CN116462669B - SOS1 and EGFR double-target compound, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a SOS1 and EGFR dual-target compound and its preparation method and application. It also discloses a pharmaceutical composition, which is composed of the above-mentioned dual-target compound or its stereoisomer, or its pharmaceutically acceptable salt. Preparations prepared with one or more pharmaceutically acceptable carriers and/or excipients. The above compounds have dual inhibitory activities of SOS1 and EGFR, can inhibit tumor cell proliferation, promote tumor cell apoptosis, and show excellent in vitro and in vivo effects. It has anti-tumor activity and has better prospects for drug development.

Description

A kind of SOS1 and EGFR dual target compound and its preparation method and application

Technical field

The present invention relates to a SOS1 and EGFR dual-target compound, in particular to a SOS1 and EGFR dual-target compound and its preparation method and application.

Background technique

Epidermal growth factor (EGF) receptor (EGFR) is a transmembrane protein with tyrosine kinase activity. The binding of growth factors to EGFR can activate EGFR, which in turn activates the downstream RAS/AKT pathway mediated by it, which inhibits cell growth. It plays a key role in apoptosis and promoting proliferation, invasion, metastasis and angiogenesis. In addition, EGFR can also mediate cell cycle progression by activating the cyclin-dependent kinase 4/6 (CDK4/6)-cyclin D (cyclin D) complex to promote cells through the G1 phase. Therefore, EGFR is an important target for prostate cancer intervention. However, numerous studies have shown that overexpression of EGFR is associated with poor prognosis in prostate cancer. Currently, EGFR-targeting drugs such as gefitinib have been approved for marketing. Gefitinib has been reported to have limited single-agent activity in androgen-refractory prostate cancer, and its combination with other drugs can improve the therapeutic effect.

SOS1 (son of sevenless 1) is a key nucleotide exchange factor for KRAS that converts GDP-bound KRAS (inactive) to GTP-bound KRAS (active form); SOS1 has been identified as a player in the development of prostate cancer. A confluence of RAS-related signaling pathways that play an important role. Therefore, SOS1 is also a key target for prostate cancer treatment. However, so far, only one SOS1 small molecule inhibitor, BI 1701963, has entered the clinical research stage, and two clinical trials of this small molecule inhibitor (NCT04835714, NCT04627142) were terminated due to severe toxicity. To date, there are no available inhibitors targeting SOS1 on the market.

Combination administration has been widely used in the treatment of complex diseases caused by abnormalities in multiple signaling pathways. Compared with single-target administration, combination therapy often has better anti-tumor efficacy. However, combined administration often has the following problems: 1) Acute toxicity and delayed toxicity may be higher during combined administration, especially when drugs with poor selectivity are used in combination; 2) Since combined administration involves multiple For each drug, the ratio and dosage of the drug need to be considered, and the medication method is often complicated, which can easily lead to poor patient compliance; 3) Combined administration may cause the drugs to interfere with each other in the metabolism of the body, making it impossible to accurately predict their pharmacokinetic properties; 4) Combined administration may cause adverse reactions caused by drug-drug interactions. In addition, the pharmacodynamic characteristics of combined administration are difficult to accurately predict.

Contents of the invention

Purpose of the invention: The purpose of the present invention is to provide a dual-target compound of SOS1 and EGFR that has good efficacy and reduces drug resistance and toxicity. The second purpose of the present invention is to provide a preparation method and application of the above-mentioned dual-target compound.

Technical solution: a SOS1 and EGFR dual target compound or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to the present invention. The compound has a structural formula as shown in formula (I):

.

The preparation method of the above compounds includes the following steps:

(1) Add raw materials 1 and 2 to isopropyl alcohol, stir and heat, then concentrate in vacuum, separate and purify to obtain intermediate 3;

(2) Dissolve intermediate 3, compound 4 and triphenylphosphine into methylene chloride, lower the temperature and stir the reaction, then add diisopropyl azodicarboxylate dropwise, stir the reaction at room temperature, and finally concentrate in vacuum, separate and purify. Obtain SOS1 and EGFR dual target compounds;

.

The present invention also provides a pharmaceutical composition, which is composed of a dual-target compound represented by formula (I) or its stereoisomer, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers. and/or excipients.

Preferably, the pharmaceutically acceptable carrier and/or excipients include diluents, binders, surfactants, wetting agents, lubricants, fillers, disintegrants, colorants, glidants, and stabilizers. , suspending agent, buffer, emulsifier, granulating agent, anti-adhesive agent, gelling agent, absorption delaying agent, dissolution inhibitor, enhancer, adsorbent, chelating agent, preservative, colorant, flavoring agent or Sweetener.

Preferably, the preparations are tablets, capsules, oral liquids, injections, freeze-dried powder injections, transdermal agents, aerosols, solid preparations, liposomes, sustained-release preparations, pills, suppositories, granules, Powder, nano preparation, syrup, wine, tincture or lotion.

The above-mentioned compound or its stereoisomer, or its pharmaceutically acceptable salt, and the use of the above-mentioned pharmaceutical composition in the preparation of SOS1/EGFR dual-target inhibitor drugs.

The above-mentioned compound or its stereoisomer, or its pharmaceutically acceptable salt, and the use of the above-mentioned pharmaceutical composition in the preparation of drugs for inhibiting the RAS signaling pathway.

The above-mentioned compounds or their stereoisomers, or their pharmaceutically acceptable salts, and the use of the above-mentioned pharmaceutical compositions in the preparation of medicaments for treating and/or preventing tumors.

Further, the tumors include prostate cancer, colon cancer, lung cancer, breast cancer, gastric cancer, esophageal cancer, cervical cancer, glioma, nasopharyngoma, liver cancer, ovarian cancer or lymphoma.

Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: (1) SOS1 and EGFR dual-target compounds can effectively inhibit cell proliferation and promote cell apoptosis. In addition, it also has good in vivo anti-tumor activity without obvious The toxic and side effects of .

Description of the drawings

Figure 1 is the ¹ H NMR spectrum of SE-9 in Example 1;

Figure 2 is the MS spectrum of SE-9 in Example 1;

Figure 3 is the HPLC spectrum of SE-9 in Example 1;

Figure 4 is the result of SE-9 inhibiting SOS1 in Example 4;

Figure 5 shows the results of SE-9 inhibiting EGFR in Example 5;

Figure 6 is the result of SE-9 promoting PC-3 cell apoptosis in Example 6;

Figure 7 is the result of SE-9 inhibiting PC-3 cell proliferation in Example 7;

Figure 8 is the result of SE-9 inhibiting PC-3 cell proliferation in Example 7;

Figure 9 shows the effect of SE-9 on tumor growth in nude mice in Example 8;

Figure 10 shows the effect of SE-9 on the body weight and blood biochemical indicators of nude mice in Example 8, indicating the toxic and side effects of SE-9 on nude mice.

Implementation

The technical solution of the present invention will be further described below with reference to the accompanying drawings.

Example 1

Preparation of SOS1 and EGFR dual target compound SE-9

(1) Dissolve 4-chloro-7-methoxyquinazolin-6-yl acetate (267mg, 1mmol) and (R) -1-(3-fluorophenyl)ethylamine (418mg, 3mmol) The reaction mixture was stirred at 60°C for 8 h in isopropanol, then concentrated in vacuo, and the crude product was purified on a silica gel column to obtain intermediate 3 (33%). ¹ H NMR (400 MHz, DMSO) δ 9.42 (s, 1H),8.23 (s, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.71 (s, 1H), 7.37 – 7.31 (m,1H ), 7.27– 7.22 (m, 2H), 7.09 (s, 1H), 7.04 – 6.99 (m, 1H), 5.57 – 5.50 (m, 1H), 3.93(s, 3H), 1.55 (d, J = 7.1 Hz, 3H).

(2) Dissolve intermediate 3 (157mg, 0.5mmol), tetrahydro-2H-pyran-4-ol (61mg, 0.6mmol) and triphenylphosphine (183mg, 0.6mmol) obtained in step (1) in in dichloromethane; the reaction mixture was stirred at 0°C for 10 min; then, diisopropyl azodicarboxylate (121 mg, 0.6 mmol) was added dropwise to the mixture. The reaction mixture was stirred at room temperature for 6 h and then concentrated in vacuo; the crude product was purified on a silica gel column to obtain compound SE-9 (21%). ¹ HNMR (300 MHz, DMSO- d ₆ ) δ 8.27 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.87 (s, 1H), 7.67 – 7.51 (m, 1H), 7.42 – 7.30 (m, 1H), 7.29 – 7.18 (m, 2H), 7.12 (s, 1H), 7.09 – 6.97 (m, 1H), 5.59 (p, J = 7.6, 7.2 Hz, 1H), 4.72 (dt, J = 8.1, 4.1 Hz,1H), 3.90 (s, 4H), 3.52 (t, J = 9.2 Hz, 2H), 2.03 (s, 1H), 2.00 (s, 1H), 1.67(ddd, J = 12.7, 8.5, 3.9 Hz, 2H), 1.59 (d, J = 7.0 Hz, 3H) (Figure 1); MS(ESI) m/z:398.2 [M+H] ⁺ (Figure 2); HPLC: 99.024% (R _t =10.080 min) (mobile phase A: MeOH, phase B: H ₂ O, ratio 70:30, column temperature: 40 ℃, flow rate: 1mL/min, detector: 254nm) (Figure 3).

Example 2

Inhibitory effect of SE-9 on SOS1 and EGFR: First, dissolve the SOS1 single-target inhibitor AX and SE-9 compounds in buffer (10 mM HEPES (pH 7.4), 5 mM MgCl ₂ , 150 mM NaCl, 1 mM DTT , 0.0025% Igepal, 0.05% BSA), then SOS1 (40 nM, 2.5 μL) was added and incubated at 25°C for 30 minutes. Then add 2.5 μL buffer (80 nM KRAS ^G12C , 1 ng/μL Mab Anti 6His-XL665, 1 ng/μL Mab AntiGSH-Eu cryptat), and incubate the mixture at 25°C for 60 minutes. Homogeneous time-resolved fluorescence (HTRF) signals were recorded at an excitation wavelength of 320 nm to determine the inhibitory effect of compounds on SOS1. In addition, different concentrations of EGFR single-target inhibitor gefitinib, SE-9 compound, and EGFR kinase were added to the assay plate, and then the ATP-substrate mixture was added to start the enzymatic reaction. After reacting for 1 hour at room temperature, EDTA buffer was added to stop the reaction. Finally, the detection solution was added to the assay plate and incubated for 1 h to determine the inhibitory effect of the compound on EGFR. The results are shown in Table 1. SE-9 has a significant dual inhibitory effect on SOS1 and EGFR, and the inhibitory effect is better than the SOS1 single-target inhibitor AXE and the EGFR single-target inhibitor gefitinib.

Example 3

In vitro inhibitory effect of SE-9 on tumor cells: human prostate cancer PC-3 cells, human colorectal adenocarcinoma DLD-1 cells, human liver cancer HepG2 cells, human chronic myeloid leukemia K-562 cells, and human lung cancer A549 cells were administered respectively Different concentrations of TN-2 compounds were incubated for 72 hours in an incubator at 37°C and 5% _CO2 , and the inhibitory rate of the compounds on tumor cells was determined using the tetramethylazolium salt (MTT) colorimetric method. The results are shown in Table 2. SE-9 has obvious in vitro inhibitory activity on PC-3, DLD-1, HepG2, K-562 and A549 cells.

Example 4

Inhibitory effect of SE-9 on cellular SOS1 activity: RAS-GTP levels were determined in PC-3 cells treated with different concentrations of SE-9 (0, 0.5, 12.5 μM), and EGF was used as a positive control for SOS1 activation. As shown in Figure 4, SE-9 can significantly reduce the level of RAS-GTP in cells, while the level of RAS-GTP in the positive control group increased.

To further confirm the inhibitory effect of SE-9 on SOS1, the levels of RAS downstream effectors p-ERK and p-AKT were measured under the same conditions. As shown in Figure 4, the changes in p-AKT and p-ERK levels are consistent with the changes in RAS-GTP, that is, the levels of p-AKT and p-ERK in cells after SE-9 treatment were reduced, and the positive control group p-AKT and p-ERK levels were elevated (Fig. 4, A and Fig. 4, B). The results showed that compound SE-9-mediated inhibition of SOS1 inhibited RAS signaling and resulted in reduction of the downstream effectors p-AKT and p-ERK.

Example 5

Inhibitory effect of SE-9 on cellular EGFR activity: PC-3 cells were treated with different concentrations of SE-9 (0, 0.5, 12.5μM) and Western blot analysis was performed. The results showed that EGF, a positive activator of EGFR, significantly induced the level of p-EGFR, while SE-9 treatment could significantly attenuate this effect. Moreover, compared with the EGF alone treatment group, SE-9 treatment significantly blocked the EGF-mediated up-regulation of p-AKT levels. The results in Figure 5 indicate that SE-9 is a highly active EGFR inhibitor.

Example 6

Effect of SE-9 on apoptosis of PC-3 cell line: cells were treated with gradient concentrations of SE-9 (0, 0.5, 2.5, 12.5 μM), prepared with DMSO as a solvent. After treating the cells, Annexin V was used -Double labeling of cells with FITC and PI. A in Figure 6 shows that SE-9 treatment can promote PC-3 cell apoptosis. Furthermore, as shown in Figure 6 B and Figure 6 C, the detection of apoptotic proteins suggests that SE-9 treatment can induce the increased expression of apoptotic proteins Cleaved PARP and Cleaved caspase 3. The results show that SE-9 can promote PC-3 cells. Apoptosis.

Example 7

The effect of SE-9 on the colony formation ability of PC-3 cells was examined through cell cloning experiments. The independent viability of cells was expressed by the cell colony formation rate and colony formation size. The specific process is as follows: Plate 500-800 cells in a 6-well plate. /well, place it in a 37°C constant-temperature incubator and culture it to make it adhere to the wall. After 48 hours, replace with 2 mL of fresh complete culture medium, then add different concentrations of SE-9 (0, 0.5, 2.5, 12.5 μM), prepared with DMSO as the solvent, and then place it in the incubator to continue culturing. Thereafter, the culture medium was replaced every 3 days. After 2 weeks, the supernatant was discarded, washed with PBS and fixed with pre-cooled methanol for 30 minutes. Then wash with PBS and add 0.1% crystal violet staining solution for 30 minutes. Finally, wash with PBS until the bottom of the well plate is transparent and colorless. Let it sit for a few days to dry naturally and take photos. The colony formation results after treatment with different concentrations of drugs are shown in Figure 7. SE-9 can inhibit the clonogenic ability and cell viability of PC-3 cell line in a concentration-dependent manner.

Use EdU to detect cell proliferation ability: Plate 1w cells/well in a 96-well plate and place them in a 37°C constant-temperature incubator overnight to allow them to adhere. The next day, replace with 200 μl of complete culture medium containing SE-9 at different concentrations (0, 0.5, 2.5, 12.5 μM), prepared with DMSO as the solvent, and then place it in the incubator to continue culturing. After 24 hours of drug action, complete culture medium containing EdU reagent (10 μM) was added and incubated at 37°C for 2 hours. After washing with PBS for 5 minutes, add 4% paraformaldehyde to fix at room temperature for 30 minutes. Neutralize with 2 mg/mL glycine for 5 minutes. After washing with PBS for 5 minutes, add 0.5% TritonX-100 to permeate for 20 minutes. Wash with PBS for 5 minutes. Use kFluor488-azide staining reaction solution and incubate at room temperature in the dark for 30 minutes, then wash with PBS for 5 minutes. Use 1× Hoechst 33342 reaction solution and incubate at room temperature in the dark for 30 minutes. After washing with PBS, place it under a fluorescence microscope for imaging, as shown in Figure 8. The results of the EdU proliferation experiment also indicate that SE-9 treatment can inhibit cell proliferation activity, indicating that SE-9 can Inhibit PC-3 cell proliferation.

Example 8

Effect of SE-9 on tumor growth in nude mice with heterotopic transplantation of PC-3 cells: 6-8 week old BALB/C female nude mice were adapted to the environment for about 1 week, and the PC-3 cells were digested with trypsin. Centrifuge at 1300 rpm for 4 min, add 5 mL of fresh FBS-free DMEM, wash once, count, adjust the concentration to 1×10 ⁸ cells/ml, and inoculate 100 μL of each mouse subcutaneously. Administration was started after the tumor volume grew to 100 mm ³ . Each type of cells was randomly divided into 3 groups, with 6 cells in each group, which were SE-9 (5mg/kg, 20mg/kg) administration groups and solvent blank control. After tumor grafting, intraperitoneal injection was performed according to the group, once every 4 days, and each mouse was given 100 μL of the drug (SE-9 dissolved in 4% DMSO + 1% Tween-80 + 95% sterile PBS) Or solvent blank control (4% DMSO + 1% Tween 80 + 95% sterile PBS) for 24 consecutive days. During the experiment, the body weight and tumor volume of the nude mice were recorded every 4 days, and the growth of the nude mice was observed. At the end of the experiment, tumor tissue was taken, the tumor weight was measured and the inhibition rate was calculated. The inhibition rate = (tumor volume in the control group - tumor volume in the drug-added group/tumor volume in the control group) × 100%. As shown in Figure 9A to Figure 9E , the tumor volume of the SE-9 administration group was dose-dependently reduced compared with the control group. In addition, the tumor weight of the SE-9 administration group was significantly lower than that of the control group (F in Figure 9). G in Figure 9 further revealed that SE-9 can induce apoptosis and inhibit angiogenesis in vivo through the measurement of TUNEL, CD31 and Ki67. Finally, Figure 10 shows the detection of nude mouse body weight and blood biochemical indicators. The results show that SE-9 affects the nude mouse body weight, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea (BUN) and creatinine. (CRE) and other biochemical indicators had no significant impact. These results indicate that SE-9 has good in vivo anti-tumor activity without obvious toxic side effects.

Claims (7)

1. A SOS1 and EGFR dual-target compound, or a pharmaceutically acceptable salt thereof, characterized in that the compound is a compound of formula (I):

。

2. a process for the preparation of a compound as claimed in claim 1, comprising the steps of:

(1) Adding the raw materials 1 and 2 into isopropanol, stirring and heating, vacuum concentrating, separating and purifying to obtain an intermediate 3;

(2) Dissolving the intermediate 3, the compound 4 and triphenylphosphine into dichloromethane, cooling, stirring and reacting, then dropwise adding diisopropyl azodicarboxylate, stirring and reacting at room temperature, concentrating in vacuum, separating and purifying to obtain a SOS1 and EGFR double-target compound;

。

3. a pharmaceutical composition characterized by: the pharmaceutical composition is a preparation prepared from the double-target compound or pharmaceutically acceptable salt thereof in claim 1 and one or more pharmaceutically acceptable carriers and/or auxiliary materials.

4. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 3 for the preparation of a SOS1/EGFR dual-target inhibitor drug.

5. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 3 in the manufacture of a medicament for inhibiting the RAS signaling pathway.

6. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 3 in the manufacture of a medicament for the treatment and/or prophylaxis of tumors.

7. The use according to claim 6, wherein the tumour is selected from prostate cancer, colon cancer, lung cancer, breast cancer, gastric cancer, oesophageal cancer, cervical cancer, glioma, nasopharyngeal carcinoma, liver cancer, ovarian cancer or lymphoma.