Protein arginine methylation-targeting proximity induction small molecule chimera platform MeTAG and application thereof

Abstract

The invention belongs to the technical field of small molecule compounds, and particularly relates to a proximity induction small molecule chimera platform MeTAG for targeting protein arginine methylation and application of the proximity induction small molecule chimera platform MeTAG, methyltransferase PRMT5 is connected to a target protein (POI), and the MeTAG promotes arginine methylation of the POI in a reversible and PRMT5-dependent mode. In addition, the MeTAG can also regulate and control downstream signals in a methylation-dependent manner, thereby causing down-regulation of AR transcriptional activity and activation of AKT. Therefore, MeTAG is proved to be a feasible scheme for regulating the methylation level of POI, and can also be used as a unique research and transformation tool to reveal the biological function of protein arginine methylation. More importantly, the research result of the invention proves that the MeTAG has the potential of benefit in clinical treatment by accurately controlling the methylation state of the specific POI.

Description

Technical Field

[0001] The present invention belongs to the technical field of small molecule compounds, and specifically relates to a proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation and its application. Background Art

[0002] Protein arginine methylation refers to the biochemical process of transferring the methyl group in the methyl donor S-adenosylmethionine (SAM) to the guanidinium group of arginine. This modification has been found on more than 4,000 proteins (including histones and non-histone substrates).

[0003] Arginine methylation is involved in a wide range of biological processes, including transcription, signal transduction, DNA damage response, RNA translation, and protein stability. Among them, dysregulation of arginine methylation mediated by protein arginine methyltransferases (PRMTs) is involved in many diseases, including cancer, neurodegenerative diseases, and inflammatory diseases.

[0004] Arginine methylation can be divided into three types: monomethylation, asymmetric dimethylation, and symmetric dimethylation. Different PRMTs can catalyze the three types of arginine methylation. Type I PRMTs, consisting of PRMT1-4, PRMT6, and PRMT8, are responsible for asymmetric dimethylation (ADMA) of arginine, type II PRMTs, PRMT5, and PRMT9 are responsible for symmetric dimethylation (SDMA) of arginine, and the only type III PRMT7 is responsible for monomethylation (MMA) of arginine. Among them, PRMT1 and PRMT5 have become valuable therapeutic targets for a variety of cancers, especially for patients with methylthioadenosine phosphorylase gene (MTAP) deletion, and their inhibitors are currently in clinical trials. In addition, PRMTs are also crucial for the survival of non-tumor cells, because mice lacking Prmt1 and Prmt5 are embryonic lethal. Therefore, precise manipulation of the methylation of the target protein (POI) is a prerequisite for deepening our understanding of the biological mechanism of protein methylation.

[0005] To this end, the emergence of genetic research methods and chemical probe methods (including methylarginine antibodies, advanced proteomics technology and small molecule probes, etc.) has made the study of arginine methylation possible.

[0006] On the one hand, in genetic research methods, arginine-lysine (R-to-K) or alanine (R-to-A) point mutations are often used to simulate the elimination of arginine methylation, but arginine-phenylalanine (R-to-F) methylation mimicking mutations are still controversial, so this type of method still has certain limitations in biological research. On the other hand, although chemical probes targeting PRMT enzymes have had a significant revolutionary impact in basic life sciences and translational medicine, PRMT inhibition inevitably introduces side effects and toxicity due to the broad spectrum of PRMT enzyme substrates.

[0007] Similarly, several inhibitors of arginine demethylases have been developed, including the deaminase peptidyl arginine deaminase 4 (PAD4), which help increase arginine methylation levels. However, due to the functional diversity of these proteins, these inhibitors inevitably interfere with other cellular processes, such as lysine methylation. Similar to PRMT inhibitors, arginine demethylase inhibitors can also induce changes in global methylation levels in cells due to their broad substrate range.

[0008] It is not difficult to see that small molecules that selectively regulate the methylation of target proteins in cells without inducing changes in global methylation levels are still very limited. Such small molecules will have superior biological properties because they can be used to explore unknown proteins modified by arginine methylation and also have the potential to trigger downstream cellular signaling pathways. This can overcome the challenges often encountered by traditional methods and provide alternative strategies for disease intervention. It has been reported that heterobifunctional molecules can bring enzymes to the vicinity of target proteins and induce ubiquitination / deubiquitination, phosphorylation / dephosphorylation, acetylation and o-glycosylation of target proteins, but current heterobifunctional molecules cannot introduce arginine methylation into target proteins. Summary of the invention

[0009] In view of this, the present invention designs a proximity-induced chimera platform MeTAG targeting protein arginine methylation, which can selectively and dynamically methylate the arginine of the target protein POI.

[0010] In a first aspect, the present invention provides a proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation, which is obtained by connecting a target protein ligand with a PRMT5 ligand via a linker;

[0011] The PRMT5 ligand is a piperidine ring substituted product of EPZ015666;

[0012] The target protein ligand is a hydrolysis product of the AR inhibitor enzalutamide, a pan-AKT inhibitor AZD5363, or a bromodomain targeting the P300 protein;

[0013] The Linker is any chemically feasible connection structure, which is used to connect two ligands via an amide bond.

[0014] The MeTAG of the present invention recruits PRMT5 and POI respectively through two warhead parts, and can effectively induce the two to approach each other to promote arginine methylation of POI.

[0015] When the target protein ligand in the first aspect is the hydrolysis product of the AR inhibitor enzalutamide, the structural formula of the MeTAG is as shown in formula (1):

[0016]

[0017] Linker is

[0018] The second aspect of the present invention provides the use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in the preparation of drugs for treating prostate cancer.

[0019] Furthermore, the MeTAG is used to prepare a drug for inhibiting the proliferation of prostate cancer cells, wherein the prostate cancer cells include VCAP and C4-2.

[0020] When the target protein ligand in the first aspect is the pan-AKT inhibitor AZD5363, the structural formula of the MeTAG is as shown in formula (2):

[0021]

[0022] Linker is

[0023] The third aspect of the present invention provides the use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in a preparation for promoting cell growth, wherein the cells include MCF7, H9C2, HDF and HUVEC cells.

[0024] When the target protein ligand of the first aspect is a bromodomain targeting the P300 protein, the structural formula of the MeTAG is as shown in formula (3):

[0025]

[0026] Linker is

[0027] The fourth aspect of the present invention provides the use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in the preparation of a P300 protein activator, wherein the MeTAG can promote the interaction between p300 and PRMT5, significantly increase the arginine methylation level of p300, and then upregulate the FOS and EGR2 mRNA levels in HCT116 cells, suggesting the possibility that P300-MeTAG 1 can mediate the activation of P300.

[0028] The present invention has the following beneficial effects:

[0029] The present invention takes two known arginine methylation substrates, androgen receptor (AR) and protein kinase B (AKT), as examples. MeTAG induces their symmetrical dimethylation in a PRMT5-dependent manner, thereby inhibiting AR signal transduction and activating AKT signal transduction, respectively. In addition, it is confirmed that E1A binding protein (p300) (a protein that has not yet been identified as a PRMT5 substrate) can be methylated by the heterobifunctional molecule p300-MeTAG 1. In summary, the MeTAG system provides a method for targeting POI arginine methylation, and can also be used as a research and transformation tool to reveal the biological functions of protein arginine methylation. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Development of the MeTAG system, which:

[0031] A is a schematic diagram of the MeTAG system.

[0032] B is a schematic diagram of the design of PRMT5-binding ligands.

[0033] Figure 2 For the development of AR-MeTAG system, among which:

[0034] A is a schematic diagram of the AR-MeTAG system consisting of PRMT5 and AR warhead.

[0035] B is the predicted binding mode diagram of the AR:AR-MeTAG 1:PRMT5 ternary complex.

[0036] C is the RMSD curve of the AR:AR-MeTAG 1:PRMT5 ternary complex.

[0037] D is the result of fluorescence polarization detection of AR-MeTAG molecules inducing the formation of AR:AR-MeTAG:PRMT5 complex.

[0038] E is the result of immunoprecipitation detection of AR-MeTAG 1 promoting the formation of ternary complex.

[0039] F is the result of immunoprecipitation detection of AR-MeTAG 1-induced SDMA modification on AR.

[0040] G is the result of immunoprecipitation detection of AR-MeTAG 1 introducing SDMA on AR in a PRMT5-dependent manner.

[0041] Figure 3 AR-MeTAG 1 reduces the transcriptional function of AR, including:

[0042] A is a schematic diagram of AR-MeTAG 1-induced reduction in transcription of AR target genes PSA, NKX3.1, and TMPRSS2.

[0043] B is the result of RT-qPCR detection of different concentrations of AR-MeTAG 1 down-regulating PSA mRNA levels in VCaP cells.

[0044] C is the result of RT-qPCR detection of the inhibition of AR-MeTAG 1-induced decrease in PSA mRNA levels in VCaP cells by knocking out PRMT5.

[0045] D is the result of the washout experiment detecting the increase in PSA mRNA level after removal of AR-MeTAG 1 in VCaP cells.

[0046] E is the result of RT-qPCR detection of downregulation of PSA mRNA levels in C4-2 cells caused by treatment with different concentrations of AR-MeTAG 1.

[0047] F is the result of RT-qPCR detection of down-regulation of TMPRSS2 mRNA level in C4-2 cells caused by treatment with different concentrations of AR-MeTAG 1.

[0048] G is the result of RT-qPCR detection of down-regulation of NKX3.1 mRNA level in C4-2 cells caused by treatment with different concentrations of AR-MeTAG 1.

[0049] Figure 4 The inhibition of prostate cancer cell proliferation by AR-MeTAG 1, wherein:

[0050] A shows the inhibition of C4-2 cell proliferation by different concentrations of AR-MeTAG 1.

[0051] B is a representative picture showing the inhibition of C4-2 cell colony formation ability by AR-MeTAG 1.

[0052] C is the statistical result of B.

[0053] D is the flow cytometry detection of AR-MeTAG 1-induced apoptosis of C4-2 cells.

[0054] E is the statistical analysis result of three independent apoptosis assays.

[0055] F is a representative picture showing the ability of AR-MeTAG 1 to inhibit C4-2 cell migration.

[0056] G is the statistical result of F.

[0057] Figure 5 For the development of AKT-MeTAG, which:

[0058] A is a schematic diagram of the AKT-MeTAG system.

[0059] B is the predicted binding mode of the AKT:AKT-MeTAG1:PRMT5 ternary complex.

[0060] C is the RMSD curve of the AKT:AKT-MeTAG 1:PRMT5 ternary complex.

[0061] D is the result of fluorescence polarization detection of AKT-MeTAG molecule-induced ternary complex formation.

[0062] E is the result of immunoprecipitation detection of AKT-MeTAG 1 promoting the formation of ternary complex.

[0063] F is the result of immunoprecipitation detection of SDMA modification on AKT induced by AKT-MeTAG 1.

[0064] G is the result of immunoprecipitation detection of AKT-MeTAG 1 introducing SDMA on AKT in a PRMT5-dependent manner.

[0065] H is the result of immunoblot analysis of global methylation in 293T cells treated with DMSO, AKT-MeTAG 1 (1.5 μM) and GSK3326595 (1.5 μM or 10 μM).

[0066] I is the result of immunoprecipitation detection of AKT-MeTAG 1 inducing SDMA of AKT on R391 residue.

[0067] Figure 6 AKT-MeTAG is the activation of AKT, where:

[0068] A is a graph showing the experimental results that AKT-MeTAG 1 activates AKT1 in a dose-dependent manner.

[0069] B is the experimental result showing that AKT-MeTAG 1 activates AKT1 in a time-dependent manner.

[0070] C is the experimental result that AKT-MeTAG 1 activates AKT1 in a PRMT5 activity-dependent manner.

[0071] D is the experimental result that AKT-MeTAG 1 activates AKT1 in a PRMT5-dependent manner.

[0072] E is the result of the elution experiment.

[0073] F is the experimental result that AKT-MeTAG 1 promotes the growth of H9C2 cells.

[0074] G is the experimental result that AKT-MeTAG 1 promotes HDF cell growth.

[0075] H is the experimental result that AKT-MeTAG 1 promotes the growth of HUVEC cells.

[0076] Figure 7 For the development of p300-MeTAG, which:

[0077] A is a schematic diagram of the p300-MeTAG system.

[0078] B is the predicted binding mode of the p300:p300-MeTAG1:PRMT5 ternary complex.

[0079] C is the RMSD curve of the p300:p300-MeTAG1:PRMT5 ternary complex.

[0080] D is the result of fluorescence polarization detection of the formation of ternary complex induced by p300-MeTAG molecule.

[0081] E is the result of immunoprecipitation detection of P300-MeTAG 1 promoting the formation of ternary complex.

[0082] F is the relative p300 SDMA arginine abundance in p300-HA 293T cells.

[0083] G is the experimental result showing that P300-MeTAG 1 induces SDMA of p300 in a PRMT5-dependent manner.

[0084] H is the experimental result that p300-MeTAG 1 up-regulates EGR2 mRNA level in a concentration-dependent manner.

[0085] I is the experimental result that p300-MeTAG 1 up-regulates FOS mRNA level in a concentration-dependent manner.

[0086] Figure 8 The synthetic route of AR-MeTAG 1-3.

[0087] Fig. 9 The synthetic route of AKT-MeTAG 1-3.

[0088] Fig.10 The synthetic route of p300-MeTAG 1-4. DETAILED DESCRIPTION

[0089] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0090] The present invention provides a proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation, and its structural design principle is as follows Figure 1 As shown in A, that is, the target protein ligand and the PRMT5 ligand are connected via an intermediate linker.

[0091] The present invention uses a reversible non-competitive PRMT5 inhibitor EPZ015666 to develop its ligand. From the co-crystal structure of the PRMT5:MEP50 and EPZ015666 complex, the oxyethane part on EPZ015666 protrudes into the solvent and hardly interacts with the PRMT5 structure, which is the best attachment point for the linker to bind. In another PRMT5 inhibitor GSK3326595, the oxyethane part is replaced by a piperidine ring that is easier to synthesize. Therefore, the present invention uses a piperidine ring-substituted compound as a PRMT5 ligand of MeTAG ( Figure 1 B).

[0092] The present invention selects the known PRMT5 substrate proteins androgen receptor (AR) and protein kinase B (AKT) and the E1A binding protein (p300) which has not been proven to be a PRMT5 substrate as target proteins. The specific implementation methods of MeTAG targeting the above three POIs will be introduced in turn below:

[0093] 1) The AR-MeTAG in the present invention uses the mature AR inhibitor enzalutamide as a ligand. Through two consecutive amidation steps, the PRMT5 binder and the enzalutamide hydrolysis product were linked to different linkers to construct three preset AR-MeTAGs ( Figure 2A). Structure-activity relationship and molecular dynamics simulation (MD), fluorescence polarization (FP) evaluation and fluorescence polarization analysis of AR-MeTAG demonstrated that AR-MeTAG 1 could induce the formation of a stable AR:AR-MeTAG1:PRMT5 ternary complex, while AR-MeTAG 2 and AR-MeTAG 3 failed to induce the formation of a ternary complex ( Figure 2 Exogenous CO-IP experiments in 293T cells and after knocking out PRMT5 in VCAP cells demonstrated that AR-MeTAG 1 promoted the binding of AR to PRMT5 and promoted the arginine methylation SDMA of AR in a PRMT5-dependent manner (2E-G).

[0094] The arginine methylation induced by AR-MeTAG 1 in various prostate cancer cells VCaP and C4-2 weakens the transcriptional function of AR. Specifically, we used AR target genes PSA, NKX3.1 and TMPRSS2 as biomarkers, and used RT-qPCR experiments, drug elution experiments, and construction of PRMT5 knockout cell lines to confirm that AR-MeTAG 1 reduces the transcriptional function of AR in prostate cancer cells in a dose-dependent manner ( Figure 3 Functional experiments such as plate clone formation assay, flow cytometry apoptosis assay and wound healing assay also support the above conclusions ( Figure 4 ).

[0095] In summary, the molecule AR-MeTAG 1 in the present invention can recruit endogenous PRMT5 methylation and inhibit the transcriptional function of AR, thereby having the potential to inhibit the proliferation of prostate cancer cells and also has the potential to be further developed for the treatment of prostate cancer.

[0096] 2) The AKT-MeTAG in the present invention uses the pan-AKT inhibitor AZD5363 as a ligand. According to the co-crystal structure analysis and structure-activity relationship (SAR) study of the complex of AKT and AZD5363, the solvent-exposed hydroxyl group is replaced by a piperazine ring and connected to the PRMT5 ligand through various linkers to obtain AKT-MeTAG 1-3 ( Figure 5 A). Structure-activity relationship, molecular dynamics simulation (MD), fluorescence polarization (FP) evaluation and fluorescence polarization analysis showed that AKT-MeTAG 1 could induce the formation of a stable AKT:AKT-MeTAG 1:PRMT5 ternary complex ( Figure 5BD). In 293T cells exogenously transfected with PRMT5 and AKT1 plasmids, the interaction between PRMT5 and AKT induced by AKT-MeTAG 1 was further verified by co-immunoprecipitation (CO-IP), and this effect could be abolished by the PRMT5 inhibitor GSK3326595. Since R391 of AKT1 has been reported as the main site that can be methylated by PRMT5 under physiological conditions, the AKT-MeTAG 1 in the present invention can only induce arginine methylation of wild-type AKT, while the R391K mutant AKT cannot. This indicates that AKT-MeTAG 1 also induces arginine methylation of AKT at the R391 residue of AKT ( Figure 5 EI).

[0097] PRMT5-mediated AKT methylation can promote its activation. After treatment with the molecule AKT-MeTAG 1 of the present invention, the levels of AKT-pT308 and AKT-pS473 in 293T and MCF7 cells increased in a dose- and time-dependent manner ( Figure 6 A and B). These findings collectively indicate that the molecule of the present invention, AKT-MeTAG 1, can act as a reversible positive regulator of AKT activation by recruiting PRMT5 to methylate AKT at its R391 residue ( Figure 6 CE). In addition, H9C2, HDF, HUVEC, and MCF7 cells treated with AKT-MeTAG 1 showed enhanced cell proliferation. These preliminary results validate the potential of AKT-MeTAG 1 to promote cell growth, and whether AKT-MeTAG 1 can be developed as a kinase activator for tissue protection and regeneration requires further study in the future ( Figure 6 of FH).

[0098] 3) The present invention can be applied to mediate arginine methylation of a novel substrate protein, p300. The present invention uses a bromodomain (BRD) ligand targeting P300 ( Figure 7 A) was connected to different types of linkers to obtain a series of heterobifunctional molecules. Structure-activity relationship, molecular dynamics simulation (MD), fluorescence polarization (FP) evaluation and fluorescence polarization analysis showed that P300-MeTAG 1 can induce the formation of a stable P300: P300-MeTAG 1: PRMT5 ternary complex ( Figure 7 BD). CO-IP experiments after the P300 and PRMT5 plasmids were transfected into 293T cells demonstrated that P300-MeTAG 1 could promote the interaction between p300 and PRMT5 and significantly increase the arginine methylation level of p300 ( Figure 7 E and G).

[0099] Since P300 has not been previously reported as a PRMT5 substrate, we further explored the potential arginine methylation sites on p300 488 mediated by PRMT5 using mass spectrometry (MS) analysis. Mass spectrometry analysis showed that P300 was already methylated at R237, R695, and R2059 residues, and treatment with P300-MeTAG 1 resulted in methylation of 12 additional arginine sites ( Figure 7 These results confirm that the molecule P300-MeTAG 1 of the present invention can induce arginine methylation on p300, providing a research tool for studying arginine methylation of other target proteins POI. Taking P300 as an example, we used the molecule P300-MeTAG 1 of the present invention as a tool and found that it can upregulate FOS and EGR2 mRNA levels in HCT116 cells, which suggests the possibility that P300-MeTAG 1 can mediate the activation of P300 ( Figure 7 of H and I).

[0100] The small molecule proximity-induced chimera platform (MeTAG) targeting arginine methylation of target protein provided by the present invention and its application are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0101] Description of experimental materials

[0102] All reagents and solvents were purchased from standard commercial sources and were of analytical grade. All synthesized compounds described in this invention were examined by analytical thin layer chromatography (Machery-Nagel pre-coated F254 aluminum plates) and observed under UV light at 254 nm and purified by column chromatography (CC) on a Reveleris X2 (Grace) automatic flash unit. The residual solvent signal was used as a reference and the results were analyzed at 298.15 K on a Bruker Avance 400 / 100MHz or Bruker Avance NMR data were recorded on a 600 / 150 MHz spectrometer. The structures of the compounds were confirmed by 1H, 13C, and HSQC NMR spectroscopy. In addition, high-resolution mass spectrometric analysis was performed on a Waters LCT Premier XETM time-of-flight (TOF) mass spectrometer equipped with standard electrospray ionization (ESI) and a modular LockSprayTM interface. LC-MS (Waters AutoPurification system: Waters Cortecs C18 column (2.7 μm, 100 × 4.6 mm); formic acid in H 2O (0.2%, v / v) / MeCN gradient system; flow rate 0.30 mL / min; gradual change from 95:5 to 0:100 in 15 min).

[0103] Example 1: Laboratory synthesis details and compound characterization of all small molecules mentioned in this invention

[0104] 1. Reference Figure 8 The route shown is for the synthesis of AR-MeTAG 1-3

[0105] Compound 1 (CAS No.: 1242137-15-0, 56 mg, 0.12 mmol), tert-butyl 2-(2-(2-aminoethoxy)ethoxy)acetate (CAS No.: 1442687-80-0, 30 mg, 0.137 mmol), DIPEA (65 μL, 0.37 mmol), HATU (50 mg, 0.13 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the reaction of the raw materials was complete, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, Na 2 SO 4 The residue was dried and concentrated in vacuo. Purification by silica gel column gave compound 2 (50 mg, 62%), named as Tert-butyl2-(2-(2-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorobenzamido)ethoxy)ethoxy)acetate.

[0106] NMR data of compound 2:

[0107] 1 H-NMR (400MHz, CDCl3-d) δppm1.47 (s, 9H), 1.62 (s, 6H), 3.69-3.76 (m, 8H), 4.02 (s, 2H), 7.15 (dd, J=11.41, 1.79Hz, 1H), 7.2 4 (dd, J=8.24, 1.92Hz, 1H), 7.84 (dd, J=8.24, 1.65Hz, 1H), 7.94-7.97 (m, 1H), 8.00 (d, J=8.24Hz, 1H), 8.23 ​​(t, J=8.38Hz, 1H).

[0108] TFA (2 ml) was added to a CH 2 Cl2 (2ml) solution, and the reaction mixture was stirred at room temperature for 2h. After the raw material was completely consumed, the volatiles were removed by vacuum. The residue, compound 3 (CAS No.: 1616391-79-7, 41mg, 0.099mmol), DIPEA (42μL, 0.24mmol), HATU (32mg, 0.084mmol) were dissolved in DMF (1mL), stirred at room temperature for 2h, and after the reaction was completed, cooled with water and extracted with ethyl acetate. The organic phase was washed with brine, Na 2 SO 4 The mixture was dried and concentrated in vacuo. ARMeTAG 1 (33 mg, 44%, two steps) was obtained by silica gel column purification and named (S)-6-((1-(2-(2-(2-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorobenzamido)ethoxy)ethoxy)acetyl)piperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide.

[0109] NMR data of ARMeTAG 1:

[0110] 1H-NMR (600MHz, DMSO-d6) δppm1.36-1.44 (m, 2H), 1.53 (s, 6H), 1.89 (br.s., 2H), 2.53-2.65 (m, 2H), 2.71-2.91 (m, 6H), 3.12 (t, J=11.28H z, 1H), 3.29-3.32 (m, 1H), 3.39-3.46 (m, 3H), 3.56 (t, J=5.78Hz, 2H), 3.58 (br.s., 4H), 3.77 (d, J=12.65Hz, 1H), 3.93 (br.s., 1H), 4.05-4 .17 (m, 3H), 4.17-4.26 (m, 2H), 5.06 (br.s., 1H), 7.03 (br.s., 1H), 7.07 (br.s., 1H), 7.11 (br.s., 3H), 7.33 (d, J=8.07Hz, 1H), 7.43 (d, J =10.45Hz, 1H), 7.75-7.84 (m, 2H), 8.08 (d, J = 7.89Hz, 1H), 8.27-8.34 (m, 2H), 8.40 (d, J = 8.25Hz, 1H), 8.55 (br.s., 1H), 8.76 (br.s., 1H).

[0111] Compound 3 (65 mg, 0.16 mmol), Boc-8-aminooctanoic acid (CAS No.: 30100-16-4, 32.4 mg, 0.125 mmol), DIPEA (97 μL, 0.55 mmol), HATU (63 mg, 0.16 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the reaction of the raw materials was complete, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4 The residue was dried and concentrated in vacuo. Purification by silica gel column gave compound 4 (84 mg, 81%), named as Tert-butyl(S)-(8-(4-((6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyrimidin-4-yl)amino)piperidin-1-yl)-8-oxooctyl)carbamate.

[0112] NMR data of compound 4:

[0113] 1H-NMR (600MHz, CD3OD-d4) δppm1.35 (d, J=8.99Hz, 8H), 1.43 (s, 9H), 1.46-1.48 (m, 2H), 1.59-1.62 (m, 2H), 1.9 8-2.04 (m, 1H), 2.09 (br.s., 1H), 2.41 (t, J=7.43Hz, 2H), 2.72-2.80 (m, 1H), 2.88 (t, J=12.56Hz, 2H), 2.94-2.9 9 (m, 3H), 3.02 (t, J = 7.06Hz, 3H), 3.23-3.29 (m, 2H), 3.50-3.53 (m, 3H), 3.84 (s, 1H), 3.97 (d, J = 12.84Hz, 1H), 4 .07-4.14 (m, 1H), 4.18 (br.s., 1H), 7.03 (d, J=7.15Hz, 1H), 7.06-7.15 (m, 4H), 8.27 (s, 1H), 8.40-8.52 (m, 1H).

[0114] To compound 4 (84 mg, 0.13 mmol) CH 2 Cl 2 TFA (2 mL) was added to the solution (2 mL), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo. The residue, compound 1 (46 mg, 0.10 mmol), DIPEA (78 μL, 0.45 mmol), and HATU (51 mg, 0.13 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4 The mixture was dried and concentrated in vacuo. Purification by silica gel column gave AR-MeTAG 2 (35 mg, 28%, two steps), named (S)-6-((1-(8-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorobenzamido)octanoyl)piperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide.

[0115] NMR data of AR-MeTAG 2:

[0116] 1H-NMR (600MHz, DMSO-d6) δppm1.31 (br.s., 8H), 1.47-1.52 (m, 4H), 1.54 (s, 6H), 1.82-1.8 9 (m, 1H), 1.89-1.96 (m, 1H), 2.31 (t, J=7.06Hz, 2H), 2.47 (br.s., 1H), 2.53 (br.s., 1H), 2 .64-2.70(m, 1H), 2.72-2.80(m, 2H), 2.80-2.85(m, 2H), 3.15(t, J=12.01Hz, 1H), 3.26(q, J=6.42Hz, 2H), 3.28-3.33(m, 1H), 3.39-3.45(m, 1H), 3.83(d, J=12.65Hz, 1H), 3.86-3.91 (m, 1H), 4.09 (br.s., 1H), 4.24 (d, J=10.09Hz, 1H), 4.98 (d, J=4.40Hz, 1H), 7.01 (d, J=6.7 9Hz, 1H), 7.06 (s, 1H), 7.07-7.12 (m, 3H), 7.33 (d, J=8.07Hz, 1H), 7.42 (d, J=10.27Hz, 1H) , 7.73-7.78 (m, 2H), 8.08 (d, J = 8.07Hz, 1H), 8.29 (d, J = 4.40Hz, 2H), 8.40 (d, J = 8.25Hz, 1H ), 8.51(t, J=5.23Hz, 1H), 8.74-8.78(m, 1H).2H(piperidyl2and6)couldnotbeobserved.

[0117] Compound 3 (72 mg, 0.14 mmol), 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid (CAS No.: 462100-06-7, 36.3 mg, 0.113 mmol), DIPEA (86 μL, 0.49 mmol), HATU (56 mg, 0.15 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4The reaction mixture was dried and concentrated in vacuo. Purification by silica gel column gave compound 5 (67 mg, 66%), named as Tert-butyl(S)-(2-(2-(3-(4-((6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyrimidin-4-yl)amino)piperidin-1-yl)-3-oxopropoxy)ethoxy)ethoxy)ethyl)carbamate.

[0118] NMR data of compound 5:

[0119] 1 H-NMR (600MHz, CDCl3-d) δppm1.43 (s, 9H), 1.54 (d, J=13.20Hz, 2H), 2.00-2.09 (m, 2H), 2.51-2.60 (m, 2H), 2.61-2.6 9(m, 2H), 2.74-2.85(m, 4H), 2.93(dt, J=11.28, 5.55Hz, 1H), 2.97-3.03(m, 1H), 3.27-3.35(m, 2H), 3.43-3.51(m, 1H) , 3.54-3.58 (m, 2H), 3.60-3.72 (m, 11H), 3.73-3.78 (m, 1H), 3.79-3.86 (m, 1H), 3.88 (d, J=14.86Hz, 1H), 3.95 (d, J=1 3.94Hz, 1H), 4.07 (d, J=3.85Hz, 1H), 7.01 (d, J=7.15Hz, 1H), 7.09-7.18 (m, 4H), 8.44 (t, J=5.96Hz, 1H), 8.49 (s, 1H).

[0120] In compound 5 (67 mg, 0.094 mmol) CH 2 Cl 2 TFA (2 mL) was added to the solution (2 mL), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo. The residue, compound 1 (34 mg, 0.075 mmol), DIPEA (49 μL, 0.28 mmol), and HATU (37 mg, 0.097 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4The mixture was dried and concentrated in vacuo. Purification by silica gel column gave AR-MeTAG 3 (59 mg, 47%, two steps), named (S)-6-((1-(1-(4-(3-(4-cyano-3-(trifluoromethyl)phenyl)-5,5-dimethyl-4-oxo-2-thioxoimidazolidin-1-yl)-2-fluorophenyl)-1-oxo-5,8,11-trioxa-2-azatetradecan-14-oyl)piperidin-4-yl)amino)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide.

[0121] AR-MeTAG 3 NMR data:

[0122] 1 H-NMR (600MHz, DMSO-d6) δppm1.32-1.42(m, 2H), 1.54(s, 6H), 1.82-1.89(m, 1H ), 1.91(br.s., 1H), 2.53-2.64(m, 4H), 2.71-2.90(m, 5H), 3.15(t, J=11.28Hz, 1 H), 3.28-293.32(m, 1H), 3.41-3.45(m, 3H), 3.48-3.52(m, 4H), 3.52-3.56(m, 8 H), 3.84 (d, J=13.02Hz, 1H), 3.93 (br.s., 1H), 4.08 (br.s., 1H), 4.19-4.26 (m, 1 H), 5.08 (br.s., 1H), 7.02 (d, J=6.79Hz, 1H), 7.07 (s, 1H), 7.08-7.14 (m, 3H), 7 .33 (dd, J=8.07, 1.65Hz, 1H), 7.43 (dd, J=10.64, 1.47Hz, 1H), 7.78 (t, J=7.98Hz , 2H), 8.06-8.11 (m, 1H), 8.28-8.33 (m, 2H), 8.40 (d, J = 8.25Hz, 1H), 8.52 (t, J = 4.95Hz, 1H), 8.76 (t, J = 5.13Hz, 1H). 2H (piperidyl2and6) could not be observed.

[0123] 2. Reference Fig. 9 Synthesis of AKT-MeTAG 1-3 by the route shown

[0124] Compound 3 (120 mg, 0.29 mmol) and K2CO3 (81 mg, 0.586 mmol) were added to a DMF (2 mL) solution of tert-butyl bromoacetate (57.1 mg, 0.29 mmol). The resulting mixture was stirred at room temperature overnight and monitored by TLC. After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4 The product was dried and concentrated in vacuo. Purification by flash silica gel column gave compound 6 (99.6 mg, 65%), named as Tert-butyl(S)-2-(4-((6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyrimidin-4-yl)amino)piperidin-1-yl)acetate.

[0125] NMR data of compound 6:

[0126] 1 H-NMR (400MHz, CDCl3-d) δppm1.45 (s, 9H), 1.57-1.70 (m, 2H), 2.02 (d, J=11.27Hz, 2H), 2.34(t, J=10.58Hz, 2H), 2.51-2.68(m, 2H), 2.77(dd, J=7.83, 5.36Hz, 1H), 2.87-2.98(m , 5H), 3.13 (s, 2H), 3.40-3.49 (m, 1H), 3.61-3.70 (m, 2H), 3.84 (d, J=14.57Hz, 1H), 4.04 (m, 1H), 5.35 (br.s., 1H), 6.99 (d, J=6.60Hz, 1H), 7.06-7.22 (m, 4H), 8.36-8.55 (m, 2H).

[0127] Compound 3 (31 mg, 0.077 mmol), 11-(tert-Butoxy)-11-oxoundecanoic acid (CAS No.: 1789702-17-5, 21 mg, 0.077 mmol), DIPEA (44.3 mg, 0.34 mmol), HATU (39 mg, 0.10 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification with a silica gel column gave compound 7 (40 mg, 61%). Named Tert-butyl(S)-11-(4-((6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyrimidin-4-yl)amino)piperidin-1-yl)-11-oxoundecanoate.

[0128] NMR data of compound 7:

[0129] 1 H-NMR (600MHz, CDCl3-d) δppm1.28-1.30 (m, 10H), 1.44 (s, 9H), 1.53-1.64 (m, 6H), 2.01-2.07 (m, 1H) , 2.07-2.15(m, 1H), 2.20(t, J=7.24Hz, 3H), 2.32(d, J=6.60Hz, 3H), 2.75-2.87(m, 2H), 2.93-3.05(m , 2H), 3.14-3.25(m, 2H), 3.43-3.50(m, 1H), 3.56-3.61(m, 1H), 3.63-3.70(m, 1H), 3.82-3.91(m, 2H) , 3.94-4.06 (m, 1H), 4.14-4.23 (m, 1H), 7.02 (d, J=6.79Hz, 1H), 7.06-7.25 (m, 4H), 8.42-8.53 (m, 2H).

[0130] In CH 2 Cl 2To a solution of compound 6 (100 mg, 0.17 mmol) in 4% paraformaldehyde (2 ml) was added TFA (2 ml), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo. The residue, tert-butyl 2-(2-(2-aminoethoxy)ethoxy)acetate (38.5 mg, 0.17 mmol), DIPEA (107 μL, 0.615 mmol), and HATU (70 mg, 0.184 mmol) were dissolved in DMF and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column gave compound 8 (50 mg, 42%, two steps), named Tert-butyl(S)-2-(2-(2-(2-(4-((6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyrimidin-4-yl)amino)piperidin-1-yl)acetamido)ethoxy)ethoxy)acetate.

[0131] NMR data of compound 8:

[0132] 1 H-NMR (400MHz, CDCl3-d) δppm1.50 (s, 9H), 1.63 (q, J=10.15Hz, 2H), 2.05 (d, J=6.11Hz, 2H), 2.37 (t, J=11.0 0Hz, 2H), 2.58-2.66 (m, 2H), 2.75-2.81 (m, 1H), 2.86 (d, J=11.62Hz, 2H), 2.89-2.99 (m, 3H), 3.05 (s, 2H), 3. 43-3.54 (m, 3H), 3.56-3.61 (m, 2H), 3.62-3.70 (m, 4H), 3.70-3.75 (m, 2H), 4.00-4.09 (m, 3H), 5.60 (d, J=7.5 8Hz, 1H), 7.01 (d, J=6.60Hz, 1H), 7.09-7.20 (m, 4H), 7.58 (br.s., 1H), 8.43 (t, J=5.20Hz, 1H), 8.49 (s, 1H).

[0133] In the CH 2 Cl 2TFA (2 mL) was added to the (2 mL) solution, and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo. The residue, glycine tert-butyl ester (25.4 mg, 0.19 mmol), DIPEA (107 μL, 0.615 mmol), and HATU (70 mg, 0.184 mmol) were dissolved in DMF and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4 The residue was dried and concentrated in vacuo. Purification by silica gel column gave compound 9 (63 mg, 62%, two steps), named Tert-butyl(S)-(2-(4-((6-((3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)carbamoyl)pyrimidin-4-yl)amino)piperidin-1-yl)acetyl)glycinate.

[0134] NMR data of compound 9:

[0135] 1 H-NMR (600MHz, CDCl3-d) δppm1.48 (s, 9H), 1.59-1.67 (m, 2H), 2.08 (d, J=11.19Hz, 2H), 2.40 ( t, J=10.82Hz, 2H), 2.66-2.72 (m, 1H), 2.87-2.97 (m, 4H), 2.98-3.05 (m, 1H), 3.08 (s, 2H), 3.4 4-3.50 (m, 1H), 3.63-3.79 (m, 4H), 3.92 (d, J = 14.86Hz, 1H), 3.98 (d, J = 5.50Hz, 2H), 4.10 (d, J =3.85Hz, 1H), 7.01 (d, J = 7.15Hz, 1H), 7.10-7.19 (m, 4H), 8.45 (t, J = 5.96Hz, 1H), 8.48 (s, 1H).

[0136] Compound 10-a ((S)-3-amino-3-(4-chlorophenyl)propan-1-ol, CAS No.: 886061-26-3, 1 g, 5.4 mmol) was dissolved in dichloromethane (DCM), and di-tert-butyl dicarbonate (CAS No.: 24424-99-5, 1.42 g, 6.5 mmol) was added. The reaction was stirred for 2 hours. After the reaction was completed, the solvent was evaporated and the resulting mixture was purified by flash column chromatography to obtain a white solid intermediate of tert-butyl ester (1.31 g, yield 85%).

[0137] Dissolve triphenylphosphine (CAS No.: 603-35-0, 1.84 g, 7 mmol) and iodine (CAS No.: 7553-56-2, 1.80 g, 7 mmol) in DCM (50 mL), add imidazole (CAS No.: 288-32-4, 0.89 g, 14 mmol). The mixture is stirred at room temperature for 30 minutes. Then, a DCM (30 mL) solution of the white solid intermediate of tert-butyl ester (1.0 g, 3.5 mmol) is added. The resulting mixture is stirred for 3 hours.

[0138] After the reaction was complete, saturated NaHCO 3 solution and 10% Na 2 S 2 O 3 The organic phase was extracted with DCM (20 mL × 3). 2 SO 4 After drying and evaporation, the obtained crude product was purified by silica gel column chromatography to obtain compound 10-b as a white solid (2.3 g, yield 65%).

[0139] Compound 10-b (1.48 g, 3.7 mmol) was dissolved in acetonitrile (30 mL) and K 2 CO 3 (2.1 g, 14.8 mmol) and benzyl piperzine-1-carboxylate (CAS: 31166-44-6, 1.65 g, 7.4 mmol). The mixture was stirred at 80 ° C overnight. After the reaction was completed, the obtained product was purified by silica gel column chromatography. The obtained product was dissolved in DCM (20 mL) and TFA (20 mL), stirred for 30 minutes, evaporated the solvent and dried to obtain compound 10-c as a white powder (1.35 g, yield 95%).

[0140] Compound 10-c (1.3 g, 3.4 mmol) and 4-((tert-butoxycarbonyl)amino)-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxylic acid (CAS No.: 956460-96-1, 1.2 g, 3.4 mmol) were dissolved in DMSO (15 mL), and EDCI (CAS No.: 25952-53-8, 0.98 g, 5.1 mmol), HOAt (CAS No.: 39968-33-7, 0.69 g, 5.1 mmol) and NMM (CAS No.: 109-02-4, 1.0 g, 10.2 mmol) were added in sequence. The mixture was stirred at room temperature overnight, and the obtained product was purified by reverse phase column chromatography, and the obtained white solid was the TFA salt of compound 10 (AKT-MeTAG Ctr, 1.71 g, yield 69%).

[0141] In the CH 2 Cl 2 TFA (2 mL) was added to the solution and stirred at room temperature for 32 h. After the starting material was consumed, the volatiles were removed in vacuo. Purification by silica gel column gave AKT-MeTAG 1N (38 mg, 88%), named benzyl(S)-4-(3-(4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)-3-(4-chlorophenyl)propyl)piperazine-1-carboxylate.

[0142] Pd / C (10 mg) was added to a solution of compound 10 (50 mg, 0.068 mmol) in MeOH (2 mL), and the mixture was stirred at room temperature for 3 hours. 2 The reaction mixture was stirred at 40 °C for 2 h. After the starting material was consumed, the reaction mixture was filtered and the filtrate was collected and evaporated to obtain the intermediate product compound 10-NH without purification.

[0143] TFA (2 ml) was added to a solution of compound 8 (50 mg, 0.075 mmol) in CH2Cl2 (2 ml), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo. The residue, compound 10-NH (60 mg, 0.082 mmol), DIPEA (46 μL, 0.26 mmol), and HATU (30 mg, 0.078 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, Na2 SO 4 The residue was dried and concentrated in vacuo. The residue was dissolved in DCM (1 ml) and TFA (2 ml) was then added. The resulting mixture was stirred at room temperature for 4 hours and concentrated in vacuo. AKT-MeTAG 1 (28 mg, 34%, three steps) was obtained by silica gel column purification and named 6-((1-(2-((2-(2-(2-(4-((S)-3-(4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)-3-(4-chlorophenyl)propyl)piperazin-1-yl)-2-oxoethoxy)ethoxy)ethyl)amino)-2-oxoethyl)piperidin-4-yl)amino)-N-((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide.

[0144] AKT-MeTAG 1 NMR data:

[0145] 1H-NMR (600MHz, DMSO-d6) δppm1.48-1.58 (m, 2H), 1.70 (d, J=12.47Hz, 1H), 1.74-1.81 (m, 1H), 1. 82-1.93(m, 4H), 2.18(d, J=7.52Hz, 2H), 2.25(br.s., 3H), 2.28-2.36(m, 5H), 2.51-2.56(m, 1H) , 2.65(br.s., 2H), 2.84(br.s., 3H), 2.88(br.s., 2H), 2.99(br.s., 2H), 3.24-3.27(m, 2H), 3.3 1(br.s., 1H), 3.42(dt, J=12.10, 6.24Hz, 8H), 3.51-3.55(m, 4H), 3.57-3.63(m, 2H), 3.78(br.s ., 1H), 3.86 (br.s., 1H), 3.96 (br.s., 1H), 4.12 (s, 2H), 4.40-4.52 (m, 2H), 4.88 (q, J=7.34Hz, 1 H), 5.19 (br.s., 1H), 6.63-6.66 (m, 1H), 7.04 (d, J=7.34Hz, 1H), 7.09 (br.s., 1H), 7.10-7.16 (m , 3H), 7.18-7.22(m, 1H), 7.29-7.31(0.5H), 7.32-7.39(m, 5H), 7.78(d, J=6.97Hz, 2H), 8.15(s, 1H), 8.16 (s, 0.5H), 8.32 (s, 1H), 8.72-8.78 (m, 1H), 8.87 (d, J=6.97Hz, 1H), 11.73 (br.s., 1H).

[0146] According to the same procedure as AKT-MeTAG 1, compound 9 (63 mg, 0.11 mmol) was reacted with CH 2 Cl 2TFA (2 ml) was added to the (2 ml) solution, and the reaction mixture was stirred at room temperature for 2 hours. After the raw material was consumed, the volatiles were removed in vacuo. The residue in DMF (1 mL), compound 10-NH (87 mg, 0.12 mmol), DIPEA (66 μL, 0.38 mmol), and HATU (43 mg, 0.11 mmol) were dissolved in DMF and stirred at room temperature for 2 hours. After the reaction was completed, it was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was dissolved in DCM (1 ml), and TFA (2 ml) was added. The resulting mixture was stirred at room temperature for 4 hours and concentrated in vacuo. AKT-MeTAG 2 (59 mg, 54%, three steps) was obtained by purification on a silica gel column and named 6-((1-(2-((2-(4-((S)-3-(4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)-3-(4-chlorophenyl)propyl)piperazin-1-yl)-2-oxoethyl)amino)-2-oxoethyl)piperidin-4-yl)amino)-N-((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide.

[0147] AKT-MeTAG 2 NMR data:

[0148] 1H-NMR (600MHz, DMSO-d6) δppm1.52 (d, J=9.90Hz, 2H), 1.64 (br.s., 2H), 1.83-1.92 (m, 4H) , 2.07-2.19(m, 2H), 2.20-2.28(m, 4H), 2.28-2.31(m, 1H), 2.31-2.40(m, 3H), 2.58(br.s., 2H), 2.77(br.s., 1H), 2.85(br.s., 5H), 2.98(br.s., 2H), 3.29-3.31(m, 1H), 3.39-3.49( m, 6H), 3.57 (br.s., 2H), 3.70 (br.s., 1H), 3.86 (br.s., 1H), 3.92 (br.s., 1H), 3.97 (d, J=4 .77Hz, 2H), 4.43-4.52(m, 2H), 4.85-4.92(m, 1H), 5.08(br.s., 1H), 6.63(br.s., 1H), 7.0 3(d, J=7.15Hz, 1H), 7.06-7.09(m, 1H), 7.09-7.14(m, 3H), 7.19(br.s., 1H), 7.20-7.24(m, 0.5H), 7.28-7.42(m, 5H), 7.78(d, J=7.34Hz, 1H), 7.90(br.s., 1H), 8.14(s, 1H), 8.30(s, 1 H), 8.74 (t, J=4.86Hz, 1H), 8.82 (br.s., 0.5H), 8.85 (d, J=7.70Hz, 1H), 11.71 (br.s., 1H).

[0149] Following the same procedure as AKT-MeTAG 1, TFA (2 ml) was added to a solution of compound 7 (40 mg, 0.061 mmol) in CH2Cl2 (2 ml), and the reaction mixture was stirred at room temperature for 2 hours. After the starting material was consumed, the volatiles were removed in vacuo. The residue, compound 10-NH (45 mg, 0.061 mmol), DIPEA (37 μL, 0.21 mmol), and HATU (24 mg, 0.064 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After the reaction was completed, it was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was dissolved in DCM (1 ml), and TFA (2 ml) was added.

[0150] The resulting mixture was stirred at room temperature for 4 hours and concentrated in vacuo. Purification by silica gel column gave AKT-MeTAG 3 (32 mg, three steps, 48%), named 6-((1-(11-(4-((S)-3-(4-amino-1-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)piperidine-4-carboxamido)-3-(4-chlorophenyl)propyl)piperazin-1-yl)-11-oxoundecanoyl)piperidin-4-yl)amino)-N-((S)-3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)pyrimidine-4-carboxamide.

[0151] AKT-MeTAG 3 NMR data:

[0152] 1 H-NMR (600MHz, DMSO-d6) δppm1.25 (d, J=4.22Hz, 10H), 1.46 (d, J=6.97Hz, 8H), 1.82-1 .87(m,2H),1.89-1.95(m,2H),2.14-2.36(m,12H),2.5(m,2H),2.64-2.71(m,1H),2.75 (td, J=11.46, 5.87Hz, 2H), 2.80-2.85(m, 2H), 3.10-3.18(m, 1H), 3.28-3.32(m, 1H), 3. 41-3.48(m, 5H), 3.49-3.57(m, 2H), 3.82(d, J=12.29Hz, 1H), 3.88(br.s., 1H), 4.09(br .s., 1H), 4.18-4.27 (m, 2H), 4.39 (t, J=14.21Hz, 2H), 4.82-4.88 (m, 1H), 5.01 (br.s., 1 H), 6.56-6.59 (m, 1H), 7.01 (d, J=6.60Hz, 1H), 7.06 (s, 1H), 7.07-7.12 (m, 3H), 7.13-7. 16 (m, 1H), 7.29 (s, 0.5H), 7.34 (q, J=8.62Hz, 4H), 7.79-7.84 (m, 1H), 8.08 (s, 0.5H), 8. 11(s, 1H), 8.28(s, 1H), 8.71-8.81(m, 2H), 11.67(br.s., 1H), 2Hcouldnotbeobserved.

[0153] 3. Reference Fig.10 Synthesis of p300-MeTAG 1-4 by the route shown

[0154] Tert-butyl (2- (2-bromoethoxy) ethyl) carbamate (CAS No.: 164332-88-1, 164 mg, 0.61 mmol), NaH (in oil, 24 mg, 0.61 mmol) and KI (18 mg, 0.11 mmol) were dissolved in DMF (3 mL) and stirred at 0 ° C for 0.5 h. Compound 11 (CAS No.: 5597-50-2, 100 mg, 0.55 mmol) was then added. After stirring at room temperature for 3 h, the reaction was quenched with ice water and extracted with ethyl acetate. The organic phase was collected and washed with brine. The mixture was washed with Na 2 SO 4 After drying, filtration and concentration, the mixture was washed with MeOH / H 2 O (v / v, 1 / 1, 10 mL) was dissolved, 2M NaOH (3 mL) was added, and the reaction was carried out at 50° C. for 2 h. The reaction solution was cooled to room temperature, the pH was adjusted to 2-3 with HCl, and extracted with ethyl acetate. The organic phase was collected to obtain the intermediate compound 12 for the next step reaction.

[0155] Pd(dppf)Cl 2 (CAS No.: 72287-26-4, 1.81 g, 2.47 mmol) was added to a DME solution (100 mL) containing 4-bromo-1-fluoro-2-nitrobenzene (CAS No.: 364-73-8, 10.87 g, 49.4 mmol) and compound 13-a (3,5-dimethylisoxazole-4-boronic acid pinacol ester, CAS No.: 832114-00-8, 12.68 g, 56.8 mmol). Saturated NaHCO 3 Aqueous solution (100 mL). Subsequently, the mixture was degassed by evacuation and nitrogen filling (repeated 3 times) and then heated at 80°C for 3 hours.

[0156] After the reaction was cooled, the mixture was extracted with ethyl acetate (100 mL) and water (100 mL). MgSO 4and activated carbon, filtered after stirring, and the filtrate was evaporated to remove the solvent. The obtained residue was redissolved with a small amount of dichloromethane and purified by silica gel column chromatography. The column chromatography was eluted with a gradient of ethyl acetate and cyclohexane, and ether (30 mL) was added to the obtained product to induce crystallization of the product. The supernatant was taken up with a pipette and the process was repeated. Finally, the obtained solid was dried under vacuum to obtain the product 13-b as a light beige solid (8.57 g, yield 73%).

[0157] 4-(2-Aminoethyl)morpholine (CAS No.: 2038-03-1, 0.787 mL, 6.00 mmol) was added to a tetrahydrofuran (THF, 25 mL) solution containing compound 13-b (1.18 g, 5.00 mmol) and DIPEA (CAS No.: 7087-68-5, 1.05 mL, 6.00 mmol). The mixture was stirred at room temperature for 16 hours, and then 4-(2-Aminoethyl)morpholine (0.262 mL, 2.00 mmol) was added and stirred for 5 hours. After the reaction was completed, the mixture was separated with ethyl acetate (20 mL) and water (20 mL). The separated organic phase was washed with water (20 mL) and saturated brine (20 mL), and then with anhydrous MgSO 4 Drying and evaporation of the solvent gave the product 13-c as an orange solid (1.63 g, yield 94%).

[0158] 1.0M Na 2 S 2 O 4 The aqueous solution (50 mL, 50 mmol) was added to a suspension of compound 13-c (3.43 g, 3.43 mmol) in ethanol (50 mL). The reaction mixture was heated under reflux for 1 hour and then cooled. The mixture was separated with 10% aqueous ammonia solution (50 mL) and ethyl acetate (50 mL). The separated aqueous phase was extracted with ethyl acetate (50 mL). The combined organic phase was washed with saturated brine (50 mL) and then precipitated with anhydrous MgSO 4 Drying and evaporation of the solvent gave compound 13 as a light yellow gum (2.737 g, 87% yield).

[0159] Compound 13 (CAS No.: 1613694-70-4, 72 mg, 0.23 mmol), intermediate compound 12 (80 mg, 0.23 mmol), T3P (145 mg, 0.45 mmol) and DIPEA (45 μL, 0.27 mmol) were dissolved in EtOAc (3 mL). The mixture was heated at 150° C. for 3 h, alkalized with 0.1 M aq. NaOH, and extracted with ethyl acetate. The organic phase was collected and washed with brine. The mixture was washed with Na2 SO 4 The product was dried, filtered and separated using a silica gel column to give Compound 14 (70 mg, 48%), named Tert-butyl(2-(2-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1Hbenzo[d]imidazol-2yl)ethyl)phenoxy)ethoxy)ethyl)carbamate.

[0160] NMR data of compound 14:

[0161] 1 H-NMR (400MHz, CDCl3-d) δppm1.45 (s, 9H), 2.30 (s, 3H), 2.43 (s, 3H), 2.45-2.51 (m, 4H), 2.61(t, J=6.85Hz, 2H), 3.15-3.21(m, 2H), 3.21-3.27(m, 2H), 3.35(q, J=5.01H z, 2H), 3.62 (t, J=5.07Hz, 2H), 3.68 (m, 4H), 3.82 (t, J=4.77Hz, 2H), 4.03-4.19 (m, 4 H), 6.87 (d, J=8.56Hz, 2H), 7.11-7.18 (m, 3H), 7.36 (d, J=8.19Hz, 1H), 7.64 (s, 1H).

[0162] Compound 13 (50 mg, 0.16 mmol), T3P (50 wt.% in EtOAc, 0.50 mL, 0.79 mmol), DIPEA (31 μL, 0.18 mmol) and 3-(4-(2-((tert-butoxycarbonyl)amino)ethoxy)phenyl)propanoic acid

[0163] (CAS No.: 2306467-18-3, 37.7 mg, 0.18 mmol) was dissolved in ethyl acetate (0.5 mL), sealed in a microwave reaction bottle, and heated in microwaves at 150° C. for 10 minutes.

[0164] The reaction mixture was alkalized by adding 1M NaOH solution and then extracted with ethyl acetate (3mL). The organic phase was washed with water (3mL) and saturated brine (3mL) in turn, filtered through a hydrophobic filter, and then the volatile solvent was blown dry with nitrogen. The crude product was purified by silica gel column chromatography to obtain compound 15 as a light yellow jelly (yield 27mg, yield 37%).

[0165] Compound 15 (127 mg, 0.26 mmol), 2,2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azaheptadecan-17-oic acid (CAS No.: 1347750-75-7, 83 mg, 0.26 mmol), DIPEA (158 μL, 0.91 mmol), HATU (104 mg, 0.27 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4 The residue was dried and concentrated in vacuo. Purification by a silica gel column gave compound 16 (98 mg, 48%), named Tert-butyl(15-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1Hbenzo[d]imidazol-2-yl)ethyl)phenoxy)-12-oxo-3,6,9-trioxa-13-azapentadecyl)carbamate.

[0166] NMR data of compound 16:

[0167] 1 H-NMR (400MHz, CDCl3-d) δppm1.46 (s, 9H), 2.28 (s, 3H), 2.41 (s, 3H), 2.48-2.58 (m, 6H), 2.64 (t, J=6. 79Hz, 2H), 3.12(m, 2H), 3.23(m, 2H), 3.29(m, 2H), 3.52(t, J=5.14Hz, 2H), 3.59-3.65(m, 10H), 3.69-3. 71 (m, 6H), 4.01 (t, J = 5.32Hz, 2H), 4.14 (t, J = 6.79Hz, 2H), 5.07 (br.s., 1H), 6.81 (m, J = 8.44Hz, 2H), 7 .10 (m, J=8.31Hz, 2H), 7.17 (d, J=8.31Hz, 1H), 7.40 (d, J=8.44Hz, 1H), 7.68 (s, 1H), 10.86 (br.s., 1H).

[0168] Compound 15 (127 mg, 0.26 mmol), 2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic acid (CAS No.: 1365655-91-9, 72 mg, 0.26 mmol), DIPEA (158 μL, 0.91 mmol), HATU (104 mg, 0.27 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine and Na 2 SO 4 The residue was dried and concentrated in vacuo. Purification by silica gel column gave compound 17 (100 mg, 51%), named as Tert-butyl(2-(2-(3-((2-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1Hbenzo[d]imidazol-2-yl)ethyl)phenoxy)ethyl)amino)-3-oxopropoxy)ethoxy)ethyl)carbamate.

[0169] NMR data of compound 17:

[0170] 1H-NMR (400MHz, CDCl3-d) δppm1.45 (s, 9H), 2.30 (s, 3H), 2.43 (s, 3H), 2.48-2.54 (m, 6H), 2.6 5(t, J=6.66Hz, 2H), 3.12(m, 2H), 3.23(m, 2H), 3.30(m, 2H), 3.51(t, J=4.95Hz, 2H), 3.60(m, 4 H), 3.64-3.70 (m, 8H), 4.02 (t, J=5.14Hz, 2H), 4.15 (t, J=6.72Hz, 2H), 5.13 (br.s., 1H), 6.84 (d, J=8.44Hz, 2H), 7.11-7.19 (m, 3H), 7.39 (d, J=8.31Hz, 1H), 7.65 (s, 1H), 10.09 (br.s., 1H).

[0171] Compound 15 (112 mg, 0.23 mmol), 8-((tert-butoxycarbonyl)amino)octanoic acid (CAS No.: 30100-16-4, 65 mg, 0.25 mmol), DIPEA (152 μL, 0.88 mmol), HATU (100 mg, 0.263 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column gave compound 18 (100 mg, 60%), named Tert-butyl(8-((2-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1Hbenzo[d]imidazol-2-yl)ethyl)phenoxy)ethyl)amino)-8-oxooctyl)carbamate.

[0172] TFA (2 mL) was added to a solution of compound 6 (46 mg, 0.088 mmol) in CH2Cl2 (2 mL), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo to give the deprotected product of compound 6.

[0173] TFA (2 ml) was added to a solution of compound 14 (70 mg, 0.11 mmol) in CH2Cl2 (2 ml), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo to obtain the deprotected product of compound 14. Compound 6 and the deprotected product of compound 14, DIPEA (57 μL, 0.33 mmol) in DMF (1 mL), and HATU (37 mg, 0.098 mmol) were dissolved in DMF and stirred at room temperature for 2 h.

[0174] After the raw material was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification with a silica gel column gave p300-MeTAG 1 (17 mg, 20%, three steps), named (S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-((1-(2-((2-(2-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1H-benzo[d]imidazol-2-yl)ethyl)phenoxy)ethoxy)ethyl)amino)-2-oxoethyl)piperidin-4-yl)amino)pyrimidine-4-carboxamide.

[0175] NMR data of p300-MeTAG 1:

[0176] 1 H-NMR (600MHz, DMSO-d6) δppm1.70 (dd, J=12.20, 6.14Hz, 2H), 2.02 (br.s., 2H), 2.23 (s, 4H), 2.31 (t, J=7.79Hz, 1H), 2.37-2.49 (m, 7H), 2.51-2.67(m, 5H), 2.69-2.75(m, 1H), 2.95-3.09(m, 4H), 3.14(d, J=4.03Hz, 2H), 3.15-3.22(m, 4H), 3.30-3.32(m, 2H), 3.41-3.44(m, 2H) , 3.50-3.56(m, 6H), 3.67-3.75(m, 3H), 4.00-4.07(m, 3H), 4.14-4.26(m, 2H), 4.31(br.s., 2H), 5.87(br.s., 1H), 6.86(d, J=8.44Hz, 2H), 7.10-7.16 (m, 2H), 7.16-7.26 (m, 6H), 7.56 (s, 1H), 7.59 (d, J=8.25Hz, 1H), 7.97 (br.s., 1H), 8.47 (br.s., 1H), 8.78 (s, 1H), 8.81 (s, 1H).

[0177] The procedure for synthesizing p300-MeTAG 2 was the same as that for synthesizing p300-MeTAG 1. Compound 16 (98 mg, 0.12 mmol) was reacted in CH 2 Cl 2TFA (2 ml) was added to the solution, and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo to obtain the deprotected product of compound 16. Compound 6 (52 mg, 0.099 mmol) and the deprotected product of compound 16, DIPEA (74 μL, 0.43 mmol), and HATU (49 mg, 0.13 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, Na 2 SO 4 The mixture was dried and concentrated in vacuo. Purification by silica gel column gave p300-MeTAG 2 (43 mg, 38%, three steps), named (S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-((1-(18-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1H-benzo[d]imidazol-2-yl)ethyl)phenoxy)-2,15-dioxo-6,9,12-trioxa-3,16-diazaoctadecyl)piperidin-4-yl)amino)pyrimidine-4-carboxamide.

[0178] NMR data of p300-MeTAG 2:

[0179] 1H-NMR(600MHz,DMSO-d6)δppm1.50(d,J=9.17Hz,2H),1.84-1.90(m,2H),2.17(t,J=7.43Hz,2H),2.23(s,3H),2.33(t,J=6.42Hz,2H),2.40(s,7H),2.54(t,J=6.33Hz,2H),2.78(br.s.,4H),2.83(br.s.,2H),2.92(br.s.,2H),3.10-3.14(m,2H),3.15-3.19(m,2H),3.25(q,J=5.81Hz,2H),3.28-3.33(m,2H),3.37-3.43(m,6H),3.46(br.s.,4H),3.48(br.s.,4H),3.49-3.53(m,4H),3.58(t,J=6.42Hz,2H),3.60-3.69(m,2H),3.83(br.s.,1H),3.87-3.95(m,3H),4.27(t,J=6.33Hz,2H),5.04(br.s.,1H),6.86(d,J=8.44Hz,2H),7.01(d,J=6.97Hz,1H),7.05-7.14(m,4H),7.16(dd,J=8.16,1.38Hz,1H),7.22(m,J=8.62Hz,2H),7.55-7.59(m,2H),7.68-7.78(m,2H),8.10(t,J=5.50Hz,1H),8.29(s,1H),8.74(t,J=5.14Hz,1H)。

[0180] The synthesis process of P300-MeTAG 3 is the same as that of p300MeTAG 1. TFA (2 mL) was added to a solution of compound 17 (100 mg, 0.13 mmol) in CH2Cl2 (2 mL), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo to obtain the deprotected product of compound 17. Compound 6 (56 mg, 0.11 mmol) and the deprotected product of compound 17, DIPEA (81 μL, 0.47 mmol), and HATU (53 mg, 0.14 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After the reaction was completed, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column gave p300-MeTAG 3 (48 mg, 41%, three steps) and named (S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-((1-(15-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1H-benzo[d]imidazol-2-yl)ethyl)phenoxy)-2,12-dioxo-6,9-dioxa-3,13-diazapentadecyl)piperidin-4-yl)amino)pyrimidine-4-carboxamide.

[0181] NMR data of p300-MeTAG 3:

[0182] 1H-NMR(600MHz,DMSO-d6)δppm1.45-1.55(m,2H),1.83-1.89(m,2H),2.14-2.21(m,2H),2.23(s,3H),2.34(t,J=6.42Hz,2H),2.40(br.s.,7H),2.54(t,J=6.42Hz,2H),2.76(br.s.,4H),2.83(br.s.,2H),2.91(br.s.,2H),3.08-3.14(m,2H),3.14-3.19(m,2H),3.24(q,J=5.81Hz,2H),3.27-3.33(m,2H),3.37-3.45(m,6H),3.46(s,4H),3.50(t,J=4.03Hz,4H),3.56-3.68(m,4H),3.83(br.s.,1H),3.87-3.96(m,3H),4.27(t,J=6.33Hz,2H),5.01(br.s.,1H),6.86(dJ=8.62Hz,2H),7.01(d,J=6.79Hz,1H),7.05-7.13(m,4H),7.16(dd,J=8.25,1.47Hz,1H),7.22(dJ=8.44Hz,2H),7.54-7.59(m,2H),7.69(br.s.,391H),7.75(d,J=7.34Hz,1H),8.12(t,J=5.41Hz,1H),8.28(s,1H),8.74(t,J=5.04Hz,1H)。

[0183] The steps for synthesizing p300-MeTAG 4 are the same as those for synthesizing p300-MeTAG 1. TFA (2 mL) was added to a solution of compound 18 (100 mg, 0.14 mmol) in CH2Cl2 (2 mL), and the reaction mixture was stirred at room temperature for 2 h. After the starting material was consumed, the volatiles were removed in vacuo to obtain the deprotected product of compound 18. Compound 6 (67 mg, 0.13 mmol) and the deprotected product of compound 18, DIPEA (79 μL, 0.45 mmol), and HATU (51 mg, 0.13 mmol) were dissolved in DMF (1 mL), stirred at room temperature for 2 h, and quenched with water after the reaction was completed, and extracted with ethyl acetate. The organic phase was washed with brine, dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column gave p300-MeTAG 4 (32 mg, 23%, three steps) and named (S)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)-2-hydroxypropyl)-6-((1-(2-((8-((2-(4-(2-(5-(3,5-dimethylisoxazol-4-yl)-1-(2-morpholinoethyl)-1H-benzo[d]imidazol-2yl)ethyl)phenoxy)ethyl)amino)-8-oxooctyl)amino)-2-oxoethyl)piperidin-4-yl)amino)pyrimidine-4-carboxamide.

[0184] NMR data of p300-MeTAG 4:

[0185] 1H-NMR (600MHz, DMSO-d6) δppm1.23 (d, J=4.77Hz, 6H), 1.38 (quin, J=6.74Hz, 2H), 1.44-1.55 ( m, 4H), 1.82-1.90 (m, 2H), 2.07 (t, J=7.43Hz, 2H), 2.15-2.21 (m, 2H), 2.23 (s, 3H), 2.40 (s, 7H) ), 2.52-2.55(m, 2H), 2.76(br.s., 4H), 2.83(br.s., 2H), 2.89(br.s., 2H), 3.06(q, J=6.60Hz , 2H), 3.09-3.14(m, 2H), 3.15-3.20(m, 2H), 3.27-3.32(m, 2H), 3.36-3.39(m, 2H), 3.39-3.46 (m, 2H), 3.50 (t, J=4.03Hz, 4H), 3.62 (br.s., 2H), 3.83 (br.s., 1H), 3.86-3.95 (m, 3H), 4.27 ( t, J=6.33Hz, 2H), 5.00 (br.s., 1H), 6.85 (m, J=8.44Hz, 2H), 7.01 (d, J=6.79Hz, 1H), 7.05-7.1 3 (m, 4H), 7.16 (dd, J=8.16, 1.56Hz, 1H), 7.22 (m, J=8.62Hz, 2H), 7.55-7.59 (m, 2H), 7.67 (br. s., 1H), 7.73 (d, J=7.15Hz, 1H), 8.01 (t, J=5.50Hz, 1H), 8.28 (s, 1H), 8.74 (t, J=5.23Hz, 1H).

[0186] Example 2: Fluorescence polarization (FP) evaluation, molecular docking and molecular dynamics simulation (MD) of MeTAC

[0187] 1) Fluorescence polarization (FP) experiment

[0188] The assay was performed using a Tecan Ultra plate reader on a Microfluor 384 black well plate (Thermo Fisher Scientific, Rochester, USA). The aim was to assess the ability of MeTAG to induce the formation of a trimeric complex involving the target protein and PRMT5. To achieve this, PRMT5 was initially labeled with Cy3. During the experiment, the target protein was mixed with MeTAG at a one-to-one concentration and incubated for 20 minutes. A mixture ranging from 5 μM to 20 nM was then used for trimer incubation. Subsequently, 100 nM cy3-labeled PRMT5 was introduced into the wells and incubated for another 20 minutes. FP values ​​were measured using a Tecan Ultra plate = reader. During analysis, PBS was used as background and cy3-labeled PRMT5 alone was used as a control.

[0189] 2) Molecular docking

[0190] Molecular docking is a method to predict the preferred orientation of a molecule (ligand) when it binds to another molecule (receptor, such as protein or enzyme). In this study, we adopted a semi-flexible docking method to form stable complexes. In order to predict reasonable ternary complexes, we divided the small molecule into three parts: two ligand parts that bind to the target protein and PRMT5 and the linker part. We docked the ligand part with the corresponding target protein and then constructed the binding conformations of the two proteins with the small molecule based on the docking site or the active region of the protein. First, we downloaded the crystal structure of the target protein from the Protein Data Bank (www.rcsb.org). Subsequently, we prepared the protein using AutoDock 1.5.2 software. During the preparation process, we removed water molecules, added polar hydrogen atoms, and calculated Gasteiger charges. Then, we performed local charge distribution on the protein and defined AutoDock atom types. In order to understand the torsion angles and rotatable bonds of the molecule, we used the torsion tree menu in ADT to view and standardize. Secondly, set the parameters in the Grid module. We defined the box size of the docking area as 40×40×40 to include the entire active site of the protein molecule. Other parameters were kept at default values. For small molecules, we used AutoDock software for preparation. AutoDock Vina software was used for molecular docking. Finally, Discover Studio software was used to build the initial model of the ternary complex, the active site of the reference protein, and the docking results.

[0191] 3) Molecular dynamics simulation (MD) technology

[0192] In order to further study the interaction and stability of the ternary complex, this study used molecular dynamics simulation (MD) technology and GROMACS2021 software to simulate the ternary complex for 30 nanoseconds. The AMBER99SB-ILDN force field was selected to establish the protein topology file, and the small molecule ligand topology file was generated using sobtop software and GAFF force field. A truncated cubic TIP3P solvent box was added at a distance of 1nm from the system, and Na / Cl was added to balance the charge. Then, 2500 steps of the steepest descent method and 2500 steps of the conjugate gradient method were used for energy minimization, respectively. While maintaining the system temperature at 298.15K, 100 picoseconds of NVT ensemble simulation and 100 picoseconds of NPT equilibrium simulation were performed. Finally, under periodic boundary conditions, 30 nanoseconds or 50 nanoseconds of dynamic simulation were performed, and the PME method was used to calculate the long-range electrostatic interaction. The non-bonded cutoff distance was set to 1nm, the system pressure was 101.325kPa, the integration time step was 2fs, and the trajectory was saved every 100 picoseconds. Through molecular dynamics simulation, the binding conformations of the two proteins and molecules were obtained, which provided a deeper understanding of the interaction mechanism. At the same time, by extracting stable molecular conformations from equilibrium trajectories and observing the interactions formed between small molecules and proteins, we further understood the role of small molecules in forming complex structures.

[0193] Example 3: Cell-level experiments of MeTAC

[0194] 1. Cell culture and transfection methods:

[0195] The cell lines used in this study were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HEK293T, H9C2, HDF, and MCF7 cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS, BI, USA), 100 U / mL penicillin, and 0.1 mg / mL streptomycin (Gibco; Thermo Fisher Scientific, Rochester, USA). VCaP and HUVEC cell lines were cultured in RMI-1640 medium containing 10% fetal bovine serum. All cells were maintained at 37°C and 5% CO. 2 Save it.

[0196] Transfection was performed using lipofectamine 2000 transfection reagent purchased from Life Technologies, following the manufacturer's protocol. Different cell lines were infected with viruses expressing lentiviral and retroviral cDNAs packaged in HEK293T cells. Stable PRMT5 knockdown cell lines were then generated by transfecting lentiviral vectors loaded with PRMT5 shRNA into the indicated cells.

[0197] 2. Cell proliferation ability detection experiment

[0198] Cell Counting Kit-8 detection method to analyze the effect of drugs on cell proliferation ability

[0199] Cell Counting Kit-8, referred to as CCK-8 kit, is a rapid and highly sensitive detection kit based on WST-8 that is widely used for cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of an electron coupling agent, it can be reduced by some dehydrogenases in mitochondria to generate an orange-yellow compound. The more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color. For the same cells, the depth of the color is linearly related to the number of cells.

[0200] To evaluate the effect of AR-MeTAG 1 on the cell viability of prostate cancer cells, C4-2 cells were plated at 4 × 10 3 In order to detect the effect of AKT-MeTAG 1 on cell proliferation, MCF7, H9C2, HDF and HUVEC cells were seeded at a density of 5×10 per well. 2 The density was plated on a 96-well plate. The drug dilution was serially diluted 2-fold in the selected culture medium, and 100 μL / well of the drug dilution was added to the 96-well plate. The plates were incubated at 37°C for the specified time, and then treated with CCK-8 reagent (NCM Biotech, China) for 4 hours, and the absorbance values ​​were read at 450 nM and 690 nm wavelength.

[0201] A=OD450-OD690.

[0202] Cell viability (%) = (A(drug addition) - A(background)) / (A(control) - A(blank)) x 100%.

[0203] Results Figure 4 A.

[0204] 3. Immunoprecipitation Assay and Immunoblotting Analysis

[0205] In order to study whether the three MeTAC molecules mentioned in the present invention can exert their proximity induction effect in cells, and whether the MeTAC function is affected in the presence of PRMT5 inhibitors or under PRMT5 knockdown conditions, immunoprecipitation and immunoblotting analysis were used, and the specific experimental process was as follows:

[0206] 1) Cell preparation

[0207] 293T, VCaP, and MCF7 cells were cultured at 3 × 10 4The cells were seeded into 6-well plates at a density of 100 μL / well and 2 ml of cell solution was added to each well. After the cells were cultured for 24 h, different drugs were added to the cells for treatment. After a specific treatment time, the cells were lysed with 100 μL of protein lysis buffer containing protease inhibitors (Sigma-Aldrich; Merck KGaA, Missouri, USA) and phosphatase inhibitors (Sigma-Aldrich; Merck, Missouri, USA) per well, and a portion was separated as WCL. The total protein content in each group of samples was quantified using a BCA quantitative kit, and the protein concentration in each group of samples was made consistent by adjusting the sample volume.

[0208] 2) Add monoclonal anti-FLAG (A2220 Sigma-Aldrich, MO, USA) or anti-HA (A2095 Sigma-Aldrich, MO, USA) antibody-conjugated M2 agarose beads and gently rock at 4°C for 4 h. Then, centrifuge at 13,000 rpm for 10 min, wash the agarose beads three times with IP buffer, add Loading Buffer, and incubate at 100°C for 10 min to completely denature the protein.

[0209] 3) Perform SDS-PAGE separation on samples from different groups. Prepare 10% polyacrylamide separation gel containing SDS and 5% polyacrylamide concentration gel. Then add the prepared samples and the same volume of pre-stained protein samples into the loading wells for electrophoresis separation experiments. The electrophoresis conditions are: the voltage is set to 70V, and the separation is about 15min until the bromophenol blue reaches the separation gel. Then adjust the voltage to 120V, separate for about 60min until the bromophenol blue reaches about 1cm from the end of the separation gel, and stop the electrophoresis.

[0210] 4) Transfer the protein sample to a membrane. All Western Blot experiments in the present invention use PVDF membranes, which are placed in the following order on a membrane transfer apparatus: three layers of filter paper, PVDF membrane, gel, and three layers of filter paper. The transfer current is set to 1 mA, and the transfer time is 1 h.

[0211] 5) Blocking: The transferred PVDF membrane was immersed in a blocking solution containing 5% BSA and incubated at room temperature for 1 hour to remove the influence of non-specific adsorption.

[0212] 6) Primary antibody incubation. Prepare different antibody dilutions as required, and then incubate overnight at 4 degrees to achieve the purpose of antibody recognition of specific antigens.

[0213] 7) Secondary antibody incubation. Prepare species-specific HRP-labeled secondary antibodies (anti-mouse or anti-rabbit) according to the species of the primary antibody, dilute 1:2000, and incubate at room temperature for 1 hour.

[0214] 8) Color development. Prepare color development solution, infiltrate the PVDF membrane incubated with secondary antibody, and use chemiluminescence instrument for color development analysis. All immunoblotting experiments were repeated at least three times with similar results. Semi-quantitative analysis was performed using ImageJ software (NIH) for immunoblotting band analysis.

[0215] The results are as follows Figure 2 EG, Figure 5 EI Figure 6 AE Figure 7 E and Figure 7 G.

[0216] 4. Cell-based PRMT5 inhibition assay

[0217] C4-2 cells were plated at 4 × 10 3 The drug was serially diluted 2-fold in the selected culture medium and 100 μL / well of the drug dilution was added to the 96-well plate. The culture dish was incubated at 37°C for 24 hours. The culture medium was collected and detected using an enzyme-linked immunosorbent assay (ELISA). The sample solution was added to the 96-well plate fixed with the PRMT5 antibody, and then incubated with the HRP-labeled detection antibody at 37°C for 1 hour, and then 3,3',5,5'tetramethylbenzidine (TMB) was used as the detection system (450nm) to detect whether the level of PRMT5 was inhibited.

[0218] 4. RT-qPCR was used to detect the mRNA levels of PSA, Tmprss2, NKX3.1, EGR2 and FOS.

[0219] Results Figure 3 BG Figure 7 HI.

[0220] 5. Plate colony formation assay and scratch assay

[0221] A) Plate colony formation assay

[0222] (1) C4-2 cells in the logarithmic growth phase were trypsinized in a 6-well plate, resuspended in complete medium (basal medium + 10% fetal bovine serum) into a cell suspension, and counted;

[0223] (2) Cell seeding: 400-1,000 cells / well (depending on the cell growth conditions, generally 700 cells / well) were seeded in each experimental group in a 6-well culture plate;

[0224] (3) Add a specific concentration of drug to each well and continue culturing until 14 days or until the number of cells in most single clones is greater than 50. Change the medium every 3 days and observe the cell status;

[0225] (4) After cloning, take photos of the cells under a microscope, wash once with PBS, add 1 mL of 4% paraformaldehyde to each well for fixation for 30-60 min, and wash once with PBS.

[0226] (5) Add 1 ml of crystal violet stain to each well and stain the cells for 10-20 min;

[0227] (6) Wash the cells several times with PBS, dry them, and take photos with a digital camera (take photos of the entire six-well plate and each well separately).

[0228] B) Scratch test

[0229] (1) Striking the culture plate: First, use a marker pen and a ruler to evenly draw horizontal lines on the back of the 6-well plate, approximately every 0.5 to 1 cm, across the wells. Make at least 5 lines in each well.

[0230] (2) Cell plating: Add about 5×10 5 C4-2 cells, based on the ability to become confluent overnight;

[0231] (3) Cell streaking: On the second day, use the tip of the gun to measure against the ruler and try to make the horizontal line on the back of the gun as vertical as possible. The tip of the gun should be vertical and not tilted.

[0232] (4) Washing cells: Wash the cells three times with PBS, remove the scratched cells, and add serum-free culture medium;

[0233] (5) Cell culture and observation: Place in 37°C, 5% CO 2 Incubator, and treat for specific time according to specific groups and concentrations; take samples at 0, 6, 12, 24, 48, 72, and 96 hours, observe cell migration at specific locations under a microscope and take photos;

[0234] (6) Result analysis: After opening the image using Image J software, 6-8 horizontal lines were randomly drawn and the mean of the distance between cells was calculated.

[0235] The results are as follows Figure 4 As shown in BG, AR-MeTAG 1 inhibits the clone formation and migration ability of prostate cancer cells.

[0236] 6. Flow cytometry to detect apoptosis

[0237] (1) Cell inoculation and treatment: 1×10 cells were inoculated in each experimental group in a 6-well culture plate. 5C4-2 cells / well were allowed to adhere to the wall for 24 hours, and drugs were administered according to specific group concentrations for 24 hours, and the cells were collected at the end of the treatment period.

[0238] (2) The collected cells were resuspended once in pre-cooled 1× PBS (4°C), centrifuged at 300×g for 5 minutes, and the supernatant was discarded to collect the cells.

[0239] (3) Add 300 μl of 1× Binding Buffer to resuspend the cells and adjust the cell concentration to 1×10 6 / ml.

[0240] (4) Take the flow tube and number the blank tube, single positive tube and sample tube in order. Add 100 μl of cell suspension filtered through a 200-mesh cell filter membrane to each flow tube, and add 5 μl of Annexin V-Alexa Fluor to each single positive tube. TM 488 or 10 μl PI propidium iodide dye and mix gently.

[0241] (5) Add 5 μl Annexin V-Alexa Fluor to the sample tube TM 488 and mix gently; then add 10 μl of PI propidium iodide and mix gently.

[0242] (6) Incubate at room temperature in the dark for 10 to 20 minutes, add 400 μl 1× Binding Buffer, then place in an ice bath and detect on a flow cytometer. The selected channels are as follows:

[0243] Annexin V-Alexa Fluor TM 488: 488nm excitation, collection band 530 / 30nm;

[0244] PI: 561nm excitation, collection band 610 / 20nm.

[0245] The results are as follows Figure 4 As shown in D, it is proved that AR-MeTAG 1 can induce apoptosis of C4-2 cells.

[0246] Example 4: Mass spectrometry analysis

[0247] Arginine methylation sites were identified using nano-LC-MS / MS equipped with electrospray ionization according to previously reported analysis. 293T cells were stably transfected with 10 μg HA-p300. 24 h after transfection, cells were treated with DMSO (control) or 20 μM p300-MeTAG1 for 24 h and lysed. To identify p300 methylation sites, the precipitate pulled down by anti-HA agarose beads of 293T cells was first dissolved in denaturing buffer (8 M urea and 0.1 M Tris-HCl, pH 8.5). It was then centrifuged at 14000 g for 20 min at 4 °C. Subsequently, urea in the protein mixture was replaced with 50 mM NH4HCO3, and centrifuged at 14000 g for 20 min at 20 °C, repeated 3 times. The proteins were then digested overnight with sequencing-grade modified trypsin (Promega) at a protease ratio of 50:1 at 37 °C.

[0248] To localize p300 arginine methylation, liquid chromatography-mass spectrometry (LC-MS) analysis was performed using a nanoflow EASY-nLC1000 system (Thermo Fisher Scientific, Odense, Denmark) and an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were analyzed on a C18 analytical column (75 μm id×20 cm, resil-pur 120C18-aq, 1.9 μm, Dr. Maisch GmbH, Germany). The mobile phases were solution A (0.1% formic acid) and solution B (0.1% formic acid + 100% acetonitrile). Peptide elution conditions: 5-28% solution B (50 minutes), 28-90% solution B (2 minutes), 90% solution B (10 minutes), flow rate 200 nL / min, spray voltage 2.0 kV, heated capillary temperature 275°C. Mass spectrometry / MS experiments were performed in an Orbitrap using high energy collision dissociation (HCD) fragmentation with an isolation window of 1.6, a resolution of 15,000, and a normalized collision energy (NCE) of 30%. The signal threshold was set to 5000 and the normalized collision energy was set to 35%.

[0249] The resulting data were processed using the UniProt human protein database (70,956 entries, downloaded on December 2, 2016) using Protein Discoverer (version 1.4.0.288, Thermo Fisher Scientific) and Mascot (version 2.3.2, Matrix Science). The mass tolerance of the precursor was set to 10 ppm and the mass tolerance of the fragment ion was set to 0.05 Da. The minimum precursor mass was 350 Da and the maximum precursor mass was 8000 Da. A maximum of two cleavages were allowed to be missed. Carbamidomethylation of cysteine ​​was designated as a fixed modification, while protein N-terminal acetylation, methionine oxidation, and arginine methylation were considered variable modifications. The false discovery rate (FDR) threshold for peptide identification was set to 0.05.

[0250] The results are as follows Figure 7 As shown in H, treatment with P300-MeTAG 1 can lead to methylation of 12 arginine sites of P300.

[0251] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation, characterized in that: The target protein ligand and the PRMT5 ligand are connected via a linker to obtain; The PRMT5 ligand is a piperidine ring substituted product of EPZ015666; The target protein ligand is a hydrolysis product of the AR inhibitor enzalutamide, a pan-AKT inhibitor AZD5363, or a bromodomain targeting the P300 protein; The Linker is any chemically feasible connection structure, which is used to connect two ligands via an amide bond.

2. According to claim 1, the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation is characterized in that: When the target protein ligand is the hydrolysis product of the AR inhibitor enzalutamide, the structural formula of the MeTAG is as shown in formula (1): Linker is 3. The proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 1, characterized in that: When the target protein ligand is the pan-AKT inhibitor AZD5363, the structural formula of the MeTAG is as shown in formula (2): Linker is 4. The proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 1, characterized in that: When the target protein ligand is a bromodomain targeting P300 protein, the structural formula of the MeTAG is shown in formula (3): Linker is 5. Use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation as described in claim 2 in the preparation of drugs for treating prostate cancer.

6. The use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in the preparation of a drug for treating prostate cancer according to claim 5, characterized in that: The MeTAG is used to prepare a drug for inhibiting the proliferation of prostate cancer cells, wherein the prostate cancer cells include VCAP and C4-2.

7. Use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation as claimed in claim 3 in the preparation of a preparation for promoting cell growth.

8. The use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in the preparation of a preparation promoting cell growth according to claim 7, characterized in that: The cells include MCF7, H9C2, HDF and HUVEC.

9. Use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation as claimed in claim 4 in the preparation of a P300 protein activator.

10. The use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in the preparation of a P300 protein activator according to claim 9, characterized in that: The MeTAG was used to prepare a preparation for activating P300 protein in HCT116 cells.

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