A fused pyrimidine compound or a pharmaceutically acceptable salt thereof, and a preparation method and application thereof

Abstract

The application discloses a fused pyrimidine compound or a pharmaceutically acceptable salt thereof, a preparation method and application thereof, wherein the fused pyrimidine compound is shown in a structural formula I, and compared with the prior art, the fused pyrimidine compound or the pharmaceutically acceptable salt thereof is a novel compound with a structure and a function of inhibiting p97 protein, and as a p97 protein inhibitor, the fused pyrimidine compound or the pharmaceutically acceptable salt thereof can block tumor cell proliferation and induce tumor cell apoptosis, thereby being used for treating and preventing various diseases of human and animals such as malignant tumors, and the effect is remarkable.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of fused pyrimidine compounds or its pharmaceutically acceptable salt and preparation method and application thereof, belong to the technical field of pharmaceutical chemistry. BACKGROUND

[0002] Cdc48 (Cell Division Cycle 48) was first isolated and identified in 1982 by Moir et al. in Saccharomyces cerevisiae, which is an ATPase. It was named because of its close relationship with cell cycle arrest. Later, Koller and Brownstein found a mammalian homolog, which was named p97 or Valosine Containing Protein (VCP). Mammalian p97 is composed of six identical subunits, each consisting of an N-terminal domain, a D1 domain, a D2 domain and a C-terminal extension, with a molecular weight of 92KD. Studies have shown that the D1 domain is involved in oligomerization and hexamer assembly, and is the main factor for the stability of the hexamer. The D1 and D2 domains of each subunit contain conserved ATP binding (Walker A) and hydrolysis (Walker B) sequences, as well as a second homologous region (SRH) necessary for efficient hydrolysis. Therefore, p97 contains 12 ATPase active sites. However, under physiological conditions (37℃), the D1 domain only shows weak ATPase activity, while the D2 domain has strong ATPase activity.

[0003] P97 is one of the most abundant proteins in the cytoplasm of eukaryotic cells, which is involved in many cellular processes, including ubiquitin proteasome system (UPS) mediated protein degradation, endoplasmic reticulum associated degradation (ERAD), nuclear membrane fusion after mitotic completion, Golgi reorganization, transcription activation and autophagy, cell cycle control, apoptosis and chaperone activity. P97 is involved in various protein quality control pathways, and the most studied is its role in ERAD. P97 transfers misfolded polyubiquitinated proteins from the endoplasmic reticulum lumen to the cytosol, and then transports them to the proteasome for degradation.

[0004] Nuclear factor kappa B (NF-κB) is an important transcription factor. NF-κB is involved in the regulation of a variety of genes and plays an important role in physiological and pathological processes through the regulation of inflammation, apoptosis and immune response. In the basic state, the NF-κB heterodimer composed of proteins p50 and p65 is combined with inhibitor protein I-κBα (NF-κB inhibitor α) or related proteins to maintain an inactive state and prevent the NF-κB dimer from entering the nucleus to bind to DNA. The activation of the transcription factor requires the degradation of I-κBα, which depends on p97. When cells are stimulated by factors such as bacteria, viruses, inflammatory factors, tumor necrosis factor, radiation and drugs, as part of the signal cascade, both p65 and I-κBα are phosphorylated. After phosphorylation, Cullin-RING ubiquitin ligase CRL1β-TrCP ubiquitinates I-κBα to recruit p97. p97 binds to polyubiquitinated I-κBα and dissociates from NF-κB, which is then degraded by 26S proteasome. The dissociated NF-κB dimer translocates to the nucleus to regulate its target genes.

[0005] Studies have shown that siRNA-induced p97 knockdown can cause ER stress and activate UPR, leading to apoptosis through UPS inhibition and caspase activation. p97 is highly expressed in various solid and hematological tumors, such as non-small cell lung cancer, pancreatic cancer, breast cancer and leukemia, and plays an important role in maintaining cellular protein homeostasis. P97 inhibition can preferentially kill cancer cells with high protein synthesis load. Given the important role of p97 in ERAD and NF-κB regulation, targeting p97 inhibition can retain most of the efficacy of proteasome inhibitors but with less toxicity compared to inhibiting proteasome function. However, although a variety of p97 inhibitors have been developed, most of them cannot be developed into drugs. Therefore, there is a need to develop compounds suitable for inhibiting p97 activity and having good drugability for the treatment of tumors. SUMMARY

[0006] The first object of the present application is to provide a fused pyrimidine compound or a pharmaceutically acceptable salt thereof; the second object of the present application is to provide a preparation method of the fused pyrimidine compound or the pharmaceutically acceptable salt thereof, and the third object of the present application is to provide an application of the fused pyrimidine compound or the pharmaceutically acceptable salt thereof in the preparation of a VCP protein inhibitor or a drug for blocking the proliferation of tumor cells.

[0007] Technical solution: The fused pyrimidine compound or the pharmaceutically acceptable salt thereof of the present application, the structure of the fused pyrimidine compound is shown in formula I,

[0008]

[0009] wherein R is selected from -COORa , -CON(R a )2, -CONHS(O) t R a , wherein each R a is independently hydrogen, hydroxyl, alkyl, haloalkyl, alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, 3-8 membered saturated or partially unsaturated monocyclic carbocyclic ring, phenyl, 4-8 membered saturated or partially unsaturated monocyclic heterocyclic ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or 8-10 membered bicyclic heteroaromatic ring having 1-5 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and any combination thereof, each t is independently an integer from 1-2; R 1 is independently hydrogen, hydroxyl, amino, or boronic acid; R 2 is independently methyl or deuterated methyl; A, B, C, X, Y, Z are each independently carbon or nitrogen; D is independently -NH, oxygen, or sulfur.

[0010] Further, R is selected from -COOR a , -CON(R a )2, -CSN(R a )2, -CONHS(O)2R a , wherein each R a is independently hydrogen, alkyl, alkoxy, 5-6 membered monocyclic heteroaromatic ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and any combination thereof; R 1 is independently hydrogen or hydroxyl; R 2 is independently methyl or deuterated methyl; A, B, C, X, Y, Z are each independently carbon or nitrogen; D is independently -NH, oxygen, or sulfur.

[0011] Still further, the fused pyrimidine derivative is selected from:

[0012]

[0013]

[0014] The method for preparing the fused pyrimidine compound or a pharmaceutically acceptable salt thereof, comprises the following steps:

[0015] (1) preparing a dichloropyrimidine mother nucleus

[0016]

[0017] B (pyrido[2,3-d]pyrimidine-2,4-diol) is obtained by reaction of A (2-aminonicotinic acid) in a solution of urea, followed by hydrolysis, B is reduced to C (5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-2,4-diol) using palladium hydroxide as catalyst in acetic acid as solvent, C is converted to the dichloride D, then the Boc protecting group is introduced on the amino group to obtain E (tert-butyl 2,4-dichloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate);

[0018] (2) Preparation of methyl 2-methyl-1H-indole-4-carboxylate

[0019]

[0020] F (4-bromo-1H-indole) is reacted with benzenesulfonyl chloride in the presence of sodium hydride to obtain the protected G (indole), G is reacted with iodomethane in the presence of LDA to obtain H (indole with methyl at position 2), H is deprotected in the presence of sodium hydroxide to obtain I (4-bromo-2-methyl-1H-indole), I is subjected to a carbonylation reaction in the presence of a catalyst to obtain J (methyl 2-methyl-1H-indole-4-carboxylate);

[0021] (3) Preparation of the target compound fused pyrimidine compound V15

[0022]

[0023] E (tert-butyl 2,4-dichloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate) is reacted with benzylamine in the presence of triethylamine as base to obtain K, K is coupled with J (methyl 2-methyl-1H-indole-4-carboxylate) in the presence of a palladium catalyst to obtain the intermediate L (methyl carboxylate as substituent at position 4 of indole), L is saponified in the presence of lithium hydroxide to obtain the carboxylic acid, which is then acylated to obtain the product N, N is deprotected in the presence of trifluoroacetic acid or HCl to obtain the fused pyrimidine compound V15.

[0024] A pharmaceutical composition comprising the fused pyrimidine compound or a pharmaceutically acceptable salt thereof according to the present application.

[0025] The present application also includes the use of the fused pyrimidine compound or a pharmaceutically acceptable salt thereof according to the present application or the pharmaceutical composition according to the present application in the preparation of a p97 inhibitor.

[0026] The present application also includes the use of the fused pyrimidine compound or a pharmaceutically acceptable salt thereof according to the present application or the pharmaceutical composition according to the present application in the preparation of a medicament for treating a disease associated with p97 activity.

[0027] The present application also includes use of the fused pyrimidine compound or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of the present application in the preparation of a medicament for treating cancer.

[0028] Further, the cancer includes, but is not limited to, prostate cancer, bladder cancer, lung cancer (including small cell or non-small cell cancer), colon cancer, kidney cancer, breast cancer, cervical cancer, endometrial or other uterine cancer, ovarian cancer, testicular cancer, penile cancer, vaginal cancer, urethral cancer, gallbladder cancer, esophageal cancer or pancreatic cancer, multiple myeloma, leukemia.

[0029] Further, the medicament can block tumor cell proliferation, induce tumor cell apoptosis.

[0030] Further, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0031] "Alkyl" refers to straight chain or branched hydrocarbon chain groups, consisting only of carbon and hydrogen atoms, which is unsaturated, having from one to ten carbon atoms (e.g., C1-C10alkyl). Whenever it appears herein, a numerical range such as "1 to 10" refers to each integer in the given range - in this example, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. 10 Alkyl). Typical alkyl groups include but are in no way limited to methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, and the like. Alkyl groups are attached to the rest of the molecule through a single bond, e.g., methyl (Me), ethyl (Et), n-propyl, 1-methylethyl (isopropyl), n-butyl, n-butyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like.

[0032] "Alkoxy" refers to the group -O-alkyl, including 1 to 8 carbon atoms in straight- chain, branched, cyclic configurations, and combinations thereof, which is attached to the parent structure by an oxygen linkage. Examples include methoxy, ethoxy, propyloxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like. "Lower alkoxy" refers to alkoxy groups containing one to six carbons. In some embodiments, C1-C4alkyl is alkyl including 1 to 4 carbon atoms of straight-chain and branched alkyl groups.

[0033] "Heteroaromatic ring" refers to a 5-, 6-, or 10-membered aromatic group (e.g., C5-C 13Heteroaryl) which includes one or more ring heteroatoms selected from nitrogen, oxygen, and sulfur and which can be a monocyclic, bicyclic, tricyclic, or tetracyclic system. Whenever it appears in a herein, a numerical range includes each integer within the given range. A "heteroaromatic" or "heteroaromatic ring" moiety containing N refers to an aromatic group in which at least one ring backbone atom is a nitrogen atom. The polycyclic heteroaromatic ring can be fused or non-fused. The heteroatoms in the heteroaromatic ring are optionally oxidized. One or more nitrogen atoms, if present, are optionally quaternized. The heteroaromatic ring is attached to the rest of the molecule through any atom of the ring.

[0034] Suitable pharmaceutically acceptable acid addition salts can be prepared from inorganic acids or from organic acids. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids can be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, taurinic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic) acid, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric, and galacturonic acids. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates. Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthoate, mesylate, gluconate, lactiobionate, lauryl sulfate, and amino acid salts, and the like.

[0035] A "prodrug" as known in the art is a substance that can be administered to a patient, wherein the substance is converted to the active pharmaceutical ingredient in the patient's body under the action of a biochemical agent, such as an enzyme. Examples of prodrugs include esters of carboxylic acid groups, which can be hydrolyzed by endogenous esterases found in the bloodstream of humans and other mammals.

[0036] If the value of a variable that must be an integer (e.g., the number of carbon atoms in an alkyl group or the number of substituents on a ring) is described as a range, e.g., 0-4, then this means that the value can be any integer between 0 and 4, inclusive, i.e., 0, 1, 2, 3, or 4.

[0037] In different embodiments, the compound or group of compounds as used in the methods of the application can be any of the combinations and / or subcombinations of the embodiments listed above.

[0038] In various embodiments, a compound as shown in any of the Examples or among the exemplary compounds is provided. Limitations can be used with any of the disclosed classes or embodiments, wherein any one or more of the other above disclosed embodiments or materials can be excluded from such classes or embodiments.

[0039] In certain embodiments, the present application relates to methods of inhibiting p97. The fused pyrimidine compounds of the present application used in the methods disclosed herein bind, for example, non-covalently or covalently, to the active site of p97. In certain such embodiments the covalent bond can be reversible or irreversible.

[0040] The compounds of the present application and their pharmaceutical compositions are capable of acting as "inhibitors" of p97, which means that they are capable of blocking or reducing the activity of the enzyme, for example, inhibiting various activities of p97. Inhibitors can act competitively, non-competitively, or un-competitively. Inhibitors can bind reversibly or irreversibly, and thus the term includes compounds of suicide enzymes, or which can cause conformational changes elsewhere on the enzyme.

[0041] The compounds of the present application and their pharmaceutical compositions are capable of acting as therapeutic agents because they are capable of preventing, ameliorating, alleviating, and affecting a disorder or condition, which means that they reduce the occurrence of the disorder or condition in a treated sample relative to an untreated control sample; or delay the onset of or reduce the severity of one or more symptoms of the disorder or condition relative to an untreated control sample in a statistical sample.

[0042] The ability to prevent, ameliorate, alleviate, and affect a relevant condition such as local recurrence (e.g., pain), a disease such as cancer, a complex syndrome such as heart failure, or any other medical condition is well known in the art and includes administering a composition that reduces the incidence of or delays the onset of symptoms of the medical condition in a subject relative to a subject that does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable cancer growths and delaying the occurrence of detectable cancer growths in a treated population relative to an untreated control population, e.g., by a statistically or clinically significant amount.

[0043] Advantages: The present application has the following significant advantages compared to the prior art:

[0044] The fused pyrimidine compounds of the present application or their pharmaceutically acceptable salts are structurally novel compounds with the function of inhibiting p97 protein, which can be used as p97 protein inhibitors, block tumor cell proliferation, and induce tumor cell apoptosis, and thus can be used for the treatment and prevention of various diseases such as malignant tumors in humans and animals, with significant effects. Compared with the existing positive drug CB-5339, it has better rat oral absorption and lower mouse toxicity. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 Figure for the body weight change of the mice continuously dosed for 15 days in Example 13. DETAILED DESCRIPTION

[0046] The technical solutions of the present application are further described below in combination with the drawings.

[0047] Preparation of intermediate 5 in Example 1

[0048] Preparation route and synthesis of intermediate 5:

[0049]

[0050] The specific preparation process is as follows:

[0051] (1) Preparation of the above compound 2 (pyrido[2,3-d]pyrimidine-2,4-diol)

[0052] In a 1L eggplant flask, urea (150g, 2497mmol) was added, then heated to 195℃ to dissolve the urea, and 2-aminonicotinic acid (69g, 499mmol) was slowly added under stirring. After the addition was completed, the reaction was continued for 1.5 hours. Then the reaction was cooled to room temperature, and an aqueous solution of NaOH (600mL, 499mmol) was added, and reacted at 100℃ for 1 hour. The reaction mixture was cooled to room temperature, and the pH was adjusted to 4 with 1 mol / L hydrochloric acid, at which time a large amount of white solid was precipitated, and the filter cake was washed with water, and dried under vacuum at 60℃ to obtain the product pyrido[2,3-d]pyrimidine-2,4-diol (66g, yield 81%).

[0053] The above pyrido[2,3-d]pyrimidine-2,4-diol was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 11.47 (s, 1H), 8.60 (dd, J = 4.8, 1.9 Hz, 1H), 8.26 (dd, J = 7.8, 1.9 Hz, 1H), 7.25 (dd, J = 7.7, 4.8 Hz, 1H). MS (ESI, m / z): 164.1 [M+H] + .

[0054] (2) Preparation of the above compound 3 (5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-2,4-diol)

[0055] The pyrido[2,3-d]pyrimidine-2,4-diol prepared in step (1) (66 g, 6 mmol) was dissolved in acetic acid (900 mL), and water (600 mL) and Pd(OH)2 / C (6.6 g, 10% wt) were added. The reaction mixture was purged with hydrogen gas for 3 times, and reacted under the action of hydrogen balloon at 70 °C for 18 hours. After the reaction was completed, it was filtered under suction while hot, and then the filtrate was concentrated to remove most of the acetic acid. It was left to stand at room temperature overnight, filtered under suction, and the filter cake was washed with water and dried under vacuum at 60 °C to obtain the product 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-2,4-diol (54 g, crude), which was directly used in the next step.

[0056] The 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-2,4-diol was subjected to nuclear magnetic hydrogen spectrum analysis, and the results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 10.10 (d, J = 6.1 Hz, 2H), 5.97 (d, J = 2.7 Hz, 1H), 3.17 (m, 2H), 2.17 (t, J = 6.2 Hz, 2H), 1.74 - 1.56 (m, 2H). MS (ESI, m / z): 168.1 [M+H] + .

[0057] (3) Preparation of the above compound 4 (2,4-dichloro-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine)

[0058] The 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine-2,4-diol prepared in step (2) (30 g, 179.4 mmol) was dissolved in POCl3 (300 mL), and PCl5 (18.7 g, 89.7 mmol) was added. The reaction mixture was stirred at 130 °C for 12 hours. After the reaction was completed, it was cooled to room temperature, and most of the solvent was removed by rotary evaporation under reduced pressure. Then ice water (300 mL) was added, and the aqueous phase was extracted with EA (100 mL x 3). The combined organic phases were dried, rotary evaporated, and then purified by column chromatography to obtain the intermediate 2,4-dichloro-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine (13.5 g, yield 36.8%).

[0059] The 2,4-dichloro-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine was subjected to nuclear magnetic hydrogen spectrum analysis, and the results were as follows: 1 H NMR (400 MHz, CDCl3) δ 7.01 (s, 1H), 3.50 (m, 2H), 2.74 (t, J = 6.4 Hz, 2H), 2.04 - 1.87 (m, 2H). MS (ESI, m / z): 204.1 [M+H] + .

[0060] (4) Preparation of compound 5 (tert-butyl 2,4-dichloro-6,7-dihydropyrido[2,3- d]pyrimidine-8(5H)-carboxylate) described above

[0061] The 2,4-dichloro-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine (13.5 g, 66.2 mmol) prepared in step (3) was dissolved in DCM (150 mL), and then DMAP (1.6 g, 13.2 mmol) and (Boc)2O (15.9 g, 72.8 mmol) were added, and the reaction was carried out at room temperature for 3 hours. After the reaction was completed, water (100 mL) was added, and the mixture was separated, and the aqueous phase was extracted with DCM (100 mL x 2), and the combined organic phase was dried, rotary evaporated, and then purified by column chromatography to obtain the intermediate tert-butyl 2,4-dichloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate (18 g, yield 89.4%).

[0062] The above tert-butyl 2,4-dichloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate was analyzed by1H NMR, and the results were as follows: 1 H NMR (400 MHz, Chloroform-d) δ 3.83-3.76 (m, 2H), 2.79 (t, J = 6.7 Hz, 2H), 2.08-1.96 (m, 2H), 1.58 (s, 9H). MS (ESI, m / z): 305.3 [M+H] + .

[0063] Preparation of intermediate 9c-9h in Example 2

[0064] Preparation route and synthesis of intermediate 9c:

[0065]

[0066] The specific preparation processes are as follows:

[0067] (1) Preparation of compound 7b (4-bromo-1-(phenylsulfonyl)-1H-indole) described above

[0068] To a solution of 6b (4-bromo-1H-indole) (25 g, 127.5 mmol) in THF (200 mL) was added NaH (7.7 g, 191.3 mmol) portionwise slowly at 0 °C. The mixture was continued to stir in ice bath for 30 min, then benzene sulfonyl chloride (27 g, 153 mmol) was added. The reaction was then allowed to warm to room temperature and stirred for another 2 h, then poured into pre-cooled 5% aqueous NH4Cl solution (200 mL). The aqueous phase was separated and extracted with ethyl acetate (100 mL x 3), the combined organic phase was dried and rotary evaporated to give the crude product, which was then recrystallized (EA, PE) to give 4-bromo-1-(phenylsulfonyl)-1H-indole (7b) (38 g, yield 89%) as off-white solid.

[0069] (2) Preparation of the above compound 8b (4-bromo-2-methyl-1-(phenylsulfonyl)-1H-indole)

[0070] In a 250 mL three-necked flask was added anhydrous THF (50 mL) and DIPA (5.9 g, 58.3 mmol), and cooled to -50 °C. Then n-BuLi (2.5 M in THF, 23.3 mL, 58.3 mmol) was added slowly. The reaction was continued to stir at -50 °C for 1 h. 4-bromo-1-(phenylsulfonyl)-1H-indole (9.8 g, 29.2 mmol) prepared in step (1) was dissolved in anhydrous THF (50 mL) and then added slowly into the three-necked flask, and the reaction was continued at -50 °C for 1 h. Then Mel (8.3 g, 58.3 mmol) was added. After 10 min, the reaction was transferred from low temperature to room temperature and continued to react for 3 h. After the reaction was completed, the reaction liquid was poured into pre-cooled 5% aqueous NH4Cl solution (100 mL) and extracted with ethyl acetate (100 mL x 3). The combined organic phase was dried, rotary evaporated and then purified by silica gel column chromatography to give the product 4-bromo-2-methyl-1-(phenylsulfonyl)-1H-indole (6.8 g, yield 66%) as a white solid.

[0071] The above 4-bromo-2-methyl-1-(phenylsulfonyl)-1H-indole was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 HNMR (400 MHz, DMSO-d6) δ 8.06 (m, 1H), 7.93-7.89 (m, 2H), 7.76-7.70 (m, 1H), 7.63-7.58 (m, 2H), 7.46 (dd, J1= 0.7 Hz, J2= 7.8 Hz, 1H), 7.24 (t, J = 8.1 Hz, 1H), 6.60 (t, J = 1.1 Hz, 1H), 2.64 (d, J = 1.1 Hz, 3H).

[0072] (3) Preparation of the above compound 9b (4-bromo-2-methyl-1H-indole)

[0073] In a 2L round bottom flask, add ethanol (600 mL) and 4-bromo-2-methyl-l- (phenylsulfonyl)-lH-indole (42.3 g, 117.8 mmol) prepared in step (2). After stirring and dissolving at room temperature, add sodium hydroxide aqueous solution (4 M, 117 mL, 471 mmol). Then, the reaction mixture is reacted at 50°C for 12 hours. After monitoring the completion of the reaction, the reaction solution is concentrated under reduced pressure to remove ethanol, then water (200 mL) is added, and extracted with ethyl acetate (100 mL x 3). After drying and rotary evaporation, the crude 4-bromo-2-methyl-lH-indole (24 g) is obtained as a yellow viscous liquid, which is directly used in the next step.

[0074] The above 4-bromo-2-methyl-lH-indole is analyzed by nuclear magnetic hydrogen spectrum, and the results are as follows: 1 H NMR (400 MHz, DMSO-d6) δ 7.61 (m, 1H), 7.43 (m, 1H), 7.13 (m, 1H), 6.30 (m, 1H), 2.44 (s, 3H). MS (ESI) m / z: 210.0 [M+H] + .

[0075] (4) Preparation of the above compound 9c (methyl 2-methyl-lH-indole-4-carboxylate)

[0076] The 4-bromo-2-methyl-lH-indole (24 g, 114.2 mmol) prepared in step (3), Pd(OAc)2(2.6 g, 11.4 mmol), 1,3-bis(diphenylphosphino)propane (4.7 g, 11.4 mmol) and TEA (23.1 g, 228.4 mmol) are dissolved in methanol (200 mL). Then, the reaction mixture is placed in a high-pressure reaction kettle and reacted at 80°C under CO atmosphere for 24 hours. After the reaction is completed, the reaction solution is concentrated under reduced pressure to remove methanol, water (200 mL) is added, and extracted with ethyl acetate (100 mL x 3). After drying and rotary evaporation, the intermediate methyl 2-methyl-lH-indole-4-carboxylate (18.2 g, yield 84%) is obtained as a brownish yellow solid after purification by silica gel column chromatography.

[0077] The above methyl 2-methyl-lH-indole-4-carboxylate is analyzed by nuclear magnetic hydrogen spectrum, and the results are as follows: 1H NMR (400 MHz, DMSO-d6) δ 11.33 (s, 1H), 7.66 (m, 1H), 7.53 (dd, J = 7.9, 1.0 Hz, 1H), 7.08 (t, J = 7.8 Hz, 1H), 6.67 - 6.64 (m, 1H), 3.86 (s, 3H), 2.42 (d, J = 0.9 Hz, 3H). MS (ESI) m / z 190.3 [M+H] + .

[0078] Preparation of intermediates 9d-9h in Example 3

[0079] Preparation route and synthesis of intermediates 9d-9h:

[0080]

[0081] The specific preparation processes are as follows:

[0082] 1. Preparation of intermediate 9d (4-chloro-2-methyl-l-(phenylsulfonyl)-lH-pyrrolo[3,2- c]pyridine)

[0083] (1) Preparation of the above compound 12 (l-(phenylsulfonyl)-lH-pyrrolo[3,2-c]pyridine 5-oxide)

[0084] The preparation method of compound 11 is the same as that of 7b in Example 2.

[0085] l-(phenylsulfonyl)-lH-pyrrolo[3,2-c]pyridine (4.2 g, 16.1 mmol) was dissolved in dioxane (200 mL), and then m-CPBA (3.1 g, 17.7 mmol) was slowly added in batches at room temperature for 5 hours. After the reaction was completed, the dioxane was removed by rotary evaporation under reduced pressure, Na2SO3 (5%, 50 mL) was added, and the aqueous phase was extracted with DCM (100 x 3). The combined organic phases were dried, rotary evaporated, and then purified by silica gel column chromatography to obtain the intermediate l-(phenylsulfonyl)-lH-pyrrolo[3,2-c]pyridine 5-oxide (3 g, yield 68%) in yellow solid.

[0086] The nuclear magnetic hydrogen spectrum analysis of the above l-(phenylsulfonyl)-lH-pyrrolo[3,2- c]pyridine 5-oxide showed the following results: 1 H NMR (400 MHz, CDCl3) δ 8.52 (dd, J = 1.7, 0.7 Hz, 1H), 8.19 (dd, J = 7.2, 1.8 Hz, 1H), 7.90 (m, 3H), 7.71 - 7.62 (m, 2H), 7.55 (m, 2H), 6.66 (dd, J = 3.7, 0.8 Hz, 1H). MS (ESI, m / z): 275.0 [M+H]+ .

[0087] (2) Preparation of the above compound 13 (4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine)

[0088] 1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine 5-oxide (3 g, 10.9 mmol) was dissolved in MeCN / dioxane (30 / 30, v / v), then POCl3(1.8 g, 12 mmol) was added, and the reaction was carried out at 90°C for 18 hours. After the reaction was confirmed to be complete, the solvent was removed by rotary evaporation under reduced pressure, water (50 mL) was added, and the aqueous phase was extracted with DCM (50 x 3). The organic phases were combined, dried, and rotary evaporated, and then purified by silica gel column chromatography to obtain the intermediate 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine (2.6 g, yield 81%) as a yellow solid.

[0089] The above 4-chloro-1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine was subjected to1H NMR analysis, and the results were as follows: 1 H NMR (400 MHz, CDCl3) δ 8.27 (d, J = 5.8 Hz, 1H), 7.92 (d, J = 1.4 Hz, 1H), 7.87 (dd, J = 5.8, 0.9 Hz, 1H), 7.68 - 7.60 (m, 2H), 7.53 (m, 2H), 6.82 (dd, J = 3.7, 0.9 Hz, 1H). MS (ESI, m / z): 292.0 [M+H] + .

[0090] (3) Preparation of the above compound 9d (4-chloro-2-methyl-1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine)

[0091] The preparation process of compound 14 was identical to the synthesis method of 9b in Example 2, and the intermediate 14 (4-chloro-2-methyl-1H-pyrrolo[3,2-c]pyridine) was obtained by first reacting compound 13 with iodomethane and then deprotecting under the action of NaOH. The above 4-chloro-2-methyl-1H-pyrrolo[3,2-c]pyridine was subjected to mass spectrometry analysis, and the results were as follows: MS (ESI, m / z): 167.1 [M+H] + .

[0092] In a 25 mL vial, 4-chloro-2-methyl-1H-pyrrolo[3,2-c]pyridine (100 mg, 0.6 mmol), zinc cyanide (78 mg, 0.66 mmol), Pd2(dba)3(114 mg, 0.13 mmol), dppf (134 mg, 0.24 mmol) and zinc powder (4 mg, 0.06 mmol) were added, then NMP (6 mL) was added, and argon was replaced for 3 times, and then the reaction was carried out at 120 °C for 18 hours. After the reaction was completed, water (30 mL) was added, and the aqueous phase was extracted with EA (15 x 3). The organic phase was combined, dried, rotary evaporated, and then purified by silica gel column chromatography to obtain the intermediate 4-chloro-2-methyl-1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine (50 mg, yield 53%).

[0093] Mass spectrometric analysis was performed on 4-chloro-2-methyl-1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine, and the result was as follows: MS (ESI, m / z): 158.1 [M+H] + .

[0094] 2, Preparation of compound 9e (2-methyl-1H-pyrrolo[2,3-c]pyridine-4-carbonitrile (9e)

[0095] Using 15a as the raw material, 16a was prepared by the same method as 9b. 16a (280 mg, 1.3 mmol) was dissolved in DMF (10 mL), water (0.3 mL) was added, and the exhaust was ultrasonically treated. dppf (72 mg, 0.13 mmol), Pd2(dba)3(60 mg, 0.065 mmol) and zinc cyanide (160 mg, 1.36 mmol) were added, argon was replaced for 3 times, the reaction was carried out at 120 °C for 1.5 hours, and after the reaction was completed, water (50 mL) was added to the reaction, and the aqueous phase was extracted with EA (20 x 3). The organic phase was combined, dried, rotary evaporated, and then purified by silica gel column chromatography to obtain the intermediate 2-methyl-1H-pyrrolo[2,3-c]pyridine-4-carbonitrile (160 mg, yield 78%).

[0096] Mass spectrometric analysis was performed on 2-methyl-1H-pyrrolo[2,3-c]pyridine-4-carbonitrile, and the result was as follows: MS (ESI, m / z): 158.1 [M+H] + .

[0097] 3, Preparation of compounds 9f-9h

[0098] Using 15b or 17 as the raw material, the same method as the preparation of compound 9e was used, first reacted with phenylsulfonyl chloride, then reacted with iodomethane, and then deprotected under the action of NaOH to obtain the intermediate 16b or 18. Using the same method as the preparation of 9e, the intermediate 16b or 18 was reacted with zinc cyanide to obtain the product 9f or 9g with a cyano group at position 4.

[0099] The same as the preparation method of compound 9e, with 19 as raw material, first reacted with phenylsulfonyl chloride, then with deuterated methyl iodide, and then deprotected under the action of NaOH to obtain intermediate 9h.

[0100] Preparation of intermediates 20a-20i in Example 4

[0101] Synthesis route of intermediates 20a-20i:

[0102]

[0103] 1. Preparation of compound 20a (tert-butyl 4-(benzylamino)-2-chloro-6,7- dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate)

[0104] Compound 5 (3.5 g, 11.5 mmol) in Example 1 was dissolved in isopropanol (50 mL), then benzylamine (1.9 g, 17.3 mmol) and TEA (3.5 g, 34.5 mmol) were added, and reacted at 70°C for 12 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, water (50 mL) was added, and then EA (50 mL x 3) was extracted. The organic phase was combined, dried, rotary evaporated, and then purified by silica gel column chromatography to obtain intermediate tert-butyl 4-(benzylamino)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate (27 g, yield 63%) as a white solid.

[0105] The above-mentioned tert-butyl 4-(benzylamino)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (400 MHz, CDCl3) δ 7.43-7.31 (m, 5H), 4.81 (t, J = 5.4 Hz, 1H), 4.71 (d, J = 5.3 Hz, 2H), 3.78-3.69 (m, 2H), 2.33 (t, J = 6.8 Hz, 2H), 2.05-1.94 (m, 2H), 1.57 (s, 9H). MS (ESI, m / z): 375.1 [M+H] + .

[0106] 2. In the same way as 20a, compound 5 was subjected to nucleophilic substitution reaction with 2-(aminomethyl)phenol, 3-(aminomethyl)phenol, 4-(aminomethyl)phenol, pyridin-2-ylmethylamine, pyridin-3-ylmethylamine, and pyridin-4-ylmethylamine to obtain 20b-20g.

[0107] 3. Preparation of compound 20h (tert-butyl 4-(benzyloxy)-2-chloro-6,7- dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate)

[0108] Compound 5 (200 mg, 0.66 mmol) in Example 1 was dissolved in DMF (5 mL), NaH (40 mg, 0.99 mmol) was added, then benzyl alcohol (78 mg, 0.73 mmol) was added, and the reaction was allowed to proceed at room temperature for 3 hours. After the reaction was detected to be complete, water (30 mL) was added, and the aqueous phase was extracted with EA (20 x 3). The organic phases were combined, dried, and rotary evaporated, and then purified by silica gel column chromatography to obtain intermediate tert-butyl 4-(benzyloxy)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate (112 mg, yield 45%) as a white solid.

[0109] The above-mentioned tert-butyl 4-(benzyloxy)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate was subjected to nuclear magnetic hydrogen spectrum analysis, and the results were as follows: 1 H NMR (400 MHz, Chloroform-d) δ 7.55-7.33 (m, 5H), 5.44 (s, 2H), 3.79-3.70 (m, 2H), 2.62 (m, 2H), 1.98-1.89 (m, 2H), 1.57 (s, 9H). MS (ESI, m / z): 376.1 [M+H] + .

[0110] 4. Preparation of compound 20i (tert-butyl 4-(benzylthio)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate (20i)

[0111] Compound 5 (200 mg, 0.66 mM) in Example 1 was dissolved in DMF (5 mL), K2CO3 (182 mg, 1.32 mM) and benzyl mercaptan (98 mg, 0.79 mM) were added, and the reaction was allowed to proceed at room temperature for 3 hours. After the reaction was detected to be complete, water (30 mL) was added, and the aqueous phase was extracted with EA (20 x 3). The organic phases were combined, dried, and rotary evaporated, and then purified by silica gel column chromatography to obtain intermediate tert-butyl 4-(benzylthio)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate (310 mg, yield 79%) as a light yellow solid.

[0112] The above-mentioned tert-butyl 4-(benzylthio)-2-chloro-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate was subjected to nuclear magnetic hydrogen spectrum analysis, and the results were as follows: 1H NMR (400 MHz, CDC13) δ 7.46 - 7.43 (m, 2H), 7.37 - 7.31 (m, 2H), 7.28 (s, 1H), 4.47 (s, 2H), 3.80 - 3.68 (m, 2H), 2.52 (t, J = 6.7 Hz, 2H), 2.01 - 1.88 (m, 2H), 1.57 (s, 9H). MS (ESI, m / z): 392.1 [M+H] + .

[0113] Preparation of target products V1-V13

[0114] 1, The synthetic route of target product V1 is as follows:

[0115]

[0116] The specific preparation process of V1 is as follows:

[0117] (1) Preparation of compound 21b (tert-butyl 2-(4-cyano-2-methyl-1H-indol-1-yl)-4- ((2-hydroxybenzyl) amino)-6, 7-dihydropyrido [2, 3-d] pyrimidine-8 (5H)-carboxylate)

[0118] In a 25 mL tomato bottle, 20b (200 mg, 0.51 mmol), 9a in example 2 (80 mg, 0.51 mmol), Cs2CO3 (249 mg, 0.76 mmol), Pd2(dba)3 (73 mg, 0.08 mmol) and X-Phos (38 mg, 0.08 mmol) were added, then dioxane (10 mL) was added. After purging Ar for 3 times, the reaction was refluxed at 105°C for 4 hours. After TLC monitoring that the reaction was completed, the reaction was cooled to room temperature. The filtrate was filtered and the solvent was evaporated under reduced pressure. The obtained solid was dissolved in EA (50 mL), washed with water (50 mL), and the aqueous phase was extracted with EA (50 mL x 2). After drying and rotary evaporation, the intermediate tert-butyl 2-(4-cyano-2-methyl-1H-indol-1-yl)-4-((2-hydroxybenzyl)amino)-6,7- dihydropyrido [2, 3-d] pyrimidine-8 (5H)-carboxylate (203 mg, yield 78%) was obtained by silica gel column chromatography.

[0119] Mass spectrum analysis was performed on tert-butyl 2-(4-cyano-2-methyl-1H-indol-1-yl)-4-((2-hydroxybenzyl)amino)-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate, and the result was: MS (ESI, m / z): 511.6 [M+H] + .

[0120] (2) Preparation of compound V1 (1-(4-((2-hydroxybenzyl)amino)-5,6,7,8- tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxamide)

[0121] In a 20 mL vial, intermediate compound 21b (203 mg, 0.4 mmol) was dissolved in DMSO (6 mL). Then urea peroxide (184 mg, 2.0 mmol) and K2CO3(55 mg, 0.4 mmol) were dissolved in water (0.6 mL) and added to the reaction vial, and stirred at 45 °C for 4 h. After the reaction was completed, water (20 mL) was added, and EA (20 mL x 3) was extracted. The organic phase was combined, dried, rotary evaporated, and then purified by silica gel column chromatography to obtain intermediate 21 (180 mg, yield 85%).

[0122] Mass spectrometry was performed on compound 21, and the mass spectrometry result was: MS (ESI, m / z): 513.2 [M+H] + The above intermediate 21 (180 mg, 0.34 mmol) was dissolved in DCM (5 mL), and TFA (1 mL) was added, and the reaction was carried out at room temperature for 4 h. After the reaction was completed, rotary evaporation was carried out under reduced pressure, EA (30 mL) was added, water (20 mL) was added, and Na2CO3(5%) was added to pH 9. The liquid was separated, the water phase was extracted with EA (20 mL x 2), the organic phase was combined, dried, rotary evaporated, and then purified by silica gel column chromatography to obtain the target compound 1-(4-((2-hydroxybenzyl)amino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxamide (102 mg, yield 70%), light yellow solid.

[0123] Compound V1 was subjected to nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, high resolution mass spectrometry, and mass spectrometry analysis, and the mass spectrometry result was: MS (ESI, m / z): 429.3 [M+H] + The results of nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, and high resolution mass spectrometry are shown in Table 1.

[0124] 2. The same preparation method as V1, compounds 20c-20i were respectively coupled with 9a, and then the cyano group was hydrolyzed into an amide, and finally the Boc protecting group was removed to obtain compounds V2-V8. Compounds 20a were respectively coupled with 9d-9h, and then the cyano group was hydrolyzed into an amide, and finally the Boc protecting group was removed to obtain compounds V9-V13.

[0125] Compounds V2-V13 were subjected to nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, and high resolution mass spectrometry analysis, and the results are shown in Table 1.

[0126] Preparation of target product V14 in Example 6

[0127] Synthesis route and preparation of target product V14

[0128]

[0129] The specific preparation process is as follows:

[0130] (1) Preparation of compound 22 (tert-butyl 4-(benzylamino)-2-(4-(methyloxycarbonyl)-2-methyl-1H-indol-1-yl)-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate)

[0131] In a 25 mL tomato bottle, 20a (200 mg, 0.53 mmol) in Example 4, 9c (100 mg, 0.53 mmol) in Example 2, Cs2CO3 (261 mg, 0.8 mmol), Pd2(dba)3 (73 mg, 0.08 mmol) and X-Phos (38 mg, 0.08 mmol) were added, then dioxane (10 mL) was added. After the reaction was replaced with Ar gas for 3 times, it was refluxed at 105°C for 4 hours. After TLC monitoring that the reaction was completed, the reaction was cooled to room temperature. The filtrate was filtered and the solvent was evaporated under reduced pressure. The obtained solid was dissolved in EA (50 mL), washed with water (50 mL), and the aqueous phase was extracted with EA (50 mL x 2). After drying and rotary evaporation, the intermediate tert-butyl 4-(benzylamino)-2-(4-(methyloxycarbonyl)-2-methyl-1H-indol-1-yl)-6,7-dihydropyrido[2,3-d]pyrimidine-8(5H)-carboxylate (218 mg, yield 78%) was obtained by silica gel column chromatography purification, which was a yellow solid.

[0132] The compound 22 was analyzed by nuclear magnetic hydrogen spectrum, and the results were as follows: 1 H NMR (400 MHz, DMSO-d6) δ 8.28 (d, J = 8.3 Hz, 1H), 7.71 (ddd, J = 8.6, 5.9, 3.4 Hz, 2H), 7.38 – 7.29 (m, 4H), 7.25 (td, J = 6.0, 2.6 Hz, 1H), 6.99 (t, J = 7.9 Hz, 1H), 6.89 – 6.86 (m, 1H), 4.67 (d, J = 5.9 Hz, 2H), 3.88 (s, 3H), 3.77 – 3.69 (m, 2H), 2.56 (s, 2H), 2.54 – 2.54 (m, 3H), 2.04 – 1.91 (m, 2H), 1.43 (s, 9H). MS (ESI, m / z): 528.3 [M+H] + .

[0133] (2) Preparation of compound 23 (1-(4-(benzylamino)-8-(tert-butoxycarbonyl)- 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxylic acid)

[0134] Compound 22 (5 g, 9.5 mmol) was dissolved in THF / MeOH (90 mL / 30 mL) in a 250 mL round-bottom flask. LiOH-H2O (2.4 g, 57 mmol) was dissolved in water (30 mL) and slowly added to the reaction flask, and the reaction was carried out at 60°C for 6 hours. After the reaction was completed, the solvent was removed by rotary evaporation under reduced pressure, water (50 mL) was added, and EA (50 mL x 5) was used for extraction. The organic phase was combined, dried, and rotary evaporated to obtain the crude product 1-(4-(benzylamino)-8-(tert-butoxycarbonyl)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxylic acid (23) (4.4 g, yield 90%), brown solid, which was directly used in the next step.

[0135] Mass spectrometric analysis of compound 23 gave the following results: MS (ESI, m / z): 514.1 [M+H] + .

[0136] (3) Preparation of target product V14 (1-(4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxylic acid)

[0137] The above intermediate compound 23 (200 mg, 0.39 mmol) was dissolved in DCM (5 mL), and TFA (1 mL) was added, and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, rotary evaporation was carried out under reduced pressure, EA (30 mL) was added, water (20 mL) was added, and Na2CO3 (5%) was added to pH 9. The liquid was separated, the aqueous phase was extracted with EA (20 mL x 2), the organic phase was combined, dried, and rotary evaporated, and then purified by silica gel column chromatography to obtain the target compound 1-(4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-1H-indole-4-carboxylic acid (120 mg, yield 75%), light brown solid.

[0138] The target product V14 was subjected to nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, high-resolution mass spectrometry, and mass spectrometry analysis, and the mass spectrometry results were as follows: MS (ESI, m / z): 413.3 [M+H] + . The results of the nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum, and high-resolution mass spectrometry are shown in Table 1.

[0139] Example 7 Preparation of target product V15

[0140] The synthesis route of the target product V15 is as follows:

[0141]

[0142] Compound 23 (200 mg, 0.39 mmol) was dissolved in THF (10 mL), methoxyamine hydrochloride (33 mg, 0.39 mmol), EDCI (112 mg, 0.59 mmol), HOBt (80 mg, 0.59 mmol) and DIPEA (202 mg, 1.56 mmol) were added, and the reaction was allowed to proceed at room temperature overnight. Water (30 mL) was added, and extraction was performed with EA (20 mL x 3). The organic phases were combined, dried, and evaporated, and the resulting product was purified by column chromatography on silica gel to obtain the Boc-protected intermediate compound 24. Subsequently, the intermediate compound 24 was deprotected with TFA, and the resulting product was purified to obtain the target compound 1-(4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-N-methoxy-2-methyl-1H-indole-4-carboxamide (80 mg, yield 62%) as a yellow powder.

[0143] The target product V15 was subjected to analysis by1H NMR,13C NMR and high resolution mass spectrometry, and the results are shown in Table 1.

[0144] Preparation of target products V16-V17

[0145] The synthetic route of the target product V16 is as follows:

[0146]

[0147] Intermediate compound 23 (100 mg, 0.2 mmol) and methanesulfonamide (37 mg, 0.4 mmol) were dissolved in DCM (2 mL), and CMPI (60 mg, 0.23 mmol), DMAP (1.2 mg, 0.01 mmol) and TEA (50 mg, 0.58 mmol) were added, and the reaction was allowed to proceed at room temperature for 1 hour. Water (30 mL) was added, and extraction was performed with DCM (20 mL x 3). The organic phases were combined, dried, and evaporated, and the resulting product was purified by column chromatography on silica gel to obtain the target compound 1-(4-(benzylamino)-5,6,7,8-tetrahydropyrido[2,3-d]pyrimidin-2-yl)-2-methyl-N-(methylsulfonyl)-1H-indole-4-carboxamide (45 mg, yield 62%) as a light yellow powder.

[0148] The target product V16 was subjected to analysis by1H NMR,13C NMR and high resolution mass spectrometry, and the results are shown in Table 1.

[0149] In the same manner as for V16, compound 23 and 5-bromo furan-2-sulfonamide were reacted to obtain the target product V17. The target product V17 was subjected to analysis by1H NMR,13C NMR and high resolution mass spectrometry, and the results are shown in Table 1.

[0150] The NMR and mass data of the target compounds V1-V17 are shown in Table 1.

[0151] Table 1. NMR and mass data of the synthesized specific compounds V1-V17.

[0152]

[0153]

[0154]

[0155]

[0156]

[0157] Example 9 In vitro p97 inhibition experiment

[0158] P97 converts the chemical energy produced by ATP hydrolysis into mechanical energy to perform its function, and is involved in a series of cellular processes. Therefore, inhibiting the ability of p97 to hydrolyze ATP can inhibit its function, thereby exerting a pharmacological effect. The synthesized compounds of the present application are ATP competitive inhibitors of the D2 domain of p97, which can competitively bind to the ATP binding site of the D2 domain to inhibit ATP hydrolysis. Therefore, we screened the inhibitory activity of the series of compounds on p97 by detecting the generation of ADP using ATP as a substrate.

[0159] 1. The determination scheme is as follows:

[0160] (1) Accurately weigh the compounds V1-V17 to be tested, dissolve in DMSO to make the concentration 10 mM. After dilution 20 times with DMSO and then 25 times with water, a drug solution with a concentration of 20 μM is obtained. Take this as the starting concentration, dilute 3 times, a total of 9 concentration points, and the tenth concentration point is the blank control group. Compound CB-5339 is the positive control, and water containing 1% DMSO is used as the solvent control.

[0161] (2) Add p97 hexamers (2 μL, 60 μg / mL) to each well of a 384-well plate, then add different concentrations of the diluted compounds (1 μL) V1-V17 or solvent controls, respectively. React at room temperature for 10 min.

[0162] (3) Add ATP (1 μL, 100 μM) to each well, vortex to mix, and react at 30°C for 60 min.

[0163] (4) Add ADP-Glo TM reagent 4 μL, and react at 25°C for 40 min.

[0164] (5) Add ADP-Glo to each well TM Max detection reagent 8 μL, continue to react at 25 °C for 60 min.

[0165] (6) Use the microplate reader to detect the absorbance value of each well at 570 nm, and use Graph Pad Prism 8.0 software to calculate the IC 50 value of each compound.

[0166] 2, The results of the in vitro P97 inhibition experiment are shown in Table 2.

[0167] Table 2 Inhibition activity of target compounds V1-V17 and CB-5339 on p97

[0168]

[0169] a All experiments were repeated three times. Data were reported as the mean ± SD. b No activity.

[0170] The results of the enzyme inhibition activity in Table 2 show that compounds V1-V2, V12-V16 exhibit good inhibition activity, which is comparable or better than CB-5339.

[0171] Example 10 Study on the proliferation inhibition activity of compounds on tumor cells

[0172] According to the experimental results of the inhibition activity of p97 in Example 9, the compounds with IC 50 values less than 0.2 μM for p97 inhibition were further tested for their proliferation inhibition effect on colorectal cancer cell line HCT-116 cells and multiple myeloma cell line RPMI-8226 cells.

[0173] 1, The determination scheme is as follows:

[0174] The HCT-116 cell line is derived from ATCC, and the culture conditions are 5A (GIBCO #16600082) + 10% FBS, 5% CO2; the A549 cell line is derived from ATCC, and the culture conditions are F-12K (GIBCO #21127022w / o HEPS) + 10% FBS, 5% CO2; the RPMI-8226 cell line is derived from ATCC, and the culture conditions are RPMI-1640 (GIBCO #11875119w / o HEPS) + 10% FBS + 25mM HEPES, 5% CO2.

[0175] HCT-116, A549, and RPMI-8226 cell suspensions in logarithmic growth phase were added to 384-well plates at a volume of 18 μL per well, with cell counts of 600, 400, and 2000 cells / well, respectively. The edges were sealed with PBS or basal medium, and the plates were incubated for 24 h. Then, 2 μL of different concentrations of the compound (initial concentration 10 μM, 3-fold dilution) were added to each well. Control wells contained no drug, and zeroing wells contained neither cells nor drug. The plates were incubated at 37°C and 5% CO2 for 72 h. Next, 20 μL of Cell Titer Glo assay reagent was added to each well, and the plates were incubated for 10 minutes. The fluorescence intensity of each well was measured using a microplate reader, and the cell inhibition rate was calculated using the following formula. The IC50 value for each compound was calculated using GraphPad Prism 8.0 software. 50 value.

[0176]

[0177] 2. The experimental results of the study on the inhibitory activity of tumor cells on proliferation are shown in Table 3.

[0178] Table 3 shows the antiproliferative activity of some compounds against HCT-116 and RPMI-8226 cell lines.

[0179]

[0180] a All experiments were repeated two times.Data were reported as themean±SD. b No activity.

[0181] The results in Table 3 show that multiple compounds have different IC50 values ​​for HCT-116 and RPMI8226 cells. 50 Compounds with concentrations below 1.0 μM all exhibited good inhibitory activity. In the proliferation inhibition experiment of the colorectal cancer cell line HCT-116, compounds V2 (0.7 μM) and V15 (0.7 μM) showed the same inhibitory effect on HCT-116 cell proliferation as CB-5339 (0.7 μM). However, compound V14 showed better inhibitory activity, with an IC50 value below 1.0 μM. 50 The value was only 0.4 μM; in the results of inhibiting the proliferation of multiple myeloma cell line RPMI-8226, compounds V2, V14, and V15 showed better inhibitory activity, with IC50 values ​​of only 0.4 μM. 50 The values ​​were 0.3 μM, 0.8 μM and 0.5 μM, respectively, which were better than the positive control CB-5339 (0.9 μM).

[0182] In the following PK experiment of V15, it was found that it was partially metabolized into V14 in vivo, so further activity screening of V14 and V15 was carried out in multiple cell lines, and the cell line culture conditions and the number of cells plated per well in 384-well plates are shown in Table 4, and the anti-proliferative activity of compounds V14 and V15 on multiple cell lines is shown in Table 5.

[0183] Table 4 Different cell line culture conditions and the number of cells plated per well in 384-well plates

[0184]

[0185] Table 5 Anti-proliferative activity of compounds V14 and V15 on multiple cell lines

[0186]

[0187]

[0188] a All experiments were repeated two times. Data were reported as the mean ± SD. b Not tested.

[0189] The results of cell proliferation inhibition activity in Table 5 show that CB-5339 has IC 50 values of 1.3 μM for ARP1 and KMS-11 cell lines, and IC 50 values of less than 1 μM for the other 7 cell lines. Compound V14 has IC 50 values of less than 1 μM for the other 8 cell lines except for SW620 cell line with IC 50 value of 2.1 μM, and shows better cell activity than CB-5339 in multiple cell lines. Compound V15 has comparable activity to CB-5339 on H929, MV-4-11 and MOLM16 cell lines.

[0190] Example 11 Liver microsomal stability study

[0191] 1. The determination scheme is as follows:

[0192] (1) Weigh a certain amount of the compound to be tested, dissolve it in DMSO to make its concentration 10 mM, then add Acetonitrile / H2O (1:1, v:v) to obtain a working solution with a concentration of 100 μM.

[0193] (2) Take the liver microsomes (20 mg protein / mL) from the -80°C refrigerator, place them on a 37°C water bath constant temperature oscillator for 3 min of pre-incubation, and thaw for use.

[0194] (3) Prepare the reaction system mixture solution (without β-NADPH) according to Table 6.

[0195] (4) Control group (without β-NADPH): Take 75 μL of the reaction system mixture solution described in step (3), add 25 μL of PBS, vortex to mix, and then incubate in a 37°C constant temperature shaker. Take samples at 0 min and 60 min, respectively.

[0196] (5) Sample group: Take 75 μL of the reaction system mixture solution described in step (3), add 25 μL of β-NADPH solution (4 mM), vortex to mix, and then incubate in a 37°C constant temperature shaker. Take samples at 0 min, 5 min, 15 min, 30 min, and 60 min, respectively.

[0197] (6) Take the sample tube at each time point, and add 300 μL of pre-cooled acetonitrile (containing midazolam) to terminate the reaction.

[0198] (7) After vortexing for 5 min, centrifuge (5500 g, 10 min), take 150 μL of supernatant, add 150 μL of water, vortex to mix, and then analyze the sample by LC-MS / MS. The incubation conditions of the positive control, midazolam, are the same as above.

[0199] Table 6 Composition of the microsomal stability incubation system

[0200]

[0201] 2. The experimental results of the liver microsomal stability study are shown in Table 7.

[0202] Table 7 Stability of compounds V14, V15, and CB-5339 in liver microsomes of different species

[0203]

[0204] As can be seen from Table 7, compound V15 is metabolized relatively quickly in liver microsomes of five species, and the T 1 / 2 of V15 in human liver microsomes is only 13.3 min, and the T 1 / 2 in mouse, dog, and monkey liver microsomes is even less than 10 min. However, the T 1 / 2 of compound V14 in human, dog, and monkey liver microsomes is greater than 120 min, showing good stability. These results show that V14 in rat plasma in the pharmacokinetic experiment is likely to be produced by the metabolism of V15 in liver microsomes.

[0205] Example 12 Pharmacokinetic study

[0206] 1. The determination scheme is as follows:

[0207] The preparation method of the drug solution is as follows: a certain amount of V14, V15 and CB-5339 is precisely weighed into a glass bottle, dissolved in a certain volume of DMSO and polyoxyethylene castor oil, after the solution is clear, add normal saline (DMSO: castor oil: normal saline = 5:5:90, v:v) and adjust pH = 4, ultrasonic dissolution, so that its final concentration is 0.5 mg / mL (for injection) and 2 mg / mL (for oral administration). 6 SD male rats, weighing 220±20g, are randomly divided into two groups, and the compounds V14, V15 and CB-5339 are given by tail vein injection at a dose of 1.00 mg / kg and oral administration at a dose of 10 mg / kg, respectively. Blood samples are collected before administration and at 2 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h after administration, about 0.20 mL of blood is taken from the orbit and placed in a centrifuge tube containing heparin, and centrifuged at 4°C (6800g, 6 min). After centrifugation, 20 μL of plasma sample is taken, 200 μL of propranolol / glibenclamide-acetonitrile solution containing 10 ng / mL is added for protein precipitation, vortexed for 5 min, then the above sample is placed in a centrifuge and centrifuged at 5500g for 10 min. 150 μL of supernatant is taken, 150 μL of water is added for dilution, and 20 μL of sample is taken after vortexing. The sample is analyzed by LC / MS / MS, and the pharmacokinetic parameters are calculated using Winonlin software. The remaining plasma samples are stored in a -80°C refrigerator.

[0208] 2. The results of the pharmacokinetic study experiment are shown in Table 8.

[0209] Table 8. PK results of SD rats after single dose oral and tail vein injection of compounds V15 and CB-5339

[0210]

[0211] As shown by the results in Table 8, the elimination half-life of compound CB-5339 by oral and tail vein is 1.23 h and 0.38 h, respectively. After single dose oral administration of 10 mg / kg CB-5339, the C max and AUC 0-inf values in plasma are 444 ng / mL and 796 ng·h / mL, respectively, and the oral bioavailability is 20%. After oral or tail vein injection of V15, V15 is rapidly metabolized to V14 in vivo. The oral half-life of V14 in plasma is 3.52 h, and the tail vein half-life is 3.83 h, which is much higher than that of CB-5339. The T max of V14 in plasma is 0.25 h, which is half of that of CB-5339, and the oral absorption speed is faster. After single dose oral administration of 10 mg / kg V15, the C max and AUC 0-infThe values were 1070 ng / mL and 1412 ng-h / mL, respectively, which were much higher than the plasma drug concentration of CB-5339. The oral bioavailability of V15 was calculated to be 29.7% based on the results of V14 alone.

[0212] Example 13 In vivo toxicity study

[0213] 1. The experimental scheme is as follows:

[0214] A certain amount of compound V15 was weighed and ultrasonically dispersed in sodium chloride injection containing 0.3% CMC-Na. The concentration of V15 was prepared to be 10 mg / mL, and the concentration of CB-5339 was prepared to be 5 mg / mL.

[0215] Each group of mice was given intragastrically (ig) once a day, respectively, as control group (blank solvent), V15 (50 mg / kg), V15 (100 mg / kg), CB-5339 (50 mg / kg), CB-5339 (100 mg / kg). The volume of administration was 0.1 mL / 10 g of animal body weight, and the administration was continuous for 15 days. The size of the tumor was measured with a vernier caliper. The tumor volume (TV) was calculated as: V = (length x width 2 ) / 2.

[0216] The results of the change in body weight of mice after continuous administration are shown in Table 2. Figure 1 The results show that no obvious toxic side effects were observed for compound CB-5339 at a dose of 50 mg / kg during the in vivo experiment. However, when the dose of CB-5339 was 100 mg / kg, significant weight loss and other side effects were observed on the 4th day, and all died on the 8th day. However, when the dose of compound V15 was 50 mg / kg and 100 mg / kg, no obvious weight loss and other side effects were observed after 15 days of continuous administration, indicating that compound V15 had no obvious toxicity at a dose of 100 mg / kg and showed good safety.

Claims

1. A fused pyrimidine compound or a pharmaceutically acceptable salt thereof, characterized in that, The fused pyrimidine compound is selected from the following structures: 。 2. A process for the preparation of the fused pyrimidine compounds or pharmaceutically acceptable salts thereof according to claim 1, characterized in that, The method comprises the following steps: (1) Preparation of dichloropyrimidine nucleus ， B is obtained by reacting A in urea solution, followed by hydrolysis, C is obtained by reduction of B in the presence of palladium hydroxide and acetic acid as solvent, C is converted into dichloride D, then a Boc protecting group is introduced on the amino group to obtain E; (2) Preparation of 2-methyl-1H-indole-4-carboxylic acid methyl ester ， F is reacted with benzenesulfonyl chloride in the presence of sodium hydride to obtain a protected G, G is then reacted with iodomethane in the presence of LDA to obtain H, H is deprotected in the presence of sodium hydroxide to obtain I, I is subjected to a carbonylation reaction in the presence of a catalyst to obtain J; (3) Preparation of the target compound, fused pyrimidine compound V15 ， K is obtained by reacting E with benzylamine in the presence of triethylamine as a base, K is coupled with J in the presence of a palladium catalyst to obtain an intermediate L, L is saponified in the presence of lithium hydroxide to obtain a carboxylic acid, then acylation is performed to obtain N, N is deprotected from the Boc protecting group in the presence of trifluoroacetic acid or HCl to obtain the fused pyrimidine compound V15.

3. A pharmaceutical composition, characterized by, The fused pyrimidine compound or a pharmaceutically acceptable salt thereof as claimed in claim 1.

4. Use of the fused pyrimidine compound or a pharmaceutically acceptable salt thereof as claimed in claim 1 or the pharmaceutical composition according to claim 3 in the preparation of a medicament for treating a disease related to p97 activity, wherein the disease related to p97 activity is pancreatic cancer, multiple myeloma or leukemia.

5. Use according to claim 4, characterized in that, The medicament can block tumor cell proliferation and induce tumor cell apoptosis. The medicament can block tumor cell proliferation and induce tumor cell apoptosis.

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