmol) in dichloromethane (8 mL) and the mixture was stirred at room temperature for 1 h. The reaction mixture was removed under reduced pressure to give the desired product 10i (hydrochloride, 370 mg, solid) in 99% yield. The product was used directly in the next step without purification.

MS m/z (ESI): 156 [M+1]

Stage 9

8-(5-Iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-one (10j)

1c (100 mg, 0.37 mmol) was added to a mixture of 10i (hydrochloride, 106 mg, 0.555 mmol), potassium carbonate (153 mg, 1.11 mmol) and acetonitrile (4 ml), and the mixture was heated to 80 °C and stirred for 16 h. The reaction mixture was cooled to room temperature, mixed with water (5 ml) and ethyl acetate (20 ml), and the phases were separated. The aqueous phase was extracted with ethyl acetate (10 ml × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/4) to give the target product 10j (70 mg, solid) in 49% yield.

MS m/z (ESI): 390 [M+l]

Stage 10

(R)-N-((S)-8-(5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (10k)

The reaction mixture of 10j (60 mg, 0.15 mmol), (R)-2-methylpropane-2-sulfinamide (36.3 mg, 0.3 mmol), tetraethyl titanate (137 mg, 0.6 mmol) and THF (5 mL) was heated to 90 °C and stirred for 4 h. The mixture was cooled to 0 °C, methanol (2 mL) and lithium borohydride in THF (2.0 M, 0.15 mL, 0.3 mmol) were added and stirred for 1 h. The reaction mixture was mixed with water (5 mL) and dichloromethane (20 mL) and the two layers were separated. The aqueous phase was extracted with dichloromethane (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the target product 10k (70 mg, solid) in 92% yield.

MS m/z (ESI): 495 [M+l]

Stage 11

(R)-N-((S)-8-(5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (101)

_Pd2 (dba) ₃ (12.8 mg, 0.014 mmol) was added to a mixture of 10k (70 mg, 0.14 mmol), 7e (37.4 mg, 0.17 mmol), DIPEA (54 mg, 0.42 mmol), XantPhos (8.1 mg, 0.014 mmol) and dioxane (4 mL) and the reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 2 h. The reaction mixture was cooled to room temperature, mixed with water (5 mL) and dichloromethane (20 mL) and the two layers were separated. The aqueous phase was extracted with dichloromethane (10 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the target product 101 (50 mg, solid) in 63% yield.

MS m/z (ESI): 565 [M+1]

Stage 12

(S)-2-(4-amino-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one (10)

101 (50 mg, 0.09 mmol) was dissolved in methanol (8 mL), HCl in dioxane (4.0 M, 2 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 10 (5.8 mg, solid) in 14% yield.

MS m/z (ESI): 461 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (s, 1H), 7.91 (s, 1H), 7.51 (d, J=9.0 Hz, 1H), 6.99 (d, J=9.0 Hz, 1H), 4.18 (s, 3H), 4.02-3.95 (m, 1H), 3.69 (d, J=8.5 Hz, 1H), 3.63 (d, J=8.5 Hz, 1H), 3.39 (s, 3H), 3.15-3.13 (m, 1H), 3.08-3.00 (m, 2H), 1.84-1.76 (m, 1H), 1.72-1.65 (m, 1H), 1.54-1.43 (m, 2H).

Example 11

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate

Stage 1

Methyl (S)-2-((tert-butyldimethylsilyl)oxy)propanoate (11b)

Methyl (S)-2-hydroxypropanoate 11a (70 g, 672 mmol) was added dropwise to imidazole (68.5 g, 1008 mmol) in dichloromethane (800 mL) at 0 °C, tert-butyldimethylsilyl chloride (120 g, 807 mmol) was slowly added and the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with water (600 mL) and extracted with dichloromethane (500 mL × 2). The organic phases were combined, washed with dilute hydrochloric acid (1 N, 300 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the desired product 11b (150 g, oil) in 99% yield.

¹ H NMR (400 MHz, CDCl ₃ ) δ 4.32 (q, J=6.7 Hz, 1H), 3.70 (s, 3H), 1.38 (d, J=6.8 Hz, 3H), 0.88 (s, 9H), 0.08 (s, 3H), 0.05 (s, 3H).

Stage 2

(S)-2-((tert-butyldimethylsilyl)oxy)propionaldehyde (11c) 11b (151.2 g, 693 mmol) in dichloromethane (1200 mL) was cooled to -78 °C, diisobutylaluminum hydride in n-hexane (1 M, 832 mL, 832 mmol) was added dropwise and the reaction mixture was warmed up to -40 °C and stirred for 1 h. The reaction mixture was poured into a saturated solution of potassium sodium tartrate (140 mL), mixed with ether (100 mL) and stirred at room temperature for 2 h. The two layers were separated and the aqueous phase was extracted with ether. The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 4/1) to give the target product 11c (62.3 g, oil) in 48% yield.

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.60 (s, 1H), 4.09 (q, J=6.8 Hz, 1H), 1.27 (d, J=6.9 Hz, 3H), 0.91 (s, 9H), 0.10 (s, 3H), 0.08 (s, 3H).

Stage 3

1-tert-Butyl 4-ethyl-4-(2S)-2-((tert-butyldimethylsilyl)oxy)-1-hydroxypropyl)piperidine-1,4-dicarboxylate (11d)

1-tert-Butyl 4-ethylpiperidine-1,4-dicarboxylate (72.8 g, 283.5 mmol) in THF (50 mL) was added dropwise to a solution of lithium diisopropylamide in THF (1 M, 425 mL, 425 mmol) and THF (800 mL) at -50 °C, and the reaction mixture was stirred at -10 °C for 1 h. 11c (53.3 g, 283.5 mmol) was added and stirred at -10 °C for 1 h. The reaction mixture was warmed to 0 °C and stirred for 1 h, and then stirred at room temperature for 1 h. A mixture of saturated sodium bicarbonate solution and water (400 mL, 1/4 v/v) was added, ethyl acetate (500 mL) was added, and the resulting mixture was separated. The aqueous phase was extracted with ethyl acetate (500 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the desired product 11d (120 g, oil, crude). The product was used directly in the next step without purification.

MS m/z (ESI): 346 [M+1-100]

Stage 4

tert-Butyl 4-(2S)-2-((tert-butyldimethylsilyl)oxy)-1-hydroxypropyl)-4-(hydroxymethyl)piperidine-1-carboxylate (11e)

Lithium borohydride in THF (2 M, 270 mL, 539.2 mmol) was added to 11d (120 g, 269.6 mmol) in THF (1200 mL) and the reaction mixture was stirred at room temperature for 16 h. After cooling to 0 °C, the reaction was quenched with saturated sodium bicarbonate solution and water (600 mL, 1/2 v/v), the mixture was diluted with ethyl acetate (800 mL) and filtered. The two layers of the filtrate were separated and the aqueous phase was extracted with ethyl acetate (500 mL x 3). The organic phases were combined, washed with brine (300 mL x 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the desired product 11e (90 g, oil, crude). The product was used directly in the next step without purification.

MS m/z (ESI): 404 [M+1]

Stage 5

tert-Butyl 4-((2S)-1,2-dihydroxypropyl)-4-(hydroxymethyl)piperidine-1-carboxylate (11f)

Tetrabutylammonium fluoride in THF (1 M, 303 mL, 303 mmol) was added to 11e (90 g, 223 mmol) in THF (1300 mL) and the reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with saturated sodium bicarbonate solution and water (800 mL, 1/2 v/v), ethyl acetate (800 mL) was added, the two layers were separated and the aqueous phase was extracted with ethyl acetate (500 mL × 3). The organic phases were combined, washed with brine (300 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel (ethyl acetate/petroleum ether=49/1) to give the desired product 11f (24.5 g, oil) in 26% yield in three steps.

MS m/z (ESI): 290 [M+1]

Stage 6

tert-Butyl (3S)-4-hydroxy-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (11g)

Sodium hydride (60%, 10.8 g, 270.0 mmol) was suspended in THF (600 mL) and cooled to 0 °C. 11f (22.3 g, 77.1 mmol) in THF (40 mL) was added dropwise and p-toluenesulfonyl chloride (14.6 g, 77.1 mmol) in THF (50 mL) was added. The reaction mixture was stirred at 0 °C for 6 h. After cooling to -20 °C, saturated ammonium chloride solution (10 mL) was slowly added to the reaction mixture and vigorously stirred until gas evolution ceased. The mixture was extracted with ethyl acetate (300 mL × 3). The organic phases were combined, washed with brine (150 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/1) to give the target product 11g (12.1 g, oil) in 58% yield.

MS m/z (ESI): 172 [M+1-100]

Stage 7

tert-Butyl (S)-3-methyl-4-oxo-2-oxa-8-azaspiro[4.5]decane-8-carboxylate (11h) Dess-Martin periodinane (37.8 g, 89.2 mmol) was slowly added to 11g (12.1 g, 44.6 mmol) in dichloromethane (140 mL) at 0 °C and the reaction mixture was stirred at room temperature for 16 h. Saturated sodium bicarbonate solution was added until the solution became neutral and the solution was extracted with dichloromethane (150 mL × 3). The organic phases were combined, washed with brine (80 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=3/7) to give the target product 11h (8.36 g, oil) in 70% yield.

MS m/z (ESI): 170 [M+1-100]

Stage 8

(S)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-one (11i)

11h (1.2 g, 4.46 mmol) was dissolved in HCl in dioxane (4 M, 20 mL) and stirred at room temperature for 2 h. The reaction mixture was removed under reduced pressure to give the desired product 11i (hydrochloride, 800 mg, solid) in 87% yield.

MS m/z (ESI): 170 [M+1]

Stage 9

(S)-8-(5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-one (11j)

11i (hydrochloride, 108 mg, 0.529 mmol) and potassium carbonate (199 mg, 1.44 mmol) were added to 2-chloro-5-iodo-3-methylpyrimidin-4(3H)-one 1c (130 mg, 0.481 mmol) in acetonitrile (20 mL) and the reaction mixture was heated to 80 °C and stirred for 16 h. The reaction mixture was cooled to room temperature, mixed with water (50 mL) and extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=1.3/1) to give the target product 11j (180 mg, solid) in 92% yield.

MS m/z (ESI): 404 [M+1]

Stage 10

(R)-N-((3S,4S)-8-(5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (11k)

(R)-2-methylpropane-2-sulfinamide (108 mg, 0.892 mmol) and tetraethyl titanate (813 mg, 3.59 mmol) were added to a solution of 11j (180 mg, 0.446 mmol) in THF (2 mL), heated to 90 °C and stirred for 16 h. After cooling to 0 °C, methanol (5 mL) and lithium borohydride in THF (2.0 M, 0.23 mL, 0.46 mmol) were added to the mixture and stirred for 1 h. The reaction was quenched with saturated ammonium chloride solution and the resulting mixture was filtered. The filtrate was extracted with ethyl acetate (25 mL × 3). The organic phases were combined, washed with saturated brine (20 mL × 3), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/16) to give the target product 11k (90 mg, solid) in 39% yield.

MS m/z (ESI): 509 [M+1]

Stage 11

(R)-N-((3S,4S)-8-(5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropane-2-sulfinamide (11l)

7e (60 mg, 0.27 mmol), DIPEA (60.7 mg, 0.471 mmol), XantPhos (14.2 mg, 0.023 mmol) and _Pd2 (dba) ₃ (14.4 mg, 0.0157 mmol) were added to 11k (80 mg, 0.157 mmol) in dioxane (5 mL). The reaction mixture was heated to 80 °C under nitrogen atmosphere and reacted for 4 h. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/13) to give the desired product 11l (55 mg, solid) in 60% yield.

MS m/z (ESI): 579 [M+1]

Stage 12

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate (11)

11l (55 mg, 0.095 mmol) was dissolved in methanol (5 ml), mixed with HCl in dioxane (4.0 M, 5 ml) and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 11 (16.8 mg, solid) in 37% yield.

MS m/z (ESI): 475 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (s, 1H), 8.21 (s, 1H), 7.91 (s, 1H), 7.51 (d, J=9.1 Hz, 1H), 6.99 (d, J=9.0 Hz, 1H), 4.18 (s, 3H), 4.11-4.05 (m, 1H), 3.68 (d,J=8.6 Hz, 1H), 3.51 (d,J=8.6 Hz, 1H), 3.47-3.43 (m, 2H), 3.41 (s, 3H), 3.20-3.03 (m, 2H), 3.00 (d, J=4.9 Hz, 1H), 1.89-1.82 (m, 1H), 1.77-1.72 (m, 1H), 1.62-1.53 (m, 2H), 1.11 (d, J=6.4 Hz, 3H).

Example 12

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate

Stage 1

(R)-N-((3S,4S)-8-(5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (12a)

11k (140 mg, 0.28 mmol) was dissolved in dioxane (16 mL), and 8h (60 mg, 0.29 mmol), DIPEA (71.5 mg, 0.56 mmol), _Pd2 (dba) ₃ (25.3 mg, 0.03 mmol), and XantPhos (32 mg, 0.06 mmol) were added. The reaction mixture was heated to 100 °C under nitrogen atmosphere and reacted for 16 h. The reaction mixture was cooled to room temperature, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/25) to give the desired product 12a (100 mg, solid) in 64% yield.

MS m/z (ESI): 565 [M+1]

Stage 2

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate (12)

12a (86 mg, 0.166 mmol) was dissolved in methanol (5 mL), mixed with HCl in dioxane (4.0 M, 1 mL) and stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 12 (16.8 mg, solid) in 19% yield.

MS m/z (ESI): 461 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.51 (d, J=7.3 Hz, 1H), 8.26 (s, 1H), 8.17 (s, 1H), 8.04 (d,.7=2.1 Hz, 1H), 6.59 (d, J=1.5 Hz, 1H), 6.48 (d, J=7.3 Hz, 1H), 4.14-4.07 (m, 1H), 3.72 (d,J=8.7 Hz, 1H), 3.57-3.51 (m, 3H), 3.39 (s, 3H), 3.21-3.07 (m, 2H), 3.06 (d, J=4.9 Hz, 1H), 1.88-1.75 (m, 2H), 1.66-1.55 (m, 2H), 1.12 (d, J=6.4 Hz, 3H).

Example 13

(R)-5-((1H-indazol-4-yl)thio)-6-amino-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one formate

Stage 1

6-Amino-5-bromo-3-methylpyrimidine-2,4(1H,3H)-dione (13b)

6-Amino-3-methylpyrimidine-2,4(1H,3H)-dione (1.0 g, 7.08 mmol) was dissolved in DMF (6 mL), NBS (1.38 g, 7.8 mmol) was added and stirred at room temperature for 16 h. The reaction mixture was diluted with water (20 mL) and filtered. The filter cake was washed with water (5 mL × 2) and dried in vacuo to give the desired product 13b (1.0 g, solid) in 64% yield.

MS m/z (ESI): 220 [M+1]

Stage 2

8-(4-Amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-one (13c)

13b (200 mg, 0.91 mmol) was dissolved in DMF (5 mL), 8-azaspiro[4,5]decan-1-one (167 mg, 1.1 mmol), Castros reagent (1.2 g, 2.73 mmol) and DBU (692 mg, 4.55 mmol) were added and stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate (50 mL) and washed with water (10 mL × 3) and brine (10 mL). The organic phase was removed under reduced pressure and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 7/3) to give the desired product 13c (170 mg, oil) in 53% yield.

MS m/z (ESI): 355 [M+1]

Stage 3

(R)-N-((R)-8-(4-amino-5-bromo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (13d)

(R)-2-methylpropane-2-sulfinamide (110 mg, 0.9 mmol) and tetraethyl titanate (411 mg, 1.8 mmol) were added to 13c (160 mg, 0.45 mmol) in THF (15 mL), heated to 90 °C and stirred overnight. After cooling to room temperature, methanol (5 mL) and lithium borohydride in THF (2.0 M, 0.5 mL) were added to the mixture and stirred for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/20) to give the desired product 13d (200 mg, oil) in 96% yield.

MS m/z (ESI): 460 [M+1]

Stage 4

(R)-N-((R)-8-(5-((1H-indazol-4-yl)thio)-4-amino-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-8-azaspiro[4.5]decan-1-yl)-2-methylpropane-2-sulfinamide (13e)

13d (100 mg, 0.22 mmol) was dissolved in dioxane (10 mL) and sodium 1H-indazole-4-thiolate 3c (75 mg, 0.44 mmol), DIPEA (84 mg, 0.66 mmol), Pd ₂ (dba) ₃ (40 mg, 0.044 mmol) and XantPhos (51 mg, 0.088 mmol) were added. The reaction mixture was heated to 100 °C under nitrogen atmosphere and stirred overnight. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by preparative thin layer chromatography to afford the desired product 13e (30 mg, solid) in 26% yield.

MS m/z (ESI): 530 [M+1]

Stage 5

(R)-5-((1H-indazol-4-yl)thio)-6-amino-2-(1-amino-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one formate (13)

13e (30 mg, 0.056 mmol) was dissolved in methanol (2 mL), HCl in dioxane (4.0 M, 2 mL) was added and stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 13 (5 mg, solid) in 21% yield.

MS m/z (ESI): 426 [M+1]

¹ H NMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 8.12 (s, 1H), 7.30 (d, J=8.3 Hz, 1H), 7.20 (t, J=7.7 Hz, 1H), 6.78 (d, J=7.0 Hz, 1H), 3.65-3.57 (m, 2H), 3.42 (s, 3H), 3.27-3.25 (m, 1H), 3.09 (t, J=12.3 Hz, 2H), 2.22-2.17 (m, 1H), 1.85-1.72 (m, 7H), 1.54 (t, J=13.5 Hz, 2H).

Example 14

6-Amino-2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate

Stage 1

(S)-8-(4-amino-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-one (14a)

6-Amino-3-methylpyrimidine-2,4(1H,3H)-dione 13a (400 mg, 2.8 mmol) was dissolved in DMF (25 mL), 11i (576 mg, 2.8 mmol), Castros reagent (6.18 g, 14 mmol) and DBU (1.29 g, 8.5 mmol) were added and stirred at room temperature overnight. The reaction was quenched with water (60 mL) and the reaction mixture was extracted with a mixed solvent of dichloromethane and methanol (10/1 v/v, 50 mL × 3). The organic phases were combined, washed with brine (20 mL × 3), dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane=1/9) to give the target product 14a (700 mg, oil) in 84% yield.

MS m/z (ESI): 293 [M+1]

Stage 2

(R)-N-((3S,4S)-8-(4-amino-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropane-2-sulfinamide (14b)

(R)-2-Methylpropane-2-sulfinamide (557 mg, 4.6 mmol) and tetraethyl titanate (2.06 g, 9.2 mmol) were added to 14a (700 mg, 2.3 mmol) in THF (30 mL) and the reaction mixture was heated to 90 °C and stirred for 16 h. After cooling to 0 °C, methanol (10 mL) and lithium borohydride in THF (2.0 M, 1.35 mL) were added to the mixture and stirred for 1 h. The reaction was quenched with ammonium chloride solution, the mixture was filtered and the filtrate was extracted with ethyl acetate (25 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane=1/10) to give the target product 14b (80 mg, solid) in 8.7% yield.

MS m/z (ESI): 398 [M+1]

Stage 3

(R)-N-((3S,4S)-8-(4-amino-5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (14c)

14b (80 mg, 0.2 mmol) was dissolved in acetonitrile (10 mL), NIS (54.2 mg, 0.24 mmol) was added and stirred at room temperature for 1.5 h. The solvent was removed from the reaction mixture under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/12) to give the target product 14c (100 mg, solid) in 95% yield.

MS m/z (ESI): 524 [M+1]

Stage 4

(R)-N-((3S,4S)-8-(4-amino-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropane-2-sulfinamide (14d)

14c (50 mg, 0.095 mmol) was dissolved in dioxane (6 mL) and 7e (22.8 mg, 0.114 mmol), DIPEA (36.7 mg, 0.285 mmol), _Pd2 (dba) ₃ (8.6 mg, 0.0095 mmol) and XantPhos (8.2 mg, 0.014 mmol) were added. The reaction mixture was heated to 80 °C under nitrogen atmosphere and stirred for 16 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane=1/14) to give the target product 14d (46 mg, solid) in 82% yield.

MS m/z (ESI): 594 [M+1]

Stage 5

6-Amino-2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate (14)

14d (46 mg, 0.077 mmol) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 14 (6.5 mg, solid) in 17% yield. MS m/z (ESI): 490 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.33 (s, 1H), 8.20 (s, 1H), 7.46 (d, J=9.0 Hz, 1H), 6.76 (d, J=9.0 Hz, 1H), 4.15 (s, 3H), 4.10-4.06 (m, 1H), 3.69 (d, J=8.5 Hz, 1H), 3.52 (d, J=8.5 Hz, 1H), 3.39-3.37 (m, 2H), 3.29 (s, 3H), 3.11-2.97 (m, 3H), 1.85-1.71 (m, 2H), 1.63-1.54 (m, 2H), 1.11 (d, J=6.4 Hz, 3N).

Example 15

6-Amino-2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate

Stage 1

(R)-N-((3S,4S)-8-(4-amino-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (15a)

14c (40 mg, 0.076 mmol) was dissolved in dioxane (4 mL) and 8h (31.3 mg, 0.152 mmol), DIPEA (29.4 mg, 0.228 mmol), _Pd2 (dba) ₃ (6.9 mg, 0.0076 mmol) and XantPhos (6.5 mg, 0.0114 mmol) were added. The reaction mixture was heated to 80 °C under nitrogen atmosphere and stirred for 16 h. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with brine. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane=1/17) to give the target product 15a (35 mg, solid) in 79% yield.

MS m/z (ESI): 580 [M+1]

Stage 2

6-Amino-2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloropyrazolo[1,5-a]pyridin-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate (15)

15a (35 mg, 0.06 mmol) was dissolved in methanol (5 mL), HCl in dioxane (4.0 M, 5 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 15 (4.2 mg, solid) in 15% yield.

MS m/z (ESI): 476 [M+1]

¹ H NMR (400 MHz, CD3OD) δ 8.52 (s, 1H), 8.28 (d, J=8.0 Hz, 1H), 7.93 (d, J=2.3 Hz, 1H), 6.55 (d, J=1.7 Hz, 1H), 6.45 (d, J=7.3 Hz, 1H), 4.28-4.23 (m, 1H), 3.89 (d,J=8.9 Hz, 1H), 3.77 (d, J=8.9 Hz, 1H), 3.63-3.56 (m, 2H), 3.43 (s, 3H), 3.24-3.02 (m, 3H), 1.97-1.90 (m, 2H), 1.79-1.69 (m, 2H), 1.26 (d, J=6.5 Hz, 3H).

Example 16

2-(4-(Aminomethyl)-4-methylpiperidin-1-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate

Stage 1

tert-Butyl ((1-benzyl-4-methylpiperidin-4-yl)methyl)carbamate (16b) A reaction mixture of 1-benzyl-4-methylpiperidine-4-carbonitrile 16a (2 g, 9.3 mmol), di-tert-butyl dicarbonate (6.1 g, 27.9 mmol), nickel chloride hexahydrate (2.2 g, 9.3 mmol) and methanol (50 mL) was stirred at room temperature for 15 min. After cooling to 0 °C, the reaction mixture was mixed with sodium borohydride (1.77 g, 46.5 mmol), warmed to room temperature and stirred for 8 h. The solvent was removed from the reaction mixture under reduced pressure and the residue was suspended in dichloromethane and filtered. The solvent was removed from the filtrate under reduced pressure, and the residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether=1/1) to give the target product 16b (1.2 g, oil) in 40% yield.

MS m/z (ESI): 319 [M+1]

Stage 2

tert-Butyl ((4-methylpiperidin-4-yl)methyl)carbamate (16c)

16b (1.2 g, 3.77 mmol) was dissolved in methanol (20 mL), palladium on carbon (10%, water content 55%, 1.2 g) was added and stirred at room temperature for 3 h under hydrogen atmosphere. The reaction mixture was filtered, the filter cake was washed with methanol, the filtrate was concentrated to give the desired product 16c (810 mg, oil) in 94% yield.

MS m/z (ESI): 229 [M+l]

Stage 3

tert-Butyl ((1-(5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-4-methylpiperidin-4-yl)methyl)carbamate (16d)

150 mg (0.55 mmol) of 1c was added to a mixture of 16c (150 mg, 0.66 mmol), potassium carbonate (228 mg, 1.65 mmol) and acetonitrile (10 ml), heated to 90°C and stirred for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the target product 16d (150 mg, solid) in 58% yield.

MS m/z (ESI): 463 [M+1]

Stage 4

tert-Butyl ((1-(5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-4-methylpiperidin-4-yl)methyl)carbamate (16e)

The reaction mixture of 16d (150 mg, 0.32 mmol), 7e (86 mg, 0.39 mmol), DIPEA (124 mg, 0.96 mmol), _Pd2 (dba) ₃ (29 mg, 0.032 mmol), XantPhos (18.5 mg, 0.032 mmol) and dioxane (4 mL) was heated to 90 °C under nitrogen atmosphere and stirred for 3 h. The solvent was removed from the reaction mixture under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 16e (130 mg, solid) in 75% yield.

MS m/z (ESI): 533 [M+1]

Stage 5

2-(4-(Aminomethyl)-4-methylpiperidin-1-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-methylpyrimidin-4(3H)-one formate (16)

16e (130 mg, 0.24 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added and stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 16 (32.4 mg, solid) in 31% yield.

MS m/z (ESI): 433 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (s, 1H), 8.37 (s, 1H), 7.91 (s, 1H), 7.51 (d, J=9.0 Hz, 1H), 6.99 (d, J=9.0 Hz, 1H), 4.18 (s, 3H), 3.40-3.36 (m, 5H), 3.17 (t, J=10.2 Hz, 2H), 2.65 (s, 2H), 1.63-1.57 (m, 2H), 1.42 (d, J=11.1 Hz, 2H), 1.01 (s, 3H).

Example 17

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)thio)pyrimidin-4(3H)-one formate

Stage 1

(R)-2-methyl-N-((3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)propan-2-sulfinamide (17b)

tert-Butyl (3S,4S)-4-((((R)-tert-butylsulfinyl)amino)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate 17a (300 mg, 0.8 mmol) was dissolved in dichloromethane (5 mL), trifluoroacetic acid (1 mL) was added and stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to give the desired product 17b (350 mg, crude product). The product was used directly in the next step without purification.

MS m/z (ESI): 275 [M+1]

Stage 2

(R)-N-((3S,4S)-8-(5-iodo-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (17c)

The reaction mixture of 1c (216 mg, 0.8 mmol), 17b (crude product, 350 mg, 0.8 mmol), potassium carbonate (562 mg, 4.05 mmol) and acetonitrile (10 mL) was heated to 80 °C and stirred for 16 h. The reaction mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the target product 17c (360 mg, solid) in 89% yield.

MS m/z (ESI): 509 [M+1]

Stage 3

(R)-2-methyl-N-((3S,4S)-3-methyl-8-(1-methyl-5-((1-methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-6-oxo-1,6-dihydropyrimidin-2-yl)-2-oxa-8-azaspiro[4.5]decan-4-yl)propan-2-sulfinamide (17d)

The reaction mixture of 17c (180 mg, 0.35 mmol), 2c (70 mg, 0.42 mmol), DIPEA (135 mg, 1.05 mmol), _Pd2 (dba) ₃ (32 mg, 0.035 mmol), XantPhos (20 mg, 0.035 mmol) and dioxane (5 mL) was heated to 90 °C under nitrogen atmosphere and stirred for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 17d (160 mg, solid) in 83% yield.

MS m/z (ESI): 545 [M+1]

Stage 4

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-methyl-5-((1-methyl-1H-pyrrolo[2,3-6]pyridin-4-yl)thio)pyrimidin-4(3H)-one formate (17)

17d (160 mg, 0.29 mmol) was dissolved in methanol (4 mL), HCl in dioxane (4.0 M, 2 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 17 (36 mg, solid) in 28% yield.

MS m/z (ESI): 441 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.22 (s, 1H), 8.14 (s, 1H), 8.03 (d, J=5.1 Hz, 1H), 7.51 (d, J=3.5 Hz, 1H), 6.58 (d, J=5.1 Hz, 1H), 6.44 (d, J=3.5 Hz, 1H), 4.12-4.04 (m, 1H), 3.80 (s, 3H), 3.70 (d, J=8.6 Hz, 1H), 3.53-3.48 (m, 3H), 3.40 (s, 3H), 3.19-3.08 (m, 2H), 3.02 (d, J=4.9 Hz, 1H), 1.88-1.84 (m, 2H), 1.64-1.54 (m, 2H), 1.11 (d, J=6.4 Hz, 3H).

Example 18

5-((1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one formate

Stage 1

1H-pyrrolo[2,3-b]pyridine-4-thiol (18a)

Potassium tert-butoxide (224 mg, 2 mmol) was added to 1 g (250 mg, 1 mmol) in THF (5 mL) and the reaction mixture was stirred at room temperature for 2 h. The reaction was quenched with water and the resulting mixture was washed with ethyl acetate. The aqueous phase was acidified with hydrochloric acid (6 N) to pH = 4 and extracted with ethyl acetate. The organic phase was dried over anhydrous sodium sulfate, the drying agent was removed by filtration, and the filtrate was freed from the solvent under reduced pressure to give the desired product 18a (100 mg, solid) in 67% yield.

MS m/z (ESI): 151 [M+1]

Stage 2

(R)-N-((3S,4S)-8-(5-((1H-pyrrolo[2,3-b]pyridin-4-yl)thio)-1-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)-3-methyl-2-oxa-8-azaspiro[4.5]decan-4-yl)-2-methylpropan-2-sulfinamide (18b)

The reaction mixture of 17c (180 mg, 0.35 mmol), 18a (63 mg, 0.42 mmol), DIPEA (135 mg, 1.05 mmol), _Pd2 (dba) ₃ (32 mg, 0.035 mmol), XantPhos (20 mg, 0.035 mmol) and dioxane (5 mL) was heated to 90 °C under nitrogen atmosphere and stirred for 2 h. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9) to give the desired product 18b (150 mg, solid) in 80% yield.

MS m/z (ESI): 531 [M+1]

Stage 3

5-((1H-pyrrolo[2,3-6]pyridin-4-yl)thio)-2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-3-methylpyrimidin-4(3H)-one formate (18)

18b (150 mg, 0.28 mmol) was dissolved in methanol (4 mL), HCl in dioxane (4.0 M, 2 mL) was added and stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by preparative RP-HPLC to give the desired product 18 (37.9 mg, solid) in 31% yield.

MS m/z (ESI): 427 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.73 (s, 1H), 8.25 (s, 1H), 8.14 (s, 1H), 7.99 (d,J=5.1 Hz, 1H), 7.47-7.45 (m, 1H), 6.54 (d, J=5.1 Hz, 1H), 6.42 (d, J=2.3 Hz, 1H), 4.15-4.05 (m, 1H), 3.71 (d, J=8.5 Hz, 1H), 3.52-3.48 (m, 3H), 3.41 (s, 3H), 3.24-3.05 (m, 3H), 1.89-1.75 (m, 2H), 1.65-1.55 (m, 2H), 1.12 (d, J=6.2 Hz, 3H).

Example 19

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-(methyl-d3)pyrimidin-4(3H)-one

Stage 1

(3S,4S)-3-methyl-2-oxa-8-azaspiro[4.5]decane-4-amine (19b)

tert-Butyl (3S,4S)-4-((((R)-tert-butylsulfinyl)amino)-3-methyl-2-oxa-8-azaspiro[4.5]decane-8-carboxylate 19a (95.0 g, 254 mmol) and HCl in methanol (4 M, 1 L) were stirred at room temperature for 4 h. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in methanol (500 mL), and a mixed solvent of petroleum ether and ethyl acetate (1/1, 4 L) was slowly added thereto. The resulting mixture was stirred at room temperature for 2 h. After filtration, the filter cake was dried to give the desired product 19b (64.1 g, solid, dihydrochloride) in >100% yield.

MS m/z (ESI): 171 [M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.12 (brs, 2H), 8.33 (s, 3H), 4.25-4.19 (m, 1H), 3.82 (d, J=9.1 Hz, 1H), 3.64 (d, J=9.1 Hz, 1H), 3.47 (brs, 1H), 3.27-3.24 (m, 1H), 3.19-3.16 (m, 1H), 2.94-2.85 (m, 2H), 2.03-1.85 (m, 2H), 1.79-1.67 (m, 2H), 1.23 (d, J=6.5 Hz, 3H).

Stage 2

2-Chloro-5-iodo-3-(methyl-d-3)pyrimidin-4(3H)-one (19c)

To 5.0 g (19.5 mmol) of 2-chloro-5-iodopyrimidin-4(3H)-one 1b in THF (100 mL) was added DBU (3.85 g, 25.3 mmol), the resulting mixture was cooled to 0 °C and supplemented with iodomethane-d-3 (3.39 g, 23.4 mmol). The reaction mixture was heated to 50 °C and stirred for 6 h. The solvent was removed from the reaction mixture under reduced pressure. The residue was mixed with ethyl acetate (3.5 L) and washed with dilute hydrochloric acid (0.5 N, 1 L × 2) and brine (800 mL × 2). The organic phase was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 1/19 to 1/4) to give the desired product 19c (3.3 g, solid) in 62% yield.

MS m/z (ESI): 274[M+1]

Stage 3

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-iodo-3-(methyl-d3)pyrimidin-4(3H)-one (19d)

The reaction mixture of 19c (1.8 g, 6.5 mmol), 19b (1.67 g, 6.9 mmol, dihydrochloride), cesium carbonate (9.5 g, 29.3 mmol) and acetonitrile (60 mL) was stirred at room temperature for 3 h under nitrogen atmosphere. The residue was purified by silica gel column chromatography (ethyl acetate/petroleum ether = 9/1) to give the desired product 19d (2.4 g, solid) in 91% yield.

MS m/z (ESI): 408[M+1]

Stage 4

2-((3S,4S)-4-amino-3-methyl-2-oxa-8-azaspiro[4.5]decan-8-yl)-5-((4-chloro-2-methyl-2H-indazol-5-yl)thio)-3-(methyl-d-3)pyrimidin-4(3H)-one (19)

The reaction mixture of 7e (1.9 g, 9.0 mmol), 19d (2.3 g, 5.65 mmol), copper iodide (430 mg, 2.26 mmol), 1,10-phenanthroline (814 mg, 4.52 mmol), potassium phosphate (2.3 g, 11.3 mmol) and dioxane (50 mL) was heated to 100 °C under nitrogen atmosphere and stirred overnight. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (methanol/dichloromethane = 1/9). The resulting crude product (2.5 g) was mixed with acetonitrile (25 ml) and stirred at 90°C for 3 h and at room temperature for 16 h. After filtration, the filter cake was dried in vacuo at 40°C for 4 h to give the desired product 19 (1.47 g, solid) in 54% yield.

MS m/z (ESI): 478[M+1]

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.42 (s, 1H), 7.91 (s, 1H), 7.50 (dd, J=9.0, 0.9 Hz, 1H), 6.98 (d, J=9.0 Hz, 1H), 4.17 (s, 3H), 4.05-4.02 (m, 1H), 3.64 (d, J=8.5 Hz, 1H), 3.47 (d,J=8.5 Hz, 1H), 3.48-3.37 (m, 2H), 3.15-3.05 (m, 2H), 2.90 (d, J=5.1 Hz, 1H), 1.86-1.80 (m, 1H), 1.74-1.68(m, 1H), 1.59-1.50 (m, 2H), 1.38 (brs, 2H), 1.07 (d, J=6.4 Hz, 3H).

Biological experiments

Measurement of SHP2 activity inhibition

The effect of the compound of the present invention on SHP2 activity was assessed by a rapid fluorescence detection method using the surrogate substrate DiFMUP.

The experimental method is described as follows: Recombinant human full-length SHP2 protein was expressed and purified by Tsinghua University Protein Purification and Identification Platform; diphosphopeptide (H2M-LN(pY)IDLDLV-(dPEG8)LST(pY)ASINFQK-amide) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; DiFMUP surrogate substrate was purchased from Thermo Fisher Scientific (Cat. No. D6567); No. D5879) The reaction buffer contained the following components: 60 mM HEPES (pH 7.2), 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% TWEEN 20, and 5 mM DTT.

The compound was dissolved in DMSO (Sigma, Cat. No. D5879) and diluted to 100 μM. The resulting solution was serially diluted 4-fold with DMSO to a minimum concentration of 6.1 nM, and each concentration point was diluted 25-fold with reaction buffer.

10 μl of compound solution and 10 μl of 0.25 nM SHP2 protein solution were added to a 384-well microplate (Corning, Cat. No. 3575), mixed well and incubated at room temperature for 30 min. 10 μl of 0.5 μM diphosphopeptide (dissolved in reaction buffer) were added, mixed well and incubated at room temperature for 30 to 60 min. Then 10 μl of 60 μM DiFMUP solution (dissolved in reaction buffer) were added, mixed well and left to stand at room temperature. After 30 min, fluorescence signals were measured at 340 nm (excitation wavelength)/450 nm (emission wavelength) using a microplate reader (EnSpire, Perkin Elmer). The fluorescence intensity value was positively correlated with the degree of substrate dephosphorylation, which reflected the catalytic activity of SHP2. In the experiment, the group without protein was used as the 100% inhibition group, and the group with protein but no compounds was used as the 0% inhibition group. The compound inhibition curve was plotted by XLfit software, and the inhibitory value IC ₅₀ was calculated. The experimental results are shown in the table below.

Compounds in embodiments of the present invention exhibit inhibitory activity on SHP2 activity with a preferred IC ₅₀ value of less than 50 nM.

NCI-H358 Cell Proliferation Inhibition Assay

The effect of the compound of the present invention on the proliferation of the human non-small cell lung cancer cell line NCI-H358 was assessed using a luminescent cell viability assay.

The experimental method is described as follows:

The compound was dissolved in DMSO (Sigma, Cat. No. D5879) and diluted to 5 mM. The resulting solution was serially diluted 4-fold with DMSO to a minimum concentration of 0.31 μM, and each concentration point was diluted 50-fold with RPMI 1640 medium (Thermo Fisher Scientific, Cat. No. 11995073). When the IC ₅₀ value of the compound was low, the initial concentration of the compound was further reduced.

NCI-H358 cells (ATCC, Cat. No. CRL-5807) were cultured in RPMI 1640 Complete Medium [RPMI 1640 medium was supplemented with 10% FBS (GBICO, Cat. No. 10099-141) and 100 units/mL Penicillin-Streptomycin (Thermo Fisher Scientific, Cat. No. 15140122)]. Cells (15,000 cells/mL) were seeded in 90 μL Complete Medium per 96-well plate. After overnight culture, 10 μL of compound solution was added to each well and cultured at 37°C in a 5% _CO2 incubator for 5 days. According to the instructions of the CellTilter-Glo (CTG) kit (Promega, Cat. No. G7572), the cell culture plate was removed from the incubator and equilibrated to room temperature. The cells were completely lysed with 50 μl of CTG reagent and incubated for 10 min at room temperature. The luminescence signal was read with a microplate reader (EnVision, Perkin Elmer). In the experiment, a group of 10 μM positive control RMC-4550 was used as a negative control (100% inhibition), and a group containing 0.2% DMSO was used as a positive control (0% inhibition). The inhibition curve of the compound was plotted using XLfit software and the inhibitory IC ₅₀ value was calculated. The experimental results are presented in the table below.

Compounds in embodiments of the present invention exhibit an inhibitory effect on cell proliferation with a preferred IC ₅₀ value of less than 1000 nM.

Determination of blockade of the potassium channel hERG

The effect of the compound of the present invention on possible arrhythmia is assessed by determining the blockade of the hERG potassium channel.

The experimental method is described as follows:

Extracellular solution: 140 mM NaCl, 3.5 mM KCl, 1 mM MgCh, 2 mM CaCl ₂ , 10 mM D-glucose, 10 mM HEPES and 1.25 mM NaH ₂ PO ₄ , pH = 7.4.

Electrode solution: 20 mM KCl, 115 mM K-aspartate, 1 mM MgCl ₂ , 5 mM EGTA, 10 mM HEPES and 2 mM Na ₂ -ATP, pH = 7.2.

Compound solution: The test compound was dissolved in DMSO as a 10 mM stock solution, which was diluted with DMSO to 3 mM and then with extracellular solution to a 3 μM solution for subsequent use.

Cell culture: HEK293 cell line stably expressing the hERG potassium channel (Creacell, Cat. No. A-0320) was cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, Cat. No. 11995-065) supplemented with 10% fetal bovine serum (Gibco, Cat. No. 1428478) and 0.8 mg/ml G418 (Amresco, Cat. No. E859-5G) at 37°C and 5% carbon dioxide. The spent medium was discarded and the cell line was washed once with PBS (Gibco, Cat. No. 1009-141). 1 ml TrypLE™ Express (Gibco, Cat. No. 12604021) was added and the cell line was incubated at 37°C for 30 s. When the cells were detached from the bottom of the cell culture dish, 5 ml Complete Medium pre-warmed to 37°C was added, the cell suspension was transferred to a sterile centrifuge tube and centrifuged at 1000 rpm for 5 min to collect the cells. The cells were seeded into 6 cm cell culture dishes, each of which was seeded with 2.5* ¹⁰⁵ cells (final volume: 5 ml). Before the patch clamp experiment, 3* ¹⁰³ cells were spread on a coverslip, cultured in a 24-well plate (final volume: 500 μl) and detected after 18 h.

The voltage stimulation method for recording hERG potassium current in whole cells using a whole-cell patch clamp was as follows: the cell membrane was clamped at -80 mV after the formation of a whole-cell seal. The clamp voltage was depolarized from -80 mV to -50 mV and held for 0.5 s (to detect leak current), increased to 30 mV and held for 2.5 s, quickly restored to -50 mV and held for 4 s to excite the peak tail current of the hERG channel, in which the hERG potassium current was recorded every 10 s. Experimental data were acquired using an EPC-10 USB Patch Clamp Amplifier (HEKA) and stored in PatchMaster software (HEKA v2x73).

Measurement: A glass capillary tube (Sutter Instruments) was converted into a recording electrode using a microelectrode puller (Sutter Instruments). A cover slip with cells taken from a 24-well plate placed in an incubator was placed under an inverted microscope. The electrode solution was poured into the recording electrode and the microelectrode puller (Sutter Instruments) was manipulated to allow the recording electrode to contact the cell surface and provide negative pressure suction to form a GΩ seal. Fast capacitance compensation was provided to continuously apply negative pressure suction until the cell membrane was disrupted, forming the whole-cell recording mode. In the whole-cell recording mode, slow capacitance compensation was performed, recording the membrane capacitance and series resistance, where leakage current compensation was not provided. When the peak hERG tail current in whole-cell recordings was stable for 3-5 min, 8 ml of compound-free extracellular solution (blank control) and 8 ml of 3 μM test compound solution were gravity-perfused through the recording bath sequentially to expose the cells for 5 min (or until the current was stable). The current of each cell measured in compound-free extracellular solution was used as a separate control group. Independently, 2 to 3 cells were measured in duplicate. All electrophysiological experiments were performed at room temperature.

Data analysis: First of all, the current after exposure to the test compound and the idle control current were normalized and the percentage of inhibition corresponding to the concentration of the compound was calculated .

Claims (24)

1. A compound represented by the general formula (II) shown below, or its pharmaceutically acceptable salts, stereoisomers, stable isotopic derivatives, mixtures thereof wherein ring A is selected from the group consisting of a phenyl ring and a 6-membered heteroaryl ring containing one heteroatom selected from N; ring B is selected from the group consisting of a 5-membered heteroaryl ring fused to ring A, wherein the 5-membered heteroaryl ring contains one or two heteroatoms selected from N; and wherein the phenyl ring and the heteroaryl ring are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, C _1-6 alkyl, -OR ^a and -NR ^a R ^b ; R ¹ is selected from the group consisting of H, D, cyano, C _1-2 alkyl and -NR ^a R ^b ; R ² is selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro; R ^4a and R ^4b are each independently selected from the group consisting of C _1-6 alkyl, wherein the alkyl is optionally substituted with one or more substituents selected from the group consisting of D or -NR ^a R ^b ; R ^4a and R ^4b optionally together with the carbon atom to which R ^4a and R ^4b are attached form a C _3-7 carbocyclic ring or a 4-7 membered heterocyclic ring containing one oxygen; R ^a and R ^b each independently represent H. 2. A compound represented by the general formula (IV) shown below, or its pharmaceutically acceptable salts, stable isotopic derivatives, stereoisomers and mixtures thereof wherein ring A is selected from the group consisting of a phenyl ring and a 6-membered heteroaryl ring containing one heteroatom selected from N; ring B is selected from the group consisting of a 5-membered heteroaryl ring fused to ring A, wherein the 5-membered heteroaryl ring contains one or two heteroatoms selected from N; and wherein the phenyl ring and the heteroaryl ring are optionally substituted with one or more substituents selected from the group consisting of D, halogen, cyano, C _1-2 alkyl, -OR ^a and -NR ^a R ^b ; X is selected from the group consisting of -O- and _-CH2- ; R ¹ is selected from the group consisting of H, D, C _1-2 alkyl and -NR ^a R ^b ; R ² is selected from the group consisting of H and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with D and fluorine; R ⁷ is selected from the group consisting of H, D and C _1-2 alkyl, wherein one or more hydrogen atoms of the alkyl are optionally substituted with one or more substituents selected from the group consisting of D and fluoro; R ^a and R ^b each independently represent H. 3. A compound or its pharmaceutically acceptable salts, stable isotopic derivatives, stereoisomers and mixtures thereof, wherein the compound is selected from the group consisting of: 4. A pharmaceutical composition for the treatment and/or prevention of diseases associated with abnormal SHP2 activity, comprising a therapeutically effective dose of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt, stable isotopic derivative, and stereoisomer, and a mixture thereof, and one or more pharmaceutically acceptable carriers and excipients. 5. Use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt, stable isotopic derivative and stereoisomer thereof, as well as a mixture thereof or a pharmaceutical composition according to claim 4 for the preparation of a medicament for the treatment and/or prevention of diseases associated with abnormal SHP2 activity, wherein the diseases are cancer selected from leukemia, Noonan syndrome, leopard syndrome, neuroblastoma, melanoma, lung cancer, breast cancer, esophageal cancer, colon cancer, head and neck cancer and gastric cancer. 6. The use of a compound according to any one of claims 1-3 or a pharmaceutically acceptable salt, stable isotopic derivative and stereoisomer thereof, as well as a mixture thereof or a pharmaceutical composition according to claim 4 for the treatment and/or prevention of diseases associated with SHP2, wherein the diseases are a cancer selected from leukemia, Noonan syndrome, leopard syndrome, neuroblastoma, melanoma, lung cancer, breast cancer, esophageal cancer, colon cancer, head and neck cancer and gastric cancer.