CN115003668A - Macrocyclic compounds as KRAS inhibitors - Google Patents
Macrocyclic compounds as KRAS inhibitors Download PDFInfo
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- CN115003668A CN115003668A CN202180010403.2A CN202180010403A CN115003668A CN 115003668 A CN115003668 A CN 115003668A CN 202180010403 A CN202180010403 A CN 202180010403A CN 115003668 A CN115003668 A CN 115003668A
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- compound
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- pharmaceutically acceptable
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- 150000002678 macrocyclic compounds Chemical class 0.000 title abstract description 6
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- 201000010099 disease Diseases 0.000 claims abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 4
- 150000001875 compounds Chemical class 0.000 claims description 267
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- 238000004519 manufacturing process Methods 0.000 claims 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
- A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
- C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
- C07D487/04—Ortho-condensed systems
Abstract
Relates to a macrocyclic compound and application of the macrocyclic compound in preparing medicaments for diseases related to KRAS inhibitors, in particular to the macrocyclic compound shown in a formula (I) and pharmaceutically acceptable salts thereof, wherein L is 1 Is selected from- (CH) 2 ) m ‑、‑(CH 2 ) m ‑NR 6 ‑、‑(CH 2 ) m ‑O‑、‑NR 6 ‑(CH 2 ) m ‑NR 6 -and the like.
Description
The invention claims the following priority:
CN202010071528.3, application date: 21 months 01, 2020;
CN202010514548.3, application date: 2020, 06 months and 08 days;
CN202010986192.3, application date: 09 and 18 days 2020.
The invention relates to a series of macrocyclic compounds and application thereof in preparing medicaments for treating diseases related to KRAS inhibitors. In particular to a compound shown as a formula (I) and pharmaceutically acceptable salts thereof.
The first RAS oncogene is found in rat sarcoma (rat sarcoma) and is therefore named. RAS proteins are products expressed by RAS genes, and refer to a closely related class of monomeric globulins consisting of 189 amino acids, with a molecular weight of 21 KDa. RAS proteins may bind to Guanine Triphosphate (GTP) or Guanine Dinucleotide Phosphate (GDP), and the active state of RAS proteins has an effect on the growth, differentiation, cytoskeleton, protein transport and secretion, etc. of cells, and its activity is regulated by binding to GTP or GDP: when the RAS protein binds to GDP, it is in a dormant, i.e., "inactivated," state; upon stimulation by specific upstream cytokines, RAS proteins are induced to exchange GDP and bind GTP, which is referred to as the "activated" state. RAS proteins bound to GTP are able to activate downstream proteins for signaling. RAS proteins themselves have weak hydrolytic GTP hydrolytic activity, capable of hydrolyzing GTP to GDP. This allows the transition from the activated state to the deactivated state. GAP (GTPase activating proteins) is also required to participate in this hydrolysis process. It can interact with RAS protein to greatly promote its ability to hydrolyze GTP to GDP. Mutations in the RAS protein affect its interaction with GAP and thus its ability to hydrolyze GTP to GDP, leaving it in an activated state. The activated RAS protein continuously imparts downstream protein growth signals, eventually leading to the incessant growth and differentiation of cells, ultimately resulting in the development of tumors. There are numerous members of the RAS gene family, among which the subfamilies closely related to various cancers are mainly the KRAS sarcoma rat sarcoma virus oncogene homolog (KRAS), the hragma sarcoma rat sarcoma virus oncogene Homolog (HRAS), and the neuroblastoma rat sarcoma oncogene homolog (NRAS). It was found that approximately 30% of human tumors carry certain mutated RAS genes, with KRAS mutations being the most prominent, accounting for 86% of all RAS mutations. For KRAS mutations, the most common mutations occur at glycine 12 (G12), glycine 13 (G13) and glutamine 61 (Q61) residues, with the G12 mutation accounting for 83%.
The G12C mutation is a common subtype in KRAS gene mutation, and refers to the mutation from No. 12 glycine to cysteine. KRAS G12C Mutations are most common in lung cancer, as predicted by data reported in the literature (Nat Rev Drug Discov 2014; 13:828- G12C Mutations account for around 10% of all lung cancer patients.
KRAS G12C The mutant protein is used as a leading-edge target, and the current research is not much.
Document J Med chem.2020jan 9; 63 (1) 52-65 report AMG510 (structure shown below) and KRAS G12C Eutectic structure of proteins (ID:6 IOM). According to the literature report, AMG510 is combined in KRAS G12C In the Swich ii pocket of the protein, the propenyl and Cys12 form covalent bonds by addition, the carbonyl forms hydrogen bonds with Lys16, the parent nucleus pyrimidinopyridine forms pi-pi interactions with Tyr96, respectively, the isopropyl picoline inserts a hydrophobic pocket at 86.8 ° dihedral angle between the parent nucleus and the parent nucleus, the fluorophenol inserts a hydrophobic pocket at 58.8 ° dihedral angle between the fluorophenol and the parent nucleus, while the phenolic hydroxyl group forms hydrogen bonds with Arg 68. (see FIGS. 1 and 2, Maestro 2017-2, Pymol 1.8.6).
Disclosure of Invention
The invention provides a compound shown in a formula (I) or a pharmaceutically acceptable salt thereof,
wherein the content of the first and second substances,
R 1 selected from H, F, Cl, Br, I and CH 3 Said CH 3 Optionally substituted by 1, 2 or 3R a Substitution;
R 2 selected from H, F, Cl, Br and I;
R 3 selected from H, F, Cl, Br, I and C 1-3 Alkyl radical, said C 1-3 Alkyl is optionally substituted by 1, 2 or 3R b Substitution;
R 4 selected from H, F, Cl, Br and I;
R 5 is selected from C 1-6 Alkyl and cyclopropyl, said C 1-6 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c Substitution;
L 1 is selected from- (CH) 2 ) m -、-(CH 2 ) m -NR 6 -、-(CH 2 ) m -O-、-NR 6 -(CH 2 ) m -NR 6 -、-NR 6 -(CH 2 ) m -O-、-O-(CH 2 ) m -O-and- (CH) 2 ) n -(CH＝CH) p -(CH 2 ) m -O-, said- (CH) 2 ) m -、-(CH 2 ) m -NR 6 -、-(CH 2 ) m -O-、-NR 6 -(CH 2 ) m -NR 6 -、-NR 6 -(CH 2 ) m -O-、-O-(CH 2 ) m -O-and- (CH) 2 ) n -(CH＝CH) p -(CH 2 ) m -O-is optionally substituted by 1, 2 or 3R d Substitution;
m is selected from 1, 2, 3, 4, 5 and 6;
n is selected from 0 and 1;
p is selected from 1 and 2;
R 6 is selected from H and CH 3 ；
Each R a 、R b And R c Each independently selected from H, F, Cl, Br and I;
R d selected from H, F, Cl, Br, I and CH 3 ；
The carbon atom with "-" is a chiral carbon atom, and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
In some embodiments of the invention, the above compound or a pharmaceutically acceptable salt thereof is selected from
Wherein R is 1 、R 2 、R 3 、R 4 、R 5 And L 1 As defined herein.
In some embodiments of the invention, R is as defined above 1 Selected from H and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 2 Selected from H and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 3 Is selected from CH 3 The other variables are as defined herein.
In some embodiments of the invention, R is as defined above 4 Selected from F, and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from C 1-4 Alkyl and cyclopropyl, said C 1-4 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c And the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
The CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
Optionally substituted by 1, 2 or 3R c And the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
Other variables are as defined herein.
In some embodiments of the invention, m is selected from 1, 2, 3, 4 and 5, and the other variables are as defined herein.
In some embodiments of the invention, L is 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH ═ CH) - (CH) 2 ) 3 -O-, said- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH ═ CH) - (CH) 2 ) 3 -O-is optionally substituted by 1, 2 or 3R d And the other variables are as defined herein.
In some embodiments of the invention, L is 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH) 2 ) 3 CH(CH 3 ) O-the other variables are as defined herein.
The invention also provides a compound shown in the formula (I) or a pharmaceutically acceptable salt thereof,
wherein, the first and the second end of the pipe are connected with each other,
R 1 selected from H, F, Cl, Br, I and CH 3 Said CH 3 Optionally substituted by 1, 2 or 3R a Substitution;
R 2 selected from H, F, Cl, Br and I;
R 3 selected from H, F, Cl, Br, I and C 1-3 Alkyl radical, said C 1-3 Alkyl is optionally substituted by 1, 2 or 3R b Substitution;
R 4 selected from H, F, Cl, Br and I;
R 5 is selected from C 1-6 Alkyl and cyclopropyl, said C 1-6 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c Substitution;
L 1 is selected from- (CH) 2 ) m -、-(CH 2 ) m -NR 6 -、-(CH 2 ) m -O-、-NR 6 -(CH 2 ) m -NR 6 -、-NR 6 -(CH 2 ) m -O-、-O-(CH 2 ) m -O-and- (CH) 2 ) n -(CH＝CH) p -(CH 2 ) m -O-, said- (CH) 2 ) m -、-(CH 2 ) m -NR 6 -、-(CH 2 ) m -O-、-NR 6 -(CH 2 ) m -NR 6 -、-NR 6 -(CH 2 ) m -O-、-O-(CH 2 ) m -O-and- (CH) 2 ) n -(CH＝CH) p -(CH 2 ) m -O-is optionally substituted by 1, 2 or 3R d Substitution;
m is selected from 1, 2, 3, 4, 5 and 6;
n is selected from 0 and 1;
p is selected from 1 and 2;
R 6 is selected from H and CH 3 ；
Each R a 、R b 、R c And R d Each independently selected from H, F, Cl, Br and I;
the carbon atom with "-" is a chiral carbon atom, and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
In some embodiments of the invention, the above compound or a pharmaceutically acceptable salt thereof is selected from
Wherein R is 1 、R 2 、R 3 、R 4 、R 5 And L 1 As defined herein.
In some embodiments of the invention, R is as defined above 1 Selected from H and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 2 Selected from H and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 3 Is selected from CH 3 The other variables are as defined herein.
In some embodiments of the invention, R is as defined above 4 Selected from F and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from C 1-4 Alkyl and cyclopropyl, said C 1-4 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c And the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
The CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
Optionally substituted by 1, 2 or 3R c And the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
Other variables are as defined herein.
In some embodiments of the invention, m is selected from 1, 2, 3, and 4, and the other variables are as defined herein.
In some embodiments of the present invention, L is 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH ═ CH) - (CH) 2 ) 3 -O-, said- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-(CH 2 ) 2 -O-and optionally substituted by 1, 2 or 3R d And the other variables are as defined herein.
In some embodiments of the invention, L is 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-and- (CH ═ CH) - (CH) 2 ) 2 -O-and the other variables are as defined herein.
The invention provides a compound shown in a formula (I) or a pharmaceutically acceptable salt thereof,
wherein, the first and the second end of the pipe are connected with each other,
R 1 selected from H, F, Cl, Br, I and CH 3 Said CH 3 Optionally substituted by 1, 2 or 3R a Substitution;
R 2 selected from H, F, Cl, Br and I;
R 3 selected from H, F, Cl, Br, I and C 1-3 Alkyl radical, said C 1-3 Alkyl is optionally substituted by 1, 2 or 3R b Substitution;
R 4 selected from H, F, Cl, Br and I;
R 5 is selected from C 1-6 Alkyl and cyclopropyl, said C 1-6 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c Substitution;
L 1 is selected from- (CH) 2 ) m -、-(CH 2 ) m -NH-、-(CH 2 ) m -O-、-NH-(CH 2 ) m -NH-、-NH-(CH 2 ) m -O-and-O- (CH) 2 ) m -O-, said- (CH) 2 ) m -、-(CH 2 ) m -NH-、-(CH 2 ) m -O-、-NH-(CH 2 ) m -NH-、-NH-(CH 2 ) m -O-and-O- (CH) 2 ) m -O-is optionally substituted by 1, 2 or 3R d Substitution;
m is selected from 2, 3, 4, 5 and 6;
each R a 、R b 、R c And R d Each independently selected from H, F, Cl, Br and I;
the carbon atom with "-" is a chiral carbon atom, and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer.
In some embodiments of the invention, the above compound or a pharmaceutically acceptable salt thereof is selected from
Wherein R is 1 、R 2 、R 3 、R 4 、R 5 And L 1 As defined herein.
In some embodiments of the invention, R is as defined above 1 Selected from H and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 2 Selected from H and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 3 Is selected from CH 3 And the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 4 Selected from F and the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from C 1-4 Alkyl and cyclopropyl, said C 1-4 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c And the other variables are as defined herein.
In some embodiments of the invention, R is as defined above 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
The CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
Optionally substituted by 1, 2 or 3R c And (4) substitution.
In some embodiments of the invention, R is as defined above 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、 
Other variables are as defined herein.
In some embodiments of the invention, m is selected from 2, 3 and 4, and the other variables are as defined herein.
In some embodiments of the invention, L is 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-and-NH (CH) 2 ) 3 O-, said- (CH) 2 ) 4 -、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-and-NH (CH) 2 ) 3 O-is optionally substituted by 1, 2 or 3R d And the other variables are as defined herein.
In some embodiments of the invention, L is 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-(CH 2 ) 4 O-、-O(CH 2 ) 3 O-and-NH (CH) 2 ) 3 O-the other variables are as defined herein.
In some embodiments of the invention, the above compound or a pharmaceutically acceptable salt thereof is selected from
Wherein the content of the first and second substances,
R 1 、R 2 、R 3 、R 4 、R 5 and R d As defined herein;
m1 is selected from 3, 4 and 5;
m2 is selected from 1, 2 and 3.
Still other aspects of the invention are derived from any combination of the above variables.
The invention also provides a compound shown in the specification or a pharmaceutically acceptable salt thereof,
in some embodiments of the invention, the above compound or a pharmaceutically acceptable salt thereof is selected from
In some embodiments of the invention, the above compound or a pharmaceutically acceptable salt thereof is selected from
The invention also provides application of the compound or the pharmaceutically acceptable salt thereof in preparing a medicament for treating KRAS-related diseases.
The compounds of the present invention can be synthesized by the following methods:
the method comprises the following steps:
the method 2 comprises the following steps:
the method 3 comprises the following steps:
the method 4 comprises the following steps:
technical effects
Compounds of the invention in KRAS G12C The lowest binding energy barrier in the protein structure has a smaller energy difference from the energy barrier of reference compound AMG510 in the active conformation in the protein structure, and therefore the compound of the present invention binds to the protein more easily, and it is possible to exhibit similar or superior binding activity to that of reference compound AMG510 in the actual binding to the protein. The compound shows better inhibitory activity on the proliferation of Mia PaCa-2 cells, has better inhibitory action on the tumor growth of Mia PaCa-2 xenografts and NCI-H358 xenografts, and has excellent pharmacokinetic properties.
Correlation definition
As used herein, the following terms and phrases are intended to have the following meanings, unless otherwise indicated. A particular term or phrase, unless otherwise defined, should not be considered as indefinite or unclear but rather construed according to ordinary meaning. When a trade name appears herein, it is intended to refer to its corresponding commercial product or its active ingredient.
The term "pharmaceutically acceptable" as used herein is intended to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salts" refers to salts of the compounds of the present invention, prepared from the compounds of the present invention found to have particular substituents, with relatively nontoxic acids or bases. When compounds of the present invention contain relatively acidic functional groups, base addition salts can be obtained by contacting such compounds with a sufficient amount of a base in neat solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amines or magnesium salts or similar salts. When compounds of the present invention contain relatively basic functional groups, acid addition salts can be obtained by contacting such compounds with a sufficient amount of acid, either in neat solution or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, and the like; and salts of organic acids including such acids as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-toluenesulfonic, citric, tartaric, methanesulfonic, and the like; also included are salts of amino acids such as arginine and the like, and salts of organic acids such as glucuronic acid and the like. Certain specific compounds of the invention contain both basic and acidic functionalities and can thus be converted to any base or acid addition salt.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, which contains an acid or base, by conventional chemical methods. In general, such salts are prepared by the following method: prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of the two.
The compounds of the present invention may exist in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) -and (+) -enantiomers, (R) -and (S) -enantiomers, diastereomers, (D) -isomers, (L) -isomers, as well as racemic and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be labelled with radioactive isotopes, such as tritium (A), (B), (C) and C) 3 H) Iodine-125 ( 125 I) Or C-14( 14 C) In that respect For example, deuterium can be used to replace hydrogen to form a deuterated drug, the bond formed by deuterium and carbon is stronger than the bond formed by common hydrogen and carbon, and compared with an undeuterated drug, the deuterated drug has the advantages of reducing toxic and side effects, increasing the stability of the drug, enhancing the curative effect, prolonging the biological half-life period of the drug and the like. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.
The terms "optional" or "optionally" mean that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.
The term "substituted" means that any one or more hydrogen atoms on the specified atom is replaced with a substituent that may include variations of deuterium and hydrogen, so long as the valence of the specified atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., ═ O), it means that two hydrogen atoms are substituted. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, and unless otherwise specified, the kind and number of substituents may be arbitrary on the basis of chemical realizability.
When any variable (e.g., R) occurs more than one time in the composition or structure of a compound, its definition in each case is independent. Thus, for example, if a group is substituted with 0-2R, the group may optionally be substituted with up to two R, and there are separate options for R in each case. Furthermore, combinations of substituents and/or variants thereof are permissible only if such combinations result in stable compounds.
When the number of one linking group is 0, e.g. - (CRR) 0 -, represents that the linking group is a single bond.
When one of the variables is selected from a single bond, it means that the two groups to which it is attached are directly linked, for example, in A-L-Z where L represents a single bond, it means that the structure is actually A-Z.
When the listed linking groups do not indicate their direction of attachment, the direction of attachment is arbitrary, for example,
the middle connecting group L is-M-W-, in this case-M-W-can be formed by connecting the ring A and the ring B in the same direction as the reading sequence from left to right
The ring A and the ring B may be connected in the reverse direction of the reading sequence from left to right
Combinations of the linking groups, substituents, and/or variants thereof are permissible only if such combinations result in stable compounds.
Unless otherwise specified, when a group has one or more attachable sites, any one or more of the sites of the group may be attached to other groups by chemical bonds. When the chemical bond is connected in an un-positioned way and H atoms exist in the connectable sites, the number of the H atoms at the connectable sites is correspondingly reduced along with the number of the connected chemical bonds and becomes phaseA group corresponding to valence number. The chemical bond linking said site to other groups may be a direct solid bond
Straight dotted line key
Or wavy lines
And (4) showing. For example-OCH 3 The straight solid line bond in (a) represents a bond to another group via an oxygen atom in the group;
the straight dotted bond in (1) represents the linkage to the other group through both ends of the nitrogen atom in the group;
the wavy line in (a) indicates that the phenyl group is bonded to other groups through the carbon atoms at the 1-and 2-positions in the phenyl group;
means that any of the linkable sites on the piperidinyl group may be linked to other groups by 1 chemical bond, including at least
These 4 linkages, even though the-N-atom is drawn as a H atom, are
Still comprise
This attachment is a group whose H at the site is reduced by 1 to the corresponding monovalent piperidinyl group, except when 1 bond is attached.
Unless otherwise specified, the term "C 1-6 Alkyl "is intended to mean a straight or branched saturated hydrocarbon group consisting of 1 to 6 carbon atoms. Said C is 1-6 The alkyl group comprising C 1-5 、C 1-4 、C 1-3 、C 1-2 、C 2-6 、C 2-4 、C 6 And C 5 Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C 1-6 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), pentyl (including n-pentyl, isopentyl and neopentyl), hexyl, and the like.
Unless otherwise specified, the term "C 1-4 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 4 carbon atoms. Said C is 1-4 The alkyl group comprising C 1-2 、C 1-3 And C 2-3 Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C 1-4 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), butyl (including n-butyl, isobutyl, s-butyl and t-butyl), and the like.
Unless otherwise specified, the term "C 1-3 Alkyl "is used to denote a straight or branched saturated hydrocarbon group consisting of 1 to 3 carbon atoms. Said C is 1-3 The alkyl group comprising C 1-2 And C 2-3 Alkyl, etc.; it may be monovalent (e.g., methyl), divalent (e.g., methylene), or multivalent (e.g., methine). C 1-3 Examples of alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (clathration)Including n-propyl and isopropyl), and the like.
Unless otherwise specified, C n-n+m Or C n -C n+m Including any one particular case of n to n + m carbons, e.g. C 1-12 Comprising C 1 、C 2 、C 3 、C 4 、C 5 、C 6 、C 7 、C 8 、C 9 、C 10 、C 11 And C 12 Also included are any ranges of n to n + m, e.g. C 1- 12 Comprising C 1-3 、C 1-6 、C 1-9 、C 3-6 、C 3-9 、C 3-12 、C 6-9 、C 6-12 And C 9-12 Etc.; similarly, n to n + m means the number of atoms on the ring is n to n + m, for example, the 3-12 membered ring includes a 3-membered ring, a 4-membered ring, a 5-membered ring, a 6-membered ring, a 7-membered ring, an 8-membered ring, a 9-membered ring, a 10-membered ring, a 11-membered ring, and a 12-membered ring, and any range of n to n + m is also included, for example, the 3-12 membered ring includes a 3-6-membered ring, a 3-9-membered ring, a 5-6-membered ring, a 5-7-membered ring, a 6-8-membered ring, and a 6-10-membered ring, etc.
The compounds of the present invention may be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combinations thereof with other chemical synthetic methods, and equivalents thereof known to those skilled in the art, with preferred embodiments including, but not limited to, examples of the present invention.
The compounds of the present invention exist in a stereoconfiguration and can be determined by single crystal diffraction, optical rotation, CD, and the like.
The compounds of the present invention may be structurally confirmed by conventional methods well known to those skilled in the art, and if the present invention relates to the absolute configuration of the compound, the absolute configuration may be confirmed by means of conventional techniques in the art. For example, single crystal X-ray diffraction method (SXRD), collecting diffraction intensity data of the cultured single crystal with Bruker D8 vehicle diffractometer, and irradiating with light sourceFor CuK α radiation, scan mode:
after scanning and collecting relevant data, the crystal structure is further analyzed by a direct method (Shelxs97), so that the absolute configuration can be confirmed.
The solvent used in the present invention can be obtained commercially. The invention employs the following abbreviations: KOAc represents potassium acetate; pd (dppf) Cl 2 DCM represents [1,1' -bis (diphenylphosphino) ferrocene]A palladium dichloride dichloromethane complex; dioxane represents 1, 4-Dioxane; DIPEA stands for N, N-diisopropylethylamine; DCM represents dichloromethane; cbz represents benzyloxycarbonyl; iPr represents an isopropyl group; KHMDS for potassium bis (trimethylsilyl) amide; POCl 3 Represents phosphorus oxychloride; CH (CH) 3 CN represents acetonitrile; TBS represents tert-butyldimethylsilyl; THF represents tetrahydrofuran.
The compounds are used according to the conventional naming principle in the field
The software names, and the commercial compounds used the supplier catalog names.
FIG. 1 shows AMG510 and KRAS G12C Binding mode of the protein (active conformation: 6 IOM).
FIG. 2 shows AMG510 and KARS G12C FIG. 2D of the binding pattern of (1).
FIG. 3 is a low energy constellation diagram of AMG 510.
Fig. 4 is a graph showing the rotational dihedral angle of AMG510 and the corresponding energy barrier change (coordinate 1 is the dihedral angle of fluorophenol with the parent nucleus, and coordinate2 is the dihedral angle of isopropylmethylpyridine with the parent nucleus).
FIG. 5 is a superposition of the low energy conformation of WX001 (i.e., compound 003 of example 2) and the active conformation of AMG 510.
FIG. 6 is a graph showing the rotational dihedral angle and the corresponding energy barrier change of WX001 (i.e., Compound 003 of example 2) (the dihedral angle between fluorophenol and the parent nucleus is shown in coordinate1, and the dihedral angle between isopropylmethylpyridine and the parent nucleus is shown in coordinate 2)
FIG. 7 is a superposition of the low energy conformation of WX002 and the active conformation of AMG 510.
Fig. 8 is a graph showing the rotational dihedral angle of WX002 and the corresponding energy barrier change (the dihedral angle of fluorophenol and the parent nucleus is shown as coordinate1, and the dihedral angle of isopropylmethylpyridine and the parent nucleus is shown as coordinate 2).
FIG. 9 is a superposition of the low energy conformation of WX003 and the active conformation of AMG 510.
Fig. 10 is a graph showing the rotational dihedral angle of WX003 and the corresponding energy barrier change (the dihedral angle of fluorophenol and the parent nucleus is shown as coordinate1, and the dihedral angle of isopropylmethylpyridine and the parent nucleus is shown as coordinate 2).
Figure 11 is a superposition of the low energy conformation of WX004 (i.e., compound 011 of example 5) and the active conformation of AMG 510.
Fig. 12 is a graph of the rotational dihedral angle and corresponding energy barrier change for WX004 (i.e., compound 011 of example 5) (coordinate 1 is the dihedral angle of fluorophenol with the parent nucleus, and coordinate2 is the dihedral angle of isopropylmethylpyridine with the parent nucleus).
FIG. 13 is a overlay of the low energy conformation of WX005 and the active conformation of AMG 510.
Fig. 14 is a graph showing the rotational dihedral angle of WX005 and the corresponding energy barrier change (the dihedral angle of fluorophenol and the parent nucleus is shown as coordinate1, and the dihedral angle of isopropylmethylpyridine and the parent nucleus is shown as coordinate 2).
Figure 15 is a superposition of the low energy conformation of WX006 (i.e., compound 005 of example 3) and the active conformation of AMG 510.
Fig. 16 is a graph showing the rotational dihedral angle and the corresponding energy barrier change of WX006 (i.e., compound 005 of example 3) (the dihedral angle of fluorophenol and the parent nucleus is shown as coordinate1, and the dihedral angle of isopropylmethylpyridine and the parent nucleus is shown as coordinate 2).
FIG. 17 is a overlay of the low energy conformation of WX007 and the active conformation of AMG 510.
FIG. 18 is a graph showing the rotational dihedral angle of WX007 and the corresponding energy barrier change (the dihedral angle of fluorophenol and the parent nucleus is shown as coordinate1, and the dihedral angle of isopropylmethylpyridine and the parent nucleus is shown as coordinate 2).
FIG. 19 is a superposition of the low energy conformation of WX008 and the active conformation of AMG 510.
FIG. 20 is a graph showing the dihedral angle rotation of WX008 and the corresponding energy barrier (the dihedral angle of fluorophenol with the core is shown as coordinate1, and the dihedral angle of isopropylpicoline with the core is shown as coordinate 2).
FIG. 21 is a overlay of the low energy conformation of WX009 and the active conformation of AMG 510.
FIG. 22 is a graph showing the rotation dihedral angle of WX009 and the corresponding energy barrier change (the dihedral angle of fluorophenol and the parent nucleus is shown as coordinate1, and the dihedral angle of isopropylmethylpyridine and the parent nucleus is shown as coordinate 2).
FIG. 23 is a superposition of the low energy conformation of WX010 and the low energy conformation of WX 006.
FIG. 24 is a schematic overlay of the low energy conformation of WX011 and the low energy conformation of WX 006.
FIG. 25 is a superposition of the low energy conformation of WX012 and the low energy conformation of WX 006.
FIG. 26 is a schematic overlay of the low energy conformation of WX013 and the low energy conformation of WX 006.
FIG. 27 is a schematic overlay of the low energy conformation of WX014 and the low energy conformation of WX 006.
FIG. 28 is a graph of tumor growth in Mia PaCa-2 xenograft tumor model tumor-bearing mice after administration of compound.
FIG. 29 is a graph of body weight changes during dosing in Mia PaCa-2 xenograft tumor model tumor-bearing mice.
FIG. 30 is a graph showing the tumor growth profile of tumor-bearing mice in the NCI-H358 xenograft tumor model after administration of the compound.
FIG. 31 is a graph showing the body weight changes of tumor-bearing mice in the NCI-H358 xenograft tumor model during administration.
Note: coordinate1 represents coordinate axis 1; coordinate2 represents coordinate axis 2.
The present invention is described in detail below by way of examples, but is not meant to be limited to any of the disadvantages of the present invention. Having described the invention in detail and having disclosed specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
Reference example 5
The low energy conformation of AMG510 was calculated by the Macromodel module of scherodinger Maestro software. In the low energy conformation, the dihedral angle (dehidal 1) of the fluorophenol with the pyridopyrimidone parent nucleus (hereinafter simply referred to as the parent nucleus) was 50.6 °, and the dihedral angle (dehidal 2) of isopropylmethylpyridine with the parent nucleus was 88.3 ° (see FIG. 3). The transition of AMG510 from its low energy conformation to its protein binding mode active conformation (rotation of fluorophenol from 50.6 ° to 58.8 °, isopropylpicoline from 88.3 ° to 86.8 °) requires overcoming the energy barrier of 0.185kcal/mol (see fig. 4).
Calculation example 1
By observing AMG510 and KRAS G12C Active conformation binding mode of protein, we find that fluorophenol fragment and isopropylmethylpyridine fragment form the lowest energy barrier conformation (low energy conformation) with the parent nucleus of pyridopyrimidone while freely rotating, and AMG510 and KRAS G12C The conformation of the protein binding mode (active conformation) in the protein cocrystal is relatively identical, which explains AMG510 and KRAS G12C High binding activity of the protein. Generally, the smaller the energy barrier difference between the lowest energy barrier conformation (low energy conformation) of a small molecule and its binding mode conformation (active conformation) in the protein means that the smaller the energy lost when the small molecule is converted from the low energy conformation to the binding active conformation with the protein, the easier a compound binds to the protein, and the higher the binding activity thereof.
To lock the active conformation of AMG510, further lowering its rotational energy barrier, we linked the fluorophenol fragment of AMG510 and isopropyl via linker chains of different lengthsThe picolyl fragment is cyclized to obtain a series of macrocyclic molecules which are connected by chains and are different from the fluorophenol fragment and the isopropylpicoline fragment, and the minimum energy barrier conformation of the macrocyclic molecules and AMG510 and KRAS are explored G12C Energy barrier differences between active conformations in protein co-crystals; meanwhile, taking a five-membered linker macrocyclic molecule as an example, the change of the lowest energy barrier conformation of the macrocyclic molecules after different substituents (methyl, ethyl, propyl, cyclopropyl, tert-butyl and the like) replace isopropyl is further explored.
The low energy conformation rotational dihedral angles and rotational energy barriers for WX001 to WX009 were calculated by a Macromodel module and the results are shown in Table 1.
TABLE 1 Low energy conformation rotational dihedral and rotational energy barrier of the compounds of the invention
| Compound (I) | Dialdral 1 (degree) | Dialdral 2 (degree) | ΔE(Kcal/mol) |
| AMG510 (active conformation) | 58.8 | 86.8 | / |
| AMG510 (Low energy conformation) | 50.6 | 88.3 | 0.185 |
| WX001 (Low energy conformation) | 54.7 | 98.8 | 0.769 |
| WX002 (Low energy conformation) | 60.6 | 96.4 | 0.434 |
| WX003 (Low energy conformation) | 52.4 | 97.4 | 0.727 |
| WX004 (Low energy conformation) | 56.2 | 102.3 | 1.139 |
| WX005 (Low energy conformation) | 56.2 | 103.2 | 1.398 |
| WX006 (Low energy conformation) | 59.2 | 90.8 | 0.003 |
| WX007 (Low energy conformation) | 69 | 92.6 | 0.256 |
| WX008 (Low energy conformation) | 67.8 | 95 | 0.531 |
| WX009 (Low energy conformation) | 66.3 | 88.3 | 0.209 |
Note: dihedral1 is the Dihedral angle of fluorobenzene and fluoropyridine, Dihedral2 is the Dihedral angle of isopropylpyridine and pyrimidinone, Δ E is the energy barrier to be consumed to switch from the low energy conformation to the protein binding mode active conformation of AMG510 (Dihedral 1 is 58.8 ° and Dihedral2 is 86.8 °); WX001 is compound 003 of example 2 and WX006 is compound 005 of example 3.
And (4) conclusion: the low energy conformations of WX 001-WX 009 are well-aligned with the active conformation of AMG 510. Compounds of the invention in KRAS G12C The lowest energy barrier for binding in the protein structure has a smaller energy difference from the energy barrier of reference compound AMG510 in the active conformation in the protein structure, and therefore the compound of the present invention binds to the protein more easily, and may exhibit similar or superior binding activity to reference compound AMG510 in actual binding to the protein.
The results of the calculation of the low energy conformation rotational dihedral angles of WX 010-WX 014 by the Macromodel module are shown in Table 2.
TABLE 2 Low energy conformational dihedral rotation of compounds of the invention
| Compound (I) | Dialdral 1 (degree) | Dialdral 2 (degree) |
| WX010 (Low energy conformation) | 59.6 | 91.1 |
| WX011 (Low energy conformation) | 59.5 | 91.6 |
| WX012 (Low energy conformation) | 59.8 | 87.8 |
| WX013 (Low energy conformation) | 60.0 | 90.8 |
| WX014 (Low energy conformation) | 61.7 | 89.6 |
Note: dihedral1 is the Dihedral angle of fluorobenzene and fluoropyridine, and Dihedral2 is the Dihedral angle of methylpyridine and pyrimidinone (WX010) or the Dihedral angle of ethylpyridine and pyrimidinone (WX011 to WX 014).
And (4) conclusion: the low energy conformation of WX 010-WX 014 was substantially identical to that of WX006 in KRAS G12C It is possible that the actual binding of the protein exhibits a similar or superior binding activity to that of the reference compound AMG 510.
Example 1
The synthetic route is as follows:
step 1: synthesis of Compound 001-2
In a 500mL three-necked flask dried in advance were charged compound 001-1(20g,122.70mmol,1.00eq), potassium isopropenyltrifluoroborate (19.06g,128.83mmol,1.05eq), potassium carbonate (25.44g,184.04mmol,1.5eq),1, 4-dioxane (200mL) and water (20 mL). After the addition was complete, the nitrogen was replaced three times. Then 1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (1.09g,1.49mmol,1.22e-2eq) was added to the system. After the addition, the reaction system was stirred at 100 ℃ for 12 hours. The reaction system was directly concentrated to dryness. The crude product was subjected to silica gel column chromatography (petroleum ether: ethyl acetate ═ 8:1 to 5:1) to give compound 001-2.
1 H NMR(400MHz,CDCl 3 )δ＝7.82(d,J＝5.2Hz,1H),7.01(d,J＝5.1Hz,1H),5.43(s,1H),5.25(s,1H), 4.28(br s,2H),2.09(s,3H)。
Step 2: synthesis of Compound 001-3
To a 500mL single-neck flask previously purged with nitrogen was added tris (triphenylphosphine) rhodium chloride (3.10g,3.35mmol,3.65e-2 eq). After the addition was completed, methanol (300mL) and compound 001-2(15.5g,91.92mmol,1eq) were added to the system. After the addition was complete, the system was replaced 3 times with hydrogen. The system was stirred for 1.0 hour at 25 ℃. The reaction was filtered, and the filtrate was concentrated. The crude product was isolated by flash column chromatography (petroleum ether: ethyl acetate: 5:1 to 3:1) and purified to give compound 001-3.
LCMS:MS m/z:171.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝7.82(d,J＝5.2Hz,1H),6.96(d,J＝5.2Hz,1H),4.03(br s,2H),3.01-2.88(m,1H),1.22(d,J＝6.8Hz,6H)。
And step 3: synthesis of Compound 001-4
In a predried 250mL three-necked flask, compound 001-3(10g,58.60mmol,1.00eq) and N, N-dimethylformamide (100mL) were added. After the addition was complete, the nitrogen was replaced three times. The compounds allyltributyltin (58.93g,177.98mmol,54.57mL,3.04 eq.) and 1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (4.29g,5.86mmol,0.10eq) were then added to the system. After the addition was complete, the nitrogen was replaced three times. The reaction system was stirred at 135 ℃ for 12 hours. After the reaction system is cooled to room temperature, a prepared saturated potassium fluoride solution (250mL) is added into the system to quench the reaction. Water (150mL) and ethyl acetate were added to the system for extraction (150 mL. times.2). The organic phases were combined, dried over anhydrous sodium sulfate, filtered and the filtrate was concentrated. The crude product was isolated by flash column chromatography (petroleum ether: ethyl acetate 10:1 to 8:1) and purified to give compound 001-4.
LCMS:MS(ESI)m/z:177.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝7.97(d,J＝4.8Hz,1H),6.81(d,J＝4.8Hz,1H),5.97-5.83(m,1H),5.19-5.04(m,2H),3.66(br s,2H),3.26(d,J＝6.4Hz,2H),3.08-2.94(m,1H),1.28(d,J＝6.8Hz,6H)。
And 4, step 4: synthesis of Compounds 001-6
To a pre-dried 250mL single-neck flask, under nitrogen, was added compound 001-5(5.0g,23.92mmol,1.00eq) and tetrahydrofuran (75 mL). After the sample had dissolved, the temperature of the system was cooled to 0 ℃ and a solution of oxalyl chloride (12.15g,95.69mmol,8.38mL,4.00eq) in tetrahydrofuran (15mL) was slowly added dropwise to the system. After the addition was complete, the system was warmed to 75 ℃ for 2 hours. The reaction was then concentrated directly under reduced pressure, and tetrahydrofuran (75mL) was added to the reaction at 0 ℃. Then, a solution of compound 001-4(4.09g,23.21mmol,0.97eq) in tetrahydrofuran (15mL) was slowly added dropwise at 0 ℃ and reacted at 0 ℃ for 2 hours. The reaction was quenched by the addition of saturated ammonium chloride solution (25 mL). Followed by extraction with ethyl acetate (50mL x 3) and separation. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 001-6. The crude product was used in the next step without purification.
LCMS:MS(ESI)m/z:411.0[M+1] + 。
And 5: synthesis of Compounds 001-7
To a pre-dried 100mL three-necked flask, under nitrogen protection, was added compound 001-6(7.6g,18.48mmol,1eq) and tetrahydrofuran (230 mL). After the solution became clear, the system was cooled to-70 to-60 ℃ and potassium hexamethyldisilazide (1M tetrahydrofuran solution, 42.50mL,2.3eq) was slowly added dropwise to the system. After the addition was complete, the system was warmed to 25 ℃ and stirred at this temperature for 2 hours. The reaction was quenched by adding saturated ammonium chloride solution (10 mL). Then, the mixture was extracted with ethyl acetate (10mL × 3) and separated. The organic phases were combined, washed with saturated brine (10mL), separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give a crude product. The crude product was isolated and purified by column chromatography (eluent: petroleum ether: ethyl acetate: 20:1 to 8:1) to give compound 001-7.
LCMS:MS(ESI)m/z:375.0[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.91(br s,1H),8.58(d,J＝5.2Hz,1H),8.19(d,J＝6.4Hz,1H),7.29(d,J＝4.8Hz,1H),6.47-6.29(m,1H),5.90(dd,J＝1.2,15.6Hz,1H),2.69-2.56(m,1H),1.74(dd,J＝1.6,6.4Hz,3H),1.15(d,J＝6.8Hz,3H),1.06(d,J＝6.8Hz,3H)。
Step 6: synthesis of Compounds 001-8
Under nitrogen protection, compound 001-7(1.00g,2.67mmol,1.00eq) and N, N' -diisopropylethylamine (1.72g,13.34mmol,2.32mL,5.00eq) were added to a pre-dried 80mL long tube. After the addition was complete, the temperature was controlled at 25 ℃ and phosphorus oxychloride (2.05g,13.34mmol,1.24mL,5.00eq) was added slowly to the system. After the addition was complete, the system was stirred at 40 ℃ for 2 hours to give compound 001-8. The reaction system can be used directly in the next step.
LCMS:MS(ESI)m/z:393.0[M+1] + 。
And 7: synthesis of Compounds 001-10
Tetrahydrofuran (40mL) was added to a 80mL long tube containing compound 001-8(1.05g,2.67mmol,1.00eq) under nitrogen, cooled to 0 ℃ and N, N' -diisopropylethylamine (5.18g,40.05mmol,6.98mL,15eq) was added to the system. After the addition was completed, the temperature was controlled to 0 ℃ and a solution of compound 001-9(802.14mg,4.01mmol,1.5eq) in tetrahydrofuran (20mL) was slowly added to the system. After the addition was complete, the system was stirred at 25 ℃ for 1 hour. The reaction was poured slowly into 10mL of ice-water and diluted with 15mL of ethyl acetate. And standing and separating the system. After separation, the organic phase was collected and the aqueous phase was extracted with ethyl acetate (10mL _ 1). The combined organic phases were dried over anhydrous sodium sulfate and concentrated under reduced pressure to give a residue. The crude product was isolated by flash column chromatography (petroleum ether: ethyl acetate 1:1 to 1:4) and purified to give compound 001-10.
LCMS:MS(ESI)m/z:557.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.53(d,J＝5.2Hz,1H),7.72(d,J＝7.6Hz,1H),7.28(dd,J＝2.8,5.2Hz,1H),6.42-6.27(m,1H),5.93-5.79(m,1H),4.90-4.55(m,1H),4.36-4.07(m,2H),4.01-3.77(m,1H),3.70-3.44(m,1H),3.36-2.90(m,2H),2.61-2.42(m,1H),1.68(td,J＝1.6,6.8Hz,4H),1.46-1.38(m,12H),1.14(dd,J＝4.4,6.8Hz,3H),1.03(t,J＝7.2Hz,3H)。
And 8: synthesis of Compounds 001-12
In a previously dried 100mL single-necked flask, were added compound 001-10(927mg,1.66mmol,1.00eq), compound 001-11(272.44mg,1.75mmol,1.05eq), potassium phosphate (706.48mg,3.33mmol,2.0eq), tetrahydrofuran (8mL) and H 2 O (2 mL). After the completion of the addition, the system was purged with nitrogen 3 times, and 1,1' -bis (di-t-butylphosphino) ferrocene dichloropalladium (II) (108.46mg, 166.41. mu. mol,0.1eq) was added to the system. After the addition, the system was purged with nitrogen 3 times. The system was heated to 80 ℃ and the reaction was stirred at 80 ℃ for 12 hours. The system was directly concentrated. The crude product was purified by flash column chromatography (petroleum ether: ethyl acetate: methanol ═ 2:1:0.5 to 4:1:0.5) to afford compound 001-12.
LCMS:MS(ESI)m/z:633.4[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝9.12(d,J＝6.0Hz,1H),8.57(d,J＝5.2Hz,1H),7.84(dd,J＝7.2,9.2Hz, 1H),7.35(dd,J＝1.2,5.2Hz,1H),7.24-7.16(m,1H),6.65-6.54(m,2H),6.47-6.34(m,1H),5.88(t,J＝16.4Hz,1H),5.12-4.67(m,1H),4.55-4.12(m,2H),4.01-3.80(m,1H),3.77-3.43(m,1H),3.36-2.94(m,2H),2.82-2.57(m,1H),1.71-1.63(m,3H),1.48-1.40(m,12H),1.24-1.13(m,6H)。
And step 9: synthesis of Compounds 001-13
In a previously dried 40mL single neck flask were added compounds 001-12(0.35g, 553.19. mu. mol,1eq), allyl bromide (80.31mg, 663.82. mu. mol,1.2eq), potassium carbonate (114.68mg, 829.78. mu. mol,1.5eq), potassium iodide (119.38mg, 719.14. mu. mol,1.3eq) and acetonitrile (20 mL). After the addition was complete, the reaction was stirred at 25 ℃ for 12 hours. 10mL of water and 10mL of ethyl acetate were added to the system. And standing and separating the system. The aqueous phase was extracted with ethyl acetate (10 mL). The organic phases were combined, washed with saturated brine (10mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give compounds 001-13. The crude product was used in the next step without purification.
LCMS:MS(ESI)m/z:673.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.42(d,J＝4.8Hz,1H),7.73(d,J＝8.0Hz,1H),7.25-7.14(m,2H),6.69-6.52(m,2H),6.34-6.15(m,1H),5.90(t,J＝16.0Hz,1H),5.78-5.54(m,1H),5.14-4.95(m,2H),4.93-4.68(m,1H),4.51-4.14(m,3H),4.13-3.78(m,2H),3.74-3.42(m,1H),3.36-2.90(m,2H),2.75-2.47(m,1H),1.73-1.62(m,3H),1.45(s,12H),1.17-1.09(m,3H),1.02-0.85(m,3H)。
Step 10: synthesis of Compounds 001-14
In a previously dried 100mL single-necked flask, compound 001-13(377mg, 560.38. mu. mol,1eq), (1, 3-bis (2,4, 6-trimethylphenyl) -2-imidazolidinylidene) dichloro (o-isopropoxybenzylidene) ruthenium (134.64mg, 158.59. mu. mol,0.283eq) and dichloromethane (45mL) were added. After the addition was complete, the reaction was stirred at 45 ℃ for 1.0 hour. The reaction system was directly concentrated. The crude product was isolated by flash column chromatography (petroleum ether: ethyl acetate: methanol: 1:0.02 to 6:1:0.02) and purified to give compounds 001-14.
LCMS:MS(ESI)m/z:631.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.53(d,J＝4.8Hz,1H),7.76(dd,J＝8.4,12.8Hz,1H),7.39-7.26(m,1H),7.00-6.82(m,2H),6.51(t,J＝10.8Hz,1H),6.04-5.68(m,1H),4.81-4.56(m,1H),4.40-4.13(m,2H),4.06-3.86(m,1H),3.84-3.45(m,1H),3.42-2.96(m,2H),2.94-2.68(m,1H),2.37-2.14(m,1H),1.96-1.66(m,2H),1.68-1.54(m,3H),1.50(s,9H),1.31-1.17(m,6H)。
Step 11: synthesis of trifluoroacetate salts of Compounds 001-15
To a pre-dried 100mL three-necked flask, under nitrogen, were added compounds 001-14(0.15g, 237.84. mu. mol,1eq) and dichloromethane (9 mL). After the addition was completed, trifluoroacetic acid (271.19mg,2.38mmol, 176.10. mu.L, 10eq) was added dropwise to the system using a syringe. After the addition was complete, the system was stirred at 25 ℃ for 3 hours. The system was concentrated under reduced pressure to give the trifluoroacetate salt of compounds 001-15. The crude product was used in the next reaction without purification.
LCMS:MS(ESI)m/z:531.3[M+1] + 。
Step 12: synthesis of Compounds 001 and 002
In a previously dried 100mL single neck flask were added compound 001-15(0.26g, 229.10. mu. mol,1.0eq,5.3TFA), N, N' -diisopropylethylamine (296.09mg,2.29mmol, 399.04. mu.L, 10eq) and dichloromethane (10 mL). After the addition was complete, the system was cooled to-60 ℃. Acryloyl chloride (20.74mg, 229.10. mu. mol,1.0eq) was added dropwise to the system using a syringe. After the addition was complete the reaction was stirred at-60 ℃ for 2 minutes. And (6) detecting. Water (10mL) was slowly added to the system, and the mixture was allowed to stand for liquid separation. After separation, the organic phase was collected and the aqueous phase was extracted with dichloromethane (10mL 1). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was purified by preparative separation (separation method: column: Phenomenex Gemini-NX 80 x 40mM x 3 μm; mobile phase: [ water (10mM ammonium bicarbonate) -acetonitrile ]; (B (acetonitrile)%: 25% -55%, 8 min). The fractions were lyophilized. The product was resolved by SFC (separation method: column: DAICEL CHIRALPAK AD (250mm x 30mm,10 μm), mobile phase: [ 0.1% ammonia-ethanol ], B (ethanol)%: 45% -45%, 12min) to obtain compounds 001 and 002.
Compound 001: peak position of chiral column: 1.16min
LCMS:MS(ESI)m/z:585.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝4.8Hz,1H),7.70(d,J＝8.4Hz,1H),7.31(dd,J＝8.4,15.2Hz,1H),6.90(d,J＝8.4Hz,1H),6.86(t,J＝8.4Hz,1H),6.80(d,J＝4.8Hz,1H),6.66-6.48(m,1H),6.44(d,J＝10.4Hz,1H),6.35(d,J＝16.8Hz,1H),5.75(d,J＝10.4Hz,1H),5.73-5.63(m,1H),4.90-4.62(m,2H),4.58(dd,J＝2.4,9.6Hz,1H),4.42(d,J＝12.0Hz,1H),4.15(t,J＝10.4Hz,1H),4.03-3.76(m,1H),3.71-3.32(m,2H),3.21-3.04(m,1H),2.87-2.73(m,1H),1.61-1.53(m,3H),1.23(d,J＝6.8Hz,3H),0.92(d,J＝6.4Hz,3H)。
Compound 002: peak position of chiral column: 1.37min
LCMS:MS(ESI)m/z:585.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝4.8Hz,1H),7.73(d,J＝8.0Hz,1H),7.31(dd,J＝8.0,15.2Hz,1H),6.91(d,J＝8.0Hz,1H),6.86(t,J＝8.4Hz,1H),6.81(d,J＝4.8Hz,1H),6.65-6.42(m,2H),6.40-6.29(m,1H),5.79-5.65(m,2H),5.31-4.41(m,3H),4.30-3.96(m,2H),3.91-3.16(m,3H),2.98-2.72(m,1H),1.38-1.31(m,3H),1.23(d,J＝6.8Hz,3H),0.98-0.84(m,3H)。
Example 2
The synthetic route is as follows:
step 1: synthesis of Compound 003-1
A35 mL hydrogenation flask, purged with argon, was charged with palladium on carbon (0.2g, 10% purity). After the addition was complete, methanol (20mL) and 001-14(0.24g, 380.54. mu. mol,1eq) were added to the system. After the addition was complete, the system was replaced 3 times with hydrogen. The system was stirred at 25 deg.C (20psi) for 12 hours under hydrogen atmosphere. The system was filtered through a layer of celite. Concentration gave compound 003-1. The crude product was used in the next reaction without purification.
LCMS:MS(ESI)m/z:633.3[M+1] + 。
Step 2: synthesis of trifluoroacetate salt of Compound 003-2
Under nitrogen, compound 003-1(0.24g, 379.33. mu. mol,1eq) and methylene chloride (5mL) were added to a previously dried 100mL single-neck flask. After the addition was complete, trifluoroacetic acid (432.51mg,3.79mmol, 280.85. mu.L, 10eq) was added dropwise to the system using a syringe. After the addition was complete, the system was stirred at 25 ℃ for 1.0 hour. The system was concentrated under reduced pressure to give the trifluoroacetate salt of compound 003-2. The crude product was used in the next reaction without purification.
LCMS:MS(ESI)m/z:533.3[M+1] + 。
And step 3: synthesis of Compounds 003 and 004
In a previously dried 100mL single neck flask were added compound 003-2(0.420g, 376.60. mu. mol,1.0eq, converted to 5.11 trifluoroacetates), N, N' -diisopropylethylamine (486.72mg,3.77mmol, 655.95. mu.L, 10eq) and dichloromethane (10 mL). After the addition was complete, the system was cooled to-60 ℃. Acryloyl chloride (34.09mg, 376.60. mu. mol,1.0eq) was added dropwise to the system using a syringe. After the addition was complete the reaction was stirred at-60 ℃ for 2 minutes. Water (10mL) was slowly added to the system, and the mixture was allowed to stand for liquid separation. After separation, the organic phase was collected and the aqueous phase was extracted with dichloromethane (10 mL). The organic phases were combined, dried over anhydrous sodium sulfate and concentrated under reduced pressure to give the crude product. The crude product was isolated by preparative separation (separation method: column: Welch Xtimate C18150 mm 25mm 5 μm; mobile phase: [ water (0.04% hydrochloric acid) -acetonitrile ]; B (acetonitrile)%: 20% -35%, 8min), and the fractions were lyophilized. The product was separated by SFC (separation method: column: REGIS (s, s) WHELK-O1(250 mm. times.50 mm,10 μm); mobile phase: [ 0.1% ammonia-ethanol ]; B (ethanol)%: 55% -55%, 6min), fraction concentration, and lyophilization with deionized water to obtain 003 (i.e., compound WX001 of calculation example 1) and 004.
Compound 003: peak position of chiral column: 3.157min
LCMS:MS(ESI)m/z:587.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝5.2Hz,1H),7.72(d,J＝8.4Hz,1H),7.29(td,J＝6.8,8.0Hz,1H),7.04(d,J＝5.2Hz,1H),6.83(d,J＝8.4Hz,1H),6.76(t,J＝8.8Hz,1H),6.65-6.39(m,1H),6.35(dd,J＝1.6,16.8Hz,1H),5.75(d,J＝1.2,10.0Hz,1H),4.88-4.36(m,3H),4.33-4.26(m,1H),4.01-3.74(m,1H),3.72-3.30(m,3H),3.24-3.03(m,1H),2.91-2.78(m,1H),2.55-2.45(m,1H),2.43-2.31(m,1H),2.25-2.01(m,2H),1.31-1.10(m,6H),0.97(d,J＝6.8Hz,3H)。
Compound 004: peak position of chiral column: 3.836min
LCMS:MS(ESI)m/z:587.2[M+1] + .
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝5.2Hz,1H),7.76(br d,J＝8.4Hz,1H),7.34-7.24(m,1H),7.04(d,J＝4.8Hz,1H),6.83(d,J＝8.0Hz,1H),6.75(t,J＝8.8Hz,1H),6.63-6.45(m,1H),6.35(dd,J＝1.6,16.8Hz,1H),5.75(dd,J＝1.6,10.4Hz,1H),5.42-4.95(m,1H),4.80-4.45(m,1H),4.34-4.25(m,1H),4.17-4.07(m,1H),3.94-3.79(m,1H),3.78-3.64(m,1H),3.63-3.52(m,1H),3.50-3.20(m,1H),2.92-2.82(m,1H),2.56-2.45(m,1H),2.44-2.30(m,1H),2.25-2.00(m,2H),1.30(d,J＝6.4Hz,3H),1.24(d,J＝6.4Hz,3H),1.18(s,1H),0.96(br d,J＝5.2Hz,3H)。
Example 3
The synthetic route is as follows:
step 1: synthesis of Compound 005-1
Under nitrogen protection, compound 001-12(0.37g, 584.80. mu. mol,1eq) and compound 4-bromo-1-butene (789.49mg,5.85mmol, 593.60. mu.L, 10eq) were dissolved in acetonitrile (20mL), and potassium carbonate (181.86mg,1.32mmol,2.25eq) and potassium iodide (189.30mg,1.14mmol,1.95eq) were added and reacted at 80 ℃ for 5 hours. The compound 4-bromo-1-butene (394.74mg,2.92mmol, 296.80. mu.L, 5 eq.) was added and reacted at 80 ℃ for 5 hours. Water (20mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (20mL × 2) and separated. The combined organic phases were washed with brine (30mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by flash column chromatography (mobile phase: methanol/dichloromethane 0.2% -0.8%), and then purified to obtain the compound 005-1.
LCMS:MS(ESI)m/z:687.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.51(d,J＝5.2Hz,1H),7.79(t,J＝8.8Hz,1H),7.34-7.28(m,2H),6.70(d,J＝8.4Hz,2H),6.40-6.31(m,1H),5.98(t,J＝16.8Hz,1H),5.65(s,1H),4.99-4.96(m,3H),4.47-4.24(m,2H),4.14-4.06(m,1H),3.98-3.89(m,2H),3.71-3.60(m,1H),3.35-3.15(m,2H),2.73-2.69(m,1H),2.32-2.27(m,2H),1.76(t,J＝6.8Hz,3H),1.60(s,6H),1.53(s,9H),1.05-0.97(m,3H)。
Step 2: synthesis of Compound 005-2
Under nitrogen protection, 005-1(0.29g, 422.25. mu. mol,1eq) was dissolved in dichloromethane (45mL), and (1, 3-bis (2,4, 6-trimethylphenyl) -2-imidazolidinylidene) dichloro (o-isopropoxybenzylidene) ruthenium (71.70mg, 84.45. mu. mol,0.2eq) was added and reacted at 45 ℃ for 1.5 hours. The reaction was completed at 45 ℃ for 1.5 hours. Directly decompressing and concentrating the reaction system to obtain a crude product. The crude product is separated by flash column chromatography (mobile phase: methanol/dichloromethane 0.2% -1.5%), and then purified to obtain a compound 005-2.
LCMS:MS(ESI)m/z:645.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.54(d,J＝5.2Hz,1H),7.77(dd,J＝19.6,8.0Hz,1H),7.38-7.32(m,2H),6.74(t,J＝8.8Hz,1H),6.66(d,J＝8.4Hz,1H),6.12-6.05(m,1H),6.02-5.97(m,1H),4.70-4.64(m,1H), 4.34-4.27(m,1H),4.19(d,J＝8.8Hz,1H),4.12-4.08(m,1H),4.01-3.96(m,1H),3.91(t,J＝9.2Hz,1H),3.85-3.74(m,1H),2.95-2.91(m,1H),2.49-2.35(m,2H),1.91-1.75(m,2H),1.59(s,6H),1.52(s,9H),1.34(d,J＝6.4Hz,3H)。
And step 3: synthesis of Compound 005-3
Compound 005-2(280.00mg, 434.30. mu. mol,1eq) was dissolved in methanol (30mL), palladium on carbon (0.6g, 10% purity) was added, hydrogen gas was substituted three times, and then the reaction was carried out at 50 ℃ for 15 hours under a hydrogen atmosphere (pressure: 50 Psi). The reaction was not complete by LCMS and was continued for 5 hours at 50 ℃. Directly filtering the reaction system, and concentrating the filtrate under reduced pressure to obtain a compound 005-3. The crude product was used in the next reaction without purification.
LCMS:MS(ESI)m/z:647.2[M+1] + .
1 H NMR(400MHz,CDCl 3 )δ＝8.48(d,J＝4.8Hz,1H),7.85-7.82(m,1H),7.35(q,J＝8.0Hz,1H),7.02(d,J＝5.2Hz,1H),6.84(d,J＝8.4Hz,1H),6.80(t,J＝8.8Hz,1H),5.01-4.84(m,1H),4.31-4.26(m,1H),3.94-3.89(m,3H),3.74-3.61(m,1H),3.39-3.13(m,3H),2.97-2.87(m,1H),2.69-2.57(m,1H),2.49-2.42(m,1H),1.58(s,6H),1.53(s,9H),1.20(dd,J＝6.8,3.6Hz,4H),0.96(t,J＝6.8Hz,3H)。
And 4, step 4: synthesis of trifluoroacetate salt of Compound 005-4
Under nitrogen, 005-3(0.24g, 371.10. mu. mol,1eq) was dissolved in dichloromethane (24mL), and trifluoroacetic acid (2.12g,18.56mmol,1.37mL,50eq) was added to react at 20 ℃ for 3 hours. The reaction system is directly concentrated under reduced pressure to obtain the trifluoroacetate of the compound 005-4. The reaction system is used directly in the next reaction without purification.
LCMS:MS(ESI)m/z:547.2[M+1] + .
And 5: synthesis of Compounds 005 and 006
Under nitrogen protection, compound 005-4(0.45g, 334.63. mu. mol,1eq, calculated as 7 trifluoroacetates) was dissolved in dichloromethane (30mL), N' -diisopropylethylamine (518.97mg,4.02mmol, 699.42. mu.L, 12eq) was added, followed by acryloyl chloride (45.43mg, 501.94. mu. mol, 40.93. mu.L, 1.5eq) and reacted at 60 ℃ for 0.5 hour. A saturated sodium bicarbonate solution (20mL) was added to the reaction system, and the mixture was separated. The aqueous phase was extracted with dichloromethane (20mL x 3) and the layers were separated. The combined organic phases were washed with brine (30mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by HPLC (column: Phenomenex luna C1880: 40 mm: 3 μm; mobile phase: [ water (0.04% hydrochloric acid) -acetonitrile ]; B (acetonitrile)%: 18% -36%, 7min), and then further separated by SFC (column: DAICEL CHIRALPAK IG (250 mm: 30mm,10 μm); mobile phase: [ 0.1% ammonia-ethanol ]; B (ethanol)%: 60% -60%, 3min), and purified to give compound 005 (i.e., compound WX006 of calculation example 1) and compound 006, respectively.
Compound 005: peak position of chiral column: 1.839min
LCMS:MS(ESI)m/z:601.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝4.8Hz,1H),7.84(d,J＝8.4Hz,1H),7.35(q,J＝8.4Hz,1H),7.02(d,J＝5.2Hz,1H),6.84(d,J＝8.4Hz,1H),6.80(t,J＝8.8Hz,1H),6.69-6.56(m,1H),6.42(dd,J＝16.8,1.2Hz,1H),5.82(dd,J＝10.4,1.6Hz,1H),5.02-4.66(m,2H),4.54-4.41(m,1H),4.06-3.88(m,3H),3.78-3.56(m,2H),3.27-3.11(m,1H),2.92-2.84(m,1H),2.68-2.58(m,1H),2.50-2.43(m,1H),1.53(d,J＝6.4Hz,3H),1.48-1.35(m,4H),1.20(d,J＝6.8Hz,3H),0.96(s,3H)。
Compound 006: peak position of chiral column: 2.180min
LCMS:MS(ESI)m/z:601.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.51(d,J＝5.2Hz,1H),7.86(d,J＝8.0Hz,1H),7.36(q,J＝6.8Hz,1H),7.07(d,J＝4.8Hz,1H),6.84(d,J＝8.4Hz,1H),6.80(t,J＝8.8Hz,1H),6.67-6.57(m,1H),6.42(dd,J＝16.4,0.8Hz,1H),5.83(dd,J＝10.4,1.6Hz,1H),4.91-4.77(m,1H),4.55-4.45(m,1H),4.32-4.07(m,1H),3.97-3.87(m,3H),3.76-3.62(m,2H),3.34-3.04(m,1H),2.92(d,J＝11.6Hz,1H),2.67-2.59(m,1H),2.52-2.45(m,1H),1.47(s,7H),1.22(d,J＝6.4Hz,3H),0.97(s,3H)。
Example 4
The synthetic route is as follows:
step 1: synthesis of trifluoroacetate salt of Compound 007-1
Under nitrogen protection, compound 005-2(160.00mg, 248.17. mu. mol,1eq) was dissolved in dichloromethane (16mL), trifluoroacetic acid (1.41g,12.41mmol, 918.75. mu.L, 50eq) was added, and the reaction was carried out at 20 ℃ for 3 hours. The reaction system is directly decompressed and concentrated to obtain the trifluoroacetate of the crude compound 007-1, and the trifluoroacetate can be directly used for the next reaction without purification.
LCMS:MS(ESI)m/z:545.2[M+1] + 。
Step 2: synthesis of Compounds 007 and 008 and 009 and 010
Under nitrogen protection, compound 007-1(0.19g, 208.91. mu. mol,1eq, reduced to 3.2 trifluoroacetate salts) was dissolved in dichloromethane (20mL), N' -diisopropylethylamine (270.01mg,2.09mmol, 363.89. mu.L, 10eq) was added, followed by acryloyl chloride (28.36mg, 313.37. mu. mol, 25.55. mu.L, 1.5eq) and reacted at 60 ℃ for 0.5 hour. A saturated sodium bicarbonate solution (20mL) was added to the reaction system, and the mixture was separated. The aqueous phase was extracted with dichloromethane (20mL x 3) and the layers were separated. The organic phases were combined, washed with brine (30mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by HPLC (column: Phenomenex luna C1880: 40 mm: 3 μm; mobile phase: [ water (0.04% hydrochloric acid) -acetonitrile ]; B (acetonitrile)%: 25% -35%, 7min), and purified to give two pure products, configuration 1 (retention time t ═ 5min) and configuration 2 (retention time t ═ 6.5 min). Configuration 1 was separated and purified by SFC (column: REGIS (s, s) WHELK-O1(250 mm. times.50 mm,10 μm); mobile phase: 0.1% ammonia-methanol; B (methanol)%: 62.5% -62.5%, 3min) to give compound 007 (chiral column peak position: 2.008min) and compound 008 (chiral column peak position: 2.516min), respectively. Configuration 2 was separated and purified by SFC (column: REGIS (s, s) WHELK-O1(250 mm. times.50 mm,10 μm); mobile phase: [ 0.1% ammonia-methanol ]; B (methanol)%: 50% -50%, 3min) to give compound 009 (chiral column peak position: 1.963min) and compound 010 (chiral column peak position: 2.151min), respectively. Compound 007: peak position of chiral column: 2.008min
LCMS:MS(ESI)m/z:599.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.54(d,J＝5.2Hz,1H),7.76(d,J＝8.0Hz,1H),7.38-7.32(m,2H),6.74(t,J＝8.8Hz,1H),6.68-6.55(m,2H),6.42(d,J＝16.8Hz,1H),6.11-6.04(m,1H),6.01-5.97(m,1H),5.83(d,J＝10.8Hz,1H),4.85-4.69(m,2H),4.59-4.49(m,1H),4.19(d,J＝9.2Hz,1H),4.04-3.89(m,2H),3.74-3.47(m,2H),3.24-3.15(m,1H),2.94-2.88(m,1H),2.48-2.35(m,2H),1.66(s,3H),1.33(d,J＝6.8Hz,3H),1.02(d,J＝6.8Hz,3H)。
Compound 008: peak position of chiral column: 2.516min
LCMS:MS(ESI)m/z:599.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.55(d,J＝5.2Hz,1H),7.80(d,J＝8.0Hz,1H),7.38-7.32(m,2H),6.74(t,J＝8.4Hz,1H),6.68-6.58(m,2H),6.42(dd,J＝18.0,1.2Hz,1H),6.12-6.05(m,1H),6.02-5.98(m,1H),5.83(dd,J＝10.4,1.6Hz,1H),5.35-5.04(m,1H),4.82-4.58(m,1H),4.19(d,J＝8.8Hz,2H),3.94-3.89(m,2H),3.78(d,J＝14.0Hz,1H),3.48-3.38(m,1H),2.92(s,2H),2.48-2.39(m,2H),1.39(d,J＝6.8Hz,3H),1.34(d,J＝6.8Hz,3H),1.01(s,3H)。
Compound 009: peak position of chiral column: 1.963min
LCMS:MS(ESI)m/z:599.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.58(d,J＝4.8Hz,1H),7.76(d,J＝8.8Hz,1H),7.35(q,J＝6.8Hz,1H),7.05(d,J＝4.8Hz,1H),6.82(d,J＝8.4Hz,1H),6.75(t,J＝8.4Hz,1H),6.70-6.56(m,1H),6.42(d,J＝ 16.8Hz,1H),6.20(s,1H),5.83(d,J＝10.4Hz,1H),5.62(t,J＝9.6Hz,1H),4.94-4.76(m,2H),4.53-4.42(m,2H),4.07-3.88(m,2H),3.72-3.51(m,2H),3.25-3.15(m,1H),2.95-2.88(m,1H),2.85-2.78(m,1H),2.23(d,J＝14.4Hz,1H),1.31(d,J＝6.8Hz,3H),1.26(s,3H),1.02(s,3H)。
Compound 010: peak position of chiral column: 2.151min
LCMS:MS(ESI)m/z:599.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.60(d,J＝4.8Hz,1H),7.78(d,J＝8.4Hz,1H),7.36(q,J＝7.2Hz,1H),7.07(d,J＝4.4Hz,1H),6.82(d,J＝8.4Hz,1H),6.75(t,J＝8.8Hz,1H),6.68-6.57(m,1H),6.42(d,J＝16.0Hz,1H),6.22(d,J＝10.4Hz,1H),5.83(dd,J＝10.8,1.6Hz,1H),5.65(t,J＝11.2Hz,1H),4.98-4.71(m,1H),4.44(d,J＝11.6Hz,1H),4.35-4.21(m,1H),4.06-3.87(m,3H),3.76-3.50(m,2H),3.05-2.82(m,3H),2.24(d,J＝16.0Hz,1H),1.33(d,J＝6.8Hz,3H),1.26(s,3H),1.03(s,3H)。
Example 5
The synthetic route is as follows:
step 1: synthesis of Compounds 011-3
Under nitrogen protection, compound 011-1(8g,41.45mmol,1eq) and compound 001-2(6.58g,37.31mmol,0.9eq) were dissolved in N, N-dimethylformamide (120mL), and cesium carbonate (27.01g,82.91mmol,2eq) was added and reacted at 60 ℃ for 12 hours. Water (150mL) was added to the reaction system, and the aqueous phase was extracted with methyl tert-ether (300mL × 2) and separated. The organic phases were combined, washed with saturated brine (200mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by a column chromatography (mobile phase: ethyl acetate/petroleum ether is 3.0% -20.0%) to obtain compound 011-3.
LCMS:MS(ESI)m/z:333.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.33(d,J＝6.0Hz,1H),7.06(d,J＝5.6Hz,1H),4.27(t,J＝4.4Hz,2H),3.97(t,J＝4.8Hz,2H),0.87(s,9H),0.06(s,6H)。
Step 2: synthesis of Compound 011-4
Under nitrogen protection, compound 011-3(10.2g,30.64mmol,1eq) and isopropenylpotassium trifluoroborate (4.76g,32.18mmol,1.05eq) were dissolved in 1, 4-dioxane (100mL) and water (10mL), potassium carbonate (6.35g,45.97mmol,1.5eq) and 1,1' -bis (diphenylphosphino) ferrocene ] dichloropalladium (448.45mg, 612.88. mu. mol,0.02eq) were added, and reaction was carried out at 100 ℃ for 12 hours. The two reaction systems were combined, water (150mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (200mL × 3) and separated. The organic phases were combined, washed with brine (200mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by a column chromatography (mobile phase: ethyl acetate/petroleum ether is 2.0% -20.0%) to obtain compound 011-4.
LCMS:MS(ESI)m/z:339.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝5.6Hz,1H),6.95(d,J＝5.6Hz,1H),5.34(s,1H),5.25(s,1H),4.23(t,J＝4.8Hz,2H),3.97(t,J＝4.8Hz,2H),2.17(s,3H),0.88(s,9H),0.07(s,6H)。
And 3, step 3: synthesis of Compound 011-5
Compound 011-4(10.1g,29.84mmol,1eq) was dissolved in ethanol (150mL), palladium on carbon (5g, 10% pure, hydrogen (60.28mg,29.84mmol,1eq) was added for three times, followed by reaction at 30 ℃ for 20 hours under hydrogen atmosphere (50Psi), the reaction was filtered directly through celite, the filter cake was washed with ethanol (400mL), and the filtrate was concentrated under reduced pressure to give compound 011-5, which was used directly in the next reaction without purification.
LCMS:MS(ESI)m/z:311.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝7.98(d,J＝5.2Hz,1H),6.62(d,J＝5.6Hz,1H),4.12(t,J＝4.8Hz,2H),4.00(t,J＝5.2Hz,2H),3.74(br s,2H),3.09-3.02(m,1H),1.32(s,3H),1.30(s,3H),0.92(s,9H),0.11(s,6H)。
And 4, step 4: synthesis of Compound 011-6
Under nitrogen protection, compound 001-5(3g,14.35mmol,1eq) was dissolved in tetrahydrofuran (30mL), a solution of oxalyl chloride (7.29g,57.41mmol,5.03mL,4eq) in tetrahydrofuran (6mL) was added at 0 ℃ and the temperature was raised to 75 ℃ for reaction for 2 hours. The reaction mixture was directly concentrated under reduced pressure, and then tetrahydrofuran (30mL) was added thereto, and a solution of compound 011-5(4.06g,13.06mmol,0.91eq) in tetrahydrofuran (1mL) was slowly added thereto at 0 ℃ to react at 0 ℃ for 2 hours. The reaction was quenched by slowly adding a saturated ammonium chloride solution (30mL) and a saturated aqueous salt solution (30mL), and the aqueous phase was extracted with ethyl acetate (100mL × 3) and separated. The organic phases were combined, washed with saturated brine (50mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. Adding petroleum ether to the crude product: ethyl acetate 3:1 organic phase (60mL), stirred for 1 hour, filtered and the filter cake dried under reduced pressure to give compound 011-6.
LCMS:MS(ESI)m/z:545.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝9.69(s,1H),8.60(d,J＝5.6Hz,1H),8.06-8.00(m,1H),7.03(s,1H),4.28(t,J＝4.4Hz,2H),4.00(t,J＝4.8Hz,2H),3.40-3.33(m,1H),1.41(d,J＝6.0Hz,6H),0.86(s,9H),0.05(s,6H)。
And 5: synthesis of Compounds 011-7
Under nitrogen protection, compound 011-6(4.2g,7.70mmol,1eq) was dissolved in tetrahydrofuran (63mL), potassium hexamethyldisilazide (1M in tetrahydrofuran, 18.48mL,2.4eq) was added slowly at 0 ℃ and then reacted at 20 ℃ for 2 hours. As the reaction was not completed, hexamethyldisilazane (1M tetrahydrofuran solution, 2.31mL,0.3eq) was added thereto at 0 ℃ and reacted at 20 ℃ for 2 hours. The reaction was quenched by adding saturated ammonium chloride solution (50mL), and the aqueous phase was extracted with ethyl acetate (3 × 100mL) and separated. The combined organic phases were washed with brine (100mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated and purified by a column chromatography (mobile phase: ethyl acetate/petroleum ether is 5.0% -75.0%) to obtain compound 011-7.
LCMS:MS(ESI)m/z:509.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.62(d,J＝6.0Hz,1H),8.42(br s,1H),8.21(d,J＝6.8Hz,1H),6.86(d,J＝5.6Hz,1H),4.12-4.05(m,2H),3.82-3.74(m,2H),2.82-2.75(m,1H),1.24(d,J＝6.8Hz,3H),1.18(d,J＝6.8Hz,3H),0.77(s,9H),-0.07(s,3H),-0.10(s,3H)。
And 6: synthesis of Compounds 011-8
Under nitrogen protection, compound 011-7(1.2g,2.36mmol,1eq) was added to phosphorus oxychloride (2.05g,13.37mmol,1.24mL,5.67eq), followed by N, N-diisopropylethylamine (1.52g,11.79mmol,2.05mL,5eq) and reacted at 40 ℃ for 3 hours. The compound 011-8 is obtained, and the reaction system is used for the next reaction without purification.
And 7: synthesis of Compounds 011-9
Under the protection of nitrogen, compounds 011 to 8(1.2g,2.27mmol,1eq) are dissolved in tetrahydrofuran (18mL) at 0 ℃, N-diisopropylethylamine (8.82g,68.25mmol,11.89mL,30eq) is added, then compounds 001 to 9(1.37g,6.82mmol,3eq) are added, and the temperature is slowly raised to 25 ℃ for reaction for 1 hour. The reaction system was added to ice water (30mL), and the aqueous phase was extracted with ethyl acetate (50mL × 3) and separated. The organic phases were combined, washed with saturated brine (60mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by a column chromatography (mobile phase: ethyl acetate/petroleum ether: 30.0% -100.0%) and purified to obtain compound 011-9.
LCMS:MS(ESI)m/z:691.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.59(d,J＝5.6Hz,1H),7.74(dd,J＝7.6,3.2Hz,1H),6.85(d,J＝5.6Hz,1H),4.86-4.77(m,1H),4.29-4.20(m,1H),4.10-3.90(m,4H),3.79-3.54(m,3H),3.32-3.07(m,2H),2.78-2.66(m,1H),1.52(s,9H),1.48-1.45(m,3H),1.27-1.23(m,3H),1.15(t,J＝6.8Hz,3H),0.75(d,J＝2.0Hz,9H),-0.10(d,J＝3.2Hz,3H),-0.15(d,J＝9.2Hz,3H)。
And 8: synthesis of Compounds 011-10
Under nitrogen protection, compounds 011-9(1.1g,1.59mmol,1eq) and 001-11(322.53mg,2.07mmol,1.3eq) were dissolved in 1, 4-dioxane (22mL) and water (5.5mL), potassium phosphate (675.52mg,3.18mmol,2eq) and 1,1' -bis (di-tert-butylphosphine) ferrocene ] dichloropalladium (II) (103.71mg, 159.12. mu. mol,0.1eq) were added and reacted at 80 ℃ for 4 hours. Water (50mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (60mL × 3) and separated. The combined organic phases were washed with brine (60mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by a column chromatography (mobile phase: methanol/dichloromethane ═ 0.5% to 3.0%) and purified to obtain compound 011-10.
LCMS:MS(ESI)m/z:767.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝9.39(d,J＝5.6Hz,1H),8.61(d,J＝6.0Hz,1H),7.86(dd,J＝11.6,9.6Hz, 1H),7.32-7.28(m,1H),7.14(q,J＝7.6Hz,1H),6.90(d,J＝6.0Hz,1H),5.17-4.92(m,1H),4.75(s,1H),4.57-4.47(m,1H),4.34-4.11(m,1H),4.09-3.89(m,4H),3.70-3.66(m,2H),3.39-3.20(m,2H),2.95-2.85(m,1H),1.53(s,9H),1.45(d,J＝6.8Hz,2H),1.30-1.26(m,4H),1.09(dd,J＝6.8,5.2Hz,3H),0.71(d,J＝2.0Hz,9H),-0.16(d,J＝3.6Hz,3H),-0.20(d,J＝9.2Hz,3H)。
And step 9: synthesis of Compounds 011-11
Under nitrogen protection, compound 011-10(1.3g,1.70mmol,1eq) was dissolved in tetrahydrofuran (60mL), and tetramethylammonium fluoride (789.40mg,8.48mmol,5eq) was added and reacted at 65 ℃ for 5 hours. The reaction system was directly concentrated under reduced pressure, then water (20mL) was added, the aqueous phase was extracted with ethyl acetate (20mL × 4), the layers were separated, the organic phases were combined, washed with saturated brine (20mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to give compound 011-11. The reaction system does not need to be purified and can be directly used for the next reaction.
LCMS:MS(ESI)m/z:653.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.59(d,J＝5.2Hz,1H),7.84(dd,J＝12.0,9.2Hz,1H),7.25-7.22(m,1H),6.93(d,J＝4.8Hz,1H),6.69(d,J＝8.4Hz,1H),6.63(t,J＝8.8Hz,1H),4.71-4.47(m,1H),4.27-4.23(m,2H),4.17-3.98(m,4H),3.84-3.73(m,2H),3.65(s,1H),3.52-3.25(m,2H),2.94-2.93(m,1H),1.52(s,9H),1.31-1.20(m,6H),1.10(dd,J＝6.8,4.0Hz,3H)。
Step 10: synthesis of Compounds 011-12
Under nitrogen protection, compound 011-11(0.94g,1.44mmol,1eq) was dissolved in tetrahydrofuran (75mL), triphenylphosphine (1.51g,5.76mmol,4eq) was added, diethyl azodicarboxylate (1.00g,5.76mmol,1.05mL,4eq) was added at 20 ℃, and reaction was carried out at 20 ℃ for 15 hours. Water (50mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (100mL × 3) and separated. The combined organic phases were washed with brine (50mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated and purified by a column chromatography (mobile phase: ethyl acetate/petroleum ether is 30.0-100.0%, methanol/dichloromethane is 2.0-10.0%) to obtain compound 011-12.
LCMS:MS(ESI)m/z:635.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(dd,J＝5.6,0.8Hz,1H),7.77(dd,J＝16.8,8.4Hz,1H),7.41-7.36(m,1H),6.90-6.86(m,2H),6.69(dd,J＝6.0,2.0Hz,1H),4.69-4.66(m,1H),4.55(d,J＝12.8Hz,1H),4.40-4.30(m,2H),4.23-4.17(m,2H),4.08-3.97(m,2H),3.47-3.14(m,3H),3.07-3.00(m,1H),1.52(s,9H),1.36-1.34(m,3H),1.29-1.27(m,3H),1.06(dd,J＝6.8,5.6Hz,3H)。
Step 11: synthesis of trifluoroacetate salt of Compounds 011-13
Compound 011-12(1.05g,1.65mmol,1eq) was dissolved in dichloromethane (40mL) under nitrogen protection, and trifluoroacetic acid (9.43g,82.72mmol,6.12mL,50eq) was added and reacted at 30 ℃ for 2 hours. The reaction system is directly concentrated under reduced pressure to obtain the trifluoroacetate of the compound 011-13. The reaction system does not need to be purified and can be directly used for the next reaction.
LCMS:MS(ESI)m/z:535.2[M+1] + 。
Step 12: synthesis of Compounds 011 and 012
Under nitrogen protection, compound 011-13(2g,1.55mmol,1eq, equivalent to 6.6 trifluoroacetate) was dissolved in dichloromethane (60mL), N-diisopropylethylamine (3.21g,24.86mmol,4.33mL,16eq) was added, followed by acryloyl chloride (281.28mg,3.11mmol, 253.40. mu.L, 2eq) and reacted at-60 ℃ for 10 min. A saturated sodium bicarbonate solution (50mL) was added to the reaction system, and the mixture was separated. The aqueous phase was extracted with dichloromethane (50mL x 3) and the layers were separated. The combined organic phases were washed with brine (50mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by HPLC (column: Phenomenex luna c18250 mm × 100mm × 10 μm; mobile phase: [ water (0.05% hydrochloric acid) -acetonitrile ];% acetonitrile: 15% -45%, 20min) and purified to give the pure product. The pure product was separated and purified by SFC (column: DAICEL CHIRALCEL OD (250 mm. times.30 mm,10 μm); mobile phase: [ 0.1% ammonia-ethanol ]; ethanol%: 50% -50%, 15min) to give compounds 011 and 012, respectively.
Compound 011: peak position of chiral column: 1.420min
LCMS:MS(ESI)m/z:589.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.54(d,J＝5.2Hz,1H),7.78(d,J＝8.4Hz,1H),7.43-7.37(m,1H),6.91-6.87(m,2H),6.80(d,J＝4.8Hz,1H),6.69-6.59(m,1H),6.41(dd,J＝16.8,1.6Hz,1H),5.82(dd,J＝10.4,1.2Hz,1H),4.87-4.69(m,2H),4.60(d,J＝13.6Hz,2H),4.49-4.34(m,2H),4.16(t,J＝10.4Hz,1H),4.03-3.87(m,1H),3.76-3.45(m,2H),3.28-3.17(m,1H),3.08-3.01(m,1H),1.67-1.58(m,3H),1.39(d,J＝6.4Hz,3H),1.11(d,J＝6.8Hz,3H)。
Compound 012: peak position of chiral column: 1.719min
LCMS:MS(ESI)m/z:589.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.55(d,J＝5.2Hz,1H),7.82(d,J＝8.0Hz,1H),7.43-7.37(m,1H),6.91-6.87(m,2H),6.82(d,J＝5.6Hz,1H),6.67-6.57(m,1H),6.42(dd,J＝16.8,1.6Hz,1H),5.82(dd,J＝10.4,1.6Hz,1H),5.38-5.10(m,1H),4.78-4.60(m,2H),4.45-4.34(m,2H),4.19-4.09(m,2H),3.99-3.75(m,2H),3.46-3.38(m,1H),3.06-2.92(m,2H),1.40(d,J＝6.8Hz,3H),1.37(d,J＝6.8Hz,3H),1.11(d,J＝5.2Hz,3H)。
Example 6
The synthetic route is as follows:
step 1: synthesis of Compound 013-1
Under nitrogen protection, the compound 001-3(5g,29.30mmol,1eq) and potassium vinyltrifluoroborate (5.89g,43.95mmol,1.5eq) were dissolved in dioxane (108mL) and water (12mL), cesium carbonate (28.64g,87.90mmol,3eq) and (2-dicyclohexylphosphino-2 ', 4', 6 '-triisopropyl-1, 1' -biphenyl) [2- (2 '-amino-1, 1' -biphenyl) ] palladium (II) chloride (1.15g,1.47mmol,0.05eq) were added, and the reaction was carried out at 90 ℃ for 6 hours. Water (50mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (100mL × 3) and separated. The combined organic phases were washed with brine (100mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by a column chromatography (mobile phase: ethyl acetate/petroleum ether 2.0% -10.0%) and purified to obtain the compound 013-1.
LCMS:MS(ESI)m/z:163.1[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.03(d,J＝4.8Hz,1H),7.01(d,J＝4.8Hz,1H),6.76(dd,J＝17.2,10.8Hz,1H),5.77(dd,J＝17.2,1.2Hz,1H),5.50(dd,J＝11.2,1.2Hz,1H),3.77(br s,2H),3.10-3.01(m,1H),1.33(s,3H),1.32(s,3H)。
Step 2: synthesis of Compound 013-2
Under nitrogen protection, compound 001-5(4g,19.14mmol,1eq) was dissolved in tetrahydrofuran (65mL), oxalyl chloride (9.72g,76.55mmol,6.70mL,4eq) was added at 0 deg.C in tetrahydrofuran (13mL), and the temperature was raised to 75 deg.C for 2 hours. The reaction mixture was directly concentrated under reduced pressure, and then tetrahydrofuran (65mL) was added, and a solution of compound 013-1(2.30g,14.16mmol,0.74eq) in tetrahydrofuran (13mL) was slowly added at 0 ℃ to react at 0 ℃ for 2 hours. The reaction was quenched by slowly adding a saturated ammonium chloride solution (50mL) and a saturated saline solution (50mL), then ethyl acetate (200mL) was added, and filtered to obtain a filter cake. After separating the filtrate, the aqueous phase was extracted with ethyl acetate (200mL 4), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was mixed with the filter cake, and concentrated under reduced pressure to give a crude product. Adding methanol (60mL) into the crude product, pulping, stirring for 1 hour, filtering, and concentrating the filtrate under reduced pressure to obtain the compound 013-2.
LCMS:MS(ESI)m/z:397.0[M+1] + 。
1 H NMR(400MHz,CD 3 OD)δ＝8.64(d,J＝6.4Hz,1H),8.23(d,J＝6.4Hz,1H),8.18(d,J＝7.6Hz,1H),7.12(dd,J＝17.2,10.8Hz,1H),6.50(d,J＝17.6Hz,1H),6.04(d,J＝11.2Hz,1H),3.69-3.62(m,1H),1.46(s,3H),1.44(s,3H)。
And 3, step 3: synthesis of Compound 013-3
Under nitrogen protection, compound 013-2(4.5g,11.33mmol,1eq) was dissolved in tetrahydrofuran (60mL), potassium hexamethyldisilazide (1M in tetrahydrofuran, 26.06mL,2.3eq) was slowly added at 0 ℃ and then reacted at 20 ℃ for 3.5 hours. The reaction was quenched by adding saturated ammonium chloride solution (200mL), then ethyl acetate (200mL) was added, filtered through celite, and then the layers were separated. The aqueous phase was extracted with ethyl acetate (3 x 200mL) and the layers were separated. The organic phases were combined, washed with saturated brine (200mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by column chromatography (mobile phase: ethyl acetate/petroleum ether: 5.0% to 50.0%) and purified to obtain compound 013-3.
LCMS:MS(ESI)m/z:361.0[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.72(d,J＝5.2Hz,1H),8.68(br s,1H),8.25(d,J＝6.8Hz,1H),7.43(d,J＝5.2Hz,1H),6.37(dd,J＝17.6,11.2Hz,1H),5.91(d,J＝17.6Hz,1H),5.46(d,J＝11.2Hz,1H),2.79-2.69(m,1H),1.24(d,J＝6.8Hz,3H),1.15(d,J＝6.4Hz,3H)。
And 4, step 4: synthesis of Compound 013-4
Phosphorus oxychloride (6.46g,42.13mmol,3.92mL,5.07eq) was added to compound 013-3(3g,8.32mmol,1eq) followed by diisopropylethylamine (5.37g,41.58mmol,7.24mL,5eq) and reacted at 40 ℃ for 3 h under nitrogen. After decompression and concentration, the compound 013-4 is obtained and can be directly used for the next reaction without purification.
LCMS:MS(ESI)m/z:379.0[M+1] + 。
And 5: synthesis of Compound 013-5
Under the protection of nitrogen, compound 013-4(3.1g,8.17mmol,1eq) was dissolved in tetrahydrofuran (50mL) at 0 ℃, diisopropylethylamine (15.85g,122.62mmol,21.36mL,15eq) was added, compound 001-9(2.46g,12.26mmol,1.5eq) was then added, and the temperature was slowly raised to 25 ℃ for reaction for 1 hour. The reaction was completed at 25 ℃ for 14 hours. The reaction system was added to ice water (50mL), and the aqueous phase was extracted with ethyl acetate (50mL × 3) and separated. The combined organic phases were washed with brine (100mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product is separated by a column chromatography (mobile phase: ethyl acetate/petroleum ether 5.0% -60.0%) and purified to obtain the compound 013-5.
LCMS:MS(ESI)m/z:543.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.67(d,J＝5.2Hz,1H),7.78(d,J＝8.0Hz,1H),7.41(dd,J＝5.2,2.8Hz,1H),6.37-6.26(m,1H),5.87(dd,J＝17.6,7.6Hz,1H),5.36(dd,J＝10.8,6.8Hz,1H),4.89-4.80(m,1H),4.26(s,2H),3.97(s,1H),3.66(br s,1H),3.31-3.09(m,2H),2.68-2.57(m,1H),1.52(s,9H),1.49-1.47(m,3H),1.24(dd,J＝6.4,4.0Hz,3H),1.12(t,J＝6.4Hz,3H)。
Step 6: synthesis of Compound 013-6
Under nitrogen protection, 013-5(1.6g,2.95mmol,1eq) and 001-11(689.11mg,4.42mmol,1.5eq) were dissolved in dioxane (48mL) and water (12mL), potassium phosphate (1.25g,5.89mmol,2eq) and 1,1' -bis (di-tert-butylphosphine) ferrocene ] dichloropalladium (II) (192.03mg, 294.64. mu. mol,0.1eq) were added, and the reaction was carried out at 80 ℃ for 5 hours. Water (50mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (60mL _ 3) and separated. The combined organic phases were washed with brine (60mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by column chromatography (mobile phase: methanol/dichloromethane ═ 0.5% to 3.0%) and purified to give compound 013-6.
LCMS:MS(ESI)m/z:619.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝9.18(d,J＝7.2Hz,1H),8.73(d,J＝5.2Hz,1H),7.91(dd,J＝9.6,6.8Hz,1H),7.48(dd,J＝4.8,1.6Hz,1H),7.31-7.25(m,1H),6.71-6.65(m,2H),6.40-6.28(m,1H),5.92(dd,J＝17.2,7.6Hz,1H),5.38(dd,J＝10.8,8.8Hz,1H),5.04-4.84(m,1H),4.48(s,1H),4.30(d,J＝12.4Hz,1H),4.02(s,2H),3.81-3.65(m,2H),3.50-3.01(m,3H),2.88-2.79(m,1H),1.53(s,9H),1.27(dd,J＝6.8,3.6Hz,3H),1.07(t,J＝6.0Hz,3H)。
And 7: synthesis of Compounds 013-8
Under nitrogen protection, 013-6(0.5g, 808.18. mu. mol,1eq) and 013-7(2.65g,16.16mmol, 166.73. mu.L, 20eq) were dissolved in acetonitrile (20mL), and potassium iodide (402.47mg,2.42mmol,3eq) and potassium carbonate (335.10mg,2.42mmol,3eq) were added and reacted at 80 ℃ for 15 hours. As the reaction was not completed, 013-7(1.33g,8.08mmol,10eq), potassium iodide (268.32mg,1.62mmol,2eq) and potassium carbonate (223.39mg,1.62mmol,2eq) were added and the reaction was carried out at 80 ℃ for 15 hours. The reaction was not completed, and the reaction was carried out at 80 ℃ for 25 hours. Water (50mL) was added to the reaction system, and the aqueous phase was extracted with ethyl acetate (100mL × 2) and separated. The combined organic phases were washed with brine (50mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by column chromatography (mobile phase: methanol/dichloromethane ═ 0.5% to 2.0%) and purified to give compound 013-8.
LCMS:MS(ESI)m/z:687.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.57(d,J＝5.2Hz,1H),7.81-7.75(m,1H),7.35-7.30(m,2H),6.69(d,J＝8.4Hz,2H),6.40-6.30(m,1H),5.84-5.77(m,1H),5.41-5.31(m,2H),5.06-4.90(m,2H),4.41-4.27(m,2H),4.18-3.93(m,3H),3.77-3.60(m,1H),3.40-3.12(m,2H),2.76(s,1H),2.29-2.09(m,2H),1.53(s,9H),1.26-1.24(m,6H),1.11-1.05(m,3H),1.00-0.93(m,3H)。
And 8: synthesis of Compounds 013-9
Under nitrogen protection, compound 013-8(0.46g, 669.78. mu. mol,1eq) was dissolved in dichloromethane (70mL), and 1, 3-bis (2,4, 6-trimethylphenyl) -2- (imidazolidinylidene) (dichlorobenzylidene) (tricyclohexylphosphine) ruthenium (113.73mg, 133.96. mu. mol,0.2eq) was added and reacted at 45 ℃ for 3 hours. Directly decompressing and concentrating the reaction system to obtain a crude product. The crude product was separated by column chromatography (mobile phase: methanol/dichloromethane ═ 0.2% to 2.0%) and purified to give compound 013-9.
LCMS:MS(ESI)m/z:659.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.58-8.52(m,1H),7.78(dd,J＝17.2,8.0Hz,1H),7.38-7.29(m,3H),6.71-6.65(m,2H),6.02-5.83(m,1H),5.55-4.89(m,1H),4.72-4.47(m,2H),4.40-4.24(m,2H),4.09-3.64(m,3H),3.51-2.92(m,3H),1.53(s,9H),1.40-1.34(m,6H),1.09-1.06(m,3H),1.03-1.00(m,3H)。
And step 9: synthesis of Compounds 013-10
Compound 013-9(0.31g, 470.60. mu. mol,1eq) was dissolved in methanol (60mL), palladium on carbon (0.2g, purity: 10% p, hydrogen (950.61. mu.g, 470.60. mu. mol,1eq) was added three times, and then reacted at 50 ℃ for 20 hours under a hydrogen atmosphere (50Psi), the reaction system was directly filtered through celite, the filtrate was concentrated under reduced pressure to give a crude product, the crude product was separated by HPLC (column: Phenomex luna C18250 mm 50mm 10. mu.m; mobile phase: [ water (0.04% hydrochloric acid) -acetonitrile ]; acetonitrile%: 40% -60%, 10min), and purified to give compound 013-10.
LCMS:MS(ESI)m/z:661.2[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.55-8.48(m,1H),7.85-7.78(m,1H),7.35-7.29(m,1H),7.00(d,J＝5.6Hz,1H),6.81-6.73(m,2H),4.83-4.51(m,1H),4.36-3.97(m,4H),3.76-3.55(m,2H),3.40-2.93(m,2H),2.78-2.66(m,1H),2.47-2.20(m,1H),1.53(s,9H),1.47(d,J＝6.8Hz,3H),1.23-1.20(m,6H),1.15(d,J＝6.0Hz,3H),1.08-1.05(m,2H),0.99-0.96(m,2H)。
Step 10: synthesis of trifluoroacetate salt of Compounds 013-11
Compound 013-10(0.22g, 332.95. mu. mol,1eq) was dissolved in dichloromethane (9mL) under nitrogen, trifluoroacetic acid (1.90g,16.65mmol,1.23mL,50eq) was added and the reaction was carried out for 1 h at 20 ℃. The reaction system is directly concentrated under reduced pressure to obtain the trifluoroacetate salt of the compound 013-11. The reaction system does not need to be purified and can be directly used for the next reaction.
LCMS:MS(ESI)m/z:561.2[M+1] + 。
Step 11: synthesis of Compounds 013,014,015 and 016
Under nitrogen protection, compound 013-11(0.3g, 295.06. mu. mol,1eq, reduced to 4 trifluoroacetate salts) was dissolved in dichloromethane (15mL), diisopropylethylamine (610.15mg,4.72mmol, 822.30. mu.L, 16eq) was added, followed by acryloyl chloride (53.41mg, 590.13. mu. mol, 48.12. mu.L, 2eq) and reacted at 60 ℃ for 10 min. A saturated sodium bicarbonate solution (20mL) was added to the reaction system, and the mixture was separated. The aqueous phase was extracted with dichloromethane (50mL x 3) and the layers were separated. The combined organic phases were washed with brine (50mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product. The crude product was separated by HPLC (column: Welch Xtimate C18100 × 25mm × 3 μm; mobile phase: [ water (0.05% hydrochloric acid) -acetonitrile ]; acetonitrile%: 15% -35%, 8min) and purified to give the pure product. The pure product was separated and purified by SFC (column: DAICEL CHIRALPAK IG (250 mm. times.30 mm,10 μm); mobile phase: [ 0.1% ammonia-ethanol ]; ethanol%: 55% -55%, min.) to give compound 013, compound 015, compound 016 and mixture A, respectively. Mixture A was again separated and purified by SFC (column: DAICEL CHIRALPAK IG (250 mm. times.30 mm,10 μm); mobile phase: [ 0.1% ammonia-ethanol ]; ethanol%: 45% -45%, min) to give compound 014 and compound 013.
013 chiral column peak position: 1.682min
LCMS:MS(ESI)m/z:615.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.55(d,J＝5.2Hz,1H),7.80(d,J＝8.4Hz,1H),7.32-7.28(m,1H),7.15(d,J＝5.2Hz,1H),6.82-6.77(m,2H),6.63(s,1H),6.42(dd,J＝16.4,1.6Hz,1H),5.83(dd,J＝10.4,1.6Hz,1H),4.87-4.71(m,2H),4.62-4.50(m,1H),4.07-3.89(m,1H),3.78-3.45(m,3H),3.29-3.18(m,1H),2.95-2.88(m,1H),2.51-2.44(m,1H),2.22-2.16(m,1H),1.87-1.80(m,1H),1.66(s,3H),1.44-1.31(m,2H),1.28(d,J＝6.4Hz,4H),1.15(d,J＝6.4Hz,3H),1.07(d,J＝6.4Hz,3H)。
014, peak position of chiral column: 1.796min
LCMS:MS(ESI)m/z:615.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.49(d,J＝4.8Hz,1H),7.83(d,J＝8.0Hz,1H),7.36-7.30(m,1H),7.00(d,J＝4.8Hz,1H),6.78-6.74(m,2H),6.70-6.56(m,1H),6.42(dd,J＝16.8,1.6Hz,1H),5.83(dd,J＝10.4,1.6Hz,1H),5.01-4.79(m,1H),4.76-4.67(m,1H),4.55-4.43(m,1H),4.37-4.31(m,1H),4.07-3.87(m,1H),3.74-3.51(m,2H),3.27-3.13(m,1H),2.71-2.65(m,2H),2.48-2.40(m,1H),1.58-1.55(m,4H),1.26-1.25(m,1H),1.22(s,3H),1.21(s,3H),1.18-1.13(m,2H),0.97(s,3H)。
015, chiral column peak position: 2.049min
LCMS:MS(ESI)m/z:615.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.58(d,J＝5.2Hz,1H),7.84(d,J＝8.0Hz,1H),7.33-7.29(m,1H),7.21(d,J＝4.4Hz,1H),6.82-6.78(m,2H),6.69-6.57(m,1H),6.43(dd,J＝16.8,1.6Hz,1H),5.83(dd,J＝10.4,1.6Hz,1H),5.41-5.10(m,1H),4.81-4.56(m,1H),4.23-4.09(m,2H),3.99-3.89(m,1H),3.82-3.75(m,2H),3.55-3.36(m,1H),3.02-2.88(m,1H),2.55-2.47(m,1H),2.28-2.21(m,1H),1.90-1.81(m,1H),1.44(s,1H),1.41(d,J＝6.8Hz,3H),1.30(d,J＝6.8Hz,3H),1.26(s,2H),1.16(d,J＝6.4Hz,3H),1.09(s,3H)。
016, peak position of chiral column: 2.342min
LCMS:MS(ESI)m/z:615.3[M+1] + 。
1 H NMR(400MHz,CDCl 3 )δ＝8.71(d,J＝5.6Hz,1H),7.92(d,J＝8.0Hz,1H),7.40(d,J＝4.4Hz,1H),7.38-7.34(m,1H),6.79-6.75(m,2H),6.62(s,1H),6.42(dd,J＝16.8,1.6Hz,1H),5.83(dd,J＝10.4,2.0Hz,1H),5.23-4.97(m,1H),4.79-4.76(m,1H),4.53-4.50(m,1H),4.44-4.32(m,2H),4.09-3.91(m,1H),3.76-3.62(m,2H),3.51-3.34(m,1H),3.10-3.00(m,1H),2.66-2.58(m,1H),1.65-1.57(m,2H),1.51-1.46(m,3H),1.34(d,J＝6.8Hz,3H),1.29-1.21(m,5H),1.08(s,3H)。
Biological test data:
experimental example 1: MIA-PA-CA-2 cell assay
1. Experimental materials:
DMEM medium, fetal bovine serum from Biosera and horse serum from Gibco. CellTiter-Glo (cell viability chemiluminescence detection reagent) reagent was purchased from Promega. MIA-PA-CA-2 cell line was purchased from Nanjing Kebai Biotech Co. EnVision multi-label analyzer (PerkinElmer).
2. The experimental method comprises the following steps:
MIA-PA-CA-2 cells were seeded in white 96-well plates, 80. mu.L of cell suspension per well, containing 1000 MIA-PA-CA-2 cells. The cell plate was placed in a carbon dioxide incubator overnight.
The test compounds were diluted 5-fold with a calandria to the 8 th concentration, i.e. from 2mM to 26nM, setting up a duplicate well experiment. Add 78. mu.L of medium to the intermediate plate, transfer 2. mu.L of each well of the gradient dilution compound to the intermediate plate according to the corresponding position, mix well and transfer 20. mu.L of each well to the cell plate. The concentration of compound transferred to the cell plate ranged from 10. mu.M to 0.13 nM. The cell plates were incubated in a carbon dioxide incubator for 3 days. A separate cell plate was prepared, and the signal values were read on the day of drug addition as maximum values (Max values in the following equation) for data analysis. To each well of this cell plate, 50. mu.L of a cell viability chemiluminescence detection reagent was added, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. Reading with a multi-label analyzer.
3. And (3) data analysis:
the raw data was converted to inhibition, IC, using the equation (sample-min)/(max-min) × 100% 50 Values of (d) can be obtained by curve fitting of four parameters (obtained from the "log (inhibitor)/response-variable scope" model in GraphPad Prism software). Table 3 provides the inhibitory activity of the compounds of the invention on MIA-PA-CA-2 cell proliferation.
TABLE 3 in vitro screening test results for the compounds of the invention
And (4) conclusion: the compound of the invention shows better inhibition activity on the cell proliferation of MIA-PA-CA-2, wherein the compound 005 (namely WX006 of the calculation example 1) has the cell inhibition activity IC on the MIA-PA-CA-2 50 2.7nM, significantly better than the in vitro activity of AMG 510. The results were compared with those obtained in calculation example 1 by the computational chemical methodConclusion (same structural compounds WX006 and KRAS with lower energy barrier difference of Delta E) G12C The actual binding of the protein, possibly exhibiting similar or superior binding activity to that of the reference compound AMG 510) was highly consistent, fully demonstrating the accuracy of the computational chemistry of the present invention. Therefore, compounds with lower delta E energy barrier differences predicted according to the computational chemistry methods of the present invention will exhibit similar or superior inhibitory activity in cell proliferation as the reference compound, AMG 510.
Test example 2: in vivo pharmacokinetic Studies
Pharmacokinetic study of oral and intravenous test Compounds in SD mice
The test compound was mixed with 10% dimethylsulfoxide/60% polyethylene glycol 400/30% aqueous solution, vortexed and sonicated to prepare a 1mg/mL clear solution, which was filtered through a microporous membrane for use. Male SD mice, two per group, were selected at 7 to 10 weeks of age. The candidate compound solution was administered intravenously at a dose of 3mg/kg for AMG510 and 2mg/kg for compound 003. Candidate compound solutions were administered orally at a dose of 10 mg/kg. Whole blood was collected for a certain period of time, plasma was prepared, drug concentration was analyzed by LC-MS/MS method, and drug parameters were calculated using Phoenix WinNonlin software (Pharsight, USA). The results of the experiment are shown in table 4:
TABLE 4 pharmacokinetic results of the test compounds
And (4) conclusion of results: the total systemic exposure, peak concentration and bioavailability of the 003 compound after oral administration were superior to that of the reference compound AMG510 at the same administration dose, exhibiting excellent pharmacokinetic properties.
Test example 3: in vivo pharmacodynamic study
In vivo pharmacodynamic study of human pancreatic cancer Mia PaCa-2 cell Nude mouse subcutaneous transplantation tumor Balb/c Nude mouse model
1. Cell culture and tumor tissue preparation
Cell culture: human pancreatic cancer Mia PaCa-2 cells (ATCC-CRL-1420) are cultured in vitro in a monolayer way under the culture condition that 20 percent of fetal bovine serum and 1 percent of double antibody are added into a DMEM/F12 culture medium and cultured in a 5 percent carbon dioxide incubator at 37 ℃. Passage was performed twice a week with conventional digestion treatment with pancreatin-EDTA. When the saturation degree of the cells is 80-90 percent and the quantity reaches the requirement, collecting the cells, counting, suspending in a proper amount of PBS (phosphate buffer solution), adding matrigel at a ratio of 1:1, and obtaining the cell density of 25 multiplied by 10 6 cells/mL of cell suspension.
Cell inoculation: 0.2mL (5X 10) 6 cells/mouse) Mia PaCa-2 cells (added with matrigel and the volume ratio of 1:1) are subcutaneously inoculated on the right back of each mouse, and the average tumor volume reaches 190mm 3 At that time, random groupings were made based on tumor volume and dosing was initiated according to the protocol in table 5.
TABLE 5 Experimental animal groups and dosing regimens
Note: PO represents oral administration; QD stands for once daily.
2. Tumor measurement and Experimental indices
Tumor diameters were measured twice weekly using a vernier caliper. The tumor volume was calculated as: v is 0.5a × b 2 And a and b represent the major and minor diameters of the tumor, respectively.
The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). The relative tumor proliferation rate T/C (%) ═ TRTV/CRTV × 100% (TRTV: treatment group RTV; CRTV: negative control group RTV). Relative Tumor Volume (RTV) is calculated from the tumor measurement results, and the calculation formula is RTV ═ Vt/V0, where V0 is the average tumor volume measured at the time of group administration (i.e. D0), Vt is the average tumor volume at a certain time of measurement, and TRTV and CRTV take the same day data.
TGI (%), reflecting the rate of tumor growth inhibition. TGI (%) × (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group))/(average tumor volume at the end of treatment in the solvent control group-average tumor volume at the start of treatment in the solvent control group) ] × 100%.
3. Results of the experiment
The results of the experiment are shown in FIGS. 28 and 29.
The results show that: when the drug is administrated for 21 days, the tumor volume of the tumor-bearing mice of the solvent control group reaches 1572mm 3 Mean tumor volume values of test substance compound 003(5mg/kg), compound 003(10mg/kg) and compound 003(30mg/kg) were 46mm, respectively 3 、111mm 3 And 14mm 3 (ii) a T/C is 2.79%, 8.27% and 0.90% respectively; the TGI was 110.37%, 105.70% and 112.71%, respectively, and significantly inhibited tumor growth at all three concentrations (p values less than 0.001). The mean tumor volumes of the test substances AMG510(5mg/kg) and AMG510(10mg/kg) were 229mm 3 And 86mm 3 T/C of 14.90% and 5.81%, TGI of 97.17% and 107.51%, p values both<0.001, also has obvious tumor inhibiting effect, and the weight of each dose group of the mouse is stable without obvious intolerance.
Test example 4: in vivo pharmacodynamic study
In vivo pharmacodynamic study of human non-small cell lung cancer NCI-H358 cell Nude mouse subcutaneous transplantation tumor Balb/c Nude mouse model
1. Cell culture and tumor tissue preparation
Cell culture: human non-small cell lung cancer NCI-H358 in vitro monolayer culture is carried out under the culture condition that 20% fetal bovine serum and 1% double antibody are added into a DMEM/F12 culture medium and cultured in a 5% carbon dioxide incubator at 37 ℃. Passage was performed twice a week with conventional digestion treatment with pancreatin-EDTA. When the cell saturation is 80% -90% and the quantity reaches the requirement, collecting cells, counting, resuspending in a proper amount of PBS, adding matrigel at a ratio of 1:1, and obtaining the cell density of 25 × 10 6 cells/mL of cell suspension.
Cell inoculation: 0.2mL (5X 10) 6 cells/mouse) NCI-H358 cells (with matrigel added, volume ratio 1:1) were subcutaneously inoculated into the right hind dorsal area of each mouse, and the mean tumor volume reached 100-150mm 3 At that time, random groupings were made based on tumor volume and dosing was initiated according to the protocol in table 6.
TABLE 6 Experimental animal groups and dosing regimens
Note: PO represents oral administration; QD stands for once daily.
2. Tumor measurement and Experimental indices
Tumor diameters were measured twice weekly using a vernier caliper. The formula for tumor volume is: v is 0.5a × b 2 And a and b represent the major and minor diameters of the tumor, respectively.
The tumor suppressor therapeutic effect of the compound was evaluated as TGI (%) or relative tumor proliferation rate T/C (%). Relative tumor proliferation rate T/C (%) (TRTV/CRTV × 100% (TRTV: treatment group RTV; CRTV: negative control group RTV). Relative Tumor Volume (RTV) is calculated from the tumor measurement results, and the calculation formula is RTV ═ Vt/V0, where V0 is the mean tumor volume measured at the time of group administration (i.e. D0), Vt is the mean tumor volume at a certain time of measurement, and TRTV and CRTV take the same day data.
TGI (%), reflecting the rate of tumor growth inhibition. TGI (%) × (1- (average tumor volume at the end of administration of a certain treatment group-average tumor volume at the start of administration of the treatment group))/(average tumor volume at the end of treatment in the solvent control group-average tumor volume at the start of treatment in the solvent control group) ] × 100%.
3. Results of the experiment
The results of the experiment are shown in FIGS. 30 and 31.
The results show that: when the drug is administrated for 28 days, the tumor volume of the tumor-bearing mice of the solvent control group reaches 559mm 3 Mean tumor volume values of test compound 003(1.5mg/kg), compound 003(5mg/kg) and compound 003(15mg/kg) were 307mm, respectively 3 、138mm 3 And 50mm 3 (ii) a T/C is 55%, 24% and 9% respectively; TGI 54%, 91% and 111%, respectively, at three concentrationsCan obviously inhibit the growth of the tumor (p values are all less than 0.001). Mean tumor volume of test substance AMG510(5mg/kg) is 248mm 3 T/C of 44%, TGI of 68%, p values<0.001, also has obvious tumor inhibiting effect, and the weight of each dose group of the mouse is stable without obvious intolerance.
Claims (17)
- A compound of formula (I) or a pharmaceutically acceptable salt thereof,
wherein the content of the first and second substances,R 1 selected from H, F, Cl, Br, I and CH 3 Said CH 3 Optionally substituted by 1, 2 or 3R a Substitution;R 2 selected from H, F, Cl, Br and I;R 3 selected from H, F, Cl, Br, I and C 1-3 Alkyl radical, said C 1-3 Alkyl is optionally substituted by 1, 2 or 3R b Substitution;R 4 selected from H, F, Cl, Br and I;R 5 is selected from C 1-6 Alkyl and cyclopropyl, said C 1-6 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c Substitution;L 1 is selected from- (CH) 2 ) m -、-(CH 2 ) m -NR 6 -、-(CH 2 ) m -O-、-NR 6 -(CH 2 ) m -NR 6 -、-NR 6 -(CH 2 ) m -O-、-O-(CH 2 ) m -O-and- (CH) 2 ) n -(CH＝CH) p -(CH 2 ) m -O-, said- (CH) 2 ) m -、-(CH 2 ) m -NR 6 -、-(CH 2 ) m -O-、-NR 6 -(CH 2 ) m -NR 6 -、-NR 6 -(CH 2 ) m -O-、-O-(CH 2 ) m -O-and- (CH) 2 ) n -(CH＝CH) p -(CH 2 ) m -O-is optionally substituted by 1, 2 or 3R d Substitution;m is selected from 1, 2, 3, 4, 5 and 6;n is selected from 0 and 1;p is selected from 1 and 2;R 6 is selected from H and CH 3 ；Each R a 、R b And R c Each independently selected from H, F, Cl, Br and I;R d selected from H, F, Cl, Br, I and CH 3 ；The carbon atom with "-" is a chiral carbon atom, and exists in the form of (R) or (S) single enantiomer or enriched in one enantiomer. - The compound according to claim 1, or a pharmaceutically acceptable salt thereof, selected from
Wherein R is 1 、R 2 、R 3 、R 4 、R 5 And L 1 As defined in claim 1. - A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, whichIn, R 1 Is selected from H.
- A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 2 Is selected from H.
- A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 3 Is selected from CH 3 。
- A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 4 Is selected from F.
- A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein R 5 Is selected from C 1-4 Alkyl and cyclopropyl, said C 1-4 Alkyl and cyclopropyl optionally substituted by 1, 2 or 3R c And (4) substitution.
- A compound according to claim 7, or a pharmaceutically acceptable salt thereof, wherein R 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、The CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、
Optionally substituted by 1, 2 or 3R c And (4) substitution.
- The compound according to claim 8 or a pharmaceutical thereofThe above acceptable salt, wherein, R 5 Is selected from CH 3 、CH 2 CH 3 、CH 2 CH 3 CH 3 、
- A compound according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein m is selected from 1, 2, 3, 4 and 5.
- A compound according to claim 10, or a pharmaceutically acceptable salt thereof, wherein L 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 4 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH ═ CH) - (CH) 2 ) 3 -O-, said- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 4 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH ═ CH) - (CH) 2 ) 3 -O-is optionally substituted by 1, 2 or 3R d Substitution。
- A compound according to claim 11, or a pharmaceutically acceptable salt thereof, wherein L 1 Is selected from- (CH) 2 ) 4 -、-(CH 2 ) 5 O-、-(CH 2 ) 4 O-、-(CH 2 ) 3 O-、-(CH 2 ) 3 NH-、-O(CH 2 ) 2 O-、-NH(CH 2 ) 2 O-、-O(CH 2 ) 3 O-、-NH(CH 2 ) 3 O-、-(CH＝CH)-CH 2 -O-、-(CH＝CH)-(CH 2 ) 2 -O-and- (CH) 2 ) 3 CH(CH 3 )O-。
- A compound according to any one of claims 1 to 12, or a pharmaceutically acceptable salt thereof, selected from
Wherein the content of the first and second substances,R 1 、R 2 、R 3 、R 4 and R 5 As defined in claim 1;m1 is selected from 3, 4 and 5;m2 is selected from 1, 2 and 3;R d as defined in claim 1. - A compound represented by the following formula or a pharmaceutically acceptable salt thereof,
- the compound according to claim 14 or a pharmaceutically acceptable salt thereof selected from
- The compound according to claim 14 or a pharmaceutically acceptable salt thereof selected from
- Use of a compound according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a disease associated with a KRAS inhibitor.
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| JP2022517222A (en) | 2019-01-10 | 2022-03-07 | ミラティ セラピューティクス， インコーポレイテッド | KRAS G12C inhibitor |
| KR20220071193A (en) | 2019-08-29 | 2022-05-31 | 미라티 테라퓨틱스, 인크. | KRAS G12D inhibitor |
| MX2022003537A (en) | 2019-09-24 | 2022-07-11 | Mirati Therapeutics Inc | Combination therapies. |
| AU2020372881A1 (en) | 2019-10-28 | 2022-06-09 | Merck Sharp & Dohme Llc | Small molecule inhibitors of KRAS G12C mutant |
| BR112022012106A2 (en) | 2019-12-20 | 2022-09-20 | Mirati Therapeutics Inc | SOS1 INHIBITORS |
| AU2021344830A1 (en) | 2020-09-03 | 2023-04-06 | Revolution Medicines, Inc. | Use of SOS1 inhibitors to treat malignancies with SHP2 mutations |
| IL301298A (en) | 2020-09-15 | 2023-05-01 | Revolution Medicines Inc | Indole derivatives as ras inhibitors in the treatment of cancer |
| WO2022133345A1 (en) | 2020-12-18 | 2022-06-23 | Erasca, Inc. | Tricyclic pyridones and pyrimidones |
| CN116249703A (en) * | 2020-12-21 | 2023-06-09 | 上海和誉生物医药科技有限公司 | Macrocyclic K-RAS G12C inhibitor, preparation method and application thereof |
| CR20230570A (en) | 2021-05-05 | 2024-01-22 | Revolution Medicines Inc | Ras inhibitors |
| AR125787A1 (en) | 2021-05-05 | 2023-08-16 | Revolution Medicines Inc | RAS INHIBITORS |
| TW202246287A (en) * | 2021-05-06 | 2022-12-01 | 大陸商南京明德新藥研發有限公司 | Twelve-membered macrocycle macrocyclic compound |
| AR127308A1 (en) | 2021-10-08 | 2024-01-10 | Revolution Medicines Inc | RAS INHIBITORS |
| TW202340214A (en) | 2021-12-17 | 2023-10-16 | 美商健臻公司 | Pyrazolopyrazine compounds as shp2 inhibitors |
| EP4227307A1 (en) | 2022-02-11 | 2023-08-16 | Genzyme Corporation | Pyrazolopyrazine compounds as shp2 inhibitors |
| WO2023172940A1 (en) | 2022-03-08 | 2023-09-14 | Revolution Medicines, Inc. | Methods for treating immune refractory lung cancer |
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