CN118221698A - KRAS G12D inhibitors - Google Patents

Abstract

The present invention relates to heterocyclic compounds as Kras G12D inhibitors and isomers thereof, or pharmaceutically acceptable salts thereof, methods of their preparation, pharmaceutical compositions and their use in the preparation of a medicament for treating cancer in a subject suffering from said cancer.

Description

KRAS G12D inhibitors

Technical Field

The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to an indole compound and its isomers, or pharmaceutically acceptable salts thereof, as a KRAS G12D inhibitor, and relates to a preparation method of the compound, a pharmaceutical composition comprising the compound as an active ingredient, and the use of the compound in treating cancer.

Background Art

RAS is a group of closely related small monomeric GTP-binding proteins composed of 189 amino acids with a molecular weight of about 21 kDa. It is associated with the plasma membrane and binds GDP or GTP, acting as a molecular switch. RAS can bind to GTP by binding to proteins of the guanine nucleotide exchange factor (GEF) (e.g., SOS1), which forces the release of the bound nucleotide and releases GDP. When RAS binds to GTP, it becomes activated (on) and recruits and activates other proteins required for the propagation of receptor signals, such as c-Raf and PI 3-kinase. RAS also has enzymatic activity that cleaves the terminal phosphate of the nucleotide and converts it to GDP. The conversion rate is usually slow, but can be significantly accelerated by proteins of the GTPase activating protein (GAP) class (e.g., RasGAP). When GTP is converted to GDP, RAS is deactivated (off).

The main members of the RAS subfamily include HRAS, KRAS and NRAS. Among them, KRAS mutations are observed in many malignant tumors: 95% of pancreatic ductal adenocarcinoma (PDAC), 45% of colon and rectal cancer (CRC), and 35% of non-small cell lung cancer (NSCLC). In 82% of PDAC, 64% of colorectal cancer, and 92% of non-small cell lung cancer, mutations often occur at the glycine residue at position 12 of KRAS. Among these mutations, it is reported that the main mutation of KRAS at position 12 in PDAC (39%) and CRC (44%) is aspartic acid mutation (see non-patent literature 1).

RAS has been considered to be untamperable for many years. However, it is reported that targeting inactive GDP-bound KRAS (G12C) is a promising method for producing new anti-RAS therapies (see non-patent literature 2). Because KRAS (G12C) retains GTPase activity and there is a nucleotide cycle in KRAS (G12C) cells, inhibitors bound to inactivated KRAS (G12C) can inhibit the activation of KRAS (G12C) in cells. Like KRAS (G12C) mutants, KRAS (G12D) is reported to retain GTPase activity (see non-patent literature 3). Therefore, a strategy that targets GDP-bound KRAS (especially G12D) and inhibits the conversion from GDP to GTP-bound states is considered to be extremely attractive.

WO2021106231, WO2021107160, CN114615981A, WO2022068921, WO2022061251 and the like disclose KRAS G12D inhibitor heterocyclic compounds for treating cancer.

There is a need to develop new KRAS G12D inhibitor compounds that exhibit sufficient efficacy for treating KRAS G12D-mediated cancers.

[List of non-patent literature]

Non-patent literature 1: Nature Reviews Drug Discovery 13(11), 828-51, 2014

Non-patent literature 2: Cancer Discov. 6(3), 316-29, 2016

Non-patent literature 3: Mol Cancer Res; 13(9), 1325-35, 2015

Summary of the invention

SUMMARY OF THE INVENTION

To solve the above technical problems, the inventors conducted extensive research and found that the compound shown in the following formula I has a strong KRAS inhibitory function, overcoming the shortcomings of KRAS G12D mutant protein inhibitors in the prior art. The indole compounds provided by the present invention have a good inhibitory effect on KRAS G12D mutant protein.

The present invention solves the above technical problems through the following technical solutions.

In one aspect, the present invention provides a compound represented by formula I and its isomers, or a pharmaceutically acceptable salt thereof,

A is selected from CH or N;

B is selected from CH or N;

E is selected from CH or N;

F is selected from CH or N;

G is selected from N or hydroxyl;

L is selected from O or N;

Ring C represents a substituted or unsubstituted, saturated or unsaturated 8- to 10-membered N-bridged ring containing at least one other heteroatom selected from N, O, S;

R ₁ is selected from 5-10 membered aryl, 5-10 membered heteroaryl with 1 to 4 heterocyclic ring members being N, O, S; each R ₁ is independently, at each occurrence, optionally substituted with one or more of the following groups: hydroxy, halogen, -NH ₂ , -CN, C ₁ -C ₆ -alkyl, C ₁ -C ₆ -alkoxy;

m = 0 or 1;

Z is selected from 5-8 membered heterocyclyl or heteroaryl with 1 to 4 heterocyclic ring members being N, O, S; each Z is independently optionally substituted at each occurrence by one or more of the following groups: hydroxy, halogen, _-NH2 , -CN, _C1 - _C6 -alkyl, _C1 - _C6 -alkoxy.

Further, the R ₁ is selected from the following structures:

The groups are optionally substituted by one or more of the following groups: hydroxy, halogen, _-NH2 , -CN, _C1 - _C6 -alkyl, _C1 - _C6 -alkoxy.

Further, the ring C is selected from The halogen is F, Cl, Br, I, preferably F; the C ₁ -C ₆ alkyl is a straight chain alkyl, branched chain alkyl or cycloalkyl, preferably a straight chain alkyl, more preferably a methyl; the C ₁ -C ₆ alkoxy is a straight chain alkyloxy, branched chain alkyloxy or cycloalkyloxy, preferably a methoxy.

Further, the ring Z is selected from

The present invention also provides compounds having the structures of formula (I-1), (I-2), and (I-3):

Wherein, the groups A, C, Z, R ₁ and m are as defined above.

In certain most preferred embodiments, the compounds of the present invention and their isomers, or pharmaceutically acceptable salts thereof are selected from the following compounds:

The present invention also provides a method for preparing the compound of formula I and its isomers, or a pharmaceutically acceptable salt thereof, the method comprising the following steps:

The starting materials are subjected to three substitution reactions and deprotection reactions to obtain the compound represented by formula (I); wherein R ₁ , A, B, E, F, G, L, Z, and C rings are defined as above.

The present invention also provides a pharmaceutical composition, which comprises the compound represented by Formula I and its isomers, or pharmaceutically acceptable salts thereof, and one or more pharmaceutical excipients.

The present invention also provides use of the compound represented by formula I and its isomers, or pharmaceutically acceptable salts or compositions thereof in the preparation of a drug for inhibiting KRAS G12D activity in cells.

The present invention also provides a compound as shown in formula I and its isomers, or a pharmaceutically acceptable salt thereof, or use of the pharmaceutical composition in preparing a drug; the drug is preferably used to prevent and/or treat cancer mediated by KRAS mutation, and the KRAS mutant protein may be a KRAS G12D mutant protein; the cancer may be pancreatic cancer, metastatic colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, or cutaneous squamous cell carcinoma.

The inventors have found that the compounds are highly effective KRAS G12D inhibitors, have extremely strong KRAS G12D inhibitory activity, and can be used to prepare drugs for preventing and/or treating KRAS G12D inhibition-related indications, including pancreatic cancer, metastatic colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, and cancers caused by skin squamous cell carcinoma. The present invention is completed based on the above findings.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects and features of the present invention are further described below.

All documents cited in the present invention are incorporated herein by reference in their entirety, and if the meanings expressed in these documents are inconsistent with the present invention, the description of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the general meanings known to those skilled in the art. Even so, the present invention still hopes to provide a more detailed description and explanation of these terms and phrases herein. If the terms and phrases mentioned are inconsistent with the known meanings, the meanings described in the present invention shall prevail. Below are the definitions of various terms used in the present invention, which apply to the terms used throughout the specification of this application, unless otherwise specified in specific circumstances.

The compounds according to the invention may exist in tautomeric forms and the present invention then includes all tautomeric forms.

The compounds of the present invention have asymmetric centers. The compounds of the present invention containing asymmetrically substituted atoms can be separated into optically active or racemic forms. Those skilled in the art know how to prepare optically active forms, such as by racemic separation or synthesis from optically active starting materials. Unless the specific stereochemistry or isomeric form is specifically stated, the present invention includes all chiral, diastereoisomers and racemates. The methods for preparing the compounds of the present invention and the intermediates thereof are part of the present invention. All tautomers of the compounds of the present invention are also part of the present invention.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced with a substituent as long as the valence state of the particular atom is normal and the resulting compound after the substitution is stable.

The terms "alkoxy" and "alkylamino" are customary expressions and refer to an alkyl group attached to the remainder of the molecule through an oxygen atom or an amino group, respectively, wherein the alkyl group is as described herein.

As used herein, the terms "halogen", "halo" and the like represent fluorine, chlorine, bromine or iodine, and particularly represent fluorine, chlorine and bromine, with fluorine and chlorine being particularly preferred.

As used herein, the term "alkyl" refers to an alkyl group having a specified number of carbon atoms, which is a linear or branched alkyl group, and which may include subgroups thereof, for example, when referring to " _C1 - _C6 alkyl", it may also include sub-ranges of groups represented by _C1 - _C4 alkyl, _C1 - _C3 alkyl, _C2 - _C6 alkyl, _C2 - _C4 alkyl, etc., as well as specific groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.

The terms "heterocycle", "heterocyclic" or "heterocyclyl" are used interchangeably and refer to substituted and unsubstituted 3 to 7-membered monocyclic groups, 7 to 11-membered bicyclic groups, and 10 to 15-membered tricyclic groups having at least one heteroatom (O, S or N) in at least one ring, the heteroatom-containing ring preferably having 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of such heteroatom-containing groups may contain one or two oxygen or sulfur atoms and/or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and further provided that the ring contains at least one carbon atom. Nitrogen and sulfur atoms may be optionally oxidized, and nitrogen atoms may be optionally quaternized. The fused rings completing the bicyclic and tricyclic groups may contain only carbon atoms, and may be saturated, partially saturated or fully unsaturated. The heterocyclic group may be attached to any available nitrogen or carbon atom. As used herein, the terms "heterocycle," "heterocycloalkyl," "heterocyclo," "heterocyclic," and "heterocyclyl" include "heteroaryl" groups, as defined below.

In addition to the heteroaryl groups described below, exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, oxetanyl, imidazolinyl, oxazolidinyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, piperidinyl, piperazinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, 2-oxoazepinyl, azepinyl, 1-pyridonyl, 4-piperidonyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl, thiomorpholinyl sulfoxide, thiomorpholinyl sulfone, 1,3-dioxolane, and tetrahydro-1,1-dioxythienyl, etc. Exemplary bicyclic heterocyclic groups include quinuclidinyl.

The term "heteroaryl" refers to substituted and unsubstituted aromatic 5- or 6-membered monocyclic groups, 9- or 10-membered bicyclic groups, and 11- to 14-membered tricyclic groups, which have at least one heteroatom (O, S or N) in at least one ring, and the heteroatom-containing ring preferably has 1, 2 or 3 heteroatoms selected from O, S and N. Each ring of the heteroaryl containing heteroatoms can contain one or two oxygen or sulfur atoms and/or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less, and each ring has at least one carbon atom. The fused rings completing the bicyclic and tricyclic groups can contain only carbon atoms and can be saturated, partially saturated or unsaturated. Nitrogen and sulfur atoms can be optionally oxidized, and nitrogen atoms can be optionally quaternized. As a bicyclic or tricyclic heteroaryl, it must include at least one completely aromatic ring, but the other one or more fused rings can be aromatic or non-aromatic. Heteroaryl can be attached to any available nitrogen or carbon atom of any ring. If the other ring is cycloalkyl or heterocycle it is additionally optionally substituted with =0 (oxo) where valence permits.

Unless otherwise indicated, when referring to a specifically named aryl (e.g., phenyl), heterocyclyl (e.g., pyrrolidinyl, piperidinyl, and morpholinyl), or heteroaryl (e.g., tetrazolyl, imidazolyl, pyrazolyl, triazolyl, thiazolyl, and furanyl) group, the reference is intended to include rings optionally having 0 to 3, preferably 0 to 2, substituents selected from the substituents listed above for arylheterocyclyl and/or heteroaryl groups.

As described herein, the term "pharmaceutically acceptable salt" means that the salt is not only physiologically acceptable to the subject, but also refers to a synthetic substance that has pharmaceutical use value, such as a salt formed as an intermediate when performing chiral resolution. Although the salt of this intermediate cannot be directly administered to the subject, the salt can play a role in obtaining the final product of the present invention.

As described herein, the term "disease" refers to a physical state of the subject, which is related to the disease described in the present invention, for example, peripheral arterial disease and neurodegenerative-related diseases described in the present invention.

The cancer treatments of the present invention include standard treatments such as surgery, radiation therapy, chemotherapy and hormone therapy.

"Cancer" or "malignancy" refers to any of a variety of diseases characterized by uncontrolled abnormal proliferation of cells, the ability of affected cells to spread locally or via the bloodstream and lymphatic system to other sites in the body (i.e., metastasis), and any of a number of characteristic structural and/or molecular features. "Cancer cell" refers to a cell that is undergoing an early, intermediate, or advanced stage of multistep neoplastic progression. Cancers include mesothelioma, neuroblastoma, rectal cancer, colon cancer, familial adenomatous polyposis cancer and hereditary nonpolyposis colorectal cancer, esophageal cancer, lip cancer, laryngeal cancer, hypopharyngeal cancer, tongue cancer, salivary gland cancer, gastric cancer, adenocarcinoma, medullary thyroid cancer, papillary thyroid cancer, kidney cancer, renal parenchymal cancer, ovarian cancer, cervical cancer, uterine corpus cancer, endometrial cancer, choriocarcinoma, pancreatic cancer, prostate cancer, bladder cancer, testicular cancer, breast cancer, urinary tract cancer, melanoma, brain tumors, head and neck cancer, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), LL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), hepatocellular carcinoma, gallbladder cancer, bronchial tumor, advanced solid tumors, small cell lung cancer, metastatic non-small cell lung cancer, multiple myeloma, basal cell tumor, teratoma, retinoblastoma, choroidal melanoma, seminoma, rhabdomyosarcoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma and plasmacytoma, lymphoma, pancreatic ductal adenocarcinoma, pancreatic cancer, metastatic colorectal cancer, non-small cell lung cancer, acute myeloid leukemia, squamous cell carcinoma of the skin.

The compound of the present invention or the pharmaceutical composition containing the same can be administered in a unit dosage form, and the administration route can be enteral or parenteral, such as oral, intravenous injection, intramuscular injection, intravenous drip, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, vagina, rectum, etc.

The dosage form can be a liquid dosage form, a solid dosage form or a semisolid dosage form. Liquid dosage forms can be solutions (including true solutions and colloidal solutions), emulsions (including o/w types, w/o types and multiple emulsions), suspensions, injections (including water injections, powder injections and infusions), eye drops, nasal drops, lotions and liniments, etc.; solid dosage forms can be tablets (including ordinary tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules, enteric-coated capsules), granules, powders, micropills, dropping pills, suppositories, films, patches, aerosols (powders), sprays, etc.; semisolid dosage forms can be ointments, gels, pastes, etc.

To achieve the purpose of medication and enhance the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

The compound or composition of the present invention can be taken alone or in combination with other therapeutic drugs or symptomatic drugs. When the compound of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.

The solvents used in this application can be obtained commercially. The abbreviations used in this application are as follows:

Table 1: Meaning of abbreviations

The compounds of the present invention were named manually or using Chemdraw 14.0 software, and commercially available compounds were named according to the supplier's catalog.

Beneficial technical effects

The inventors found that the compounds of the present invention have good KRAS G12D inhibitory activity, and the IC50 is less than that of the positive control drug MRTX-1133. The present invention provides a class of KRAS G12D inhibitor compounds with novel structure and strong activity, which have good application prospects in preventing and/or treating KRAS G12D inhibition-related indications such as reduction or loss of MTAP expression, loss of MTAP gene, reduction of MTAP protein function, etc.

DETAILED DESCRIPTION

The following embodiments are intended to help those skilled in the art better understand the technical solutions of the present invention, but are not intended to limit the present invention in any way.

For all the following examples, standard procedures and methods known to those skilled in the art can be used. Unless otherwise indicated, all temperatures are expressed in ° C (Celsius). The structures of the compounds are determined by nuclear magnetic resonance spectroscopy (NMR) and/or mass spectroscopy (MS).

The structure of the compound of the present invention is determined by nuclear magnetic resonance (NMR) or/and liquid chromatography-mass spectrometry (LC-MS). The NMR chemical shift (δ) is in parts per million (ppm). The NMR is measured using a Bruker avance-400 NMR spectrometer, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated methanol (CD ₃ OD) and deuterated chloroform (CDCl ₃ ) as the solvent, and tetramethylsilane (TMS) as the internal standard.

Liquid phase mass spectrometry LC-MS measurement The liquid phase part uses ACQUITY UPLC ultra-high pressure liquid chromatography, and the mass spectrometry part uses Xevo G2-S Qtof mass spectrometer.

The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

Example 1: 1-{3-(cyclohex-1-en-1-yl)-6-(4-methoxyphenyl)-7-oxo-2-phenyl-4,7-dihydropyrazolo[1,5-a]pyrimidin-5-yl)-3-(pyridin-2-yl}urea (Compound 1)

Step 1: Synthesis of compound 1-1

2-Bromo-4-fluoro-1-nitrobenzene (48.0 g, 219.2 mmol, 1.0 eq.) and tetrabutylammonium bromide (288.0 mg, 1.0 mmol, 0.005 eq.) were added to a mixed solvent of dichloromethane (260 mL) and methanol (340 mL), and sodium hydroxide aqueous solution (30.7 g, 768.0 mmol, 3.5 eq, 1 M) was added at room temperature, and the reaction was carried out at 40°C for 16 hours. After the reaction was complete, the reaction solution was concentrated, extracted with ethyl acetate and water, the organic layer was collected, and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, separated by normal phase column, petroleum ether and ethyl acetate system, and the polarity was 20%, and yellow solid 2-bromo-4-methoxy-1-nitrobenzene (46.1 g, 91.0% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 232.0. ¹ H NMR (400MHz, DMSO-d6) δ8.09 (d, J = 9.1 Hz, 1H), 7.46 (d, J = 2.7 Hz, 1H), 7.17 (dd, J = 9.1, 2.7 Hz, 1H), 3.91 (s, 3H).

Step 2: Synthesis of compound 1-2

2-Bromo-4-methoxy-1-nitrobenzene (46.1 g, 199.6 mmol, 1.0 eq.) and ammonium chloride (52.9 g, 998.0 mmol, 5.0 eq.) were added to a mixed solvent of ethanol (460 ml) and water (92 ml), and reduced iron powder (55.9 g, 998.0 mmol, 5.0 eq.) was added under stirring at room temperature, and the mixture was reacted at 50°C for 16 hours. After the reaction was completed, the mixture was filtered while hot, and the filter cake was washed three times with hot ethyl acetate. The filtrate was concentrated under reduced pressure, extracted with ethyl acetate, separated, dried over anhydrous sodium sulfate, filtered, concentrated, and separated by a normal phase column. The dichloromethane and methanol system was used to obtain an orange-red liquid 2-bromo-4-methoxyaniline (44.0 g, 99.7% yield) when the polarity was 7%.

LCMS (ESI) [M+H] ⁺ : 202.0.

Step 3: Synthesis of Compound 1-3

After adding 2-bromo-4-methoxyaniline (40.0 g, 199.0 mmol, 1.0 eq.) to 1,2-dichloroethane (500 mL) solution, the nitrogen atmosphere was replaced three times, and boron trichloride (1 M, 298.5 mL, 1.5 eq.), chloroacetonitrile (28.4 g, 378.1 mmol, 1.9 eq.), and titanium tetrachloride (101.2 g, 298.5 mmol, 1.5 eq.) were added in sequence at 0°C. After the addition, the mixture was heated under reflux for 16 hours. After the reaction, water was added to quench the reaction under ice bath, and the pH was adjusted to about 4 with dilute hydrochloric acid. The mixture was extracted with dichloromethane, separated, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The mixture was separated by normal phase column, and the polarity of petroleum ether and ethyl acetate system was 10%, and yellow solid 1-(2-amino-3-bromo-5-methoxyphenyl)-2-chloroethane-1-one (11.5 g, 20.9% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 278.0. ¹ H NMR (400MHz, DMSO-d6) δ7.48 (d, J＝2.8 Hz, 1H), 7.36 (d, J＝2.8 Hz, 1H), 5.16 (s, 2H), 3.74 (s, 3H).

Step 4: Synthesis of Compound 1-4

1-(2-amino-3-bromo-5-methoxyphenyl)-2-chloroethane-1-one (11.5 g, 41.5 mmol, 1.0 eq.) was added to a mixed solution of 1,4-dioxane (120 mL) and water (12 mL), and sodium borohydride (1.7 g, 46.5 mmol, 1.1 eq) was slowly added at room temperature, and refluxed at 90°C for 16 hours. After the reaction was completed, the reaction solution was concentrated, extracted with ethyl acetate and water, and the organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The filtrate was separated by a normal phase column, and the polarity of petroleum ether and ethyl acetate system was 10%, and yellow solid 7-bromo-5-methoxy-1H-indole (6.5 g, 69.6% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 226.0. ¹ H NMR (400MHz, DMSO-d6) δ11.13(s,1H),7.35(dd,J＝3.1,2.0Hz,1H),7.09(d,J＝2.2Hz,1H),6.98(d,J＝2.2Hz,1H),6.47(dd,J＝3.1,1.7Hz,1H),3.76(s,3H).

Step 5: Synthesis of Compound 1-5

7-bromo-5-methoxy-1H-indole (5.8 g, 25.8 mmol, 1.0 eq.) was added to N, N-dimethylformamide (60 mL) solution, sodium hydride (60%, 1.2 g, 30.9 mmol, 1.2 eq.) was added under ice bath and stirred for 1 hour, and then iodomethane (7.3 g, 51.6 mmol, 2.0 eq.) was added, and the reaction was carried out at room temperature for 1 hour after the ice bath was removed. After the reaction was completed, water was added under ice bath to quench the excess sodium hydride, and the mixture was extracted with ethyl acetate, separated, and the organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, separated by normal phase column, petroleum ether and ethyl acetate system, and the polarity was 10%, and yellow solid 7-bromo-5-methoxy-1-methyl-1H-indole (4.9 g, 79.5% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 240.0. ¹ H NMR (400MHz, DMSO-d6) δ7.31(d,J＝3.0Hz,1H),7.08(d,J＝2.3Hz,1H),6.97(d,J＝2.3Hz,1H),6.37(d,J＝3.0Hz,1H),4.05(s,3H),3.75(s,3H).

Step 6: Synthesis of Compound 1-6

After replacing _N2 in the three-necked flask, boron tribromide (33% v in DCM, 40 mL) was added. After cooling to -78°C, 7-bromo-5-methoxy-1-methyl-1H-indole (4.9 g, 20.5 mmol, 1.0 eq.) was dissolved in dichloromethane (30 mL) and slowly added to the reaction system. After the addition was complete, the reaction was maintained at -50°C for 6 hours. After the reaction was complete, water was added at 0°C to quench, ethyl acetate was extracted, the liquid was separated, the organic layer was collected, and it was washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The normal phase column was separated, and the petroleum ether and ethyl acetate system was used. When the polarity was 20%, a yellow solid 7-bromo-1-methyl-1H-indole-5-ol (1.6 g, 34.7% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 226.0. ¹ H NMR (400MHz, DMSO-d6) δ9.07(s,1H),7.25(d,J＝3.1Hz,1H),6.88–6.83(m,2H),6.28(d,J＝3.0Hz,1H),4.03(s,3H).

Step 7: Synthesis of Compound 1-7

7-Bromo-1-methyl-1H-indole-5-ol (1.6 g, 7.1 mmol, 1.0 eq.) and N,N-diisopropylethylamine (1.8 g, 14.2 mmol, 2.0 eq.) were added to a dichloromethane (20 mL) solution, and chloromethyl methyl ether (1.0 g, 12.8 mmol, 1.8 eq.) was added at 0°C, and the reaction was carried out at room temperature for 16 hours. After the reaction was completed, dichloromethane and water were extracted, the organic layer was collected, and washed with dilute brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated, separated by a normal phase column, and a petroleum ether and ethyl acetate system was used. When the polarity was 10%, a colorless liquid 7-bromo-5-(methoxymethoxy)-1-methyl-1H-indole (1.1 g, 57.6% yield) was obtained.

LCMS (ESI) [M+H] ⁺ : 269.0.

Step 8: Synthesis of Compound 1-8

2,4,6-trichloropyrimidine-5-carboxaldehyde (29.0 g, 138.2 mmol, 1.0 eq.) and triethylamine (13.9 g, 138.2 mmol, 1.0 eq.) were added to a methanol (200 mL) solution, and ammonia water (7.1 g, 142.3 mmol, 1.03 eq.) was added dropwise at -15 °C and stirred for 4 hours. After the reaction was completed, the reaction solution was concentrated and separated by a normal phase column. When the polarity of petroleum ether and ethyl acetate system was 10%, a yellow solid 4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (18.4 g, 70.8% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 189.0. ¹ H NMR (400 MHz, DMSO-d6) δ 14.62 (s, 1H), 8.50 (s, 1H).

Step 9: Synthesis of Compound 1-9

4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidine (7.8 g, 41.5 mmol, 1.0 eq.) and N,N-diisopropylethylamine (10.7 g, 83.0 mmol, 2.0 eq.) were added to a dichloromethane (80 mL) solution, and 2-(trimethylsilyl)ethoxymethyl chloride (8.3 g, 49.8 mmol, 1.2 eq.) was added at 0°C, and the mixture was reacted at room temperature for 16 hours. After the reaction was completed, the reaction solution was concentrated and separated by a normal phase column. When the polarity of the petroleum ether and ethyl acetate system was 25%, a white solid 4,6-dichloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine (6.8 g, 51.6% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 319.0. ¹ H NMR (400MHz, DMSO-d6) δ8.62 (s, 1H), 5.74 (s, 2H), 3.58 (t, J = 8.6Hz, 2H), 0.89–0.79 (m, 2H), -0.08 (s, 9H).

Step 10: Synthesis of Compound 1-10

4,6-Dichloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidine (6.8 g, 21.4 mmol, 1.0 eq.) and N,N-diisopropylethylamine (5.5 g, 42.8 mmol, 2.0 eq.) were added to a dichloromethane (60 mL) solution, and tert-butyl 3,8-diazabicyclo[3.2.1]octane-8-carboxylate hydrochloride (5.3 g, 21.4 mmol, 1.0 eq.) was added at 0°C, and stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was concentrated and separated by a normal phase column. When the polarity of the petroleum ether and ethyl acetate system was 25%, a white solid tert-butyl 3-(6-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (9.9 g, 70.8% yield) was obtained. LCMS (ESI) [M+H] ⁺ : 495.2. ¹ H NMR (400MHz, CDCl ₃ -d6) δ7.94(s,1H),5.67(s,2H),4.44(s,2H),3.69–3.57(m,2H),2.01(d,J＝7.2Hz,2H),1.70(s,2H),1.49(s,9H),1.34–1.17(m,2 H),0.96–0.89(m,2H),0.87–0.77(m,2H),-0.04(s,9H).

Step 11: Synthesis of Compound 1-11

(1-Methyl-1H-imidazol-4-yl)methanol (2.0 g, 18.2 mmol, 1.3 eq.) was added to N,N-dimethylformamide (80 mL) solution, and sodium hydride (60%, 1.4 g, 36.4 mmol, 2.0 eq.) was added at 0°C. After stirring at 0°C for 1 hour, tert-butyl 3-(6-chloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (6.9 g, 14.0 mmol, 1.0 eq.) was added and reacted at room temperature for 16 hours. After the reaction was completed, water was added at 0°C to quench the reaction, and the mixture was extracted with ethyl acetate. The organic layer was collected and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The mixture was separated by a normal phase column. When the polarity of the dichloromethane and methanol system was 10%, a white solid 3-(6-((1-methyl-1H-imidazol-4-yl)methoxy)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (3.8 g, 36.6% yield) was obtained.

LCMS (ESI) [M+H] ⁺ : 571.3.

Step 12: Synthesis of Compound 1-12

3-(6-((1-methyl-1H-imidazol-4-yl)methoxy)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (3.8 g, 6.7 mmol, 1.0 eq.) was added to a tetrabutylammonium fluoride (50 mL) solution and reacted at 100°C for 16 hours. After the reaction was complete, the liquid was extracted with ethyl acetate and water and collected. The organic layers were collected and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. The mixture was separated by a normal phase column. When the polarity of the dichloromethane and methanol system was 10%, a white solid tert-butyl 3-(6-((1-methyl-1H-imidazol-4-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (1.6 g, 54.6% yield) was obtained. LCMS (ESI) [M+H] ⁺ :441.2. ¹ H NMR (400MHz, DMSO-d6) δ13.21(s,1H),8.13(s,1H),7.54(s,1H),7.16(s,1H),5.13(s,2H),4.29(s,2H),3.63(s,3H),1.85(s,2H),1.61(d,J＝7.6Hz,2 H),1.44(s,9H).

Step 13: Synthesis of Compound 1-13

3-(6-((1-methyl-1H-imidazol-4-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (1.2 g, 2.7 mmol, 2.0 eq.), 7-bromo-5-(methoxymethoxy)-1-methyl-1H-indole (365 mg, 1.35 mmol, 1.0 eq.), cuprous iodide (257 mg, 1.35 mmol, 1.0 eq.), 1,10-phenanthroline (432 mg, 2.7 mmol, 2.0 eq.), potassium carbonate (373 mg, 2.7 mmol, 2.0 eq.) were added to N,N-dimethylformamide (20 mL) solution and reacted at 150°C for 16 hours. After the reaction was completed, the solid was filtered, and the filtrate was separated by reverse phase column, and water (0.1% Formic acid) and acetonitrile system, polarity is 45%, to give 130 mg brown crude product, by HPLC purification, white solid 3-(1-(5-(methoxy-methoxy)-1-methyl-1H-indol-7-yl)-6-((1-methyl-1H-imidazol-4-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (10 mg, 1.2% yield) was obtained.

LCMS (ESI) [M+H] ⁺ : 630.3.

Step 14: Synthesis of Compound 1

3-(1-(5-(methoxy-methoxy)-1-methyl-1H-indol-7-yl)-6-((1-methyl-1H-imidazol-4-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (10 mg, 0.0159 mmol, 1.3 eq.) was added to a methanol (0.5 mL) solution, and concentrated 1 drop of hydrochloric acid was added, and the mixture was stirred at 60°C for 2 hours. After the reaction was complete, the mixture was purified by HPLC to obtain a white solid 7-(4-(3,8-diazabicyclo[3.2.1]octan-3-yl)-6-((1-methyl-1H-imidazol-4-yl)methoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-1-methyl-1H-indol-5-ol (1.7 mg, 22.0% yield). LCMS (ESI) [M+H] ⁺ :486.2. ¹ H NMR (400MHz, DMSO-d6) δ9.09(s,1H),8.36(s,1H),8.21(s,1H),7.48(s,1H),7.19(d,J＝3.0Hz,1H),7.04(d,J＝2.2Hz,1H),6.90(s,1H),6.58(d,J＝2.2 Hz,1H),6.36(d,J=3.0Hz,1H),5.00(s,2H),4.47(s,1H),4.04(s,1H),3.62(s,3H),3.56(s,4H),3.02(s,3H),1.81–1.58(m,4H).

Example 2: 4-((1R,5S)-3,8-diazabicyclo[3.2.1]oct-3-yl)-6-(((S)-1-methylpyrrolidin-2-yl)methoxy)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine (Compound 2)

Step 1: Synthesis of 2-1

At -70°C, phenylhydrazine (256.0 mg, 2.36 mmol, 1 eq.) and N,N-diisopropylethylamine (986.0 ml, 2.36 mmol, 1 eq.) were added to a solution of 2,4,6-trichloro-5-pyrimidinecarboxaldehyde (500 mg, 2.36 mmol, 1 eq.) in anhydrous ethanol (5.0 ml) in order, and the mixture was incubated at 0°C for 1 hour. After the reaction was completed, the mixture was diluted with water and extracted with ethyl acetate, and dried to obtain a white solid 4,6-dichloro-1-phenyl-1-hydrogen-pyrazolo[3,4-D]pyrimidine (300.0 mg, 47.9% yield). LCMS (ESI) [M+H] ⁺ : 265.0. ¹ H NMR (500MHz, Chloroform-d) δ8.52(s,1H),8.15–8.08(m,2H),7.53–7.46(m,2H),7.39–7.31(m,1H).

Step 2: Synthesis of compound 2-2

To a solution of 4,6-dichloro-1-phenyl-1hydrogen-pyrazolo[3,4-D]pyrimidine (200.0 mg, 7.6 mmol, 1.0 eq.) in N-dimethylformamide (3.0 ml) were added tert-butyl (1R,5S)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (160 mg, 7.6 mmol, 1.0 eq.) and N,N-diisopropylethylamine (88.0 mg, 8.3 mmol, 1.1 eq.), and the mixture was stirred at room temperature overnight. After the reaction was completed, water was added for dilution, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride, and the organic phases were combined and dried over anhydrous sodium sulfate. The mixture was separated by a normal phase column, and the polarity of petroleum ether and ethyl acetate system was 20%, to obtain white solid tert-butyl (1R, 5S)-3-(6-chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (260.0 mg, 78.2% yield). LCMS (ESI) [M+H]+: 441.17. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.59 (s, 1H), 8.02 (d, J = 8.6Hz, 2H), 7.58 (t, J = 8.0Hz, 2H), 7.40 (t, J = 7.4Hz, 1H), 4.64–3.83 (m, 4H), 1.79 (d, J = 79.0Hz, 4H), 1.45 (s,9H).

Step 3: Synthesis of compound 2-3

To a solution of the compound tert-butyl (1R, 5S)-3-(6-chloro-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (200.0 mg, 0.45 mmol, 1 eq.) in DMF (5.0 ml) were added the compound N-methyl-L-prolinol (100 mg, 1.84 mmol, 4.0 eq.), cesium carbonate (440.0 mg, 2.3 mmol, 5.0 eq.) and triethylenediamine (200.0 mg, 0.69 mmol, 1.5 eq.), and the mixture was stirred at 100°C for 6 hours. After the reaction was completed, water was added for dilution, and the mixture was extracted with ethyl acetate, washed with saturated sodium chloride, and the organic phases were combined and separated by a normal phase column (neutral alumina). When the polarity of the petroleum ether and ethyl acetate system was 50%, a white solid tert-butyl (1R, 5S)-3-(6-(S)-1-methylpyrrolidin-2-yl)methoxy)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (130.0 mg, 55.2% yield) was obtained. LCMS (ESI) [M+H]+: 520.3. ¹ H NMR (400MHz, DMSO-d ₆ ) δ8.17(t,J＝12.6Hz,1H),7.55(t,J＝7.9Hz,1H),7.46(s,3H),4.38(d,J＝27.9Hz,3H),3.04–2.96(m,1H),2.05–1.99(m,1H),1.9 1(s,1H),1.75(s,1H),1.72–1.66(m,2H),1.64(s,23H),1.60–1.50(m,3H),1.47(s,4H),0.91(m,J＝18.6,10.4Hz,1H).

Step 4: Synthesis of compound 2

To the compound tert-butyl (1R, 5S) -3- (6- (S) -1-methylpyrrolidin-2-yl) methoxy) -1-phenyl -1H- pyrazolo [3, 4-d] pyrimidin-4-yl) -3, 8-diazabicyclo [3.2.1] octane -8-carboxylate (120.0 mg,), add 4M hydrogen chloride-dioxane solution and stir at room temperature for 0.5 hours. After the reaction is completed, spin dry to obtain a brown solid 4- ((1R, 5S) -3, 8-diazabicyclo [3.2.1] octane -3-yl) -6- (((S) -1-methylpyrrolidin-2-yl) methoxy) -1-phenyl -1H- pyrazolo [3, 4-d] pyrimidine (90.0 mg, 92.9%). LCMS (ESI) [M + H] +: 420.24. ¹ H NMR(400MHz,DMSO-d ₆ )δ8.41(s,1H),8.20–8.11(m,4H),7.57–7.50(m,2H),7.36–7.30(m,1H),4.39(m,J＝10.8,4.7Hz,2H),4.18–4.10(m,2H),3.81(d,J＝4.5Hz,4H),3.00(m,J＝9.1,4.4Hz,1H),2.71–2.66(m,1H),2.39(s,3H),2.29–2.21(m,1H),2.00–1.92(m,1H),1.85–1.79(m,2H),1.73–1.60(m,5H).

Example 3: 6-(3,8-diazabicyclo[3.2.1]oct-3-yl)-2-(((S)-1-methylpyrrolidin-2-yl)methoxy)-9-phenyl-9H-purine (Compound 3)

Step 1: Synthesis of compound 3-1

Compound 2,6-dichloro-9H-purine (1.00 g, 5.29 mmol), triethylamine (0.803 mg, 7.94 mmol), 3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (1.24 g, 5.82 mmol) in ethanol (10 ml) was reacted at 60 degrees for 12 hours. The system was cooled to room temperature, solid precipitated, water (10 mL) was added, stirred and filtered, and the filter cake was dried to obtain a yellow-white solid (1R, 5S)-3-(2-chloro-9H-purine-6-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (1.60 g, yield 82.9%).

LCMS (ESI) [M+H] +: 309.08

Step 2: Synthesis of compound 3-2

Dissolve the compound (1R,5S)-3-(2-chloro-9H-purine-6-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (400 mg, 1.09 mmol), phenylboronic acid (200 mg, 1.64 mmol), pyridine (432 mg, 5.48 mmol), and copper acetate (298 mg, 1.64 mmol) in dioxane (20 ml) and react at 40 degrees for 12 hours. Cool to room temperature, add water and ethyl acetate, and then add a few drops of ammonia until the aqueous phase is no longer blue, separate and extract twice, combine the organic phases and wash once with saturated brine, spin dry the organic phase and purify it through a column to obtain a yellow solid (1R, 5S)-3-(2-chloro-9-phenyl-9H-purine-6-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (850 mg, yield 87.9%). LCMS (ESI) [M+H] +: 441.17.

Step 3: Synthesis of compound 3-3

Compound (1R,5S)-3-(2-chloro-9-phenyl-9H-purine-6-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (100 mg, 0.226 mmol), (S)-(1-methylpyrrolidin-2-yl)methanol (78.36 mg, 0.680 mmol), cesium fluoride (103 mg, 0.680 mmol) were dissolved in dimethyl sulfoxide (2 ml), heated to 13 degrees and reacted for 16 hours. After cooling to room temperature, solids precipitated. Water was added, stirred and filtered. The filter cake was dissolved in ethyl acetate, mixed, column-filtered and purified to obtain yellow solid (1R,5S)-3-(2-((S)-1-methylpyrrolidin-2-yl)methoxy)-9-phenyl-9H-purin-6-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (310 mg, yield 65.7%). LCMS (ESI) [M+H]+: 520.30.

Step 4: Synthesis of compound 3

Trifluoroacetic acid (138 mg, 1.21 mmol) was added to a solution of tert-butyl (1R, 5S)-3-(2-(((S)-1-methylpyrrolidin-2-yl)methoxy)-9-phenyl-9H-purin-6-yl)-3,8-diazabicyclo[3.2.1]octane-8-carboxylate (210 mg, 0.404 mmol) in dichloromethane (5 ml), and the mixture was reacted at room temperature for 3 hours. The pH of the reaction solution was adjusted to 8, and the mixture was extracted with dichloromethane three times. The organic phases were concentrated and combined to obtain a crude product, which was dissolved in methanol to obtain a white solid 6-(3,8-diazabicyclo[3.2.1]oct-3-yl)-2-(((S)-1-methylpyrrolidin-2-yl)methoxy)-9-phenyl-9H-purine (25 mg, purity 99.15%, yield 14.7%).

LCMS(ESI)[M+H]+:420.25; ¹ H NMR(400MHz,DMSO-d6):δ8.45(s,1H),8.20(s,2H),7.87–7.80(m,2H),7.59 (t,J＝7.9Hz,2H),7.50–7.42(m,1H),5.17(d,J＝334.9Hz,2H),4.32(dd,J＝10.9,4.8Hz,1H),4.08(dd, J＝10.9,6 .5Hz,1H),4.01(s,2H),3.41(s,2H),3.01(dt,J＝9.4,4.5Hz,1H),2.68(p,J＝6.4Hz,1H),2.39(s, 3H), 2.27 (q, J＝8.7Hz, 1H), 2.02–1.85 (m, 3H), 1.67 (dddd, J＝36.4, 18.8, 9.5, 6.0Hz, 5H). ¹ H NMR (400 MHz, D ₂ O): δ8.43 (s, 2H), 8.22 (s, 1H), 7.70–7.55 (m, 5H), 5.24 (s, 2H), 4.58 (dd, J=12.9 ,6.4Hz,1H),4.33(s,2H),3.86(d,J＝7.7Hz,1H),3.68(d,J＝13.5Hz,3H),3.26–3.15(m,1H),2.96(s , 3H), 2.43–2.31 (m, 1H), 2.27–1.94 (m, 7H). The compounds in Table 2 were obtained by referring to the method of Example 1:

Table 2: Structure and characterization of compounds 4-7

Experimental Example 1: Binding ability test of the compound of the present invention to KRAS G12D protein

Experimental purpose: To detect the binding ability of the compound to KRAS G12D protein by CETSA experimental method

Background principle: The CETSA experiment is a molecular detection method to measure the affinity between drugs and target proteins. The principle is that after the drug binds to the target protein, its structure becomes more stable. If the candidate drug is an inhibitor of KRAS G12D, the candidate drug will be able to bind to KRAS G12D, making the KRAS G12D protein more stable. After the sample is heated, the KRAS G12D protein in the sample will be more easily detected by specific antibodies, and the KRAS G12D protein will be more easily detected by Western blot experiments; conversely, the stability of the KRAS G12D protein will be worse after heating, and the amount of protein detected will be lower. In this way, the binding ability of the drug to the target protein is evaluated, which is used for the screening of MAT2A protein inhibitors.

Specific experimental process:

KRAS G12D protein expression: Full-length KRAS G12D was cloned into the pET24N vector with an n-terminal (His) 6x tag and a tobacco etch virus (TEV) protease cleavage site. The constructed vector was transformed into Escherichia coli ArcticExpress (DE3), and the cells were shaken to an OD of 0.6, 1 mM IPTG was added, and cultured at 12°C for 16 h. The cells were collected, ultrasonically disrupted, and the supernatant was taken by centrifugation. The protein was purified by Ni-NTA, and the protein concentration and purity were determined after dialysis.

CETSA sample preparation: Incubate the samples with candidate drugs, control drugs and control reagents for 30 minutes, heat each group of samples at about 10 set temperature points; return to room temperature, centrifuge the samples at 20,000g, and collect the supernatant; use sample buffer to denature the protein samples at 100°C for 10 minutes. After the samples return to room temperature, perform western blot on the samples; the protein loading amount is controlled at 20ug. After determining the mutation temperature, set the concentration gradient of the compound to generally 9 points, incubate the samples, perform the same operation as above, and perform western blot detection. Protein electrophoresis: Set the voltage of the concentrated gel to 60v and the voltage of the separation gel to 120v; after the electrophoresis, start electrotransfer. The conditions of electroporation were set at 250 mA for 2 hours; 5% BSA was blocked for 1 hour; specific antibody 1 was added and incubated overnight on a shaker at 4°C; TBST was washed 4 times, 2.5 minutes each time; 2 antibodies were incubated on a shaker at room temperature for 1 hour; TBST was washed 4 times, 2.5 minutes each time; ECL was used for development to detect the expression of TYK2 protein in different groups and at each temperature point. Western blot bands were converted and processed by image J and GraphPad software to calculate EC50.

EC50 is the concentration for 50% of maximal effect (EC50), which refers to the drug concentration that can cause 50% of individuals to be effective. The compound MRTX-1133 was prepared using the synthesis method reported in Example 252 of CN114615981A as a positive reference compound.

The MRTX-1133 structure is as follows:

The test results are shown in Table 3 below. The EC50 values of each compound are classified according to the following description:

“+” indicates that the EC50 value is greater than 1 μM;

“++” indicates that the EC50 value is less than 1 μM and greater than 100 nM;

“+++” indicates that the EC50 value is less than 100 nM and greater than 10 nM;

"++++" indicates that the EC50 value is less than 10 nM.

The lower the EC50, the stronger the binding affinity of the compound to the Kras G12D protein.

Table 3: EC50 results

EC50 experimental data show that the compounds of the present invention have good activity in stimulating KRAS G12D cytokines and have good KRAS G12D protein agonist function. Compared with the positive control compound, the compounds of the present invention have comparable or even higher activity in stimulating immune cells to produce cytokines.

Experimental Example 2: Determination of the effect of the compounds of the present invention on AGS cell proliferation

Experimental purpose: The purpose of this test example is to test the effect of compounds on AGS cell proliferation.

Background principle: KRAS protein maintains normal biological function by responding to extracellular signals and switching between the activated state (ON) bound to GTP and the inactivated state (OFF) bound to GDP. For mutated KRAS proteins, GTP entry into GFP is restricted, thereby preventing hydrolysis, forming a continuously activated GTP-bound state. KRAS activity therefore becomes independent of extracellular stimuli, leading to excessive stimulation of downstream pathways and induction of cell proliferation, migration, and metastasis signals, which promotes the proliferation, survival, and metastasis of cancer cells. AGS is a cell line with KRAS G12D mutation. Therefore, the inhibition rate of compounds on AGS cell proliferation is detected for screening of KRAS G12D protein inhibitors.

Specific experimental process:

AGS cells in the logarithmic growth phase were inoculated into 96-well plates, 90uL per well, 3000 cells/well, and left to stand overnight in a 37°C incubator. On the second day, 10μl of compounds of different concentrations (DMSO final concentration was 1%) were added and incubated in a 37°C incubator for 3 days. On the third day, the old culture medium was removed, 110ul of culture medium (the ratio of culture medium to CCK8 was 100:10) was added, and the cells were incubated at 37°C for 1-4h. The absorbance was detected at 450nM, and the IC50 was calculated by GraphPad software. The compounds were screened by comparing with positive drugs.

IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It can indicate the half amount of a drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). The compound MRTX-1133 was prepared using the synthesis method reported in Example 252 of CN114615981A as a positive reference compound.

The test results are shown in Table 4 below, where the IC50 values of each compound are classified according to the following description:

+++ less than 100 nM, ++ between 100 nM and 1 μM, + greater than 1 μM.

The test results are shown in Table 4 below:

Table 4: IC50 experimental data

Compound No. IC50 for 24h IC50 at 48h IC50 at 72h 1 +++ ++ +++ 2 ++ ++ +++ 3 ++ ++ +++ 4 ++ ++ +++ 5 ++ +++ +++ 6 ++ ++ +++ 7 + ++ +++ 8 + ++ ++ 9 + ++ +++ 10 + ++ +++ MRTX-1133 + ++ ++

The results show that the compounds of the present invention have a very strong inhibitory effect on KRAS G12D, and the IC50 values all reach nM level, which is lower than the positive control drug MRTX-1133. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to KRAS G12D inhibition.

Experimental Example 3: Determination of the effect of the compounds of the present invention on cellular ERK protein phosphorylation

Experimental purpose: The purpose of this test case is to test the inhibitory ability of the compound on cellular ERK protein phosphorylation.

Background and Principle: After KRAS protein is activated, it can activate multiple downstream signaling pathways, including the MAPK-ERK signaling pathway. After ERK phosphorylation, it activates downstream signals and participates in cell survival, proliferation and cytokine release. Therefore, after compound incubation, WB detection of ERK phosphorylation level in AGS cells can be used for the screening of KRAS G12D protein inhibitors.

Specific experimental process:

AGS cells in the logarithmic growth phase were inoculated into 24-well plates, 500uL per well, 1x10^5 cells/well, and left in a 37°C incubator overnight. The next day, different concentrations of compounds were added (DMSO final concentration was 1%), and the cells were incubated in a 37°C incubator for 3h. After the incubation, the cells were collected and tested by WB. Protein electrophoresis: The voltage of the concentrated gel was set to 60v, and the voltage of the separation gel was set to 120v; after the electrophoresis, electrotransfer was started. The conditions for electrotransfer were set to 250mA, 2h; 5% BSA was blocked for 1h; specific 1 antibody was added and incubated on a shaker at 4°C overnight; TBST was washed 4 times, 2.5min each time; 2 antibodies were incubated on a shaker at room temperature for 1h; TBST was washed 4 times, 2.5min each time; ECL was used for development. Western blot bands were converted and processed by image J and GraphPad software, IC50 was calculated, and compounds were screened.

IC50 (half maximal inhibitory concentration) refers to the half inhibitory concentration of the measured antagonist. It can indicate the half amount of a drug or substance (inhibitor) in inhibiting certain biological processes (or certain substances contained in this process, such as enzymes, cell receptors or microorganisms). The compound MRTX-1133 was prepared using the synthesis method reported in Example 252 of CN114615981A as a positive reference compound.

The test results are shown in Table 5 below, where the IC50 values of the compounds are classified according to the following descriptions:

+++ is less than 100 nM, ++ is between 100 nM and 1 μM, and + is greater than 1 μM.

The test results are shown in Table 5 below:

Table 5: Inhibition test results of the compounds of the present invention on cellular ERK protein phosphorylation

The results show that the compounds of the present invention have a very strong inhibitory effect on KRAS G12D, and the IC50 values all reach nM level, which is lower than the positive control drug MRTX-1133. This strong inhibitory effect has important therapeutic significance for the treatment of symptoms or diseases related to KRAS G12D inhibition.

The above examples are only representative. It can be seen from the above examples that the compounds of the present invention are ideal and highly effective KRAS G12D inhibitors, and can be expected to be used for treating or preventing disorders or diseases associated with KRAS G12D inhibition.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, and they should be covered by the protection scope of the present invention.

Claims (10)

1. A compound represented by the formula (I) and an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that,

A is selected from CH or N;

b is selected from CH or N;

e is selected from CH or N;

F is selected from CH or N;

G is selected from N or hydroxyl;

l is selected from O or N;

Ring C represents a substituted or unsubstituted, saturated or unsaturated 8-to 10-membered N-containing bridged ring containing at least one additional heteroatom selected from N, O, S;

R ₁ is selected from 5-10 membered aryl, 5-10 membered heteroaryl with 1 to 4 heterocyclic ring members N, O, S; each R ₁ is independently at each occurrence optionally substituted with one or more of the following groups: hydroxy, halogen, -NH ₂、-CN、C₁-C₆ -alkyl, C ₁-C₆ -alkoxy;

m=0 or 1;

Z is selected from 5-8 membered heterocyclyl or heteroaryl having 1 to 4 heterocyclic ring members N, O, S; each Z is independently at each occurrence optionally substituted with one or more of the following groups: hydroxy, halogen, -NH ₂、-CN、C₁-C₆ -alkyl, C ₁-C₆ -alkoxy.

2. The compound of claim 1, wherein R ₁ is selected from the following structures:

The groups are optionally substituted with one or more of the following groups: hydroxy, halogen, -NH ₂、-CN、C₁-C₆ -alkyl, C ₁-C₆ -alkoxy.

3. The compound of claim 1, wherein ring C is selected from the group consisting of isomers thereof, and pharmaceutically acceptable salts thereofThe halogen is F, cl, br, I, preferably F; the C ₁-C₆ alkyl is a linear alkyl, branched alkyl, or cycloalkyl, preferably a linear alkyl, more preferably methyl; the C ₁-C₆ alkoxy group is a linear alkyl oxy group, a branched alkyl oxy group or a cycloalkyl oxy group, preferably methoxy.

4. The compound according to claim 1, wherein the ring Z is selected from the group consisting of isomers thereof, and pharmaceutically acceptable salts thereof

5. The compound of claim 1, wherein the compound has the structure of formula (I-1), (I-2), (I-3):

the radicals A, ring C, Z, R ₁, m are as defined in claim 1.

6. The compound of claim 1, wherein the compound is selected from the group consisting of:

7. A process for the preparation of a compound according to any one of claims 1 to 6, or an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that the process comprises the steps of:

the initial raw materials are subjected to three substitution reactions and deprotection reactions to prepare a compound shown in a formula (I);

wherein R ₁, A, B, E, F, G, L, Z, C rings are as defined in claim 1.

8. A pharmaceutical composition comprising a compound of formula I according to any one of claims 1 to 6, and isomers thereof, or pharmaceutically acceptable salts thereof, and one or more pharmaceutical excipients.

9. Use of a compound according to any one of claims 1 to 6, or an isomer thereof, or a pharmaceutically acceptable salt thereof, or a composition according to claim 8 in the manufacture of a medicament for inhibiting KRAS G12D activity in a cell.

10. Use of a compound according to any one of claims 1 to 6 as shown in formula I and isomers thereof, or pharmaceutically acceptable salts thereof, or a pharmaceutical composition according to claim 8 for the manufacture of a medicament; the medicament is preferably for the prevention and/or treatment of KRAS mutation mediated cancer, which KRAS mutein may be a KRAS G12D mutein; the cancer is preferably pancreatic cancer, metastatic colorectal cancer, non-small cell lung cancer, acute myelogenous leukemia, cutaneous squamous cell carcinoma.