CN112939965A

CN112939965A - Compound for simultaneously inducing degradation of EGFR (epidermal growth factor receptor) and PARP (para-amyloid peptide) proteins as well as preparation method and application thereof

Info

Publication number: CN112939965A
Application number: CN202110170636.0A
Authority: CN
Inventors: 李华; 陈丽霞; 周宜荣; 霍峻锋; 刘洋; 顾小霞; 张文波
Original assignee: Shenyang Pharmaceutical University
Current assignee: Shenyang Pharmaceutical University
Priority date: 2021-02-08
Filing date: 2021-02-08
Publication date: 2021-06-11
Anticipated expiration: 2041-02-08
Also published as: WO2022166570A1; CN112939965B

Abstract

A compound for simultaneously inducing degradation of EGFR and PARP proteins, a preparation method and application thereof, belonging to the field of pharmaceutical chemistry; the invention provides a series of novel dual-targeting degradation compounds with two independent inhibitor units and an E3 ligase ligand, or application of pharmaceutically acceptable salts, hydrates, stereoisomers or prodrugs thereof in preparing medicaments for treating or preventing tumors. The compound provided by the method can simultaneously effectively induce E3 ligase dependent degradation of EGFR and PARP in pancreatic cancer cell strains and 1299 cells, and effectively inhibit the growth of cancer cells. Can resolve tumor heterogeneity and reverse target diversity of chemotherapy-resistant advanced cancers. The methods of the invention provide novel therapeutic modalities for the treatment of EGFR and PARP mediated tumors and/or other diseases.

Description

Compound for simultaneously inducing degradation of EGFR (epidermal growth factor receptor) and PARP (para-amyloid peptide) proteins as well as preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a bifunctional compound for simultaneously inducing degradation of EGFR (epidermal growth factor receptor) and PARP (para-amyloid peptide) proteins based on a PROTAC (procoat peptide) technology, and a preparation method and application thereof.

Background

Late-stage cancer chemotherapy resistance is mediated by a number of factors, including Epidermal Growth Factor Receptor (EGFR) overexpression and DNA repair enzyme (PARP). Overexpression of the epidermal growth factor receptor tyrosine kinase is associated with increased DNA repair activity and activation of anti-apoptotic signals.

EGFR is a transmembrane protein tyrosine kinase, and a receptor which is a member of the EGF family triggers the signaling pathway of the human epidermal growth factor receptor, thereby regulating cell proliferation, invasion, metastasis, and apoptosis. Increased epidermal growth factor receptor activity results in excessive, mutated, or amplified epidermal growth factor receptor genes leading to activating mutations, such as in-frame deletion of exon 19 and L858R mutations, which are classified as carcinogenic factors for non-small cell lung cancer. EGFR is overexpressed in many tumors, including lung, brain, colon, prostate, and is often associated with poor prognosis. EGFR inhibitors have been developed and approved by the FDA for the treatment of non-small cell lung cancer, but their efficacy may be compromised by acquired resistance due to mutant EGFR variants. Therefore, the development of new small molecule inhibitors or therapeutic approaches to overcome the multiple point mutations of EGFR remains an unmet medical need.

PARP, as a DNA-dependent ribozyme, plays a critical role in the signaling and repair of DNA damage. PARP in turn is a cleavage substrate for the apoptotic core member caspase (caspase). Therefore, it plays an important role in DNA damage repair and apoptosis.

Over the past decades, the development of cancer-targeting drugs has been directed primarily to the design of monofunctional molecules. In cancer, tumor cells often up-regulate different growth-promoting factors that can act independently or interfere with each other intracellularly through a signaling network. Tumor cells readily acquire resistance by up-regulating replacement factors or switching signaling pathways that promote proliferation. Thus, treatment directed to only a single target has limitations.

In addition to drug resistance, single-target therapeutic drugs can also cause reduced efficacy and reduced quality of life for patients due to side effects, tissue toxicity, and the like. In order to overcome the defects of single-target drugs, the combined administration of two different approaches related to the development of diseases has become a recognized effective method. The combination can obtain additive or synergistic effect, and reduce drug resistance, and because the combination improves the curative effect, the combination usually requires smaller dosage of each single drug, thereby reducing side effect.

Another strategy to improve efficacy is to design a single hybrid molecule that fuses two or more pharmacophores to target two or more anti-tumor epitopes or targets simultaneously. These hybrid molecules are generally more effective and have fewer side effects because they can modulate multiple targets or pathways simultaneously. The single-molecule double-target or multi-target medicine is superior to single-target medicine or combined treatment and also shows the following aspects; less susceptibility to drug resistance than single target drugs, predictable pharmacokinetics, reduced risk of drug interaction, simpler dosing regimens, increased patient compliance, fewer potential intellectual property conflicts, simpler regulatory approval procedures, etc. compared to combination administration. These hybrid molecules have attracted considerable interest and have enjoyed considerable success over the past decades because of their advantages in treating complex diseases, and have become an alternative to combination therapies or the use of cocktails, including bispecific antibodies and other dual-or multi-target small molecule drugs.

Proteolytic targeting chimeras (PROTACs) are bifunctional small molecules in which the target protein ligand and the E3 ubiquitin ligase ligand are joined together by a linker arm to form a triplet compound. As a potential therapeutic approach, proteolytic targeting chimeras (PROTACs) are capable of degrading specific proteins. The proteolytic targeting chimera is a specific biofunctional molecule, usually a compound molecule that binds to a protein target, a small molecule ligand that recruits the E3 ligase, and a linker. Induced by PROTACs, leading to selective polyubiquitination of the target protein and subsequent degradation at the proteasome. Compared with the traditional small molecule inhibitor, the PROTAC has various advantages, including the functions without being combined with the active site of a target protein, degradation of a target point which is difficult to form a drug, event-driven action, catalytic property and capability of acting at a lower dosage, thereby having great potential particularly in the development of anticancer drugs.

However, the combination of drugs has some problems or disadvantages, such as the change of the drug action caused by the direct physical or chemical reaction due to the unreasonable preparation when the combination is used; the medicine has a plurality of varieties, so that the incidence of the interaction of the medicines is increased, the curative effect or toxicity of the medicines is influenced, the curative effect is weakened, and even serious adverse reaction can be caused; the drugs with the same pharmacological effect or toxicity reaction are combined for use, and if the drugs are not used in a decrement way, the possibility of drug poisoning is generated; it is difficult to determine the optimal dosage of the drugs for combination; combination therapy also has the problem of timing of combination, and proper combination and reasonable sequential administration can improve the curative effect.

Disclosure of Invention

In view of the existing problems, the invention provides a bifunctional compound capable of simultaneously inducing degradation of EGFR and PARP proteins based on the PROTAC technology, or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or prodrug of the compound, and further provides a preparation method of the compound and application of the compound in preparing a medicament for treating and/or preventing tumors.

Specifically, the invention takes PROTAC technology as support, takes the existing EGFR inhibitor (Gefitinib) and PARP inhibitor (Olaparib) as raw materials, and synthesizes double targeting degradation chimeric molecules (Dual Protacs) of EGFR and PARP proteins recruited by different Linker lengths and different E3 ligase (CRBN-and VHL-). The invention takes amino acid as a star-shaped linking unit (linker), and different inhibitors and E3 ligand small molecules are connected through the star-shaped linking unit. Amino acid is a non-toxic and harmless multifunctional molecule of natural origin, and has very high biocompatibility. Besides common amino and carboxyl, the multiple amino acid molecules also have a third reaction site, and three different small molecules can be artificially, controllably and as required introduced according to different reaction activities of the three reaction sites.

The invention aims at providing a compound for simultaneously inducing degradation of EGFR and PARP proteins, which is a compound shown in formula (I) or (II) or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or a prodrug of the compound shown in formula (I) or (II).

The invention also aims to provide a preparation method of the compound shown in the formula (I) or (II).

The invention also aims to provide application of the compound shown in the formula (I) or (II) in preparing a medicament for treating or preventing tumors.

The purpose of the invention is realized by the following technical scheme:

the formulas (I) and (II) are respectively as follows:

wherein:

a is the PARP selective inhibitor Olaparib (Olaparib); the structure is as follows:

b is an EGFR selective inhibitor Gefitinib (Gefitinib); the structure is as follows:

e3 is a CRBN or VHL small molecule ligand in an E3 ubiquitin ligase complex, and specifically is thalidomide and derivatives thereof, lenalidomide and derivatives thereof or pomalidomide and derivatives thereof; the structure of E3 is

Wherein:

w is CH₂、C＝O、SO₂NH or N-alkyl;

x is O or S;

z is-alkyl, -cycloalkyl, -Cl, -F or-H;

g and G' are each independently-H, alkyl, -OH or-CH₂-a heterocycle;

R¹is-H, -D, -F, -Cl, -Br, -I, -NO₂、-CN、-NH₂、-OH、-CH₃、-CH₂F、-CHF₂、-CF₃、-CH₂D、-CHD₂、-CD₃or-CH₂CH₃；

L is a linking arm which is an aliphatic chain, an aromatic chain, an ether chain or an amide chain; a, B and E3 are respectively connected by covalent bonds to form the compound shown in the formula (I) or (II) or the stereoisomer, hydrate and pharmaceutically acceptable salt or prodrug of the compound shown in the formula (I) or (II); the structure is as follows

And n is more than or equal to 1 and less than or equal to 10.

A compound for simultaneously inducing degradation of EGFR and PARP proteins is any one of the compounds shown in formulas (a) to (d) or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or a prodrug of the compound;

wherein:

L is any of the foregoing structures; and n is more than or equal to 1 and less than or equal to 10.

A compound for simultaneously inducing degradation of EGFR and PARP proteins is any one of the compounds shown in formulas (a ') to (d') or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or a prodrug of the compound;

wherein L is

And n is more than or equal to 1 and less than or equal to 5.

A compound for simultaneously inducing degradation of EGFR and PARP proteins, which is any one of the following compounds or a stereoisomer, hydrate and pharmaceutically acceptable salt or prodrug thereof;

the compound for simultaneously inducing the degradation of EGFR and PARP proteins contains asymmetric or chiral centers and exists in different stereoisomeric forms. The present invention includes all stereoisomeric forms including, but not limited to, diastereomers, enantiomers, and atropisomers, as well as mixtures thereof, such as racemates.

The pharmaceutically acceptable salt of the compound for simultaneously inducing the degradation of EGFR and PARP proteins is an addition salt formed by the compound and hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, pyruvic acid or succinic acid.

The prodrug of a compound that induces degradation of both EGFR and PARP proteins is a derivative of a compound of formula (a), formula (b), formula (c) or formula (d), which may itself have a weak or even no activity, but which is converted to the corresponding biologically active form under physiological conditions (e.g., by metabolism, solvolysis or otherwise) after administration.

A pharmaceutical composition comprising one or more of the compounds of the present invention, stereoisomers of the compounds, hydrates of the compounds, and pharmaceutically acceptable salts or prodrugs of the compounds; further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or combination thereof.

Wherein the dosage form of the pharmaceutical composition is injection, tablet or capsule.

A method for preparing a compound that simultaneously induces degradation of EGFR and PARP proteins, comprising the steps of:

step 1: dissolving N-Boc amino acid methyl ester and propyne bromide in a solvent, adding potassium carbonate or sodium hydride as alkali, and performing hydroxyl etherification protection to obtain an intermediate A;

step 2: carrying out amide condensation on the intermediate A and the olaparib under the condition of adding EDCI, HOBt and DIPEA, and then removing protection to obtain a product which is subjected to the same amide condensation with gefitinib to obtain an intermediate B;

and step 3: and dissolving the intermediate B and the azide-linked E3 ligand in a solvent, and linking through a Click reaction to obtain the Dual Protacs compound.

In the preparation method, the solvent is one or two of DMF, dichloromethane, THF and water; the amino acid is tyrosine, serine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, tryptophan, aspartic acid, glutamic acid or hydroxyproline.

The reaction route starting from N-Boc tyrosine or serine methyl ester is as follows:

application of a compound capable of simultaneously inducing degradation of EGFR and PARP proteins or a stereoisomer thereof, a hydrate thereof, a pharmaceutically acceptable salt or a prodrug thereof or a pharmaceutical composition of the stereoisomer thereof in preparation of medicines for treating and/or preventing tumors.

The tumor is multiple myeloma, gastric cancer, lung cancer, breast cancer, esophageal cancer, colon cancer, medulloblastoma, acute myelogenous leukemia, chronic leukemia, melanoma, prostate cancer, hepatoma, renal cell tumor, cervical cancer, skin cancer, ovarian cancer, colon cancer, glioma, thyroid cancer or pancreatic cancer.

In order to verify the simultaneous degradation effect of the compounds of the present invention on EGFR and PARP proteins in cancer cells, the present invention proposes the following 4 compounds for comparing the degradation effects.

The protein degradation activity evaluation system is used for evaluating the targeted degradation activity of 8 Dual Protacs (compound DP-1-DP-8) synthesized by the method and 4 single PROTACs (compound MP-1-MP-4) corresponding to VHL-and CRBN-ligands on EGFR and PARP proteins, and the results of comparative experiments show that the compound DP-1-DP-8 has obvious effect on the targeted degradation of the EGFR and PARP proteins and the effect is superior to that of the single PROTAC.

The invention has the advantages that:

the invention designs a series of novel Dual targeting degradation molecules Dual Protacs (compound DP-1-DP-8) with two independent inhibitor units (inhibitors of EGFR and PARP) and an E3 ligase ligand (CRBN or VHL), which can assemble multiple functions (such as inhibiting EGFR phosphorylation, inducing DNA damage and preventing the repair of the EGFR) into one molecule. Compared with a negative control, the compounds can simultaneously effectively induce E3 ligase dependent degradation of EGFR and PARP in pancreatic cancer cell strains and 1299 cells, and effectively inhibit the growth of cancer cells.

The Dual targeting degradation molecules (Dual Protacs) of the invention are superior to single PROTAC in that the Dual targeting degradation molecules (Dual Protacs) as EGFR-PARP Dual targeting PROTAC molecules can degrade EGFR and PARP proteins in cancer cells simultaneously, and the method can possibly stimulate the design of new generation PROTAC molecules and can solve the tumor heterogeneity and the target diversity of late stage cancer with reversed chemotherapy drug resistance.

The Dual targeting degradation molecule (Dual Protacs) of the invention is superior to the combined administration, and has the characteristics of effectively targeting and degrading EGFR and PARP proteins; similar to catalytic reaction, the medicament has low effective dose; only provides binding activity, is event-driven, is different from the traditional occupation drive, and does not need to directly inhibit the functional activity of the target protein; the drug does not require long-term and high-strength binding to the target protein.

The methods of the invention provide novel therapeutic modalities for the treatment of EGFR and PARP mediated tumors and/or other diseases.

Drawings

FIG. 1 DP-1 mediated degradation properties of EGFR and PARP proteins at various concentrations 24h after administration in SW1990 cells;

FIG. 2 compares the DP-1, DP-2, DP-3, DP-4 mediated degradation of EGFR and PARP proteins in SW1990 cells at 24h post-administration;

FIG. 3 degradation effects of EGFR and PARP proteins after 6-48 hours of DP-1 action at different concentrations (1. mu.M, 3. mu.M, 5. mu.M) in SW1990 cells;

FIG. 4700 nM MG132 reverses effects on DP-1 mediated degradation of EGFR and PARP proteins at different concentrations;

FIG. 5 the degradation effect of MP-3 and MP-4 on EGFR and PARP proteins in H1299 cells after 36H of administration;

FIG. 6 compares the DP-5, DP-6, DP-7, DP-8 mediated degradation of EGFR and PARP proteins in H1299 cells after 36H of administration;

FIG. 7 in H1299 cells, the effect of different concentrations of DP-8 on the degradation of EGFR and PARP proteins at 36H;

FIG. 8 the effect of 4 μ M DP-8 on the degradation of EGFR and PARP proteins in H1299 cells after 6-48H;

FIG. 9 in H1299 cells, the reversal effect of 1 μ M MG132 on DP-8 mediated degradation of EGFR and PARP proteins at different concentrations;

FIG. 10 compares the DP-5, DP-6, DP-7, DP-8 mediated degradation of EGFR and PARP proteins in A431 cells after 36h of administration.

FIG. 11 evaluation of the effect of gefitinib, olaparib, DP-8 mediated proliferation of antitumor cells in H1299 cells.

Detailed Description

The above-mentioned aspects of the present invention will be further described in detail with reference to the accompanying drawings and examples.

The preparation process of the compound DP-1-DP-8 and the single PROTAC compound MP-1-MP-4 for simultaneously inducing the degradation of EGFR and PARP proteins according to the present invention is shown below.

The starting materials and reagents mentioned in all the examples below are commercially available.

Example 1

A method for preparing a compound DP-1 which can simultaneously induce the degradation of EGFR and PARP proteins, comprising the following steps:

step 1: preparation of intermediate a: dissolving N-Boc tyrosine methyl ester (19) (5.90g, 20mmol) and 2 equivalents of propynylbromide (20) in 20mL of anhydrous DMF, adding 2 equivalents of potassium carbonate while stirring, stirring at room temperature for 6h for hydroxyl etherification protection, adding 200mL of ethyl acetate and 200mL of saturated aqueous sodium chloride solution after the reaction is completed, extracting and layering, drying the organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to obtain 6.50g of yellow oily product (21) with the yield of 98%. Dissolving 2mmol of compound (21) in 30mL of mixed solvent of methanol and tetrahydrofuran with volume ratio of 1:1, adding 5 equivalents of saturated aqueous solution of sodium hydroxide under stirring, stirring overnight at room temperature, acidifying after complete reaction, and concentrating to obtain yellow oily intermediate A (22) with yield of 91%.

Step 2: the intermediate A is sequentially subjected to amide condensation with Olaparib and Gefitinib to prepare an intermediate B;

(1) binding to olaparib: dissolving olaparib (7) (0.73g, 2mmol) and 1 equivalent of intermediate A (22) in 50mL of anhydrous dichloromethane, sequentially adding 1.1 equivalent of EDCI, HOBt and 2 equivalents of DIPEA at 0 ℃ under stirring, reacting at room temperature overnight, adding 50mL of dichloromethane and 50mL of saturated aqueous sodium bicarbonate solution after reaction is complete, extracting and layering, drying the organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to obtain 0.60g of white solid compound (25) with a yield of 45%;

(2) dissolving 0.2mmol of compound (25) in 5mL of anhydrous dichloromethane, adding 2mL of hydrogen chloride saturated ethyl acetate solution under the stirring condition, stirring at room temperature overnight to remove protection, and directly removing the solvent by spin-drying after complete reaction to obtain a white solid compound (26) with the yield of 92%;

(3) in combination with gefitinib: in order to perform amide condensation, a hydroxyl carboxylation reaction is firstly performed, specifically, gefitinib (1) (0.64g, 2mmol) is dissolved in 10mL acetone, 1.1 equivalent of bromate (2a) and 2 equivalent of potassium carbonate are sequentially added under the stirring condition, heating reflux is performed overnight, after the reaction is completed, natural cooling is performed to room temperature, solid is removed by suction filtration, the filtrate is dried and concentrated by spinning to obtain a crude product, and the crude product is purified by silica gel column chromatography to obtain a white solid compound (3a) with the yield of 42%. Then, 1mmol of the compound (3a) is dissolved in 20mL of mixed solvent of methanol and tetrahydrofuran with the volume ratio of 1:1, 5 equivalents of saturated aqueous solution of sodium hydroxide is added under the condition of stirring, the mixture is stirred at room temperature overnight, the mixture is acidified after the reaction is completed, and the white solid product (4a) containing carboxyl is obtained after concentration, and the yield is 90%. Compound (4a) (75mg, 0.2mmol) and 1 equivalent of compound (26) were dissolved in 15mL of anhydrous dichloromethane, 1.1 equivalents EDCI, HOBt and 2 equivalents DIPEA were added in this order with stirring at 0 ℃, stirred overnight at room temperature, 20mL dichloromethane and 20mL of saturated aqueous sodium bicarbonate were added after completion of the amide condensation reaction, the layers were extracted, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 81mg of intermediate B (27) as a white solid in 44% yield.

And step 3: connecting the intermediate B with an azide-linked E3 ligand to obtain a compound DP-1;

(1) preparation of azide-linked E3 ligand: dissolving the compound (13) (0.27g and 1mmol) and 1 equivalent of the compound (14a) in 5mL of anhydrous DMF, adding 2 equivalents of DIPEA under stirring, reacting at 85 ℃ and stirring overnight, adding 50mL of ethyl acetate and 50mL of saturated aqueous sodium chloride solution after the reaction is completed, extracting and layering, drying an organic layer with anhydrous sodium sulfate, concentrating and purifying by silica gel column chromatography to obtain 0.11g of a yellow solid product, namely an azide-linked E3 ligand (15a) with the yield of 29%;

(2) linkage of intermediate B and azide-linked E3 ligand: intermediate B (27) (18mg, 0.02mmol) and 1 equivalent of azide-linked E3 ligand (15a) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalents of copper sulfate and 2 equivalents of aqueous sodium ascorbate were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 11mg of DP-1 as a yellow solid in 42% yield.

The compound obtained in the preparation process is subjected to nuclear magnetic resonance hydrogen spectrum and mass spectrum detection, and the method specifically comprises the following steps:

3a.¹H NMR(400MHz,DMSO-d₆)δ9.66(brs,1H),8.48(d,J＝4.4Hz,1H),8.10(d,J＝6.8Hz,1H),7.87(s,1H),7.74(d,J＝5.0Hz,1H),7.15(td,J＝13.2,2.8Hz,1H),7.22(d,J＝3.2Hz,1H),4.94(s,2H),4.21(q,J＝7.2Hz,2H),3.96(s,3H),1.24(t,J＝7.2Hz,3H).

4a.¹H NMR(400MHz,DMSO-d₆)δ13.15(brs,1H),12.06(s,1H),8.85(s,1H),8.78(s,1H),8.12(dd,J＝6.8,2.4Hz,1H),7.86(ddd,J＝9.0,4.4,2.6Hz,1H),7.60(s,1H),7.53(t,J＝13.2Hz,1H),5.09(s,2H),4.00(s,3H).

15a.¹H NMR(400MHz,CDCl₃)δ8.31(s,1H),7.50(t,J＝8.0Hz,1H),7.11(d,J＝7.2Hz,1H),6.94(d,J＝8.4Hz,1H),6.50(t,J＝5.6Hz,1H),4.92(dd,J＝12.0,5.2Hz,1H),3.73(t,J＝5.2Hz,2H),3.68(t,J＝5.2Hz,2H),3.50(q,J＝5.6Hz,2H),3.41(t,J＝5.2Hz,2H),2.87-2.72(m,3H),2.14-2.10(m,1H).

21.¹H NMR(400MHz,CDCl₃)δ7.06(d,J＝8.4Hz,2H),6.91(d,J＝8.8Hz,2H),4.98(d,J＝8.0Hz,1H),4.67(d,J＝2.4Hz,2H),4.55(dd,J＝14.0,6.0Hz,1H),3.72(s,3H),3.03(qd,J＝14.0,6.0Hz,2H),2.53(t,J＝2.4Hz,1H),1.42(s,9H).

25.¹H NMR(400MHz,CDCl₃)δ10.76(brs,1H),8.49-8.46(m,1H),7.80-7.69(m,3H),7.30-7.28(m,2H),7.13(t,J＝8.8Hz,2H),7.02(dd,J＝15.2,8.8Hz,1H),6.99(dd,J＝15.6,8.4Hz,2H),5.44(t,J＝8.0Hz,1H),4.84-4.71(m,1H),4.67(dd,J＝10.8,6.0Hz,2H),4.28(s,2H),3.83(brs,1H),3.60-3.35(m,3H),3.27-3.21(m,1H),3.08-3.01(m,2H),2.97(dd,J＝13.6,5.6Hz,1H),2.89(dd,J＝12.8,5.6Hz,1H),2.53(t,J＝2.4Hz,1H),1.42(s,9H).

27.¹H NMR(400MHz,CDCl₃)δ12.58(s,1H),9.50(d,J＝2.4Hz,1H),8.53(s,1H),8.25(d,J＝6.4Hz,1H),8.22(t,J＝6.8Hz,1H),8.11(dd,J＝5.2,2.0Hz,1H),7.96-7.93(m,1H),7.90-7.86(m,1H),7.84-7.77(m,3H),7.47-7.42(m,2H),7.33(d,J＝4.0Hz,1H),7.22(t,J＝7.2Hz,1H),7.11(dd,J＝11.2,6.4Hz,2H),6.81(dd,J＝11.2,6.4Hz,2H),5.01(ddd,J＝44.8,13.8,6.6Hz,1H),4.71-4.69(m,4H),4.32(s,2H),3.94(s,3H),3.56-3.44(m,7H),3.11-3.08(m,2H),2.97-2.84(m,3H).

DP-1.¹H NMR(400MHz,DMSO-d₆)δ12.58(s,1H),11.08(s,1H),9.49(s,1H),8.53(s,1H),8.25(d,J＝5.2Hz,1H),8.21(t,J＝6.8Hz,1H),8.11-8.10(m,2H),7.94(t,J＝7.2Hz,1H),7.87(t,J＝7.2Hz,1H),7.84-7.82(m,1H),7.80-7.76(m,1H),7.54(t,J＝6.0Hz,1H),7.44-7.42(m,2H),7.35(brs,1H),7.22(t,J＝6.8Hz,1H),7.13-7.07(m,3H),7.01(d,J＝6.0Hz,1H),6.84(t,J＝7.6Hz,2H),6.57(t,J＝4.8Hz,1H),5.04-4.99(m,4H),4.70(brs,2H),4.54(brs,2H),4.32(brs,2H),4.06-4.02(m,1H),3.96(s,3H),3.84(t,J＝4.0Hz,2H),3.60(t,J＝4.0Hz,2H),3.51(brs,2H),3.43(q,J＝4.0Hz,4H),3.10(brs,2H),2.94-2.81(m,4H),1.99-1.97(m,2H),1.76-1.71(m,1H).

HRMS(pos.ESI):m/z[M+H]⁺for C₆₆H₆₀ClF₂N₁₄O₁₂calcd:1313.4166,found:1313.4152.

example 2

A method for preparing a compound DP-2 which induces the degradation of EGFR and PARP proteins simultaneously, comprising the steps of:

step 1: preparation of intermediate a: dissolving N-Boc serine methyl ester (23) (4.10g, 20mmol) and 2 equivalents of propynylbromide (20) in 20mL of anhydrous DMF, adding 2 equivalents of sodium hydride under stirring at 0 ℃, naturally raising the temperature of the reaction to room temperature and stirring overnight, performing hydroxyl etherification protection, after the reaction is completed, dropwise adding saturated aqueous sodium chloride solution to quench the reaction, adding 200mL of diethyl ether, performing extraction and separation, drying an organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to obtain 3.61g of yellow oily intermediate A (24) with the yield of 74%.

(1) binding to olaparib: dissolving olaparib (7) (0.73g, 2mmol) and 1 equivalent of intermediate A (24) in 50mL of anhydrous dichloromethane, sequentially adding 1.1 equivalent of EDCI, HOBt and 2 equivalents of DIPEA at 0 ℃ under stirring, reacting at room temperature overnight, adding 50mL of dichloromethane and 50mL of saturated aqueous sodium bicarbonate solution after reaction, extracting and layering, drying the organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to obtain 0.59g of white solid compound (28) with 50% yield;

(2) dissolving 0.2mmol of compound (28) in 5mL of anhydrous dichloromethane, adding 2mL of hydrogen chloride saturated ethyl acetate solution under the stirring condition, stirring at room temperature overnight to remove protection, and directly removing the solvent by spin-drying after complete reaction to obtain a white solid compound (29) with the yield of 90%;

(3) in combination with gefitinib: dissolving gefitinib (1) (0.64g, 2mmol) in 10mL acetone, sequentially adding 1.1 equivalent of bromate (2b) and 2 equivalents of potassium carbonate under stirring, heating and refluxing overnight, naturally cooling to room temperature after complete reaction, removing solids by suction filtration, drying and concentrating the filtrate to obtain a crude product, and purifying by silica gel column chromatography to obtain an ester group-containing white solid compound (3b) with a yield of 51%. Then, 1mmol of the compound (3b) was dissolved in 20mL of a mixed solvent of methanol and tetrahydrofuran in a volume ratio of 1:1, and 5 equivalents of a saturated aqueous solution of sodium hydroxide was added with stirring, and the mixture was stirred at room temperature overnight, and after the reaction was completed, the product was acidified and concentrated to obtain a white solid product (4b) containing a carboxyl group in a yield of 92% (see example 1 for a specific reaction route). The prepared compound (4B) (81mg, 0.2mmol) and 1 equivalent of the compound (29) were dissolved in 15mL of anhydrous dichloromethane, 1.1 equivalents of EDCI, HOBt and 2 equivalents of DIPEA were added in this order with stirring at 0 ℃ and stirred overnight at room temperature, 20mL of dichloromethane and 20mL of saturated aqueous sodium bicarbonate were added after completion of the amide condensation reaction, the layers were extracted and separated, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to obtain 81mg of intermediate B (30) as a white solid in 64% yield.

And step 3: connecting the intermediate B with an azide-linked E3 ligand to obtain a compound DP-2;

(1) preparation of azide-linked E3 ligand: azide-linked E3 ligand (15a) was prepared according to the preparation method in example 1;

(2) linkage of intermediate B and azide-linked E3 ligand: intermediate B (30) (18mg, 0.02mmol) and 1 equivalent of azide-linked E3 ligand (15a) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalent of copper sulfate and 2 equivalent of aqueous sodium ascorbate were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 12mg of DP-2 as a yellow solid in 48% yield.

24.¹H NMR(400MHz,CDCl₃)δ9.51(brs,1H),5.42(d,J＝8.4Hz,1H),4.50(dd,J＝5.2,3.2Hz,1H),4.19-4.18(m,2H),4.00(dd,J＝9.2,3.2Hz,1H),3.81(dd,J＝9.2,3.2Hz,1H),2.48(t,J＝2.4Hz,1H),1.46(s,9H).

28.¹H NMR(400MHz,CDCl₃)δ10.86(brs,1H),8.49-8.47(m,1H),7.79-7.71(m,3H),7.35-7.32(m,2H),7.05(t,J＝8.8Hz,2H),5.51(d,J＝8.0Hz,1H),4.83(d,J＝28.8Hz,1H),4.30(s,2H),4.15-4.11(m,2H),3.98(brs,1H),3.81-3.36(m,9H),2.44(d,J＝11.2Hz,1H),1.43(s,9H).

4b.¹H NMR(400MHz,DMSO-d₆)δ12.03(s,2H),8.84(s,1H),8.69(s,1H),8.10(dd,J＝6.8,2.4Hz,1H),7.87-7.84(m,1H),7.56(s,1H),7.53(t,J＝9.2Hz,1H),4.32(t,J＝6.4Hz,2H),3.99(s,3H),2.46(t,J＝7.2Hz,2H),2.03(p,J＝6.8Hz,2H).

30.¹H NMR(400MHz,CDCl₃)δ12.60(s,1H),9.57(brs,1H),8.51(s,1H),8.37(d,J＝8.0Hz,1H),8.28-8.25(m,2H),8.13(dd,J＝6.8,2.4Hz,1H),7.98-7.91(m,3H),7.87-7.78(m,3H),7.40-7.36(m,1H),7.24(t,J＝9.2Hz,1H),5.00-4.90(m,1H),4.32(s,2H),4.16(s,4H),3.95(s,3H),3.63-3.53(m,8H),3.47(s,2H),3.16-3.13(m,2H),2.39(q,J＝6.8Hz,2H),2.05(q,J＝6.0Hz,2H).

DP-2.¹H NMR(400MHz,DMSO-d₆)δ12.59(s,1H),11.09(s,1H),9.52(s,1H),8.50(s,1H),8.32(d,J＝8.0Hz,1H),8.26-8.24(m,1H),8.11(dd,J＝6.8,2.4Hz,1H),8.01-7.99(m,1H),7.94-7.92(m,1H),7.88(td,J＝7.2,1.2Hz,1H),7.82-7.76(m,2H),7.53(t,J＝8.0Hz,1H),7.45-7.41(m,2H),7.35(brs,1H),7.20(brs,2H),7.08-7.02(m,2H),7.01(d,J＝7.2Hz,1H),6.56(brs,1H),5.05(dd,J＝12.8,5.2Hz,1H),4.97-4.92(m,1H),4.50(brs,4H),4.32(brs,2H),4.14-4.13(m,2H),3.94(s,3H),3.83(brs,2H),3.58-3.42(m,12H),3.12(brs,2H),2.93-2.85(m,1H),2.51-2.50(m,2H),2.49-2.36(m,2H),2.04(brs,2H),1.83-1.81(m,1H).

HRMS(pos.ESI):m/z[M+H]⁺for C₆₂H₆₀ClF₂N₁₄O₁₂calcd:1265.4166,found:1265.4172.

example 3

A method for preparing compounds DP-3 and DP-4 which simultaneously induce the degradation of EGFR and PARP proteins, comprising the steps of:

step 1: preparation of intermediate a: intermediate a (24) was prepared using the procedure of example 2.

(1) binding to olaparib: dissolving olaparib (7) (0.73g and 2mmol) and 1 equivalent of compounds (8b) and (8c) in 50mL of anhydrous dichloromethane, respectively adding 1.1 equivalent of EDCI, HOBt and 2 equivalents of DIPEA under stirring at 0 ℃, stirring overnight at room temperature, respectively adding 50mL of dichloromethane and 50mL of saturated aqueous sodium bicarbonate solution after reaction is completed, extracting and layering, drying an organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to respectively obtain white solid compounds (9b) and (9c), wherein the yield is respectively (9b) and 76%; (9c) 85 percent. Then respectively dissolving 0.2mmol of compounds (9b) and (9c) in 5mL of anhydrous dichloromethane, respectively adding 2mL of hydrogen chloride saturated ethyl acetate solution under the condition of stirring, stirring at room temperature overnight, directly removing the solvent by spin-drying after complete reaction to respectively obtain white solid products (10b) and (10c), wherein the yield is respectively (10b) and 91%; (10c) 92 percent. Dissolving 2mmol of compounds (10b) and (10c) and 1 equivalent of intermediate A (24) in 30mL of anhydrous dichloromethane, reacting at 0 ℃ under stirring, sequentially adding 1.1 equivalent of EDCI, HOBt and 2 equivalents of DIPEA respectively, reacting at room temperature overnight under stirring, after the reaction is completed, adding 30mL of dichloromethane and 30mL of saturated aqueous sodium bicarbonate solution respectively, extracting and layering, drying an organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to obtain white solid products (31b) and (31c), wherein the yield is (31b) and 61% respectively; (31c) 76%;

(2) respectively dissolving 0.2mmol of compounds (31b) and (31c) in 5mL of anhydrous dichloromethane, respectively adding 2mL of hydrogen chloride saturated ethyl acetate solution under the condition of stirring, stirring at room temperature overnight to remove protection, directly removing the solvent by spin-drying after complete reaction to respectively obtain white solid compounds (32b) and (32c), wherein the yield is respectively (32b) and 91%; (32c) 94%;

(3) in combination with gefitinib: compounds (4b) and (4c) were prepared by the method of example 2, and compound (4c) was obtained in a yield of 96%. Dissolving compound (4B) (75mg, 0.2mmol) and 1 equivalent of compound (32B), compound (4c) (75mg, 0.2mmol) and 1 equivalent of compound (32c) in 15mL of anhydrous dichloromethane respectively, adding 1.1 equivalent of EDCI, HOBt and 2 equivalents of DIPEA respectively at 0 ℃ under stirring, stirring overnight at room temperature, adding 20mL of dichloromethane and 20mL of saturated aqueous sodium bicarbonate solution after amide condensation reaction is completed, extracting and layering, drying the organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to obtain white solid intermediates B (33a) and (33c) respectively, wherein the yield is (33a) and 38%; (33c) and 52 percent.

And step 3: connecting the intermediate B with an azide-linked E3 ligand to obtain compounds DP-3 and DP-4;

(1) preparation of azide-linked E3 ligand: azide-linked E3 ligands (15a) and (15b) were prepared according to the preparation method in example 1, wherein the yield of compound (15b) was 33%;

(2) linkage of intermediate B and azide-linked E3 ligand: dissolving 0.02mmol of intermediate B (33a) and intermediate B (33c) with E3 ligands (15a) and (15B) respectively connected with 1 equivalent of azide in 2mL of tetrahydrofuran, respectively dropwise adding 0.5mL of 1 equivalent of copper sulfate and 2 equivalent of sodium ascorbate aqueous solution under stirring at room temperature, stirring for 0.5h at room temperature after reaction is completed, respectively adding 10mL of ethyl acetate and 10mL of saturated sodium chloride aqueous solution after reaction is completed, extracting and layering, drying an organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to respectively obtain yellow solid products DP-3 and DP-4, wherein the yield is respectively DP-3 and DP-45%; DP-4, 41%.

4c.¹H NMR(400MHz,DMSO-d₆)δ12.13(s,2H),8.84(s,1H),8.73(s,1H),8.12(dd,J＝6.8,2.4Hz,1H),7.89-7.86(m,1H),7.56(s,1H),7.52(t,J＝8.8Hz,1H),4.29(t,J＝6.4Hz,2H),3.98(s,3H),2.26(t,J＝7.2Hz,2H),1.81(p,J＝7.2Hz,2H),1.60(p,J＝7.2Hz,2H),1.48(p,J＝7.2Hz,2H).

9b.¹H NMR(400MHz,CDCl₃)δ10.94(brs,1H),8.49-8.46(m,1H),7.78-7.73(m,3H),7.34-7.27(m,2H),7.05(t,J＝8.8Hz,1H),4.81(brs,1H),4.30(s,2H),4.38(brs,3H),3.57-3.56(m,2H),3.43(brs,1H),3.30(brs,2H),3.20-3.17(m,2H),2.42(t,J＝6.8Hz,1H),2.35(t,J＝6.8Hz,1H),1.88-1.81(m,2H),1.44(s,9H).

9c.¹H NMR(400MHz,CDCl₃)δ10.80(brs,1H),8.49-8.47(m,1H),7.78-7.72(m,3H),7.33(brs,2H),7.05(t,J＝8.8Hz,1H),4.60(brs,1H),4.30(s,2H),3.78(brs,3H),3.57(brs,2H),3.43(brs,1H),3.29(brs,2H),3.11(brs,2H),2.37-2.30(m,2H),1.66-1.65(m,2H),1.51-1.50(m,2H),1.44(s,9H),1.37-1.36(m,2H).

31b.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),8.26(d,J＝7.6Hz,1H),7.97(d,J＝7.6Hz,1H),7.90(t,J＝7.6Hz,2H),7.84(t,J＝7.6Hz,1H),7.44(brs,1H),7.37(t,J＝6.4Hz,1H),7.24(t,J＝9.2Hz,1H),6.81(t,J＝6.4Hz,1H),4.33(s,2H),4.12(d,J＝12.4Hz,2H),4.06-4.02(m,1H),3.64(brs,1H),3.58-3.46(m,5H),3.41-3.38(m,1H),3.19(brs,1H),3.14(brs,1H),3.08(brs,2H),2.33(t,J＝6.8Hz,1H),2.26(t,J＝6.8Hz,1H),1.63(q,J＝5.6Hz,1H),1.35(d,J＝24.8Hz,9H),1.24(s,2H).

31c.¹H NMR(400MHz,CDCl₃)δ11.06(d,J＝46.8Hz,1H),8.49-8.46(m,1H),7.79-7.70(m,3H),7.34-7.32(m,2H),7.05(t,J＝9.2Hz,1H),6.57(brs,1H),5.51-5.46(m,1H),4.30(s,2H),4.28-4.23(m,1H),4.19-4.11(m,2H),3.93-3.89(m,1H),3.70-3.66(m,4H),3.55(brs,2H),3.43(brs,1H),3.28(brs,4H),2.48(brs,1H),2.37(t,J＝7.2Hz,1H),2.29(t,J＝7.2Hz,1H),1.66(p,J＝7.2Hz,2H),1.55(p,J＝7.2Hz,2H),1.45(s,9H),1.38(p,J＝7.2Hz,2H).

33a.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),9.56(s,1H),8.51(s,1H),8.26(dd,J＝7.6,1.2Hz,1H),8.15(t,J＝7.6Hz,1H),8.13(dd,J＝7.2,2.4Hz,1H),8.04-7.99(m,1H),7.95(t,J＝8.4Hz,1H),7.88(t,J＝7.6Hz,1H),7.85-7.77(m,3H),7.46-7.42(m,2H),7.36(brs,1H),7.25-7.21(m,2H),4.43(p,J＝6.8Hz,1H),4.32(s,2H),4.15-4.11(m,4H),3.94(s,3H),3.60-3.57(m,4H),3.50-3.99(m,4H),3.30(brs,1H),3.19(brs,1H),3.13(brs,1H),3.09-3.03(m,2H),2.43-2.41(m,2H),2.31(t,J＝7.2Hz,1H),2.24(t,J＝7.2Hz,1H),2.05(p,J＝7.2Hz,2H),1.61(brs,2H).

33c.¹H NMR(400MHz,CDCl₃)δ11.91(d,J＝53.2Hz,1H),9.15(s,1H),8.58(s,1H),8.40(d,J＝7.2,1H),7.88-7.86(m,1H),7.74-7.67(m,4H),7.64-7.62(m,2H),7.30-7.28(m,2H),7.23-7.19(m,2H),7.09-6.99(m,2H),4.70-4.64(m,1H),4.25(s,2H),4.15-4.00(m,2H),3.99(brs,2H),3.94(s,3H),3.82-3.65(m,6H),3.51(brs,3H),3.38-3.21(m,6H),2.45(s,1H),2.35-2.23(m,4H),1.81(t,J＝6.8Hz,2H),1.66(t,J＝6.8Hz,2H),1.57-1.43(m,5H).

DP-3.¹H NMR(400MHz,DMSO-d₆)δ12.59(s,1H),11.10(s,1H),9.56(s,1H),8.50(s,1H),8.25(d,J＝7.6Hz,1H),8.12(dd,J＝6.8,2.4Hz,2H),8.00-7.76(m,6H),7.53(t,J＝8.0Hz,1H),7.43(t,J＝8.8Hz,2H),7.35(brs,1H),7.24-7.19(m,2H),7.07(d,J＝8.8Hz,1H),7.01(d,J＝7.2Hz,1H),6.56(t,J＝5.6Hz,1H),5.05(dd,J＝12.8,5.2Hz,1H),4.50-4.42(m,4H),4.31(d,J＝7.6Hz,2H),4.12(t,J＝5.2Hz,2H),3.94(s,3H),3.83(brs,2H),3.58-3.57(m,5H),3.49-3.42(m,5H),3.29(brs,1H),3.17(brs,1H),3.12(brs,1H),3.05-3.04(m,2H),2.93-2.85(m,1H),2.51-2.50(m,2H),2.42(t,J＝8.0Hz,2H),2.28(t,J＝7.2Hz,1H),2.21(t,J＝7.2Hz,1H),2.03(brs,3H),1.59(brs,2H),1.23(brs,2H).

HRMS(pos.ESI):m/z[M+H]⁺for C₆₆H₆₇ClF₂N₁₅O₁₃calcd:1350.4694,found:1350.4681.

DP-4.¹H NMR(400MHz,CDCl₃)δ11.06(d,J＝16.08Hz,1H),10.01(s,1H),8.99(s,1H),8.59(s,1H),8.41(d,J＝8.4Hz,1H),7.85(d,J＝6.4Hz,1H),7.74-7.70(m,4H),7.59-7.57(m,2H),7.46(t,J＝8.0Hz,1H),7.33-7.28(m,3H),7.08-7.04(m,4H),6.86(dd,J＝8.8,4.4Hz,1H),6.50(d,J＝4.4Hz,1H),4.94-4.89(m,1H),4.54-4.49(m,5H),4.25(s,2H),4.02(t,J＝7.2Hz,2H),3.98(s,3H),3.84(brs,3H),3.68-3.64(m,5H),3.60(s,4H),3.52(brs,2H),3.36(brs,2H),3.28(brs,2H),3.21(brs,3H),2.80-2.70(m,2H),2.32-2.29(m,3H),2.22-2.21(m,1H),1.84(brs,2H),1.69(brs,2H),1.57-1.43(m,7H).

HRMS(pos.ESI):m/z[M+H]⁺for C₇₂H₇₉ClF₂N₁₅O₁₄calcd:1450.5582,found:1450.5536.

example 4

A method for preparing a compound DP-5 which induces the degradation of EGFR and PARP proteins simultaneously, comprising the steps of:

step 1: preparation of intermediate a: intermediate a (22) was prepared using the procedure of example 1;

step 2: preparation of intermediate B: intermediate B (27) was prepared using the procedure of example 1;

and step 3: connecting the intermediate B with an azide-linked E3 ligand to obtain a compound DP-5;

intermediate B (27) (18mg, 0.02mmol) and 1 equivalent of azide-linked E3 ligand (18) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalents of copper sulfate and 2 equivalents of aqueous sodium ascorbate were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 14mg of DP-5 as a yellow solid in 48% yield.

The prepared compound DP-5 is subjected to nuclear magnetic resonance hydrogen spectrum and mass spectrum detection, and specifically comprises the following steps:

DP-5.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),9.54(s,1H),8.98(s,1H),8.57(t,J＝6.0Hz,1H),8.54(s,1H),8.25(d,J＝7.6Hz,2H),8.18(d,J＝7.6Hz,1H),8.11(dd,J＝6.8,2.4Hz,1H),8.02(d,J＝9.2Hz,1H),7.94(t,J＝7.2Hz,1H),7.89-7.78(m,4H),7.47-7.37(m,6H),7.26(brs,1H),7.23(t,J＝8.8Hz,1H),7.11(t,J＝8.8Hz,2H),6.86(t,J＝8.4Hz,2H),5.14(d,J＝3.2Hz,1H),5.06-4.98(m,3H),4.71(brs,2H),4.54(d,J＝9.2Hz,1H),4.46-4.41(m,2H),4.35-4.33(m,5H),4.21(dd,J＝16.0,5.2Hz,1H),3.96(s,3H),3.66-3.43(m,7H),3.11(brs,2H),2.93-2.85(m,3H),2.44(s,3H),2.30-2.15(m,2H),2.04-2.02(m,3H),0.94(s,9H).

HRMS(pos.ESI):m/z[M+H]⁺for C₇₅H₇₇ClF₂N₁₅O₁₁S calcd:1468.5299,found:1468.5287.

example 5

A method for preparing a compound DP-6 which induces the degradation of EGFR and PARP proteins simultaneously, comprising the following steps:

step 1: preparation of intermediate a: intermediate a (24) was prepared using the procedure of example 2;

step 2: preparation of intermediate B: intermediate B (30) was prepared using the procedure of example 2;

and step 3: connecting the intermediate B with an azide-linked E3 ligand to obtain a compound DP-6;

intermediate B (30) (17mg, 0.02mmol) and 1 equivalent of azide-linked E3 ligand (18) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalents of copper sulfate and 2 equivalents of aqueous sodium ascorbate were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 10mg of DP-6 as a yellow solid in 36% yield.

The prepared compound DP-6 is subjected to nuclear magnetic resonance hydrogen spectrum and mass spectrum detection, and specifically comprises the following steps:

DP-6.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),9.57(s,1H),8.98(s,1H),8.57(t,J＝6.0Hz,1H),8.51(s,1H),8.34(d,J＝8.0Hz,1H),8.25(d,J＝7.6Hz,1H),8.11(dd,J＝6.8,2.4Hz,1H),8.05(d,J＝13.6Hz,1H),7.99(d,J＝9.2Hz,1H),7.97-7.93(m,1H),7.88(td,J＝8.0,1.2Hz,1H),7.84-7.76(m,3H),7.46-7.35(m,7H),7.21(s,2H),5.13(d,J＝3.6Hz,1H),4.97-4.92(m,1H),4.55-4.50(m,3H),4.46-4.41(m,2H),4.32(brs,5H),4.22(dd,J＝16.0,5.6Hz,1H),4.14(d,J＝5.6Hz,2H),3.94(s,3H),3.65-3.41(m,10H),3.13(brs,2H),2.44(s,3H),2.38(d,J＝6.4Hz,2H),2.28-2.15(m,2H),2.03-2.01(m,5H),0.92(s,9H).

HRMS(pos.ESI):m/z[M+H]⁺for C₇₁H₇₇ClF₂N₁₅O₁₁S calcd:1420.5299,found:1420.5281.

example 6

A method for preparing compounds DP-7 and DP-8 which simultaneously induce the degradation of EGFR and PARP proteins, comprising the steps of:

step 1: preparation of intermediate a: intermediate a (24) was prepared using the procedure of example 3;

step 2: preparation of intermediate B: intermediate B (33B) and intermediate B (33c) were prepared using the procedure of example 3;

intermediates B (33B) and (33c) were prepared using the reaction route in example 3, intermediate B (33B) in 45% yield. Dissolving 0.02mmol of intermediate B (33B) and intermediate B (33c) in 2mL of tetrahydrofuran, respectively adding 0.5mL of 1 equivalent of copper sulfate and 2 equivalent of sodium ascorbate aqueous solution dropwise under stirring at room temperature, stirring at room temperature for 0.5h, respectively adding 10mL of ethyl acetate and 10mL of saturated sodium chloride aqueous solution after reaction is completed, extracting and layering, drying an organic layer with anhydrous sodium sulfate, concentrating, and purifying by silica gel column chromatography to respectively obtain yellow solid products DP-7 and DP-8 with the yields of DP-7 and 41%; DP-4, 35%.

33b.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),9.77(s,1H),8.54(s,1H),8.26(d,J＝7.6Hz,1H),8.14-8.10(m,1H),8.02(t,J＝4.8Hz,1H),7.97-7.95(m,1H),7.90-7.78(m,3H),7.45(t,J＝8.8Hz,2H),7.36(brs,1H),7.24(brs,1H),7.21(s,1H),4.56(brs,1H),4.33(s,2H),4.16-4.13(m,3H),3.99(brs,1H),3.95(s,3H),3.63-3.44(m,6H),3.19(brs,1H),3.13(brs,1H),3.05-2.99(m,1H),2.75(d,J＝4.4Hz,1H),2.22(t,J＝6.0Hz,2H),1.86-1.82(m,2H),1.61(t,J＝6.4Hz,2H),1.49(p,J＝7.6Hz,2H).

DP-7.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),10.05(brs,1H),8.98(s,1H),8.59(brs,2H),8.25(d,J＝7.6Hz,1H),8.13(d,J＝7.6Hz,1H),8.11-8.08(m,2H),8.03-7.99(m,2H),7.97-7.93(m,2H),7.89(brs,1H),7.83(d,J＝7.6Hz,1H),7.79-7.76(m,1H),7.45-7.36(m,6H),7.22(s,2H),5.15(brs,1H),4.55-4.52(m,4H),4.45-4.50(m,2H),4.34-4.30(m,5H),4.21(dd,J＝16.0,5.6Hz,1H),4.14(t,J＝6.4Hz,2H),3.99(brs,2H),3.95(s,3H),3.64-3.57(m,6H),3.51-3.46(m,2H),3.19(brs,1H),3.13(brs,1H),2.43(s,3H),2.28-2.15(m,5H),2.06-2.01(m,3H),1.83-1.81(m,3H),1.60(brs,2H),1.46(brs,2H),0.92(s,9H).

HRMS(pos.ESI):m/z[M+H]⁺for C₇₅H₈₄ClF₂N₁₆O₁₂S calcd:1505.5826,found:1505.5829.

DP-8.¹H NMR(400MHz,DMSO-d₆)δ12.60(s,1H),10.29(brs,2H),8.98(s,1H),8.65(s,1H),8.59(t,J＝6.0Hz,1H),8.25(d,J＝7.6Hz,1H),8.08(dd,J＝6.8,2.0Hz,1H),8.05(brs,1H),8.02-7.94(m,4H),7.88(t,J＝7.6Hz,1H),7.82(t,J＝7.6Hz,1H),7.78-7.76(m,1H),7.49(t,J＝8,8Hz,1H),7.42-7.36(m,5H),7.21-7.21(m,2H),5.15(brs,1H),4.55-4.40(m,5H),4.32(brs,5H),4.21(dd,J＝15.6,5.6Hz,1H),4.14(t,J＝5.2Hz,2H),3.96(s,3H),3.65-3.62(m,3H),3.55-3.49(m,5H),3.15(d,J＝16.4Hz,3H),3.02(brs,3H),2.43(s,3H),2.28-2.19(m,7H),2.06-2.00(m,3H),1.89-1.83(m,1H),1.82(brs,2H),1.58(brs,2H),1.44-1.34(m,7H),0.92(s,9H).

HRMS(pos.ESI):m/z[M+H]⁺for C₇₉H₉₂ClF₂N₁₆O₁₂S calcd:1561.6452,found:1561.6462.

comparative example 1

The preparation process of the single PROTAC compound MP-1 comprises the following steps:

compound (6) (22mg, 0.05mmol) and 1 equivalent of azide-linked E3 ligand (15a) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalents of copper sulfate and 2 equivalents of aqueous sodium ascorbate solution were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride solution were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 13mg of yellow solid product MP-1 with a yield of 32%.

The prepared compound MP-1 is subjected to nuclear magnetic resonance hydrogen spectrum and mass spectrum detection, and specifically comprises the following steps:

MP-1.¹H NMR(400MHz,DMSO-d₆)δ11.10(s,1H),9.59(s,1H),8.51(s,1H),8.42(t,J＝5.6Hz,1H),8.13(dd,J＝6.8,2.4Hz,1H),7.89(s,1H),7.82(s,1H),7.80-7.77(m,1H),7.55(t,J＝8.0Hz,1H),7.44(t,J＝9.2Hz,1H),7.20(s,1H),7.07(d,J＝8.8Hz,1H),7.02(d,J＝7.2Hz,1H),6.56(t,J＝6.0Hz,1H),5.06(dd,J＝12.8,5.6Hz,1H),4.49(t,J＝4.8Hz,2H),4.31(d,J＝5.6Hz,2H),4.15(t,J＝6.0Hz,2H),3.94(s,3H),3.80(t,J＝5.2Hz,2H),3.58(t,J＝5.2Hz,2H),3.41(dd,J＝10.8,5.2Hz,2H),2.94-2.84(m,1H),2.61-2.54(m,2H),2.36(t,J＝7.2Hz,2H),2.10-2.02(m,3H).

HRMS(pos.ESI):m/z[M+H]⁺for C₃₉H₃₈ClFN₁₀O₈calcd:829.2619,found:829.2631.

comparative example 2

The preparation process of the single PROTAC compound MP-2 comprises the following steps:

compound (12) (22mg, 0.05mmol) and 1 equivalent of azide-linked E3 ligand (15a) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalents of copper sulfate and 2 equivalents of aqueous sodium ascorbate were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 16mg of MP-2 as a yellow solid product in 39% yield.

The prepared compound MP-2 is detected by nuclear magnetic resonance hydrogen spectrum and mass spectrum, and the method comprises the following steps:

MP-2.¹H NMR(400MHz,DMSO-d₆)δ12.61(s,1H),11.11(s,1H),8.26(d,J＝8.0Hz,1H),7.96(d,J＝8.0Hz,1H),7.89(td,J＝8.0,1.6Hz,1H),7.85-7.80(m,2H),7.56(dd,J＝12.8,7.6Hz,1H),7.46-7.43(m,1H),7.37(d,J＝6.4Hz,1H),7.24(t,J＝8.8Hz,1H),7.11(dd,J＝8.4,5.6Hz,1H),7.03(dd,J＝6.8,4.4Hz,1H),6.58(d,J＝4.4Hz,1H),5.07(dd,J＝12.8,5.6Hz,1H),4.49(t,J＝4.4Hz,2H),4.34(s,2H),3.82(d,J＝3.2Hz,2H),3.62-3.61(m,4H),3.52(brs,2H),3.45(brs,2H),3.37(brs,1H),3.30(brs,1H),3.16(brs,2H),2.90-2.82(m,3H),2.72-2.54(m,4H),2.06-2.03(m,1H).

HRMS(pos.ESI):m/z[M+H]⁺for C₄₂H₄₂FN₁₀O₈calcd:833.3166,found:833.3161.

comparative example 3

The preparation process of the single PROTAC compound MP-3 comprises the following steps:

compound (6) (22mg, 0.05mmol) and 1 equivalent of azide-linked E3 ligand (18) were dissolved in 2mL of tetrahydrofuran, 0.5mL of 1 equivalents of copper sulfate and 2 equivalents of aqueous sodium ascorbate were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL of ethyl acetate and 10mL of saturated aqueous sodium chloride were added after completion of the reaction, the layers were separated by extraction, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 17mg of MP-3 as a yellow solid product in 35% yield.

The prepared compound MP-3 is detected by nuclear magnetic resonance hydrogen spectrum and mass spectrum, and the method comprises the following steps:

MP-3.¹H NMR(400MHz,CDCl₃)δ8.95(brs,1H),8.67(s,1H),8.61(s,1H),8.04(d,J＝4.4Hz,1H),7.96(brs,1H),7.88(s,1H),7.46(s,1H),7.36-7.29(m,5H),7.20(s,1H),7.14(t,J＝8.8Hz,1H),7.07(d,J＝5.6Hz,1H),6.53(d,J＝8.0Hz,1H),4.74(t,J＝4.8Hz,1H),4.59-4.47(m,4H),4.44(d,J＝8.8Hz,1H),4.38-4.34(m,2H),4.25-4.22(m,1H),4.19-4.15(m,2H),4.07(d,J＝12.0Hz,1H),3.97(s,3H),3.62(dd,J＝11.2,3.2Hz,1H),2.50(s,3H),2.45(t,J＝4.8Hz,3H),2.22-2.01(m,7H),0.93(s,9H).

HRMS(pos.ESI):m/z[M+H]⁺for C₄₈H₅₆ClFN₁₁O₇S calcd:984.3752,found:984.3759.

comparative example 4

The preparation process of the single PROTAC compound MP-4 comprises the following steps:

compound (12) (22mg, 0.05mmol) and 1 equivalent azide-linked E3 ligand (18) were dissolved in 2mL tetrahydrofuran, 0.5mL1 equivalent copper sulfate and 2 equivalent sodium ascorbate aqueous solution were added dropwise with stirring at room temperature, the reaction was stirred at room temperature for 0.5h, 10mL ethyl acetate and 10mL saturated sodium chloride aqueous solution were added after completion of the reaction, the layers were extracted and separated, the organic layer was dried over anhydrous sodium sulfate, concentrated and purified by silica gel column chromatography to give 14mg yellow solid product MP-4 in 36% yield.

The prepared compound MP-4 is detected by nuclear magnetic resonance hydrogen spectrum and mass spectrum, and the method comprises the following steps:

MP-4.¹H NMR(400MHz,CDCl₃)δ11.33(brs,1H),8.69(s,1H),8.43(s,1H),7.78-7.71(m,4H),7.44-7.29(m,6H),7.24-7.19(m,2H),7.07-7.00(m,1H),4.78-4.72(m,1H),4.64-4.55(m,2H),4.34-4.22(m,5H),4.15(t,J＝11.2Hz,1H),3.70-3.67(m,2H),3.48(brs,2H),3.31(brs,1H),3.17(brs,1H),3.02(brs,2H),2.76(brs,2H),2.48(s,3H),2.43-2.07(m,10H),0.99(s,9H).

HRMS(pos.ESI):m/z[M+Na]⁺for C₅₁H₅₈FN₁₁NaO₇S calcd:1010.4118,found:1010.4128.

comparative example 5

Compounds MP-3, MP-4 and DP-1-DP-8 were tested for EGFR and PARP protein degradation in tumor cells (SW1990, H1299 or A431).

1. Experimental methods and procedures

(1) SW1990, H1299 or A431 cells in log phase were made in fresh medium to a concentration of 2X 10⁵Mixing the cell suspension solution with the cell suspension solution of/mL, and adding the cell suspension solution into a 6-hole plate;

(2) adding compounds (DP-1-DP-8) with different concentrations after the cells adhere to the wall, and centrifugally collecting the cells after treating for 24 hours;

adding DP-1 or DP-8 with different concentrations after the cells adhere to the wall, and centrifugally collecting the cells after 6,12,24,36 and 48 hours of treatment;

③ after the cells are attached to the wall, the cells are pretreated for 12 hours without adding or adding MG132 (proteasome inhibitor) with given concentration, then DP-1 or DP-8 with different concentrations are added, and after 36 hours of treatment, the cells are collected by centrifugation;

(3) detecting protein degradation of the cells collected in (2) by using EGFR and PARP antibodies in a western method;

(4) log phase grown H1299 cells were trypsinized, plated in 96 well plates at 5000/well density and after 24 hours treated with DP-8 in different concentration gradients (0-200 μ M) and gelitinib, Olaparib. Viable cell numbers were measured 24 hours after administration with CCK-8 at 450nm wavelength. IC calculation Using GraphPad Prism₅₀The value is obtained.

2. Results of the experiment

The Western Blot method was used to detect the degradation of both EGFR and PARP proteins by compound DP-1-DP-4 24 hours after administration in SW1990 cells. As shown in FIG. 1, the expression of EGFR and PARP decreased with increasing concentrations of DP-1 after 2 hours of action on SW1990 cells. As shown in FIG. 2, it is known that DP-1, DP-2, DP-3 and DP-4 all degrade EGFR and PARP, wherein DP-1 and DP-3 have the strongest degradation ability to EGFR, and DP-1 has the best degradation ability to PARP among 4 compounds. The kinetics of degradation of compound DP-1 in the SW1990 cell line was further examined. As shown in FIG. 3, it can be seen that 5 μ M DP-1 starts to degrade EGFR and PARP at 6 hours, and the degradation of EGFR and PARP is most significant at 24 and 36 hours. To further demonstrate that compound DP-1 is proteolytically degraded by the proteasome system, the proteasome inhibitor MG132 (700nM) was introduced and the DP-1 was co-acted for 24 hours before protein extraction and the change in the corresponding protein was detected using the Western Blot assay. As shown in FIG. 4, it was found that the expression level of each drug-added concentration protein was consistent with that of MG132 alone, indicating that the degradation of compound DP-1 was completely reversed by MG 132.

The degradation of both EGFR and PARP proteins by the compound DP-5-DP-8 was examined by Western Blot using the H1299 cell line. As shown in FIG. 6, after 36h of administration, a significantly stronger degrading activity of DP-8 was observed than the remaining three compounds, especially the ability to degrade the EGFR protein. DP-8 has a similar effect on PARP degradation to the other 3 compounds, but has a certain degradation effect. As shown in FIG. 7, DP-8 at different concentrations had a degradation effect on EGFR and PARP proteins at 36 h. As shown in FIG. 8, compound DP-8 showed the best degradation of both proteins EGFR and PARP at 6 hours and 12 hours. As shown in FIG. 9, the same addition of MG132 (1. mu.M) completely reversed DP-8 degradation. In addition, as shown in FIG. 10, DP-8 was also found to have a significant effect of degrading EGFR and PARP in A431 cells.

In addition, the degradation effect of single PROTAC (compounds MP-3, MP-4) on EGFR and PARP proteins in H1299 cells after 36H administration is shown in FIG. 5. As is clear from the results, the degradation effect of MP-3 or MP-4 was not as significant as that of DP-1-DP-8, and was much less effective than that of DP-1-DP-4 or DP-8.

Measurement of killing of cancer cells by DP-8 using the H1299 cell line, IC of DP-8, as shown in FIG. 11₅₀19.92 ± 1.08 μ M, better than olaparib (IC50 ═ 35.93 ± 1.05 μ M), slightly worse than gefitinib (IC50 ═ 6.56 ± 1.07 μ M). DP-8 is a good lead compound because its molecular weight is much greater than Olaparib and gefitinib, and it still maintains good effect of killing tumor cells.

In conclusion, the dual-targeting degradation compound DP-1-DP-8 designed by the invention can simultaneously degrade EGFR and PARP through a proteasome pathway, is superior to MP-3 and MP-4 which can only degrade single target protein, can complete the function of degrading two targets by a single drug and single molecule, and provides a new idea for the research and development of antitumor drugs.

Claims

1. A compound for simultaneously inducing degradation of EGFR and PARP proteins is characterized in that the compound is a compound shown as a formula (I) or (II) or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or a prodrug of the compound shown as the formula (I) or (II);

wherein:

Wherein:

w is CH₂、C＝O、SO₂NH or N-alkyl;

x is O or S;

z is-alkyl, -cycloalkyl, -Cl, -F or-H;

g and G' are each independently-H, alkyl, -OH or-CH₂-a heterocycle;

And n is more than or equal to 1 and less than or equal to 10.

2. The compound for simultaneously inducing degradation of EGFR and PARP proteins as claimed in claim 1, which is any one of the compounds represented by formulas (a) - (d) or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or a prodrug thereof;

wherein:

3. The compound for simultaneously inducing degradation of EGFR and PARP proteins as claimed in claim 1, which is any one of the compounds represented by formulas (a ') to (d') or a stereoisomer, a hydrate and a pharmaceutically acceptable salt or a prodrug thereof;

wherein L is

And n is more than or equal to 1 and less than or equal to 5.

4. The compound for simultaneously inducing degradation of EGFR and PARP proteins as claimed in claim 1, which is any one of the following compounds or a stereoisomer, hydrate and pharmaceutically acceptable salt or prodrug thereof;

5. the compound capable of inducing degradation of both EGFR and PARP proteins as claimed in any one of claims 1 to 4, wherein the pharmaceutically acceptable salt of the compound is an addition salt of the compound with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, acetic acid, propionic acid, lactic acid, trifluoroacetic acid, maleic acid, citric acid, fumaric acid, oxalic acid, tartaric acid, benzoic acid, pyruvic acid or succinic acid.

6. A pharmaceutical composition comprising one or more of the compound of any one of claims 1-4 that simultaneously induces degradation of EGFR and PARP proteins, a stereoisomer of the compound, a hydrate of the compound, and a pharmaceutically acceptable salt or prodrug of the compound; further comprising a pharmaceutically acceptable carrier, diluent, adjuvant, vehicle, or combination thereof; wherein the dosage form of the pharmaceutical composition is injection, tablet or capsule.

7. A method of preparing a compound for simultaneously inducing degradation of EGFR and PARP proteins as claimed in any one of claims 1 to 4, comprising the steps of:

and step 3: dissolving the intermediate B and an azide-linked E3 ligand in a solvent, and linking through a Click reaction to obtain a Dual Protacs compound;

8. the method for preparing a compound capable of simultaneously inducing degradation of EGFR and PARP proteins as claimed in claim 7, wherein the solvent is one or two of DMF, dichloromethane, THF and water; the amino acid is tyrosine, serine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, tryptophan, aspartic acid, glutamic acid or hydroxyproline.

9. Use of a compound of any one of claims 1-4, or a stereoisomer, hydrate, pharmaceutically acceptable salt or prodrug thereof, or a pharmaceutical composition of claim 6, for simultaneously inducing degradation of EGFR and PARP proteins, in the manufacture of a medicament for the treatment and/or prevention of tumors.

10. The use of claim 9, wherein the tumor is multiple myeloma, gastric cancer, lung cancer, breast cancer, esophageal cancer, colon cancer, medulloblastoma, acute myelogenous leukemia, chronic leukemia, melanoma, prostate cancer, hepatoma, renal cell tumor, cervical cancer, skin cancer, ovarian cancer, colon cancer, glioma, thyroid cancer, or pancreatic cancer.