CN117143175A - Compound for targeted SOS1 protein ubiquitination regulation and preparation method and application thereof - Google Patents

Compound for targeted SOS1 protein ubiquitination regulation and preparation method and application thereof Download PDF

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CN117143175A
CN117143175A CN202311093648.3A CN202311093648A CN117143175A CN 117143175 A CN117143175 A CN 117143175A CN 202311093648 A CN202311093648 A CN 202311093648A CN 117143175 A CN117143175 A CN 117143175A
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秦冲
张思琪
吕燕
马学沛
杨子瑄
李卓悦
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Qingdao Putaike Biomedical Technology Co ltd
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Qingdao Putaike Biomedical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
    • C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a compound for targeted SOS1 protein ubiquitination regulation, a preparation method and application thereof. The compound is composed of an E3 ligase ligand and an SOS1 protein ligand, and optionally further comprises a linker structure, and has good degradation capability on SOS1 protein through in vitro activity tests.

Description

Compound for targeted SOS1 protein ubiquitination regulation and preparation method and application thereof
Technical Field
The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a difunctional compound for targeted SOS1 protein ubiquitination regulation, and a preparation method and application thereof.
Background
Ras proteins (including H-Ras, N-Ras, K-Ras) belong to the small GTPase superfamily, are important nodes in multiple cell signaling pathways, and are closely related to proliferation and differentiation of cells. Ras protein function depends on its GTP-binding state, i.e., the active state of GTP binding and the inactive state of GDP binding. These two states are regulated by GTPase Activating Proteins (GAPs) and Guanylate Exchange Factors (GEFs). By switching between the two states, ras acts as a "switch" in the signal pathway, effecting signal transduction. Ras protein itself has weak GTPase hydrolysis activity, which can be enhanced by GAPs, promoting hydrolysis of GTP to GDP, and converting Ras protein to an inactive state; GEFs can then catalyze the release of GDP from the Ras protein, thereby binding to higher intracellular concentrations of GTP, converting the Ras protein to an inactive state.
Activating mutation of Ras protein is one of the most common oncogenic driving factors in human cancers, especially the K-Ras mutation is mainly about 75% of the Ras mutation, and various cancers such as pancreatic cancer, colorectal cancer, lung adenocarcinoma and the like are involved. After oncogenic mutation of the Ras protein, GTP hydrolysis is impaired, stabilizing it in its active form of GTP binding, leading to sustained activation of downstream signaling pathways, the most common mutation sites of which are amino acids 12, 13, 61. Although Ras proteins are potential research targets for many cancer treatments, they are termed "non-patentable" targets because of their relatively smooth surface, lacking pockets that can bind small molecule compounds. Currently, covalent inhibitors AMG510 and MRTX849 that selectively target KRAS-G12C have entered the clinical research stage, however, to date, there are a variety of mutant Ras-induced cancers for which no drug is available.
SOS1 protein is a GEFs acting on Ras protein, and can promote the exchange of GDP and GTP to activate signal molecule Ras. SOS1 protein has two Ras binding sites, a catalytic site for binding to GDP-Ras and an allosteric site for binding to GTP-Ras, respectively (Jeng, H.H.; taylor, L.J.; bar-Sagi, D.; sos-media cross-activation of wild-type Ras by oncogenic Ras is essential for turigenesis. Nat Commun 2012,3,1168.). Notably, oncogenic mutated Ras promotes SOS 1-mediated activation of wild-type Ras by binding at the allosteric site of the SOS1 protein. Thus, SOS1 protein plays a very important regulatory role in the activation of Ras signaling pathway, especially in the overactivation of signaling pathway caused by oncogenic Ras. Thus, inhibition of proliferation and differentiation of tumor cells by blocking Ras-related signaling pathway through inhibition of SOS1 action may be an effective strategy for treating Ras-driven cancers. SOS1 inhibitors that have been reported to date are BAY-293 and BI-3406, wherein the BI-3406 series of compounds has entered the clinical stage of research, however the therapeutic effect of the compounds alone has not reached the intended goal, which may be related to negative feedback modulation of the signaling pathway and compensatory effects of SOS2, and therapeutic strategies for co-administration are currently being attempted with the compounds (Kessler, D.; gerlach, D.; kraut, N.; mcConnell, D.B.; targeting Son of Sevenless 1:The pacemaker of KRAS.Curr Opin Chem Biol 2021,62,109-118.).
The protein degradation targeting chimera (PROTAC) technology is an emerging drug development strategy in recent years, and the protein targeting degradation agent designed by the strategy can exert the similar catalyst effect in vivo, achieves the aim of treating diseases by degrading pathogenic proteins, has the characteristics of micro high efficiency, is expected to solve the defects of large toxic and side effects, easy drug resistance generation and the like of the traditional small molecule inhibitor, can act on a 'non-patent drug' protein target point, and becomes a bright point for drug innovation research. The protoc molecule is typically composed of three parts, an E3 ubiquitin ligase ligand, a target protein ligand, and a linking structure linking the two, and can pull up the target protein and the E3 ubiquitin ligase in vivo to form a ternary complex, label the target protein with ubiquitin, and then degrade the target protein by the ubiquitin-proteinase system (UPS). The disease is treated by directly regulating and controlling the protein content in the body, and a wide new idea is provided for the research of innovative medicines.
In view of the above, the present application aims to design a degradation agent for synthesizing a targeted SOS1 protein, which can inhibit the Ras-related signal pathway by reducing the content of SOS1 in vivo, thereby inhibiting the development of cancer, in order to solve the problems of no drug availability and poor therapeutic effect of Ras-related diseases.
Disclosure of Invention
The application provides a compound for targeting ubiquitination regulation of SOS1 protein, which is composed of an E3 ligase ligand and an SOS1 protein ligand or further comprises a linker structure and is composed of three parts. In-vitro activity tests prove that the compound has good degradation capability on SOS1 protein.
The present application provides a compound of formula (I), a stereoisomer or a pharmaceutically acceptable salt thereof, having the structure shown below:
wherein: x is X 1 、X 2 Each independently selected from N or C; y is selected from N, S, O, si or P;
the R group is selected from:
wherein: x is selected from N or C; w is selected from S or O; m1 and m2 are each independently integers of 0 to 5;
the R is 1 Selected from H; -OH; -NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Halogen; a substituted or unsubstituted linear or branched C1-C3 alkyl group; a substituted or unsubstituted straight or branched C1-C3 alkenyl group; substituted or unsubstituted, straight or branched C1-C3 alkynyl; a substituted or unsubstituted linear or branched C1-C3 carbonyl group; substituted or unsubstituted C1-C10 cycloalkyl; a substituted or unsubstituted C1-C10 heterocyclyl; substituted or unsubstituted C1-C10 aryl or heteroaryl;
the linking group 1 is selected from: absence of; or a substituted or unsubstituted, linear or branched C1-C12 alkylene group; the optional carbon chain contains 1-4 heteroatoms selected from N, S, O, si or P;
wherein: n1, n2, n3 are each independently integers from 0 to 5; x is X 1 、X 2 、X 3 、X 4 Each independently selected from N or C;
the E3 ligand is selected from:
wherein: m is an integer of 0 to 3;
the R is 2 、R 3 、R 4 And R is 5 Each independently selected from: h is formed; -OH; -NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Halogen; a substituted or unsubstituted linear or branched C1-C6 alkyl group; a substituted or unsubstituted linear or branched C1-C6 cyano group; a substituted or unsubstituted linear or branched C1-C6 alkenyl group; substituted or unsubstituted, straight or branched chain C1-C6 alkynyl; a substituted or unsubstituted linear or branched C1-C6 alkoxy group; a substituted or unsubstituted linear or branched C1-C6 secondary, tertiary or quaternary amine group; a substituted or unsubstituted linear or branched C1-C6 ester group; a substituted or unsubstituted linear or branched C1-C6 amide group; a substituted or unsubstituted linear or branched C1-C6 ketone group; substituted or unsubstituted C1-C10 cycloalkyl; a substituted or unsubstituted C1-C10 heterocyclyl; substituted or unsubstituted C1-C10 aryl or heteroaryl.
The application specifically provides a compound, a stereoisomer or a salt thereof, which is characterized by being selected from the following compounds:
some embodiments of the application relate to a compound of formula I or a stereoisomer, a solvent, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal thereof.
The application also provides a preparation method of the compound.
The application relates to a pharmaceutical composition, which comprises a compound or stereoisomer, solvent compound, prodrug, metabolite, pharmaceutically acceptable salt or eutectic crystal of the compound or the stereoisomer, solvent compound, prodrug, metabolite, pharmaceutically acceptable salt or eutectic crystal of the compound.
The application relates to an application of a compound or a stereoisomer, a solvent compound, a prodrug, a metabolite, a pharmaceutically acceptable salt or a eutectic thereof in preparing a medicament for treating diseases related to SOS1 activity or expression quantity.
The application relates to an application of a compound or stereoisomer, solvent compound, prodrug, metabolite, pharmaceutically acceptable salt or eutectic crystal thereof in preparing a medicine for inhibiting SOS1 activity or reducing SOS1 expression.
The compound or stereoisomer, solvent compound, prodrug, metabolite, pharmaceutically acceptable salt or eutectic crystal thereof can be used as SOS1 degradation agent, and can be used for preventing and/or treating colon cancer, non-small cell lung cancer, pancreatic cancer, rectal cancer, melanoma and multiple myeloma.
The term convention:
"stereoisomers" are compounds having the same chemical composition but different arrangements of atoms or groups in space. It includes "diastereoisomers" and "enantiomers"
"diastereomers" are stereoisomers which have two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, for example: melting point, boiling point, spectral characteristics and reactivity. Mixtures of diastereomers can be separated under high resolution analytical steps such as electrophoresis, crystallization, using, for example, chiral HPLC columns in the presence of resolving agents or chromatography.
"enantiomer" refers to two stereoisomers of a compound that are not overlapping mirror images of each other. The 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur during chemical reactions or treatments without having been stereoselective or stereotactic.
"alkyl" includes both branched and straight chain saturated aliphatic hydrocarbon groups and has the indicated number of carbon atoms, typically from 1 to about 12 carbon atoms. The term C1-C6 alkyl as used herein denotes an alkyl group having from 1 to about 6 carbon atoms. When C0-Cn alkyl is used herein in combination with another group, the named group is exemplified by (phenyl) C0-C4 alkyl, in which case phenyl is directly bonded by a single covalent bond (C0) or linked by an alkyl chain having the named number of carbon atoms (in this case, 1 to about 4 carbon atoms). Examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, and sec-pentyl.
"alkenyl" or "alkenyl" refers to straight and branched hydrocarbon chains that include one or more unsaturated carbon-carbon bonds, which may occur at any stable point along the chain. Alkenyl groups described herein typically have 2 to about 12 carbon atoms. Preferred alkenyl groups are lower alkenyl groups, those alkenyl groups having from 2 to about 8 carbon atoms, such as: C2-C8, C2-C6, and C2-C4 alkenyl. Examples of alkenyl groups include ethenyl, propenyl, and butenyl.
"alkoxy" refers to an alkyl group as defined above having the indicated number of carbon atoms attached through an oxygen bridge. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, 3-hexyloxy, and 3-methylpentyloxy.
The term "heterocycle" means a 5-to 8-membered saturated ring, a partially unsaturated ring, or an aromatic ring containing 1 to about 4 heteroatoms selected from N, O and S and the remaining ring atoms being carbon, or a 7-to 11-membered saturated ring, a partially unsaturated ring, or an aromatic heterocyclic ring system and a 10-to 15-membered tricyclic ring system containing at least 1 heteroatom in a polycyclic system selected from N, O and S and up to about 4 heteroatoms independently selected from N, O and S in each ring in the polycyclic system. Unless otherwise indicated, a heterocycle may be attached to a group that it is substituted at any heteroatom and carbon atom that results in a stable structure. When indicated, the heterocycles described herein may be substituted on carbon or nitrogen atoms, provided that the resulting compounds are stable. The nitrogen atoms in the heterocycle may optionally be quaternized. Preferably the total number of heteroatoms in the heterocyclyl is not more than 4 and preferably the total number of S and O atoms in the heterocyclyl is not more than 2, more preferably not more than 1. Examples of heterocyclyl groups include: pyridyl, indolyl, pyrimidinyl, pyridazinyl (pyridizinyl), pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benzo [ b ] thiophenyl (benzob ] thiophenyl), isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, dihydroisoindolyl, 5,6,7, 8-tetrahydroisoquinolinyl, pyridyl, pyrimidinyl, furanyl, thienyl, pyrrolyl, pyrazolyl, pyrrolidinyl, morpholinyl, piperazinyl, piperidinyl, and pyrrolidinyl.
"aryl" or "heteroaryl" means a stable 5-or 6-membered monocyclic or multicyclic ring comprising 1 to 4, or preferably 1 to 3 heteroatoms selected from N, O and S, and the remaining ring atoms being carbon. When the total number of S and O atoms in the heteroaryl group exceeds 1, these heteroatoms are not adjacent to each other. Preferably, the total number of S and O atoms in the heteroaryl group is not greater than 2. It is particularly preferred that the total number of S and O atoms in the heteroaryl group is not greater than 1. The nitrogen atoms in the heterocycle may optionally be quaternized. These heteroaryl groups may also be substituted with carbon or non-carbon atoms or groups when indicated. Such substitution may include fusion with a 5-to 7-membered saturated cyclic group optionally containing 1 or 2 heteroatoms independently selected from N, O and S, thereby forming, for example, a [1,3] dioxa [4,5-c ] pyridinyl group. Examples of heteroaryl groups include, but are not limited to: pyridyl, indolyl, pyrimidinyl, pyridazinyl, pyrazinyl, imidazolyl, oxazolyl, furanyl, thiophenyl, thiazolyl, triazolyl, tetrazolyl, isoxazolyl, quinolinyl, pyrrolyl, pyrazolyl, benzo [ b ] thiophenyl, isoquinolinyl, quinazolinyl, quinoxalinyl, thienyl, isoindolyl, and 5,6,7, 8-tetrahydroisoquinolinyl.
A "pharmaceutically acceptable salt" or "salt of a compound" is a derivative of the disclosed compound in which the parent compound is prepared by preparing a non-toxic acid or base addition salt thereof.
Drawings
Figure 1 is a dose-dependent of compound 2 on SOS1 degradation.
FIG. 2 is a time dependence of compound 2 on SOS1 degradation.
FIG. 3 shows the proliferation inhibition of NCI-H358 by Compound 2.
Detailed Description
The application will be further illustrated by the following specific examples for a better understanding of the application, but are not to be construed as limiting the application.
The preparation method of the compound or the pharmaceutically acceptable salt thereof comprises the following steps:
synthetic route I
Synthesis of scheme II
The preparation and properties of the compounds of the application are further illustrated below with reference to the examples.
Example 1 preparation of Compound 1
Compound 1 formula: (2S, 4R) -1- ((S) -2- (4- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) butylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
The synthesis process refers to the route I, and the specific synthesis steps are as follows:
s-1-1 (2.26 g,9.7mmol;1.0 eq.) was dissolved in 20mL tetrahydrofuran. (R) -2-methylpropane-2-sulfoxide amide (1.8 g,14.5mmol;1.5 eq.) and Ti (OEt) were then added at room temperature 4 (5.5 g,24.3mmol;2.5 eq.). After stirring overnight at 80 ℃, the reaction mixture was cooled to room temperature and quenched with ice water, extracted with ethyl acetate, the organic phases combined, anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure to obtain a crude product. Purifying the crude product by flash chromatography to obtainPale yellow solid S-1-2, yield: 75%.
S-3-2 (2.45 g,7.3mmol;1.0 eq.) was dissolved in a mixed solution of tetrahydrofuran (50 mL) and water (1 mL) and NaBH was slowly added at-78deg.C 4 (497 mg,13.1mmol;1.8 eq.) then stirred at room temperature for 3h. Quench the reaction with ice water, extract with ethyl acetate, combine the organic phases, anhydrous Na 2 SO 4 And (5) drying. The diastereomer mixture generated by the reaction was separated by flash chromatography to give white solid S-1-3, yield: 87%.
To S-1-3 (2.0 g,6.0 mmol) was added 10mL of a 1, 4-dioxane solution of 4M HCl and stirred at room temperature for 1h. The solvent was removed under reduced pressure to give intermediate S-1-4. Yield: 95%.
To a solution of S-2-1 (14.4 g,50.0mmol;1.0 eq.) in acetonitrile (100 mL) was added methanesulfonic acid (38.0 g,400.0mmol;8.0 eq.). The reaction mixture was stirred at 100 ℃ overnight. After completion of the reaction by UPLC-MS, the mixture was cooled to room temperature. Dropwise adding NaHCO into the reaction mixture 3 And (3) regulating the pH of the aqueous solution to be alkaline, standing for a while, generating precipitate, carrying out suction filtration, and freeze-drying to obtain a crude product S-2-2. Yield: 91%.
To a suspension of S-2-2 (12.7 g,43.0mmol;1.0 eq.) in methylene chloride (100 mL) was added 2,4, 6-triisopropylbenzenesulfonyl chloride (15.6 g,51.6mmol;1.2 eq.), DMAP (254 mg,4.3mmol;0.1 eq.) and Et 3 N (12.9 g,127.8mmol;3.0 eq.). The reaction mixture was stirred at room temperature for 12h. Diluting the solvent with dichloromethane, adding NaHCO 3 Aqueous solution and extracted with DCM. The combined organic layers were treated with anhydrous Na 2 SO 4 Dried, filtered, and the solvent removed in vacuo. The crude product was purified by flash chromatography to give the desired product S-2-3, yield: 60%.
S-2-3 (8.4 g,15.0mmol;1.0 eq.) and S-2-4 (4.1 g,15.0mmol;1.0 eq.) were dissolved in 50mL of isopropanol and stirred overnight at 95 ℃. After UPLC-MS demonstrated complete conversion of the starting material, the mixture was cooled to room temperature. The solvent was removed in vacuo. The crude product was purified by flash chromatography to give intermediate S-2-4, yield: 91%.
Dissolving S-2-4 in 10mL of methanol, adding Pd/C, stirring overnight at room temperature in a hydrogen atmosphere, filtering to remove Pd/C by using diatomite after the reaction is detected by UPLC-MS, evaporating the solvent under reduced pressure, purifying by using a flash chromatographic column to obtain S-2-5, and obtaining the yield: 74%.
S-2-5 (500 mg,1.27mmol;1.0 eq.) was dissolved in 10mL acetonitrile and K was added 2 CO 3 (440 mg,3.19mmol;2.5 eq.) and methyl 4-bromobutyrate (274 mg,1.52mmol;1.2 eq.) were stirred overnight at 70 ℃, after completion of the reaction by UPLC-MS detection, the solvent was evaporated under reduced pressure, diluted with ethyl acetate and water, the organic layer was separated, washed with brine, and dried over anhydrous Na 2 SO 4 Drying and purifying by using a flash chromatographic column to obtain S-2-6, and the yield is: 57%.
S-2-6 (400 mg,0.813mmol;1.0 eq.) was dissolved in 3mL tetrahydrofuran, lithium hydroxide (78 mg,3.25mmol;4.0 eq.) previously dissolved in 1mL water was added, and stirred at room temperature for 2h. After the reaction, the pH of the reaction solution was adjusted to 4-6 with acetic acid, extracted with water and ethyl acetate, and the organic phase was separated with anhydrous Na 2 SO 4 Drying and the solution was removed under vacuum. Intermediate S-2-7 is obtained, yield: 95%.
V-1-1 (100 mg,0.215mmol;1.0 eq.), S-2-7 (52 mg,0.215mmol;1.0 eq.)), HATU (122 mg,0.322mmol;1.5 eq.), and DIPEA (111 mg,0.858mmol;4 eq.)) were dissolved in 3mL of N, N-dimethylformamide, stirred at room temperature for 6h, and after completion of the UPLC-MS detection reaction, the reaction mixture was diluted with water and ethyl acetate. The organic layer was separated, washed with brine, and dried over anhydrous Na 2 SO 4 And (5) drying. The solution was removed under vacuum. Purification by prep-HPLC then gave example 1, yield: 69%. 1 H NMR(400MHz,DMSO-d 6 )δ9.65(d,J＝8.0Hz,1H),8.95(s,1H),8.55(t,J＝6.1Hz,1H),8.01(d,J＝9.3Hz,1H),7.93(s,1H),7.36(q,J＝8.3Hz,4H),7.06(s,1H),6.82(d,J＝4.4Hz,2H),6.71(s,1H),5.66(q,J＝7.2Hz,2H),5.11(s,1H),4.53(d,J＝9.3Hz,1H),4.39(dt,J＝15.2,7.3Hz,2H),4.32(s,1H),4.19(d,J＝5.5Hz,1H),4.15(d,J＝5.2Hz,1H),4.13–4.04(m,2H),3.94(s,3H),3.64(d,J＝30.0Hz,2H),2.54(s,3H),2.40(s,3H),2.29(s,1H),1.97(d,J＝19.6Hz,4H),1.92–1.81(m,1H),1.59(d,J＝7.0Hz,3H),0.88(s,9H)。
Example 2 preparation of Compound 2
The formula of compound 2: (2S, 4R) -1- ((S) -2- (5- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) pentylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme I, the specific synthetic procedure was the same as example 1 except that methyl 4-bromobutyrate was replaced with methyl 5-bromovalerate in example 1. Total yield: 1.3%. 1 H NMR(400MHz,DMSO-d 6 )δ9.64(d,J＝7.9Hz,1H),8.95(s,1H),8.56(t,J＝6.2Hz,1H),8.03–7.77(m,2H),7.36(q,J＝8.4Hz,4H),7.06(s,1H),6.84(d,J＝8.7Hz,2H),6.72(d,J＝1.9Hz,1H),5.68(p,J＝7.1Hz,2H),5.10(s,1H),4.52(d,J＝9.3Hz,1H),4.40(dt,J＝12.7,7.2Hz,2H),4.31(s,1H),4.19(d,J＝5.4Hz,1H),4.15(d,J＝5.4Hz,1H),4.09(d,J＝8.4Hz,2H),3.93(s,3H),3.62(d,J＝10.7Hz,2H),2.54(s,3H),2.40(s,3H),2.33(d,J＝6.5Hz,1H),2.20(dt,J＝14.1,7.0Hz,1H),1.99(q,J＝10.9,9.8Hz,1H),1.86(ddd,J＝12.9,8.8,4.5Hz,1H),1.76(d,J＝6.6Hz,2H),1.71–1.61(m,2H),1.59(d,J＝7.0Hz,3H),0.90(s,9H).
Example 3 preparation of Compound 3
The formula of compound 3: (2S, 4R) -1- ((S) -2- (6- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) hexylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme I, the specific synthetic procedure was the same as example 1 except that methyl 4-bromobutyrate was replaced with methyl 6-bromohexanoate in example 1. Total yield: 1.9%. 1 H NMR(400MHz,DMSO-d 6 )δ9.63(d,J＝7.9Hz,1H),8.95(s,1H),8.56(t,J＝6.1Hz,1H),7.96–7.85(m,2H),7.41–7.31(m,4H),7.08(s,1H),6.83(d,J＝7.0Hz,2H),6.71(s,1H),5.67(p,J＝7.2Hz,1H),4.51(d,J＝9.3Hz,1H),4.44–4.35(m,2H),4.31(s,1H),4.17(dd,J＝15.9,5.5Hz,1H),4.11–4.04(m,2H),3.92(s,3H),3.65–3.58(m,2H),2.54(s,3H),2.40(s,3H),2.33–2.23(m,1H),2.19–2.08(m,1H),2.04–1.75(m,4H),1.59(d,J＝7.0Hz,3H),1.56–1.49(m,1H),1.45–1.36(m,2H),0.89(s,9H)。
Example 4 preparation of Compound 4
The formula of compound 4: (2S, 4R) -1- ((S) -2- (7- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) heptanamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme I, the specific synthetic procedure was the same as example 1 except that methyl 4-bromobutyrate was replaced with methyl 7-bromoheptanoate in example 1. Total yield: 2.1%. 1 H NMR(400MHz,DMSO-d 6 )δ8.95(s,1H),8.56(t,J＝6.1Hz,1H),7.91–7.84(m,2H),7.43–7.31(m,4H),7.08(s,1H),6.85–6.80(m,2H),6.70(s,1H),5.65(p,J＝7.5Hz,1H),4.51(d,J＝9.4Hz,1H),4.45–4.35(m,2H),4.31(s,1H),4.17(dd,J＝16.0,5.4Hz,1H),4.09–4.04(m,2H),3.91(s,3H),2.51(s,3H),2.40(s,3H),2.31–2.20(m,1H),2.15–1.82(m,3H),1.80–1.71(m,2H),1.57(d,J＝7.0Hz,3H),1.46–1.38(m,3H),1.33–1.27(m,2H),0.89(s,9H)。
Example 5 preparation of Compound 5
The formula of compound 5: (2S, 4R) -1- ((S) -2- (8- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) octylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme I, the specific synthetic procedure was the same as example 1 except that methyl 4-bromobutyrate was replaced with methyl 8-bromooctanoate in example 1. Total yield: 2.7%. 1 H NMR(400MHz,DMSO-d 6 )δ9.62(d,J＝8.1Hz,1H),8.95(s,1H),8.55(t,J＝6.1Hz,1H),7.92(s,1H),7.85(d,J＝9.4Hz,1H),7.36(q,J＝8.3Hz,4H),7.04(s,1H),6.83(d,J＝8.0Hz,2H),6.71(s,1H),5.68(t,J＝7.3Hz,2H),5.10(s,1H),4.51(d,J＝9.4Hz,1H),4.39(dt,J＝16.2,7.2Hz,2H),4.31(s,1H),4.19(d,J＝5.3Hz,1H),4.15(d,J＝5.2Hz,1H),4.08(d,J＝3.1Hz,2H),3.92(s,3H),3.61(d,J＝7.1Hz,2H),2.54(s,3H),2.40(s,3H),2.09(dd,J＝13.8,6.8Hz,1H),1.96(q,J＝6.9,6.1Hz,2H),1.91–1.82(m,1H),1.76(d,J＝7.6Hz,2H),1.59(d,J＝7.0Hz,3H),1.42(q,J＝7.0Hz,4H),1.36–1.22(m,4H),0.89(s,9H)。
EXAMPLE 6 preparation of Compound 6
Compound 6 has the formula: (2S, 4R) -1- ((S) -2- (9- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) nonylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme I, the specific synthetic procedure was the same as example 1 except that methyl 4-bromobutyrate was replaced with methyl 9-bromononanoate in example 1. Total yield: 2.1%. 1 H NMR(400MHz,DMSO-d 6 )δ9.61(s,1H),8.95(s,1H),8.55(t,J＝6.1Hz,1H),7.91(s,1H),7.85(d,J＝9.3Hz,1H),7.42–7.31(m,4H),7.03(s,1H),6.86–6.79(m,2H),6.71(s,1H),5.74–5.65(m,1H),4.51(d,J＝9.3Hz,1H),4.45–4.34(m,2H),4.31(s,1H),4.17(dd,J＝16.0,5.4Hz,1H),4.08(s,2H),3.92(s,3H),3.65–3.57(m,2H),2.54(s,3H),2.40(s,3H),2.28–2.18(m,1H),2.12–2.01(m,1H),2.00–1.84(m,2H),1.81–1.73(m,2H),1.59(d,J＝7.0Hz,3H),1.52–1.36(m,5H),1.35–1.25(m,4H),0.89(s,9H)。
EXAMPLE 7 preparation of Compound 7
Compound 7 has the formula: (2S, 4R) -1- ((S) -2- (10- ((R) -1- (3-amino-5- (trifluoromethyl) phenyl) ethyl) amino) -7-methoxy-2-methyl quinazolin-6-yl) oxy) decylamino) -3, 3-dimethylbutyryl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme I, the specific synthetic procedure was the same as example 1 except that methyl 4-bromobutyrate was replaced with methyl 10-bromodecanoate in example 1. Total yield: 3.6%. 1 H NMR(400MHz,DMSO-d 6 )δ9.64(d,J＝7.7Hz,1H),8.95(s,1H),8.56(t,J＝6.1Hz,1H),7.92(s,1H),7.84(d,J＝9.4Hz,1H),7.36(q,J＝8.4Hz,4H),7.08(s,1H),6.95–6.78(m,2H),6.72(s,1H),5.68(t,J＝7.3Hz,2H),5.10(s,1H),4.51(d,J＝9.4Hz,1H),4.39(dt,J＝12.7,7.2Hz,2H),4.33–4.26(m,1H),4.19(d,J＝5.5Hz,1H),4.15(d,J＝5.4Hz,1H),4.08(td,J＝6.4,3.4Hz,2H),3.92(s,3H),3.75–3.53(m,2H),2.54(s,3H),2.40(s,3H),2.23(dt,J＝14.8,7.7Hz,1H),2.15–1.94(m,2H),1.86(ddd,J＝12.9,8.7,4.5Hz,1H),1.77(t,J＝7.4Hz,2H),1.59(d,J＝7.0Hz,3H),1.43(dd,J＝15.1,7.7Hz,4H),1.30(s,2H),1.28–1.13(m,6H),0.89(s,9H)。
Example 8 preparation of Compound 8
The formula of compound 8: (2S, 4R) -4-hydroxy-1- ((S) -2- (5- (4- (7-methoxy-2-methyl-4- (((R) -1- (4- (2-)) ((methylamino) methyl) phenyl) thiophen-2-yl) ethyl) amino) quinazolin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -5-oxopentanoylamino) -3, 3-dimethylbutyryl) -N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to synthetic scheme II, the specific synthetic steps are as follows:
s-3-1 (2 g,9.7mmol;1.0 eq.) was dissolved in 20mL tetrahydrofuran. (R) -2-methylpropane-2-sulfoxide amide (1.8 g,14.5mmol;1.5 eq.) and Ti (OEt) were then added at room temperature 4 (5.5 g,24.3mmol;2.5 eq.). After stirring overnight at 80 ℃, the reaction mixture was cooled to room temperature and quenched with ice water, extracted with ethyl acetate, the organic phases combined, anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure to obtain a crude product. The crude product was purified by flash chromatography to give S-3-2 as a pale yellow solid in yield: 79%.
S-3-2 (2.4 g,7.77mmol;1.0 eq.) was dissolved in a mixed solution of tetrahydrofuran (50 mL) and water (1 mL) and NaBH was slowly added at-78deg.C 4 (531 mg,14.0mmol;1.8 eq.) and then stirred at room temperature for 3h. Quench the reaction with ice water, extract with ethyl acetate, combine the organic phases, anhydrous Na 2 SO 4 And (5) drying. The diastereomer mixture generated by the reaction was separated by flash chromatography to give white solid S-3-3, yield: 85%.
To S-3-3 (2.0 g,6.6 mmol) was added 9mL of a 1, 4-dioxane solution of 4M HCl and stirred at room temperature for 1h. Removing the solvent under reduced pressure to obtainIntermediate S-3-4. Yield: 96%. 1 H NMR(400MHz,DMSO-d 6 )δ8.72(s,3H),7.67(d,J＝1.5Hz,1H),7.31(d,J＝1.5Hz,1H),4.64(q,1H),1.54(d,J＝6.8Hz,3H).
To a solution of S-4-1 (12.3 g,47.4mmol;1.0 eq.) in acetonitrile (100 mL) was added methanesulfonic acid (36.8 g,379.2mmol;8.0 eq.). The reaction mixture was stirred at 100 ℃ overnight. After completion of the reaction by UPLC-MS, the mixture was cooled to room temperature. Dropwise adding NaHCO into the reaction mixture 3 And (3) regulating the pH of the aqueous solution to be alkaline, standing for a while, generating precipitate, carrying out suction filtration, and freeze-drying to obtain a crude product S-4-2. Yield: 90%.
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To a suspension of S-4-2 (11.46 g,42.6mmol;1.0 eq.) in methylene chloride (100 mL) was added 2,4, 6-triisopropylbenzenesulfonyl chloride (15.4 g,51.1mmol;1.2 eq.), DMAP (254 mg,4.3mmol;0.1 eq.) and Et 3 N (12.9 g,127.8mmol;3.0 eq.). The reaction mixture was stirred at room temperature for 12h. Diluting the solvent with dichloromethane, adding NaHCO 3 Aqueous solution and extracted with DCM. The combined organic layers were treated with anhydrous Na 2 SO 4 Dried, filtered, and the solvent removed in vacuo. The crude product was purified by flash chromatography to give the desired product S-4-3, yield: 67%.
S-4-3 (7.7 g,14.4mmol;1.0 eq.) and S-3-4 (3.5 g,14.4mmol;1.0 eq.) were dissolved in 50mL of isopropanol and stirred overnight at 95 ℃. After UPLC-MS demonstrated complete conversion of the starting material, the mixture was cooled to room temperature. The solvent was removed in vacuo. The crude product was purified by flash chromatography to give intermediate S-4-4, yield: 91%.
S-4-4 (2.64 g,5.87mmol;1.0 eq.), (2- (hydroxymethyl) phenyl) boronic acid (981 mg,6.45mmol;1.1 eq.) Pd (PPh) 3 ) 4 (678 mg,0.587mmol;0.1 eq.) and Cs 2 CO 3 (5739 mg,17.6mmol;3.0 eq.) in a mixed solution of tetrahydrofuran and water, under argon, stirring overnight at 70 ℃. After completion of the TLC detection, the reaction solution was cooled to room temperature and extracted with ethyl acetate and water. The combined organic layers were washed with brine and dried over anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure. The crude product was purified by flash chromatography to give the desired compound S-4-5, yield: 76%.
To a solution of S-4-5 (53 mg,1.1mmol;1.0 eq.) in methylene chloride (10 mL) was slowly added dess-martin oxidant (1462 mg,3.45mmol;3.0 eq.). Stirred at room temperature for 5h and tlc detection was complete. Na is added to the reaction mixture 2 S 2 O 4 And NaHCO 3 And stirred at room temperature for a further 30min. The solution was extracted with ethyl acetate and washed with brine. The combined organic layers were treated with anhydrous Na 2 SO 4 Dried and concentrated in vacuo. The crude product was further purified by flash chromatography to give compound S-5-1, yield: 59%.
S-5-1 (1000 mg,2.07mmol;1.0 eq.) and methylamine (64 mg,2.07mmol;1.0 eq.) were dissolved in 1, 2-dichloroethane (20 mL) and stirred at room temperature for 1h before NaBH (OAc) was slowly added 3 (877 mg,4.14mmol;2.0 eq.) and stirring at room temperature was continued overnight, after which the reaction was detected by UPLC-MS, the reaction mixture was diluted with water and taken up in dichloroMethane extraction and extraction with anhydrous Na 2 SO 4 Drying and solvent removal in vacuo. The crude product was purified by flash chromatography, the resulting product was dissolved in 10mL of ethanol, boc anhydride (243 mg,1.10mmol;1.2 eq.) was slowly added, stirred for 3h at room temperature, after completion of TLC detection the reaction solvent was removed under pressure and purified by flash chromatography to give the target compound S-5-2, yield: 42%.
S-5-2 (500 mg,0.787mmol;1 eq.), (1- (tert-butoxycarbonyl) -1,2,3, 6-tetrahydropyridin-4-yl) boronic acid (197mg, 0.866mmol;1.1 eq.), pd (PPh 3 ) 4 (9 mg,0.079mmol;0.1 eq.) and Cs 2 CO 3 (770 mg,2.36mmol;3.0 eq.) in a mixed solution of tetrahydrofuran and water, under argon protection, stirring overnight at 70 ℃. After completion of the TLC detection, the reaction solution was cooled to room temperature and extracted with ethyl acetate and water. The combined organic layers were washed with brine and dried over anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure. The crude product was purified by flash chromatography to give the desired compound S-5-3, yield: 72%.
V-1-1 (150 mg,0.348mmol;1.0 eq.) and monomethyl glutarate (56 mg,0.348mmol;1.0 eq.) and HATU (159 mg,0.418mmol;1.2 eq.) and DIPEA (270 mg,2.09mmol;6.0 eq.) were dissolved in 5mL of N, N-dimethylformamide and stirred at room temperature for 6h, after the UPLC-MS detection reaction was completed, the reaction mixture was diluted with water and ethyl acetate. The organic layer was separated, washed with brine, and dried over anhydrous Na 2 SO 4 And (5) drying. The solution was removed under vacuum. The intermediate obtained by flash column purification was dissolved in tetrahydrofuran, and lithium hydroxide previously dissolved in water was added thereto and stirred at room temperature for 2 hours. After the reaction, the pH of the reaction solution is adjusted to 4-6 with acetic acid, extracted with water and ethyl acetate, and combinedAnhydrous Na for organic phase 2 SO 4 Drying and the solution was removed under vacuum. Intermediate V-1-3 is obtained. The yield of the two steps is 58%.
V-1-3 (30 mg,0.050mmol;1.0 eq.) S-5-3 (27 mg,0.05mmol;1.0 eq.), HATU (29 mg,0.075mmol;1.5 eq.) and DIPEA (39 mg,0.075mmol;6.0 eq.) were dissolved in 3mL of N, N-dimethylformamide and stirred at room temperature for 6h, after the UPLC-MS detection reaction was completed, the reaction mixture was diluted with water and ethyl acetate. The organic layer was separated, washed with brine, and dried over anhydrous Na 2 SO 4 And (5) drying. The solution was removed under vacuum. And purifying by a flash chromatography column to obtain a crude product. The crude product was dissolved in dichloromethane, 5mL of a 4M HCl 1, 4-dioxane solution was added, and the mixture was stirred at room temperature for 1h. The solvent was removed under reduced pressure and the crude product was purified by prep-HPLC to afford example 8. The yield of the two steps is 43%. 1 H NMR(400MHz,DMSO-d 6 )δ8.94(s,1H),8.77(s,1H),8.56(t,J＝6.0Hz,1H),8.11(s,1H),7.94–7.87(m,1H),7.65–7.55(m,1H),7.45–7.30(m,9H),7.17(s,1H),7.05(s,1H),6.01–5.91(m,1H),5.85(d,J＝6.4Hz,1H),5.14(s,1H),4.51(d,J＝9.2Hz,1H),4.43–4.35(m,2H),4.32(s,1H),4.17(dd,J＝15.9,5.4Hz,1H),4.11(s,2H),4.07(s,2H),3.84(s,3H),3.83–3.79(m,1H),3.63(s,3H),3.58–3.54(m,3H),2.46(s,3H),2.40(s,3H),2.36–2.09(m,6H),2.05–1.71(m,4H),1.69(d,J＝7.0Hz,3H),0.91(s,9H)。
Example 9 preparation of Compound 9
The formula of compound 9: (2S, 4R) -4-hydroxy-1- ((S) -2- (3- (4- (7-methoxy-2-methyl-4- (((R) -1- (4- (2-)) ((methylamino) methyl) phenyl) thiophen-2-yl) ethyl) amino) quinazolin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -3-oxopropanamido) -3, 3-dimethylbutyryl) -N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to synthesis scheme II, the specific synthesis procedure was to replace monomethyl glutarate in example 8 with monomethyl malonate, and the remainder was identical to example 8, overall yield: 2.1%. 1 H NMR(400MHz,DMSO-d 6 )δ8.66(s,1H),8.25(s,1H),8.11(d,J＝12.8Hz,1H),8.00(t,J＝9.1Hz,1H),7.68(d,J＝9.1Hz,1H),7.55–7.48(m,2H),7.40–7.28(m,8H),7.24(d,J＝1.5Hz,1H),6.24(tt,J＝6.3,1.0Hz,1H),5.38(tq,J＝6.7,3.2Hz,1H),5.09(dq,J＝9.1,6.8Hz,1H),4.61(d,J＝12.8Hz,1H),4.44–4.31(m,4H),4.13(d,J＝7.3Hz,1H),4.11–3.98(m,2H),3.86(s,3H),3.70(ddt,J＝6.1,2.1,1.0Hz,2H),3.68–3.61(m,3H),3.49(dd,J＝12.3,7.0Hz,1H),3.24(d,J＝12.5Hz,1H),3.18(d,J＝12.3Hz,1H),3.14–3.05(m,1H),3.03(ddt,J＝7.2,6.1,1.0Hz,1H),2.52(s,3H),2.48(d,J＝3.1Hz,3H),2.37(s,3H),2.19–2.04(m,2H),1.78(d,J＝6.8Hz,3H),0.97(s,9H)。
Example 10 preparation of Compound 10
The formula of compound 10: (2S, 4R) -4-hydroxy-1- ((S) -2- (4- (4- (7-methoxy-2-methyl-4- (((R) -1- (4- (2-)) ((methylamino) methyl) phenyl) thiophen-2-yl) ethyl) amino) quinazolin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -4-oxobutanamido) -3, 3-dimethylbutyryl) -N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme II, the specific synthesis procedure was the same as that of example 8 except that monomethyl glutarate in example 8 was replaced with monomethyl succinate. Total yield: 2.5%. 1 H NMR(400MHz,DMSO-d 6 )δ8.66(s,1H),8.25(s,1H),8.07–7.95(m,2H),7.68(d,J＝9.1Hz,1H),7.55–7.48(m,2H),7.40–7.28(m,8H),7.24(d,J＝1.5Hz,1H),6.24(tt,J＝6.3,1.0Hz,1H),5.38(tq,J＝6.6,3.2Hz,1H),5.09(dq,J＝9.1,6.8Hz,1H),4.59(d,J＝12.5Hz,1H),4.42–4.34(m,4H),4.34–4.24(m,1H),4.11–4.00(m,2H),3.98(dt,J＝6.3,1.0Hz,1H),3.86(s,3H),3.73(dt,J＝6.3,1.0Hz,1H),3.68–3.53(m,3H),3.47(dd,J＝12.4,7.0Hz,1H),3.02(tdq,J＝7.0,5.9,1.0Hz,2H),2.63–2.54(m,2H),2.52(s,3H),2.50–2.39(m,5H),2.37(s,3H),2.20(dt,J＝12.3,6.9Hz,1H),2.06(dt,J＝12.4,7.0Hz,1H),1.78(d,J＝6.8Hz,3H),0.97(s,9H)。
EXAMPLE 11 preparation of Compound 11
The formula of compound 11: (2S, 4R) -4-hydroxy-1- ((S) -2- (6- (4- (7-methoxy-2-methyl-4- (((R) -1- (4- (2-)) ((methylamino) methyl) phenyl) thiophen-2-yl) ethyl) amino) quinazolin-6-yl) -3, 6-dihydropyridin-1 (2H) -yl) -6-oxohexanamido) -3, 3-dimethylbutyryl) -N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide.
Referring to scheme II, the specific synthesis procedure was the same as that of example 8 except that monomethyl glutarate in example 8 was replaced with monomethyl adipate. Total yield: 2.3%. 1 H NMR(400MHz,DMSO-d 6 )δ8.94(s,1H),8.57(t,J＝6.1Hz,1H),8.37(d,J＝8.2Hz,1H),8.18(s,1H),8.05(d,J＝2.6Hz,1H),7.87(d,J＝9.3Hz,1H),7.56–7.50(m,1H),7.42–7.28(m,9H),7.20(s,1H),7.00(s,1H),5.96–5.87(m,1H),5.83(d,J＝10.5Hz,1H),4.51(d,J＝9.4Hz,1H),4.43–4.35(m,2H),4.31(s,1H),4.17(dd,J＝15.9,5.4Hz,1H),4.08(d,J＝21.5Hz,2H),3.83(d,J＝5.9Hz,6H),3.63–3.59(m,5H),2.40(d,J＝1.7Hz,6H),2.33(s,3H),2.31–2.21(m,2H),2.17–2.06(m,1H),2.03–1.92(m,1H),1.90–1.81(m,1H),1.66(d,J＝6.9Hz,3H),1.52–1.43(m,4H),1.16–1.10(m,1H),0.89(s,9H)。
Example 12: evaluation of SOS1 expression level reducing Effect of the Compound of the present application on PANC-1 and K562 cells
Human pancreatic cancer cells PANC-1 or human chronic myelogenous leukemia cells K562 were subjected to 3X 10 in DMEM high-sugar medium (hereinafter referred to as evaluation medium) containing 10% PBS and 2.5% horse serum 5 Amount per well was inoculated into 6-well microwell plates (Corning) and incubated overnight. To this culture, an evaluation medium containing the compound of the example was added so that the final concentrations of the compound of the example reached 0.1, 1 and 10. Mu. Mol/L, and the culture was continued for 24 hours. After 24 hours of culture, the culture medium was removed, after washing the cells with PBS, RIPA lysate containing 1% Protease Inhibitor Cocktail was added, and after lysis and centrifugation, total protein extract was obtained, and the protein concentration in the extract was detected by BCA method; protein electrophoresis was performed by SDS-PAGE, after which 200mA constant current was transferred to PVDF (MilliporeSigma IPVH 00010) membrane for 120 min; placing the PVDF film in skimmed milk containing 5%, and sealing for 1h at room temperature; using Anti-SOS1 antibody [12409 ] respectively](CST) performing an immune reaction; and (3) dripping ECL luminous liquid after washing the film, and exposing. Banding Using software Image JAnd (5) performing row gray level analysis. Each sample was tested simultaneously for GAPDH protein bands as an internal control. The protein degradation rate of the compound SOS1 of the example was calculated according to the protein band gray scale, and NA represents no measurement.
The results of compounds inducing SOS1 degradation in PANC-1 and K562 cells are shown in Table 1.
TABLE 1
Of all the compounds of the examples, the compound of example 2 showed the best SOS1 degradation, with a degradation rate of approximately 90% for SOS1 in PANC-1 and K562 cells at the measured concentrations. We selected Compound 2 for a time and dose dependent study of SOS1 degradation activity in both cells as described above, as shown in FIGS. 1A and B, compound 2 was significantly dose dependent on SOS1 degradation in both cell lines, DC 50 129.8nM and 43.1nM, respectively (FIG. 1 is C and D). In FIG. 2, A and B show the time dependence of SOS1 degradation of compound 2, and the SOS1 degradation rate of compound 2 can reach more than 70% after 24 hours of administration at a dose of 1. Mu. Mol/L (C and D in FIG. 2).
Example 13: compound 2 3D proliferation inhibitory Activity on NCI-H358 cells
NCI-H358 cells were seeded in 96-well Nunclon Sphera Plates and incubated with serial dilutions of compound 2 and inhibitor BI3406, respectively, for 9 days. Cell viability was determined using CellTiter-Glo 3D Cell Viability Assay according to the manufacturer's instructions. IC (integrated circuit) 50 The value, half inhibition concentration, was determined by nonlinear regression (curve fitting) using variable slopes (four parameters) in Graphpad Prism.
The cell proliferation inhibition results are shown in FIG. 3, wherein it is shown that compound 2 exhibits proliferation inhibition activity on NCI-H358 cells, the inhibition rate reaches 50% at 3.0. Mu. Mol/L, and inhibitor BI3406 is used as a positive control, which has slightly poorer inhibition activity than compound 2.
The above description is a general description of the application. Variations in form and value may be substituted for the purpose of illustration and not limitation, as the terms are used herein, depending on the circumstances or actual requirements. Various changes and modifications may be made by one skilled in the art, and such equivalents are intended to fall within the scope of the application as defined in the following claims.

Claims (10)

1. A compound for degrading SOS1 protein has a structure shown in a formula I,
wherein: x is X 1 、X 2 Each independently selected from N or C; y is selected from N, S, O, si or P;
the R group is selected from:
wherein: x is selected from N or C; w is selected from S or O; m1 and m2 are each independently integers of 0 to 5;
the R is 1 Selected from H; -OH; -NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Halogen; a substituted or unsubstituted linear or branched C1-C3 alkyl group; a substituted or unsubstituted straight or branched C1-C3 alkenyl group; substituted or unsubstituted, straight or branched C1-C3 alkynyl; a substituted or unsubstituted linear or branched C1-C3 carbonyl group; substituted or unsubstituted C1-C10 cycloalkyl; a substituted or unsubstituted C1-C10 heterocyclyl; substituted or unsubstituted C1-C10 aryl or heteroaryl;
the linking group 1 is selected from: absence of; or a substituted or unsubstituted, linear or branched C1-C12 alkylene group; the optional carbon chain contains 1-4 heteroatoms selected from N, S, O, si or P;
wherein: n1, n2, n3 are each independently integers from 0 to 5; x is X 1 、X 2 、X 3 、X 4 Each independently selected from N or C;
the E3 ligand is selected from:
wherein: m is an integer of 0 to 3;
the R is 2 、R 3 、R 4 And R is 5 Each independently selected from: h is formed; -OH; -NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Halogen; a substituted or unsubstituted linear or branched C1-C6 alkyl group; a substituted or unsubstituted linear or branched C1-C6 cyano group; a substituted or unsubstituted linear or branched C1-C6 alkenyl group; substituted or unsubstituted, straight or branched chain C1-C6 alkynyl; a substituted or unsubstituted linear or branched C1-C6 alkoxy group; a substituted or unsubstituted linear or branched C1-C6 secondary, tertiary or quaternary amine group; a substituted or unsubstituted linear or branched C1-C6 ester group; a substituted or unsubstituted linear or branched C1-C6 amide group; a substituted or unsubstituted linear or branched C1-C6 ketone group; substituted or unsubstituted C1-C10 cycloalkyl; a substituted or unsubstituted C1-C10 heterocyclyl; substituted or unsubstituted C1-C10 aryl or heteroaryl.
2. The compound of claim 1, wherein the compound has the following structural formula:
wherein: x is X 1 、X 2 Each independently selected from N or C; y is selected from N or O;
m1 is an integer of 0 to 5, and m2 is an integer of 0 to 4;
the R is 1 Selected from H; -OH; -NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Halogen; a substituted or unsubstituted linear or branched C1-C3 alkyl group; a substituted or unsubstituted straight or branched C1-C3 alkenyl group; substituted or unsubstituted, straight or branched C1-C3 alkynyl; a substituted or unsubstituted linear or branched C1-C3 carbonyl group; substituted or unsubstituted C1-C10 cycloalkyl; a substituted or unsubstituted C1-C10 heterocyclyl; substituted or unsubstituted C1-C10 aryl or heteroaryl;
R 2 、R 3 、R 4 and R is 5 Each independently selected from: h is formed; -OH; -NH 2 The method comprises the steps of carrying out a first treatment on the surface of the Halogen; a substituted or unsubstituted linear or branched C1-C6 alkyl group; a substituted or unsubstituted linear or branched C1-C6 cyano group; a substituted or unsubstituted linear or branched C1-C6 alkenyl group; substituted or unsubstituted, straight or branched chain C1-C6 alkynyl; a substituted or unsubstituted linear or branched C1-C6 alkoxy group; a substituted or unsubstituted linear or branched C1-C6 secondary, tertiary or quaternary amine group; a substituted or unsubstituted linear or branched C1-C6 ester group; a substituted or unsubstituted linear or branched C1-C6 amide group; a substituted or unsubstituted linear or branched C1-C6 ketone group; substituted or unsubstituted C1-C10 cycloalkyl; a substituted or unsubstituted C1-C10 heterocyclyl; substituted or unsubstituted C1-C10 aryl or heteroaryl.
The linking group is selected from: absence of; or a substituted or unsubstituted, linear or branched C1-C12 alkylene group; the optional carbon chain contains 1-4 heteroatoms selected from N, S, O, si or P;
wherein: n1, n2, n3 are each independently integers from 0 to 5; x is X 1 、X 2 、X 3 、X 4 Each independently selected from N or C.
3. The compound of claim 2, wherein the compound has the following structural formula:
4. a stereoisomer, solvate, prodrug, metabolite, pharmaceutically acceptable salt or co-crystal of a compound of claim 1 or 2 or 3.
5. A pharmaceutical composition comprising a compound according to claim 1 or 2 or 3, or a stereoisomer, a solvent compound, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal thereof.
6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable carrier.
7. Use of a compound according to claim 1 or 2 or 3, or a stereoisomer, a solvent compound, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal thereof, for the manufacture of a medicament for the treatment or prophylaxis of a disease associated with SOS1 activity or expression level.
8. The use according to claim 7, wherein the compound or a stereoisomer, a solvate, a prodrug, a metabolite, a pharmaceutically acceptable salt or a co-crystal thereof acts as a SOS1 degrading agent.
9. Use according to claim 8 for the prevention or treatment of colon cancer, non-small cell lung cancer, pancreatic cancer, rectal cancer, melanoma or multiple myeloma.
10. A process for the preparation of a compound according to claim 1 or 2 or 3, wherein the compound is obtained according to one of the following synthetic schemes I or II:
synthetic route I
Synthesis of scheme II
CN202311093648.3A 2023-08-29 2023-08-29 Compound for targeted SOS1 protein ubiquitination regulation and preparation method and application thereof Pending CN117143175A (en)

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