Patent Application
20240270732
The present invention belongs to the field of chemical medicine, and specifically relates to a receptor-interacting protein (RIP) inhibitor, a preparation method therefor, and uses thereof.
Programmed necrosis, also known as necroptosis, is a new and controllable cell death mode with necrotic morphological characteristics. Programmed necrosis plays a key role in embryonic development and the dynamic balance of the adult body, and is widely involved in the pathophysiological processes of many diseases. Receptor-interacting protein 1 (RIP1) is located at a critical position in the programmed necrosis pathway, and is an upstream kinase that has a regulatory effect. As an upstream regulatory molecule of the signaling pathway, its abnormal activation can cause a series of reactions. RIP1 has become the “central controller” determining cell fate in the death receptor signaling pathway.
Numerous studies have shown that cell death and inflammatory responses mediated by the activation of RIP1 and programmed necrosis pathways involve various human diseases, including stroke, myocardial infarction, retinal injury, fatal systemic inflammatory response syndrome, chronic enteritis, and malignant tumors. RIP1 kinase serves as a potential target in the programmed necrosis pathway, and thus suppression of its kinase activity can retard the progression of the disease. RIP1 inhibitors have important application value in treating diseases such as inflammation, cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and tumors. Therefore, RIP inhibitors, especially RIP1 inhibitors, have received great attention from pharmaceutical chemistry researchers.
Philip A. Harris et al. have reported a class of RIP inhibitors with representative structures and found that they have potential applications in treating inflammation or tumors. However, its therapeutic effect still needs to be further improved, and RIP inhibitors with better therapeutic effects are still urgently needed in clinical practice.
The present invention aims to provide a receptor-interacting protein (RIP) inhibitor, a preparation method therefor, and uses thereof.
The present invention provides the compound as represented by formula (I), or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof:
R0′ is selected from the group consisting of H, C1-C5 alkyl or cycloalkyl;
R0 is selected from the group consisting of H, C1-C5 alkyl or cycloalkyl;
R0 is selected from the group consisting of H, C1-C5 alkyl or cycloalkyl;
Further, X is a structure represented by formula (Ib′), Y and Y′ is , and the compound has a structure represented by following formula (II):
wherein, W is selected from the group consisting of —NR7—, O, S, 1,3-succinynyl, —(OCH2CH2)n3O—, a saturated or unsaturated C3-C10 cyclic hydrocarbon group, a saturated or unsaturated 3-10 membered heterocyclic hydrocarbon group, a C5-C10 aryl, a 5-10 membered heteroaryl or a structure as represented by formula (Ia):
Further, Z1, Z2, Z1′, and Z2′ are CH; Z3 and Z3′ are O;
rings A and A′ are each independently selected from 5-membered unsaturated heterocyclyls; the heteroatoms contained in the heterocyclic group are N, O, or S, and the number of heteroatoms is 1, 2, 3, or 4;
Further, R3, R3′, R4, R4′, R5, R5′, R6, and R6′ are H; rings B and B′ are phenyl; the compound has the structure as represented by following formula (IIb):
Further, L is the following structures:
—CH2—(OCH2CH2)n3O—CH2—,
Further, X is a structure represented by formula (Ib′), Y and Y′ are CH2, and the compound has a structure represented by following formula (III):
wherein R7 is H or C1-C3 alkyl; n3 is selected from an integer of 1 to 10; n4 is selected from an integer of 1 to 6;
Further, Z1, Z2, Z1′, and Z2′ are CH; Z3 and Z3′ are O;
R1, R2, R1′ and R2′ are each independently selected from the group consisting of H, C1-C6 alkyl or C1-C6 deuteroalkyl;
R3 and R3′ are each independently selected from the group consisting of H, C1-C6 alkyl or C1-C6 deuteroalkyl; R4 and R4′ are each independently selected from the group consisting of H, C1-C6 alkyl, C1-C6 deuteroalkyl, C1-C6 alkoxy, hydroxyl, amino, cyano, or thiol;
Further, R3, R3′, R4, R4′, R5, R5′, R6, and R6′ are H; rings B and B′ are phenyl; the compound has the structure as represented by following formula (IIIb):
Further, L is the following structures:
—CH2—(OCH2CH2)n4O—CH2—,
wherein, n4 is selected from an integer of 1 to 6.
Further, X is not a structure represented by formula (Ib′), R2 is methyl, Y is , and the compound has a structure represented by following formula (IV):
Further, Z1 and Z2 are CH, Z3 is O; the compound has a structure as represented by following formula (IVa):
the substituted substituents are selected from the group consisting of halogen, alkyl, cyano, amino, and hydroxyl;
R1 is selected from the group consisting of H, C1-C6 alkyl, and C1-C6 deuteroalkyl;
or R3 and R4 are linked to form a ring; or R4 and R5 are linked to form a ring;
Further, R3, R4, R5, and R6 are H; ring B is phenyl; the compound has a structure as represented by following formula (IVb):
Further, X is not a structure represented by formula (Ib′), R2 is H, Y is , and the compound has a structure represented by following formula (V):
or R3 and R4 are linked to form a ring; or R4 and R5 are linked to form a ring;
Further, Z1 and Z2 are CH, Z3 is O; the compound has a structure as represented by following formula (Va):
Further, R3, R4, R5, and R6 are H; ring B is phenyl; the compound has a structure as represented by following formula (Vb):
Further, the salt is hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromate, hydrofluorate, phosphate, acetate, propionate, succinate, oxalate, lactate, malate, succinate, fumarate, maleate, tartrate, trifluoroacetate, sodium salt or potassium salt.
Further, the compound is one of the following compounds:
The present invention also provides above compounds, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof for use in the manufacture of RIP inhibitors; preferably, the RIP inhibitor is receptor-interacting protein 1 (RIP1) inhibitors.
The present invention also provides the use of above compounds, or a salt thereof, or a stereoisomer thereof, or a solvate thereof, or a prodrug thereof in the manufacture of medicaments for anti-inflammation, anti-tumor, prevention and/or treatment of psoriasis;
The present invention also provides a medicament, which is manufactured from a compound according to any one of claims 1 to 17, or a salt thereof, or a stereoisomer thereof, as active ingredients, in combination with pharmaceutically acceptable excipients or auxiliary ingredients.
The definition of terms in the present invention:
In the present invention, the minimum and maximum values of the carbon numbers in hydrocarbon groups are represented by prefixes. For example, the C1-C8 alkyls or C1-8 alkyls refer to C1, C2, C3, C4, C5, C6, C7, C8 alkyls, i.e. straight or branched alkyls having 1-8 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, hexyl, heptyl, octyl, etc. Similarly, the C1-C3 alkoxys refer to C1, C2, C3 alkoxys; C3-C8 cycloalkyls refer to the cycloalkyls having 3, 4, 5, 6, 7, and 8 carbons.
The term “substitution” refers to substituting one, two, or more hydrogens in a molecule with other different atoms, molecules, or groups, including one, two, or more substitutions at the same or different atoms in the molecule.
The term “halogen” refers to fluorine, chlorine, bromine, or iodine; the term “alkyl” refers to the hydrocarbon group formed by lossing one hydrogen from a straight or branched alkane molecule;
The term “cyclic hydrocarbon group” refers to a saturated or unsaturated cyclic monovalent substituent group only containing carbons and hydrogens, which can be a group formed by removal of one H atom from saturated alicyclic hydrocarbons, cycloalkenes, or cycloalkynes, and can be further substituted. The C3˜C6 cyclic hydrocarbon groups refer to that having 3-6 carbons.
The term “heterocyclic hydrocarbon group” refers to a cyclic hydrocarbon group in which at least one carbon is substituted with a heteroatom, and the heteroatom is O, N, or S.
The term “aryl” refers to all-carbon monocyclic or fused polycyclic (i.e., the rings sharing adjacent carbon atom pairs) groups with conjugated π electron systems, such as phenyl and naphthyl. The aromatic ring can condense with other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as O, N, or S, and the point connecting the parent must be the carbon atom in the ring with a conjugated π electron system.
The term “heteroaryl” refers to an aryl in which at least one carbon atom in the ring with conjugated π electron system is substituted with a heteroatom, and the heteroatom is O, N, or S.
The term “heterocyclyl” refers to both heterocyclic hydrocarbon groups and heteroaromatic groups, and the heteroatom is O, N, or S.
Obviously, based on the above content of the present invention, according to the common technical knowledge and the conventional means in the field, other various modifications, alternations, or changes can further be made, without department from the above basic technical spirits.
With reference to the following specific examples of the embodiments, the above content of the present invention is further illustrated. But it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples. The techniques realized based on the above content of the present invention are all within the scope of the present invention.
The raw materials and equipment used in the specific examples of the present invention were known products obtained by purchasing those commercially available.
Step 1: 20 mL of ethanol was cooled to −5° C., to which was added 10 g of acetyl chloride (0.13 mol) dropwise, and after addition, the mixture was allowed to react at low temperature (0-5° C.) for half an hour, to obtain freshly prepared HCl gas in ethanol. The mixed solution of ethyl cyanoformate (12.5 g, 0.13 mol) and dichloromethane (30 mL) was added dropwise at low temperature (0-5° C.), and then the reaction was stirred overnight at 0-5° C., followed by filtering to collect the solids formed in the reaction, that is the imine intermediate hydrochloride. The hydrochloride was suspended in 50 mL of methyl tert-butyl ether, to which was added 20 g of triethylamine, and then the reaction was stirred at room temperature for 5 h. The reaction solution was filtered to remove triethylamine hydrochloride, and then concentrated under reduced pressure to remove methyl tert-butyl ether and obtain 12.3 g of intermediate AM1-1.
Step 2: 9.6 g of intermediate AM1-1 (66.1 mmol) prepared in step 1 was dissolved in a mixed solution of ethanol (40 mL) and methyl tert-butyl ether (120 mL), to which was added 10 g of phenylacetic hydrazide (66.6 mmol), and then the reaction was stirred overnight at room temperature. The reaction solution was filtered, and the collected solids were washed with ethanol twice, followed by drying, to obtain 8.7 g of intermediate AM1-2, which was used in the next reaction.
Step 3: 5 g of intermediate AM1-2 (20.0 mmol) was suspended in 100 mL of xylene, and reacted under reflux at 170° C. for 24 h. Then, the reaction solution was cooled to room temperature, and filtered to obtain 4.3 g of intermediate AM1-3.
Step 4: 4 g of intermediate AM1-3 (17.3 mmol) was suspended in 40 mL of water, to which was added 1.2 g of LiOH (50.1 mmol), and then the mixture was stirred at room temperature for 3 h. The reaction solution was adjusted to pH 2 with 2M dilute hydrochloric acid, and then the solids precipitated, which were collected by filtration. The solids were washed twice with water, and then dried, to obtain 3.3 g of intermediate A-1.
Step 1: 5 g of commercially available methyl 1,2,4-triazol-3-carboxylate (39.3 mmol) was dissolved in 100 mL of acetone, to which were added 8.1 g of potassium carbonate (58.6 mmol) and 6.7 g of benzyl bromide (39.1 mmol), and then the mixture was stirred overnight at room temperature. The complete reaction of the raw material was detected by TLC. Then, 100 ml of water was added, and the resultant solution was extracted three times with 150 mL of ethyl acetate. The organic layers were combined, washed once with saturated NaCl aqueous solution, and dried over 50 g of anhydrous sodium sulfate. Sodium sulfate was removed by filtration. The filtrate was concentrated under reduced pressure to dry, and then 50 mL of petroleum ether was added to crystallize. 6.6 g of wet product was obtained, which was recrystallized in 30 mL mixed solution of ethyl acetate and petroleum ether (EA:PE=1:1). The crystals were collected by filtration and dried to obtain a total of 4.1 g of intermediate AM2-1.
Step 2: 2 g of intermediate AM2-1 (9.2 mmol) was dissolved in 20 mL of methanol, to which was added the solution of LiOH (0.7 g, 29.2 mmol) in 20 mL of water, and the mixture was stirred at room temperature for 5 h. TLC indicated the complete reaction of the raw material. The reaction solution was adjusted to pH 2-3 with 2 M dilute hydrochloric acid. Solids precipitated, were collected by filtration, washed with water to neutral, and dried to obtain 1.7 g of intermediate A-2.
Using methyl 1H-imidazol-4-carboxylate as the raw material, the intermediate A-10 was obtained by the synthesis method similar to that of intermediate A-2.
The other intermediates A-3, A-4, A-5, A-6, A-7, A-8, and A-9 were commercially available and had the following structures:
Substituting phenylacetic hydrazide in the method of synthesizing A-1 with pyridine-4-carbohydrazide, the intermediate A-11 was prepared by the synthesis method similar to that of intermediate A-1.
Similar to the above method, using pyridine-3-carbohydrazide, 3-hydroxyphenylacetic hydrazide, and 3-aminophenylacetic hydrazide as raw materials, intermediates A-12, A-13, A-14, A-15, and A-16 were synthesized, with the following structures:
The specific procedures were as follows:
5 g of BOC-L-threonine (MS1-2, CAS: 2592-18-9, 22.8 mmol) was dissolved in 50 mL of DMF, and then the reaction solution was cooled to 0° C. in an ice salt bath. 1.4 g of NaH (57.0 mmol) was suspended in 10 mL of DMF, and added to the reaction solution dropwise. The ice salt bath was removed, and the reaction solution was stirred at room temperature until bubbles disappeared, to which was added 5.0 g of 4-bromo-1-fluoro-2-nitrobenzene (MS-1-1, 22.7 mmol) dropwise, and the resultant solution was stirred at room temperature for 4 h. Ice water was added to quench the reaction. 2M dilute hydrochloric acid was added to the reaction system, and the pH value of the system was adjusted to be 2-3. The reaction solution was extracted three times with 50 mL of ethyl acetate. The organic layers were combined, washed with water, dried, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 7.6 g of intermediate MM1-1 (molecular weight 417.25) as yellow semi-solids.
The above intermediate MM1-1 was dissolved in 60 mL of methanol, and then subjected to the catalytic hydrogenation in the presence of the catalyst Pd/C. After completion of the reaction, the reaction solution was filtered to remove Pd/C, and the filtrate was concentrated under reduced pressure. The residue was stirred in methyl tert-butyl ether to crystallize. The solid was filtered, dried, and then dissolved in 25 mL of DMSO, to which were added 6.4 g of TBTU (19.9 mmol) and 2.6 g of DIPEA (20.1 mmol). The reaction solution was stirred overnight at room temperature, and then the reaction was quenched with 25 mL of ice water. The reaction solution was extracted three times with 30 mL of ethyl acetate. The organic layers were combined, washed with water, dried, and concentrated under reduced pressure. The residue was crystallized in petroleum ether, and then the crystals were filtered and dried, to obtain 5.1 g of intermediate MM1-2 (molecular weight 371.23) as white solids.
2.5 g of intermediate MM1-2 (6.7 mmol) was dissolved in 50 mL of DMF, to which were added 3.3 g of cesium carbonate (10.2 mmol) and 1.4 g of iodomethane (10.2 mmol), and then the mixture was stirred at room temperature until the reaction was completed. The reaction was quenched with 50 mL of ice water. The reaction solution was extracted three times with 80 mL of ethyl acetate. The organic layers were combined, washed with water, dried, and concentrated under reduced pressure. The residue was crystallized in petroleum ether, and then filtered to collect the solids, to which was added 20 mL of methyl tert-butyl ether saturated with hydrochloric acid gas. The resultant solution was stirred at room temperature for 6 h, and then concentrated under reduced pressure to remove the solvent. The residue was crystallized in petroleum ether to obtain 1.9 g of intermediate MM1-3 (molecular weight 321.60) as light yellow solids.
1.7 g of intermediate MM1-3 (5.3 mmol), 3.4 g of TBTU (10.6 mmol), and 1.0 g of DIPEA (7.7 mmol) were dissolved in 20 mL of DCM, to which was added 1.3 g of intermediate A-1 (5-benzyl-4H-1,2,4-triazol-3-carboxylic acid, 6.4 mmol), and then the mixture was stirred overnight at room temperature. Ice water was added to quench the reaction. The organic layer was washed with water, dried, and concentrated under reduced pressure. The residue was separated by column chromatography to obtain 1.8 g of intermediate M-1.
Using 4-bromo-1-fluoro-2-nitrobenzene and BOC-L-serine as raw materials, the intermediate M-2 was obtained by the synthesis method similar to that of intermediate M-1.
Using MM1-2 as the raw material and substituting iodomethane with deuterated iodomethane, the intermediate M-3 was prepared by the synthesis method similar to that of steps 3 and 4 for intermediate M-1.
MM1-2 was used as the raw material, to which was added methyl tert-butyl ether saturated with hydrochloric acid gas, and then the solution was stirred at room temperature for 6 h. The solvent was removed by concentration under reduced pressure. The residue was crystallized in petroleum ether to obtain the intermediate MM4-3 (molecular weight 307.57).
Using above intermediate MM4-3 and intermediate A-1 as raw materials, compound M-4 was obtained by the synthesis method similar to that of step 4 for intermediate M-1.
Using BOC-D-threonine as raw material, compound M-5 was obtained by the synthesis method similar to that of intermediate M-1.
Similar to the above synthesis method, corresponding intermediates were synthesized from different raw materials, and the intermediates have the following structures:
15 g of piperazine (174.1 mmol) was added to a flask, to which was added 50 mL of DMF to dissolve, and then the flask was cooled to 0° C. in an ice salt bath. 10.4 g of NaH (433.3 mmol) was slowly added, and after addition, the mixture was stirred at 0° C. for 5 min. 80% solution of propargyl bromide (60 g) in toluene was slowly added. After that, the ice salt bath was removed. Then, the mixture was stirred at room temperature for 5 h. TLC detection showed that the raw material disappeared. The reaction solution was slowly added to 200 ml of water, and then extracted twice with 150 mL of ethyl acetate. The ethyl acetate layers were combined, dried with 10 g of anhydrous sodium sulfate for 30 minutes, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and then the intermediate M-31 was obtained as sheet-like solids.
400 mg of intermediate M-1 (0.85 mmol) was added to a flask, to which were added 20 mg of palladium acetate, 20 mg of CuI, 40 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 400 mg of triphenylphosphine, 151 mg of intermediate M-31 (0.93 mmol), 5 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 150 mL of water was slowly added, and then the resultant solution was extracted twice with 100 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 70 mL of saturated NaCl aqueous solution, dried with 10 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-1.
ESI-MS m/z: 550.26 [M-1]−
Compound I-2 could be obtained by the following method 1:
100 mg of intermediate I-1 (0.18 mmol) was added to a flask, to which were added 5 mg of palladium acetate, 5 mg of CuI, 8 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 100 mg of triphenylphosphine, 85 mg of intermediate M-1 (0.18 mmol), 3 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 50 mL of water was slowly added, and then the resultant solution was extracted twice with 50 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 30 mL of saturated NaCl aqueous solution, dried with 5 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-2.
ESI-MS m/z: 939.41 [M-1]−
Compound I-2 could be obtained by the following method 2:
400 mg of intermediate M-1 (0.85 mmol) was added to a flask, to which were added 20 mg of palladium acetate, 20 mg of CuI, 40 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 400 mg of triphenylphosphine, 76 mg of intermediate M-31 (0.47 mmol), 5 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 150 mL of water was slowly added, and then the resultant solution was extracted twice with 100 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 70 mL of saturated NaCl aqueous solution, dried with 10 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-2.
Compound I-1 and intermediate M-2 were used as raw materials, to obtain compound I-3, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 925.40 [M-1]−
Compound I-1 and intermediate M-7 were used as raw materials, to obtain compound I-4, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 939.41 [M-1]−
Compound I-1 and intermediate M-8 were used as raw materials, to obtain compound I-5, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 925.40 [M-1]−
Compound I-1 and intermediate M-19 were used as raw materials, to obtain compound I-6, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 940.41 [M-1]−
Compound I-1 and intermediate M-20 were used as raw materials, to obtain compound I-7, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 926.39 [M-1]−
Compound I-1 and intermediate M-21 were used as raw materials, to obtain compound I-8, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 938.42[M-1]−
Compound I-1 and intermediate M-22 were used as raw materials, to obtain compound I-9, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 924.40 [M-1]−
Intermediate M-2 was used as raw material, to obtain compound I-10, by the synthesis method similar to that of Example 1.
ESI-MS m/z: 536.25 [M-1]−
Compound I-10 and intermediate M-2 were used as raw materials, to obtain compound I-11, by the synthesis method similar to Method 1 of Example 4.
1HNMR (400 MHZ, DMSO-d6) δ: 8.40 (d, J=7.8 Hz, 2H), 7.55-7.50 (m, 2H), 7.38-7.20 (m, 16H), 4.91-4.83 (m, 4H), 4.65-4.55 (m, 2H), 4.45-4.35 (m, 4H), 3.49 (s, 4H), 3.35 (s, 6H), 2.60-2.40 (m, 8H).
ESI-MS m/z: 911.38[M-1]−
Alternatively, intermediates M-2 and M-31 were used as raw materials, to obtain compound I-11, by the synthesis method similar to Method 2 of Example 4.
Compound I-10 and intermediate M-7 were used as raw materials, to obtain compound I-12, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 925.40 [M-1]−
Compound I-10 and intermediate M-8 were used as raw materials, to obtain compound I-13, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 911.38 [M-1]−
Intermediate M-3 was used as raw material, to obtain compound I-14, by the synthesis method similar to that of Example 1.
ESI-MS m/z: 553.28 [M-1]−
Compound I-10 and intermediate M-2 were used as raw materials, to obtain compound I-15, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 945.45 [M-1]−
Alternatively, intermediates M-3 and M-31 were used as raw materials, to obtain compound I-15, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-4 was used as raw material, to obtain compound I-16, by the synthesis method similar to that of Example 1.
ESI-MS m/z: 536.25 [M-1]−
Compound I-16 and intermediate M-4 were used as raw materials, to obtain compound I-17, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 911.38 [M-1]−
Alternatively, intermediates M-4 and M-31 were used as raw materials, to obtain compound I-17, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-5 was used as raw material, to obtain compound I-18, by the synthesis method similar to that of Example 1.
ESI-MS m/z: 550.26 [M-1]−
Compound I-18 and intermediate M-5 were used as raw materials, to obtain compound I-19, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 939.41 [M-1]−
Alternatively, intermediates M-5 and M-31 were used as raw materials, to obtain compound I-19, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-6 was used as raw material, to obtain compound I-20, by the synthesis method similar to that of Example 1.
ESI-MS m/z: 522.23 [M-1]−
Compound I-20 and intermediate M-6 were used as raw materials, to obtain compound I-21, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 883.35 [M-1]−
Alternatively, intermediates M-6 and M-31 were used as raw materials, to obtain compound I-21, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-7 was used as raw material, to obtain compound I-22 by the synthesis method similar to that of Example 1.
1HNMR (400 MHZ, DMSO-d6) δ: 8.83 (s, 1H), 7.95-7.90 (m, 1H), 7.55-7.50 (m, 1H), 7.40-7.22 (m, 7H), 5.54-5.41 (m, 2H), 4.90-4.78 (m, 2H), 3.49 (s, 2H), 3.33 (s, 3H), 3.26-3.20 (m, 2H), 3.16-3.09 (m, 1H), 2.60-2.40 (m, 8H), 1.28-1.23 (m, 3H).
ESI-MS m/z: 550.26 [M-1]−
Compound I-22 and intermediate M-7 were used as raw materials, to obtain compound I-23, by the synthesis method similar to Method 1 of Example 4.
1HNMR (400 MHZ, CDCl3) δ: 8.09 (d, J=7.0 Hz, 2H), 7.99 (s, 2H), 7.43-7.33 (m, 6H), 7.33-7.23 (m, 8H), 7.12 (d, J=8.2 Hz, 2H), 5.37 (s, 4H), 5.10 (t, J=6.7 Hz, 2H), 5.05-4.93 (m, 2H), 3.54 (s, 4H), 3.40 (s, 6H), 2.90-2.60 (m, 8H), 1.40 (d, J=6.3 Hz, 6H).
ESI-MS m/z: 939.41 [M-1]−
Alternatively, intermediates M-7 and M-31 were used as raw materials, to obtain compound I-23, by the synthesis method similar to Method 2 of Example 4.
Compound I-22 and intermediate M-8 were used as raw materials, to obtain compound I-24, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 925.40 [M-1]−
Compound I-22 and intermediate M-19 were used as raw materials, to obtain compound I-25, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 940.41 [M-1]−
Compound I-22 and intermediate M-20 were used as raw materials, to obtain compound I-26, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 926.39 [M-1]−
Compound I-22 and intermediate M-21 were used as raw materials, to obtain compound I-27, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 938.42 [M-1]−
Compound I-22 and intermediate M-22 were used as raw materials, to obtain compound I-28, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 924.40 [M-1]−
Intermediate M-8 was used as raw material, to obtain compound I-29 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 536.25 [M-1]−
Compound I-29 and intermediate M-8 were used as raw materials, to obtain compound I-30, by the synthesis method similar to Method 1 of Example 4.
1HNMR (400 MHZ, DMSO-d6) δ: 8.84 (s, 2H), 8.47 (d, J=8.0 Hz, 2H), 7.70-7.50 (m, 2H), 7.49-7.25 (m, 12H), 7.21 (d, J=8.0 Hz, 2H), 5.48 (s, 4H), 4.86-4.79 (m, 2H), 4.62-4.56 (m, 2H), 4.44-4.39 (m, 2H), 3.52 (s, 4H), 3.40 (s, 6H), 2.78-2.48 (m, 8H).
ESI-MS m/z: 911.38 [M-1]−
Alternatively, intermediates M-8 and M-31 were used as raw materials, to obtain compound I-30, by the synthesis method similar to Method 2 of Example 4.
Compound I-29 and intermediate M-19 were used as raw materials, to obtain compound I-31, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 926.39 [M-1]−
Compound I-29 and intermediate M-20 were used as raw materials, to obtain compound I-32, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 912.38 [M-1]−
Compound I-29 and intermediate M-21 were used as raw materials, to obtain compound I-33, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 924.40 [M-1]−
Compound I-29 and intermediate M-22 were used as raw materials, to obtain compound I-34, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 910.39 [M-1]−
Intermediate M-9 was used as raw material, to obtain compound I-35 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 553.28 [M-1]−
Compound I-35 and intermediate M-9 were used as raw materials, to obtain compound I-36, by the synthesis method similar to Method 1 of Example 4.
1HNMR (400 MHZ, CDCl3) δ: 8.10 (d, J=7.0 Hz, 2H), 8.00 (s, 2H), 7.43-7.34 (m, 6H), 7.34-7.23 (m, 8H), 7.14-7.10 (m, 2H), 5.37 (s, 4H), 5.11 (t, J=6.7 Hz, 2H), 5.05-4.93 (m, 2H), 3.55 (s, 4H), 2.88-2.68 (m, 8H), 1.41 (d, J=6.3 Hz, 6H).
ESI-MS m/z: 945.45 [M-1]−
Alternatively, intermediates M-9 and M-31 were used as raw materials, to obtain compound I-36, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-10 was used as raw material, to obtain compound I-37 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 536.25 [M-1]−
Compound I-37 and intermediate M-10 were used as raw materials, to obtain compound I-38, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 911.38 [M-1]−
Alternatively, intermediates M-10 and M-31 were used as raw materials, to obtain compound I-38, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-11 was used as raw material, to obtain compound I-39 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 550.26 [M-1]−
Compound I-39 and intermediate M-11 were used as raw materials, to obtain compound I-40, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 939.41 [M-1]−
Alternatively, intermediates M-11 and M-31 were used as raw materials, to obtain compound I-40, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-12 was used as raw material, to obtain compound I-41 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 522.23 [M-1]−
Compound I-41 and intermediate M-12 were used as raw materials, to obtain compound I-42, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 883.35 [M-1]−
Alternatively, intermediates M-12 and M-31 were used as raw materials, to obtain compound I-42, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-13 was used as raw material, to obtain compound I-43 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 550.25 [M-1]−
Compound I-43 and intermediate M-13 were used as raw materials, to obtain compound I-44, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 939.39 [M-1]−
Alternatively, intermediates M-13 and M-31 were used as raw materials, to obtain compound I-44, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-14 was used as raw material, to obtain compound I-45 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 550.25 [M-1]−
Compound I-45 and intermediate M-14 were used as raw materials, to obtain compound I-46, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 939.39 [M-1]−
Alternatively, intermediates M-14 and M-31 were used as raw materials, to obtain compound I-46, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-15 was used as raw material, to obtain compound I-47 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 549.27 [M-1]−
Compound I-47 and intermediate M-15 were used as raw materials, to obtain compound I-48, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 937.42 [M-1]−
Alternatively, intermediates M-15 and M-31 were used as raw materials, to obtain compound I-48, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-16 was used as raw material, to obtain compound I-49 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 551.25 [M-1]−
Compound I-49 and intermediate M-16 were used as raw materials, to obtain compound I-50, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 941.38 [M-1]−
Alternatively, intermediates M-16 and M-31 were used as raw materials, to obtain compound I-50, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-17 was used as raw material, to obtain compound I-51 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 551.25 [M-1]−
Compound I-51 and intermediate M-17 were used as raw materials, to obtain compound I-52, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 941.38 [M-1]−
Alternatively, intermediates M-17 and M-31 were used as raw materials, to obtain compound I-52, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-18 was used as raw material, to obtain compound I-53 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 550.26 [M-1]−
Compound I-53 and intermediate M-18 were used as raw materials, to obtain compound I-54, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 939.41 [M-1]−
Alternatively, intermediates M-18 and M-31 were used as raw materials, to obtain compound I-54, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-19 was used as raw material, to obtain compound I-55 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 551.26 [M-1]−
Compound I-55 and intermediate M-19 were used as raw materials, to obtain compound I-56, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 941.40 [M-1]−
Alternatively, intermediates M-19 and M-31 were used as raw materials, to obtain compound I-56, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-20 was used as raw material, to obtain compound I-57 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 537.24 [M-1]−
Compound I-57 and intermediate M-20 were used as raw materials, to obtain compound I-58, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 913.37 [M-1]−
Alternatively, intermediates M-20 and M-31 were used as raw materials, to obtain compound I-58, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-21 was used as raw material, to obtain compound I-59 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 549.27 [M-1]−
Compound I-59 and intermediate M-21 were used as raw materials, to obtain compound I-60, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 937.42 [M-1]−
Alternatively, intermediates M-21 and M-31 were used as raw materials, to obtain compound I-60, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-22 was used as raw material to obtain compound I-61 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 535.25 [M-1]−
Compound I-61 and intermediate M-22 were used as raw materials, to obtain compound I-62, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 909.39 [M-1]−
Alternatively, intermediates M-22 and M-31 were used as raw materials, to obtain compound I-62, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-23 was used as raw material to obtain compound I-63 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 549.27 [M-1]−
Compound I-63 and intermediate M-23 were used as raw materials, to obtain compound I-64, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 937.42 [M-1]−
Alternatively, intermediates M-23 and M-31 were used as raw materials, to obtain compound I-64, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-24 was used as raw material to obtain compound I-65 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 551.26 [M-1]−
Compound I-65 and intermediate M-24 were used as raw materials, to obtain compound I-66, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 941.40 [M-1]−
Alternatively, intermediates M-24 and M-31 were used as raw materials, to obtain compound I-66, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-25 was used as raw material to obtain compound I-67 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 551.26 [M-1]−
Compound I-67 and intermediate M-25 were used as raw materials, to obtain compound I-68, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 941.40 [M-1]−
Alternatively, intermediates M-25 and M-31 were used as raw materials, to obtain compound I-68, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-26 was used as raw material to obtain compound I-69 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 566.26 [M-1]−
Compound I-69 and intermediate M-26 were used as raw materials, to obtain compound I-70, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 971.40 [M-1]−
Alternatively, intermediates M-26 and M-31 were used as raw materials, to obtain compound I-70, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-27 was used as raw material to prepare compound I-69 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 565.28 [M-1]−
Compound I-71 and intermediate M-27 were used as raw materials, to prepare compound I-72, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 969.44 [M-1]−
Alternatively, intermediates M-27 and M-31 were used as raw materials, to prepare compound I-72, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-28 was used as raw material to prepare compound I-73 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 551.26 [M-1]−
Compound I-73 and intermediate M-28 were used as raw materials, to prepare compound I-74, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 941.40 [M-1]−
Alternatively, intermediates M-28 and M-31 were used as raw materials, to prepare compound I-74, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-29 was used as raw material to prepare compound I-75 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 566.26 [M-1]−
Compound I-75 and intermediate M-29 were used as raw materials, to prepare compound I-76, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 971.40 [M-1]−
Alternatively, intermediates M-29 and M-31 were used as raw materials, to prepare compound I-76, by the synthesis method similar to Method 2 of Example 4.
Intermediate M-30 was used as raw material to prepare compound I-77 by the synthesis method similar to that of Example 1.
ESI-MS m/z: 565.28 [M-1]−
Compound I-77 and intermediate M-30 were used as raw materials, to prepare compound I-78, by the synthesis method similar to Method 1 of Example 4.
ESI-MS m/z: 969.44 [M-1]−
Alternatively, intermediates M-30 and M-31 were used as raw materials, to prepare compound I-78, by the synthesis method similar to Method 2 of Example 4.
400 mg of intermediate M-1 (0.85 mmol) was transferred to a flask, to which were added 20 mg of palladium acetate, 20 mg of CuI, 40 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 400 mg of triphenylphosphine, 50 mg of 1,7-octanediyne (S-1, 0.47 mmol), 5 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° ° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 150 mL of water was slowly added, and then the resultant solution was extracted twice with 100 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 70 mL of saturated NaCl aqueous solution, dried with 10 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-79.
ESI-MS m/z: 883.38 [M-1]−
Intermediates M-30 and S-1 were used as raw materials, to prepare compound I-80, by the synthesis method similar to that of Example 81.
ESI-MS m/z: 855.34 [M-1]−
10 mL of 50% NaOH aqueous solution was placed in a flask, to which were added 10 mL of toluene, 0.1 g of TBTU, and 1 g of tetra-polyethylene glycol, and then the mixture was stirred to dissolve. Then, 80% solution of propargyl bromide (2 g) in toluene was added, and the reaction solution was allowed to react in an oil bath at 60° C. for 20 h. The reaction system was cooled to room temperature, to which were added 20 mL of ethyl acetate and 20 mL of water, and then the resultant solution was shaken and stood for layering. The ethyl acetate layer was collected, and the water layer was extracted twice with 10 mL of ethyl acetate. The ethyl acetate layers were combined, washed once with saturated NaCl aqueous solution, dried with anhydrous sodium sulfate for 30 min, and filtered to remove anhydrous sodium sulfate. The filtrate was concentrated to dryness to obtain intermediate M-32.
400 mg of intermediate M-1 (0.85 mmol) was transferred to a flask, to which were added 20 mg of palladium acetate, 20 mg of CuI, 40 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 400 mg of triphenylphosphine, 127 mg of intermediate M-32 (0.47 mmol), 5 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 150 mL of water was slowly added, and then the resultant solution was extracted twice with 100 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 70 mL of saturated NaCl aqueous solution, dried with 10 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-81-1:
ESI-MS m/z: 1047.44 [M-1]−
and compound I-81-2:
ESI-MS m/z: 658.30 [M-1]−.
Intermediates M-2 and M-32 were used as raw materials, to prepare compound I-82-1 and I-82-2, by the synthesis method similar to that of Example 84.
I-82-1, ESI-MS m/z: 1019.40 [M-1]−
I-82-2, ESI-MS m/z: 644.23 [M-1]−
Intermediates M-1, M-2 and M-32 were used as raw materials, to prepare compound I-83, by the synthesis method similar to that of Example 84.
ESI-MS m/z: 1033.43 [M-1]−
Intermediates M-7 and M-32 were used as raw materials, to prepare compounds I-84-1 and
I-84-2, by the synthesis method similar to that of Example 84.
I-84-1, ESI-MS m/z: 1047.43 [M-1]−
I-84-2, ESI-MS m/z: 658.28 [M-1]−
Intermediates M-8 and M-32 were used as raw materials, to prepare compounds I-85-1 and I-85-2, by the synthesis method similar to that of Example 84.
1HNMR spectral data of I-85-1:
1HNMR (400 MHZ, DMSO-d6) δ: 8.84 (s, 2H), 8.47 (d, J=7.8 Hz, 2H), 7.66-7.58 (m, 2H), 7.43-7.27 (m, 12H), 7.26-7.20 (m, 2H), 5.49 (s, 4H), 4.90-4.78 (m, 2H), 4.65-4.55 (m, 2H), 4.46-4.38 (m, 4H), 3.68-3.56 (m, 4H), 3.56-3.46 (m, 10H), 3.44-3.39 (m, 2H), 3.31 (s, 6H).
ESI-MS m/z: 1019.40 [M-1]−
I-85-2, ESI-MS m/z: 644.27 [M-1]−
Intermediates M-7, M-8 and M-32 were used as raw materials, to prepare compound I-86, by the synthesis method similar to that of Example 84.
ESI-MS m/z: 1033.43 [M-1]−
200 mg of intermediate I-81-2 (0.30 mmol) was transferred to a flask, to which were added 10 mg of palladium acetate, 10 mg of CuI, 20 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 200 mg of triphenylphosphine, 3 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 70 ml of water was slowly added, and then the resultant solution was extracted twice with 50 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 30 mL of saturated NaCl aqueous solution, dried with 5 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-87:
ESI-MS m/z: 1315.58 [M-1]−
Intermediate I-82-2 was used as raw material, to prepare compound I-88, by the synthesis method similar to that of Example 89.
ESI-MS m/z: 1287.54 [M-1]−
Compound I-84-2 was used as raw material, to prepare compound I-89, by the synthesis method similar to that of Example 89.
1HNMR (400 MHZ, DMSO-d6) δ: 8.85 (s, 2H), 8.66-8.60 (m, 2H), 8.20-8.12 (m, 2H), 7.42-7.24 (m, 14H), 5.56-5.43 (m, 4H), 4.90-4.82 (m, 4H), 4.44-4.38 (m, 2H), 4.36-4.28 (m, 6H), 3.68-3.62 (m, 8H), 3.62-3.48 (m, 20H), 3.38-3.32 (m, 2H), 2.71 (s, 6H), 1.42-1.22 (m, 6H).
ESI-MS m/z: 1315.58 [M-1]−
Compound I-85-2 was used as raw material, to prepare compound I-90, by the synthesis method similar to that of Example 89.
ESI-MS m/z: 1287.54 [M-1]−
Compound I-1 was used as raw material, to prepare compound I-91, by the synthesis method similar to that of Example 89.
ESI-MS m/z: 1109.51 [M-1]−
Compound I-10 was used as raw material, to prepare compound I-92, by the synthesis method similar to that of Example 89.
ESI-MS m/z: 1071.48 [M-1]−
Compound I-22 was used as raw material, to prepare compound I-93, by the synthesis method similar to that of Example 89.
ESI-MS m/z: 1109.51 [M-1]−
Compound I-29 was used as raw material, to prepare compound I-94, by the synthesis method similar to that of Example 89.
1HNMR (400 MHZ, DMSO-d6) δ: 8.84 (s, 2H), 8.47 (d, J=8.0 Hz, 2H), 7.58 (s, 2H), 7.39-7.28 (m, 12H), 7.21 (d, J=8.0 Hz, 2H), 5.48 (s, 4H), 4.86-4.79 (m, 2H), 4.61-4.56 (m, 2H), 4.44-4.39 (m, 2H), 3.51-3.47 (m, 8H), 3.43 (s, 6H), 2.66-2.46 (m, 16H).
ESI-MS m/z: 1071.48 [M-1]−
Intermediates M-1 and S-2 were used as raw materials, to prepare compound I-95, by the synthesis method similar to that of Example 81.
ESI-MS m/z: 940.43 [M-1]−
Intermediates M-2 and S-2 were used as raw materials, to prepare compound I-96, by the synthesis method similar to that of Example 81.
ESI-MS m/z: 912.40 [M-1]−
Intermediates M-7 and S-2 were used as raw materials, to prepare compound I-97, by the synthesis method similar to that of Example 81.
ESI-MS m/z: 940.43 [M-1]−
Intermediates M-8 and S-2 were used as raw materials, to prepare compound I-98, by the synthesis method similar to that of Example 81.
ESI-MS m/z: 912.40 [M-1]−
10 mL of 50% NaOH aqueous solution was placed in a flask, to which were added 10 mL of toluene, 0.1 g of TBTU, and 1 g of tetra-polyethylene glycol, and then the mixture was stirred to dissolve. Then, 80% solution of propargyl bromide (1.1 g) in toluene was added, and the reaction solution was allowed to react in an oil bath at 60° C. for 20 h. The reaction system was cooled to room temperature, to which were added 20 mL of ethyl acetate and 20 mL of water, and then the resultant solution was shaken and stood for layering. The ethyl acetate layer was collected, and the water layer was extracted twice with 10 mL of ethyl acetate. The ethyl acetate layers were combined, washed once with saturated NaCl aqueous solution, dried with anhydrous sodium sulfate for 30 min, and filtered to remove anhydrous sodium sulfate. The filtrate was concentrated to dryness to obtain intermediate M-34.
400 mg of intermediate M-1 (0.85 mmol) was transferred to a flask, to which were added 20 mg of palladium acetate, 20 mg of CuI, 40 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 400 mg of triphenylphosphine, 209 mg of intermediate M-34 (0.85 mmol), 5 mL of triethylamine, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 150 mL of water was slowly added, and then the resultant solution was extracted twice with 100 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 70 mL of saturated NaCl aqueous solution, dried with 10 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-99:
ESI-MS m/z: 634.30 [M-1]−
Intermediates M-2 and M-34 were used as raw materials, to prepare compound I-100, by the synthesis method similar to that of Example 101.
ESI-MS m/z: 620.28 [M-1]−
Intermediates M-7 and M-34 were used as raw materials, to prepare compound I-101, by the synthesis method similar to that of Example 101.
1HNMR (400 MHZ, DMSO-d6) δ: 8.85 (s, 1H), 7.95 (d, J=5.8 Hz, 1H), 7.68-7.53 (m, 1H), 7.43-7.24 (m, 7H), 5.55-5.45 (m, 2H), 4.93-4.81 (m, 2H), 4.42 (s, 2H), 3.68-3.56 (m, 4H), 3.56-3.46 (m, 10H), 3.44-3.39 (m, 2H), 3.35 (s, 3H), 3.23 (s, 3H), 1.32-1.25 (m, 3H).
ESI-MS m/z: 634.30 [M-1]−
Intermediates M-8 and M-34 were used as raw materials, to prepare compound I-102, by the synthesis method similar to that of Example 101.
1HNMR (400 MHZ, DMSO-d6) δ: 8.84 (s, 1H), 8.47 (d, J=7.9 Hz, 1H), 7.72-7.58 (m, 1H), 7.43-7.20 (m, 7H), 5.49 (s, 2H), 4.66-4.54 (m, 1H), 4.48-4.36 (m, 2H), 3.69-3.56 (m, 4H), 3.56-3.47 (m, 10H), 3.47-3.37 (m, 2H), 3.36-3.27 (m, 3H), 3.23 (s, 3H).
ESI-MS m/z: 620.28 [M-1]−
Intermediates M-1 and S-3 were used as raw materials, to prepare compound I-103, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 458.19 [M-1]−
Intermediates M-1 and S-3 were used as raw materials, to prepare compound I-104, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 458.19 [M-1]−
Intermediates M-1 and S-4 were used as raw materials, to prepare compound I-105, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 458.19 [M-1]−
Intermediates M-1 and S-4 were used as raw materials, to prepare compound I-106, by the synthesis method similar to Method 2 of Example 4.
1HNMR (400 MHz, DMSO-d6) δ: 8.85 (s, 1H), 7.98-7.92 (m, 1H), 7.49 (d, J=2.0 Hz, 1H), 7.43-7.27 (m, 5H), 7.26-7.20 (m, 2H), 5.56-5.43 (m, 2H), 4.94-4.88 (m, 1H), 4.88-4.78 (m, 2H), 3.64-3.54 (m, 2H), 3.23 (s, 3H), 1.25 (d, 3H).
ESI-MS m/z: 458.19 [M-1]−
Intermediates M-1 and S-5 were used as raw materials, to prepare compounds I-107, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 514.22 [M-1]−
Intermediates M-7 and S-5 were used as raw materials, to prepare compound I-108, by the synthesis method similar to Method 2 of Example 4.
1HNMR (400 MHZ, DMSO-d6) δ: 11.88 (s, 1H), 8.72 (s, 1H), 7.82 (d, J=6.0 Hz, 1H), 7.38-7.33 (m, 1H), 7.29-7.14 (m, 6H), 7.13-7.09 (m, 1H), 5.39-5.34 (m, 2H), 4.77-4.68 (m, 2H), 3.21 (s, 3H), 2.32 (t, J=6.8 Hz, 2H), 2.24-2.04 (m, 2H), 1.60-1.30 (m, 4H), 1.18-1.12 (m, 3H).
ESI-MS m/z: 514.22 [M-1]−
Intermediates M-1 and S-6 were used as raw materials, to prepare compound I-109, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 491.19 [M-1]−
Intermediates M-2 and S-6 were used as raw materials, to prepare compound I-110, by the synthesis method similar to Method 2 of Example 4.
1HNMR (400 MHZ, DMSO-d6) δ: 8.84 (s, 1H), 8.66-8.60 (m, 2H), 7.98-7.92 (m, 1H), 7.76-7.71 (m, 1H), 7.57-7.49 (m, 3H), 7.41-7.26 (m, 6H), 5.54-5.52 (m, 2H), 4.93-4.84 (m, 2H), 3.37 (s, 3H), 1.28 (d, J=5.2 Hz, 3H).
ESI-MS m/z: 491.19 [M-1]−
Intermediates M-1 and S-7 were used as raw materials, to prepare compound I-111, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 491.19 [M-1]−
Intermediates M-2 and S-7 were used as raw materials, to prepare compound I-112, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 491.19 [M-1]−
Intermediates M-1 and S-8 were used as raw materials, to prepare compound I-113, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 570.15 [M-1]−
Intermediates M-2 and S-8 were used as raw materials, to prepare compound I-114, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 570.15 [M-1]−
Intermediates M-1 and S-9 were used as raw materials, to prepare compound I-115, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 535.18 [M-1]−
Intermediates M-2 and S-9 were used as raw materials, to prepare compound I-116, by the synthesis method similar to Method 2 of Example 4.
ESI-MS m/z: 535.18 [M-1]−
Compound I-115 was used as raw material and dissolved in ethanol/water, to which were added NH4Cl and iron powder, and then the solution was allowed to react under reflux until the reaction of the raw material was complete. After concentration, the residue was separated by silica gel column chromatography to obtain I-117.
ESI-MS m/z: 505.21 [M-1]−
Compound I-116 was used as raw material to prepare compound I-118, using the synthesis method similar to that of Example 119.
ESI-MS m/z: 535.18[M-1]−
400 mg of intermediate M-1 (0.85 mmol) was transferred to a flask, to which were added 20 mg of palladium acetate, 20 mg of CuI, 40 mg of 2-dicyclohexylphosphino-2,4,6-triisopropylbiphenyl, 400 mg of triphenylphosphine, 250 mg of CaCO3, 5 mL of triethylamine, catalytic amount of water, and 10 mL of acetonitrile; the mixture was allowed to react overnight in an oil bath at 80° C. under nitrogen protection. TLC indicated the disappearance of raw materials. 150 mL of water was slowly added, and then the resultant solution was extracted twice with 100 mL of ethyl acetate. The ethyl acetate layers were combined, washed twice with 70 mL of saturated NaCl aqueous solution, dried with 10 g of anhydrous sodium sulfate for 30 min, and then filtered to remove sodium sulfate. The filtrate was concentrated to dry, and the residue was purified over column chromatography, to provide compound I-119.
ESI-MS m/z: 414.21 [M-1]−
Compound M-7 was used as starting material to prepare compound I-120, using the synthesis method similar to that of Example 121.
ESI-MS m/z: 414.16 [M-1]−
50 mg of compound I-30 was placed into a hydrogenation bottle, and then dissolved by adding 15 mL of methanol. To the solution, was added 5 mg of 10% Pd/C, and then hydrogen gas at atmospheric pressure was introduced and purged three times to completely replace the air in the bottle. The reaction solution was allowed to react for 4 h at room temperature. TLC showed that the reaction of the raw material was complete, and thus the reaction was stopped. The solvent was removed by concentration under reduced pressure. The residue was separated by column chromatography to obtain compound I-121.
ESI-MS m/z: 919.47 [M-1]−
Compound I-23 was used as starting material to prepare compound I-122, as described in Example 123.
ESI-MS m/z: 947.49 [M-1]−
Compound I-11 was used as starting material to prepare compound I-123, as described in Example 123.
ESI-MS m/z: 919.45 [M-1]−
Compound I-2 was used as starting material to prepare compound I-124, as described in Example 123.
ESI-MS m/z: 947.48 [M-1]−
The beneficial effects of the present invention were demonstrated with reference to specific Experimental Examples.
The compound of the present invention and the control compound were dissolved in the assay buffer (50 mM Hepes pH 7.5, 50 mM NaCl, 30 mM MgCl2, 1 mM DTT, 0.02% CHAPS, 0.5 mg/mL BSA), and diluted in a ratio of 1:1.5 using a 22-point titration (high concentration 2 μM), and then added to a 384-well plate. Each concentration of inhibitor (3.5 μL) and 3.5 μL of human RIP kinase (25 nM) dissolved in the assay buffer were added to the wells. After warming at 37° C. for 1 h, 3.5 μL of ATP (15 μM to 1.5 mM) in buffer was added to initiate the reaction, and then allowed to react at room temperature for 5 h. After completion of the reaction, 5 μL of ADP-Glo reagent containing 0.02% CHAPS was added to each well and incubated at room temperature for 1 h to terminate the kinase reaction and use up all remaining ATP. Then, 5 μL of ADP-Glo detection solution containing 0.02% CHAPS was added to each well and incubated at room temperature for 30 min to measure the emittance intensity. For each ATP concentration, the activity data was obtained by the emittance intensity minus the control intensity.
The IC50 value for each ATP concentration was determined by calculation, and the results are shown in Table 1:
The experimental results showed that the compound of the present invention had significant inhibitory activity against human RIP1 kinase, indicating that the compound of the present invention could be used in the manufacture of RIP inhibitors.
Human histiocytic lymphoma cells U937 (purchased from ATCC) in logarithmic growth phase were collected and resuspended in 1640 medium, and then the cell concentration was adjusted to 60 cells/μL, to obtain cell suspension, which was seeded in 384-well plate, with 20 μL cell suspension for each well. After the cells were incubated in a 5% CO2 incubator at 37° C. for 24 h, the test compound of the present invention and the control compound were diluted to the corresponding pre-determined concentration using the medium, and added to the 384-well plate at 5 μL/well. The final concentration of the compound ranged from 0 nM to 1000 nM, which was obtained by successive dilution in a ratio of 1:10. Moreover, the necrosis-inducing agent was added to the 384-well plate at 5 μL/well, which was consisted of TNF-α (100 ng/ml), Smac Mimetics (AT-406, 1 μM), and z-VAD-FMK (20 μM). After addition of test compound and the necrosis-inducing agent, the cells were cultured in a 5% CO2 incubator at 37° ° C. for 24 h. Then, 30 μL of CellTiter-Glo luminescent solution was added to each well, and shaken for 2 min. After standing at room temperature for 10 min, the absorbance was measured using an enzyme-linked immunosorbent assay (ELISA). The necrosis induction rate and the necrosis inhibition rate were calculated by the following formula:
The necrosis induction rate (%)=100×(1−B/A); A is the group without necrosis-inducing agents, and B is the group with necrosis-inducing agents.
The inhibition rate (%)=100−(A−X); the
A is the group without necrosis-inducing agents, B is the group with necrosis-inducing agents, and X is the group with compounds and necrosis-inducing agents.
The inhibitory activities (IC50) of test compounds on the necrosis of U937 cells induced by TNF-α were calculated by GraphPad, and the results are shown in following Table 2 (3 parallel wells were set for each group in the experiment):
Based on the above experimental results, the inhibitory activities (IC50) of the compounds according to the present invention on the necrosis of U937 cells induced by TNF-α were significant.
Human colon cancer cells HT-29 and human pancreatic cancer cells PANC-1 (purchased from ATCC) in logarithmic growth phase were collected and resuspended in 1640 medium, and then the cell concentration was adjusted to 20 cells/μL, to obtain cell suspension, which was seeded in 384-well plate, with 20 μL cell suspension for each well. After the cells were incubated in a 5% CO2 incubator at 37° C. for 24 h, the test compound of the present invention and the control compound were diluted to the corresponding pre-determined concentration using the medium, and added to the 384-well plate at 5 μL/well. The final concentration of the compound ranged from 0 nM to 1000 nM, which was obtained by successive dilution in a ratio of 1:10. Moreover, the necrosis-inducing agent was added to the 384-well plate at 5 μL/well, which was consisted of TNF-α (100 ng/ml), Smac Mimetics (AT-406, 1 μM), and z-VAD-FMK (20 μM). After addition of test compound and the necrosis-inducing agent, the cells were cultured in a 5% CO2 incubator at 37° C. for 24 h. Then, 30 μL of CellTiter-Glo luminescent solution was added to each well, and shaken for 2 min. After standing at room temperature for 10 min, the absorbance was measured using ELISA. The necrosis induction rate and the necrosis inhibition rate were calculated by the following formula:
The necrosis induction rate (%)=100×(1−B/A); A is the group without necrosis-inducing agents, and B is the group with necrosis-inducing agents.
The inhibition rate (%)=100−(A−X); the
A is the group without necrosis-inducing agents, B is the group with necrosis-inducing agents, and X is the group with compounds and necrosis-inducing agents.
The inhibitory activities (IC50) of test compounds on the necrosis of human colon cancer cells and human pancreatic cancer cells induced by TNF-α were calculated by GraphPad, and the results are shown in following Table 3 (3 parallel wells were set for each group in the experiment):
From the above experimental results, the inhibitory activities of the compounds according to the present invention on the necrosis of human colon cancer cells HT-29 and human pancreatic cancer cells PANC-1 induced by TNF-α were significant.
HacaT cells (purchased from ATCC) in logarithmic growth phase were collected and resuspended in 1640 medium, and then the cell concentration was adjusted to 20 cells/μL, to obtain cell suspension, which was seeded in 384-well plate, with 20 μL cell suspension for each well. After the cells were cultured in a 5% CO2 incubator at 37° C. for 24 h, the test compound of the present invention and the control compound were diluted to the corresponding pre-determined concentration using the medium, and added to the 384-well plate at 5 L/well. The final concentration of the compound ranged from 0 nM to 1000 nM, which was obtained by successive dilution in a ratio of 1:10. Moreover, the necrosis-inducing agent was added to the 384-well plate at 5 μL/well, which was consisted of TNF-α (100 ng/ml), Smac Mimetics (AT-406, 1 μM), and z-VAD-FMK (20 UM). After addition of test compound and the necrosis-inducing agent, the cells were cultured in a 5% CO2 incubator at 37 ºC for 24 h. Then, 30 μL of CellTiter-Glo luminescent solution was added to each well, and shaken for 2 min. After standing at room temperature for 10 min, the absorbance was measured using ELISA. The necrosis induction rate and the necrosis inhibition rate were calculated by the following formula:
The necrosis induction rate (%)=100×(1−B/A); A is the group without necrosis-inducing agents, and B is the group with necrosis-inducing agents.
The inhibition rate (%)=100−(A−X); the
A is the group without necrosis-inducing agents, B is the group with necrosis-inducing agents, and X is the group with compounds and necrosis-inducing agents.
The inhibitory activities (IC50) of test compounds on the necrosis of HacaT cells induced by TNF-α were calculated by GraphPad, and the results are shown in following Table 4 (3 parallel wells were set for each group in the experiment):
Based on the experimental results, the compound of the present invention had significant inhibitory activity on the necrosis of HacaT cells induced by TNF, and could be used for the prevention and treatment of psoriasis and other diseases.
28 8-week-old C57BL/6J male mice were purchased from Chengdu Dossy Experimental Animal Co., Ltd. After one week of adaptive feeding, the mice were randomly divided into 4 groups, including blank group, model group, test group, and positive control group, with 7 mice in each group. The blank group and the model group received 100 μL RO water per day, the test group was given compound I-30 of the present invention (50 mg/kg/day) dispersed in 100 μL of RO water, and the positive control group was given control compound 3 (Comp3) (50 mg/kg/d) in Experimental Example 1 every day, which was also dispersed in 100 μL RO water. The blank group had normal drinking water every day, while the model group, I-30 compound group, and control compound 3 (Comp3) group were given 3% DSS aqueous solution for 5 days, and then 3% DSS solution was changed to normal drinking water. The mice were euthanized on day 9 to collect colon tissues. The body weight, the water intake, and the disease index score (DAI) were observed daily for each mouse, and the results were recorded. The scoring method is shown in the table below:
After measuring the length of the colon tissue collected, the samples were taken and subjected to HE staining for pathological scoring, and the results were recorded. The scoring method is shown in the table below:
The weight records of mice during the experiment are shown in
The experimental results showed that in a DSS-induced inflammatory bowel disease model in mice, compound I-30 significantly alleviated the weight loss, significantly reduced the disease index (DAI) score, and significantly protected the colon tissue compared to the model group and th positive drug group, indicating a significant therapeutic effect of compound I-30.
In summary, the compound of the present invention had good inhibitory activity against human RIP1 kinase, whose effect was superior to that of compounds in the prior art, and thereby could be used in the manufacture of RIP inhibitors; moreover, the compound of the present invention had significant inhibitory activity against TNF-α induced necrosis in various cells, and so it could be used in the manufacture of medicaments for preventing and treating pancreatic cancer, colon cancer, lymphatic cancer, psoriasis and other diseases, as well as be better used in the treatment of inflammatory diseases, such as ulcerative colitis, atopic dermatitis, etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110546873.2 | May 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/093320 | 5/17/2022 | WO |
