Helicase inhibitor as well as preparation method and application thereof

Abstract

The invention provides a novel small molecule compound with Whara syndrome RecQ DNA helicase inhibitory activity, relates to a compound shown in a formula (II), a stereoisomer, a tautomer or a pharmaceutically acceptable salt thereof, a preparation method of the stereoisomer, the tautomer or the pharmaceutically acceptable salt, and application of the compound, and belongs to the field of medical chemistry.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal chemistry, and particularly relates to a kind of small molecule compounds with Werner syndrome RecQ DNA helicase inhibitory activity, and the purposes of such compounds. BACKGROUND

[0002] Werner syndrome RecQ DNA helicase (WRN) is a RecQ DNA helicase involved in maintaining genome integrity, WRN is composed of 1432 amino acids and has a molecular weight of 160KD. In undamaged human cells, WRN is mainly located in the nucleolus, but after DNA damage, it quickly moves to other nuclear regions. WRN enzyme has helicase and exonuclease activity. Cancer caused by the loss of mismatch repair (MMR) ability has a high mutation load, and deletions and insertions in repetitive DNA sequences are frequent, a phenotype called microsatellite instability (MSI). WRN plays a role in maintaining genome stability in MSI cells, and the loss of its activity causes the accumulation of DNA double-strand breaks in MSI cells, leading to apoptosis, and is a synthetic lethal target in MSI cells.

[0003] In colon cancer, endometrial cancer and gastric cancer, MSI-type tumors have a high incidence, and colorectal cancer is highly prevalent in China. Taking colon cancer as an example, MSI-type tumors account for a relatively high proportion of colon cancer, about 12-15%. Although most patients have sustained response to PD-L1, some patients still do not respond to immunotherapy or eventually develop drug resistance, which is an opportunity for WRN inhibitors. Or can become the preferred drug for the treatment of drug-resistant MSI-H tumors.

[0004] Currently, most WRN inhibitors are in the preclinical research stage, but several companies have disclosed WRN inhibitor patents, such as WO2023062575, WO2022249060, CN118459466, WO2024120378, WO2024010782, WO2024028169, CN118271323. SUMMARY

[0005] The first aspect of the present application relates to a compound represented by formula (II), a stereoisomer, a tautomer thereof or a pharmaceutically acceptable salt thereof,

[0006]

[0007] wherein ring A is a 5-10 membered heterocyclic ring;

[0008] ring B is a 5-10 membered aromatic or heteroaromatic ring;

[0009] R 1 each independently is oxo, -OH, -C1-10 Alkyl, amino, or -CN, wherein the -C 1-10 The alkyl group may optionally be substituted with one or more halogens within the range allowed by the valence;

[0010] R 2 Each independently is -C 1-10 alkyl;

[0011] L 1 For key or -O-;

[0012] L 2 -C(=O)NH-, -C(=O)NH-CHR a -or

[0013] R a -C 1-5 Alkyl or 3-6 membered cycloalkyl;

[0014] Ring C is a 4-6 membered heterocycle, optionally bounded by one or more -C groups within the range allowed by valence. 1-3 Alkyl substitution;

[0015] R 3 -CH=CH-R 4 Or a 3-10 membered heterocyclic alkyl group, wherein the heterocyclic alkyl group is optionally replaced by one or more -CN groups within the range allowed by the valence;

[0016] R 4 For -NO2 or

[0017] X is O or NR c ;

[0018] R b -C 1-5 Alkyl or amino group, wherein the amino group is optionally marked with one or two -C. 1-5 Alkyl groups are substituted;

[0019] R c H or -C 1-5 alkyl;

[0020] m and n are each independently 0, 1, 2, 3 or 4;

[0021] When ring A is n is 1, L 2 When R is -C(=O)NH-, 4 It is not -S(=O)2CH3.

[0022] In some embodiments of the present application, ring A is a 5-8 membered aromatic heterocycle or an unsaturated heterocycle; preferably, the heteroatom of the 5-8 membered aromatic heterocycle or the unsaturated heterocycle is N atom; preferably, the number of the heteroatoms is 1 or 2.

[0023] In some embodiments of the present application, n is 0, 1, 2 or 3, preferably 0, 1 or 2.

[0024] In some embodiments of the present application, ring A is Preferably, ring A is

[0025] In some embodiments of the present application, is

[0026] In some embodiments of the present application, R 1 each independently is oxo, -OH, -C 1-5 alkyl, amino or -CN, the -C 1-5 alkyl is optionally substituted with one or more F within the allowed valence;

[0027] Preferably, R 1 is -CF2CH3, isopropyl, oxo, tert-butyl, amino or -OH.

[0028] In some embodiments of the present application, ring B is a 5-6 membered aromatic ring or an aromatic heterocycle.

[0029] In some embodiments of the present application, m is 0, 1, 2 or 3, preferably 0 or 1.

[0030] In some embodiments of the present application, ring B is a benzene ring or a thiophene ring.

[0031] In some embodiments of the present application, ring B is

[0032] In some embodiments of the present application, is

[0033] In some embodiments of the present application, R 2 each independently is -C 1-5 alkyl, preferably -C 1-3 alkyl, more preferably -CH3.

[0034] In some embodiments of the present application, R a is -C 1-5 alkyl or 3-6 membered cycloalkyl, preferably -C 1-3Alkyl or 3-5 membered cycloalkyl, more preferably -CH3 or cyclopropyl.

[0035] In some embodiments of the invention, the ring C is optionally a plurality of -Cs within a range permitted by one or more valences. 1-3 An alkyl-substituted 5-membered heterocycle; preferably, the 5-membered heterocycle is... More preferably More preferably, the ring C is

[0036] In some embodiments of the present invention, L 2 For -C(=O)NH-,

[0037] In some embodiments of the present invention, R 3 -CH=CH-R 4 Or 5-6 membered heterocyclic alkyl groups, R 4 For -NO2 or The heterocyclic alkyl group is optionally substituted with one or more -CN groups within the range allowed by the valence; preferably, the 5-6 membered heterocyclic alkyl group is piperidinyl; more preferably, the piperidinyl group is substituted with one -CN group; and / or

[0038] R 4 For -NO2 or The R b -C 1-3 Alkyl or amino group, wherein the amino group is optionally marked with one or two -C. 1-3 Alkyl-substituted; preferably, the -C 1-3 The alkyl group is methyl or ethyl; more preferably, R b It is -CH3 or -NHCH3; and / or X is O or NR. c R c X is H, Me, or Et, preferably O or NH; more preferably, R 4 For -NO2, -S(=O)2CH3, -S(=O)2NHCH3 or

[0039] In some embodiments of the present invention, R 3 For -CH=CHNO2, -CH=CHS(=O)2CH3,

[0040] In some embodiments of the present invention, the compound represented by formula (II), its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof have structures as shown in formula (II-1) or (II-2):

[0041]

[0042] wherein E is C or N, ring A is 5-6 membered aromatic heterocycle or unsaturated heterocyclyl, X, R 1 , R 2 , R a , R b , m, n are each independently as described above.

[0043] In some embodiments of the present application, in the structure of formula (II-1) or formula (II-2), ring A is 6-membered aromatic heterocycle or unsaturated heterocyclyl; preferably, ring A is

[0044] In some embodiments of the present application, in the structure of formula (II-1) or formula (II-2), ring A is 5-membered aromatic heterocycle; preferably, ring A is

[0045] In some embodiments of the present application, the compound represented by formula (II), stereoisomer, tautomer thereof or pharmaceutically acceptable salt thereof, includes the following structure:

[0046]

[0047] The second aspect of the present application provides a preparation method of the compound represented by formula (II), stereoisomer, tautomer thereof or pharmaceutically acceptable salt thereof, comprising the steps of scheme one, scheme two, scheme three or scheme four:

[0048] Scheme one: when L 1 is -O-, L 2 is -C(=O)NH-CHR a , R 3 is -CH=CH-R 4

[0049]

[0050] Scheme two: when L 2 is R 3 is -CH=CH-R 4

[0051]

[0052] Scheme three: when L 1 is a bond, L 2 is -C(=O)NH-CHR a , R 3 is -CH=CH-R 4

[0053] ​​​

[0054] Option 4: When L 2 When it is -C(=O)NH-

[0055]

[0056] The definitions of each group are as described above.

[0057] A third aspect of the present invention provides a pharmaceutical composition comprising at least one compound described in the first aspect of the present invention, its stereoisomers, tautomers or pharmaceutically acceptable salts thereof, and optionally one or more pharmaceutically acceptable carriers and / or additives.

[0058] A fourth aspect of the present invention provides the use of a compound of formula (II), its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, or the pharmaceutical composition of the present invention, in the preparation of a medicament for the prevention or treatment of cancer; preferably, the cancer is characterized by high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR); more preferably, the cancer characterized by high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) is selected from colorectal cancer, gastric cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, and ovarian cancer; even more preferably, the cancer characterized by high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) is selected from colorectal cancer, gastric cancer, prostate cancer, and endometrial cancer.

[0059] The fifth aspect of the present invention provides the use of a compound of formula (II), its stereoisomers, tautomers or pharmaceutically acceptable salts thereof or the pharmaceutical composition of the present invention in the preparation of a WRN inhibitor. Detailed Implementation

[0060] Unless otherwise stated, conventional methods within the scope of the art, such as mass spectrometry, NMR, and pharmacological methods, are employed. Unless specifically defined, the terminology used herein in the relevant descriptions of analytical chemistry, organic synthetic chemistry, and pharmaceutical and medicinal chemistry is known in the art. Standard techniques can be used in chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and in the treatment of patients. For example, reactions and purifications can be carried out using the manufacturer's instructions for use of reagent kits, or in accordance with methods known in the art or the descriptions of this invention. The techniques and methods described herein are generally carried out according to conventional methods well known in the art, based on the descriptions in the various summary and more specific literatures cited and discussed herein.

[0061] Unless otherwise stated, the terms used in the specification and claims have the following meanings.

[0062] The term "optionally" includes both "optional" and "not optional". For example, "optionally substituted methyl" means "unsubstituted methyl" or "substituted methyl".

[0063] The term "substitution" refers to the selective replacement of one or more hydrogen atoms on a specified atom or group by a specified group, provided that the replacement does not exceed the normal valence state of the specified atom. When the substituent is an oxo or ketone group (i.e., =O), then two hydrogen atoms on the atom are replaced.

[0064] The term "hydrocarbon group" refers to a group containing only carbon and hydrogen atoms, including saturated or unsaturated aliphatic or aromatic hydrocarbon groups.

[0065] The term "cyclic hydrocarbon group" refers to a cyclic hydrocarbon group, which can be monocyclic or polycyclic. The term "aromatic ring" refers to a cyclic hydrocarbon group that is aromatic.

[0066] The term "alkyl", either on its own or as part of another substituent, refers to an alkyl group having a specified number of carbon atoms (e.g., C10, C20, C30, C40, C50, C60, C70, C80, C9 ... 1-20 This refers to a saturated aliphatic hydrocarbon group (meaning one to twenty carbon atoms), which is an uncyclic straight-chain or branched carbon chain (or carbon), or a combination thereof, preferably an alkyl group containing 1 to 12 carbon atoms (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12), more preferably an alkyl group containing 1 to 6 carbon atoms, and even more preferably an alkyl group containing 1 to 3 carbon atoms. For example, C 1-3 Alkyl refers to methyl, ethyl, n-propyl, and isopropyl.

[0067] The term "alkenyl" refers to a compound containing at least one unsaturated site, i.e., a carbon-carbon sp group. 2 The double-bonded hydrocarbon group can represent a straight-chain and / or branched alkenyl group, where branching refers to one or more alkyl groups such as methyl, ethyl, or propyl attached to the straight-chain alkenyl group. It can be monovalent, divalent, or polyvalent. Unless otherwise specified in the specification, the alkenyl group may optionally be substituted.

[0068] An alkoxy group is an alkyl group that is attached to the rest of the molecule via an oxygen linker (-O-).

[0069] The term "cycloalkyl" refers to a saturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 8 (e.g., 3, 4, 5, 6, 7 and 8) carbon atoms, and even more preferably 3 to 6 carbon atoms.

[0070] The term "heterocycle" refers to a group in which at least one ring atom on a cyclic hydrocarbon group is replaced by a heteroatom (e.g., a nitrogen, oxygen, or sulfur atom). The heterocycle may be saturated, unsaturated, or aromatic. The term "aromatic heterocycle" refers to an aromatic heterocycle. The term "heterocyclic alkyl" is a group in which at least one ring atom on a cyclic alkyl group is replaced by a heteroatom (e.g., a nitrogen, oxygen, or sulfur atom). For example, examples of saturated heterocycles include, but are not limited to, piperidinyl groups. Examples of unsaturated heterocycles include, but are not limited to, […]. Examples of aromatic heterocyclic compounds include, but are not limited to, pyridinyl, pyrimidinyl, or pyridazinyl.

[0071] The term "aryl" includes aromatic hydrocarbon groups with 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracene.

[0072] Unless otherwise specified, the term “halogenated” or “halogen”, either by itself or as part of another substituent, refers to a fluorine, chlorine, bromine, or iodine atom.

[0073] The term "isotope derivative" refers to a derivative of a compound in which one or more atoms are replaced by their isotopes (atoms with the same atomic number but different atomic masses or mass numbers from the dominant atomic mass or mass number found in nature). Examples of isotopes include, but are not limited to, isotopes of hydrogen (e.g., 2 H, 3 H), carbon isotopes (e.g.) 11 C 13 C and 14 C) Isotopes of fluorine (e.g.) 18 F), nitrogen isotopes (e.g.) 13 N and 15 N), isotopes of oxygen (e.g. 15 O、 17 O and 18 O).

[0074] In this invention, unless specifically specified, the term "compound" encompasses its free base, pharmaceutically acceptable salt, stereoisomer, tautomer, isotopic derivative, etc. The compounds of this invention may also exist as hydrates or solvates.

[0075] The terms "pharmaceutical-grade salt" and "pharmaceutically acceptable salt" have the same meaning, referring to salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds in their neutral form with a sufficient amount of the desired base (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts or similar salts. When the compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds in their neutral form with a sufficient amount of the desired acid (pure or in a suitable inert solvent). Examples of pharmaceutically acceptable acid addition salts can be found, for example, Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19. Certain specific compounds of the present invention contain both basic and acidic functional groups, which allows the compounds to be converted into base or acid addition salts.

[0076] "Pharmaceutically acceptable carriers and / or additives" refer to substances that facilitate the administration and absorption of compounds to an individual and can be included in the compositions of the present invention without causing significant adverse toxicity to the patient. Such formulations are sterilizable and, if desired, can be mixed with adjuvants that do not react harmfully with the compounds of the present invention, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring and / or aromatic substances, etc. Those skilled in the art will recognize the usefulness of pharmaceutical carriers and / or diluents in the present invention.

[0077] The term "treatment" refers to any indication of successfully treating or improving an injury, disease, pathology, or symptom, including any objective or subjective parameter such as symptom relief; remission; reduction or making the injury, pathology, or symptom more tolerable for the patient; slowing the rate of degeneration or decline; reducing the degree of weakness at the degenerative endpoint; or improving the patient's physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The term "treatment" and its variations may include prevention of injury, pathology, symptom, or disease. In this implementation plan, treatment is prevention. In this implementation plan, treatment does not include prevention.

[0078] As used herein, “treatment” (and as is known in the art) also broadly includes any method that can achieve a favorable or desired outcome (including clinical outcomes) in an individual’s condition. Favorable or desired clinical outcomes may include, but are not limited to, the reduction or improvement of one or more symptoms or conditions, a reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), prevention of the spread or diffusion of the disease, delay or slowing the progression of the disease, improvement or mitigation of the disease state, reduction of disease recurrence, and remission, whether partial or complete, and whether detectable or undetectable. In other words, as used herein, “treatment” includes any cure, improvement, or prevention of a disease. Treatment can prevent the occurrence of the disease; inhibit the spread of the disease; alleviate the symptoms of the disease; completely or partially eliminate the root cause of the disease; shorten the duration of the disease; or a combination of these events.

[0079] As used herein, “treatment” includes prophylactic treatment. Treatment methods include administering a therapeutically effective amount of the compound described herein to an individual. Administration may include a single dose or a series of doses. The duration of treatment depends on various factors, such as the severity of symptoms, the patient's age, the concentration of the compound, the activity of the composition used for treatment, and their combination. It should also be understood that the effective amount of the agent used for treatment or prevention may increase or decrease as a particular treatment or prevention regimen progresses. Dosage changes can be obtained and become apparent through standard diagnostic analyses known in the art. In some instances, prolonged administration may be required. For example, administering the composition to an individual in a dose and for a duration sufficient to treat the patient.

[0080] The term "prevention" refers to reducing the occurrence of disease symptoms in a patient. As mentioned above, prevention can be complete (no detectable symptoms) or partial prevention, resulting in fewer observed symptoms than would be possible without treatment. In implementation, prevention means slowing the progression of a disease, symptom, or illness, or inhibiting its progression to a harmful or other undesirable state.

[0081] "Patient" or "individual in need" means a living organism that suffers from or is susceptible to a disease or symptom that can be treated by administration of the pharmaceutical compositions provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, dairy cows, deer, and other non-mammalian animals. In some embodiments, the patient is a human.

[0082] "Effective amount" is the amount of a compound sufficient to achieve its intended purpose (e.g., to achieve the effect of administration, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce signaling pathways, or to alleviate one or more symptoms of a disease or condition) relative to the absence of such an effective amount. An example of an "effective amount" is an amount sufficient to promote the treatment, prevention, or relief of one or more symptoms of a disease, also referred to as a "therapeutic effective amount." "Relief" (and its grammatical equivalent) of one or more symptoms means a reduction in the severity or frequency of the symptoms, or the elimination of the symptoms. A "preventive effective amount" of a drug is the amount of the drug that, when administered to an individual, would have the intended preventive effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology, or condition, or reducing the likelihood of the onset (or recurrence) of such an injury, disease, pathology, or condition or its symptoms. A complete preventive effect does not necessarily occur with the administration of a single dose and may only occur after a series of doses. Therefore, a preventive effective amount can be administered over a single or multiple administrations. As used herein, "activity reduction amount" refers to the amount of antagonist required to reduce enzyme activity relative to the absence of an antagonist. As used herein, “functionally disruptive dose” refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of an antagonist. The exact dose will depend on the therapeutic purpose and will be determined by someone skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (Vols. 1–3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). Therapeuticly effective doses can be determined by measuring the relevant physiological effects and can be adjusted in conjunction with individual symptom-specific dosing regimens and diagnostic analyses.

[0083] The term "cancer" refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. This article describes examples of various cancers, including but not limited to colorectal cancer, stomach cancer, endometrial cancer, prostate cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, and ovarian cancer.

[0084] The terms "tumor" and "cancer" are used interchangeably in this invention; for example, both terms include solid and liquid tumors, such as diffuse or circulating tumors. As used in this invention, the terms "cancer" or "tumor" include pre-existing and malignant cancers and tumors.

[0085] The terms “microsatellite unstable cancer,” “high microsatellite unstable cancer,” “high microsatellite cancer,” and “high MSI cancer” and “MSI-H” are used interchangeably in this document to describe cancers with a high number of alterations to simple repetitive genomic sequences within microsatellites.

[0086] As used herein, “WRN inhibitor” or “WRN helicase inhibitor” refers to a compound that inhibits the Werner syndrome RecQ DNA helicase (WRN). As used herein, the term “WRN” refers to the protein of the Werner syndrome RecQ DNA helicase. The term “WRN” includes mutants, fragments, variants, isotypes, and homologs of the full-length wild-type WRN.

[0087] The invention is further illustrated by the following examples, which are illustrative and do not limit the invention in any way. Any modifications or alterations to the invention that are readily achievable by those skilled in the art will fall within the scope of the invention.

[0088] Unless otherwise specified, all raw materials or reagents used in the embodiments of this invention are commercially available.

[0089] The abbreviations used in this invention have their conventional meanings in the art. For example, the meanings of the following abbreviations are as follows:

[0090]

[0091]

[0092] Preparation methods of compounds

[0093] Example 1: Synthesis of N-[(1S,2E)-1-cyclopropyl-3-(methyldioxo-λ6-thio)prop-2-enyl]-2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxamide (1)

[0094]

[0095] Step 1: Synthesis of 2-methyl-2-propyl{[(S)-cyclopropyl(formyl)methyl]amino}carbamate 1b

[0096] Take a single-necked flask, purge with nitrogen three times at room temperature, and successively add (S)-(1-cyclopropyl-2-hydroxyethyl)carbamate tert-butyl ester 1a (2.0 g, 9.9 mmol) dissolved in DCM (20 mL), followed by the addition of Dys-Martin oxidant (8.4 g, 20 mmol). React at 25 °C for 1 h. Add water to the system, extract with dichloromethane, dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the residue by silica gel column chromatography to give compound 1b (1.3 g).

[0097] Step 2: Synthesis of 2-methylpropyl-2-yl{[(1S,2E)-1-cyclopropyl-3-(methyldioxo-λ6-thio)propyl-2-enyl]amino}carbamate 1c

[0098] In a single-necked flask, compound 1b (1.2 g, 6.0 mmol) was dissolved in 5.0 mL of THF. Diethyl(methylsulfonyl)phosphine (1.5 g, 6.6 mmol) and potassium carbonate (2.1 g, 15 mmol) were added, and the mixture was reacted at 60 °C for 3 h. The mixture was poured into ice water and extracted with ethyl acetate. The solution was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by silica gel column chromatography yielded compound 1c (840 mg).

[0099] LCMS(ESI-MS)m / z=175.12[M-Boc]

[0100] The third step involves the synthesis of (1S,2E)-1-cyclopropyl-3-(methyldioxo-λ6-thio)prop-2-en-1-amine 1d.

[0101] Take a single-necked flask and add compound 1c (840 mg, 3.0 mmol) dissolved in acetonitrile (5.0 mL), followed by p-toluenesulfonic acid (690 mg, 3.7 mmol). React at 50 °C for 2 h. Concentrate under reduced pressure to obtain compound 1d (1100 mg).

[0102] LCMS(ESI-MS) m / z = 176.15 [M+H] +

[0103] Step 4: Synthesis of 1-aza-2,2-difluoro-1-propylamine 1f

[0104] Take a three-necked flask and add ammonium chloride (2.3 g, 42 mmol) and toluene (0.050 mL) sequentially. Purge the mixture three times with nitrogen at room temperature. Add 2.0 M trimethylaluminum n-hexane solution (42 mL, 84 mmol) dropwise under ice-water bath conditions. Return to room temperature and react until no more bubbles emerge. Add ethyl 2,2-difluoropropionate 1e (2.0 g, 14 mmol) dropwise and react at 80 °C for 16 h. After 16 h, LC-MS is indistinct, and no obvious spots are observed on TLC. Add methanol dropwise under ice-water bath conditions and react at room temperature for 1 h. Filter with diatomaceous earth, wash with methanol, combine the organic phases, and concentrate under reduced pressure to obtain crude compound 1f (3.1 g).

[0105] LCMS(ESI-MS) m / z = 108.9 [M+H] +

[0106] Step 5: Synthesis of 1 g of 2-(1,1-difluoroethyl)-4-hydroxypyrimidine-5-carboxylic acid ethyl ester

[0107] In a single-necked flask, compound 1f (1.0 g, 9.2 mmol) and anhydrous ethanol (20 mL) were added sequentially. Diethyl ethoxymethylene malonate (2.0 g, 9.2 mmol) was added dropwise under ice-water bath conditions, and the reaction was carried out at 90 °C for 3 h. After 3 h, the reaction was not obvious on LC-MS, but a product peak was visible on MS. The mixture was concentrated under reduced pressure, water was added, the pH was adjusted to 5-6 with citric acid, and the mixture was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 1c (2.3 g).

[0108] LCMS(ESI-MS) m / z = 233.10 [M+H] +

[0109] Step 6: Synthesis of ethyl 4-chloro-2-(1,1-difluoroethyl)pyrimidine-5-carboxylate (1 h)

[0110] Take a single-necked flask and add 1 g (3.0 g, 12 mmol) of compound and 10 mL of phosphorus oxychloride sequentially. React at 80 °C for 2 h. After 2 hours of reaction, concentrate under reduced pressure to obtain compound 1 h (3.5 g), which is used directly for the next step.

[0111] LCMS(ESI-MS) m / z = 251.08 [M+H] +

[0112] Step 7: Synthesis of ethyl 2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxylate 1i

[0113] In a single-necked flask, compound 1h (1.8 g, 7.0 mmol), phenol (1.3 mL, 14 mmol), potassium carbonate (4.8 g, 35 mmol), and acetonitrile (10 mL) were added sequentially, and the mixture was reacted at 80 °C for 5 h. The mixture was concentrated under reduced pressure, water was added, the pH was adjusted to neutral with hydrochloric acid, and the mixture was extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 1i (2.5 g).

[0114] LCMS(ESI-MS) m / z = 309.30 [M+H] +

[0115] Step 8: Synthesis of 2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxylic acid 1j

[0116] In a single-necked flask, compound 1i (1.0 g, 3.2 mmol) was added sequentially, dissolved in tetrahydrofuran (10 mL). Lithium hydroxide (0.23 g, 9.7 mmol) was added at room temperature, followed by water (1.0 mL). The reaction was carried out at 25 °C for 5 h. The system was cooled to room temperature, extracted with ethyl acetate to remove impurities, then the pH was adjusted to 5-6 with 2.0 M hydrochloric acid aqueous solution, extracted with dichloromethane, and dried. The solution was concentrated under reduced pressure to obtain compound 1j (130 mg).

[0117] LCMS(ESI-MS) m / z = 281.22 [M+H] +

[0118] Step 9: Synthesis of N-[(1S,2E)-1-cyclopropyl-3-(methyldioxo-λ6-thio)prop-2-enyl]-2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxamide 1

[0119] In a single-necked flask, compound 1j (14 mg, 0.051 mmol) was added sequentially to a solution of DMF (3.0 mL). Then, DIEA (170 mg, 1.3 mmol) and HATU (240 mg, 0.64 mmol) were added at room temperature, followed by compound 1d (190 mg, 0.56 mmol). The reaction was carried out at 25 °C under a nitrogen atmosphere for 2 h. Saturated brine was added to the system, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The solution was purified by preparative HPLC to obtain compound 1 (52 mg).

[0120] LCMS(ESI-MS) m / z = 438.41 [M+H] +1H NMR(400MHz,Chloroform-d)δ9.46(s,1H),7.77(d,J=7.3Hz,1H),7.54–7.47(m,2H),7.41–7.35(m,1H),7.25–7.21(m,2H),7.02(dd,J=15.2,4.9Hz, 1H),6.60(dd,J=15.2,1.7Hz,1H),4.28-4.20(m,1H),2.96(s,3H),1.85(t ,J=18.5Hz,3H),1.13-1.02(m,1H),0.78–0.63(m,2H),0.54–0.45(m,2H).

[0121] Example 2: N-[(3E)-4-(methyldioxy-λ6-thioalkyl)but-3-en-2-yl]-6-(5-methylthiophen-2-yl)-2-oxo-1H-pyridine-3-carboxamide (2)

[0122]

[0123] Step 1: Synthesis of 2b of 2-methylpropyl-2-yl{[(3E)-4-(methyldioxy-λ6-thioalkyl)but-3-en-2-yl]amino}carbamate

[0124] 2-Methylpropyl-2-yl[(1-formylethyl)amino]carbamate 2a (200 mg, 1.2 mmol), diethyl (methyldioxy-λ6-thioalkyl)methylphosphonate (400 mg, 1.7 mmol), and potassium carbonate (400 mg, 2.9 mmol) were dispersed in anhydrous THF (15 mL). The mixture was stirred at 60 °C for 2 hours. After reacting for 1 hour, the mixture was filtered, and the filtrate was concentrated. The crude product was purified by FLASH column chromatography to give compound 2b (250 mg).

[0125] LCMS (ESI-MS) m / z: 194 [M-55] +

[0126] The second step involves the synthesis of (3E)-4-(methyldioxy-λ6-thioalkyl)but-3-ene-2-amine 2c.

[0127] Compound 2b (250 mg, 1.0 mmol) was dispersed in dichloromethane (6.0 mL), and trifluoroacetic acid (0.77 mL, 10 mmol) was added. The mixture was stirred at room temperature for 1 hour. After 1 hour of reaction, the starting material disappeared (iodine tank) as monitored by TLC (PE:EA = 3:1). The mixture was concentrated, and the crude compound 2c was used directly in the next reaction step.

[0128] Step 3: Synthesis of 2e-6-bromo-2-methoxypyridine-3-carboxylic acid

[0129] 1.0 g (4.1 mmol) of methyl 6-bromo-2-methoxypyridine-3-carboxylate 2d was dispersed in methanol (10 mL), and LiOH (0.85 g, 20 mmol) and water (20 mL) were added. The mixture was stirred at room temperature for 3 hours. After 3 hours of reaction, TLC showed that the starting material disappeared. The mixture was concentrated, acidified with dilute hydrochloric acid, filtered, the filter cake was washed with water, filtered and dried to give compound 2e (910 mg).

[0130] LCMS (ESI-MS) m / z: 232 [M+H] +

[0131] Step 4: Synthesis of 2,3,4,5,6-pentafluorophenyl 6-bromo-2-methoxypyridine-3-carboxylate 2f

[0132] Compound 2e (0.80 g, 3.5 mmol) and 2,2,2-trifluoroacetic acid 2,3,4,5,6-pentafluorophenyl ester (1.9 g, 6.9 mmol) were dispersed in anhydrous pyridine (15 mL) and stirred at 40 °C for 2 hours. After 2 hours of reaction, the mixture was quenched in water, and the compound precipitated. The precipitate was filtered, washed twice with water, and dried. The crude product was purified by FLASH column chromatography to give compound 2f (500 mg).

[0133] LCMS (ESI-MS) m / z: 398 [M+H] +

[0134] Step 5: Synthesis of 2g of 6-bromo-2-methoxy-N-[(3E)-4-(methyldioxy-λ6-thioalkyl)but-3-en-2-yl]pyridine-3-carboxamide

[0135] Compound 2f (400 mg, 1.0 mmol) and DIEA (320 mg, 2.5 mmol) were dispersed in anhydrous DMF (10 mL). Compound 2c (260 mg, 1.0 mmol) was added at room temperature, and the mixture was stirred at room temperature for 2 hours. The mixture was then quenched with water, extracted three times with ethyl acetate, washed twice with water, dried, and filtered. The solution was concentrated. The crude product was purified by FLASH column chromatography to obtain compound 2 g (200 mg).

[0136] LCMS (ESI-MS) m / z: 363.1 [M+H] +

[0137] Step 6: 2-hour synthesis of 2-hydroxy-6-iodo-N-[(3E)-4-(methyldioxy-λ6-thioalkyl)but-3-en-2-yl]pyridine-3-carboxamide

[0138] 2 g (200 mg, 0.55 mmol) of the compound was dispersed in acetonitrile (8.0 mL), and trimethyliodosilane (440 mg, 2.2 mmol) was added at room temperature. The mixture was stirred at room temperature for 2 hours. After 2 hours of reaction, the reaction was monitored by LCMS. The mixture was filtered, and the filter cake was washed twice with methyl tert-butyl ether and dried. Compound 2 h (160 mg) was obtained without further purification.

[0139] LCMS (ESI-MS) m / z: 397.19 [M+H] +

[0140] Step 7: Synthesis of N-[(3E)-4-(methyldioxy-λ6-thioalkyl)but-3-en-2-yl]-6-(5-methylthiophen-2-yl)-2-oxo-1H-pyridine-3-carboxamide 2

[0141] Compound 2h (100 mg, 0.25 mmol), 4,4,5,5-tetramethyl-2-(5-methylthiophen-2-yl)-1,3,2-dioxaborane 2i (85 mg, 0.38 mmol), Pd(dppf)Cl2 (18 mg, 0.025 mmol), and potassium carbonate (69 mg, 0.51 mmol) were dispersed in dioxane (10 L) and water (0.20 mL). The mixture was stirred at 90 °C for 2 hours under nitrogen protection. After 2 hours of reaction, the product was detected by LCMS. The mixture was filtered, and the filtrate was concentrated. The crude product was purified by preparative HPLC to give compound 2 (27 mg).

[0142] LCMS (ESI-MS) m / z: 367.27 [M+H] +

[0143] 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),9.87(s,1H),8.26(d,J=7.7Hz,1H),7.83(d,J= 3.7Hz,1H),6.94(dd,J=4.0,1.3Hz,1H),6.82(dd,J=15.3,4.4Hz,1H),6.70(dd,J=15 .3,1.6Hz,1H), 6.64(s,1H), 4.79(q,J=6.6Hz,1H), 3.00(s,3H), 2.48(s,3H), 1.33(d,J=7.0Hz,3H). Example 3: N-[(3R)-1-cyanohexahydropyridin-3-yl]-2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxamide (3)

[0144]

[0145] The first step was the synthesis of 2-methyl-2-propyl{[(3R)-1-cyanohexahydropyridin-3-yl]amino}carbamate 3b.

[0146] In a single-necked flask, (R)-3-Boc-aminopiperidine 3a (2.0 g, 10 mmol), dichloromethane (33 mL), water (6.0 mL), and sodium bicarbonate (2.5 g, 30 mmol) were added sequentially. Under ice-water bath conditions, 0.89 mL of bromonitrile (12 mmol) was added to dichloromethane (3.0 mL). The reaction was allowed to proceed at room temperature for 3 h. Water was added, followed by extraction with dichloromethane, washing with saturated sodium bicarbonate solution, and the organic phases were combined. The mixture was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Crude compound 3b (2.1 g) was obtained and used directly in the next step.

[0147] LCMS(ESI-MS) m / z = 226.22 [M+H] +

[0148] Step 2: Synthesis of (3R)-3-aminohexahydropyridine-1-carboxynitrile 3c

[0149] Take a single-necked flask and add compound 3b (560 mg, 2.5 mmol) and dioxane hydrochloride (10 mL) sequentially. React at room temperature for 1 h. Discard the supernatant, add dioxane, sonicate to disperse, and discard the supernatant. Add acetonitrile, sonicate to disperse, and discard the supernatant. Lyophilize to obtain compound 3c (300 mg), which can be used directly in the next step.

[0150] Step 3: Synthesis of N-[(3R)-1-cyanohexahydropyridin-3-yl]-2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxamide 3

[0151] In a single-necked flask, compound 1j (54 mg, 0.19 mmol) was added sequentially to a solution of DMF (3.0 mL). Then, DIEA (74 mg, 0.58 mmol) and HATU (110 mg, 0.29 mmol) were added at room temperature. Finally, compound 3c (38 mg, 0.23 mmol) was added. The reaction was carried out at room temperature under nitrogen protection for 2 hours. Saturated brine was added to the system, and the mixture was extracted with ethyl acetate, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The mixture was purified by preparative HPLC (acetonitrile:water, ammonium bicarbonate system) to obtain compound 3 (12 mg).

[0152] LCMS(ESI-MS) m / z = 388.40 [M+H] +1H NMR(400MHz, DMSO-d6)δ8.93(s,1H),8.69(d,J=7.3Hz,1H),7.50–7.44(m,2H),7.34–7.23(m,3H),4.05-3.95(m ,1H),3.42(dd,J=12.4,3.8Hz,1H),3.26–3.20(m,1H),3.15-3.00(m,2H),1.91-1.74(m,5H),1.63–1.51(m,2H).

[0153] Example 4: N-[(1S,2E)-1-cyclopropyl-3-(methyldioxo-λ6-thio)prop-2-enyl]-6-oxo-3-(phenyloxy)-1-(prop-2-yl)-1,2-diazine-4-carboxamide (4)

[0154]

[0155] Synthesis of methyl 6-hydroxy-3-benzyloxy-1,2-diazine-4-carboxylate 4b (Step 1)

[0156] Take a single-necked flask and add 270 mg (1.2 mmol) of 6-hydroxy-3-benzyloxy-1,2-diazine-4-carboxylic acid 4a in 10 mL of dichloromethane. Add 150 mg (1.2 mmol) of oxaloyl chloride and two drops of DMF (0.090 mL, 1.2 mmol) under ice-water bath conditions. React at room temperature for 2 h. Add methanol. Purify by silica gel column chromatography to obtain compound 4b (270 mg).

[0157] LCMS(ESI-MS) m / z = 247.16 [M+H] +

[0158] Step 2: Synthesis of methyl 6-oxo-3-(phenyloxy)-1-(propyl-2-yl)-1,2-diazine-4-carboxylic acid ester 4c

[0159] In a single-necked flask, compound 4b (250 mg, 1.0 mmol), 2-iodopropane (0.15 mL, 1.5 mmol), acetonitrile (5.0 mL), and potassium carbonate (280 mg, 2.0 mmol) were added sequentially, and the mixture was reacted at 80 °C for 6 h. Water was added, the mixture was adjusted to neutral with hydrochloric acid, extracted with dichloromethane, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 4c (290 mg).

[0160] LCMS(ESI-MS) m / z = 289.27 [M+H] +

[0161] Step 3: Synthesis of 6-oxo-3-(phenoxy)-1-(propyl-2-yl)-1,2-diazine-4-carboxylic acid 4d

[0162] In a single-necked flask, compound 4c (290 mg, 1.0 mmol), tetrahydrofuran (6.0 mL), and an aqueous solution of lithium hydroxide (47 mg, 2.0 mmol) (1.5 mL) were added sequentially. The reaction was allowed to proceed at room temperature for 1 h. The system was then adjusted to pH 5 with 2.0 N hydrochloric acid solution, extracted with dichloromethane, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain compound 4d (270 mg).

[0163] LCMS(ESI-MS) m / z = 275.25 [M+H] +

[0164] Step 4: Synthesis of N-[(1S,2E)-1-cyclopropyl-3-(methyldioxo-λ6-thio)prop-2-enyl]-6-oxo-3-(phenyloxy)-1-(prop-2-yl)-1,2-diazine-4-carboxamide 4

[0165] In a single-necked flask, compound 4 (200 mg, 0.73 mmol) was added sequentially and dissolved in DMF (3.0 mL). Then, DIEA (280 mg, 2.2 mmol), HATU (420 mg, 1.1 mmol), and finally compound 1 (260 mg, 0.73 mmol) were added at room temperature. The reaction was carried out at 25 °C under a nitrogen atmosphere for 2 h. Water was added to the system, and the mixture was extracted with ethyl acetate, dried, filtered, and concentrated under reduced pressure. The mixture was purified by preparative HPLC (acetonitrile:water, ammonium bicarbonate system) to give compound 4 (53 mg).

[0166] LCMS(ESI-MS) m / z = 432.44 [M+H] +1 H NMR(400MHz,Chloroform-d)δ7.81(d,J=7.4Hz,1H),7.69(s,1H),7.49–7.43( m,2H),7.34-7.27(m,1H),7.21–7.16(m,2H),6.96(dd,J=15.2,4.8Hz,1H),6.5 4(dd,J=15.2,1.7Hz,1H),5.20-5.05(m,1H),4.21-4.13(m,1H),2.92(s,3H),1 .12(t,J=6.2Hz,6H),1.07-0.98(m,1H),0.74–0.60(m,2H),0.51–0.41(m,2H).

[0167] Example 5: N-[(1S,2E)-3-[aza-ylidene(methyl)(oxo)-λ6-thio]-1-cyclopropyl-2-propenyl]-2-

[0168] (1,1-Difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxamide (5)

[0169]

[0170] Step 1: Synthesis of diethyl phosphonate 5b of {[methyl(oxo)-λ4-thio]methyl}phosphonate

[0171] Diethyl thiomethoxyphosphate 5a (2.7 mL, 15 mmol) was dissolved in a mixture of acetone (90 mL) and water (60 mL), cooled to 0 °C, and an aqueous solution of sodium periodate (160 mL, 16 mmol) was slowly added dropwise. The mixture was stirred in an ice-water bath for 3 hours. The reaction was allowed to proceed at room temperature for 16 hours. The precipitated sodium iodate was filtered off, and the solvent was removed under reduced pressure. The solution was diluted with ethyl acetate, washed with a saturated aqueous solution of NaCl, and dried over Na₂SO₄. The solution was concentrated under reduced pressure. The final product was purified by silica gel column chromatography to give compound 5b (2.6 g).

[0172] LCMS(ESI-MS) m / z = 215.10 [M+H] +

[0173] Step 2: Synthesis of diethyl phosphonate 5c of {[methyl({[(2-methylprop-2-yl)oxy]carbonyl}azaide)(oxo)-λ6-thio]methyl}phosphonate

[0174] In a three-necked flask, tert-butyl carbamate (1.1 g, 9.4 mmol), dichloromethane (25 mL), rhodium dimer acetate (0.060 g, 0.14 mmol), and magnesium oxide (2.5 g, 19 mmol) were added sequentially. The mixture was purged with nitrogen three times at room temperature. Then, compound 5b (1.0 g, 4.7 mmol) was added, and the reaction was carried out at room temperature for 20 min. Finally, iodophenyldiacetic acid (2.3 g, 7.0 mmol) was added. The reaction was carried out at 50 °C for 12 h. The mixture was filtered through diatomaceous earth and concentrated under reduced pressure. The final product was purified by silica gel column chromatography to obtain compound 5c (680 mg).

[0175] LCMS(ESI-MS) m / z = 330.19 [M+H] +

[0176] Step 3: Synthesis of 2-methyl-2-propyl{[(1S,2E)-1-cyclopropyl-3-[methyl({[(2-methylprop-2-yl)oxy]carbonyl}azaide)(oxo)-λ6-thio]prop-2-enyl]amino}carbamate 5d

[0177] Take a single-necked flask and add compound 1b (1.0 g, 5.0 mmol) dissolved in THF (20 mL), then add compound 5c (130 mg, 0.55 mmol) and potassium carbonate (1.7 g, 13 mmol). React at 60 °C for 3 h. Add water to the system, extract with dichloromethane, dry to anhydrous sodium sulfate, filter, and concentrate under reduced pressure. The crude compound 5d (3.0 g) is used directly in the next step.

[0178] LCMS(ESI-MS) m / z = 375.38 [M+H] +

[0179] Step 4: Synthesis of (1S,2E)-3-[aza-ylidene(methyl)(oxo)-λ6-thio]-1-cyclopropyl-2-propen-1-amine 5e

[0180] In a single-necked flask, compound 5d (600 mg, 1.6 mmol) was added sequentially and dissolved in acetonitrile (10 mL), followed by p-toluenesulfonic acid (300 mg, 1.6 mmol). The reaction was carried out at 50 °C for 3 h. The mixture was then concentrated under reduced pressure to give compound 1f (560 mg).

[0181] LCMS(ESI-MS) m / z = 175.18 [M+H] +

[0182] Step 5: Synthesis of N-[(1S,2E)-3-[aza-ylidene(methyl)(oxo)-λ6-thio]-1-cyclopropyl-2-propenyl]-2-(1,1-difluoroethyl)-4-(phenyloxy)pyrimidine-5-carboxamide 5

[0183] In a single-necked flask, compound 1j (100 mg, 0.36 mmol) was added sequentially to a solution of DMF (5.0 mL). Then, DIEA (230 mg, 1.8 mmol) and HATU (160 mg, 0.43 mmol) were added at room temperature, followed by compound 5e (370 mg, 1.1 mmol). The reaction was carried out at 25 °C under nitrogen for 2 h. The mixture was extracted with water and ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Preparative HPLC (acetonitrile / water, sodium bicarbonate system) was used to purify the compound, yielding compound 5 (23 mg).

[0184] LCMS(ESI-MS) m / z = 437.40 [M+H] +1H NMR(400MHz,Chloroform-d)δ9.45(d,J=2.7Hz,1H),7.83(dd,J=17.8,7.3Hz,1H ),7.53–7.45(m,2H),7.37(td,J=7.3,1.2Hz,1H),7.26–7.22(m,2H),7.06-6.94 (m,1H),6.80-6.72(m,1H),4.30-4.20(m,1H),3.09(d,J=8.2Hz,3H),2.21(br,1 H),1.90-1.76(m,3H),1.15–1.06(m,1H),0.80-0.60(m,2H),0.55–0.44(m,2H).

[0185] Example 6: (S,E)-5-amino-6-(tert-butyl)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-2-phenoxynicotinamide (6)

[0186]

[0187]

[0188] Step 1: Synthesis of 5-bromo-6-tert-butyl-2-hydroxynicotinonitrile 6b

[0189] Compound 6a (800 mg, 4.5 mmol) and NBS (1.2 g, 6.8 mmol) were dissolved in DCE (12 mL) and refluxed at 85 °C with stirring for 3 hours. After cooling to room temperature, the solution was quenched with water, extracted with DCM, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain the crude product. The crude product was purified by FLASH column chromatography to obtain compound 6b (1.0 g).

[0190] LCMS (ESI-MS) m / z: 255.11 [M+H] +

[0191] Step 2: Synthesis of 5-bromo-6-tert-butyl-2-chloronicotinonitrile 6c

[0192] Compound 6b (1.5 g, 5.9 mmol) and PCl5 (3.7 g, 18 mmol) were dissolved in a sealed tube containing 15 mL of POCl3. The mixture was then heated to 120 °C and stirred for 8 hours. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to remove phosphorus oxychloride. The solution was then dissolved in DCM, and ice-cold sodium bicarbonate solution was added dropwise to adjust the pH to approximately 7. The organic phase was concentrated to dryness under reduced pressure to obtain the crude product. The crude product was purified by FLASH column chromatography to obtain compound 6c (1.2 g).

[0193] LCMS (ESI-MS) m / z: 273.12 [M+H]+

[0194] Step 3: Synthesis of 5-bromo-6-tert-butyl-2-phenoxynicotinonitrile 6d

[0195] Compound 6c (1.2 g, 4.4 mmol), phenol (0.51 mL, 5.7 mmol), and K₂CO₃ (0.91 g, 6.6 mmol) were dissolved in ACN (60 mL), and then stirred at 85 °C for 4 hours under nitrogen protection. The reaction solution was concentrated to dryness under reduced pressure to obtain a crude product, which was purified by FLASH column chromatography to obtain compound 6d (1.3 g).

[0196] LCMS (ESI-MS) m / z: 331.24 [M+H] +

[0197] Step 4: Synthesis of tert-butyl (2-(tert-butyl)-5-cyano-6-phenoxypyridin-3-yl)carbamate 6e

[0198] Compound 6d (200 mg, 0.6 mmol), tert-butyl carbamate (140 mg, 1.2 mmol), RuPhosPdG3 (76 mg, 0.091 mmol), RuPhos (42 mg, 0.091 mmol), and Cs₂CO₃ (590 mg, 1.8 mmol) were dissolved in dioxane (10 mL) and reacted at 110 °C for 8 hours under argon protection. After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain the crude product. The crude product was purified by FLASH column chromatography to obtain compound 6e (400 mg).

[0199] LCMS (ESI-MS) m / z: 368.46 [M+H] +

[0200] Step 5: Synthesis of 5-amino-6-tert-butyl-2-phenoxynicotinic acid 6f

[0201] KOH (920 mg, 16 mmol) was dissolved in H₂O (4.0 mL) and added to an ethanol (4.0 mL) solution of compound 6e (50 mg, 0.14 mmol). The mixture was stirred at 100 °C for 8 hours (with the tube sealed). After the reaction was complete, the mixture was cooled to room temperature, and the pH was adjusted to 4-5 by dissolving it in 1.0 N HCl. The solution was then extracted with DCM, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compound 6f (35 mg).

[0202] LCMS (ESI-MS) m / z: 287.20 [M+H] +

[0203] Step 6: Synthesis of (S,E)-5-amino-6-(tert-butyl)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-2-phenoxynicotinamide 6

[0204] Compound 6f (35 mg, 0.12 mmol), compound 1d (64 mg, 0.37 mmol), and HATU (70 mg, 0.18 mmol) were dissolved in DMF (7.0 mL). Under nitrogen protection, DIEA (0.22 mL, 1.2 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the reaction solution was extracted with ethyl acetate, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by prep-HPLC and lyophilized to obtain compound 6 (4.0 mg).

[0205] LCMS (ESI-MS) m / z: 444.48 [M+H] +

[0206] 1 H NMR (400MHz, DMSO-d6) δ8.43(d,J=8.2Hz,1H),7.44(s,1H),7.37–7.29(m,2H),7.12–7.04(m,3H),6.84–6.70(m,2H),4.94(s,2H),4.15(td,J=8 .5,4.6Hz,1H),2.93(s,3H),1.19(s,9H),1.06(td,J=8.5,4.2Hz,1H),0 .54–0.46(m,1H),0.40(ddd,J=9.4,6.8,3.6Hz,2H),0.32–0.23(m,1H).

[0207] Example 7: (S,E)-N-(1-cyclopropyl-3-(N-methylaminosulfonyl)allyl)-2-(1,1-difluoroethyl)-4-phenoxypyrimidine-5-carboxamide (7)

[0208]

[0209] The first step was the synthesis of 2-methylpropyl-2-yl({dioxo[(oxodiphenyl-λ5-phosphono)methyl]-λ6-thioalkyl}(methyl)amino)carbamate 7b.

[0210] 2-Methylpropyl-2-yl({dioxo[(oxodiphenyl-λ5-phosphono)methyl]-λ6-thioalkyl}amino)carbamate 7a (500 mg, 1.3 mmol), PPh3 (500 mg, 1.9 mmol), and methanol (0.15 mL, 3.8 mmol) were dissolved in THF (20 mL), and DIAD (510 mg, 2.5 mmol) was added at room temperature. After stirring at room temperature for 2 hours, a new spot was observed by TLC, which was quenched with water, extracted with ethyl acetate, dried over the organic phase, and concentrated. The crude product was purified by FLASH column chromatography to give compound 7b (510 mg).

[0211] LCMS (ESI-MS) m / z: 410.35 [M+H] +

[0212] Step 2: Synthesis of methyl 2-methylpropyl-2-yl{[(1S,2E)-1-cyclopropyl-3-[(2,2-dimethyl-4-oxo-5-aza-3-oxahex-5-yl)dioxo-λ6-thioalkyl]propyl-2-enyl]amino}carbamate 7c

[0213] Compound 1b (300 mg, 0.73 mmol) was dissolved in DMF (4.0 mL), and sodium hydride (60%, 88 mg, 2.2 mmol) was added under ice bath conditions. After stirring for 30 minutes, a DMF solution of compound 7b (300 mg, 0.73 mmol) was added to the mixture at -30 °C. Stirring continued for 12 hours. After 12 hours of reaction, the product was detected by LCMS. The reaction was quenched with water, extracted three times with ethyl acetate, dried over the organic phase, and concentrated. The crude product was purified by FLASH column chromatography to give 7c (140 mg).

[0214] LCMS (ESI-MS) m / z: 413 [M+Na] +

[0215] The third step involves the synthesis of (S,E)-3-amino-3-cyclopropyl-N-methylprop-1-ene-1-sulfonamide 7d.

[0216] Compound 7c (100 mg, 0.26 mmol) and p-toluenesulfonic acid (73 mg, 0.38 mmol) were dissolved in acetonitrile (10 mL) and reacted at room temperature for 12 hours. The mixture was then concentrated at low temperature. The crude compound 7d was used directly in the next reaction step.

[0217] Step 4: Synthesis of (S,E)-N-(1-cyclopropyl-3-(N-methylaminosulfonyl)allyl)-2-(1,1-difluoroethyl)-4-phenoxypyrimidine-5-carboxamide 7

[0218] Compound 1j (88 mg, 0.32 mmol), compound 7d (60 mg, 0.32 mmol), HATU (120 mg, 0.32 mmol), and DIEA (120 mg, 0.95 mmol) were dissolved in DMF (10 mL), and the reaction mixture was stirred at room temperature for 2 hours. After 2 hours of reaction, the product was detected by LCMS. The reaction mixture was quenched with water, extracted three times with ethyl acetate, washed with water, dried, and concentrated. Preparative column purification gave compound 7 (1.8 mg).

[0219] LCMS (ESI-MS) m / z: 453.4 [M+H] +

[0220] 1 H NMR (400MHz, Methanol-d4) δ8.98 (s, 1H), 7.53–7.42 (m, 2H), 7.34 (d, J = 7.3Hz, 1 H),7.32–7.26(m,2H),6.78(dd,J=15.2,5.3Hz,1H),6.51(dd,J=15.2,1.6Hz,1H ),4.17(dd,J=9.0,5.5Hz,1H),2.54(s,3H),1.81(t,J=18.6Hz,3H),1.17(dt,J= 8.5,4.4Hz,1H),0.89(d,J=7.1Hz,1H),0.73–0.61(m,2H),0.50(t,J=5.2Hz,1H).

[0221] Example 8: (S,E)-6-(tert-butyl)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-5-hydroxy-2-phenoxynicotinamide (8)

[0222]

[0223]

[0224] Step 1: Synthesis of 6-tert-butyl-5-hydroxy-2-phenoxynicotinonitrile 8a

[0225] Compound 6d (200 mg, 0.60 mmol), Pd2(dba)3 (280 mg, 0.30 mmol), and t-BuXphos (130 mg, 0.30 mmol) were dissolved in 1,4-dioxane (10 mL), and argon gas was purged. A solution of KOH (100 mg, 1.8 mmol) in water (10 mL) was added under argon protection and stirred at 85 °C for 3 hours. After cooling to room temperature, the mixture was extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain the crude product. Purification by FLASH column chromatography yielded compound 8a (70 mg).

[0226] LCMS (ESI-MS) m / z: 269.26 [M+H] +

[0227] Step 2: Synthesis of 6-tert-butyl-5-hydroxy-2-phenoxynicotinic acid 8b

[0228] Compound 8a (50 mg, 0.19 mmol) was dissolved in concentrated hydrochloric acid (5.0 mL, 0.037 mmol) and stirred at 100 °C for 3 hours (with the tube sealed). After the reaction was complete, the mixture was cooled to room temperature and concentrated under reduced pressure to obtain crude compound 8b (60 mg).

[0229] LCMS (ESI-MS) m / z: 288.29 [M+H] +

[0230] Step 3: Synthesis of (S,E)-6-(tert-butyl)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-5-hydroxy-2-phenoxynicotinamide 8

[0231] Compound 8b (50 mg, 0.17 mmol), compound 1d (120 mg, 0.70 mmol), and HATU (100 mg, 0.26 mmol) were dissolved in DMF (6.0 mL). Under nitrogen protection, DIEA (0.31 mL, 1.7 mmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was complete, the mixture was extracted with ethyl acetate, washed twice with saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by prep-HPLC and lyophilized to give compound 8 (9.0 mg).

[0232] LCMS (ESI-MS) m / z: 445.47 [M+H] +

[0233] 1 H NMR (400MHz, DMSO-d6) δ9.85 (s, 1H), 8.48 (d, J = 8.2Hz, 1H), 7.53 (s, 1H), 7.3 9–7.32(m,2H),7.15–7.08(m,3H),6.85–6.73(m,2H),4.17(td,J=8.4,4.3Hz, 1H),2.94(s,3H),1.20(s,9H),1.08(dt,J=7.8,4.9Hz,1H),0.51(td,J=9.3,8 .2,3.9Hz,1H),0.42(qq,J=8.9,4.8,4.2Hz,2H),0.30(dt,J=8.2,3.9Hz,1H).

[0234] Example 9: 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethane-1-ol (9)

[0235]

[0236]

[0237] Synthesis of (R)-2-(2-(1,1-difluoroethyl)-4-phenoxypyrimidine-5-carbamate)-3-hydroxy-2-methylpropionate 9b

[0238] Compound 1j (800 mg, 2.9 mmol) and compound 1b (460 mg, 3.4 mmol) were added to a single-necked flask, dissolved in 10 mL of DCM, and then PyBOP (1.9 g, 3.7 mmol) was added. The mixture was stirred at room temperature, and then triethylamine (1.6 mL, 11 mmol) was added. The mixture was stirred at room temperature for 2 h. The mixture was then directly mixed with silica gel and purified by FLASH column chromatography to obtain compound 9b (600 mg).

[0239] LCMS (ESI-MS) m / z: 396.38 [M+H] +

[0240] The second step involves the synthesis of (R)-2-(2-(1,1-difluoroethyl)-4-phenoxypyrimidin-5-yl)-4-methyl-4,5-dihydrooxazol-4-carboxylic acid methyl ester 9c.

[0241] Compound 9b (600 mg, 1.5 mmol) was added to a single-necked flask, dissolved in DCM (10 mL), cooled to -78 °C, and DAST (490 mg, 3.0 mmol) was added dropwise. The mixture was kept at -78 °C and stirred for 2 hours. The solution was quenched with sodium bicarbonate aqueous solution at -78 °C, stirred at room temperature for 10 minutes, extracted with DCM, washed once with sodium chloride aqueous solution, and concentrated by flash column chromatography to obtain compound 9c (440 mg).

[0242] LCMS (ESI-MS) m / z: 378.34 [M+H] +

[0243] The third step involves the synthesis of (S)-(2-(1,1-difluoroethyl)-4-phenoxypyrimidin-5-yl)-4-methyl-4,5-dihydrooxazol-4-yl)methanol 9d.

[0244] Compound 9c (400 mg, 1.1 mmol) was added to a single-necked flask, and THF (10 mL) was added and stirred to dissolve. Sodium borohydride (160 mg, 4.2 mmol) and lithium chloride (180 mg, 4.2 mmol) were added, and the mixture was stirred at room temperature for 24 hours. The mixture was then cooled in an ice-water bath, quenched with ammonium chloride aqueous solution, extracted with ethyl acetate, concentrated, and passed through a flash column to obtain compound 9d (250 mg).

[0245] LCMS (ESI-MS) m / z: 350.33 [M+H] +

[0246] Step 4: Synthesis of (R)-2-(2-(1,1-difluoroethyl)-4-phenoxypyrimidin-5-yl)-4-methyl-4,5-dihydrooxazol-4-carboxaldehyde 9e

[0247] DMSO (0.11 mL, 1.6 mmol) and DCM (3.0 mL) were added to a three-necked flask, nitrogen gas was purged, and the temperature was lowered to -78 °C. Oxaloyl chloride (100 mg, 0.8 mmol) was added, followed by compound 9d (50 mg, 0.14 mmol). The mixture was stirred at -78 °C for half an hour, followed by the addition of triethylamine (0.28 mL, 2.0 mmol). The mixture was stirred at -78 °C for half an hour, quenched with water, extracted with DCM, washed once with saturated sodium chloride solution of the organic phase, dried, filtered, and then anhydrous acetonitrile was added. The mixture was then rotary evaporated to obtain crude compound 9e, which was directly added to the next step.

[0248] LCMS (ESI-MS) m / z: 348.38 [M+H] +

[0249] Step 5: Synthesis of 2-((R)-3-(1-(1-(R)-1-(4,6-dichloropyridin-3-yl)ethyl)-3-(trifluoromethyl)-1H-pyrazolo[3,4-b]pyrazin-6-yl)azacyclobutane-3-yl)piperidin-1-yl)ethane-1-ol 9

[0250] Compound 9e (49 mg, 0.14 mmol) and compound 9f (130 mg, 0.56 mmol) were added to a single-necked flask. Potassium carbonate (78 mg, 0.56 mmol), CuI (34 mg, 0.18 mmol), and acetonitrile (3.0 mL) were added, and the atmosphere was purged with nitrogen. The mixture was heated to 70 °C for 2 hours, diluted with ethyl acetate, quenched with water, and the organic phase was washed three times with saturated sodium chloride solution. The solution was concentrated and passed through a flash column to obtain the crude product. Pre-HPLC yielded compound 9 (7.0 mg).

[0251] LCMS (ESI-MS) m / z: 424.40 [M+H] +

[0252] 1 H NMR(400MHz,Chloroform-d)δ9.18(s,1H),7.46–7.42(m,2H),7.32–7.28(m,1H),7.22–7.18(m,2H),7.04(d,J=14.9Hz,1H ),6.69(d,J=14.9Hz,1H),4.36(d,J=8.7Hz,1H),4.29(d,J=8.6Hz,1H),2.95(s,3H),1.86(d,J=18.5Hz,3H),1.60(s,3H).

[0253] Example 10: (S,E)-4-cyano-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-2-(1,1-difluoroethyl)-6-phenoxypyrimidine-5-carboxamide (10)

[0254]

[0255] Step 1: Ethyl 4-chloro-2-(methylthio)-6-phenoxypyrimidine-5-carboxylate (10b)

[0256] Ethyl 4,6-dichloro-2-(methylthio)pyrimidine-5-carboxylic acid ester 10a (2.7 g, 10 mmol) and phenol (0.95 g, 10 mmol) were dissolved in acetonitrile (40 mL). Potassium carbonate (2.8 g, 20 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 8 hours. The reaction solution was filtered, washed with methyl tert-butyl ether, and the filtrate was concentrated. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 50:1) to give compound 10b (3.0 g).

[0257] LCMS (ESI-MS) m / z: 325.21 [M+H] +

[0258] Step 2: Ethyl 4-cyano-2-(methylthio)-6-phenoxypyrimidine-5-carboxylate (10c)

[0259] Compound 10b (2.5 g, 7.7 mmol) was dissolved in anhydrous N-methylpyrrolidone (25 mL), and cuprous cyanide (1.4 g, 15 mmol) and cuprous iodide (1.5 g, 7.7 mmol) were added at room temperature, followed by nitrogen purging. The mixture was heated at 155 °C for 8 hours. The reaction was quenched with dilute ammonia, extracted with ethyl acetate, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give compound 10c (1.3 g).

[0260] LCMS (ESI-MS) m / z: 316.26 [M+H] +

[0261] Step 3: Ethyl 2-chloro-4-cyano-6-phenoxypyrimidine-5-carboxylate (10 days)

[0262] Compound 10c (1.3 g, 4.0 mmol) was dissolved in anhydrous acetonitrile (20 mL) and purged with nitrogen. Anhydrous dichlorosulfonyl chloride (3.3 mL, 41 mmol) solution was slowly added dropwise under ice bath conditions. After addition was complete, the mixture was incubated under ice bath conditions for 1 hour. The reaction was quenched with ice-cold sodium bicarbonate solution, extracted with dichloromethane, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give compound 10d (0.90 g).

[0263] LCMS (ESI-MS) m / z: 304.21 [M+H] +

[0264] Step 4 (4-cyano-2-(1-ethoxyvinyl)-6-phenoxypyrimidine-5-carboxylic acid ethyl ester (10f))

[0265] Compound 10d (0.90 g, 3.0 mmol) was dissolved in anhydrous 1,4-dioxane (15 mL) with tributyl(1-ethoxyvinyl)tin (1.3 g, 3.6 mmol) and purged with nitrogen. Tetraphenylphosphine palladium (0.34 mg, 0.30 mmol) was added at room temperature and purged with nitrogen. The mixture was heated at 100 °C for 4 hours. The reaction was quenched with ice water, extracted with dichloromethane, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give compound 10f (0.75 g).

[0266] LCMS (ESI-MS) m / z: 340.33 [M+H] +

[0267] Step 5: Ethyl 2-acetyl-4-cyano-6-phenoxypyrimidine-5-carboxylate (10g)

[0268] Compound 10f (0.75 g, 2.2 mmol) was dissolved in 0.1 M hydrochloric acid-1,4-dioxane (10 mL) and reacted overnight at room temperature. The reaction was quenched with ice-cold sodium bicarbonate solution, extracted with dichloromethane, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give compound 10 g (0.28 g).

[0269] LCMS (ESI-MS) m / z: 312.31 [M+H] +

[0270] Step 6: Ethyl 4-cyano-2-(1,1-difluoroethyl)-6-phenoxypyrimidine-5-carboxylate (10 h)

[0271] 10 g (0.28 g, 0.90 mmol) of compound was dissolved in anhydrous dichloromethane (10 mL), and diethylaminotrifluoride (4.0 mL) was added under ice bath conditions. The mixture was reacted overnight at room temperature. The reaction was quenched with ice-cold sodium bicarbonate solution, extracted with dichloromethane, dried over the organic phase, filtered, concentrated, and the crude product was purified by silica gel column chromatography to give compound 10h (0.20 g).

[0272] LCMS (ESI-MS) m / z: 334.32 [M+H] +

[0273] Step 7: 4-Cyano-2-(1,1-Difluoroethyl)-6-phenoxypyrimidine-5-carboxylic acid (10i)

[0274] Compound 10h (80 mg, 0.24 mmol) was dissolved in anhydrous pyridine (4 mL) with lithium iodide (0.48 g, 3.6 mmol) and the mixture was purged with nitrogen. The reaction was heated at 100 °C for 16 hours. The mixture was concentrated, and the crude product was purified by silica gel column chromatography to give compound 10i (45 mg).

[0275] LCMS (ESI-MS) m / z: 306.26 [M+H] +

[0276] Step 8 (S,E)-4-cyano-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-2-(1,1-difluoroethyl)-6-phenoxypyrimidine-5-carboxamide (10)

[0277] Compound 10i (25 mg, 0.08 mmol), 1d (29 mg, 0.16 mmol), and pyridine (0.25 mL, 3.1 mmol) were dissolved in anhydrous dichloromethane (3.0 mL) and purged with nitrogen. Phosphorus oxychloride (0.050 mL, 0.080 mmol) was slowly added dropwise under ice bath conditions. The mixture was kept in an ice bath for 10 minutes. The reaction was quenched with ice water, extracted with dichloromethane, dried over the organic phase, filtered, concentrated, and the crude product was purified by preparative liquid chromatography (ammonium bicarbonate) to give compound 10 (6.9 mg).

[0278] LCMS (ESI-MS) m / z: 463.41 [M+H] +

[0279] 1H NMR (400MHz, DMSO-d6) δ10.56 (s, 1H), 7.52 (d, J = 7.8Hz, 2H), 7.37 (d, J = 7.4Hz, 1H),7.33(d,J=8.2Hz,2H),7.05(d,J=3.6Hz,2H),4.38(dd,J=10.4,3.2Hz,1H) ,3.01(s,3H),1.92(d,J=19.0Hz,3H),1.82(dd,J=11.8,7.6Hz,1H),0.74(h,J= 4.2Hz,1H),0.65–0.60(m,1H),0.52(dd,J=8.6,4.6Hz,1H),0.34–0.28(m,1H).

[0280] Example 11: (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-5-phenoxy-1H-pyrazole-1-carboxamide (11) and (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-3-phenoxy-1H-pyrazole-1-carboxamide (13)

[0281]

[0282] The first step was the synthesis of ethyl 5-iodo-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-carboxylate 11b.

[0283] Take a 100 mL flask, add ethyl 5-iodo-1H-pyrazole-4-carboxylate 11a (5.0 g, 19 mmol), THF (40 mL), K2CO3 (5.2 g, 38 mmol), and 1-chloro-5,5-dimethyl-2-oxa-5-silhexane (4.3 mL, 24 mmol), stir at room temperature under nitrogen protection for 3 hours, pour into water (100 mL), extract with ethyl acetate (30 mL * 3), wash with saturated sodium chloride aqueous solution (100 mL), dry with anhydrous sodium sulfate, filter, add silica gel and mix, and purify by Flash column to obtain compound 11b (5.6 g).

[0284] LCMS (ESI-MS) m / z: 397.30 [M+H] +

[0285] Step 2: Synthesis of ethyl 5-phenoxy-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazole-4-carboxylic acid ester 11c

[0286] Take 25 mL of the sealed tube, add compound 11b (1.0 g, 2.5 mmol), DMF (10 mL), phenol (0.27 mL, 3.0 mmol), and K2CO3 (520 mg, 3.8 mmol), and heat in a microwave at 140 °C for 20 hours. Pour the reaction solution into water (50 mL), extract three times with EA (20 mL * 3), wash with saturated sodium chloride aqueous solution (60 mL), dry with anhydrous sodium sulfate, filter, add silica gel and mix, evaporate the solvent, and purify by Flash column to obtain compound 11c (1.1 g).

[0287] LCMS (ESI-MS) m / z: 363.43 [M+H] +

[0288] Step 3: Synthesis of 5-(phenoxy)-1H-pyrazole 11d

[0289] Take 15 mL of the sealed tube, add compound 11c (800 mg, 2.2 mmol), concentrated hydrochloric acid (6.0 mL), and under nitrogen protection, react at 110 °C for 12 hours. Slowly add the reaction solution to a saturated sodium bicarbonate aqueous solution, extract with ethyl acetate, wash with a saturated sodium chloride aqueous solution, dry with anhydrous sodium sulfate, evaporate the solvent, and prepare by reverse phase HPLC to obtain compound 11d (80 mg).

[0290] LCMS (ESI-MS) m / z: 161.11 [M+H] +

[0291] The fourth step involves the synthesis of (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-5-phenoxy-1H-pyrazole-1-carboxamide (11) and (S,E)-N-(1-cyclopropyl-3-(methanesulfonyl)allyl)-3-phenoxy-1H-pyrazole-1-carboxamide (13).

[0292] Take a 100 mL flask, add CDI (56 mg, 0.34 mmol) and THF (5.0 mL), under nitrogen protection, add compound 11d (100 mg, 0.68 mmol) in an ice bath, keep stirring in an ice bath for 0.5 hours, add compound 1d (160 mg, 0.31 mmol), and bring to room temperature overnight. Extract with water (40 mL) and EA (10 mL * 3), wash with saturated sodium chloride aqueous solution (20 mL), dry with anhydrous sodium sulfate, filter, evaporate the solvent, and prepare by reverse-phase HPLC to obtain compound 11 (3.0 mg) and compound 13.

[0293] 11LCMS (ESI-MS) m / z: 362.20 [M+H] + , 13LCMS(ESI-MS)m / z:362.20[M+H] +

[0294] 1 H NMR(400MHz,Chloroform-d)δ8.09(d,J=2.9Hz,1H),7.40(t,J=7.9Hz,2H),7.24–7.15(m,3H),7.1 2(d,J=8.1Hz,1H),7.00(dd,J=15.2,4.6Hz,1H),6.59(dd,J=15.2,1.7Hz,1H),5.98(d,J=2.9Hz,1H ),4.01–3.92(m,1H),2.96(s,3H),1.05(dtt,J=13.1,8.5,4.8Hz,1H),0.75(dq,J=8.6,4.6,4.2Hz, 1H), 0.67 (ddt, J=13.9, 9.0, 5.0Hz, 1H), 0.51 (dq, J=10.1, 5.0Hz, 1H), 0.44 (dq, J=9.4, 4.9Hz, 1H).

[0295] Example 12: (S,E)-N-(1-cyclopropyl-3-nitroallyl)-2-(1,1-difluoroethyl)4-phenoxypyrimidine-5-carboxamide (12)

[0296]

[0297]

[0298] Synthesis of tert-butyl ((1S)-1-cyclopropyl-2-hydroxy-3-nitropropyl)carbamate 12a (Step 1)

[0299] Compound 1b (100 mg, 0.50 mmol) was dissolved in DCM (1.0 mL), and nitromethane (0.027 mL, 0.50 mmol) and TEA (0.21 mL, 1.5 mmol) were added. The mixture was stirred overnight at room temperature. TLC monitoring showed that the reaction proceeded to completion, and LCMS monitored the target product. 20 mL of saturated ammonium chloride aqueous solution was added, and the mixture was extracted with ethyl acetate (20 mL * 3). The organic phase was washed once with saturated brine (20 mL), and the organic phase was concentrated under vacuum at 45 °C using a diaphragm pump to obtain compound 12a (120 mg).

[0300] LCMS (ESI-MS) m / z: 161.1 [M+H + -boc + ]

[0301] Step 2: Synthesis of (1S)-1-amino-1-cyclopropyl-3-nitropropane-2-ol 12b

[0302] Compound 12a (120 mg, 0.46 mmol) was dissolved in acetonitrile (2.0 mL), and TsOH (87 mg, 0.51 mmol) was added. The mixture was reacted at 80 °C for 2 hours. After cooling to room temperature, the mixture was concentrated under vacuum at 45 °C using a diaphragm pump to obtain crude compound 12b (180 mg), which was then directly proceeded to the next step.

[0303] LCMS (ESI-MS) m / z: 161.1 [M+H] +

[0304] Step 3: Synthesis of N-(1S)-1-cyclopropyl-2-hydroxy-3-nitropropyl)-2-(1,1-difluoroethyl)-4-phenoxypyrimidine-5-carboxamide 12c

[0305] Compound 12b (180 mg, 1.1 mmol) was dissolved in DMF (2.0 mL), and compound 1j (310 mg, 1.1 mmol), HOBt (230 mg, 1.7 mmol), EDCI (320 mg, 1.7 mmol), and DIEA (440 mg, 3.4 mmol) were added. The mixture was stirred at room temperature for 2 hours. The solution was quenched with 20 mL of saturated ammonium chloride solution, concentrated, extracted with EA, and the organic phase was washed once with saturated brine (20 mL). The solution was dried over anhydrous Na₂SO₄ powder, filtered, and the filtrate was evaporated to dryness to obtain crude compound 12c (600 mg). The crude compound was dissolved in ethyl acetate, mixed with 1.0 g of silica gel powder, and purified by column chromatography to obtain compound 12c (100 mg).

[0306] LCMS (ESI-MS) m / z: 423.4 [M+H] +

[0307] Step 4: Synthesis of N-(1S)-1-cyclopropyl-2-hydroxy-3-nitropropyl)-2-(1,1-difluoroethyl)-4-phenoxypyrimidine-5-carboxamide 12

[0308] Compound 12c (100 mg, 0.24 mmol) was dissolved in DCM (2.0 mL), and TEA (0.030 mL, 0.21 mmol) and MsCl (120 mg, 1.1 mmol) were added in an ice bath. The mixture was stirred in an ice bath for 2 hours and purified by preparative column chromatography to obtain compound 12 (6.3 mg).

[0309] LCMS (ESI-MS) m / z: 405.3 [M+H] +

[0310] 1H NMR (400MHz, Methanol-d4) δ9.02 (s, 1H), 7.48 (t, J = 7.9 Hz, 2H), 7.39–7.34 (m, 2H), 7.34–7.26 (m, 3H), 4.22 (dd, J = 9. 2,3.8Hz,1H),3.32–3.29(m,1H),1.81(t,J=18.6Hz,3H),1.31–1.16(m,1H),0.76–0.62(m,2H),0.53(p,J=4.6Hz,2H).

[0311] Biological Examples

[0312] Experiment 1: Determination of WRN helicase binding activity

[0313] The compound was dissolved in DMSO (Mce) and diluted with phosphate buffer. 20 nM hWRN protein (hWRN(517-1238), ICE) was added to the compound dilution buffer to a final volume of 10 μL, and pre-incubated at room temperature for 30 minutes. ATP (Ark Pharm) was prepared to 4 nM using 5 μL of phosphate buffer and vortexed at room temperature. The protein-compound dilution and ATP dilution were added to 384-well plates (Nunc) and incubated at room temperature for 4 hours. 5 μL of 100 nM dsDNA-secquence 2 (Genscript), 5 μL of 1 μM capture DNA (Genscript), and 5 μL of 4 mM ATP were added to 384-well plates and incubated at room temperature for 30 minutes. Fluorescence signals were detected at Ex 620 nm and Em 685 nm using a fully automated microplate reader (BMG), and IC50 was calculated. 50 The helicase binding activities of each embodiment are shown in the table below: A represents IC50. 50 ≤250nM, B represents IC 50 ≤500nM, C represents >500nM.

[0314] Compounds Helicase binding activity 1 B 5 B 6 B 8 A D1 (CAS: 2923008-89-1) C

[0315] Experiment 2: Assay for the inhibition of SW48 and HCT116 cell proliferation

[0316] SW48 (ATCC) and HCT116 (ATCC) cell lines were cultured in RPM1 Medium 1640 (Corning+) 10% FBS (Gibco) and McCoy's 5A (Gibco) + 10% FBS, respectively, in a cell culture incubator at 37°C and 5% CO2. The compound was dissolved using DMSO and serially diluted 3-fold with culture medium. Cells in good growth condition were collected, and 60 μL of a suspension (500 cells total) was prepared and added to 384-well plates. Compound dilution buffer was then added, and the cells were cultured at 37°C and 5% CO2 for 72 hours. After 72 hours, the culture medium was removed, resuspended in PBS, and fresh culture medium was added for an additional 72 hours of culturing. Fluorescence signals were measured using a CellTiter-Glo assay kit (Promega), and IC50 was calculated. 50 .

[0317] The compounds of this invention have an inhibitory effect on the proliferation of SW48 and HCT116 cells.

Claims

1. The compound represented by formula (II), its stereoisomers, tautomers, or pharmaceutically acceptable salts thereof, Its features are, Ring A is a 5-10 membered heterocyclic ring; Ring B is a 5-10 membered aromatic ring or a heterocyclic aromatic ring; R 1 Each is independently an oxo, -OH, or -C group. 1-10 Alkyl, amino, or -CN, wherein the -C 1-10 The alkyl group may optionally be substituted with one or more halogens within the range allowed by the valence; R 2 Each independently is -C 1-10 alkyl; L 1 For key or -O-; L 2 -C(=O)NH-, -C(=O)NH-CHR a -or R a -C 1-5 Alkyl or 3-6 membered cycloalkyl; Ring C is a 4-6 membered heterocycle, optionally bounded by one or more -C groups within the range allowed by valence. 1-3 Alkyl substitution; R 3 -CH=CH-R 4 Or a 3-10 membered heterocyclic alkyl group, wherein the heterocyclic alkyl group is optionally replaced by one or more -CN groups within the range allowed by the valence; R 4 For -NO2 or X is O or NR c ; R b -C 1-5 Alkyl or amino group, wherein the amino group is optionally marked with one or two -C. 1-5 Alkyl groups are substituted; R c H or -C 1-5 alkyl; m and n are each independently 0, 1, 2, 3 or 4; When ring A is n is 1, L 2 When R is -C(=O)NH-, 4 It is not -S(=O)2CH3.

2. The compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to claim 1, characterized in that, Ring A is a 5-8 membered aromatic heterocycle or an unsaturated heterocyclic group; preferably, the heteroatom of the 5-8 membered aromatic heterocycle or unsaturated heterocyclic group is a nitrogen atom; preferably, the number of heteroatoms is 1 or 2; and / or n can be 0, 1, 2 or 3, preferably 0, 1 or 2; Preferably, ring A is More preferably, ring A is More preferably, for and / or R 1 Each is independently an oxo, -OH, or -C group. 1-5 Alkyl, amino, or -CN, wherein the -C 1-5 The alkyl group is optionally substituted with one or more Fs within the range allowed by the valence; preferably, R 1 It can be -CF2CH3, isopropyl, oxo, tert-butyl, amino, or -OH.

3. The compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to claim 1 or 2, characterized in that, Ring B is a 5-6 membered aromatic ring or a heterocyclic aromatic ring; and / or m can be 0, 1, 2 or 3, preferably 0 or 1. Preferably, ring B is a benzene ring or a thiophene ring; More preferably, ring B is More preferably, for and / or R 2 Each independently is -C 1-5 Alkyl group, preferably -C 1-3 Alkyl, more preferably -CH3.

4. The compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to any one of claims 1-3, characterized in that, R a -C 1-5 Alkyl or 3-6 membered cycloalkyl, preferably -C 1-3 Alkyl or 3-5 membered cycloalkyl, more preferably -CH3 or cyclopropyl; and / or The ring C can be optionally represented by one or more -C groups within the range allowed by the valence. 1-3 An alkyl-substituted 5-membered heterocycle; preferably, the 5-membered heterocycle is... More preferably More preferably, the ring C is Preferably, L 2 For -C(=O)NH-, 5. The compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to any one of claims 1-4, characterized in that, R 3 -CH=CH-R 4 Or 5-6 membered heterocyclic alkyl groups, R 4 For -NO2 or The heterocyclic alkyl group is optionally substituted with one or more -CN groups within the range allowed by the valence; preferably, the 5-6 membered heterocyclic alkyl group is piperidinyl; more preferably, the piperidinyl group is substituted with one -CN group; and / or R 4 For -NO2 or The R b -C 1-3 Alkyl or amino group, wherein the amino group is optionally marked with one or two -C. 1-3 Alkyl-substituted; preferably, the -C 1-3 The alkyl group is methyl or ethyl; more preferably, R b It is -CH3 or -NHCH3; and / or X is O or NR. c R c X is H, Me, or Et, preferably O or NH; more preferably, R 4 For -NO2, -S(=O)2CH3, -S(=O)2NHCH3 or Preferably, R 3 For -CH=CHNO2, -CH=CHS(=O)2CH3, 6. The compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to claim 1, characterized in that, It has a structure as shown in equation (II-1) or equation (II-2): E is C or N, ring A is a 5-6 membered aromatic heterocycle or an unsaturated heterocyclic group, R 1 R 2 R a R b m and n are each independently claimed according to any one of claims 1-5; Preferably, ring A is a 6-membered aromatic heterocycle or an unsaturated heterocyclic group; more preferably, ring A is... or Preferably, ring A is a 5-membered aromatic heterocycle; more preferably, ring A is...

7. The compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to any one of claims 1-6, characterized in that, Includes the following structure:

8. A method for preparing the compound, its stereoisomers, tautomers, or pharmaceutically acceptable salts according to any one of claims 1-7, characterized in that, The steps include Option 1, Option 2, Option 3, or Option 4: Option 1: When L 1 For -O-, L 2 -C(=O)NH-CHR a -, R 3 -CH=CH-R 4 hour Option 2: When L 2 for R 3 -CH=CH-R 4 hour Option 3: When L 1 For key, L 2 -C(=O)NH-CHR a -, R 3 -CH=CH-R 4 hour Option 4: When L 2 When it is -C(=O)NH- The definitions of each group are as described above.

9. A pharmaceutical composition comprising at least one compound according to any one of claims 1-7, its stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, and optionally one or more pharmaceutically acceptable carriers and / or additives.

10. Use of the compound, stereoisomer, tautomer, or pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 1-7, in the preparation of a medicament for the prevention or treatment of cancer; Preferably, the cancer is characterized by high microsatellite instability or mismatch repair defects; More preferably, the cancers characterized by high microsatellite instability or mismatch repair defects are selected from colorectal cancer, gastric cancer, endometrial cancer, adrenocortical cancer, uterine cancer, cervical cancer, esophageal cancer, breast cancer, kidney cancer, prostate cancer, and ovarian cancer. More preferably, the cancer characterized by high microsatellite instability or mismatch repair defects is selected from colorectal cancer, gastric cancer, prostate cancer, and endometrial cancer.

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