EGFR-MEK-TZ triple combination molecule LN-1 and its preparation method and use

Abstract

The present invention discloses an EGFR-MEK-TZ triple combination molecule LN-1, a preparation method and uses thereof. The structure of the EGFR-MEK-TZ triple combination molecule LN-1 is shown below. The present invention provides an EGFR-MEK-TZ triple combination molecule LN-1 with anti-tumor effect, which can inhibit the proliferation of prostate cancer Lancap and PC3 cells and induce their apoptosis, and exerts anti-tumor effects through multiple mechanisms, suggesting that triple combination targeted therapy can provide a new treatment strategy for CPRC chemotherapy resistance.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an EGFR-MEK-TZ triple combination molecule LN-1 and a preparation method and use thereof. Background Art

[0002] Domestic and foreign studies have shown that epidermal growth factor receptor (EGFR) is highly expressed in castration-resistant prostate cancer cells, and EGFR activation leads to upregulation of cell proliferation gene expression. Blocking the EGFR signaling pathway in tumor cells can inhibit tumor growth and reduce its invasiveness. Quinazoline drugs are common EGFR-TKI inhibitors that inhibit EGFR-related signal transduction by specifically and competitively binding to the ATP site. Compounds with different substituents introduced at the 6-site of the quinazoline ring have significant EGFR inhibitory activity. Similar drugs such as gefitinib and erlotinib hydrochloride have shown strong EGFR inhibitory activity, but a single inhibition of EGFR cannot permanently and completely block the EGFR downstream pathway.

[0003] DNA is often used as a direct target for cytotoxic antitumor drugs. Dazolid has been used as the most effective cytotoxic drug for the treatment of malignant melanoma for many years. Its mechanism of action is to decompose the monoalkyl triazene into a monomeric amine and a methyl diazonium salt molecule through oxidative demethylation. The methyl diazonium salt molecule causes alkylation of the O6- or N7-site of DNA guanine and the N3-site of adenine, causing molecular toxic damage. However, DNA damage in tumor cells will activate the rescue signaling pathway, leading to upregulation of cell proliferation gene expression and preventing cell apoptosis.

[0004] With the continuous deepening of research on intracellular signal transduction pathways, it has been recognized that when tumor cells such as prostate cancer, especially advanced tumors, are damaged by a kinase inhibitory drug, they can change the conduction pathway and use other conduction signal pathways (rescue signal pathways) to reduce or escape the damage caused by the drug, leading to continued tumor proliferation or recurrence, which makes the treatment of advanced tumors very difficult.

[0005] In order to overcome the drug resistance mechanism of tumors, the concept of "combined targeted therapy" that highly selectively blocks multiple key signal protein molecules at multiple levels has gradually been accepted. In our previous studies, we synthesized the EGFR-TZ dual-targeting molecule JDF12, which is a dual-combination molecule of EGFR inhibitor I and DNA alkylating agent TZ. Subsequent studies have shown that JDF12 has a stronger effect of inhibiting castration-resistant prostate cancer (CRPC) cell proliferation, inducing cancer cell apoptosis and DNA damage. However, this dual-targeting molecule cannot prevent DNA repair induced by the salvage signaling pathway, and the DNA damage repair genes XRCC1 and ERCC1 cannot be completely inhibited. The reason is that although the dual-targeting molecule strengthens the cell apoptosis signal, the salvage signaling pathway can weaken this effect by activating MEK to phosphorylate (re-phosphorylate) the serine residue at position 12 of BAD1.

[0006] In the study of how to block EGFR and rescue signaling pathways at the same time, it was found that MEK is a key factor in the EGFR rescue signaling pathway. Banerjee R et al. synthesized EGFR inhibitors and DNA alkylating agents into an EGFR-TZ combined targeting molecule RB24. After RB24 was combined with the MEK-specific inhibitor PD98059, XRCC1 was completely unexpressed, the effect of inhibiting prostate cancer cell proliferation was significantly increased, and the effect of promoting cancer cell apoptosis was significantly improved. However, current research mainly focuses on the design and synthesis of dimeric molecules, and there are still few reports on triplet drug molecules that connect the three fragments of EGFR, MEK, and TZ. Summary of the invention

[0007] The first object of the present invention is to provide an EGFR-MEK-TZ triple combination molecule LN-1 having anti-tumor effect and a preparation method and use thereof.

[0008] The EGFR-MEK-TZ triple combination molecule LN-1 of the present invention has the following structural formula:

[0009]

[0010] The second object of the present invention is to provide a method for preparing the above-mentioned EGFR-MEK-TZ triple combination molecule LN-1, and the preparation method thereof is shown in the following formula:

[0011]

[0012] Using nitro compound 10 as the starting material, it is coupled with m-chloroaniline 11 through a nucleophilic substitution reaction to obtain diarylamine 12. 12 is further reduced under iron powder conditions to obtain EGFR fragment 4. Methylated fragment 14 can obtain active acylating agent alkylated fragment 3 under dichlorothionyl reflux conditions. The synthesis of connecting fragment 5 can be completed by TBS single protection of Boc-protected diethanolamine 13. Using MEK fragment 2 as the starting material, in the presence of triphosgene, carbonic acid amidation and esterification are successively carried out to obtain carbonate amide intermediate 6. In the presence of TBAF, 6 selectively removes the TBS protecting group to obtain primary alcohol compound 7. Compound 7 is further connected with triphosgene and EGFR fragment 4 to obtain a dimer intermediate. The Boc protecting group is further removed under hydrochloric acid / dioxane conditions to obtain amino hydrochloride intermediate 9. Intermediate 9 is amidated with acyl chloride 3 under triethylamine conditions to obtain the target product EGFR-MEK-TZ triple joint molecule LN-1.

[0013] The third object of the present invention is to provide the use of the EGFR-MEK-TZ triple combination molecule LN-1 in the preparation of anti-tumor drugs.

[0014] The fourth object of the present invention is to provide an anti-tumor drug containing the EGFR-MEK-TZ triple combination molecule LN-1 as an active ingredient.

[0015] Preferably, the anti-tumor drug is an anti-prostate cancer drug.

[0016] Preferably, the anti-tumor drug further contains medically acceptable excipients.

[0017] The present invention provides an EGFR-MEK-TZ triple combination molecule LN-1 with anti-tumor effect. The EGFR-MEK-TZ triple combination molecule LN-1 can inhibit the proliferation of prostate cancer Lancap and PC3 cells and induce their apoptosis, and exerts anti-tumor effects through multiple mechanisms, indicating that the triple combination targeted therapy can provide a new treatment strategy for CPRC chemotherapy resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the design of EGFR-MEK-TZ triple combination molecule (LN-1);

[0019] Figure 2 It is the fitting analysis of the EGFR-MEK-TZ triple combination molecule (LN-1);

[0020] Figure 3 It is the synthesis of the EGFR-MEK-TZ triple combination molecule (LN-1);

[0021] Figure 4 The CCK-8 test showed that LN-1 inhibited the proliferation of prostate cancer cells (*the difference was statistically significant);

[0022] Figure 5 The clone formation experiment shows the inhibitory effect of LN-1 on prostate cancer cells (*The difference is statistically significant);

[0023] Figure 6 The results of EdU staining show the inhibitory effect of LN-1 on prostate cancer cell proliferation (*The difference is statistically significant);

[0024] Figure 7 RT-PCR test shows that LN-1 induces apoptosis of prostate cancer cells (*The difference is statistically significant). DETAILED DESCRIPTION

[0025] The present invention is described in detail below in conjunction with embodiments:

[0026] Embodiment 1:

[0027] 1. Design and synthesis of EGFR-MEK-TZ triple combination molecule (LN-1)

[0028] We intend to construct an EGFR-MEK-TZ triple molecule (LN-1) in the hope that the triple molecule can combine the anti-tumor properties of EGFR, MEK, and TZ, and then simultaneously block different signaling pathways at different molecular levels, prevent the reactivation of cell survival channels, and maximize the apoptosis of prostate tumor cells. The specific design and synthesis principles are as follows: Figure 1 Shown:

[0029] The synthesis steps of LN-1 are as follows Figure 2 As shown:

[0030] According to the designed molecular structure of LN-1, LN-1 can be quickly reversed into four small molecular fragments: EGFR fragment 4, MEK fragment 2, alkylation fragment 3 and connection fragment 5. The three main active fragments are interconnected through diethanolamine fragment 5, and EGFR fragment 4 and MEK fragment 2 are coupled through carbonate amide bonds.

[0031] After completing the fitting analysis, this patent conducted a synthetic study on compound LN-1. First, this patent synthesized the key EGFR fragment 4. Using nitro compound 10 as the starting material, it was coupled with m-chloroaniline 11 through a nucleophilic substitution reaction to obtain diarylamine 12. 12 was further reduced under iron powder conditions to obtain EGFR fragment 4. Methylated fragment 14 can obtain active acylating agent alkylated fragment 3 under thionyl chloride reflux conditions. The synthesis of linker fragment 5 can be completed by TBS single protection of Boc-protected diethanolamine 13, with a yield of 47.5%.

[0032]

[0033] After obtaining the above three independent fragments, this project further carried out the synthesis of LN-1 ( Figure 3 ). Using MEK fragment 2 as the starting material, in the presence of triphosgene, carbonic acid amidation and esterification were carried out successively to obtain carbonate amide intermediate 6. In the presence of TBAF, the TBS protecting group was selectively removed to obtain primary alcohol compound 7 with an approximately equivalent yield (98.3%). 7 was further connected with triphosgene and EGFR fragment 4 to successfully obtain dimer intermediate 8 with a yield of 34.7%. The Boc protecting group was further removed under hydrochloric acid / dioxane conditions to obtain the amino hydrochloride intermediate 9. Intermediate compound 9 has a high purity, so it can be directly used in the next reaction without further purification. After obtaining the key intermediate 9, it was further amidated with the acyl chloride 3 under triethylamine conditions to successfully obtain the target product LN-1 with a yield of 43.3%.

[0034] The synthesis process specifically includes the following experimental steps:

[0035] Synthesis of intermediate 5

[0036]

[0037] The starting material 13 (18.0 g, 87.7 mmol, 16.5 mL, 1.00 eq) was dissolved in 120 mL of tetrahydrofuran, and 5.97 g (87.7 mmol, 1.00 eq) of imidazole was added. At 0°C, 70 mL of tetrahydrofuran solution containing TBSCl (10.5 g, 70.1 mmol, 8.60 mL, 0.800 eq) was added dropwise to the reaction mixture, and the mixture was reacted at this temperature for half an hour. TLC (petroleum ether: ethyl acetate = 1:1) was used for monitoring until most of the raw material 13 was reacted and a new point was generated; water was then added to the reaction system to quench the reaction, and then extracted with dichloromethane three times, 100 mL each time. The organic layers were combined and washed with saturated brine (200 mL), then dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure to obtain a crude product, which was separated by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the target product 5 (11.4 g, 33.3 mmol, 47.5% yield). 1 HNMR, LC-MS confirmed.

[0038] 1 H NMR: (400MHz,CDCl3).

[0039] δ3.87(s,1H),3.65–3.81(m,4H),3.28–3.50(m,4H),1.45(s,9H),0.90(s,9H),0.07(s,6H).

[0040] LC-MS: m / z 220.2 [M–100] + (calcd for C 10 H 26 NO2Si,220.2).

[0041] 1. Preparation of Intermediate 6

[0042]

[0043] Compound 2 (3.00 g, 11.2 mmol, 1.00 eq) was dissolved in 30.0 mL of dichloromethane, triphosgene (1.17 g, 3.93 mmol, 0.35 eq) and triethylamine (3.41 g, 33.6 mmol, 4.69 mL, 3.00 eq) were added at 0 ° C, and stirred at this temperature for 1 hour. Compound 5 (7.17 g, 22.4 mmol, 2.00 eq) dissolved in 30.0 mL of dichloromethane was added to the mixture and stirred at 25 ° C for 4 hours. LC-MS showed that a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain a crude product. HPLC showed that about 58.5% of the desired compound was detected. The residue was purified by preparative HPLC (neutral conditions; column: Kromasil Eternity XT 250×80 mm×10 um; mobile phase: [water (NH4HCO3)-ACN]; B%: 65%-95%, 20 minutes). Compound 6 (4.10 g, 6.69 mmol, 59.6% yield) was obtained as a yellow solid and purified by 1 HNMR, 13 C NMR confirmed.

[0044] 1 H NMR: (400 MHz, DMSO-d6).

[0045] δ H 8.86(s,1H),8.06(dd,J1=8.0Hz,J2=2.0Hz,1H),7.47–7.54(m,1H),7.61(t,J=8.0Hz,

[0046] 1H),7.76–7.88(m,1H),7.43(t,J=8.0Hz,1H),7.25–7.37(m,2H),6.56(s,1H),3.96(t,J=8.0Hz,2H),3.8 4(s,3H),3.52(s,2H),3.24–3.32(m,2H),3.04–3.22(m,2H),1.27–1.38(m,9H),0.81(s,9H),0.03(s,6H).

[0047] 13 C NMR: (100 MHz, DMSO-d6).

[0048] δ C176.8,163.2,156.0,155.5,154.4,134.3,131.1,127.8,125.5,124.8,124.4,123.2,120.7,118.4,114.3,110.7,78.7,62.5,60.8,56.0,49.8,47.1,40.1,28.0,25.7,17.8,1.19,-5.50.

[0049] LC-MS: m / z 613.1[M+H] + (calcd for C 32 H 45 N2O8Si,613.3).

[0050] 2. Preparation of Intermediate 7

[0051]

[0052] At 0°C, compound 6 (2.00 g, 3.26 mmol, 1.00 eq) was dissolved in 20.0 mL of tetrahydrofuran, TBAF (1.00 M, 4.90 mL, 1.5 eq) was added, and the mixture was stirred at 25°C for 1 hour. LC-MS showed that compound 6 was completely consumed, and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (SiO2, dichloromethane: methanol = 10: 1, petroleum ether: ethyl acetate = 1: 0 to 0: 1). Compound 7 (1.60 g, 3.21 mmol, 98.3% yield) was obtained as a yellow solid, and was purified by 1 H NMR confirmed.

[0053] 1 H NMR: (400MHz,CDCl3).

[0054] δ H 8.22(dd,J1=8.0Hz,J2=2.0Hz,1H),7.69(t,J=4.0Hz,1H),7.47–7.59(m,1H),7.42(t,

[0055] J=8.0Hz,1H),7.28–7.34(m,2H),7.03–7.12(m,1H),6.84–7.00(m,1H),6.64(s,1H) ,4.00–4.18(m,2H),3.92(s,3H),3.66(s,2H),3.15–3.50(m,5H),1.36–1.49(m,9H).

[0056] LC-MS: m / z 499.1[M+H] + (calcd for C 26 H 31 N2O8,499.2).

[0057] 3. Preparation of Intermediate 12

[0058]

[0059] Compound 10 (5.00 g, 23.8 mmol, 1.00 eq), compound 11 (3.35 g, 26.2 mmol, 2.79 mL, 1.10 eq), and HCl (12.0 M, 695 μL, 0.350 eq) were mixed in 100 mL of acetonitrile, and the mixture was stirred at 85 ° C for 0.5 hours under N2 atmosphere. LC-MS showed that compound 10 was completely consumed, and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain a crude product, namely compound 12 (8.60 g, crude product), as a yellow solid.

[0060] LC-MS: m / z 301.1[M+H] + (calcd for C 14 H 10 ClN4O2,301.0).

[0061] 4. Preparation of Intermediate 4

[0062]

[0063] Compound 12 (8.60 g, 28.6 mmol, 1.00 eq), NH4Cl (764 mg, 14.3 mmol, 0.500 eq), Fe (7.99 g, 143 mmol, 5.00 eq) and H2O (5.00 mL) were mixed in 120 mL of isopropanol, and the mixture was stirred at 100 ° C for 2 hours under N2 atmosphere. LC-MS showed that compound 12 was completely consumed, and a main peak with the desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a crude product. The yellow solid compound 4 (6.80 g, 25.0 mmol, 87.6% yield, 99.8% purity) was obtained by LC-MS.

[0064] LC-MS: m / z 271.1[M+H] + (calcd for C 14 H 12 ClN4,271.1).

[0065] 5. Preparation of Intermediate 8

[0066]

[0067] Compound 4 (2.60 g, 9.60 mmol, 1.00 eq) was dissolved in 90.0 mL of dichloromethane solution, triphosgene (2.28 g, 7.68 mmol, 0.800 eq) and TEA (4.86 g, 48.0 mmol, 6.68 mL, 5.00 eq) were added at 0 ° C, and stirred at this temperature for 1 hour. Compound 7 (1.44 g, 2.88 mmol, 0.300 eq) was dissolved in 20.0 mL of dichloromethane solution, added to the mixture and stirred at 25 ° C for 4 hours. LC-MS showed that compound 4 was completely consumed and a main peak with the desired mass was detected. The reaction mixture was diluted with 100 mL of dichloromethane and washed with 300 mL of water, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to obtain a crude product. HPLC showed that the desired compound was detected in a yield of about 34.7%. The crude product was purified by preparative HPLC (neutral conditions; column: Waters Xbridge C 18 150×50mm×10um; mobile phase: [water (NH4HCO3)-ACN]; B%: 46%-76%, 10 minutes).

[0068] LC-MS: m / z 795.5[M+H] + (calcd for C 41 H 40 ClN6O9,795.3).

[0069] 6. Preparation of Intermediate 9

[0070]

[0071] Hydrochloric acid / dioxane (4.00M, 4.24mL, 30.0eq) was added to a 5.00mL dichloromethane solution of compound 8 (0.450g, 565μmol, 1.00eq), and the mixture was stirred at 0°C for 0.5 hours. LC-MS showed that compound 8 was completely consumed, and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain a crude product. Compound 9 (0.420g, crude product, hydrochloride) was obtained as a yellow solid.

[0072] LC-MS: m / z 695.2[M+H] + (calcd for C 36 H 32 ClN6O7,695.2).

[0073] 7. Preparation of Intermediate 3

[0074]

[0075] To a solution of compound 14 (0.500 g, 2.56 mmol, 1.00 eq) in 5.00 mL of dichloromethane was added SOCl2 (457 mg, 3.84 mmol, 278 μL, 1.50 eq) and DMF (18.7 mg, 256 μmol, 19.7 μL, 0.100 eq) at 0°C. The mixture was stirred at 45°C for 0.5 hours. LC-MS showed that compound 13 was completely consumed. The reaction mixture was concentrated under reduced pressure to obtain a crude product, compound 3 (0.540 g, crude), as a white solid.

[0076] 8. Preparation of LN-1

[0077]

[0078] Compound 9 (420 mg, 574 μmol, 1.00 eq), compound 3 (367 mg, 1.72 mmol, 3.00 eq), triethylamine (116 mg, 1.15 mmol, 159 μL, 2.00 eq) were mixed in 5.00 mL of dichloromethane solution, and the mixture was stirred at 25 ° C for 2 hours under N2 atmosphere. LC-MS showed that compound 9 was completely consumed, and a main peak with the desired mass was detected. The reaction mixture was concentrated under reduced pressure to obtain a crude product. The crude product was purified by preparative HPLC (HCl conditions; column: 3_Phenomenex LunaC 18 75×30mm×3um; mobile phase: [water (HCl)-ACN]; B%: 26%-46%, 8 minutes). The mixture was concentrated under reduced pressure to remove ACN and adjusted to pH=7 with saturated NaHCO3 aqueous solution and filtered. Concentrated under reduced pressure to obtain the product. Yellow solid LN-1 (210 mg, 248 μmol, 43.3% yield) and was purified by 1 Confirmed by H NMR, LC-MS, and Special NMR. 1 H NMR: (400 MHz, DMSO-d6).

[0079] δ H 9.77–10.25(m,2H),8.28–9.13(m,4H),7.87–8.10(m,2H),7.71–7.86(m,3H),7.53–7.68(m,2H),7 .12–7.52(m,6H),6.54(d,J=20.0Hz,1H),3.93–4.43(m,5H),3.67–3.90(m,7H),3.51–3.65(m,2H).

[0080] Special NMR: (400MHz, DMSO-d6, T=80℃).

[0081] δ C 9.41–10.14(m,2H),8.30–8.80(m,4H),7.91–8.08(m,2H),7.67–7.84(m,4H),7.53–7.64(m,1H),7.37–7.49(m,3H),7.29(dd,J 1=16.0Hz, J2=4.0Hz,2H),7.18(d,J=8.0Hz,1H),6.54(s,1H),4.10–4.40(m,4H)(m,4H),3.81–3.92(m,6H),3.71–3.80(m,4H).

[0082] LC-MS m / z 872.2[M+H] + (calcd for C 42 H 35 C1N 11 O9,872.2).

[0083] II. Study on the anti-tumor activity of the EGFR-MEK-TZ triple molecule (LN-1)

[0084] 1. Changes in the proliferation function of Lancap and PC3 cells in each group

[0085] After completing the synthesis of LN-1, this study further tested whether LN-1 could disrupt the balance between apoptosis and proliferation of CPRC cells, induce apoptosis of tumor cells, and play an anti-cancer role. This study used CCK-8 assay, clone formation assay, and EdU staining to evaluate the proliferation potential of each group of cells. The results showed that the proliferation function of prostate cancer Lancap and PC3 cell lines could be inhibited by LN-1, and showed significant time dependence, indicating that LN-1 has a significant inhibitory effect on prostate cancer cells. Figure 4-6 As shown in the figure, the results of CCK-8 experiment, clone formation experiment and EdU staining all showed that compared with the control group, LN-1 treatment could significantly reduce the activity of Lancap and PC3 cells and inhibit their proliferation, and the differences were statistically significant (p<0.05).

[0086] 2. Changes in mRNA levels of apoptosis-related genes in each group of cells

[0087] In addition, this study also used RT-PCR to detect the expression levels of apoptosis-related genes (Caspase-3, Caspase-9, Bax and p53) in each group of cells. Figure 7 As shown, Figure 7It was shown that LN-1 could significantly upregulate the mRNA expression levels of Caspase-3 and Caspase-9 in Lancap and PC3 cells, indicating that LN-1 could increase the activity of Caspase-3 and Caspase-9 in prostate cancer cells. In addition, p53 is an important apoptosis signaling pathway, which is mainly involved in inducing programmed cell death and other life activities, while Bax is a pro-apoptotic gene in the Bcl-2 family, which can antagonize the protective effect of Bcl-2 and cause cells to die, and is also an important pathway for inducing apoptosis. This study also found that after treating prostate cancer cells with LN-1, the expression levels of p53 and Bax in the cells were significantly increased, further indicating that LN-1 could significantly promote apoptosis of Lancap and PC3 cell lines. Figure 7 As shown, compared with the common culture medium, the mRNA expression levels of apoptosis-related genes (Caspase-3, Caspase-9, Bax and p53) in PC-1 and Lancap cells were significantly increased after adding LN-1, and the differences were statistically significant (p<0.05).

[0088] In summary, LN-1 can inhibit the proliferation of prostate cancer Lancap and PC3 cells and induce their apoptosis, exerting anti-tumor effects through multiple mechanisms, suggesting that triple combined targeted therapy can provide a new treatment strategy for CPRC chemotherapy resistance.

[0089] 3. Experimental materials and experimental procedures for antitumor activity

[0090] 3.1 Experimental subjects and reagents used:

[0091] Human castration-resistant prostate cancer cell lines Lancap and PC3 were obtained from the cell bank of the Chinese Academy of Sciences and cultured in a cell culture incubator at 5% CO2 and 37°C using high-glucose DMEM complete medium (containing 4.5 g / L high-glucose medium, 10% fetal bovine serum, 100 mg / mL streptomycin and 100 U / mL penicillin). When cell fusion reached 70%-80%, the cells were passaged or plated for further experimental studies.

[0092] 3.2 Experimental Grouping:

[0093] The experiment was divided into two groups, a control group and an experimental group. The control group did not contain LN-1; the experimental group was treated with LN-1 (50 mmol). After the cells in each group were attached to the wall, they were pretreated with serum-free DMEM medium, and then complete medium containing / not containing LN-1 was added for subsequent experiments.

[0094] 3.3 Experimental steps

[0095] 1) CCK-8 cell proliferation assay:

[0096] CCK-8 method was used to detect the inhibitory effect of LN-1 on the proliferation of Lancap and PC3 cell lines. Each cell line was cultured at 2×10 4 The cells were inoculated into a 96-well plate at a density of 10 cells / 100 μL / well. The culture plate was pre-cultured in an incubator for 24 hours (at 37°C, 5% CO2); PBS or LN-1 was added to the culture plate, and the culture plate was incubated in an incubator. The proliferation of the two cell lines was detected on days 1-5: 10 uL CCK-8 solution was added to each well [be careful not to generate bubbles in the well, which will affect the reading of the absorbance (OD) value], the culture plate was incubated in the incubator for 2 hours, and the OD value at 450 nm was measured with an enzyme marker. The calculation formula is: Inhibition rate (%) = [1-(experimental group OD-standard OD) / (control group OD-standard OD)] × 100%.

[0097] 2) Clone formation experiment:

[0098] The adherent cells were trypsinized and resuspended, seeded in a 6-well plate at a density of 1000 cells / 2000 μL per well, and cultured for 14 days. When colony formation was observed with the naked eye, the medium was removed, fixed with 4% formaldehyde for 15 minutes, stained with crystal violet, and observed and recorded under a microscope.

[0099] 3) EdU staining:

[0100] Each group of Lancap and PC3 cell lines were inoculated in 24-well plates for culture. After the cell fusion reached 70%-80%, the cells were treated according to the above grouping design, and then an appropriate amount of EdU reagent (Keygen Biotechnology, Jiangsu, China) was added. Finally, 1×Hoechst33342 solution was added and incubated at room temperature in the dark for 15 minutes for cell nucleus staining.

[0101] 4) RT-PCR detection:

[0102] RT-PCR was used to detect the effect of LN on the mRNA expression level of apoptosis-related genes. First, cells were collected and the total mRNA of each group of cells was extracted using Trizol reagent. Then, cDNA was synthesized by reverse transcription according to the steps of the reverse transcription kit (PrimeScript RT MasterMix, TaKaRa, Japan), and fluorescent quantitative PCR reaction was performed. The primer sequences involved are detailed in Table 1.

[0103] Table 1. RT-PCR primer sequences

[0104]

Claims

1. EGFR-MEK-TZ triple combination molecule LN-1, its structural formula is as follows:

2. The method for preparing the EGFR-MEK-TZ triple combination molecule LN-1 according to claim 1, characterized in that: Its preparation method is shown in the following formula: Using nitro compound 10 as the starting material, it is coupled with m-chloroaniline 11 through a nucleophilic substitution reaction to obtain diarylamine 12. 12 is further reduced under iron powder conditions to obtain EGFR fragment 4. Methylated fragment 14 can obtain active acylating agent alkylated fragment 3 under dichlorothionyl reflux conditions. The synthesis of connecting fragment 5 can be completed by TBS single protection of Boc-protected diethanolamine 13. Using MEK fragment 2 as the starting material, in the presence of triphosgene, carbonic acid amidation and esterification are successively carried out to obtain carbonate amide intermediate 6. In the presence of TBAF, 6 selectively removes the TBS protecting group to obtain primary alcohol compound 7. Compound 7 is further connected with triphosgene and EGFR fragment 4 to obtain a dimer intermediate. The Boc protecting group is further removed under hydrochloric acid / dioxane conditions to obtain amino hydrochloride intermediate 9. Intermediate 9 is amidated with acyl chloride 3 under triethylamine conditions to obtain the target product EGFR-MEK-TZ triple joint molecule LN-1.

3. Use of the EGFR-MEK-TZ triple combination molecule LN-1 or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of anti-tumor drugs.

4. The use according to claim 3, characterized in that: The anti-tumor drug is a drug against prostate cancer.

5. The use according to claim 3, characterized in that: The anti-tumor drug also contains medically usable auxiliary materials.

6. An anti-tumor drug, characterized in that: Contains the EGFR-MEK-TZ triple combination molecule LN-1 or a pharmaceutically acceptable salt thereof as claimed in claim 1 as an active ingredient.

7. The anti-tumor drug according to claim 6, characterized in that: The anti-tumor drug is a drug against prostate cancer.

8. The anti-tumor drug according to claim 6, characterized in that: The anti-tumor drug also contains medically usable auxiliary materials.

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