Isoindolinone-containing spleen tyrosine kinase inhibitors, methods of making and using the same

Abstract

The application discloses a kind of containing isoindole ketone spleen tyrosine kinase inhibitor and its preparation method and application, belong to the technical field of medicinal chemistry.The application uses active group splicing method, retains the trimethoxy phenyl and pyrimidine of Fostamatinib structure as parent nucleus, replaces pyridoxylidene oxazinone with isoindole ketone, designs and synthesized 16 target compounds (general formula is shown below), and test the antitumor, antibacterial and anti-inflammatory activity of the compound, wherein, compound 6a is superior to positive control drug on the inhibition of human B lymphocyte (Raji), 6l on human breast cancer cell (MCF-7) and 6j human hepatoma cell (HepG2);Compound 6l has the best antibacterial activity, 6i and 6j are better than positive control drug Fostamatinib in anti-inflammatory activity, therefore, the isoindole ketone-containing spleen tyrosine kinase inhibitor of the application has good development prospect.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, and particularly relates to an isoindole-containing spleen tyrosine kinase inhibitor, its preparation method, and its application. Background Technology

[0002] Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase that participates in multiple signal transduction pathways and plays an important role in controlling cell differentiation, proliferation, and spread. It is an effective target for the treatment of autoimmune diseases and inflammation (Mócsai, A. et al. Nat. Rev. Immunol. 2010, 10, 387~402.), and a potential target for the treatment of rheumatoid arthritis, allergic rhinitis, asthma, kidney disease, and liver fibrosis. Currently, it has been confirmed that SYK can destroy platelets in patients with ITP (immune thrombocytopenia), leading to a decrease in platelet count (Crowley, MT et al. J. Exp. Med. 1997, 186, 1027~39). In addition to hematopoietic cells, SYK is also expressed in non-hematopoietic cells, including fibroblasts, epithelial cells, hepatocytes, neurons, and vascular endothelial cells (Bradshaw, JM Cell. Signal. 2010, 22, 1175~84; Yanagi, S. et al. Biochem. Biophys. Res. Commun. 2001, 288, 495~498).

[0003] Spleen tyrosine kinase (SYK) inhibitors are a class of targeted therapies that regulate immune cell signaling pathways by inhibiting SYK kinase activity, and have significant potential in the treatment of autoimmune diseases and cancer. Fostamatinib (structural formula shown below) is currently the only approved small molecule SYK inhibitor, with its main targets being SYK, FLT3, and MDR-1. In 2018, the FDA approved it for the treatment of immune thrombocytopenic purpura (Markham, A. Drugs. 2018, 78, 959~963). However, fostamatinib has adverse reactions such as gastrointestinal disturbances, neutropenia, and hypertension (Currie, KSet al. Med. Chem. 2014, 57, 3856~73).

[0004]

[0005] Isoindolones are the basic skeletal structure of many compounds with important biological and pharmaceutical activities. As a class of heterocyclic alkaloids, they are mainly found in natural products, such as lenalidomide, which is used to treat multiple myeloma. Therefore, this invention aims to develop a spleen tyrosine kinase inhibitor containing isoindolone, with the intention of using it in drugs related to abnormal SYK expression and for anti-tumor, antibacterial, and anti-inflammatory diseases. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a class of spleen tyrosine kinase inhibitors containing isoindole, their preparation methods, and applications. This invention employs an active group splicing method, retaining the trimethoxyphenyl and pyrimidine cores of the fostamatinib structure, and replacing pyridoxazinone with isoindole. Sixteen target compounds were designed and synthesized, and their antitumor, antibacterial, and anti-inflammatory activities were tested. It was found that some compounds exhibited superior antitumor, antibacterial, and anti-inflammatory activities compared to the positive control group, demonstrating promising development prospects.

[0007] The technical solution of the present invention is as follows: having an isoindole-containing spleen tyrosine kinase inhibitor as shown in Formula 6, a pharmaceutically acceptable salt, solvate or prodrug.

[0008]

[0009] In general formula 6, R is any one of hydrogen, methyl, ethyl, n-propyl, cyclopropyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, tetrahydropyranyl, 2-hydroxyethyl, and 3-piperidin-2,6-dione.

[0010] Pharmaceutically acceptable salts include conventional, non-toxic salts, including quaternary ammonium salts formed from the base of such compounds and inorganic or organic acids. The compounds of this invention can form solvates. Methods for forming solvates by concentrating compounds with suitable organic solvents are known to those skilled in the art. A prodrug is a compound obtained by chemically modifying a drug, which is inactive or has low activity in vitro but releases its active drug effect in vivo through enzymatic or non-enzymatic conversion.

[0011] The isoindole-containing spleen tyrosine kinase inhibitor described in this invention is one of the following compounds: 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2,3-dihydro-isoindol-1-one (6a); 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-methyl-2,3-dihydro-isoindol-1-one (6b); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-ethyl-2,3-dihydro-isoindol-1-one (6c); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-propyl-2,3-dihydro-isoindol-1-one (6d); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-isopropyl-2,3-dihydro-isoindol-1-one (6e); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-cyclopropyl-2,3-dihydro-isoindol-1-one (6f); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-butyl-2,3-dihydro-isoindol-1-one (6g); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-isobutyl-2,3-dihydro-isoindol-1-one (6h); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-tert-butyl-2,3-dihydro-isoindol-1-one (6i); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-pentyl-2,3-dihydro-isoindol-1-one (6j); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-cyclopentyl-2,3-dihydro-isoindol-1-one (6k); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-hexyl-2,3-dihydro-isoindol-1-one (6l); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-cyclohexyl-2,3-dihydro-isoindol-1-one (6m); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-tetrahydropyrano-2,3-dihydro-isoindol-1-one (6n); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-(2-hydroxy-ethyl)-2,3-dihydro-isoindol-1-one (6o); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-(2,6-dione-3-piperidinyl)-2,3-dihydro-isoindol-1-one (6p).

[0012] Furthermore, the synthetic route for compound 6 is as follows:

[0013] As shown above, its preparation method is as follows: 1) Using DCM (dichloromethane) as a solvent, methyl 2-methyl-3-nitrobenzene (1) and... N - Bromosuccinimide (NBS) undergoes a bromination reaction under the catalysis of azobisisobutyronitrile (AIBN) to generate intermediate (2); 2) Using DMF as a solvent, triethylamine (TEA) is added. Intermediate (2) reacts with different primary amines to undergo nucleophilic substitution and amine-ester exchange reactions to generate intermediates (3a~3o); 3) Then, using methanol and tetrahydrofuran as solvents, in a system of zinc powder and ammonium chloride, the nitro group of intermediate (3a~3o) was reduced to an amino group to obtain intermediate (4a~4o); 4) The intermediate (4a~4o) or lenalidomide reacts with 2,4-dichloro-5-fluoropyrimidine in the presence of triethylamine and methanol, and the amino group of the indoleone undergoes a substitution reaction with the chlorine at the 4-position of the pyrimidine ring to generate the intermediate (5a~5p). 5) Finally, using the classic Buchwald reaction, with 1,4-dioxane as solvent, under nitrogen protection, in the presence of Xantphos (4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene) and Cs2CO3, and catalyzed by Pd2(dba)3, 3,4,5-methoxyaniline undergoes CN coupling with the 2-position chlorine of the pyrimidine ring of the intermediate (5a~5p) to give the final product 6a~6p. Intermediate 5, used to prepare the isoindole-containing spleen tyrosine kinase inhibitor, comprises: 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2,3-dihydro-isoindol-1-one (5a); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-methyl-2,3-dihydro-isoindol-1-one (5b); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-ethyl-2,3-dihydro-isoindol-1-one (5c); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-n-propyl-2,3-dihydro-isoindol-1-one (5d); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-isopropyl-2,3-dihydro-isoindol-1-one (5e); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-cyclopropyl-2,3-dihydro-isoindol-1-one (5f); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-n-butyl-2,3-dihydro-isoindol-1-one (5g); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-isobutyl-2,3-dihydro-isoindol-1-one (5h); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-tert-butyl-2,3-dihydro-isoindol-1-one (5i); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-n-pentyl-2,3-dihydro-isoindol-1-one (5j); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-cyclopentyl-2,3-dihydro-isoindol-1-one (5k); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-n-hexyl-2,3-dihydro-isoindol-1-one (5l); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-cyclohexyl-2,3-dihydro-isoindol-1-one (5m); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-tetrahydropyrano-2,3-dihydro-isoindol-1-one (5n); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-(2-hydroxy-ethyl)-2,3-dihydro-isoindol-1-one (5o); 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-(2,6-dione-3-piperidinyl)-2,3-dihydro-isoindol-1-one (5p).

[0014] This invention also provides the use of these compounds in pharmaceuticals for the prevention or treatment of mammalian diseases associated with abnormal SYK expression and possessing antitumor, antiviral, antibacterial, and anti-inflammatory properties. The mammalian diseases associated with abnormal SYK expression and possessing antitumor, antiviral, antibacterial, and anti-inflammatory properties include: autoimmune diseases, cancer, inflammation, hypertension, diabetic nephropathy, infectious viral diseases, and age-related diseases.

[0015] Therefore, the present invention also relates to pharmaceutical compositions comprising a compound of Formula 6, the pharmaceutical composition comprising a compound of Formula 6 and one or more pharmaceutically acceptable carriers and / or excipients. The composition may be formulated as nanoparticles. Oral formulations may include standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. The pharmaceutical carrier used may be solid or liquid: liquid carriers include, for example, saline, buffered saline, glucose, water, glycerol, ethanol, and combinations thereof. A wide variety of pharmaceutical forms may be used when administering the compounds of the present invention. If a solid carrier is used, the formulation may be a tablet, a powder or pill encapsulated in a hard capsule, or a lozenge or sugar tablet. If a liquid carrier is used, the formulation may be a syrup, emulsion, soft capsule, sterile injectable solution or suspension in an ampoule or vial, or a non-aqueous liquid suspension. The technical advantages of this invention are as follows: This invention employs a reactive group splicing method, retaining the trimethoxyphenyl and pyrimidine groups of the fostamatinib structure as the parent nucleus, and replacing the pyridoxazinone with isoyindolone. Sixteen target compounds were designed and synthesized, and the antitumor, antibacterial, and anti-inflammatory activities of these compounds were tested. The results showed that: 1. Based on the inhibitory activity assay results, it can be seen that the spleen tyrosine kinase inhibitors containing isoindolone not only have good inhibitory activity against human B lymphocytes, but also against other tumor cells. Among them, compound 6a showed the best inhibitory effect on human B lymphocytes (Raji), 6l on human breast cancer cells (MCF-7), and 6j on human liver cancer cells (HepG2), which was superior to the positive control drug. This indicates that these three compounds have potential advantages in treating tumors and are potential drugs for tumor treatment.

[0016] 2. In vitro antibacterial activity tests have shown that compounds 6a, 6b, 6c, 6d, 6i, 6l, and 6o have certain antibacterial effects; compounds 6a, 6c, and 6i have comparable antibacterial activity; and compound 6l has the best antibacterial activity and can be developed as a potential antibacterial drug.

[0017] 3. In vitro anti-inflammatory activity tests have demonstrated that all compounds shown in Formula 6 have certain anti-inflammatory effects. In particular, the anti-inflammatory activities of 6i and 6j are better than those of the positive control Fostamatinib, showing good development prospects and potential for development as anti-inflammatory drugs. Detailed Implementation

[0018] The present invention will be further described below with reference to embodiments, but is not limited thereto.

[0019] Example 1. Synthesis of intermediate 2 of the present invention The synthesis of intermediate 2 is described in references (Cao, RX and Jia, YP). Chin .J . Syn . Chemistry 2022, 30 , 90~97).

[0020] Example 2. Synthesis of intermediates 5a-5p of the present invention Intermediate 2 (7.3 mmol), primary amines with different substituents (8.8 mmol), and triethylamine (8.8 mmol) and 30 mL DMF were added to a reaction flask and heated to 80 °C (detected by TLC). After the reactants had reacted completely, the mixture was cooled to room temperature and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous Na₂SO₄, concentrated to dryness, and the residue was subjected to silica gel column chromatography (using ethyl acetate / petroleum ether as eluent). V : V =1:3) Purification yields intermediates 3a~3o.

[0021] Intermediates 3a-3o (5.2 mmol), zinc powder (52.0 mmol), ammonium chloride (52.0 mmol), 20 mL of methanol, and 20 mL of tetrahydrofuran were added to a reaction flask and stirred overnight at room temperature (TLC detection). After the reactants had reacted completely, the mixture was filtered through a diatomaceous earth filter, the filtrate was evaporated to dryness, extracted with ethyl acetate, and the organic phase was washed three times with saturated brine, dried over anhydrous Na₂SO₄, concentrated, and evaporated to dryness. The residue was then subjected to silica gel column chromatography (using methanol / dichloromethane as eluent). V : V =1:15) The purified intermediates 4a~4o were used directly in the next reaction.

[0022] 2,4-Dichloro-5-fluoropyrimidine (3.0 mmol), intermediates 4a-4o, lenalidomide (3.0 mmol), triethylamine (3.6 mmol), and 20 mL of methanol were added to a reaction flask and heated to 60 °C. o Stir overnight at C (TLC detection). After the reactants have reacted completely, evaporate the reaction solution to dryness, extract with ethyl acetate, wash the organic phase three times with saturated brine, dry with anhydrous Na2SO4, concentrate and evaporate to dryness, and then perform silica gel column chromatography (using ethyl acetate / petroleum ether as eluent). V : V =1:5) Purification yielded intermediates 5a~5p.

[0023] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2,3-dihydro-isoindol-1-one (5a): White solid, yield 87%, mp 295~297℃; 1 H NMR (DMSO- d 6, 400 MHz): d4.37 (s, 2H), 7.53~7.61 (m, 2H), 7.65(d, J =7.6 Hz, 1H), 8.36 (d, J =3.2 Hz, 1H), 8.63 (s, 1H), 10.05 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 44.7, 121.0, 128.7 (d, J =57.0 Hz), 133.0, 134.5, 139.1,142.4 (d, J =21.0 Hz), 144.6, 147.2, 152.0 (d, J =12.0 Hz), 153.5, 169.9; HR-MS(ESI) m / z: Calcd for C 12 H9ClFN4O, [M+H] + 279.0443, found 279.0438.

[0024] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-methyl-2,3-dihydro-isoindol-1-one (5b): White solid, yield 68%, mp 289~292℃; 1 H NMR (DMSO- d 6, 400 MHz): d 3.07 (s, 3H), 4.45 (s, 2H), 7.55~7.60 (m, 2H), 7.64 (dd, J =1.2 Hz, J =7.6 Hz, 1H), 8.37 (d, J =3.6 Hz, 1H), 10.10 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 29.5, 51.2, 120.9, 128.8 (d, J =68.0Hz), 132.6, 134.3, 136.8, 142.5 (d, J= 21.0 Hz), 144.6, 147.2, 152.0 (d, J=8.0Hz), 153.6, 167.3; HR-MS(ESI) m / z: Calcd for C 13 H 11 ClFN4O, [M+H] + 293.0600, found293.0597.

[0025] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-ethyl-2,3-dihydro-isoindol-1-one (5c): Pale yellow solid, yield 73%, mp 118~120℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.16 (t, J= 7.2 Hz, 3H), 3.55 (dd, J =7.2 Hz, J =14.4 Hz, 2H), 4.48 (s, 2H), 7.53~7.60 (m, 2H), 7.64 (dd, J =1.6 Hz, J =7.6 Hz, 1H), 8.37 (d, J =3.6 Hz, 1H), 10.09 (s, 1H); 13 C NMR (DMSO- d 6,100 MHz): d 13.8, 36.8, 48.6, 120.9, 128.7 (d, J =78.0 Hz), 132.7, 134.5, 136.8,142.4 (d, J =21.0 Hz), 144.6, 147.2, 151.9 (d, J =12.0 Hz), 153.5, 166.8; HR-MS(ESI) m / z: Calcd for C 14 H 13 ClFN4O, [M+H] + 307.0756, found 307.0757.

[0026] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-n-propyl-2,3-dihydro-isoindol-1-one (5d): White solid, yield 83%, mp 215~218 °C; 1 H NMR (DMSO- d 6, 400 MHz): d 0.87 (t, J =7.6 Hz,3H),1.61 (m,2H), 3.48 (t, J =7.2 Hz,2H), 4.47 (s,2H), 7.53~7.60 (m, 2H), 7.64 (dd, J =1.2 Hz, J =7.6 Hz, 1H), 8.36 (d, J =3.2 Hz, 1H), 10.07 (s, 1H); 13 C NMR (DMSO- d 6,100 MHz): d 11.6, 21.5, 43.7, 49.3, 120.9, 128.7 (d, J =83.0 Hz), 132.7, 134.4,136.8, 142.4 (d, J =20.0 Hz), 144.6, 147.2, 151.9 (d, J =12.0 Hz), 153.5, 167.3;HR-MS(ESI) m / z: Calcd for C 15 H 15 ClFN4O, [M+H] + 321.0913, found 321.0913.

[0027] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-isopropyl-2,3-dihydro-isoindol-1-one (5e): Pale yellow solid, yield 83%, mp 106~108 °C; 1 H NMR (DMSO- d 6, 400 MHz): d 1.22 (d, J =6.8 Hz, 6H),4.46 (m, 3H), 7.53~7.59 (m, 2H), 7.64 (dd, J =1.2 Hz, J= 7.2 Hz, 1H), 8.37 (d, J =3.2 Hz, 1H), 10.09 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 20.9, 42.7, 44.7,120.8, 128.6 (d,J =88.0 Hz), 132.8, 134.7, 136.7, 142.4 (d, J =21.0 Hz), 144.6,147.2, 149.4 (d, J =19.0 Hz), 151.8, 153.5, 166.5; HR-MS(ESI) m / z: Calcd forC 15 H 15 ClFN4O, [M+H] + 321.0913, found 321.0910.

[0028] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-cyclopropyl-2,3-dihydro-isoindol-1-one (5f): white solid, yield 73%, mp 250~253℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.81 (d, J =5.6 Hz, 4H), 2.93~2.99 (m, 1H), 4.39 (s, 2H), 7.52~7.58 (m, 2H), 7.64 (dd, J =1.6 Hz, J =7.2 Hz, 1H), 8.36 (d, J =3.6 Hz, 1H), 10.05 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 5.53,25.5, 49.4, 120.7, 128.8 (d, J =71.0 Hz), 132.6, 134.7, 136.6, 142.4 (d, J =21.0Hz), 144.6, 147.2, 151.8, 153.5, 168.2; HR-MS(ESI) m / z: Calcd for C 15 H 13 ClFN4O, [M+H] + 319.0756, found 319.0757.

[0029] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-n-butyl-2,3-dihydro-isoindol-1-one (5 g): White solid, yield 63%, mp 230~232℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.91 (t, J =8.0 Hz, 3H),1.24~1.32 (m, 2H), 1.55~1.62 (m, 2H), 3.53 (t, J =7.2 Hz, 2H), 4.47 (s, 2H),7.53~7.60 (m, 2H), 7.63 (d, J =7.6 Hz, 1H), 8.37 (d, J =3.2 Hz, 1H), 10.09 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 14.0, 19.9, 30.2, 41.6, 49.2, 120.9, 128.6 (d, J =83.9 Hz), 132.7, 134.4, 136.8, 142.4 (d, J =20.6 Hz), 144.6, 147.2, 151.9(d, J =12.2 Hz), 153.5 (d, J =3.0 Hz), 167.2; HR-MS(ESI) m / z: Calcd for C 16 H 17 ClFN4O, [M+H] + 335.1069, found 335.1064.

[0030] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-isobutyl-2,3-dihydro-isoindol-1-one (5h): Pale yellow solid, yield 45%, mp 181~184℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.88 (d, J =6.4 Hz, 6H),1.97~2.05 (m, 1H), 3.33 (d, J=8.8 Hz, 2H), 4.47 (s, 2H), 7.53~7.60 (m, 2H),7.63(dd, J =1.2 Hz, J =7.6 Hz, 1H), 8.35 (d, J =3.2 Hz, 1H), 10.06(s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 20.4, 27.6, 49.7, 50.0, 121.0, 128.7 (d, J =85.0 Hz),132.7, 134.3, 136.8, 142.4 (d, J =21.0 Hz), 144.6, 151.9, 153.5, 167.5; HR-MS(ESI) m / z: Calcd for C 16 H 17 ClFN4O, [M+H] + 335.1069, found 335.1063.

[0031] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-tert-butyl-2,3-dihydro-isoindol-1-one (5i): Pale yellow solid, yield 55%, mp 91~94℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.49 (s, 9H), 4.57 (s, 2H), 7.52 (m, 2H), 7.62 (t, J =4.4 Hz, 1H), 8.35 (d, J =3.6 Hz, 1H), 10.01(s, 1H); 13 CNMR (DMSO- d 6, 100 MHz): d 28.1, 47.9, 54.3, 120.4, 128.4(d, J =92.0 Hz), 132.5,135.8, 136.1, 142.4(d, J =20.0 Hz), 147.2, 152.0, 153.5, 167.5; HR-MS(ESI) m / z:Calcd for C 16 H 17ClFN4O, [M+H] + 335.1069, found 335.1067. 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-n-pentyl-2,3-dihydro-isoindol-1-one (5j): White solid, yield 72%, mp 238~241℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.86 (t, J =6.8 Hz, 3H),1.23~1.32 (m, 4H), 1.60 (m, 2H), 3.51 (t, J =6.8 Hz, 2H), 4.47 (s, 2H), 7.53~7.60 (m, 2H), 7.63 (d, J =7.6 Hz, 1H), 8.36 (d, J =3.2 Hz, 1H), 10.09 (s, 1H); 13 CNMR (DMSO- d 6, 100 MHz): d 14.3, 22.3, 27.9, 28.9, 41.9, 49.2, 120.9, 128.2,129.1, 132.7, 134.4, 136.8, 142.4 (d, J =21.0 Hz), 144.6, 147.2, 151.8 (d, J =12.0 Hz), 153.5 (d, J =3.0 Hz), 167.2; HR-MS(ESI) m / z: Calcd for C 17 H 19 ClFN4O, [M+H] + 349.1226, found 349.1226.

[0032] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-cyclopentyl-2,3-dihydro-isoindol-1-one (5k): pale yellow solid, yield 65%, mp 217~220℃; 1 H NMR (CDCl3, 400 MHz): d 1.64 (m, 4H), 1.77 (m, 2H), 1.96 (m, 2H), 4.44 (s, 2H), 4.74 (m, 1H), 7.49 (t,J =7.6 Hz, 1H), 7.69 (m,3H), 8.11 (d, J =2.0 Hz, 1H); 13 C NMR (CDCl3, 100 MHz): d 24.1, 33.1, 45.7, 52.8,121.4, 126.1, 129.1, 131.5, 134.7, 135.0, 141.6 (d, J =20.0 Hz), 143.9, 146.5,151.1 (d, J =11.0 Hz), 154.4 (d, J =3.0 Hz), 167.9; HR-MS(ESI) m / z: Calcd forC 17 H 17 ClFN4O, [M+H] + 347.1069, found 347.1068.

[0033] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-hexyl-2,3-dihydro-isoindol-1-one (5 l): Yellow solid, yield 63%, mp 228~231 °C; 1 H NMR (DMSO- d 6, 400 MHz): d 0.85 (t, J =6.8 Hz, 3H),1.27 (s, 6H), 1.59 (m, 2H), 3.51 (t, J =7.2 Hz, 2H), 4.45 (s, 2H), 7.52~7.59(m, 2H), 7.62 (dd, J =1.6 Hz, J =7.6 Hz, 1H), 8.35 (d, J =3.2 Hz, 1H), 10.06 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 14.4, 22.5, 26.4, 28.2, 31.4, 41.9, 49.2,120.9, 128.2, 129.1, 132.7, 134.4, 136.8, 142.5, 151.9, 167.2; HR-MS(ESI) m / z: Calcd for C18 H 21 ClFN4O, [M+H] + 363.1382, found 363.1383.

[0034] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-cyclohexyl-2,3-dihydro-isoindol-1-one (5m): White solid, yield 73%, mp 125~127℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.09~1.18 (m, 1H), 1.33~1.42 (m, 2H), 1.47~1.56 (m, 2H), 1.65 (d, J =12.8 Hz, 1H), 1.72 (d, J =11.2 Hz, 2H), 1.79 (d, J =12.8 Hz, 2H), 4.03 (m, 1H), 4.46 (s, 2H), 7.54~7.59 (m, 2H),7.63 (d, J =7.6 Hz, 1H), 8.37 (d, J =3.2 Hz, 1H), 10.08 (s, 1H); 13 C NMR (DMSO- d 6,100 MHz): d 25.7, 31.2, 45.7, 49.1, 50.6, 120.8, 128.1, 129.0, 132.8, 134.6,136.8, 142.3 (d, J =21.0 Hz), 144.6, 147.2, 151.9 (d, J =13 Hz), 153.5, 166.5;HR-MS(ESI) m / z: Calcd for C 18 H 19 ClFN4O, [M+H] + 361.1226, found 361.1223.

[0035] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-tetrahydropyrano-2,3-dihydro-isoindol-1-one (5n): Yellow solid, 70% yield, mp 202~205℃; 1 H NMR (CDCl3, 400 MHz): d 1.79~1.92 (m, 4H), 3.53~3.60 (m, 2H), 4.07 (dd, J =3.6 Hz, J =11.2 Hz, 2H), 4.43 (s, 2H), 4.49~4.55 (m,1H), 7.19 (s, 1H), 7.55 (t, J =7.6 Hz, 1H), 7.73 (d, J =7.6 Hz, 1H), 7.77 (d, J =7.6 Hz, 1H), 8.14 (d, J =2.4 Hz, 1H); 13 C NMR (CDCl3, 100 MHz): d 31.1, 45.6, 48.1,67.2, 121.7, 126.1, 129.3, 131.5, 134.7, 134.8, 141.7 (d, J =80.0 Hz), 143.9,146.5, 150.9 (d, J =11.0 Hz), 153.5 (d, J =4 Hz), 167.4; HR-MS(ESI) m / z: Calcdfor C 17 H 17 ClFN4O2, [M+H] + 363.1019, found 363.1018.

[0036] 4-(2-Chloro-5-fluoro-pyrimidin-4-amino)-2-(2-hydroxy-ethyl)-2,3-dihydro-isoindol-1-one (5o): pale yellow solid, yield 78%, mp 222~225℃; 1 H NMR (DMSO- d 6, 400 MHz): d 3.58~3.62 (m,4H), 4.56 (s, 2H),4.84 (t, J =4.8 Hz, 1H), 7.54~7.61 (m, 2H), 7.65 (d, J =7.6 Hz, 1H), 8.36 (d, J =3.2 Hz, 1H), 10.08(s, 1H); 13 C NMR (DMSO- d6, 100 MHz): d 45.1,50.3, 59.7, 120.9, 128.4, 129.0, 132.6, 134.4, 137.2, 142.4 (d, J =21.0 Hz),144.6, 147.2, 153.6, 167.3; HR-MS(ESI) m / z: Calcd for C 14 H 13 ClFN4O2, [M+H] + 323.0706, found 323.0705.

[0037] 4-(2-chloro-5-fluoro-pyrimidin-4-amino)-2-(2,6-dione-3-piperidinyl)-2,3-dihydro-isoindol-1-one (5p): White solid, yield 72%, mp 186~189℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.98 (t, J =5.3Hz, 1H), 2.51 (m, 1H),2.59 (d, J =17.2Hz, 1H), 4.42 (dd, J =18.0 Hz, J =31.6 Hz, 2H), 5.17 (dd, J =4.8 Hz, J =13.2 Hz, 1H), 7.59 (t, J =7.6 Hz, 1H), 7.67 (dd, J =7.6Hz, J =10.0 Hz, 2H), 8.36 (d, J =3.6 Hz, 1H), 10.09 (s, 1H), 11.02 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 22.9, 31.6, 46.9, 51.9, 121.3, 129.1(d, J =30.0 Hz),132.9, 133.5, 137.3, 142.3 (d, J =20.0 Hz), 144.6, 147.2, 152.0 (d, J=12.0 Hz),153.5, 168.1, 171.4, 173.3; HR-MS(ESI) m / z: Calcd for C 17 H 14 ClFN5O3, [M+H] + 390.0764, found 390.0764.

[0038] Example 3: Synthesis of intermediates 6a-6p of the present invention Intermediates 5a-5p (1.7 mmol), 3,4,5-trimethoxyaniline (1.7 mmol), Pd2(dba)3 (0.17 mmol), Xantphos (0.34 mmol), and Cs2CO3 (2.0 mmol), along with 20 mL of 1,4-dioxane, were added to a reaction flask. The mixture was then purged with nitrogen and heated to 80°C overnight (TCL detection). After the reactants had reacted completely, the mixture was cooled to room temperature and extracted with ethyl acetate. The organic phase was washed three times with saturated brine, dried over anhydrous Na2SO4, concentrated to dryness, and the residue was subjected to silica gel column chromatography (using ethyl acetate / petroleum ether as eluent). V : V =1:3) Purification yielded the target compounds 6a~6p.

[0039] product: 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2,3-dihydro-isoindol-1-one (6a): Purple solid, yield 58%, mp 268~271℃; 1 H NMR (DMSO- d 6, 400 MHz): d 3.49 (s, 6H), 3.56 (s, 3H), 4.35 (s, 2H), 6.97 (s, 2H), 7.49~7.55 (m, 2H), 7.67 (d, J =7.2Hz, 1H), 8.14 (d, J =3.6 Hz, 1H), 8.52 (s, 1H), 9.05 (s, 1H), 9.39 (s, 1H); 13 CNMR (DMSO- d 6, 100 MHz): d 45.1, 55.9, 60.6, 96.8, 119.9, 127.8, 128.8, 132.5,134.4 (d, J=7.0 Hz), 137.4, 138.8, 139.3, 140.4, 141.3 (d, J =1.2 Hz), 141.8,150.1 (d, J =6.0 Hz), 153.0, 156.0, 170.1; HR-MS(ESI) m / z: Calcd for C 21 H 21 FN5O4, [M+H] + 426.1572, found 426.1570.

[0040] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-methyl-2,3-dihydro-isoindol-1-one (6b): Purple solid, yield 45%, mp 193~196 °C; 1 H NMR (DMSO- d 6, 400 MHz): d 2.98 (s,3H), 3.49 (s, 6H), 3.57 (s, 3H), 4.42 (s, 2H), 6.93 (s, 2H), 7.48~7.55 (m,2H), 7.64 (d, J =7.2 Hz, 1H), 8.15 (s, 1H), 9.05 (s, 1H), 9.46 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 29.4, 51.8, 55.9, 60.6, 97.3, 119.9, 127.7, 128.9, 132.6,134.2 (d, J =36.0 Hz), 136.5, 137.3, 140.0, 141.3 (d, J =19.0 Hz), 142.5, 150.1 (d, J =11.0 Hz),153.0, 156.0, 167.4; HR-MS(ESI) m / z: Calcd for C 22 H 23 FN5O4, [M+H] + 440.1729, found 440.1728.

[0041] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-ethyl-2,3-dihydro-isoindol-1-one (6c): Purple solid, yield 55%, mp 130~133℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.00 (t, J =6.8 Hz, 3H), 3.44 (s, 8H), 3.55 (s, 3H), 4.43 (s, 2H), 6.90 (s, 2H), 7.48~7.55 (m, 2H), 7.64 (d, J =7.2 Hz, 1H), 8.15 (d, J =3.2 Hz, 1H), 9.07 (s, 1H),9.47 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 13.6, 36.7, 49.2, 55.9, 60.5, 97.2,119.9, 127.6, 128.9, 132.6, 134.2 (d, J =45.0 Hz), 136.4, 137.3, 141.4 (d, J =12.0 Hz), 142.0, 149.9 (d, J =7.0 Hz),153.0, 156.0, 166.9; HR-MS(ESI) m / z:Calcd for C 23 H 25 FN5O4, [M+H] + 454.1885, found 454.1884.

[0042] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-n-propyl-2,3-dihydro-isoindol-1-one (6d): Purple solid, yield 55%, mp 117~120℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.75(t, J =7.2 Hz, 3H), 1.38~1.45 (m, 2H), 3.38 (t, J=6.8 Hz, 2H), 3.43 (s, 6H), 3.55 (s, 3H), 4.41 (s, 2H), 6.89 (s, 2H), 7.48~7.55 (m, 2H), 7.63 (d, J =7.2Hz, 1H), 8.14 (d, J =3.6 Hz, 1H), 9.07 (s, 1H), 9.48 (s, 1H); 13 C NMR (DMSO- d 6,100 MHz): d 11.5, 21.4, 43.6, 49.8, 55.8, 60.5, 97.0, 119.9, 127.6, 128.9,132.5, 134.2 (d, J =38.0 Hz), 136.4, 137.3, 140.0, 141.4 (d, J =19.3 Hz), 142.4, 149.8 (d, J =11.4 Hz),153.0, 156.0, 167.3; HR-MS(ESI) m / z: Calcd for C 24 H 27 FN5O, [M+H] + 468.2042, found 468.2042.

[0043] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-isopropyl-2,3-dihydro-isoindol-1-one (6e): grayish-white solid, yield 45%, mp 118~121℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.06(d, J =6.4 Hz, 6H), 3.42 (s, 6H), 3.55 (s, 3H), 4.38 (m, 3H), 6.88 (s, 2H),7.48~7.55 (m, 2H), 7.65 (d, J =7.2 Hz, 1H), 8.15 (d, J =2.8 Hz, 1H), 9.08 (s,1H), 9.48 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 20.7, 42.6, 45.2, 55.9, 60.5,97.2, 119.8, 127.5, 128.9, 132.6, 134.5 (d, J =58.0 Hz), 136.4, 137.3, 140.0,141.4 (d, J =19.0 Hz), 142.4, 149.9 (d, J =11.0 Hz),153.0, 156.0, 166.0; HR-MS(ESI) m / z: Calcd for C 24 H 27 FN5O4, [M+H] + 468.2042, found 468.2040.

[0044] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-cyclopropyl-2,3-dihydro-isoindol-1-one (6f): Yellow solid, yield 23%, mp 182~185℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.65~0.71 (m, 4H), 3.80 (m, 1H), 3.46 (s, 6H), 3.57 (s, 3H), 4.34 (s, 2H), 6.90(s, 2H), 7.48~7.53 (m, 2H), 7.63 (d, J =6.8 Hz, 1H), 8.15 (d, J =2.8 Hz, 1H),9.08 (s, 1H), 9.47 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 5.4, ​​25.3, 50.2, 55.9,60.6, 97.3, 119.7, 127.6, 128.9, 132.7, 134.0, 134.6, 137.2, 140.0, 141.4 (d, J =19.0 Hz), 142.4, 149.8 (d, J =11.0 Hz), 153.0, 156.0 (d, J =3.0 Hz),168.2; HR-MS(ESI) m / z: Calcd for C24 H 25 FN5O4, [M+H] + 466.1885, found 466.1883.

[0045] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-n-butyl-2,3-dihydro-isoindol-1-one (6 g): Purple solid, yield 46%, mp 158~161℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.81(t, J =7.2 Hz, 3H), 1.14~1.20 (m, 2H), 1.37~1.40 (m, 2H), 3.42 (s, 6H), 3.57(m, 5H), 4.41 (s, 2H), 6.91 (s, 2H), 7.48~7.55 (m, 2H), 7.63 (d, J =7.2 Hz, 1H), 8.15 (d, J =2.8 Hz, 1H), 9.09 (s, 1H), 9.50 (s, 1H); 13 C NMR (DMSO- d 6, 100MHz): d 13.9, 19.9, 30.1, 41.6, 49.8, 55.8, 60.5, 97.9, 119.9, 127.6, 128.9,132.5, 134.1, 134.5, 136.4, 137.3, 140.0, 141.4 (d, J =19.0 Hz), 142.4, 149.8 (d, J =11.0 Hz), 153.0, 156.0 (d, J =2.0 Hz),167.2; HR-MS(ESI) m / z: Calcd forC 25 H 29 FN5O4, [M+H] + 482.2198, found 482.2197.

[0046] 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-isobutyl-2,3-dihydro-isoindol-1-one (6h): purple solid, yield 42%, mp 128~131℃; 1H NMR (DMSO- d 6, 400 MHz): d 0.75(d, J =6.4 Hz, 6H), 1.81~1.88 (m, 1H), 3.22 (d, J =7.2 Hz, 2H), 3.43 (s, 6H), 3.54 (m, 3H), 4.42 (s, 2H), 6.90 (s, 2H), 7.48~7.56 (m, 2H), 7.65 (d, J =7.2Hz, 1H), 8.15 (d, J =3.6 Hz, 1H), 9.07 (s, 1H), 9.48 (s, 1H); 13 C NMR (DMSO- d 6,100 MHz): d 20.3, 27.6, 49.7, 50.4, 55.8, 60.5, 96.9, 119.9, 127.7, 128.9,132.5, 134.1, 134.4, 136.5, 137.3, 140.0, 141.4 (d, J =19.0 Hz), 142.4, 149.9 (d, J =11.0 Hz),153.0, 156.0,167.6; HR-MS(ESI) m / z: Calcd for C 25 H 29 FN5O4, [M+H] + 482.2198, found 482.2198.

[0047] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-tert-butyl-2,3-dihydro-isoindol-1-one (6i): Purple solid, yield 40%, mp 54~58℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.36 (s,9H), 3.43 (s, 6H), 3.55 (m, 3H), 4.52 (s, 2H), 6.90 (s, 2H), 7.46~7.50 (m,2H), 7.63 (d, J =2.8 Hz, J =5.6 Hz, 1H), 8.14 (d, J=3.6 Hz, 1H), 9.11 (s, 1H),9.45 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 27.9, 48.5, 54.0, 55.9, 60.5, 97.1,119.3, 127.1, 128.7, 132.6, 133.9, 135.6, 135.9, 137.3, 140.0, 141.5 (d, J =20.0 Hz), 142.4, 149.8,153.0, 155.9,167.6; HR-MS(ESI) m / z: Calcd forC 25 H 29 FN5O4, [M+H] + 482.2198, found 482.2195.

[0048] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-n-pentyl-2,3-dihydro-isoindol-1-one (6j): Purple solid, yield 40%, mp 170~173℃; 1 H NMR (DMSO- d 6, 400 MHz): d 0.80(t, J =6.8 Hz, 3H), 1.15~1.23 (m, 4H), 1.41 (m, 2H), 3.43 (s, 8H), 3.55 (s,3H), 4.42 (s, 2H), 6.91 (s, 2H), 7.48~7.55 (m, 2H), 7.64 (d, J =7.2 Hz, 1H),8.15 (s, 1H), 9.09 (s, 1H), 9.49 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 14.2,22.2, 27.7, 28.9, 41.9, 49.7, 55.8, 60.5, 96.9, 119.9, 127.6, 128.9, 132.5,134.1, 134.5, 136.4, 137.3, 140.0, 141.4 (d, J =19.0 Hz), 142.4, 149.9 (d, J=11.0 Hz),153.0, 156.0,167.2; HR-MS(ESI) m / z: Calcd for C 26 H 31 FN5O4, [M+H] + 496.2355, found 496.2354.

[0049] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-cyclopentyl-2,3-dihydro-isoindol-1-one (6k): Purple solid, yield 52%, mp 130~133℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.40~1.44 (m, 2H), 1.46~1.53 (m, 2H), 1.55~1.60 (m, 2H), 1.71 (d, J =5.6 Hz, 2H),3.41 (s, 8H), 3.55 (s, 3H), 4.40 (s, 2H), 4.46~4.54 (m, 1H), 6.86 (s, 2H),7.47~7.53 (m, 2H), 7.64 (d, J =7.2 Hz, 1H), 8.14 (d, J =3.6 Hz, 1H), 9.09 (s,1H), 9.48 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 24.4, 23.0, 46.4, 52.4, 55.8,60.5, 97.1, 119.7, 127.4, 128.9, 132.6, 134.2, 134.6, 136.1, 137.2, 140.0,141.5 (d, J =19.0 Hz), 142.4, 149.7 (d, J =11.0 Hz),153.0, 155.9,167.1; HR-MS(ESI) m / z: Calcd for C 26 H 29 FN5O4, [M+H] + 494.2198, found 494.2195.

[0050] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)-pyrimidin-4-amino]-2-n-hexyl-2,3-dihydro-isoindol-1-one (6l): purple solid, yield 42%, mp 187~140 °C; 1 H NMR (DMSO- d 6, 400 MHz): d 0.81(d, J =6.4 Hz, 3H), 1.17 (m, 6H), 1.38 (m, 2H), 3.43 (s, 6H), 3.55 (m, 5H), 4.41 (s, 2H), 6.91 (s, 2H), 7.48~7.55 (m, 2H), 7.63 (d, J =7.2 Hz, 1H), 8.15 (d, J =3.2 Hz, 1H), 9.08 (s, 1H), 9.49 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 14.3,22.4, 26.4, 28.0, 31.4, 41.9, 49.8, 55.8, 60.5, 96.8, 119.9, 127.6, 128.9,132.5, 134.1, 134.5, 136.4, 137.3, 140.0, 141.4 (d, J =19.0 Hz), 142.4, 149.9,153.0, 155.9, 156.0, 167.2; HR-MS(ESI) m / z: Calcd for C 27 H 33 FN5O4, [M+H] + 510.2511, found 510.2512.

[0051] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-cyclohexyl-2,3-dihydro-isoindol-1-one (6m): Brown solid, yield 46%, mp 138~141℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.06(brs, 1H), 1.27 (brs, 4H), 1.50 (brs, 2H), 1.58 (d, J=12.0 Hz, 1H), 1.68 (brs,2H), 3.38 (s, 6H), 3.54 (s, 3H), 3.63 (m, 1H), 4.36 (s, 2H), 6.82 (s, 2H),7.48~7.55 (m, 2H), 7.62 (d, J =7.2 Hz, 1H), 8.15 (d, J =2.8 Hz, 1H), 9.09 (s,1H), 9.52 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 25.4, 25.6, 30.8, 46.4, 49.1,50.5, 55.8, 60.4, 97.1, 119.8, 127.4, 128.8, 132.6, 134.2, 134.8, 136.4,137.2, 139.9, 141.5 (d, J =19.0 Hz), 142.4, 149.7 (d, J =11.0 Hz), 153.0, 155.9 (d, J =3.0 Hz), 166.6; HR-MS(ESI) m / z: Calcd for C 27 H 31 FN5O4, [M+H] + 508.2355, found 508.2354.

[0052] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-tetrahydropyrano-2,3-dihydro-isoindol-1-one (6n): purple solid, yield 42%, mp 151~154℃; 1 H NMR (DMSO- d 6, 400 MHz): d 1.42(d, J =10.4 Hz, 2H), 1.49~1.59 (m, 2H), 3.39 (s, 6H), 3.53 (s, 3H), 3.82 (dd, J =3.6 Hz, J =11.2 Hz, 2H), 4.13~4.19 (m, 1H), 4.39 (s, 2H), 6.84 (s, 2H), 7.49~7.56 (m, 2H), 7.63 (d,J =7.6 Hz, 1H), 8.15 (d, J =3.6 Hz, 1H), 9.10 (s, 1H),9.53 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 30.8, 46.5, 48.1, 55.8, 60.5,66.8,97.1, 119.8, 127.5, 128.9, 132.6, 134.2, 134.6, 136.3, 137.2, 140.0, 141.6(d, J =19.0 Hz), 142.4, 149.7 (d, J =11.0 Hz),153.0, 155.9, 166.8; HR-MS(ESI) m / z: Calcd for C 26 H 29 FN5O5, [M+H] + 510.2147, found 510.2150.

[0053] 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-(2-hydroxy-ethyl)-2,3-dihydro-isoindol-1-one (6o): purple solid, yield 33%, mp 182~185℃; 1 H NMR (DMSO- d 6, 400 MHz): d 3.49 (brs, 10H), 3.57 (s, 3H), 4.53 (s, 2H), 4.78 (d, J =4.8 Hz, 1H), 6.94 (s,2H), 7.50~7.56 (m, 2H), 7.67 (d, J =7.6 Hz, 1H), 8.15 (d, J =3.2 Hz, 1H), 9.02(s, 1H), 9.45 (s, 1H); 13 C NMR (DMSO- d 6, 100 MHz): d 45.0, 50.8, 56.0, 59.7,60.6,97.2, 119.9, 127.7, 128.9, 132.6, 134.0, 134.4, 136.8, 137.3, 140.0,141.3 (d,J =20.0 Hz), 142.5, 150.1 (d, J =12.0 Hz),153.0, 156.0, 167.5; HR-MS(ESI) m / z: Calcd for C 23 H 25 FN5O5, [M+H] + 470.1834, found 470.1833.

[0054] 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-(2,6-dione-3-piperidinyl)-2,3-dihydro-isoindol-1-one (6p): gray solid, yield 39%, mp 183~186 °C; 1 H NMR (DMSO- d 6,400 MHz): d 1.75 (brs, 1H), 2.21 (m, 1H), 2.87 (t, J =12.9 Hz, 1H), 3.45 (s, 6H), 3.55 (s, 3H), 4.39 (dd, J =17.2 Hz, J =78.9 Hz, 2H), 5.08 (d, J =8.8 Hz, 1H), 6.88(s, 2H), 7.55 (d, J =7.4 Hz, 1H), 7.60 (d, J =6.5 Hz, 1H), 7.73 (d, J =7.2 Hz, 1H),8.14 (s, 1H), 9.04 (s, 1H), 9.44 (s, 1H), 10.96 (s, 1H); 13 C NMR (DMSO- d 6, 100MHz): d 22.7, 31.6, 47.2, 51.8, 55.9, 60.5,97.0, 120.2, 128.4, 129.1, 132.3,133.5, 134.2, 137.2 (d, J =42.0 Hz), 141.0, 150.1 (d, J=12.0 Hz),152.9, 154.4,168.1, 171.4, 173.3; HR-MS(ESI) m / z: Calcd for C 26 H 26 FN6O6, [M+H] + 537.1892, found 537.1891.

[0055] Example 4: In vitro antiproliferative activity assay of the target compound (In in vitro ) Human B lymphocytes (Raji), human breast cancer cells (MCF-7), and human liver cancer cells (HepG2) were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The MTT assay was used to test the inhibitory effect of the target compound on the proliferation of different tumor cells. The target compound (6a-6p) was prepared at five different concentrations (50 μM, 10 μM, 2 μM, 0.4 μM, and 0.08 μM). After digestion and counting, human B lymphocytes (Raji), human breast cancer cells (MCF-7), and human liver cancer cells (HepG2) were added to 96-well plates at a rate of 5000 cells / well and cultured overnight. Different concentrations of the compound were then added, and the plates were incubated at 37°C with 5% CO2 for 44 h. MTT was then added, and the plates were incubated for another 4 h. The supernatant was then centrifuged, and 150 μL of DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader, and the inhibition rate was calculated. Fostamatitinib was used as a positive control. This class of compounds exhibits inhibitory effects on three types of tumor cells: human B lymphocytes (Raji), human breast cancer cells (MCF-7), and human liver cancer cells (HepG2). Its IC50 value... 50 The values ​​are shown in Table 1.

[0056] Inhibition rate (%) = [1 - (OD)] 实验 -OD 空白 ) / (OD 阴性 -OD 空白 )]×100% Table 1. In vitro antiproliferative activity of compounds 6a-6p against three types of tumor cells (IC50) 50 (μM)

[0057] Table 1 shows that most compounds exhibited some inhibitory activity against Raji, MCF-7, and HepG2 tumor cells, while a few compounds showed superior inhibitory activity against tumor cells compared to the positive control. Compounds 6a and 6o demonstrated good inhibitory activity against Raji cells, superior to the positive control fostamatinib, with compound 6a showing the best inhibitory activity (IC50). 50The concentration was 1.56 μmol / L. By analyzing the order of Raji cell inhibitory activity, the effect of the substituent R on its structure-activity relationship was examined. When R was H and 2-hydroxyethyl, the inhibitory activity was more than twice that of the positive control drug, and better than that of R with alkyl, cycloalkyl, and tetrahydropyranyl groups. This suggests that the R group is suitable for introducing polar or water-soluble substituents, which can enhance binding to the target and increase the inhibitory activity against Raji cells. Based on the inhibitory activity assay results, it can be seen that the spleen tyrosine kinase inhibitor containing isoindolone not only has good inhibitory activity against human B lymphocytes, but also against other tumor cells, providing experimental reference data for research on broad-spectrum anti-tumor therapy using this type of inhibitor.

[0058] In this invention, compound 6a showed the best inhibitory effect on human B lymphocytes (Raji), 6l on human breast cancer cells (MCF-7), and 6j on human liver cancer cells (HepG2), which was superior to the positive control drug. This indicates that these three compounds have potential advantages in treating tumors and are potential drugs for treating tumors with good development prospects. They can also serve as lead compounds for the discovery of novel and highly effective spleen tyrosine kinase inhibitors.

[0059] Example 5: In vitro antibacterial activity test of the target compound (In in vitro ) Determination of antibacterial activity of compounds using a modified broth dilution method: MRSA strains preserved in our laboratory were inoculated into broth culture medium, incubated overnight on a shaker, and then the OD was measured using a microplate reader. 600 The value was adjusted to 0.09-0.13, and after dilution 100 times, approximately 1×10⁻⁶ was obtained. 5 CFU / mL bacterial culture was inoculated into 96-well plates at 100 µL / well. Different concentrations (50 μM, 10 μM, 2 μM, 0.4 μM, 0.08 μM) of the compound were added to the 96-well plates, with three replicates for each concentration. The 96-well plates were then incubated at 37 °C for 18 h, and the OD values ​​were measured afterward. 600 Fostamatinib was used as a positive control. The inhibition rate was calculated according to the following formula, and its MIC was... 50 The values ​​are shown in Table 2.

[0060] Inhibition rate (%) = [1 - (OD)] 实验 -OD 空白 ) / (OD 阴性 -OD 空白 )]×100% Table 2. In vitro antibacterial activity (MIC) of compounds 50 (μM)

[0061] "—" indicates no inhibitory effect. As shown in Table 2, compounds 6a, 6b, 6c, 6d, 6i, 6l, and 6o have certain antibacterial effects; 6a, 6c, and 6i have comparable antibacterial activities; and 6l has the best antibacterial activity and can be developed as a potential antibacterial drug.

[0062] Example 6: In vitro anti-inflammatory test of the target compound (In in vitro ) The Griess method was used to determine NO release (a linear relationship was observed between the optical density (OD) value of the reaction product and the NO concentration): The mouse mononuclear macrophage leukemia cell line RAW 264.7 (Wuhan Pronosei Life Sciences Co., Ltd.) was routinely cultured in high-glucose DMEM medium. When the cell confluence reached over 80%, the cells were digested and counted using 0.25% trypsin-EDTA digestion solution at a concentration of 2 × 10⁻⁶ cells / mL. 4 The samples were evenly seeded into 96-well plates at a density of [number] samples / well. The experiment included a negative control group, an LPS induction group (1 μg / mL LPS treatment), and a compound group (10 μmol / L). The compound group included 16 compounds and fostamatinib. After adding the target compounds, the plates were incubated at 37°C with 5% CO2 for 24 hours. The NO content in the supernatant of each well was determined according to the NO detection kit instructions. The optical density (OD value) of each well was measured at 540 nm using a microplate reader. The IC50 of the NO content was calculated using the standard curve method. 50 .

[0063] Table 3. In vitro anti-inflammatory activity (μM) of compounds

[0064] As shown in Table 3, all compounds have certain anti-inflammatory effects. The anti-inflammatory activities of 6a and 6e are comparable to those of fostamatinib. In particular, the anti-inflammatory activities of 6i and 6j are better than those of the positive control fostamatinib, showing good development prospects and potential for development as anti-inflammatory drugs.

Claims

1. A pharmaceutically acceptable salt, solvate, or prodrug of an isoindole-containing spleen tyrosine kinase inhibitor as shown in Formula 6; in, R is any one of hydrogen, methyl, ethyl, n-propyl, cyclopropyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, cyclopentyl, n-hexyl, cyclohexyl, tetrahydropyranyl, 2-hydroxyethyl, and 3-piperidin-2,6-dione.

2. The isoindole-containing spleen tyrosine kinase inhibitor as described in claim 1, characterized in that, It is one of the following compounds: 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2,3-dihydro-isoindol-1-one (6a); 4-[5-fluoro-2-(3,4,5-methoxy-anilino)pyrimidin-4-amino]-2-methyl-2,3-dihydro-isoindol-1-one (6b); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-ethyl-2,3-dihydro-isoindol-1-one (6c); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-propyl-2,3-dihydro-isoindol-1-one (6d); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-isopropyl-2,3-dihydro-isoindol-1-one (6e); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-cyclopropyl-2,3-dihydro-isoindol-1-one (6f); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-butyl-2,3-dihydro-isoindol-1-one (6g); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-isobutyl-2,3-dihydro-isoindol-1-one (6h); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-tert-butyl-2,3-dihydro-isoindol-1-one (6i); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-pentyl-2,3-dihydro-isoindol-1-one (6j); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-cyclopentyl-2,3-dihydro-isoindol-1-one (6k); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-n-hexyl-2,3-dihydro-isoindol-1-one (6l); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-cyclohexyl-2,3-dihydro-isoindol-1-one (6m); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-tetrahydropyrano-2,3-dihydro-isoindol-1-one (6n); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-(2-hydroxy-ethyl)-2,3-dihydro-isoindol-1-one (6o); 4-[5-fluoro-2-(3,4,5-methoxy-aniline)-pyrimidin-4-amino]-2-(2,6-dione-3-piperidinyl)-2,3-dihydro-isoindol-1-one (6p).

3. The method for preparing the isoindolone-containing spleen tyrosine kinase inhibitor according to claim 2, characterized in that, Includes the following steps: ； 1) Using dichloromethane as a solvent, methyl 2-methyl-3-nitrobenzene and... N - Bromosuccinimide undergoes a bromination reaction under the catalysis of azobisisobutyronitrile to generate intermediate (2); 2) Using DMF as a solvent, triethylamine is added. Intermediate (2) reacts with different primary amines, undergoing nucleophilic substitution and amine-ester exchange reactions to generate intermediates (3a~3o); 3) Then, using methanol and tetrahydrofuran as solvents, in a system of zinc powder and ammonium chloride, the nitro group of intermediate (3a~3o) was reduced to an amino group to obtain intermediate (4a~4o); 4) The intermediate (4a~4o) or lenalidomide reacts with 2,4-dichloro-5-fluoropyrimidine in the presence of triethylamine and methanol, and the amino group of the indoleone undergoes a substitution reaction with the chlorine at the 4-position of the pyrimidine ring to generate the intermediate (5a~5p). 5) Using the classic Buchwald reaction, with 1,4-dioxane as solvent, under nitrogen protection, in the presence of Xantphos and Cs2CO3, and catalyzed by Pd2(dba)3, 3,4,5-methoxyaniline undergoes CN coupling with the 2-position chlorine of the pyrimidine ring of the intermediate (5a~5p) to give the final product 6a~6p.

4. The use of the isoindolone-containing spleen tyrosine kinase inhibitor of claim 1 in the prevention or treatment of mammalian diseases associated with abnormal SYK expression, as well as those related to antitumor, antiviral, antibacterial, and anti-inflammatory effects.

5. The application as described in claim 4, characterized in that, The mammalian diseases mentioned include: autoimmune diseases, cancer, inflammation, hypertension, diabetic nephropathy, infectious viral diseases, and age-related diseases.

6. A pharmaceutical composition comprising the isoindolone spleen tyrosine kinase inhibitor of claim 1, characterized in that, The pharmaceutical composition comprises a compound of Formula 6 and one or more pharmaceutically acceptable carriers and / or excipients.

7. The pharmaceutical composition of claim 6, characterized in that, The pharmaceutical composition is in the form of nanoparticles.

8. The pharmaceutical composition of claim 6, characterized in that, Using a solid carrier, the formulation is a tablet or a hard capsule, wherein the substance placed in the hard capsule is in the form of a powder, a small pill, an ingot, or a sugar tablet.

9. The pharmaceutical composition of claim 6, characterized in that, Liquid carriers are used, and the formulations are syrups, emulsions, soft capsules, sterile injectable solutions, or suspensions.

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