CN111909101B - EGFR kinase inhibitor and application thereof in preparation of anti-cancer drugs - Google Patents

EGFR kinase inhibitor and application thereof in preparation of anti-cancer drugs Download PDF

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CN111909101B
CN111909101B CN201910538865.6A CN201910538865A CN111909101B CN 111909101 B CN111909101 B CN 111909101B CN 201910538865 A CN201910538865 A CN 201910538865A CN 111909101 B CN111909101 B CN 111909101B
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gefitinib
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CN111909101A (en
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金洪传
冯利锋
应士龙
杨丽贤
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Zhejiang University ZJU
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Abstract

The EGFR kinase inhibitor can effectively improve EGFR TKI acquired drug resistance caused by SQSTM1 accumulation in a mode of weakening alkalinity of EGFR TKI, and has important significance for improving clinical treatment benefit of EGFR TKI.

Description

EGFR kinase inhibitor and application thereof in preparation of anti-cancer drugs
Technical Field
The present invention relates to the field of medicine; in particular, the present invention relates to a novel EGFR kinase inhibitor and its use in the treatment of cancer.
Background
Lung cancer is a malignant tumor with the highest incidence and the largest number of deaths worldwide, and poses serious threat to human health. However, conventional chemotherapy has limited efficacy in treating advanced lung cancer. The Epidermal Growth Factor Receptor (Epidermal Growth Factor Receptor, EGFR, ErbB1) belongs to a member of the ErbB family of Receptor tyrosine kinases. Evidence from previous studies suggests that high EGFR expression, gene amplification or activation-type mutations (L858R) are prevalent in a variety of solid tumors. EGFR overexpression or activation type mutation can cause the excessive activation of cell proliferation and survival signal pathways such as downstream Ras/Raf/MAPK, PI3K/Akt and the like. Therefore, EGFR is considered to be a key oncogene that drives the development and progression of malignant tumors such as lung cancer, pancreatic cancer, breast cancer, and the like. The clinical benefit of EGFR tyrosine kinase inhibitor (EGFR TKI) in the treatment of the above-mentioned EGFR abnormally activated malignant tumor is remarkable, and once the great hope has been brought to people.
The first generation of EGFR-TKIs such as Gefitinib (Gefitinib) and Erlotinib (Erlotinib) inhibit the activation of EGFR signaling pathway in tumor cells by reversibly binding to the ATP binding site of EGFR. However, clinical practice has found that gefitinib can only maintain a half-year to one-year effective remission period, and resistance develops quickly. Currently, research reveals that the drug resistance mechanism of gefitinib mainly has the following aspects: mutations in the EGFR gene: secondary mutation of specific EGFR exon 20, causing threonine at position 790 to be replaced by a bulky methionine (T790M), preventing gefitinib and erlotinib binding to the ATP site; other secondary resistance mutations include L747S, D761Y, and T854A, etc.; the change of EGFR signal transduction path, such as Ras activation causing the up-regulation of Raf-MEK-MAPK, loss of coupling with EGFR, high expression of ERK and Met, PTEN inactivation causing the over-activation of AKT pathway, and the like; ③ EGFR bypass Effect: growth factor receptors such as Platelet Derived Growth Factor Receptor (PDGFR) and insulin-like growth factor receptor-1 (IGFR-1) receptor directly activate their downstream signaling pathways. The second generation EGFR-TKI Afatinib (Afatinib, BIBW2992) and the third generation EGFR-TKI axitinib (Osimetinib, AZD9291) developed aiming at the T790M mutation of EGFR are already applied to clinic, but various drug resistance is inevitably generated by the mechanisms including generation of C797S mutation, EGFR, HER2, C-Met gene amplification and the like.
Autophagy is a biological process in which cells maintain homeostasis by degrading macromolecular substances and damaged organelles to maintain energy supply and eliminate harmful substances under stress. In recent years, a great deal of research has found that autophagy is closely related to the progression of malignancy and the development of EGFR TKI acquired resistance. Autophagy can prevent normal cells from canceration, can help the cancerated cells resist adverse living environment, and promotes drug resistance. SQSTM1 (Sequestosome-1, also called p62) as a key ligand protein mediating multiple signal pathways such as autophagy, energy induction and oxidative stress plays a very important role in the development and resistance of tumors. As a ligand protein, SQSTM1 has an important regulatory role on multiple oncogenic signaling pathways, and not only mediates activation of mTORC1 in lysosomes, but also can dissociate its interaction with Nrf2 through direct action with keap1, thereby inhibiting keap1 from mediating ubiquitination degradation of Nrf 2. The increased protein level of SQSTM1 not only means that autophagy-related degradation pathways are attenuated, but also helps tumor cells resist apoptosis. The research shows that the expression of SQSTM1 in the platinum drug resistant cells is up-regulated, and conversely, the protein level of SQSTM1 is reduced, so that the sensitivity of the tumor to the drugs can be effectively improved. In addition, SQSTM1 can carry out plasma nuclear shuttle, thereby participating in the repair of DNA damage caused by radiotherapy, chemotherapy and the like, and the SQSTM1 is also proved to be a key regulatory factor for promoting the generation of tumor drug resistance. However, it is still unknown what role SQSTM1 plays in the development of EGFR TKI resistance. By clarifying the important action of SQSTM1 in the EGFR TKI drug resistance generation process and by aiming at the action mechanism of SQSTM1 for regulating EGFR TKI drug resistance, the reasonable modification of EGFR TKI is carried out, the problem of clinical EGFR TKI acquired drug resistance is expected to be overcome, and the method has important significance for improving the clinical treatment benefit of EGFR TKI.
Disclosure of Invention
The invention aims to provide a novel EGFR kinase inhibitor with strong anticancer effect and no influence on autophagy process and lysosome function, and application thereof in preparing a medicament for preventing and/or treating diseases and symptoms (such as cancers, particularly non-small cell lung cancer) related to receptor tyrosine protein kinase.
The invention provides a compound shown as a formula I and pharmaceutically acceptable salts thereof,
Figure GDA0003576518700000021
in the formula I, the compound has the following structure,
ring a is a5 to 18 membered ring, or ring a is absent;
x is-O-, -S-, -N (R)4) -or- (CHR)4)n-n is 0 or 1;
R1selected from: aryl, aralkyl, aromatic or non-aromatic heterocyclic and heterocycloalkyl groups;
R2selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR4、-C(O)OR4、-C(O)NR4R5、-CH＝NR4、-CN、-OR4、-OC(O)R4、-S(O)t-R4、-NR4R5、-NR4C(O)R5、 -NO2、-N＝CR4R5And a halogen;
R3independently at each occurrence, is selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR4、-C(O)OR4、-C(O)NR4R5、-CH＝NR4、-CN、-OR4、-OC(O)R4、-S(O)t-R4、 -NR4R5、-NR4C(O)R5、-NO2、-N＝CR4R5And halogen;
m is an integer of 0-3 (specifically 0, 1, 2, 3);
t is 1, 2 or 3;
R4and R5Each independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic groups, alkoxy, aryloxy and halogen;
and the compound shown in the formula I contains at least one acidic substituent in the structure, and the acidic substituent contains at least one acidic functional group.
In one embodiment of the invention, the acidic functional group is selected from: one or more of a phenolic hydroxyl group, a carboxyl group, a sulfonic acid group and a phosphoric acid group.
In one embodiment of the invention, the acidic substituent is selected from the group consisting of: one or more of hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphophenyl, hydroxybenzoyloxy-substituted alkyl, sulfo-substituted alkyl, carboxy-substituted alkyl, phospho-substituted alkyl, sulfophenyl-substituted alkyl, carboxyphenyl-substituted alkyl, phosphophenyl-substituted alkyl, hydroxybenzoyloxy-substituted alkoxy, sulfo-substituted alkoxy, carboxy-substituted alkoxy, phospho-substituted alkoxy, sulfophenyl-substituted alkoxy, carboxyphenyl-substituted alkoxy, and phosphophenyl-substituted alkoxy.
In one embodiment of the present invention, in the hydroxyphenyl group, the hydroxyl group is located in at least one of the 2-5 positions of the phenyl group.
In one embodiment of the invention, in the carboxyphenyl group, the carboxyl group is located in at least one of the 2-5 positions of the phenyl group.
In one embodiment of the present invention, in the sulfophenyl group, the sulfonic acid group is located in at least one of the 2-5 positions of the phenyl group.
In one embodiment of the present invention, the phosphate group is located in at least one of the 2-5 positions of the phenyl group.
In one embodiment of the invention, the hydroxybenzoyloxy group is a dihydroxybenzoyloxy group (e.g., 2, 3-dihydroxybenzoyloxy, 2, 4-dihydroxybenzoyloxy, 2, 5-dihydroxybenzoyloxy, 2, 6-dihydroxybenzoyloxy, 3, 4-dihydroxybenzoyloxy, 3, 5-dihydroxybenzoyloxy, 3, 6-dihydroxybenzoyloxy, 4, 5-dihydroxybenzoyloxy, 4, 6-dihydroxybenzoyloxy, or 5, 6-dihydroxybenzoyloxy) or a trihydroxybenzoyloxy group (e.g., 2,3, 4-trihydroxybenzoyloxy, 2,3, 5-trihydroxybenzoyloxy, 2,3, 6-trihydroxybenzoyloxy, 2,4, 5-trihydroxybenzoyloxy, 2,4, 6-trihydroxybenzoyloxy, 2,5, 6-trihydroxybenzoyloxy, 3,4, 5-trihydroxybenzoyloxy, 3,4, 6-trihydroxybenzoyloxy or 4,5, 6-trihydroxybenzoyloxy).
In one embodiment of the invention, the alkyl group is a C1-C6 alkyl group, preferably a C1-C3 alkyl group.
In one embodiment of the invention, alkoxy is C1-C6 alkoxy, preferably C1-C3 alkoxy.
In one embodiment of the present invention, the compound of formula i contains at least one substituent in the structure:
Figure GDA0003576518700000031
in one embodiment of the invention, X is-O-.
In another embodiment of the present invention, X is-NH-.
In another embodiment of the invention, X is- (CHR)4)nN is 0, i.e. X is absent.
In one embodiment of the invention, R1Is a substituted aryl group, such as a substituted phenyl group.
In another embodiment of the present invention, R1Is a substituted heterocyclic group, such as a substituted indolyl group (e.g., 3-indolyl group).
In one embodiment of the invention, m is 2.
In another embodiment of the present invention, m is 0.
In one embodiment of the invention, ring a is an aromatic ring, preferably a benzene ring, which forms a quinazoline ring structure with the attached pyrimidine ring.
In another embodiment of the invention, ring a is a heteroaromatic ring, preferably a single heteroatom five membered aromatic ring, for example, a pyrrole ring, furan ring or thiophene ring, fused to a pyrimidine ring to form a five membered single heteroaromatic [3,2-d ] pyrimidine ring structure.
In another embodiment of the invention, ring a is absent.
In one embodiment of the invention, in formula I, the A ring is aromatic and R is1The compound is substituted phenyl, and has a structure shown in a general formula II:
Figure GDA0003576518700000032
in the formula II, X is-O-, -S-, -N (R)17) -or- (CHR)17)n-n is 0 or 1;
R11、R12and R13Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR17、-C(O)OR17、-C(O)NR17R18、-CH＝NR17、-CN、-OR17、-OC(O)R17、-S(O)t-R17、 -NR17R18、-NR17C(O)R18、-NO2、-N＝CR17R18And halogen;
R14selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR17、-C(O)OR17、-C(O)NR17R18、-CH＝NR17、-CN、-OR17、-OC(O)R17、-S(O)t-R17、-NR17R18、 -NR17C(O)R18、-NO2、-N＝CR17R18And halogen;
R15and R16Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR17、-C(O)OR17、-C(O)NR17R18、-CH＝NR17、-CN、-OR17、-OR19-R18、-OC(O)R17、 -S(O)t-R17、-NR17R18、-NR17C(O)R18、-NO2、-N＝CR17R18And halogen; or R15And R16Form an optionally substituted fused ring system with the benzene ring to which it is attached;
t is 1, 2 or 3;
R17and R18Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic groups, alkoxy, aryloxy, halogen and amino;
R19is an alkylene group;
and the compound shown in the formula II contains at least one acidic substituent group in the structure, and the acidic substituent group contains at least one acidic functional group. The acidic functional group and the acidic substituent have the above definitions of the present invention.
In one embodiment of the invention, R11、R12、R13、R14、R15And R16At least one of which contains an acidic functional group.
In one embodiment of the invention, R15Contains at least one acidic functional group.
In one embodiment of the invention, R15Selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphophenyl, hydroxybenzoyloxy substituted alkyl, sulfo substituted alkyl, carboxy substituted alkyl, phospho substituted alkyl, sulfophenyl substituted alkyl, carboxyphenyl substituted alkyl, phospho phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfo substituted alkoxy, carboxy substituted alkoxy, phospho substituted alkoxy, sulfophenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phospho phenyl substituted alkoxy.
In one embodiment of the invention, R15Is composed of
Figure GDA0003576518700000041
In one embodiment of the invention, X is-NH-.
In one embodiment of the invention, R11、R12And R13Independently selected from: hydrogen, halogen (e.g. F, Cl), -NO2Alkenyl, alkynyl and-O (CH)2)xAr, x is 0 or 1, Ar is an aryl group (e.g., phenyl) or a heterocyclic group (e.g., a nitrogen-containing heterocyclic group).
In one embodiment of the invention, R11Is F, R12Is Cl, R13Is hydrogen.
In another embodiment of the invention, R11Is hydrogen, R12Is Cl, R13Is F.
In one embodiment of the invention, R11Is hydrogen, R13Is hydrogen, R12Selected from the group consisting of: halogen, vinyl and ethynyl.
In one embodiment of the invention, R14Selected from: hydrogen, alkyl, -CN and halogen.
In one embodiment of the invention, R14Is hydrogen.
In one embodiment of the invention, R16is-OR19-R18Wherein R is19Is methylene, ethylene, propylene or butylene, R18Selected from the group consisting of: aromatic or non-aromatic heterocyclic groups (e.g., morpholinyl, piperazinyl, piperidinyl, pyrrolidinyl, and the like), alkoxy groups (e.g., methoxy, ethoxy, propoxy), aryloxy groups (e.g., phenoxy), and substituted amino groups (e.g., methylamino, dimethylamino).
In one embodiment of the invention, R16is-OR19-R18Wherein R is19Is ethylene or propylene, R18Selected from: morpholinyl, piperazinyl, dimethylamino, pyrrolidinyl, piperidinyl, methoxy and ethoxy.
Specifically, R16Can be selected from: 3- (4-morpholinyl) propoxy, 3- (1-piperazinyl) propoxy, 3-dimethylaminopropoxy, 3- (1-pyrrolidinyl) propoxy, 3-(1-piperidinyl) propoxy, 3- (4-piperidinyl) propoxy, methoxyethoxy and ethoxyethoxy.
In one embodiment of the invention, R16Is composed of
Figure GDA0003576518700000042
In one embodiment of the invention, R16is-NHC (O) R18Wherein R is18Selected from: substituted or unsubstituted alkenyl (e.g., ethenyl, 1-propenyl, 1-butenyl), the substituents on the alkenyl being selected from: aromatic or non-aromatic heterocyclic groups (e.g., pyrrolidinyl, piperidinyl), alkoxy groups (e.g., methoxy, ethoxy), and substituted amino groups (e.g., methylamino, dimethylamino).
In one embodiment of the invention, R16Selected from: 4-methoxy-2-butenamido, 4-pyrrolidinyl-2-butenamido, 4-dimethylamino-2-butenamido and 4-piperidinyl-2-butenamido.
In one embodiment of the invention, the compound has the following structure:
Figure GDA0003576518700000043
in another embodiment of the invention, in formula I, the ring A is a single heteroatom five-membered aromatic ring, R1The compound is substituted phenyl and has a structure shown in a general formula III:
Figure GDA0003576518700000044
in the formula (III), the reaction solution is,
x is-O-, -S-, -N (R)29) -or- (CHR)29)n-n is 0 or 1;
y is-O-, -S-or-N (R)29)-；
Z is-O-, -S-or-N (R)29)-；
R21、R22And R23Independent of each otherIs selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR29、-C(O)OR29、-C(O)NR29R28、-CH＝NR29、-CN、-OR29、-OC(O)R29、-S(O)t-R29、 -NR29R210、-NR29C(O)R210、-NO2、-N＝CR29R210And halogen;
R24、R25and R26Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR29、-C(O)OR29、-C(O)NR29R28、-CH＝NR29、-CN、-OR29、-OC(O)R29、-S(O)t-R29、 -NR29R210、-NR29C(O)R210、-NO2、-N＝CR29R210And halogen;
R27and R28Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR29、-C(O)OR29、-C(O)NR29R28、-CH＝NR29、-CN、-OR29、-OC(O)R29、-S(O)t-R29、 -NR29R210、-NR29C(O)R210、-NO2、-N＝CR29R210And a halogen; r is27And R28Form an optionally substituted fused ring system with the heterocycle to which it is attached;
t is 1, 2 or 3;
R29and R210Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic groups, alkoxy, aryloxy, halogen and amino;
the compound shown in the formula III contains at least one acidic substituent group in the structure, and the acidic substituent group contains at least one acidic functional group. The acidic functional group and the acidic substituent have the above definitions of the present invention.
In one embodiment of the invention, R21、R22、R23、R24、R25、R26、R27And R28At least one of which contains an acidic functional group.
In one embodiment of the invention, R26Contains at least one acidic functional group.
In one embodiment of the invention, R26Selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphophenyl, hydroxybenzoyloxy substituted alkyl, sulfo substituted alkyl, carboxy substituted alkyl, phospho substituted alkyl, sulfophenyl substituted alkyl, carboxyphenyl substituted alkyl, phospho phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfo substituted alkoxy, carboxy substituted alkoxy, phospho substituted alkoxy, sulfophenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phospho phenyl substituted alkoxy.
In one embodiment of the invention, R26Is composed of
Figure GDA0003576518700000051
In one embodiment of the invention, X is-NH-.
In another embodiment of the present invention, X is-O-.
In one embodiment of the invention, Y is-S-.
In one embodiment of the invention, Z is-NH-.
In one embodiment of the invention, R21And R23Independently selected from: hydrogen, alkyl and halogen.
In one embodiment of the invention, R21Is hydrogen.
In one embodiment of the invention, R23Is hydrogen.
In one embodiment of the invention, R22is-NHC (O) R210Wherein R is210Is substituted or unsubstituted alkenyl, such as ethenyl, 3-propenyl. In particular, R22(may be)
Figure GDA0003576518700000052
In one embodiment of the invention, R24Is a heterocyclic group such as a substituted or unsubstituted nitrogen-containing non-aromatic heterocyclic group (specifically, piperazinyl, piperidinyl), wherein the substituents are selected from: alkyl (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclyl (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.), the substitution site being at the nitrogen position of the nitrogen-containing non-aromatic heterocyclyl.
In one embodiment of the invention, R24Selected from: 4- (1-acetyl) piperazinyl, 4- (1-methyl) piperazinyl, 4- (1-pyrrolidinyl) piperazinyl, 4- (1-piperidinyl) piperazinyl, and 4-methylpiperazinyl-1-piperidinyl.
In one embodiment of the invention, R24Is composed of
Figure GDA0003576518700000061
In another embodiment of the invention, R24is-NR27R28，R27And R28Independently selected from: hydrogen and substituted or unsubstituted alkyl (e.g., methyl, 3- (methylamino) propyl).
In one embodiment of the invention, R24Is N, N-dimethylamino or N1,N1,N2-trimethylethylenediamine.
In one embodiment of the invention, R25Is hydrogen.
In one embodiment of the invention, R27And R28Independently selected from: hydrogen, alkyl and halogen.
In one embodiment of the invention, R27Is hydrogen.
In one embodiment of the invention, R28Is hydrogen.
In one embodiment of the invention, the compound has the following structure:
Figure GDA0003576518700000062
in another embodiment of the invention, in formula I, ring A is absent and R is1Is substituted phenyl, the compound has a structure shown in a general formula IV:
Figure GDA0003576518700000063
in the formula IV, the reaction is carried out,
x is-O-, -S-, -N (R)39) -or- (CHR)39)n-n is 0 or 1;
z is-O-, -S-or-N (R)39)-；
R31Is hydrogen, substituted or unsubstituted aryl or heterocyclyl;
R32、R33、R34、R35and R36Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR39、-C(O)OR39、-C(O)NR39R310、-CH＝NR39、-CN、-OR39、-OC(O)R39、 -S(O)t-R39、-NR39R310、-NR39C(O)R310、-NO2、-N＝CR39R310And halogen;
R37and R38Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, aralkyl, aromatic or non-aromatic heterocyclyl, heterocycloalkyl, -COR39、-C(O)OR39、-C(O)NR39R310、-CH＝NR39、-CN、-OR39、-OC(O)R39、-S(O)t-R39、 -NR39R310、-NR39C(O)R310、-NO2、-N＝CR39R310And a halogen;
t is 1, 2 or 3;
R39and R310Independently selected from: hydrogen, alkyl, cycloalkyl, alkenyl, aryl, aromatic or non-aromatic heterocyclic groups, alkoxy, aryloxy, halogen and amino;
and the structure of the compound shown in the formula IV contains at least one acidic substituent group, and the acidic substituent group contains at least one acidic functional group. The acidic functional group and the acidic substituent have the above definitions of the present invention.
In one embodiment of the invention, R31、R32、R33、R34、R35、R36、R37And R38At least one of which contains an acidic functional group.
In one embodiment of the invention, R36Contains at least one acidic functional group.
In one embodiment of the invention, R36Selected from: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphophenyl, hydroxybenzoyloxy substituted alkyl, sulfo substituted alkyl, carboxy substituted alkyl, phospho substituted alkyl, sulfophenyl substituted alkyl, carboxyphenyl substituted alkyl, phospho phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfo substituted alkoxy, carboxy substituted alkoxy, phospho substituted alkoxy, sulfophenyl substituted alkoxy, carboxyphenyl substituted alkoxy and phospho phenyl substituted alkoxy.
In one embodiment of the invention, R36Is composed of
Figure GDA0003576518700000071
In one embodiment of the invention, X is- (CHR)39)nN is 0, i.e. X is absent.
In one embodiment of the invention, Z is-NH-.
In one embodiment of the invention, R31Is substituted aryl, e.g. substituted phenyl, such as
Figure GDA0003576518700000072
Wherein R is310Is substituted or unsubstituted alkenyl, such as ethenyl, 3-propenyl, and the substituents on the alkenyl are selected from: alkoxy (e.g., methoxy, ethoxy), heterocyclyl (e.g., piperidinyl, pyrrolidinyl), and substituted amino (e.g., methylamino, dimethylamino).
In one embodiment of the invention, R31Selected from: 3-acrylamidophenyl, 3- (4-methoxy-2-butenamido) phenyl, 3- (4-piperidinyl-2-butenamido) phenyl, 3- (4-pyrrolidinyl-2-butenamido) phenyl, 3- (4-dimethylamino-2-butenamido) phenyl.
In another embodiment of the present invention, R31Is a substituted or unsubstituted heterocyclic radical, e.g. pyrazolo [1,5-a ]]And pyridyl or substituted indolyl, the substituents of the indolyl being selected from: hydrogen, alkyl and acyl.
In one embodiment of the invention, R31Is composed of
Figure GDA0003576518700000073
In one embodiment of the invention, R32Is hydrogen.
In one embodiment of the invention, R33Is composed of
Figure GDA0003576518700000074
Wherein X3is-NH-or-O-, R39Is a substituted or unsubstituted alkenyl group, such as ethenyl, 3-propenyl, the substituents on the alkenyl group being selected from: alkoxy (e.g., methoxy, ethoxy), heterocyclyl (e.g., piperidinyl, pyrrolidinyl), and substituted amino (e.g., methylamino, dimethylamino).
In one embodiment of the invention, R33Selected from: acryloxy group, acrylamido group, 4-methoxy-2-butenyloxy group, 4-methoxy-2-butenoyloxy group, 4-piperidyl-2-butenyloxy group, 4-piperidyl-2-butenoyloxy group, 4-pyrrolidinyl-2-butenyloxy group, 4-pyrrolidinyl-2-butenoyloxy group, 4-dimethylamino-2-butenoyloxy groupAnd 4-dimethylamino-2-butenamido.
In one embodiment of the invention, R33Is composed of
Figure GDA0003576518700000075
In one embodiment of the invention, R35Is hydrogen.
In one embodiment of the invention, R34Is a substituted or unsubstituted heterocyclic group such as a substituted nitrogen-containing non-aromatic heterocyclic group (specifically, piperazinyl, piperidinyl), wherein the substituent is selected from: alkyl (such as methyl, ethyl), acyl (such as acetyl, propionyl), heterocyclyl (such as pyrrolidinyl, piperidinyl, piperazinyl, etc.), the substitution site being at the nitrogen position of the nitrogen-containing non-aromatic heterocyclyl.
In one embodiment of the invention, R34Selected from: 4- (1-acetyl) piperazinyl, 4- (1-methyl) piperazinyl, 4- (1-pyrrolidinyl) piperazinyl, 4- (1-piperidinyl) piperazinyl, and 4-methylpiperazinyl-1-piperidinyl.
In another embodiment of the invention, R34is-NR27R28，R27And R28Independently selected from: hydrogen and substituted or unsubstituted alkyl (e.g., methyl, 3- (methylamino) propyl).
In one embodiment of the invention, R34Is N, N-dimethylamino or N1,N1,N2-trimethylethylenediamine.
In one embodiment of the invention, R34Is composed of
Figure GDA0003576518700000076
In one embodiment of the invention, R37And R38Independently selected from: hydrogen, halogen, nitro, cyano and trifluoromethyl.
In one embodiment of the invention, R37Is hydrogen.
In one embodiment of the invention, R38Is hydrogen.
In one embodiment of the invention, the compound has the following structure:
Figure GDA0003576518700000081
the present invention also provides a method for preparing the above compound, which comprises the step of introducing at least one acidic functional group into the structure of a compound having an EGFR inhibitory activity (e.g., a known EGFR TKI compound) by reaction.
In one embodiment of the invention, the acidic functional group is selected from: one or more of a phenolic hydroxyl group, a carboxyl group, a sulfonic acid group and a phosphoric acid group.
In one embodiment of the present invention, the above preparation method comprises introducing one or more groups selected from the group consisting of: hydroxyphenyl, carboxyphenyl, sulfophenyl, phosphophenyl, hydroxybenzoyloxy substituted alkyl, sulfo substituted alkyl, carboxy substituted alkyl, phospho substituted alkyl, sulfophenyl substituted alkyl, carboxyphenyl substituted alkyl, phospho phenyl substituted alkyl, hydroxybenzoyloxy substituted alkoxy, sulfo substituted alkoxy, carboxy substituted alkoxy, phospho substituted alkoxy, sulfophenyl substituted alkoxy, carboxyphenyl substituted alkoxy, and phospho phenyl substituted alkoxy.
In one embodiment of the present invention, in the above preparation method, the EGFR TKI compound is gefitinib.
In one embodiment of the present invention, the above preparation method comprises the following reaction steps:
Figure GDA0003576518700000082
wherein, X, R11、R12、R13、R14、R16Having the above-mentioned definitions of the present invention,
ra is a halogen atom, and Ra is a halogen atom,
rp is a hydroxyl protecting group;
the reaction in the step 2 is a hydroxyl deprotection reaction.
In another embodiment of the present invention, the above preparation method comprises the following reaction steps:
Figure GDA0003576518700000083
wherein, X, Y, Z, R21、R22、R23、R24、R25、R27、R28Having the above-mentioned definitions of the present invention,
ra is a halogen atom, and Ra is a halogen atom,
rp is a hydroxyl protecting group;
the reaction in the step 2 is a hydroxyl deprotection reaction.
In another embodiment of the present invention, the above preparation method comprises the following reaction steps:
Figure GDA0003576518700000091
wherein, X, Z, R31、R32、R33、R34、R35、R37、R38Having the above-mentioned definition of the present invention,
ra is a halogen atom, and Ra is a halogen atom,
rp is a hydroxyl protecting group;
the reaction in the step 2 is a hydroxyl deprotection reaction.
For the purpose of protecting and deprotecting hydroxyl groups, the hydroxyl protecting groups and hydroxyl deprotecting reactions can be selected from the hydroxyl protecting groups and hydroxyl deprotecting reactions that are conventional in the art, and the invention is not particularly limited thereto.
The invention also provides a method of engineering an EGFR TKI, comprising the step of attenuating the basicity of the EGFR TKI, e.g., the step of introducing at least one acidic functional group in the structure of the EGFR TKI.
The acidic functional group has the above definition of the present invention.
The invention also provides a stereoisomer, a geometric isomer, a tautomer, a racemate, a solvate, a hydrate, a metabolic precursor and a prodrug of the compound or the pharmaceutically acceptable salt thereof.
The invention also provides a pharmaceutical composition, which comprises the compound or pharmaceutically acceptable salts, stereoisomers, geometric isomers, tautomers, racemates, solvates, hydrates, metabolic precursors or prodrugs thereof, and one or more pharmaceutically acceptable auxiliary materials.
The pharmaceutically acceptable carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field. For example: diluents, excipients such as water, etc., fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone, etc.; humectants such as glycerol and the like; disintegrating agents such as agar, calcium carbonate and sodium bicarbonate; absorption accelerators such as quaternary ammonium compounds and the like; surfactants such as cetyl alcohol and the like; adsorption carriers such as kaolin and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, and the like. In addition, other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.
The pharmaceutical composition according to the present invention may be a tablet (including sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), a pill, a powder, a granule, a capsule (including soft capsules, microcapsules), a troche, a syrup, a liquid, an emulsion, a suspension, a controlled release preparation (e.g., an instantaneous release preparation, a sustained release preparation, sustained release microcapsules), an aerosol, a film (e.g., an orally disintegrating film, an oral mucosa-adhering film), an injection (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), an intravenous drip, a transdermal absorption preparation, an ointment, a lotion, an adhering preparation, a suppository (e.g., a rectal suppository, a vaginal suppository), a pellet, a nasal preparation, a pulmonary preparation (inhalant), an eye drop, etc., an oral or parenteral preparation (e.g., via intravenous injection), a parenteral preparation, a pharmaceutical composition, a tablet, a, Intramuscular, subcutaneous, intraorgan, intranasal, intradermal, instillation, intracerebral, intrarectal, etc. administration to the vicinity of tumors and directly to lesions). In one embodiment of the present invention, the pharmaceutical composition is an injection.
Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient may be mixed with one or more excipients and then formulated into the desired dosage form.
The pharmaceutical composition of the present invention preferably contains 0.1 to 99.5% by weight of the active ingredient, more preferably 0.5 to 95% by weight of the active ingredient.
The amount of the compound of the present invention to be administered may vary depending on the route of administration, the age, weight, type and severity of the disease to be treated, etc., and may be administered one or more times.
The invention also provides application of the compound or pharmaceutically acceptable salts, stereoisomers, geometric isomers, tautomers, racemates, solvates, hydrates, metabolic precursors and prodrugs thereof in preparing medicines for preventing and/or treating diseases and symptoms related to receptor tyrosine protein kinase.
The present invention also provides a method for preventing and/or treating diseases and disorders associated with receptor tyrosine protein kinases, comprising the step of administering the compound of the present invention or a pharmaceutically acceptable salt, stereoisomer, geometric isomer, tautomer, racemate, solvate, hydrate, metabolic precursor and prodrug thereof, or the pharmaceutical composition to a subject in need thereof.
The diseases and conditions associated with receptor tyrosine protein kinases are tumors including, but not limited to: lung, pancreatic, breast or colon cancer, particularly lung cancer, more particularly non-small cell lung cancer.
The inventor of the invention discovers through a large number of experiments that the anti-tumor effect of EGFR TKIs is closely related to the acid-base property of the EGFR TKIs, and the EGFR TKI (such as gefitinib and AZD9291) clinically used at present can weaken the lysosome acidification process of tumor cells, further cause the accumulation of oncogenic protein SQSTM1 and finally cause the generation of acquired tumor drug resistance. The inventor develops a series of EGFR TKI compounds by weakening the alkalinity of EGFR TKI, can effectively improve EGFR TKI acquired resistance caused by SQSTM1 accumulation, and has important significance for improving the clinical treatment benefit of EGFR TKI.
Drawings
FIG. 1 shows the results of the measurement of the expression level of SQSTM1 in A549, HCC827 and H460 cells treated with gefitinib in example 1.
FIG. 2 shows the results of measurement of the expression level of SQSTM1 in A549, HCC827 and H460 cells treated with AZD9291 in example 1.
FIG. 3 is a graph showing the results of measurement of the expression amounts of LC3 and SQSTM1 in A549 cells and H460 cells in example 2.
FIG. 4 shows the results of the test in example 3; FIG. 4A is a photograph of spots detected by immunofluorescence microscopy on GFP-LC3 and RFP-LC3, wherein the spots in the first row are green fluorescent spots, the spots in the second row are red fluorescent spots, and the spots in the third row are yellow fluorescent spots; b in fig. 4 shows the result of LC3 yellow spot/red spot (%).
FIG. 5 shows the results of the test in example 4; wherein, the first column shows the result of detecting cell viability by MTS method after the SQSTM1-Flag plasmid is transiently transferred in A549 cells, and then stable transfected monoclonal cell masses are obtained by screening with antibiotic G418 and treated for 48 hours by Gefitinib and AZD9291 with different concentrations. The second panel shows the results of MTS assay for cell viability after SQSTM 148H knockdown in H460 cells, treated with different concentrations of Gefitinib and AZD9291 for 24H.
FIG. 6 shows the results of detection in example 5; a in FIG. 6 is the result of detecting the change of LC3 protein expression by immunofluorescence after treating A549 for different time with gefitinib and AZD 9291; the statistical analysis of the results in a in fig. 6 is shown in B in fig. 6, and 10 random fields are counted, respectively, with 10 μm in the scale and asterisks (x) representing P <0.05 as statistical differences.
FIG. 7 shows the results of the test in example 6; in fig. 7, a is a photograph of immunofluorescence detection of LAMP1 and LC3, and B in fig. 7 is a statistical result of co-localization coefficients of each experimental group.
FIG. 8 shows the results of measurement of CTSB expression levels of mature bodies in A549 cells and HCC827 cells after treatment with combination of Gefitinib, AZD9291, CQ and Baf A1 in example 7.
FIG. 9 shows the results of the test in example 8; wherein, A in FIG. 9 is a fluorescence microscope photograph, wherein the scale bar is 10 μ M, the spots in the first and third rows of images are all red fluorescence spots, the spots in the second and fourth rows of images are all green fluorescence spots, and B in FIG. 9 is the detection result of AO-red spots of each experimental group.
FIG. 10 shows the synthetic route for compound II-1 (Gefi-2 OH).
FIG. 11 shows the synthetic route for compound III-1.
FIG. 12 shows the synthesis route of compound IV-1 (AZD-2 OH).
FIG. 13 shows the results of the test in example 13. Wherein, A in figure 13 is the result of measuring the expression level of SQSTM1 and mature CTSB after Gefi-2OH treatment of gefitinib and compound in H460 cells, and B in figure 13 is the result of measuring the phosphorylation modification degree of EGFR and downstream Akt, Erk1/2 protein and the expression level of the protein itself after Gefi-2OH treatment of gefitinib and compound in A549 cells and HCC827 cells.
FIG. 14 shows the same results of the measurement in example 13. Wherein, A in figure 14 is the result of measuring the expression of SQSTM1 and mature CTSB after AZD9291 and AZD-2OH compound treatment in H460 cells, and B in figure 14 is the result of measuring the phosphorylation modification degree of EGFR and downstream Akt, Erk1/2 protein and the expression of the protein itself after AZD9291 and AZD-2OH compound treatment in HCC827 cells.
FIG. 15 shows the results of the test in example 14. In the figure, the left side shows the photograph of fluorescence microscopy, wherein the scale bar is 10 μ M, the spots in the picture are all blue fluorescence spots formed by Lysotracker-blue, and the right side shows the detection results of the acid vesicle spots (number of blue spots) of each experimental group.
FIG. 16 is a graph showing the results of the assay in example 15, which is the results of the inhibition of proliferation of A549 (left) and H460 (right) cells by gefitinib and the compound Gefi-2OH, respectively.
FIG. 17 shows the results of the same assay as that of example 15, which is the results of the inhibition of the proliferation of H460 cells by AZD9291 and AZD-2OH compound.
FIG. 18 shows the results of the test in example 16; the left side of the graph is the detection result of flow cytometry, and the right side of the graph is the result of apoptosis rate of each experimental group.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. In describing and claiming the present invention, the following terminology will be used.
"halogen" means fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.
"alkyl" means a hydrocarbon chain radical that is straight or branched and free of unsaturation, typically an alkyl group containing 1 to 12, 1 to 8, or 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, and the like. If an alkyl group is substituted with an aryl group, it is correspondingly an "aralkyl" radical, such as benzyl or phenethyl. If an alkyl group is substituted with a heterocyclyl group, it is correspondingly a "heterocycloalkyl" radical.
"alkenyl" refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms, at least one unsaturated bond, and which is attached to the rest of the molecule by a single bond. Typical alkenyl groups contain 2-12, 2-8, or 2-6 carbon atoms, for example, ethenyl, 1-propenyl, or butenyl.
"alkynyl" refers to a straight or branched hydrocarbon chain radical containing at least two carbon atoms, at least one carbon-carbon triple bond, and which is attached to the rest of the molecule by a single bond. Typical alkynyl groups contain 2-12, 2-8 or 2-6 carbon atoms, e.g., ethynyl, propynyl (e.g., 1-propynyl, 2-propynyl).
"cycloalkyl" refers to alicyclic hydrocarbons. Typical cycloalkyl groups contain 1 to 3 single and/or fused rings, 3 to 18 carbon atoms, preferably 3 to 10 carbon atoms, such as cyclopropyl, cyclohexyl or adamantyl. In a particular embodiment, the cycloalkyl group contains 3 to 6 carbon atoms.
"aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing monoaryl groups and/or fused aryl groups. Typical aryl groups contain 1-3 single or fused rings and 6-18 carbon ring atoms, such as phenyl, naphthyl (e.g., 2-naphthyl) radicals.
"heterocyclyl" refers to a stable, typically 3-to 18-membered ring radical consisting of carbon atoms and one to five heteroatoms selected from nitrogen, oxygen and sulfur, preferably a 4-to 8-membered ring with one or more heteroatoms, more preferably a 5-or 6-membered ring with one or more heteroatoms, which may be aromatic or non-aromatic. For the purposes of the present invention, a heterocycle may be a monocyclic, bicyclic or tricyclic ring system, which may include fused ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclic radical may optionally be oxidized; the nitrogen atom may optionally be quaternized; and the heterocyclic radical may be partially or fully saturated or aromatic. Examples of heterocyclyl groups include, but are not limited to: benzimidazole, benzothiazole, furan, isothiazole, imidazole, indole, piperidine, piperazine, purine, quinoline, thiadiazole, tetrahydrofuran, coumarin, morpholine, pyrrole, pyrazole, oxazole, isoxazole, triazole, imidazole, and the like.
"alkoxy" means a group of the formula-ORaWherein R isaIs an alkyl radical as defined above, having one or more (e.g., 1, 2,3, or 4) oxygen linkages, and typically containing from 1 to 12, 1 to 8, or 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, and the like.
"aryloxy" refers to a radical of formula-O-aryl, wherein aryl is as defined above. Some examples of aryloxy compounds are-O-phenyl, -O-p-tolyl, -O-m-tolyl, -O-O-tolyl, or-O-naphthyl.
Unless specifically stated in the present invention, all groups may be optionally substituted, if applicable.
The invention will be described in detail below with reference to the following examples and the accompanying drawings.
The compounds listed here are only for better illustration of the class of compounds and structural forms of the invention and do not limit the invention.
The invention is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Since the synthesis of the compounds of each of the general formulae (II) to (IV) employs the same method and differs only in the reaction raw materials, examples 10 to 12 are represented by only compounds of the general formulae II-1, III-1 and IV-1, and the synthesis of the compounds of the general formulae (II) to (IV) is summarized.
Some experimental materials and equipment information used in the experiment are as follows:
a549 (wild-type EGFR), HCC827 (exon 19 deleted EGFR) and NCI-H460(H460, wild-type EGFR), purchased from the chinese academy of sciences cell bank (shanghai, china);
RPMI1640 medium, DMEM medium and Fetal Bovine Serum (FBS) were purchased from Hangzhou Sijiqing Co., Ltd. (Hangzhou, China);
PBS buffer, EBSS buffer, 0.25% trypsin with 0.02% EDTA, and Opti-MEM medium (Ginno Biotechnology technologies, Inc., China);
dimethylsulfoxide DMSO (AMRESCO, usa);
cell culture dishes and plates (Corning Costar, usa);
gefitinib, ocitinib (AZD9291), bafilomycin a1(Baf a1), available from Selleck (shanghai, china);
rapamycin (Rapamycin), purchased from Beyotime (shanghai, china);
chloroquine (CQ), acridine orange dye (AO) purchased from Sigma Aldrich (shanghai, china);
BCA protein assay kit, purchased from Bio-Rad Laboratories (Calif., USA);
horseradish peroxidase conjugated secondary antibody, purchased from Jackson ImmunoResearch (shanghai, china);
chemiluminescence detection kit, available from Biological Industries (U.S.);
the Amersham Imager 600 System, available from the department of Life sciences of the general electric medical group (Shanghai, China);
siRNA transfection reagent: lipofectamineTMRNAiMAX (Invivogen, usa);
plasmid transfection reagent: x-tremagene HP DNA transformation Reagent (Roche, Switzerland);
CellTiter
Figure GDA0003576518700000121
AQueous nonradioactive cell proliferation assay kit purchased from Promega (Beijing, China);
BD Pharmingen FITC Annexin V apoptosis detection kit, purchased from BD Biosciences (shanghai, china);
BD FACSCalibur (TM) flow cytometer, purchased from BD Biosciences (Shanghai, China).
The inventors previously performed the following experiments:
in the following step of the cell biology experiment, "treatment" means that a test compound is diluted to a specific concentration with a cell culture solution, and then added to a culture dish to be incubated with cells for a specific time.
Example 1
Non-small cell lung carcinoma cells (NSCLC) were treated with gefitinib at the indicated concentrations (shown in fig. 1) for 24 hours (for a549 and H460 cells) or 36 hours (for HCC827 cells), and expression of SQSTM1 was determined by Western blotting (Western Blot). Actin was used as an internal reference protein. The results of the experiment are shown in FIG. 1.
A549, HCC827 and H460 cells were treated with AZD9291 at the indicated concentrations (as shown in FIG. 2) for 24 hours and expression of SQSTM1 was determined by Western Blot. Actin was used as an internal reference protein. The results of the experiment are shown in FIG. 2.
Western Blot operation:
the cells were lysed with lysis buffer containing 2% SDS, 0.1% bromophenol blue, 10% glycerol, 1.5% DTT (dithiothreitol) and 0.1M tris-HCl (pH 6.8). Protein concentration in cell lysates was determined by BCA method protein quantification kit. The boiled denatured lysates were separated by SDS-PAGE, transferred to polyvinylidene fluoride membranes and blocked with 5% nonfat milk (dissolved in TBST). The membranes were then incubated with the indicated primary antibodies overnight at 4 ℃, washed 3 times with TBST (TBS containing 0.1% Tween-20), and then incubated with horseradish peroxidase (HRP) -conjugated secondary antibodies for 2 hours at room temperature. TBST wash 3 times. Finally, the level of HRP on the membrane was detected by a chemiluminescence kit in the Amersham Imager 600 system to characterize the content of the corresponding antigen.
From the results in fig. 1 and 2, gefitinib and AZD9291 all up-regulated SQSTM1 protein levels in a dose-dependent manner in three NSCLC cells with different genetic backgrounds (a549, HCC827 and H460).
Example 2
A549 and H460 cells were treated with DMSO, rapamycin, gefitinib for 7 hours, and then cultured for 5 hours with or without Baf A1 (bafilomycin A1). LC3 and SQSTM1 expression were detected by Western Blot (see Western Blot procedure in example 1). The results of the experiment are shown in FIG. 3.
From the results in fig. 3, it can be seen that gefitinib alone acts similarly to the autophagy inhibitor Baf a1, in contrast to the autophagy inducer rapamycin; the protein levels of LC3-II and SQSTM1 were significantly increased after the combination of rapamycin and BafA1, whereas gefitinib and BafA1 did not show significant enhancement. The above experimental results indicate that gefitinib is likely to block the autophagic degradation of SQSTM1 by inhibiting autophagy, resulting in its accumulation in cells.
Example 3
A549 cells were transfected with the GFP-RFP-LC3 plasmid for 48 hours and then treated with EBSS, CQ, gefitinib or AZD9291, respectively. Spots of GFP-LC3 and RFP-LC3 were detected by immunofluorescence microscopy and the results are shown in FIG. 4, A.
Quantification of LC3 yellow spot/red spot (%) is expressed as mean ± SD of 3 independent experiments, n ═ 10 cells. The statistical results are shown in B in fig. 4, where the asterisks (—) indicate statistical differences (student t-test, P < 0.05).
From the results of examples 2 and 3, it can be seen that gefitinib and AZD9291 inhibit autophagy similarly to known autophagy inhibitors bafosfomycin a1(Baf a1) and Chloroquine (CQ), and although up-regulated on the expression of LC3 ii (as shown in fig. 3), negatively regulated the progress of autophagy efflux in cells (as shown in fig. 4).
Example 4
High expression of SQSTM-1 gene: cells in logarithmic growth phase were seeded into corresponding well plates and cultured overnight to allow cells to adhere. Before transfection, the culture solution is replaced by a corresponding culture medium without antibody and containing 10% fetal calf serum, an appropriate volume of Opti-MEM culture medium is taken and put into an EP tube, an appropriate amount of plasmid and a transfection reagent X-trememe GENE HD are added, and the mixture is evenly blown and stirred and then is kept stand for 20-25min at room temperature. The mixture was added to the cell culture medium uniformly in the form of drops and gently shaken to mix them well. Transfection was carried out for 48-72h as required for the experiment. And evaluating the over-expression effect by using a Western blot method.
Knocking-down of SQSTM-1 gene: cells in logarithmic growth phase were seeded into corresponding well plates and cultured overnight to allow cells to adhere (different coverage was required for different cells to be transfected). Before transfection, the culture solution was replaced with a corresponding medium containing no 10% fetal bovine serum, an appropriate amount of Opti-MEM was cultured in an EP tube, an appropriate amount of Lipofectamine RNAIMAMAX transfection reagent was added according to the protocol, mixing was performed, an appropriate amount of siRNA was added to an equal volume of Opti-MEM medium, and mixing was performed (siRNA and negative control siRNA were synthesized by Shanghai Jima pharmaceutical technology Co., Ltd.). Mixing the two tubes of liquid, blowing and stirring uniformly, and standing at room temperature for about 15 min. The mixture was added to the cell culture medium uniformly in the form of drops and gently shaken to mix them well. Transfection was carried out for 48-72h as required for the experiment. And the knockdown effect was evaluated by Western blot.
The results of the experiment are shown in FIG. 5. From the results in fig. 5, it was demonstrated that high expression of SQSTM1 resulted in cell tolerance to gefitinib, AZD9291, while knock-down of SQSTM1 increased cell sensitivity to both.
From the experimental results of examples 1 to 4, the inventors concluded that EGFR TKIs such as gefitinib, AZD9291, etc. promote the cells to develop acquired resistance to EGFR TKIs by inhibiting autophagic degradation of the cells, leading to accumulation of SQSTM 1.
Example 5
Immunofluorescence experiment steps: the sterilized coverslips were placed in a well plate, seeded at cell density from conventional 1/5 to 1/3, incubated overnight, and removed from the incubator after a specified time of drug addition. 1 XPBS washing, then washing with anhydrous methanol 1 times, room temperature fixation for 10 minutes. PBS wash 3 times. Permeabilization was then performed with 0.25% Triton X-100 for 25 minutes. PBS was washed 3 more times. Blocking was performed for 30 min at room temperature by adding 3% BSA (1 XPBS). The diluted primary antibody (diluted with 3% BSA) was added and placed in a wet box overnight at 4 ℃. The next day, after washing 3 times with PBS, diluted fluorescent secondary antibody (diluted with 3% BSA, 1: 200) was added and incubated for 1 hour at room temperature, protected from light. PBS was washed 3 more times. The specimen was mounted with a mounting agent containing DAPI, and after mounting, the specimen was allowed to stand at room temperature for 1 hour, and the fluorescence intensities of DAPI and LC3 were measured under a fluorescence microscope. The results of the experiment are shown in FIG. 6.
It is known that autophagy can be divided into 3 major processes, namely autophagosome formation, fusion of autophagosome and lysosome, degradation of autophagosomes.
As can be seen from the results in fig. 6, gefitinib and AZD9291 did not inhibit formation of autophagosomes, but rather promoted formation of autophagosomes, using fluorescence from LC3 ii expressed in cells as a detection signal.
Example 6
By taking the co-localization of the lysosomal membrane proteins LAMP1 and LC3 II as evaluation indexes, treating A549 cells with rapamycin or gefitinib for 24 hours, and detecting the co-localization of LAMP1 and LC3 through immunofluorescence, the experimental result is shown as A in FIG. 7.
The co-localization coefficient (Colocalization coeffient) is expressed as the ratio of the co-localization points of LC3 and LAMP1 to the total LC3 points. The co-localization coefficients were quantified as mean ± SD of 3 independent experiments, n ═ 10 cells. The statistical results are shown in B in fig. 7, where NS indicates no significance (student t-test, P > 0.05).
From the results in fig. 7, it is clear that gefitinib did not affect the fusion of autophagosome with lysosome.
From examples 5 and 6, the inventors believe that EGFR-TKI such as gefitinib, AZD9291, etc. does not affect autophagosome formation (rather, it acts as a promoter, with the expression level of LC3 ii as a marker, as shown in fig. 6) and fusion of autophagosome to lysosome (with the co-localization of lysosomal membrane proteins LAMP1 and LC3 ii as an evaluation index, as shown in fig. 7), but rather, it may inhibit lysosomal function.
Example 7
CTSB is a characteristic hydrolase of lysosomes, and it needs to be cleaved into mature bodies in the acidic environment of lysosomes to exert the function of hydrolyzing proteins. Therefore, many studies have used the expression level of CTSB mature bodies as an index of the strength of lysosomal function.
A549 and HCC827 cells were treated with gefitinib, AZD9291, CQ or Baf a1, respectively, for 24 hours, and expression of CTSB mature bodies was evaluated by Western Blot (refer to the Western Blot procedure in example 1) with actin as a control. The results of the experiment are shown in FIG. 8.
As can be seen from the results of fig. 8, gefitinib and AZD9291 significantly down-regulated the expression of the CTSB mature body, thereby indicating that these two compounds do have the effect of weakening the function of the lysosome.
Example 8
Acridine orange dye (AO) is a protonophilic fluorescent dye, and an aprotic monomer of the dye can emit green fluorescence, and the dye enters a lysosome to be protonated to form a polymer and emit red fluorescence.
A549 and HCC827 cells were treated with DMSO, gefitinib, AZD9291, CQ, or BafA1, respectively, for 12 hours, and then a549 and HCC827 cells were incubated with AO for 15 minutes. The number of AO-red fluorescent spots was examined by fluorescence microscopy. The experimental results are shown as a in fig. 9.
Quantification of AO-red fluorescent spots per cell is expressed as mean ± SD of 3 independent experiments, n ═ 10 cells. The statistical results are shown in fig. 9B, where asterisks (@) indicate statistical differences (student t-test, P <0.05) and ND indicates no detection.
From the results in fig. 9, gefitinib and AZD9291 both inhibited the acidification process of lysosomes.
From the experimental results of examples 4-8, the inventors speculate that EGFR TKI such as gefitinib, AZD9291, etc. causes the function of autophagosomes to be weakened and the degradation of the substrate encapsulated therein to be hindered by inhibiting lysosome acidification.
Example 9
Acid-base analysis of the chemical structure of representative EGFR TKI: the 8 representative drugs most commonly used in the clinic at present were selected from the first to third generation EGFR TKIs, and their physicochemical properties were retrieved in a free-source drug database (https:// www.drugbank.ca /). The number of nitrogen atoms in the chemical structures of 8 representative EGFR TKI is three or more, and Lewis acid acidic groups without electron deficiency belong to Lewis base basic drugs. Among them, gefitinib, afatinib, dacomitinib (dacomitinib), ocitinib, omalitinib (Olmutinib), etc. all have strong basic fatty amine groups, and the basic pKa values thereof are shown in table 1 below.
TABLE 1 basic pKa values for representative EGFR TKIs
Figure GDA0003576518700000141
Figure GDA0003576518700000151
According to the above experimental results, the inventor concludes that the root cause of inhibition of lysosome acidification by EGFR TKI such as gefitinib, AZD9291 and the like and further inhibition of autophagosomal degradation may be that since EGFR TKI such as gefitinib, AZD9291 and the like has a functional group with strong basicity in a chemical structure, the EGFR TKI can easily enter lysosomes in an acidic microenvironment, and by reducing acidification of the lysosomes, further functional blockade on autophagososomes and accumulation of oncogenic protein sq 1 are caused, and finally, tumor cells generate drug resistance to EGFR TKI such as gefitinib, AZD9291 and the like. On the basis, the EGFR TKI structurally containing a strong basic group is subjected to basic neutralization modification by the inventor to obtain a series of EGFR TKI basic neutralization analogs, such as the compounds synthesized in the following examples 10-12.
EXAMPLE 10 Synthesis of Compound II-1 of general formula (the synthetic route is shown in FIG. 10)
Step I, 3-bromopropylPreparation of 3, 5-Dimethoxybenzoate (intermediate 1-3): 3, 5-Dimethoxybenzoyl chloride (intermediate 1-1) (2g, 10mmol) and 3-bromo-1-propanol (intermediate 1-2) (1.07mL, 5mmol) were weighed, dissolved in 20mL dichloromethane, stirred until dissolved, triethylamine (1.5mL, 15mmol) was added slowly dropwise, stirred at room temperature, TLC monitored the progress of the reaction, and after about 5 hours the reaction was complete. After adding 20mL of ultrapure water to quench the reaction, 20mL of dichloromethane was added to extract the reaction mixture, the mixture was washed with saturated NaCl solution 3 times, and the dichloromethane layer was separated with anhydrous MgSO4Drying, filtering, rotary evaporating to remove, concentrating to obtain crude product, purifying by silica gel column chromatography to obtain colorless oily liquid intermediate 1-3, yield: 80 percent.
Step II, preparation of 3-bromopropyl-3, 5-dihydroxybenzoate (intermediate 1-4): intermediate 1 to 3(604mg, 2mmol) was weighed out and dissolved in 20mL of dichloromethane, and aluminum trichloride (1333mg, 10mmol, three times) and sodium iodide (150mg, 1mmol) were added successively with stirring and reacted at room temperature overnight. After the reaction was completed, 20mL of saturated ammonium chloride solution was added to neutralize the reaction, 20mL of dichloromethane was added to extract the reaction solution, and the dichloromethane layer was washed 3 times with saturated NaCl solution and with anhydrous MgSO4And (5) drying. After concentrating the filtrate, purification by flash silica gel column chromatography gave the intermediates 1-4 as a pale yellow oily liquid, in yield: 30 percent.1H-NMR(CDCl3,600MHz)δ:7.087(d,J＝2.4Hz,2H, H-2,H-6),6.572(t,J＝2.4Hz,1H,H-4),5.486(s,2H,-OH×2),4.449(t,J＝6.0Hz,2H,-OCH 2CH2CH2Br), 3.676(t,J＝6.6Hz,2H,-OCH2CH2CH 2Br),2.212(t,J＝6.0Hz,2H,-OCH2CH 2CH2Br).
Step III, preparation of 3-bromopropyl-3, 5-bistetrahydropyranopyranyl benzoate (intermediate 1-6): intermediates 1 to 4(550mg, 2mmol), dihydropyran (intermediates 1 to 5) (841.2mg, 10mmol) and pyridinium p-toluenesulfonate (502mg, 0.2mmol) were weighed out, dissolved in 20mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction was complete, 20mL of water and 20mL of dichloromethane were added, the dichloromethane extracted and spun dry. The intermediate 1-6 is obtained after silica gel column chromatography separation and purification, and the yield is as follows: and 63 percent.1H-NMR(CDCl3,500MHz)δ:7.348(t,J＝ 2.5Hz,2H,H-2,H-6),6.984(q,J＝2.5Hz,1H,H-4),5.435-5.463(m,2H,-O-pyranyl-H-2×2),4.447(t,J＝6.0 Hz,2H,-OCH 2CH2CH2Br),3.886(td,J1＝2.5Hz,J2＝10.5Hz,2H,-O-pyranyl-H-6×2),3.688(t,J＝6.5Hz,2H, -OCH2CH2CH 2Br),3.596-3.632(m,2H,-O-pyranyl-H-6×2),2.196-2.246(m,2H,-OCH2CH 2CH2Br), 1.958-2.016(m,2H,-O-pyranyl-H-3×2),1.844-1.894(m,4H,-O-pyranyl-H-3×2,-O-pyranyl-H-4×2), 1.644-1.712(m,4H,-O-pyranyl-H-4×2,-O-pyranyl-H-5×2),1.584-1.613(m,2H,-O-pyranyl-H-5×2).
Step IV, preparation of 4- ((3-chloro-4-fluorophenyl) amino) -6- (3-morpholinopropoxy) quinazolin-7-ol (intermediates 1-8): the bulk drugs gefitinib (intermediate 1-7) (89.4mg, 0.2mmol) and L-methionine (49.23mg, 0.33mmol) were weighed out and dissolved in 5mL of methanesulfonic acid and reacted at 165 ℃ under reflux for about 24 hours. After completion of the reaction, the reaction was quenched by adding 5mL of ultrapure water and allowed to cool at room temperature. And gradually dropwise adding 40% NaOH aqueous solution until the pH value of the system is about 7, adding 20mL ethyl acetate for extraction, spin-drying the solvent, and separating and purifying by silica gel column chromatography to obtain white solid powder intermediates 1-8, wherein the yield is as follows: 52 percent.1H-NMR(CD3OD,400MHz)δ:8.367(s,1H,H-2), 7.964(dd,J1＝2.8Hz,J2＝6.8Hz,1H,H-8),7.762(s,1H,H-5),7.616-7.656(m,1H,H-2’),7.230(t,J＝8.8Hz, 1H,H-6’),7.033(s,1H,H-5’),4.267(t,J＝6.0Hz,2H,-NCH2CH2CH 2O-),3.783(t,J＝4.8Hz,4H,-OCH2×2), 2.871(t,J＝7.2Hz,2H,-NCH 2CH2CH2O-),2.784(s,4H,-NCH2×2),2.145-2.208(m,2H,-NCH2CH 2CH2O-). MS(ESI)m/z:432.97(M+1)+,calculated for C21H22ClFN4O3:432.14.
The steps V-VI, 3- ((4- ((3-chloro-4-fluorophenyl) amino) -6- (3-morpholinopropoxy) quinazolin-7-yl) oxy) propyl-3, 5-dihydroxybenzoate (compound of general formula II)-1, Gefi-2 OH): intermediate 1 to 8(43.2mg, 0.1mmol) and intermediate 1 to 6(88.4mg, 0.2mmol) were weighed, dissolved in 5mL of N, N-dimethylformamide, and after addition of potassium carbonate (55.2mg, 0.4mmol) and sodium iodide (15mg, 0.1mmol), reacted at 80 ℃ overnight. After completion of the reaction, 5mL of ultrapure water and 20mL of ethyl acetate were added for extraction, and then the solvent was spin-dried. Purifying by column chromatography to obtain colorless oily liquid. The oil was redissolved in 5mL of absolute ethanol, 1mL of 10% HCl solution was added, and the reaction was stirred at room temperature for 2 h. After the absolute ethanol in the system was dried by spinning, 4mL of ultrapure water and 10mL of ethyl acetate were added to conduct extraction. After ethyl acetate is dried by spinning, a white powdery target product Gefi-2OH is obtained by separation and purification through a flash column chromatography, and the yield is as follows: 30 percent.1H-NMR(DMSO-d6,600MHz)δ:8.471(s,1H,H-2),8.116(d,J ＝4.8Hz,1H,H-8),7.849(s,1H,H-5),7.785(s,1H,H-2’),7.410(t,J＝9.0Hz,1H,H-6’),7.222(s,1H, H-5’),6.803(d,J＝2.4Hz,2H,H-2”,H-6”),6.410(t,J＝2.4Hz,1H,H-4”),4.400(t,J＝6.0Hz,2H, O＝COCH 2),4.280(t,J＝6.0Hz,2H,O＝COCH2CH2CH 2),4.177(s,2H,-NCH2CH2CH 2O-),3.556(s,4H, -OCH2×2),2.475(t,J＝1.8Hz,2H,-NCH 2CH2CH2O-),2.359(s,4H,-NCH2×2),2.218(m,J＝6.0Hz, 2H,O＝COCH2CH 2CH2),1.983(m,J＝7.8Hz,2H,-NCH2CH 2CH2O-).MALDI-TOF MS m/z:627.475 (M+1)+,calculated for C31H32ClFN4O7:626.19.
EXAMPLE 11 Synthesis of Compound III-1 of general formula (the synthetic route is shown in FIG. 11)
Step I, preparation of 4-fluoro-2- ((tetrahydro-2H-pyran-2-yl) oxy) aniline (intermediate 2-2): 2-amino-5-fluorophenol (intermediate 2-1) (1.27g, 10mmol), dihydropyran (2.52g, 30mmol) and pyridinium p-toluenesulfonate (502mg, 1mmol) were dissolved in 20mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction was complete, 20mL of water and 20mL of dichloromethane were added, the dichloromethane extracted and spun dry. Silica gel columnThe intermediate 2-2 is obtained after chromatography separation and purification, and the yield is as follows: 77 percent. MS (ESI) M/z 212.10(M +1)+,calculated for C11H14FNO2:211.10.
Step II, preparation of 4-methylpiperazino-2- ((tetrahydro-2H-pyran-2-yl) oxy) aniline (intermediate 2-3): intermediate 2-2(1.05g, 5mmol) and N-methylpiperazine (1.36mL, 15mmol) were weighed out and dissolved in 20mL 2-pentanol and reacted at 120 ℃ for 3 hours. After the reaction is completed, the solvent is dried by spinning, ethyl acetate is added for extraction, and after concentration, the red powdery intermediate 2-3 is obtained after separation and purification by silica gel column chromatography, and the yield is as follows: 71 percent. MS (ESI) M/z 292.30(M +1)+,calculated for C16H25N3O2:291.19.
Step III, 2-chlorothiophene [3,2-d]Preparation of pyrimidin-4 (3H) -one (intermediate 2-5): weighing 2, 4-chlorothiophene [3,2-d ]]Pyrimidine (intermediate 2-4) (1.025g, 5mmol) was dissolved in 10mL of a mixed solvent of ethanol/water 4:1, and 500mg of sodium hydroxide was further dissolved in 3mL of distilled water, and the solution was added dropwise to the reaction system and reacted at 40 ℃ for 4 hours. After the reaction is completed, adding 1mL of acetic acid, continuing the reaction for 2 hours at 35 ℃, filtering the obtained precipitate, washing the precipitate with distilled water to obtain a white powdery intermediate 2-5, wherein the yield is as follows: 89 percent. MS (ESI) M/z 187.96(M +2)+,calculated for C6H3ClN2OS:185.97.
Step IV, 2- ((4- (4-methylpiperazin-1-yl) -2- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) thiophene [3,2-d]Preparation of pyrimidin-4 (3H) -one (intermediates 2-6): intermediate 2-5(930mg, 5mmol) was weighed out and dissolved in 50mL of ethanol, and intermediate 2-3(1.75g, 6mmol) and 5mL of acetic acid were added and reacted under reflux for 6 hours. After the reaction is completed, cooling the reactant to room temperature, slowly dropwise adding 6mL of triethylamine into the reactant, then performing suction filtration, washing for 2 times by using 20mL of ethanol, and performing vacuum drying to obtain a yellow solid powdery intermediate 2-6, wherein the yield is as follows: and 69 percent. MS (ESI) M/z 442.30(M +1)+,calculated for C22H27N5O3S:441.18.
Step V, 4-chloro-N- (4- (4-methylpiperazin-1-yl) -2- ((tetrahydro-2H-pyran-2-yl) oxy)) Phenyl) thiophene [3,2-d]Preparation of pyrimidin-2-amine (intermediate 2-7): intermediate 2-6(1.76g, 4mmol) was weighed into 5mL acetonitrile and the reaction temperature was raised to 75 ℃. A mixed solution of 1mL of phosphorus oxychloride and 1mL of acetonitrile was added and the reaction was carried out at 75 ℃ for 1 hour. After the reaction is completed, 10mL of ice water and 40% of sodium hydroxide solution are respectively added, stirred for 1 hour at room temperature, filtered, washed for 2 times by 20mL of distilled water, and dried to obtain a crude product. Then, the crude product was redissolved in 20mL of a mixed solvent of dichloromethane and methanol at a volume ratio of 3:2, stirred at room temperature for 1 hour, and then the solvent was spin-dried. And dissolving the product in 10mL of acetonitrile/water mixed solvent with the volume ratio of 8:2 again, stirring for 2 hours at room temperature, and performing vacuum filtration to obtain a white solid powdery intermediate 2-7 with the yield: 77 percent. MS (ESI) M/z 461.20(M +2)+,calculated for C22H26ClN5O2S:459.15.
Step VI, N- (3- ((2- ((4- (4-methylpiperazin-1-yl) -2- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) thieno [3, 2-d)]Preparation of pyrimidin-4-yl) oxy) phenyl) acrylamide (intermediates 2-8): intermediate 2-7(1.76g, 4mmol), N- (3-hydroxyphenyl) acrylamide (815mg, 5mmol) and potassium carbonate (1.66g, 12mmol) were weighed out and dissolved in 25mL of an acetonitrile/water mixed solvent at a volume ratio of 8:2, followed by reflux reaction for 6 hours. After completion of the reaction, the reaction mixture was cooled to room temperature, 50mL of distilled water was added, and the reaction was allowed to proceed at room temperature overnight. And (3) carrying out suction filtration on the reaction solution, washing for 1 time by using 10mL of acetonitrile/water mixed solvent with the volume ratio of 1:2 to obtain a white solid powder intermediate 2-8, wherein the yield is as follows: 84 percent. MS (ESI) M/z 587.30(M +1)+,calculated for C31H34N6O4S:586.24.
Step VII-VIII, N- (3- ((2- ((4- (4-methylpiperazin-1-yl) -2- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) amino) thieno [3,2-d]Preparation of pyrimidin-4-yl) oxy) phenyl) acrylamide (compound of general formula III-1): referring to the procedures of step V and step VI in example 10, intermediates 2-8 and 2-6 were reacted to give the objective compound III-1 as a white solid powder in yield: 52 percent. MS (ESI) M/z 697.30(M +1)+,calculated for C36H36N6O7S:696.24.
EXAMPLE 12 Synthesis of Compound IV-1 of the general formula (the synthetic route is shown in FIG. 12)
Step I, preparation of 3- (2-chloropyrimidin-4-yl) -1-methyl-1H-indole (intermediate 3-3): after suspending 2, 4-dichloropyrimidine (intermediate 3-2) (1.49g, 10mmol) and aluminum chloride (1.33g, 10mmol) in 20mL of N, N-Dimethylacetamide (DME) and stirring at room temperature for 5 minutes, 1-methylindole (intermediate 3-1) (1.25mL, 10mmol) was added to the reaction system, and the reaction was carried out at 80 ℃ for 2 hours, and the progress of the reaction was monitored by TLC. Within 5 minutes, 50mL of ultrapure water is slowly dropped into the reaction system to quench the reaction, the reaction system is stirred for 30 minutes, the obtained solid is filtered under reduced pressure, and the intermediate 3-3 is obtained by flash silica gel column chromatography purification, wherein the yield is 93%. MS (ESI) M/z 244.10(M +1)+,calculated for C13H10ClN3:243.06.
Step II, preparation of 2-amino-4-nitro-5-fluorophenol (intermediate 3-5): 4-fluoro-2-methoxy-5-nitroaniline (intermediate 3-4) (372mg, 2mmol) was weighed and dissolved in 20mL of dichloromethane, and aluminum trichloride (1333mg, 10mmol, three times) and sodium iodide (150mg, 1mmol) were added successively with stirring and reacted at room temperature overnight. After the reaction was completed, 20mL of saturated ammonium chloride solution was added to neutralize the reaction, 20mL of dichloromethane was added to extract the reaction solution, and the dichloromethane layer was washed 3 times with saturated NaCl solution and with anhydrous MgSO4And (5) drying. After concentrating the filtrate, purification by flash silica gel column chromatography gave the intermediate 3-5 as a pale yellow oily liquid, yield: 37 percent. MS (ESI) M/z 173.10(M +1)+, calculated for C6H5FN2O3:172.03.
Step III, preparation of 4-fluoro-5-nitro-2- ((tetrahydro-2H-pyran-2-yl) oxy) aniline (intermediate 3-6): intermediate 3-5(860mg, 5mmol), dihydropyran (1680mg, 20mmol) and pyridinium p-toluenesulfonate (125.5mg, 0.5mmol) were weighed out and dissolved in 20mL of dichloromethane and reacted at room temperature for 6 hours. After the reaction was complete, 20mL of water and 20mL of dichloromethane were added, the dichloromethane was extracted and spun dry. The intermediate 3-6 is obtained after the separation and purification of silica gel column chromatography, and the yield is as follows: 70 percent. MS (ESI) M/z 257.13(M +1)+,calculated for C11H13FN2O4:256.09.
Step IV, preparation of N- (4-fluoro-5-nitro-2- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) -4- (1-methyl-1H-indol-3-yl) pyrimidin-2-amine (intermediate 3-7): intermediate 3-6(1280mg, 5mmol) and intermediate 3-3(1215mg, 5mmol) were weighed out and dissolved in 20mL 2-pentanol, p-toluenesulphonic acid monohydrate (1140mg, 6mmol) was added, and the reaction was carried out at 105 ℃ for 3 hours. After the reaction was complete the reaction was cooled to room temperature, filtered with suction and washed 2 times with 10mL 2-pentanol. Washed 1 more times with 5mL acetonitrile and dried in vacuo to afford intermediates 3-7 as yellow solid powders in yields: 95 percent. MS (ESI) M/z 464.30(M +1)+,calculated for C24H22FN5O4:463.17.
Step V, 4-fluoro-N1Preparation of- (4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) -6- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl-1, 3-diamine (intermediate 3-8): intermediate 3-7(927mg, 2mmol), iron powder (560mg, 10mmol) and ammonium chloride (80.25mg, 1.5mmol) were weighed and dissolved in 16mL of a mixed solvent of ethanol/water (3: 1) and reacted at 78 ℃ for 2 hours under reflux. After the reaction is completed, the solution is preliminarily purified by a sulfonic ion exchange column, and then separated and purified by a rapid silica gel column chromatography to obtain a light brown oily intermediate 3-8, wherein the yield is as follows: 82 percent. MS (ESI) M/z 434.30(M +1)+,calculated for C24H24FN5O2:433.19.
Step VI, preparation of N- (2-fluoro-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) -4- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) acrylamide (intermediate 3-9): intermediate 3-8(867mg, 2mmol) and N, N-diisopropylethylamine (310mg, 2.4mmol) were weighed, dissolved in 10mL of dichloromethane, and acryloyl chloride (181mg, 2mmol) was slowly added dropwise to the reaction solution, followed by reaction in an ice-water bath for about 2 hours. After the reaction is completed, 50mL of dichloromethane and 50mL of saturated sodium bicarbonate are respectively added, extraction is carried out for 3 times, and a white powder intermediate 3-9 is obtained after separation and purification by silica gel column chromatography, wherein the yield is as follows: 32 percent. MS (ESI) M/z 488.30(M +1)+,calculated for C27H26FN5O3:487.20.
Preparation of step vii-viii, N- (2-fluoro-5- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) -4- ((tetrahydro-2H-pyran-2-yl) oxy) phenyl) acrylamide (intermediate 3-10): referring to the procedure of step V and step VI in example 10, intermediates 3-9 and intermediates 1-6 were reacted to give intermediates 3-10 as white powders in yields: and 63 percent. MS (ESI) M/z 598.30(M +1)+,calculated for C32H28FN5O6:597.20.
Step IX, preparation of 3- (4-acrylamido-5- ((2- (dimethylamino) ethyl) (methyl) amino) -2- ((4- (1-methyl-1H-indol-3-yl) pyrimidin-2-yl) amino) phenoxy) propyl-3, 5-dihydroxybenzoate (compound of general formula IV-1, AZD-2 OH): intermediate 3-10(597.6mg, 1mmol) and N, N-diisopropylethylamine (306mg, 3mmol) were weighed, dissolved in 10mL of 2-pentanol, and reacted at 120 ℃ for about 3 hours. After the reaction is completed, the solvent is dried by spinning, 10mL of ethyl acetate and 10mL of saturated sodium sulfate are respectively added, extraction is carried out for 3 times, and a white solid powder target compound IV-1 is obtained by separation through a rapid silica gel column chromatography, wherein the yield is as follows: and 72 percent. MS (ESI) M/z 680.30 (M +1)+,calculated for C37H41N7O6:679.31.
Example 13 immunoblotting to detect the Effect of Gefi-2OH Compound on the expression level of p62, CTSB, and other proteins in cells
A549 cells and H460 cells were cultured at a cell size of 1X 105Hole sum 0.8X 105The density per well was determined by inoculating the cells in a 6-well plate, overnight culturing, diluting gefitinib and Gefi-2OH compound in complete medium to specific concentrations, adding the diluted solutions to the 6-well plate, incubating for 24 hours, adding lysis buffer to lyse the cells, boiling the cell lysate to denature the cells, and performing Western Blot assay (see Western Blot method in example 1), as shown in fig. 13.
The experimental results are shown in a in fig. 13, and the modified basic neutralizing analogue Gefi-2OH (prepared in example 10) has greatly reduced accumulation effect on SQSTM1 and reduced influence on the lysosome function (expression level of CTSB) compared to gefitinib, which suggests that the modification strategy of basic neutralization effectively reduces the blocking of the lysosome function by gefitinib. As shown in B in FIG. 13, both Gefi-2OH and gefitinib at the same concentration can effectively inhibit the phosphorylation degree of EGFR and downstream Akt and Erk1/2, which indicates that the modification does not affect the original EGFR kinase inhibitory activity of EGFR TKI such as gefitinib. In addition, the influence of the modified analog AZD-2OH on the expression level of SQSTM1 and CTSB and the inhibition effect of the modified analog AZD-2OH on an EGFR signaling pathway are also detected. As shown in A in FIG. 14 and B in FIG. 14, the effect of AZD-2OH on SQSTM1 accumulation and on lysosome function was also significantly reduced and the same inhibitory effect on EGFR signaling pathway as AZD9291 was exhibited, using AZD9291 as a control.
Example 14 detection of the Effect of Gefi-2OH Compound on the function of lysosomes in cells Using lysosomal Tracer fluorescent probes (Lysotracker)
The detection principle and the specific steps of the fluorescence experiment of the fluorescent dye Lysotracker are as follows: lysotracker is a weakly basic probe labeled with a blue-fluorescent chemical group, in which a weak base moiety can donate a proton to maintain pH at neutral and can selectively reside in acidic lysosomes, thereby achieving specific fluorescent labeling of the lysosome. The method comprises the following specific steps: placing the cover glass in a pore plate, inoculating the cell density to be 1/5-1/3 at ordinary times, and carrying out corresponding treatment after the cover glass is attached to the wall. Lysotractor dye was diluted 1:1000 with culture medium and added to cells and incubated for 2 hours. The culture medium was decanted and washed 3 times with 1 XPBS for 5 minutes each. Live cells were mounted directly and fluorescence was observed under a microscope using a 20% glycerol/PBS mixture as mounting solution.
In previous studies, the inventors observed that compounds such as gefitinib and AZD9291 inhibit degradation from lysosomes by affecting lysosomal function. Thus, the inventors also examined the effect on lysosome function for the modified analogue Gefi-2 OH. Cells from different drug-treated groups were labeled with lysosome-specific probe Lysotracker and subjected to fluorescence detection, with the results shown in fig. 15. The experimental result shows that the gefitinib obviously increases the volume of lysosomes and obviously reduces the quantity, which indicates that the gefitinib obviously weakens the functions of the lysosomes in cells; compared with the gefitinib group, the alkaline neutralizing analogue Gefi-2OH has a weaker influence on the volume of lysosomes, and the change of the quantity of the lysosomes is weaker than that of the gefitinib group. Based on the results, the blocking of the lysosome function by the analogue Gefi-2OH modified by alkaline neutralization is weaker than that of gefitinib, and the effect is expected to improve the killing effect of the Gefi-2OH on tumor cells.
EXAMPLE 15 proliferation inhibition of non-Small cell Lung cancer by Gefi-2OH
Taking A549 cells and H460 cells of logarithmic growth phase cells to be inoculated into a 96-well plate at the density of 5000 cells/well and 6000 cells/well respectively, and setting 5 parallel repeat wells for each experimental group; after culturing for 24h, adding the prepared culture solution with the drug concentration gradient, and continuously culturing for 48 h; the culture medium in each well is completely sucked before detection, 100 mul of prepared 1640 culture medium containing 10% MTS is added, incubation is carried out in an incubator at 37 ℃ for 20-60 minutes in the absence of light, the absorbance value is read by 490nm wavelength after shaking for 1 minute on an enzyme linked immunosorbent assay, detection is stopped when the net OD value reaches the range of 0.5-0.8, the relative proliferation rate of the cells is calculated, and a proliferation curve is drawn by using Graphpad prism 6, and the result is shown in figure 16.
The results in FIG. 16 show that Gefi-2OH, which is a basic neutralized gefitinib derivative, has significantly better proliferation inhibition effect on A549 and H460 of non-small cell lung cancer cells than gefitinib, and half Inhibitory Concentration (IC) of gefitinib derivative50) Compared with gefitinib, the increase is about 1.8 times (in A549 cells) and 3 times (in H460 cells). In addition, the proliferation inhibitory effect of the compound AZD-2OH of the present invention on H460 cells was also examined according to the above method, and the proliferation curve thereof is shown in FIG. 17. As can be seen from the results in FIG. 17, the proliferation inhibition effect of the compound AZD-2OH on H460 cells is also obviously better than that of the compound AZD9291 before modification, and the half Inhibitory Concentration (IC) of the compound AZD-2OH is higher than that of the compound AZD9291 before modification50) Only half of the AZD 9291.
Example 16 flow cytometry to examine the Effect of Gefi-2OH Compound on apoptosis in NSCLC cells
The a549 and H460 cell supernatants were collected separately in a centrifuge tube, washed once with pre-cooled PBS and similarly collected in the centrifuge tube described above. In cellsAdding proper amount of pancreatin without EDTA and 0.25%, neutralizing with 2 times of pancreatin volume of 1640 culture solution containing 10% FBS after a certain time, and collecting cell suspension to the previous centrifuge tube. Centrifuging at 1500rpm for 5min, pouring out the supernatant, resuspending the cell pellet with PBS, centrifuging again at 1500rpm for 5min, and discarding the supernatant. Add 100. mu.l of 1 XBinding Buffer to resuspend the cells and adjust the cell density to 5-10 × 105and/mL. Add 5. mu.l each of Annexin V-FITC and 5. mu.l of PI stabilizing Solution and gently mix the cell suspensions. Incubate for 15min at room temperature in the dark. Adding 400 μ l of 1 × Binding Buffer for dilution, mixing gently, and detecting on the machine within 1 hour. 10000events were collected for each sample. The results are shown in FIG. 18.
The results in FIG. 18 show that Gefi-2OH significantly induces apoptosis of A549 cells and H460 cells, and that Gefi-2OH has an apoptosis inducing effect obviously superior to that of gefitinib under the action of the same concentration.
Example 17 screening of H460 cell proliferation inhibitory Activity of different Compounds of general formula
In order to illustrate the proliferation inhibition effect of the compounds of the general formula of the present invention on non-small cell lung cancer cells, typical compounds of the general formula (I) were synthesized and tested in a cell proliferation assay, the specific synthesis method of the compounds was the same as in examples 10-12, the test method was the same as in example 15, and the test results are shown in tables 2-4; the results in tables 2,3 and 4 show the effect of compounds II, III and IV of the general formulae on the proliferation activity of H460 cells, respectively. From the results in tables 2 to 4, it can be seen that the basic neutralization modification of gefitinib, omicinib and ocitinib greatly improves the killing effect on H460 cells. The modified compounds II, III and IV of the general formulas show good lung cancer cell proliferation inhibition activity, wherein the modified compound (general formula IV-1-general formula IV-8) of ocitinib has optimal activity, and the IC of the modified compound50The value is between 1.21 and 5.13. mu.M. In addition, through the analysis of the structure-activity relationship of the three compounds, the acidity of the R substituent plays a decisive role in the activity, the stronger the acidity is, the better the antitumor activity of the whole compound is, and the specific expression is that the contribution of different substituents to the improvement of the proliferation inhibition activity of the compound is ranked: alkyl sulfonic acid>Alkyl carboxylic acids>Benzene sulfonic acid>Benzoic acid>Ternary phenol>The dihydric phenol further indicates that the alkalinity of the molecular structure of the EGFR TKIs is indeed an important reason for reducing the sensitivity of tumor cells to the EGFR TKIs and generating drug resistance, and the invention performs the alkalinity neutralization modification on the EGFR TKIs for the first time to obtain an unexpected good effect and explore a new way for improving the activity of the EGFR TKIs.
TABLE 2
Figure GDA0003576518700000201
Compound II-1-Compound II-8
Figure GDA0003576518700000202
TABLE 3
Figure GDA0003576518700000203
Compound III-1-Compound III-8
Figure GDA0003576518700000211
TABLE 4
Figure GDA0003576518700000212
Compound IV-1-compound IV-8
Figure GDA0003576518700000213
Figure GDA0003576518700000221

Claims (8)

1. A compound having one of the following structural formulae:
Figure FDA0003608965250000011
wherein: r1, R2 or R3 is selected from
Figure FDA0003608965250000012
Figure FDA0003608965250000013
2. The compound of claim 1, wherein the compound has one of the following structures:
Figure FDA0003608965250000014
Figure FDA0003608965250000021
3. a method for preparing a compound according to any one of claims 1 or 2, comprising the step of introducing at least one acidic functional group into the structure of the compound having EGFR inhibitory activity.
4. A pharmaceutical composition comprising a compound of any one of claims 1 or 2, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable excipients.
5. Use of a compound according to any one of claims 1 or 2 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 4 for the manufacture of a medicament for the prevention and/or treatment of diseases and conditions associated with receptor tyrosine protein kinases.
6. The use according to claim 5, wherein the diseases and disorders related to receptor tyrosine protein kinases are selected from lung cancer, pancreatic cancer, breast cancer or colon cancer.
7. The use according to claim 6, wherein the diseases and conditions are lung cancer.
8. The use of claim 6, wherein the diseases and disorders are non-small cell lung cancer.
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