BENZOPYRIDONE HETEROCYCLIC COMPOUND AND USE THEREOF
20220064141 · 2022-03-03
Inventors
- Huaxiang Fang (Wuhan, CN)
- Fangfang Li (Wuhan, CN)
- Yong XU (Wuhan, CN)
- Lu Huang (Wuhan, CN)
- Jinfeng Tao (Wuhan, CN)
Cpc classification
C07D405/10
CHEMISTRY; METALLURGY
C07D413/10
CHEMISTRY; METALLURGY
C07D401/10
CHEMISTRY; METALLURGY
C07D401/04
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
C07D401/10
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
C07D401/04
CHEMISTRY; METALLURGY
C07D405/10
CHEMISTRY; METALLURGY
C07D413/10
CHEMISTRY; METALLURGY
Abstract
A compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a prodrug thereof. R.sub.1-R.sub.5 and group A are as defined in the description. The compound is used for the preparation of drugs for treating diseases caused by KRAS G12C mutation and for treating and/or preventing cancers.
##STR00001##
Claims
1. A compound represented by formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a prodrug thereof, ##STR00112## wherein, R.sub.1 is independently selected from an aryl optionally substituted with a plurality of R.sub.6, or a heteroaryl optionally substituted with a plurality of R.sub.6; and when R.sub.1 is substituted with a plurality of R.sub.6, each R.sub.6 may be the same as or different from each other; R.sub.2 is independently selected from an aryl optionally substituted with a plurality of R.sub.7, or a heteroaryl optionally substituted with a plurality of R.sub.7; and when R.sub.2 is substituted with a plurality of R.sub.7, each R.sub.7 may be the same as or different from each other; R.sub.3 is selected from H, halogen, cyano, amide group, hydroxy, amino, C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl; said C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl is optionally substituted with 0 to 3 R.sub.7, and when said C.sub.1-C.sub.3 alkyl, C.sub.1-C.sub.3 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl is substituted with a plurality of R.sub.7, each R.sub.7 may be the same as or different from each other; R.sub.4 and R.sub.5 are each independently selected from H, halogen, C.sub.1-C8 alkyl optionally substituted with 0 to 3 R.sub.7, or C.sub.1-C.sub.8 heteroalkyl optionally substituted with 0 to 3 R.sub.7; ##STR00113## is selected from C.sub.4-C.sub.8 monoheterocycloalkyl optionally substituted with 0 to 3 R.sub.8, C.sub.6-C.sub.12 bridged heterocycloalkyl optionally substituted with 0 to 3 R.sub.8, or C.sub.6-C.sub.12 spiroheterocycloalkyl optionally substituted with 0 to 3 R.sub.8; R.sub.6 is selected from halogen, OH, CN, NH.sub.2, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy, C.sub.3-C.sub.8 cycloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl; wherein said C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy, C.sub.3-C.sub.8 cycloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl may be substituted with a plurality of the following groups: F, Cl, Br, I, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CF.sub.3, CHF.sub.2, CH.sub.2F, cyclopropyl, isopropyl, N(CH.sub.3).sub.2, and NH(CH.sub.3).sub.2; R.sub.7 is selected from halogen, OH, CONH.sub.2, CN, NH.sub.2, C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy, C.sub.3-C.sub.8 cycloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl, wherein said C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.8 heteroalkyl, C.sub.1-C.sub.3 haloalkyl, C.sub.1-C.sub.3 haloalkoxy, C.sub.3-C.sub.8 cycloalkyl, or C.sub.3-C.sub.8 heterocycloalkyl may be substituted with a plurality of the following groups: F, Cl, Br, I, OH, CN, NH.sub.2, CH.sub.3, CH.sub.3CH.sub.2, CH.sub.3O, CF.sub.3, CHF.sub.2, CH.sub.2F, cyclopropyl, isopropyl, N(CH.sub.3).sub.2, and NH(CH.sub.3).sub.2; and R.sub.8 is selected from H, halogen, CN, OH, C.sub.1-C.sub.3 alkyl, halogen-substituted C.sub.1-C.sub.3 alkyl, or cyano-substituted C.sub.1-C.sub.3 alkyl.
2. The compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1, wherein said R.sub.1 and R.sub.2 are each independently selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, pyrrolyl, piperazinyl, piperidinyl, phenyl, pyridyl, pyrimidinyl, pyrazolyl, thiazolyl, oxazolyl, imidazolyl, or indazolyl; said R.sub.3 is selected from hydrogen, chlorine, fluorine, amino, cyano, hydroxy, isopropyl, cyclopropyl, methyl, difluoromethyl, trifluoromethyl, methoxy, trifluoromethoxy, —OCH.sub.2CH.sub.3, —OCH.sub.2CHF.sub.2, or —OCH.sub.2CF.sub.3; said R.sub.4 and R.sub.5 are each independently selected from hydrogen, chlorine, fluorine, methyl, or —CH.sub.2N(CH.sub.3).sub.2; said R.sub.6 is selected from hydrogen, chlorine, fluorine, bromine, amino, cyano, hydroxy, methyl, ethylmethyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, methoxy, trifluoromethoxy, —OCH.sub.2CH.sub.3, —OCH.sub.2CHF.sub.2, —OCH.sub.2CF.sub.3, and —CH.sub.2N(CH.sub.3).sub.2; said R.sub.7 is selected from hydrogen, chlorine, fluorine, bromine, amino, carboxamido, cyano, hydroxy, methyl, ethylmethyl, isopropyl, cyclopropyl, difluoromethyl, trifluoromethyl, methoxy, trifluoromethoxy, —OCH.sub.2CH.sub.3, —OCH.sub.2CHF.sub.2, and —OCH.sub.2CF.sub.3; and said R.sub.8 is selected from hydrogen, methyl, —CH.sub.2OH or —CH.sub.2CN.
3. The compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1, wherein heteroatoms or heteroatom radicals in said C.sub.1-C.sub.8 heteroalkyl, C.sub.3-C.sub.8 heterocycloalkyl, heteroaryl, monoheterocycloalkyl, C.sub.6-C.sub.12 bridged heterocycloalkyl, and C.sub.6-C.sub.12 spiroheterocycloalkyl are each independently selected from —O—, —S—, —CN, —NH—, ═O, —O—N═, —C(═O)O—, —C(═O)—, —S(═O)—, —S(═O).sub.2—, —C(═O)NH—, —S(═O).sub.2NH—, or —NHC(═O)NH—, and the number of the heteroatoms or heteroatom radicals is independently selected from 1, 2 or 3.
4. The compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1, wherein said ##STR00114## is selected from ##STR00115##
5. The compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1, wherein the compound represented by formula I is selected from ##STR00116## wherein, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7 and R.sub.8 are as defined in claim 1, and n is 0, 1, 2, 3 or 4.
6. The compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 5, wherein the compound represented by formula I is selected from ##STR00117## ##STR00118## ##STR00119## ##STR00120## ##STR00121## wherein, R.sub.3 is selected from H, F, Cl, OH, CF.sub.3, CH.sub.3, cyclopropyl, OCF.sub.3, CHF.sub.2 or OCH.sub.3; R.sub.4 and R.sub.5 are independently selected from H, F or CH.sub.3; R.sub.6 is selected from H, F, Cl, Br, methyl, ethyl, isopropyl, methoxy, cyclopropyl or —CH.sub.2N(CH.sub.3).sub.2; R.sub.7 is selected from H, F, Cl, Br, NH.sub.2, OH, OCH.sub.3, CN, CF.sub.3, CONH.sub.2, methyl, ethyl, isopropyl, cyclopropyl or CHF2; R.sub.8 is selected from H or methyl; and n is 0, 1, 2, 3or4.
7. A pharmaceutical composition comprising an effective dose of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1, and at least one pharmaceutically acceptable excipient.
8. A method for treating diseases caused by KRAS G12C mutation, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1 to a subject in need thereof.
9. A method for inhibiting KRAS G12C, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1, to a subject in need thereof.
10. A method for treating and/or preventing cancer, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 1 to a subject in need thereof.
11. A compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a prodrug thereof, wherein the compound is selected from ##STR00122## ##STR00123## ##STR00124## ##STR00125## ##STR00126## ##STR00127## ##STR00128## ##STR00129## ##STR00130## ##STR00131## ##STR00132## ##STR00133## ##STR00134## ##STR00135## ##STR00136## ##STR00137## ##STR00138## ##STR00139## ##STR00140## ##STR00141## ##STR00142## ##STR00143## ##STR00144## ##STR00145## ##STR00146## ##STR00147## ##STR00148## ##STR00149## ##STR00150## ##STR00151##
12. A method for treating diseases caused by KRAS G12C mutation, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 11 to a subject in need thereof.
13. A method for inhibiting KRAS C12C, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 11 to a subject in need thereof.
14. A method for treating and/or preventing cancer, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 11 to a subject in need thereof.
15. A compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a prodrug thereof, wherein the compound is ##STR00152##
16. A method for treating diseases caused by KRAS G12C mutation, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 15 to a subject in need thereof.
17. A method for inhibiting KRAS G12C, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 15 to a subject in need thereof.
18. A method for treating and/or preventing cancer, comprising administering an effective amount of the compound, or the pharmaceutically acceptable salt thereof, or the stereoisomer thereof, or the tautomer thereof, or the hydrate thereof, or the solvate thereof, or the metabolite thereof, or the prodrug thereof according to claim 15 to a subject in need thereof.
Description
DETAILED DESCRIPTION
[0067] Preparation of the compounds of the present disclosure, or pharmaceutically acceptable salts thereof, or stereoisomers thereof, or tautomers thereof, or hydrates thereof, or solvates thereof, or metabolites thereof, or prodrugs thereof can be accomplished by the exemplary methods described in the following Examples and operations in related publications used by a person skilled in the art, but these Examples are not intended to limit the scope of the present disclosure.
[0068] The structures of the compounds of the present disclosure are determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR determination is carried out by using Bruker AVANCE-400 or Varian Oxford-300 Nuclear Magnetic Resonance Spectrometer, deuterated dimethyl sulfoxide (DMSO-d.sub.6), deuterated chloroform (CDCl.sub.3) or deuterated methanol (CD.sub.3OD) is used as solvent, and tetramethylsilane (TMS) is used as the internal standard; and the chemical shift is provided in the unit of 10.sup.−6 (ppm).
[0069] MS determination is carried out by using Agilent SQD (ESI) Mass Spectrometer (manufacturer: Agilent, model: 6110) or Shimadzu SQD (ESI) mass spectrometer (manufacturer: Shimadzu, model: 2020).
[0070] HPLC determination is carried out by using Agilent 1200DAD High-pressure Liquid Chromatograph (Sunfirc C18, 150×4.6 mm, 5 μm, column) and Waters 2695-2996 High-pressure Liquid Chromatograph (Gimini C18, 150×4.6 mm, 5 μm column).
[0071] GF254 silica gel plates supplied by Qingdao Haiyang Chemical Co Ltd are used as the silica gel plates for the thin-layer chromatography. The specifications of the silica gel plates used in thin-layer chromatography (TLC) are 0.15 mm to 0.2 mm, and the specifications of the silica gel plates used for the separation and purification of products by thin-layer chromatography are 0.4 mm to 0.5 mm.
[0072] 200-300 mesh silica gel supplied by Qingdao Haiyang Chemical Co Ltd is generally adopted as the carrier for column chromatography.
[0073] The known starting materials in the present disclosure may come from companies such as Accela ChemBio Inc. and Beijing Ouhe Technology Co. Ltd, etc., or may be synthesized according to methods known in the art.
[0074] Unless otherwise specified in the Examples, the reactions were all carried out under an argon atmosphere or a nitrogen atmosphere.
[0075] An argon atmosphere or a nitrogen atmosphere means that the reaction flask is connected to an argon or nitrogen balloon with a volume of about 1 L.
[0076] A hydrogen atmosphere means that the reaction flask is connected to a hydrogen balloon with a volume of about 1 L. A hydrogenation reaction is usually carried out by evacuating, filling the reaction flask with hydrogen, and repeating the operations three times.
[0077] Unless otherwise specified in the Examples, the reaction temperature is room temperature, and the temperature ranges from 20° C. to 30° C.
[0078] Thin-layer chromatography (TLC) is adopted to monitor the process of reactions in the Examples. Systems used as developing solvents in the reactions include: A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system, wherein the volume ratio of the solvents is adjusted according to the polarity of compounds.
[0079] Unless otherwise specified in the Examples, using preparative HPLC (formic acid) method means that the compound is separated and obtained by chromatography in a formic acid system (phase A: H.sub.2O+0.225% formic acid, phase B: acetonitrile).
[0080] The eluent systems of the column chromatography used to purify the compound and the developing solvent systems of thin-layer chromatography include: A: dichloromethane and methanol system; B: petroleum ether and ethyl acetate system, wherein the volume ratio of the solvents is adjusted according to the polarity of compounds, or may be adjusted by adding a small amount of triethylamine and an acidic or basic reagent.
[0081] The present disclosure is described in detail below by Examples, but it does not imply any disadvantageous limitation on the present disclosure. The compounds of the present disclosure may be prepared by a variety of synthetic methods well known to a person skilled in the art, including the specific embodiments listed below, embodiments formed by combining said specific embodiments with other chemical synthesis methods, and equivalent alternatives well known to a person skilled in the art. Preferred embodiments include, but are not limited to, the Examples of the present disclosure. Various changes and improvements made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure will be apparent to a person skilled in the art. The following synthetic schemes describe the steps for preparing the compounds disclosed in the present disclosure. Unless otherwise specified, each substituent has the definition as described in the present disclosure.
##STR00012## ##STR00013##
[0082] Compound A1 is reacted with thionyl chloride or oxalyl chloride to obtain A2, and then A2 is reacted with an amine-based compound to obtain A3. Compound A4 is reacted with thionyl chloride or oxalyl chloride to obtain A5. Compounds A5 and A3 undergo intramolecular ring closure under the action of a suitable strong base, such as sodium hydride or LiHMDS, to obtain A6. Compound A6 is reacted with a suitable chlorinating reagent (such as phosphorus oxychloride) to obtain A7. Compound A7 is reacted with a Boc-protected amine under the action of a suitable base (such as TEA or DIPEA) to obtain Compound A8. Compound A8 is subjected to a suzuki reaction with the corresponding boronic acid or boronic ester under a suitable condition with a palladium catalyst (such as Pd(dppf).sub.2Cl.sub.2 dichloromethane complex) to obtain Compound A9. Compound A9 undergoes a deprotection reaction under an acidic condition to obtain A10. Compound A10 is reacted with a suitable acylation reagent (such as acryloyl chloride) in the presence of a suitable base (such as TEA or DIPEA) to obtain Compound (I).
##STR00014## ##STR00015##
[0083] Compound A8 undergoes a deprotection reaction under an acidic condition to obtain B 1. Compound A9 undergoes a deprotection reaction under an acidic condition to obtain A10. Compound A10 is reacted with a suitable acylation reagent (such as acryloyl chloride) in the presence of a suitable base (such as TEA or DIPEA) to obtain Compound B2. Compound B2 is subjected to a suzuki reaction with the corresponding boronic acid or boronic ester to obtain Compound (I).
##STR00016##
[0084] Compound B2 is reacted with bis(pinacolato)diboron under a suitable condition with a palladium catalyst (such as Pd(dppf).sub.2Cl.sub.2 dichloromethane complex) to obtain intermediate C1 as a boronic ester. Compound C1 is reacted with the corresponding halide such as bromide or chloride under a suitable condition with a palladium catalyst (such as Pd(dppf).sub.2Cl.sub.2 dichloromethane complex) to obtain Compound (I).
Example 1: Preparation of Compound I-1
[0085] ##STR00017##
[0086] Synthesis Route (Refer to Scheme A):
##STR00018## ##STR00019## ##STR00020##
[0087] Preparation Method:
[0088] Step 1: Synthesizing Compound 1D-2
[0089] Oxalyl chloride (15.3 g, 120 mmol) and DMF (0.1 ml) were added to a mixture of 2-cyanoacetic acid 1D-1 (8.5 g, 100 mmol) in dichloromethane (DCM) (100 mL), and then the resultant was stirred at room temperature for 3 hours. After TLC showed that the reaction was complete, the solvent was removed under reduced pressure to obtain Compound 1D-2 (11 g) as a white solid, which was used directly in the next step without further purification.
[0090] Step 2: Synthesizing Compound 1D
[0091] Compound 1D-2 (11 g) obtained in the previous step was dissolved in DCM (100 mL), then 2-isopropylaniline ID-3 (14.8 g, 110 mmol) and triethylamine (20.2 g, 200 mmol) were added thereto, and stirring was then performed at room temperature for 3 hours. After TLC showed that the reaction was complete, the solvent was removed under reduced pressure, and the resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10:1 (V:V volume ratio)) to obtain Compound 1D (17.8 g, white solid), yield: 88%.
[0092] MS m/z (ESI): 203[M+1].
[0093] Step 3: Synthesizing Compound 1H-2
[0094] The compound 4-bromo-5-methyl-1H-indazole 1H-1 (3 g, 14.2 mmol) was added to DCM (30 mL). Then, 3,4-dihydro-2H-pyran (2.39 g, 28.4 mmol, 2.60 mL) and p-toluenesulfonic acid monohydrate (270 mg, 1.42 mmol) were successively added thereto, and the mixture was stirred at room temperature for 2 hours. After TLC showed the completion of the reaction, the reaction mixture was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10:1 (V:V volume ratio)) to obtain Compound 1H-2 (4 g, white solid), yield: 95.3%.
[0095] MS m/z (ESI): 297[M+1].
[0096] Step 4: Synthesizing Compound 1H
[0097] Compound 1H-2 (550 mg, 1.85 mmol) was added to dioxane (10 ml). KOAc (364 mg, 3.7 mmol) and bis(pinacolato)diboron (705 mg, 2.8 mmol) were added thereto. After the reaction solution was purged with nitrogen gas three times, a dichloromethane complex of Pd(dppf)Cl.sub.2 (159 mg, 0.19 mmol) was added successively. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10:1 (V:V volume ratio)) to obtain Compound 1H (429 mg, light yellow transparent liquid), yield: 67.3%.
[0098] MS m/z (ESI): 345[M+1].
[0099] Step 5: Synthesizing Compound 1B
[0100] Compound 1A (22 g, 100 mmol) was added to concentrated sulfuric acid (250 ml) at room temperature. The mixture was stirred and dissolved, and then the resulting solution was cooled to 0° C. to 5° C. Thereafter, N-chlorosuccinimide (NCS) (13.3 g, 100 mmol) was added in portions. After the addition, the resulting solution was warmed up to room temperature and allowed to react overnight. TLC showed that the reaction was complete. Stirring was performed while the reaction solution was slowly added into a large amount of ice water (1 L), and a large amount of solid precipitated. Filtering was carried out and the solid was washed with water. The obtained solid was further purified by silica gel column chromatography (dichloromethane/methanol/formic acid=100/1/0.1 (V:V:V volume ratio)) to obtain Compound 1B (11.4 g, light yellow solid), yield: 45%.
[0101] MS m/z(ESI): 253[M+1].
[0102] Step 6: Synthesizing Compound 1C
[0103] Compound 1B (10 g, 39.5 mmol) was dissolved in dichloromethane (150 ml) at room temperature, then thionyl chloride (9.4 g, 79 mmol) was added thereto, and thereafter the mixture was warmed up to 45° C. to react for 2 hours. TLC showed that the reaction was complete. The solvent was removed by rotary evaporation, so as to obtain Compound 1C (13 g), which was used directly in the next step without purification.
[0104] Step 7: Synthesizing Compound 1E
[0105] Compound 1D (8 g, 40 mmol) was added to DMF (100 ml) at room temperature, the mixture was stirred and dissolved, and then the resulting solution was cooled to 0° C. to 5° C. in an ice-water bath. Thereafter, 60% sodium hydride (4 g, 100 nmol) was added in portions. After the addition, the reaction solution was warmed up to room temperature and allowed to continue the reaction for half an hour. Then, the DMF solution (50 ml) of Compound 1C (13 g) obtained in the previous step was added dropwise into a reaction flask. After the addition, stirring was performed at room temperature for 1 hour, and then the solution was warmed up to 120° C. for reacting for 4 hours. TLC showed that the reaction was complete, and LCMS showed the target MS. After the reaction solution was cooled to room temperature, water (500 ml) was slowly added for dilution, and the pH was adjusted to 3 to 4 with hydrochloric acid (6 N), and extraction was conducted with dichloromethane (3×150 mL). The organic phases were combined and dried over anhydrous sodium sulfate; the desiccant was removed by filtration; and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio)) to obtain Compound 1E (2.14 g, light yellow solid), yield: 13%.
[0106] MS m/z(ESI): 417[M+1].
[0107] Step 8: Synthesizing Compound 1F
[0108] Compound 1E (2 g, 4.8 mmol) was added to toluene (30 ml) at room temperature, and then phosphorus oxychloride (1.5 g, 9.6 mmol) was added thereto. After the addition, the reaction solution was warmed up to 110° C. and allowed to react overnight. After TLC showed that the reaction was complete, the reaction solution was cooled to room temperature. Stirring was performed while the reaction solution was poured into a large amount of ice water (100 ml) to quench the reaction. Then, a saturated sodium bicarbonate solution (100 ml) was added thereto, and extraction was conducted with ethyl acetate (3×50 mL). The organic phases were combined and dried over anhydrous sodium sulfate; the desiccant was removed by filtration; and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10/1 (V:V volume ratio)) to obtain Compound 1F (1.4 g, light yellow solid), yield: 67%.
[0109] MS m/z(ESI): 435[M+1].
[0110] Step 9: Synthesizing Compound 1G
[0111] Compound 1F (435 mg, 1 mmol) and N,N-diisopropylethylamine (DIPEA) (258 mg, 2 mmol) were added to DMF (5 ml) at room temperature. Then, N-Boc piperazine (372 mg, 2 mmol) was added thereto, the temperature was controlled to be 110° C., and the resultant was allowed to react overnight. TLC showed that the reaction was complete. The reaction solution was added to 20 ml of water and extraction was conducted with dichloromethane (3×10 ml). The resulting solution was successively washed with a saturated sodium carbonate solution (2×10 mL), water (2×10 mL) and brine (2×10 mL). The organic phase was dried over anhydrous sodium sulfate. The desiccant was removed by filtration. After the resultant was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio) to obtain Compound 1G (423 mg, light yellow solid), yield: 73%.
[0112] MS m/z(ESI): 585[M+1].
[0113] Step 10: Synthesizing Compound 1I Compound 1G (1.17 g, 2 mmol) was added to a solution of dioxane (10 mL) and water (2 mL) at room temperature. Then, potassium phosphate (848 mg, 4 mmol), Compound 1H (460 mg, 3 mmol) and a dichloromethane complex of Pd(dppf)Cl.sub.2 (162 mg, 0.2 mmol) were added thereto. The reaction solution was allowed to react at 100° C. for 12 hours under nitrogen protection. TLC showed that the reaction was complete. A saturated sodium bicarbonate solution (50 mL) was added and extraction was conducted with dichloromethane (2×20 mL). The organic phases were combined and then dried over anhydrous sodium sulfate; the desiccant was removed by filtration; and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1 (V:V volume ratio)) to obtain Compound 1I (822 mg, light yellow solid), yield: 57%.
[0114] MS m/z(ESI): 721[M+1].
[0115] Step 11: Synthesizing the Hydrochloride of Compound 1J
[0116] Compound 1I (820 mg, 1.14 mmol) was dissolved in ethyl acetate (5 mL) at room temperature. Then, a solution of hydrochloric acid in ethyl acetate (4 N, 5 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The solution changed from clear to turbid, and solid precipitated. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to 0° C., allowed to stand for 1 hour, and then filtered. The solid was washed with ethyl ether and then dried to obtain the hydrochloride of Compound 1J (599 mg, white solid), yield: 92%.
[0117] MS m/z(ESI): 537[M+1].
[0118] Step 12: Synthesizing the Compound Represented by Formula I-1
[0119] The hydrochloride (599 mg) of Compound 1J obtained in the previous step was dissolved in dichloromethane (10 ml), and then the resultant was cooled to −10° C. Triethylamine (202 mg, 2 mmol) and acryloyl chloride (100 mg, 1.1 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction was quenched by adding MeOH (1 mL). The residue obtained after drying the reaction mixture by rotary evaporation was separated and purified by preparative HPLC (formic acid) to obtain the target compound represented by formula I-1 (33 mg, white solid).
[0120] MS m/z(ESI): 591[M+1].
[0121] .sup.1H NMR (400 MHz, MeOD) 8.20 (s, 1H), 7.50-7.45 (m, 3H), 7.36-7.27 (m, 3H), 7.143 (d, J=8 Hz, 1H), 6.92-6.99(m, 1H), 6.47(s,1H), 6.31 (dd, J=2.0, 16.8 Hz, 1H), 5.84 (dd, J=1.6, 10.4 Hz, 1H), 4.03(brs, 4H), 3.90(brs, 4H), 2.64-2.62 (m, 1H), 2.08(s, 3H), 1.17(d, J=7.2 Hz, 3H), 1.00(d, J=6.8 Hz,3H).
Example 2: Preparation of Compound I-2-1 and Compound I-2-2
[0122] ##STR00021## ##STR00022##
[0123] Step 1: Synthesizing Compound 2A
[0124] Compound 1G (435 mg, 1 mmol) and N,N-diisopropylethylamine (DIPEA) (258 mg, 2 mmol) were added to DMF (5 ml) at room temperature. Then, (S)-4-N-tert-butoxycarbonyl-2-methylpiperazine (400 mg, 2 mmol) was added thereto. The temperature was controlled to be 110° C. and the resultant was allowed to react overnight. TLC showed that the reaction was complete. The reaction solution was added to 20 ml of water and extraction was conducted with dichloromethane (3×10 ml). The resulting solution was successively washed with a saturated sodium carbonate solution (2×10 mL), water (2×10 mL) and brine (2×10 mL). The organic phase was dried over anhydrous sodium sulfate. The desiccant was removed by filtration. After the resultant was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio)) to obtain Compound 2A (480 mg, light yellow solid), yield: 80.2%.
[0125] MS m/z(ESI): 599[M+1].
[0126] Step 2: Synthesizing Compound 2B
[0127] Compound 1G (1.2 g, 2 mmol) was added to a solution of dioxane (10 mL) and water (2 mL) at room temperature. Then, potassium phosphate (848 mg, 4 mmol), Compound 1H (460 mg, 3 mmol) and a dichloromethane complex of Pd(dppf)Cl.sub.2 (162 mg, 0.2 mmol) were added thereto. The reaction solution was allowed to react at 100° C. for 12 hours under nitrogen protection. TLC showed that the reaction was complete. A saturated sodium bicarbonate solution (50 mL) was added and extraction was conducted with dichloromethane (2×20 mL). The organic phases were combined and then dried over anhydrous sodium sulfate; the desiccant was removed by filtration; and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3:1 (V:V volume ratio)) to obtain Compound 2B (926 mg, light yellow solid), yield: 63%.
[0128] MS m/z(ESI): 735[M+1].
[0129] Step 3: Synthesizing the Hydrochloride of Compound 2C
[0130] Compound 2B (837 mg, 1.14 mmol) was dissolved in ethyl acetate (5 mL) at room temperature. Then, a solution of hydrochloric acid in ethyl acetate (4 N, 5 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The solution changed from clear to turbid, and solid precipitated. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to 0° C., allowed to stand for 1 hour, and then filtered. The solid was washed with ethyl ether and then dried to obtain the hydrochloride of Compound 2C (602 mg, white solid).
[0131] MS m/z(ESI): 551[M+1].
[0132] Step 4: Synthesizing the Compound Represented by Formula I-2
[0133] The hydrochloride (551 mg) of Compound 2C obtained in the previous step was dissolved in dichloromethane (10 ml), and then the resultant was cooled to −10° C. Triethylamine (202 mg, 2 mmol) and acryloyl chloride (100 mg, 1.1 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction was quenched by adding MeOH (1 mL). The residue obtained after drying the reaction mixture by rotary evaporation was separated and purified by preparative HPLC to obtain the target compound represented by Formula I-2 (130 mg, light yellow solid). MS m/z(ESI):605[M+1].
[0134] Step 5: Synthesizing the Compounds Represented by Formula I-2-1 and Formula I-2-2
[0135] Compound I-2 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-2-1 (t.sub.R=3.55 min) and Compound I-2-2 (t.sub.R=4.49 min).
[0136] Compound Represented by Formula I-2-1:
[0137] MS m/z(ESI): 605[M+1]
[0138] .sup.1H NMR (400 MHz, MeOD) 8.23 (s, 1H), 7.52-7.43 (m, 3H), 7.38-7.27 (m, 3H), 7.21-7.12 (m, 1H), 6.95-6.83(m, 1H), 6.48(s,1H), 6.33 (d, J=16.4 Hz, 1H), 5.85 (dd, J=1.6, 10.4 Hz, 1H), 4.42-4.14(m, 2H), 4.10-3.90(m, 2H), 3.72-3.45 (m, 3H), 2.70-2.56(m, 1H), 2.09(s, 3H), 1.40-1.33(m, 3H), 1.22-1.15(m, 3H), 1.03-0.92(m, 3H).
[0139] Compound represented by Formula I-2-2:
[0140] MS m/z(ESI):605[M+1].
[0141] .sup.1H NMR (400 MHz, MeOD) 8.22 (s, 1H), 7.51-7.43 (m, 3H), 7.37-7.29 (m, 3H), 7.27-7.24(m, 1H), 6.95-6.83(m, 1H), 6.50(s, 1H), 6.33 (d, J=16.4 Hz, 1H), 5.84 (dd, J=1.6, 10.4 Hz, 1H), 4.42 (brs, 2H), 4.13-4.09(m, 2H), 3.65-3.50 (m, 3H), 2.72-2.51(m, 1H), 2.09(s, 3H), 1.38-1.35(m, 3H), 1.20-1.15(m, 3H), 1.04-0.90(m, 3H).
Example 3: Preparation of Compound I-3-1 and Compound I-3-2
[0142] ##STR00023##
[0143] Step 1: Compound I-3 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0144] Step 2: Compound I-3 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-3-1 (t.sub.R=3.22 min) and Compound I-3-2 (t.sub.R=4.25 min).
[0145] Compound Represented by Formula I-3-1:
[0146] MS m/z(ESI):605[M+1].
[0147] .sup.1H NMR (400 MHz, MeOD) 8.23 (s, 1H), 7.52-7.24 (m, 7H), 6.90-6.89 (m, 1H), 6.51 (m, 1H), 6.36-6.30 (m, 1H), 5.87-5.84 (m, 1H), 4.49-4.08 (m, 5H), 3.55-3.48 (m, 2H), 2.55-2.52 (m, 1H), 2.10 (s, 3H), 1.37 (s, 3H), 1.17 (s, 3H), 1.00 (s, 3H).
[0148] Compound represented by Formula I-3-2:
[0149] MS m/z(ESI):605[M+1].
[0150] .sup.1H NMR (400 MHz, MeOD) 8.23 (s, 1H), 7.47-7.21 (m, 7H), 6.85 (m, 1H), 6.50-6.33 (m, 2H), 5.88-5.85 (m, 1H), 4.46-3.54 (m, 7H), 2.57-2.56 (m, 1H), 2.08 (m, 3H), 1.37 (s, 3H), 1.17 (s, 3H), 1.00 (s, 3H).
Example 4: Preparation of Compound I-4-1 and Compound I-4-2
[0151] ##STR00024##
[0152] Step 1: Compound I-4 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0153] Step 2: Compound I-4 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-4-1 (t.sub.R=3.31 min) and Compound I-4-2 (t.sub.R=4.31 min).
[0154] Compound represented by Formula I-4-1:
[0155] MS m/z(ESI):619[M+1].
[0156] .sup.1H NMR (400 MHz, MeOD) 8.20 (m, 1H), 7.51 (m, 3H), 7.47-7.25 (m, 5H), 6.50 (s, 1H), 6.32 (m, 1H), 5.85 (m, 1H), 4.60-4.40 (m, 5H), 4.52 (m, 2H), 2.51-2.40 (m, 1H), 2.08 (s, 3H), 1.53-1.47 (m, 8H), 1.26-1.21 (m, 3H), 1.03-0.96 (m, 3H).
[0157] Compound represented by Formula I-4-2:
[0158] MS m/z(ESI):619[M+1].
[0159] .sup.1H NMR (400 MHz, MeOD) 8.17-8.15 (m, 1H), 7.55-7.31 (m, 7H), 6.90 (m, 1H), 6.54-6.51 (m, 1H), 6.36-6.33 (m, 1H), 5.85 (m, 1H), 4.90 (m, 3H), 4.59-4.45 (m, 3H), 3.55 (m, 2H), 2.71 (m, 1H), 2.13-2.12 (m, 3H), 1.52-1.50 (m, 7H), 1.22-1.21 (m, 3H), 1.05 (m, 3H).
Example 5: Preparation of Compound I-5-1 and Compound I-5-2
[0160] ##STR00025##
[0161] Step 1:
[0162] Compound 1-5 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0163] Step 2:
[0164] Compound I-5 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-5-1 (t.sub.R=3.43 min) and Compound I-5-2 (t.sub.R=4.51 min).
[0165] Compound represented by Formula I-5-1:
[0166] MS m/z(ESI):631[M+1].
[0167] .sup.1H NMR (400 MHz, MeOD) 8.15 (m, 1H), 7.45-7.26 (m, 7H), 6.44-6.26 (m, 3H), 5.76-5.75 (m, 1H), 5.48-5.47 (m, 1H), 4.20-4.18 (m, 2H), 2.63-2.58 (m, 1H), 2.18-2.15 (m, 10H), 1.16-1.14 (m, 3H), 1.01-0.99 (m, 3H).
[0168] Compound represented by Formula I-5-2:
[0169] MS m/z(ESI):631[M+1].
[0170] .sup.1H NMR (400 MHz, MeOD) 8.16 (m, 1H), 7.42-7.24 (m, 7H), 6.43-6.25 (m, 3H), 5.75-5.74 (m, 1H), 5.49-5.47(m, 1H), 4.22-4.20 (m, 2H), 2.64-2.59(m, 1H), 2.18-2.15 (m, 10H), 1.17-1.15 (m, 3H), 1.01-0.98(m, 3H).
Example 6: Preparation of Compound I-6-1 and Compound I-6-2
[0171] ##STR00026## ##STR00027## ##STR00028## ##STR00029##
[0172] Step 1: Synthesizing Compounds 6A and 6B
[0173] Compound 2A was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound 6A (t.sub.R=2.756 min) and Compound 6B (t.sub.R=4.203 min).
[0174] Step 2: Synthesizing Compound 6C
[0175] Compound 6A (681 mg, 1.14 mmol) was dissolved in ethyl acetate (5 mL) at room temperature. Then, a solution of hydrochloric acid in ethyl acetate (4 N, 5 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The solution changed from clear to turbid, and solid precipitated. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to 0° C., allowed to stand for 1 hour, and then filtered. The solid was washed with ethyl ether and then dried to obtain the hydrochloride of Compound 6C (450 mg, white solid).
[0176] MS m/z(ESI): 499[M+1].
[0177] Step 3: Synthesizing Compound 6D
[0178] The hydrochloride (400 mg) of Compound 6C obtained in the previous step was dissolved in DMF (10 ml). At room temperature, triethylamine (202 mg, 2 mmol), HATU (760 mg, 2 mmol) and 2-fluoroacrylic acid (100 mg, 1.1 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction solution was added to 20 ml of water and extraction was conducted with dichloromethane (3×10 ml). The resulting solution was successively washed with a saturated sodium carbonate solution (2×10 mL), water (2×10 mL) and brine (2×10 mL). The organic phase was dried over anhydrous sodium sulfate. The desiccant was removed by filtration. After the resultant was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio) to obtain Compound 6D (280 mg, light yellow solid).
[0179] MS m/z(ESI): 571[M+1].
[0180] Step 4: Synthesizing Compound I-6-1
[0181] Compound 6D (200 mg, 0.35 mmol) was added to dioxane (2 ml) and water (0.5 ml). K.sub.3PO.sub.4 (212 mg, 1.00 mmol) and 5-methyl-1H-indazol-4-yl-4-boronic acid (176 mg, 1 mmol) were added thereto. After the reaction solution was purged with nitrogen gas three times, a dichloromethane complex of Pd(dppf)Cl.sub.2 (25 mg, 0.03 mmol) was added. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by preparative silica gel plate (prepar-TLC) (developing solvent system: petroleum ether/ethyl acetate=1:2 (V:V volume ratio)) to obtain Compound I-6-1 (29 mg, light yellow solid).
[0182] MS m/z(ESI):623[M+1].
[0183] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.01 (d, J=4.4 MHz, 1H), 7.45-7.39 (m, 4H), 7.33-7.29 (m, 2H), 7.14-7.09 (m, 1H), 6.62 (s, 1H), 5.48-5.22 (m, 2H), 4.32-3.43 (m, 7H), 2.55-2.50 (m, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 1.42 (d, J=6.4 MHz, 3H), 1.18 (d, J=6.8 MHz, 3H), 1.01 (d, J=6.8 MHz, 3H).
[0184] Step 5: Synthesizing Compound 6E
[0185] Compound 6B (681 mg, 1.14 mmol) was dissolved in ethyl acetate (5 mL) at room temperature. Then, a solution of hydrochloric acid in ethyl acetate (4 N, 5 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The solution changed from clear to turbid, and solid precipitated. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was cooled to 0° C., allowed to stand for 1 hour, and then filtered. The solid was washed with ethyl ether and then dried to obtain the hydrochloride of Compound 6E (446 mg, white solid).
[0186] MS m/z(ESI): 499[M+1].
[0187] Step 6: Synthesizing Compound 6F
[0188] The hydrochloride (400 mg) of Compound 6E obtained in the previous step was dissolved in DMF (10 ml). At room temperature, triethylamine (202 mg, 2 mmol), HATU (760 mg, 2 mmol) and 2-fluoroacrylic acid (100 mg, 1.1 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction solution was added to 20 ml of water and extraction was conducted with dichloromethane (3×10 ml). The resulting solution was successively washed with a saturated sodium carbonate solution (2×10 mL), water (2×10 mL) and brine (2×10 mL). The organic phase was dried over anhydrous sodium sulfate. The desiccant was removed by filtration. After the resultant was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio) to obtain Compound 6F (272 mg, light yellow solid).
[0189] MS m/z(ESI): 571[M+1].
[0190] Step 7: Synthesizing Compound I-6-2
[0191] Compound 6F (200 mg, 0.35 mmol) was added to dioxane (2 ml) and water (0.5 ml). K.sub.3PO.sub.4 (212 mg, 1.00 mmol) and 5-methyl-1H-indazol-4-yl-4-boronic acid (176 mg, 1 mmol) were added thereto. After the reaction solution was purged with nitrogen gas three times, a dichloromethane complex of Pd(dppf)Cl.sub.2 (25 mg, 0.03 mmol) was added. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by preparative silica gel plate (prepar-TLC) (developing solvent system: petroleum ether/ethyl acetate=1:2 (V:V volume ratio)) to obtain Compound I-6-2 (25 mg, light yellow solid).
[0192] MS m/z(ESI):623[M+1].
[0193] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.03 (s, 1H), 7.47-7.29 (m, 6H), 7.12-7.11 (m, 1H), 6.61 (s, 1H), 5.48-5.21 (m, 2H), 4.33-3.43 (m, 7H), 2.51-2.46 (m, 1H), 2.10 (s, 3H), 2.09 (s, 3H), 1.42 (d, J=6.4 MHz, 3H), 1.18 (d, J=6.8 MHz, 3H), 0.98 (d, J=6.8 MHz, 3H).
Example 7: Preparation of Compound I-7-1 and Compound I-7-2
[0194] ##STR00030## ##STR00031##
[0195] Step 1: Synthesizing Compound 7A
[0196] Compound 6A (681 mg, 1.14 mmol) was dissolved in DCM (5 mL) at room temperature. Then, TFA (2 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was dried by rotary evaporation. The resulting yellow liquid compound was redissolved in DCM (5 mL), and then the resultant was cooled to −10° C. Triethylamine (303 mg, 3 mmol) and acryloyl chloride (150 mg, 1.5 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction was quenched by adding MeOH (1 mL). After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio)) to obtain Compound 7A (272 mg, light yellow solid).
[0197] MS m/z(ESI): 553[M+1].
[0198] .sup.1HNMR (400 MHz, CDCl.sub.3) 7.92 (s, 1H), 7.57-7.56 (m, 2H), 7.44-7.40 (m, 1H), 7.11-7.09 (m, 1H), 6.88 (s, 1H), 6.64-6.60 (m, 1H), 6.42-6.37 (m, 1H), 5.83-5.80 (m, 1H), 4.29-4.00 (m, 4H), 3.75-3.56 (m, 3H), 2.43-2.36 (m, 1H), 1.32 (d, J=6.0 MHz, 3H), 1.18 (d, J=6.8 MHz, 3H), 1.04 (d, J=6.8 MHz, 3H).
[0199] Step 2: Synthesizing Compound I-7-1
[0200] Compound 7A (100 mg, 0.18 mmol) was added to dioxane (2 ml) and water (0.5 ml). K.sub.3PO.sub.4 (106 mg, 0.5 mmol) and 3-chloro-2-fluoro-6-methoxyphenylboronic acid (102 mg, 0.5 mmol) were added thereto. After the reaction solution was purged with nitrogen gas three times, a dichloromethane complex of Pd(dppf)Cl.sub.2 (25 mg, 0.03 mmol) was added thereto. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by preparative silica gel plate (prepar-TLC) (developing solvent system: petroleum ether/ethyl acetate=1:2 (V:V volume ratio)) to obtain Compound I-7-1 (21 mg, light yellow solid).
[0201] MS m/z(ESI): 633[M+1].
[0202] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.98 (s, 1H), 7.53-7.48 (m, 3H), 7.39-7.33 (m, 1H), 7.13-7.11 (m, 1H), 6.70-6.38 (m, 4H), 5.83-5.80 (m, 1H), 4.42-3.92 (m, 5H), 3.74 (s, 3H), 3.56-3.38 (m, 2H), 2.51-2.44 (m, 1H), 1.36 (d, J=6.0 MHz, 3H), 1.17 (d, J=6.8 MHz, 3H), 1.04 (d, J=6.8 MHz, 3H).
[0203] Step 3: Synthesizing Compound 7B
[0204] Compound 6B (681 mg, 1.14 mmol) was dissolved in DCM (5 mL) at room temperature. Then, TFA (2 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was dried by rotary evaporation. The resulting yellow liquid compound was redissolved in DCM (5 mL), and then the resultant was cooled to −10° C. Triethylamine (303 mg, 3 mmol) and acryloyl chloride (150 mg, 1.5 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction was quenched by adding MeOH (1 mL). After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio)) to obtain Compound 7B (252 mg, light yellow solid).
[0205] MS m/z(ESI): 553[M+1].
[0206] .sup.1HNMR (400 MHz, CDCl.sub.3) 7.93 (s, 1H), 7.58-7.57 (m, 2H), 7.44-7.41 (m, 1H), 7.12-7.09 (m, 1H), 6.87 (s, 1H), 6.65-6.60 (m, 1H), 6.43-6.37 (m, 1H), 5.84-5.81 (m, 1H), 4.29-4.00 (m, 4H), 3.76-3.57 (m, 3H), 2.44-2.36 (m, 1H), 1.31 (d, J=6.0 MHz, 3H), 1.19 (d, J=6.8 MHz, 3H), 1.05 (d, J=6.8 MHz, 3H).
[0207] Step 2: Synthesizing Compound I-7-2
[0208] Compound 7B (100 mg, 0.18 mmol) was added to dioxane (2 ml) and water (0.5 ml). K.sub.3PO.sub.4 (106 mg, 0.5 mmol) and 3-chloro-2-fluoro-6-methoxyphenylboronic acid (102 mg, 0.5 mmol) were added thereto. After the reaction solution was purged with nitrogen gas three times, a dichloromethane complex of Pd(dppf)Cl.sub.2 (25 mg, 0.03 mmol) was added thereto. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by preparative silica gel plate (prepar-TLC) (developing solvent system: petroleum ether/ethyl acetate=1:2 (V:V volume ratio)) to obtain Compound I-7-2 (23 mg, light yellow solid).
[0209] MS m/z(ESI): 633[M+1].
[0210] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.98 (s, 1H), 7.52-7.48 (m, 2H), 7.39-7.35 (m, 2H), 7.13-7.11 (m, 1H), 6.69-6.60 (m, 2H), 6.54 (s, 1H), 6.42-6.38 (m, 1H), 5.83-5.80 (m, 1H), 4.30-3.99 (m, 4H), 3.67 (s, 3H), 3.74-3.37 (m, 3H), 2.53-2.46 (m, 1H), 1.37 (d, J=6.4 MHz, 3H), 1.19 (d, J=6.8 MHz, 3H), 1.00 (d, J=6.8 MHz, 3H).
Example 8: Preparation of Compound I-8-1 and Compound I-8-2
[0211] ##STR00032##
[0212] Synthesis of Compound I-8-1:
[0213] Compound 6A (t.sub.R=2.745 min) and 2-chloro-6-fluoro-4-methoxyphenylboronic acid were used as the starting materials to obtain Compound I-8-1 (refer to the synthesis of Compound I-7-1).
[0214] MS m/z(ESI): 633[M+1].
[0215] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.96 (s, 1H), 7.52-7.47 (m, 2H), 7.37-7.32 (m, 1H), 7.12-7.09 (m, 1H), 6.80-6.60 (m, 3H), 6.51-6.38 (m, 2H), 5.83-5.79 (m, 1H), 4.39-3.99 (m, 4H), 3.77 (s, 3H), 3.64-3.35 (m, 3H), 2.50-2.43 (m, 1H), 1.36 (d, J=6.0 MHz, 3H), 1.17 (d, J=6.8 MHz, 3H), 1.02 (d, J=6.8 MHz, 3H).
[0216] Synthesis of Compound I-8-2:
[0217] Compound 6B (t.sub.R=4.203 min) and 2-chloro-6-fluoro-4-methoxyphenylboronic acid were used as the starting materials to obtain Compound I-8-2 (refer to the synthesis of Compound I-7-2).
[0218] MS m/z(ESI): 633[M+1].
[0219] .sup.1H NMR (400 MHz, CDCl.sub.3)7.97 (s, 1H), 7.52-7.48 (m, 2H), 7.38-7.34 (m, 1H), 7.12-7.10 (m, 1H), 6.80-6.63 (m, 3H), 6.52-6.38 (m, 2H), 5.83-5.80 (m, 1H), 4.42-3.75 (m, 5H), 3.64 (s, 3H), 3.56-3.36 (m, 2H), 2.52-2.47 (m, 1H), 1.36 (d, J=6.4 MHz, 3H), 1.19 (d, J=6.8 MHz, 3H), 1.01 (d, J=6.8 MHz, 3H).
[0220] Compound 6B (t.sub.R=4.203 min) and commercially available boronic acid or boronic acid ester were used as the starting materials to obtain Example Compounds I-9 to I-77 in Table 1 (refer to the synthesis of Compound I-7-2).
TABLE-US-00001 TABLE 1 LCMS Examples Structures [M + H].sup.+ NMR I-9
Preparation of Compound I-78
[0221] ##STR00101## ##STR00102##
[0222] Step 1: Synthesizing Compound 78A
[0223] Compound 6B (681 mg, 1.14 mmol) was dissolved in DCM (5 mL) at room temperature. Then, TFA (2 ml) was added thereto. The mixture was stirred at room temperature for 2 hours. The reaction was monitored by TLC. After the reaction was complete, the reaction solution was dried by rotary evaporation. The resulting yellow liquid compound was redissolved in DCM (5 mL), and then the resultant was cooled to −10° C. Triethylamine (303 mg, 3 mmol) and acryloyl chloride (150 mg, 1.5 mmol) were added thereto successively. Then, after the mixture was naturally warmed up to room temperature and allowed to react for 1 hour, TLC showed the completion of the reaction. The reaction was quenched by adding MeOH (1 mL). After the reaction mixture was concentrated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol=100/1 (V:V volume ratio)) to obtain Compound 78A (252 mg, light yellow solid).
[0224] MS m/z(ESI): 553[M+1].
[0225] Step 2: Synthesizing Compound 78B
[0226] Compound 78A (1.0 g, 1.85 mmol) was added to dioxane (10 ml). KOAc (364 mg, 3.7 mmol) and bis(pinacolato)diboron (705 mg, 2.8 mmol) were added thereto. The reaction solution was purged with nitrogen gas three times, and then a dichloromethane complex of Pd(dppf)Cl.sub.2 (159 mg, 0.19 mmol) was added thereto successively. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=10:1 (V:V volume ratio)) to obtain Compound 78B (750 mg, light yellow solid).
[0227] MS m/z (ESI): 601[M+1].
[0228] Step 3: Synthesizing Compound I-78
[0229] Compound 78B (100 mg, 0.16 mmol) was added to dioxane (2 ml) and water (0.5 ml). K.sub.3PO.sub.4 (106 mg, 0.5 mmol) and 2-bromo-4-chloro-6-fluoroaniline (111 mg, 0.5 mmol) were added thereto. After the reaction solution was purged with nitrogen gas three times, a dichloromethane complex of Pd(dppf)Cl.sub.2 (25 mg, 0.03 mmol) was added thereto. The resulting reaction solution was again purged with nitrogen gas three times and then stirred overnight at 100° C. After TLC showed that the reaction was complete, the reaction solution was concentrated under vacuum, and the resulting residue was purified by preparative silica gel plate (prepar-TLC) (developing solvent system: petroleum ether/ethyl acetate=1:2 (V:V volume ratio)) to obtain Compound I-78 (26 mg, light yellow solid).
[0230] MS m/z(ESI): 618[M+1].
[0231] Compound 6B (t.sub.R=4.203 min) and commercially available substituted aniline bromide were used as the starting materials to obtain Example Compounds I-79 to I-82 in Table 2 (refer to the synthesis of Compound I-78).
TABLE-US-00002 TABLE 2 LCMS Examples Structures [M + H].sup.+ I-79
Preparation of Compound I-83-1 and Compound I-83-2
[0232] ##STR00107##
[0233] Step 1:
[0234] Compound I-83 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0235] Step 2:
[0236] Compound I-83 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-83-1 (t.sub.R=2.72 min) and Compound I-83-2 (t.sub.R=3.85 min).
[0237] Compound represented by Formula I-83-1:
[0238] MS m/z(ESI):620[M+1].
[0239] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.51 (d, J=4.8 MHz, 1H), 8.07 (s, 1H), 7.52-7.41 (m, 2H), 7.30-7.28 (m, 1H), 7.11-7.10 (m, 1H), 6.68-6.60 (m, 1H), 6.49 (s, 1H), 6.45-6.40 (m, 1H), 5.84-5.82 (m, 1H), 4.47-3.45 (m, 7H), 2.62-2.53 (m, 1H), 2.12 (s, 3H), 2.09 (s, 3H), 1.48 (d, J=6.8 MHz, 3H), 1.18 (d, J=6.8 MHz, 3H), 1.01 (d, J=6.8 MHz, 3H).
[0240] Compound represented by Formula I-83-2:
[0241] MS m/z(ESI):620[M+1].
[0242] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.59-8.57 (m, 1H), 8.08-8.06 (m, 1H), 7.52-7.30 (m, 4H), 6.67-6.62 (m, 2H), 6.45-6.40 (m, 1H), 5.85-5.82 (m, 1H), 4.65-3.46 (m, 7H), 2.82-2.75 (m, 1H), 2.24-1.97 (m, 6H), 1.44-1.43 (m, 3H), 1.26-1.21 (m, 3H), 1.16 (m, 3H).
Preparation of Compound I-84-1 and Compound I-84-2
[0243] ##STR00108##
[0244] Step 1:
[0245] Compound I-84 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0246] Step 2:
[0247] Compound I-84 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-84-1 (t.sub.R=3.32 min) and Compound I-84-2 (t.sub.R=4.16 min).
[0248] Compound represented by Formula I-84-1:
[0249] MS m/z(ESI):648.2[M+1].
[0250] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.59 (d, J=4.8 MHz, 1H), 8.01 (d, J=4.8 MHz, 1H), 7.39 (t, J=8.4 MHz, 1H), 7.18-7.17 (m, 1H), 6.70-6.66 (m, 2H), 6.43-6.39 (m, 2H), 5.82 (d, J=8.4 MHz, 1H), 4.37-4.32 (m, 1H), 4.04 (m, 2H), 3.69 (s, 3H), 3.68-3.57 (m, 2H), 2.74-2.69 (m, 1H), 1.96 (s, 3H), 1.39 (d, J=6.4 MHz, 3H), 1.26-1.25 (m, 1H), 1.23 (d, J=6.4 MHz, 3H), 1.10 (d, J=6.4 MHz, 3H).
[0251] Compound represented by Formula I-84-2:
[0252] MS m/z(ESI):648.2[M+1].
[0253] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.60 (d, J=4.8 MHz, 1H), 8.00 (brs, 1H), 7.39 (t, J=8.4 MHz, 1H), 7.15-7.14 (m, 1H), 6.70-6.66 (m, 2H), 6.44-6.39 (m, 2H), 5.84-5.81 (m, 1H), 4.37-4.04 (m, 4H), 3.69 (s, 3H), 3.45-3.42 (m, 2H), 2.72-2.68 (m, 1H), 1.97 (s, 3H), 1.39 (d, J=6.4 MHz, 3H), 1.26-1.25 (m, 1H), 1.22 (d, J=6.4 MHz, 3H), 1.10 (d, J=6.4 MHz, 3H).
[0254] Preparation of Compound I-85-1 and Compound I-85-2
##STR00109##
[0255] Step 1:
[0256] Compound I-85 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0257] Step 2:
[0258] Compound I-85 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-85-1 (t.sub.R=3.45 min) and Compound I-85-2 (t.sub.R=4.67 min). to Compound represented by Formula I-85-1:
[0259] MS m/z(ESI):625[M+1].
[0260] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.00 (s, 1H), 7.61-7.60 (m, 1H), 7.50-7.48 (m, 2H), 7.39-7.36 (m, 2H), 6.70-6.68 (m, 2H), 6.51-6.50 (m, 1H), 5.83-5.81 (m, 1H), 4.31-4.30 (m, 2H), 3.76-3.69 (m, 3H), 3.64 (s, 3H), 3.40 (m, 1H), 1.37-1.35 (m, 4H).
[0261] Compound represented by Formula I-85-2:
[0262] MS m/z(ESI):625[M+1].
[0263] .sup.1H NMR (400 MHz, CDCl.sub.3) 7.99 (s, 1H), 7.65-7.63 (m, 1H), 7.48-7.35 (m, 4H), 6.70-6.68 (m, 2H), 6.51 (s, 1H), 6.42-6.38 (m, 1H), 5.83-5.80 (m, 1H), 4.30-3.98 (m, 4H), 3.74-3.70 (m, 4H), 3.59 (m, 1H), 3.42-3.40 (m, 1H), 1.37-1.36 (d, J=6.0 MHz, 3H).
Preparation of Compound I-86-1 and Compound I-86-2
[0264] ##STR00110##
[0265] Step 1:
[0266] Compound I-86 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0267] Step 2:
[0268] Compound I-86 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-86-1 (t.sub.R=2.72 min) and Compound I-86-2 (t.sub.R=3.65 min).
[0269] Compound represented by Formula I-86-1:
[0270] MS m/z(ESI):649.2[M+1].
[0271] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.90 (s, 1H), 7.68-7.66 (m, 1H), 7.41 (t, J=8.8 MHz, 1H), 7.00 (m, 1H), 6.73-6.62 (m, 2H), 6.44-6.40 (m, 1H), 5.85-5.82 (m, 1H), 4.39-3.86 (m, 4H), 3.76-3.72 (m, 4H), 3.53-3.46 (m, 2H), 3.08-3.01 (m, 2H), 1.44-1.42 (m, 3H), 1.22-1.20 (m, 12H).
[0272] Compound represented by Formula I-86-2:
[0273] MS m/z(ESI):649.2[M+1].
[0274] .sup.1H NMR (400 MHz, CDCl.sub.3) 8.91 (s, 1H), 7.55 (m, 1H), 7.42 (t, J=8.8 MHz, 1H), 7.18-7.16 (m, 1H), 6.73-6.58 (m, 2H), 6.44-6.39 (m, 1H), 5.84-5.82 (m, 1H), 4.68-3.95 (m, 4H), 3.73-3.70 (m, 3H), 3.52-3.49 (m, 2H), 3.09-3.07 (m, 3H), 1.43-1.41 (m, 3H), 1.24-1.23 (m, 12H).
Preparation of Compound I-87-1 and Compound I-87-2
[0275] ##STR00111##
[0276] Step 1:
[0277] Compound I-87 was synthesized by referring to the synthesis method of Compound I-2 in Example 2.
[0278] Step 2:
[0279] Compound I-87 was subjected to chiral resolution by SFC (column model: CHIRALPAK IC, 250 mm*30 mm, 5 μm; mobile phase A: n-hexane/dichloromethane (75/25, containing 10 mM methylamine); mobile phase B: methanol, detection wavelength: 254 nm) to obtain Compound I-87-1 (t.sub.R=3.34 min) and Compound I-87-2 (t.sub.R=4.09 min).
[0280] Compound represented by Formula I-87-1:
[0281] MS m/z(ESI):677.2[M+1].
[0282] .sup.1H NMR (400 MHz, MeOD) HNMR: (400 MHz, CDCl.sub.3) 8.90 (s, 1H), 7.68-7.66 (m, 1H), 7.41 (t, J=8.8 MHz, 1H), 7.00 (m, 1H), 6.73-6.62 (m, 2H), 6.44-6.40 (m, 1H), 5.85-5.82 (m, 1H), 4.39-3.86 (m, 4H), 3.76-3.72 (m, 4H), 3.53-3.46 (m, 2H), 3.08-3.01 (m, 2H), 1.44-1.42 (m, 3H), 1.22-1.20 (m, 12H).
[0283] Compound represented by Formula I-87-2:
[0284] MS m/z(ESI):677.2[M+1].
[0285] .sup.1H NMR (400 MHz, MeOD) HNMR: (400 MHz, CDCl.sub.3) 8.88 (s, 1H), 7.63-7.57 (m, 1H), 7.48-7.43 (m, 2H), 7.33-7.28 (m, 1H), 7.24-7.22 (m, 1H), 6.64-6.59 (m, 1H), 6.45-6.40 (m, 1H), 5.85-5.81 (m, 1H), 4.58-3.45 (m, 7H), 3.12-3.06 (m, 2H), 2.20 (s, 3H), 1.46-1.45 (m, 3H), 1.24 (d, J=6.8 MHz, 12H).
[0286] Effect Example 1: Pharmacodynamic Assay 1 (Determination of the efficacy of the compounds of the present disclosure on NCI-H358 human non-small cell lung cancer cell with KRAS G12C mutation)
[0287] The following method was used to determine the effect of the compounds of the present disclosure on tumor cell proliferation.
[0288] For the KRAS G12C subtype, NCI-H358 non-small cell lung cancer cells with KRAS G12C mutation were used to determine the efficacy of the compounds in inhibiting cancer cell viability. NCI-H358 cells were cultured in a DMEM medium containing 10% fetal bovine serum, 100 U penicillin and 100 ng/mL streptomycin. The cells were cultured in an incubator with 5% CO.sub.2 at 37° C. Cancer cell viability was evaluated by determining the content of ATP with Cell Titer-Glo® kit (Luminescent Cell Viability Assay kit, please refer to the manufacturer's instructions for use) and evaluating the inhibition of cell growth.
[0289] The experimental method was proceeded in accordance with the steps in the instructions for the kit, and was described briefly as follows. The test compounds were first dissolved in DMSO to prepare stock solutions, and then the stock solutions were subjected to gradient dilution with the culture media of the corresponding cells, thereby preparing test samples. The final concentrations of the compounds were in the range of 30 μM to 0.01 nM. Tumor cells in logarithmic growth phase were seeded into a 96-well cell culture plate at an appropriate density. After the cell culture plate was left overnight in an incubator with 5% CO.sub.2 at 37° C., the test compound samples were added thereto and then the cells were further cultured for 72 hours. After the incubation was complete, an appropriate volume of Cell Titer-Glo® reagent was added into each well, and the cells were incubated at 37° C. for 1 to 4 hours. Then, the absorbance value of each sample well at 450 nM was read on a microplate reader. The percentage inhibition rates of the compounds at each concentration point were calculated by comparing the absorbance values of the sample wells with that of the control group (0.3% DMSO), and then non-linear regression analysis of the concentration of the compound vs inhibition rate was performed by using GraphPad Prism 5 software, thereby obtaining IC.sub.50 values indicating the inhibitory effects of the compounds on cell proliferation, where A represented IC.sub.50 of less than 100 nM, B represented IC.sub.50 of between 100 nM and 1000 nM, and C represented IC.sub.50 of greater than 1000 nM. The specific experimental results were as shown in Table 3.
TABLE-US-00003 TABLE 3 IC.sub.50 data indicating the inhibitory effects of the compounds represented by Formula I of the present disclosure on NCI-H358 human non-small cell lung cancer cells Compound No. IC.sub.50 (nM)/NCI-H358 Control Compound 226 ARS-1620 I-1 1172 I-2-1 1150 I-2-2 50 I-3-1 1980 I-3-2 250 I-4-1 C I-4-2 B I-5-1 C I-5-2 B I-6-1 C I-6-2 A I-7-1 C I-7-2 B I-8-1 C I-8-2 B I-9 B I-10 C I-11 C I-12 B I-13 B I-14 B I-15 B I-16 B I-17 C I-18 B I-19 C I-20 B I-21 B I-22 A I-23 B I-24 A I-25 B I-26 B I-27 B I-28 C I-29 B I-30 C I-31 A I-32 B I-34 C I-35 A I-36 B I-37 B I-38 B I-39 B I-40 C I-41 B I-42 B I-43 A I-44 B I-45 B I-46 C I-47 B I-48 B I-49 B I-50 B I-51 B I-52 B I-53 B I-54 B I-55 B I-56 B I-57 A I-58 B I-59 C I-60 B I-61 A I-62 B I-63 B I-64 B I-65 B I-66 A I-67 B I-68 B I-69 A I-70 A I-71 B I-72 A I-73 B I-74 C I-75 B I-76 A I-77 B I-78 A I-79 A I-80 A I-81 B I-82 A I-83-1 A I-83-2 B I-84-1 A I-84-2 B I-85-1 B I-85-2 B I-86-1 A I-86-2 B I-87-1 A I-87-2 B
[0290] Conclusion: as could be seen from Table 3, the compounds of the present disclosure had very good inhibitory effects on NCI-H358 human non-small cell lung cancer cells, in which KRAS G12C was highly expressed, and the compounds of the present disclosure could be used as drugs prepared as KRAS G12C inhibitors.
[0291] Effect Example 2: Pharmacodynamic Assay 2 (Determination of the efficacy of the compounds of the present disclosure on HCT116 human colon cancer cells with high expression of KRAS G13D and A549 human non-small cell lung cancer cells with high expression of KRAS G12S)
[0292] The following method was used to determine the effects of the compounds of the present disclosure on tumor cell proliferation.
[0293] For KRAS G13D subtype, HCT116 human colon cancer cell was used. For KRAS G12S subtype, A549 human non-small cell lung cancer cell was used. The efficacy of the compounds in inhibiting cancer cell viability was determined. HCT116 cells or A549 cells were cultured in a DMEM medium containing 10% fetal bovine serum, 100 U penicillin and 100 ng/mL streptomycin. The cells were cultured in an incubator with 5% CO.sub.2 at 37° C. Cancer cell viability was evaluated by determining the content of ATP with Cell Titer-Glo® kit (Luminescent Cell Viability Assay kit, please refer to the manufacturer's instructions for use) and evaluating the inhibition of cell growth.
[0294] The experimental method was proceeded in accordance with the steps in the instructions for the kit, and was described briefly as follows. The test compounds were first dissolved in DMSO to prepare stock solutions, and then the stock solutions were subjected to gradient dilution with the culture media of the corresponding cells, thereby preparing test samples. The final concentrations of the compounds were in the range of 30 μM to 0.01 nM. Tumor cells in logarithmic growth phase were seeded into a 96-well cell culture plate at an appropriate density. After the cell culture plate was left overnight in an incubator with 5% CO.sub.2 at 37° C., the test compound samples were added thereto and then the cells were further cultured for 72 hours. After the incubation was complete, an appropriate volume of Cell Titer-Glo® reagent was added into each well, and the cells were incubated at 37° C. for 1 to 4 hours. Then, the absorbance value of each sample well at 450 nM was read on a microplate reader. The percentage inhibition rates of the compounds at each concentration point were calculated by comparing the absorbance values of the sample wells with that of the control group (0.3% DMSO), and then non-linear regression analysis of the concentration of the compound vs inhibition rate was performed by using GraphPad Prism 5 software, thereby obtaining IC.sub.50 values indicating the inhibitory effects of the compounds on cell proliferation. The experimental results were as shown in Table 4.
TABLE-US-00004 TABLE 4 IC.sub.50 data indicating the inhibitory effects of some of the compounds of the present disclosure on HCT116 human colon cancer cells and A549 human non-small cell lung cancer cells Examples IC.sub.50 (nM)/HCT116 IC.sub.50 (nM)/A549 Control compound >3000 >3000 ARS-1620 I-1 >3000 >3000 I-2-1 >3000 >3000 I-2-2 >3000 >3000
[0295] Conclusion: The compounds of the present disclosure had no inhibitory effect on cells without G12C mutation, such as HCT116 human colon cancer cells and A549 human non-small cell lung cancer cells, which indicated that the compounds of the present disclosure had extremely high selectivity.
[0296] Effect Example 3: Pharmacokinetic Experiment
[0297] (1) Compound I-2-2 of the present disclosure prepared in the above Example was used to be formulated into a clear solution (0.3 mg/mL) (2% DMSO+30% PEG 300+2% Tween 80+66% H.sub.2O) as a drug for oral administration; and was used to be formulated into a clear solution (0.2 mg/mL) (2% DMSO+30% PEG 300+2% Tween 80+66% H.sub.2O) as a drug for intravenous administration.
[0298] (2) Male CD-1 mice (three mice in each group, weighing 27 g to 28 g) were provided by Shanghai Slack Laboratory Animal Co., Ltd. The test mice were given an environmental accommodation period of 2 to 4 days before the experiment. The mice were fasted for 8 to 12 hours before administration, and they were provided access to water (2 hours after administration) and food (4 hours after administration).
[0299] (3) 12 hours after the mice were fasted but had free access to water, blank plasma at time 0 was taken.
[0300] (4) The mice in step 1) were taken; and they were orally (PO) administered 3 mg/kg of the compound to be tested, or intravenously (IV) administered 1 mg/kg of the compound to be tested.
[0301] (5) At 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 10 h, and 24 h after oral administration, blood was successively taken from the fundus oculi venous plexus and placed in EP tubes with heparin. After centrifugation at 8000 rpm/min for 5 min, the plasma in upper layer was collected and frozen at −20° C., waiting for LC-MS/MS analysis.
[0302] (6) WinNonlin software was used to calculate the pharmacokinetic parameters based on the blood concentration-time data obtained in step 3). The specific data were as shown in Table 5.
TABLE-US-00005 TABLE 5 Pharmacokinetic data of the compound of the Example of the present disclosure Groups Parameters Example IV (1 mg/kg) Cl (mL/kg/min) 16.7 V.sub.d (L/kg) 1.17 AUC (ng .Math. h/mL) 867 T.sub.1/2 (h) 0.718 PO (3 mg/kg) C.sub.max (ng/mL) 220 T.sub.max (h) 1.0 AUC (ng .Math. h/mL) 479 F (%) 18.9%
[0303] Conclusion: In the pharmacokinetic evaluation experiment conducted in mice, the compound of the present disclosure showed relatively high exposure and very ideal oral bioavailability at a relatively low dose.
[0304] Effect Example 4: In-Vivo Pharmacodynamic Experiment
[0305] Purpose of the experiment: To evaluate the anti-tumor effect of the test drug in female BALB/c nude mice model subcutaneously xenografted with MIAPaCa-2 human-derived pancreatic cancer cells.
[0306] Experimental operation: BALB/c nude mice, female, 6- to 8-weeks old, weighing about 17.6 g to 21.1 g. Each mouse was subcutaneously inoculated with 5×10.sup.6 MIAPaCa-2 cells (with Matrigel; the volume ratio was 1:1) in the right flank. The administration was initiated when the average tumor volume reached about 100 cubic millimeters. The test compounds were orally administered daily by intragastric injection (6 mice in each group) for a total of 16 or 23 days. The doses were as shown in Table 5. Tumor volumes were measured three times a week. The volume was measured in cubic millimeters and calculated by the following formula: tumor volume (mm.sup.3)=½×(a×b.sup.2) (where a represented the long diameter and b represented the short diameter). The anti-tumor efficacy of the compound was evaluated by TGI (%).The calculation formula was as follows: TGI %=(1−T/C)×100% (T and C were the relative tumor volumes (RTV) of a mouse in the treatment group and a mouse in the control group at a specific time point), respectively. Safety was evaluated according to the changes of the body weight of the animals and animal deaths.
[0307] The experimental results were as shown in Table 6 and Table 7.
TABLE-US-00006 TABLE 6 Tumor Tumor volumes volumes (mm.sup.3) (mm.sup.3) P Groups (Day 0) (Day 23) TGI (%) TIC Value Solvent control group 100.3 631.29 — — — Example I-2-2 100.25 438.75 36.25 63.75 0.517 30 mg/kg (Day 0 to Day 9) + 15 mg/kg (Day 10 to Day 16) Example I-2-2 100.06 179.60 85.02 14.98 0.001 100 mg/kg (Day 0 to Day 9) + 50 mg/kg (Day 10 to Day 16) + 100 mg (Day 17 to Day 23)
[0308] Experimental conclusion: The compound of the present disclosure showed good in-vivo efficacy in a tumor model subcutaneously xenografted with MIAPaCa-2 human pancreatic cancer cells. 23 days after the initiation of the administration, the compound of the present disclosure, as compared with the solvent control group, showed significant inhibitory effect on tumor and an obvious dose-response relationship.
TABLE-US-00007 TABLE 7 Weight at the Initiation of Weight on Weight Administration Day 23 Change Groups (g) (g) Rate (%) Solvent control group 20.1 21.1 +4.64 Example I-2-2 19.8 21.3 +7.53 30 mg/kg (Day 0 to Day 9) + 15 mg/kg (Day 10 to Day 16) Example I-2-2 20.0 22.4 +12.32 100 mg/kg (Day 0 to Day 9) + 50 mg/kg (Day 10 to Day 16) + 100 mg (Day 17 to Day 23)
[0309] Experimental conclusion: In the treatment groups of the compound of the present disclosure, there was no animal death, no weight loss, and no obvious drug toxicity during the administration period, and the compound of the present disclosure was well tolerated during the treatment period.
[0310] In the description of this specification, descriptions with reference to the terms “one Example”, “some Examples”, “examples”, “a specific example”, “some examples” or the like mean that the specific features, structures, materials, or characteristics described in conjunction with the Example or example are included in at least one Example or example of the present disclosure. In this specification, the exemplary description of the above-mentioned terms does not necessarily refer to the same Example or example. Those described are merely preferred Examples of the present disclosure and are not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements and the like made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.