MeTAG, a proximity-induced small molecule chimera platform targeting protein arginine methylation and its applications

Abstract

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. By connecting the methyltransferase PRMT5 to the target protein (POI), MeTAG promotes arginine methylation of POI in a reversible and PRMT5-dependent manner. In addition, MeTAG can also regulate downstream signals in a methylation-dependent manner, causing downregulation of AR transcriptional activity and activation of AKT. Therefore, MeTAG has been proven to be a feasible solution for regulating the methylation level of POI, and can also be used as a unique research and translation tool to reveal the biological function of protein arginine methylation. More importantly, the research results of the present invention demonstrate that MeTAG has the potential to benefit from clinical treatment by precisely controlling the methylation status of specific POIs.

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 from 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, and different PRMTs 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 various cancers, especially those with methylthioadenosine phosphorylase (MTAP) gene deletions, and their inhibitors are currently in clinical trials. In addition, PRMTs are also crucial for the survival of non-tumor cells, as mice lacking Prmt1 and Prmt5 are embryonically lethal. Therefore, precise manipulation of the methylation of target proteins (POIs) is a prerequisite for deepening our understanding of the biological mechanisms 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-to-lysine (R-to-K) or alanine (R-to-A) point mutations are often used to mimic the effects of eliminating arginine methylation, but arginine-to-phenylalanine (R-to-F) methylation mimicking mutations remain controversial, so these methods still have certain limitations in biological research. On the other hand, although chemical probes targeting PRMT enzymes have had a significant impact on basic life sciences and translational medicine, due to the broad spectrum of PRMT enzyme substrates, PRMT inhibition inevitably introduces side effects and toxicity.

[0007] Similarly, inhibitors targeting arginine demethylases, including the deaminase peptidylarginine deaminase 4 (PAD4), have been developed to 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 also have a broad substrate range and can induce changes in global methylation levels in cells.

[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 the first aspect of the present invention, a proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation is provided, which is obtained by connecting the target protein ligand and the PRMT5 ligand through 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 P300 protein;

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

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

[0015] When the target protein ligand of 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 aforementioned 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 aforementioned 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 P300 protein, the structural formula of the MeTAG is shown in formula (3):

[0025]

[0026] linker is

[0027] The fourth aspect of the present invention provides the use of the above-mentioned proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation in the preparation of a P300 protein activator. 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, which suggests 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 uses two known arginine methylation substrates, the androgen receptor (AR) and protein kinase B (AKT), as examples. MeTAG induces their symmetrical dimethylation in a PRMT5-dependent manner, thereby inhibiting AR signaling and activating AKT signaling, respectively. In addition, it was 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 translational 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 Development of the AR-MeTAG system, 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 AR transcriptional function, including:

[0042] A is a schematic diagram of AR-MeTAG 1-induced reduced 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 PRMT5 knockout on the reduction of PSA mRNA levels in VCaP cells induced by AR-MeTAG 1.

[0045] D is the result of the elution experiment detecting the increase of PSA mRNA level after removing 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 downregulation 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 downregulation 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 is as follows:

[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 AR-MeTAG 1 on the colony-forming ability of C4-2 cells.

[0052] C is the statistical result of B.

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

[0054] E is the statistical analysis results 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-MeTAG 1: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 ternary complex formation induced by AKT-MeTAG molecules.

[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 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-induced 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 of AKT-MeTAG 1 activating 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 showing that AKT-MeTAG 1 activates AKT1 in a PRMT5 activity-dependent manner.

[0071] D is the experimental result showing 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 of AKT-MeTAG 1 promoting the growth of H9C2 cells.

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

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

[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-MeTAG 1:PRMT5 ternary complex.

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

[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 showing that p300-MeTAG 1 up-regulated EGR2 mRNA level in a concentration-dependent manner.

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

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

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

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

[0089] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they 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, the structural design principle of which is as follows Figure 1 As shown in A, 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 portion on EPZ015666 protrudes into the solvent and hardly interacts with the PRMT5 structure, making it the best attachment point for linker binding. In another PRMT5 inhibitor GSK3326595, the oxyethane portion 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 for MeTAG ( Figure 1 B).

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

[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 with different linkers to construct three pre-set AR-MeTAGs ( Figure 2A). AR-MeTAG structure-activity relationship and molecular dynamics simulation (MD), fluorescence polarization (FP) evaluation and fluorescence polarization analysis 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 PRMT5 knockout in VCAP cells demonstrated that AR-MeTAG 1 promoted the binding of AR to PRMT5 and promoted AR arginine methylation SDMA in a PRMT5-dependent manner (Figures 2E-G).

[0094] The AR-MeTAG 1 of the present invention induces arginine methylation in various prostate cancer cells VCaP and C4-2, which 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 the 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 colony 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 of 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 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). This effect can 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, but not of R391K mutant AKT. 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 transfection of P300 and PRMT5 plasmids into 293T cells demonstrated that P300-MeTAG 1 could promote the interaction between p300 and PRMT5, significantly increasing 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 used mass spectrometry (MS) analysis to explore potential arginine methylation sites on p300 488 mediated by PRMT5. Mass spectrometry analysis showed that P300 was already arginine methylated at residues R237, R695, and R2059, 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 H and I).

[0100] The small molecule proximity-induced chimera platform (MeTAG) targeting arginine methylation of target proteins and its applications provided by the present invention are described in detail below with reference to 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) observed at 254 nm UV light and purified by column chromatography (CC) on a Reveris X2 (Grace) automated flash unit. The residual solvent signal was used as a reference and the chromatograms were taken at 298.15 K on a Bruker Avance 400 / 100MHz or Bruker Avance NMR data were recorded on a 600 / 150 MHz spectrometer. The compound's structure was confirmed by 1H, 13C, and HSQC NMR spectroscopy. In addition, high-resolution mass spectrometric analysis was performed on a Waters LCT Premier XE™ time-of-flight (TOF) mass spectrometer equipped with standard electrospray ionization (ESI) and a modular LockSpray™ interface. LC-MS was performed using a Waters AutoPurification system: a Waters Cortecs C18 column (2.7 μm, 100 × 4.6 mm); a gradient of formic acid in H2O (0.2%, v / v) / MeCN; a flow rate of 0.30 mL / min; a gradient from 95:5 to 0:100 over 15 minutes.

[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), and HATU (50 mg, 0.13 mmol) were stirred in DMF (1 mL) at room temperature for 2 h with TLC monitoring. After the starting materials reacted completely, the reaction was quenched with water and extracted with ethyl acetate. The organic phase was washed with saturated brine, dried over Na2SO4, and concentrated in vacuo. Purification by silica gel column gave compound 2 (50 mg, 62%), named Tert-butyl 2-(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 solution of compound 2 (50 mg, 0.077 mmol) in CH2Cl2 (2 ml), and the reaction mixture was stirred at room temperature for 2 h. After complete consumption of the starting material, the volatiles were removed in vacuo. The residue, compound 3 (CAS No.: 1616391-79-7, 41 mg, 0.099 mmol), DIPEA (42 μL, 0.24 mmol), and HATU (32 mg, 0.084 mmol) were dissolved in DMF (1 mL) and stirred at room temperature for 2 h. After completion of the reaction, the mixture was cooled 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 ARMeTAG 1 (33 mg, 44% over two steps) 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), and HATU (63 mg, 0.16 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After the starting materials were completely reacted, 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 on a silica gel column afforded compound 4 (84 mg, 81%), designated 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 a solution of compound 4 (84 mg, 0.13 mmol) in CH2Cl2 (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, 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 completion of the reaction, 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 AR-MeTAG 2 (35 mg, 28% over two steps), which was 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] AR-MeTAG 2 NMR data:

[0116] 1H-NMR(600MHz,DMSO-d6)δppm1.31(br.s.,8H),1.47-1.52(m,4H),1.54(s,6H),1.82-1.89(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=6.79Hz,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), and HATU (56 mg, 0.15 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After completion of the reaction, 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 5 (67 mg, 66%), named as Tert-butyl(S)-(2-(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] To a solution of compound 5 (67 mg, 0.094 mmol) in CH2Cl2 (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, 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 completion of the reaction, 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 AR-MeTAG 3 (59 mg, 47% over two steps), which was 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] NMR data of AR-MeTAG 3:

[0122] 1H-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 Figure 9 The route shown is used to synthesize AKT-MeTAG 1-3

[0124] To a solution of compound 3 (120 mg, 0.29 mmol) and K2CO3 (81 mg, 0.586 mmol) in DMF (2 mL) was added 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 materials were 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 on a flash silica gel column gave compound 6 (99.6 mg, 65%), named 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), and HATU (39 mg, 0.10 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After completion of the reaction, 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 on a silica gel column afforded compound 7 (40 mg, 61%). This compound was designated 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] 1H-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] To a solution of compound 6 (100 mg, 0.17 mmol) in CH2Cl2 (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 completion, 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% over 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] 1H-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] To a solution of compound 6 (100 mg, 0.17 mmol) in CH2Cl2 (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 glycine (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 completion, 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 9 (63 mg, 62% over two steps), which was designated 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] 1H-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 completion of the reaction, the solvent was evaporated, and the resulting mixture was purified by flash column chromatography to obtain the tert-butyl ester as a white solid intermediate (1.31 g, 85% yield).

[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), and add imidazole (CAS No. 288-32-4, 0.89 g, 14 mmol). The mixture is stirred at room temperature for 30 minutes. A solution of the white solid intermediate tert-butyl ester (1.0 g, 3.5 mmol) in DCM (30 mL) is then added. The resulting mixture is stirred for 3 hours.

[0138] After the reaction was complete, saturated NaHCO₃ solution and 10% aqueous Na₂S₂O₃ were added, and the organic phase was extracted with DCM (20 mL x 3). The organic phase was dried over anhydrous Na₂SO₄ and evaporated. The crude product was purified by silica gel column chromatography to afford compound 10-b as a white solid (2.3 g, 65% yield).

[0139] Compound 10-b (1.48 g, 3.7 mmol) was dissolved in acetonitrile (30 mL), and K2CO3 (2.1 g, 14.8 mmol) and benzyl piperzine-1-carboxylate (CAS: 31166-44-6, 1.65 g, 7.4 mmol) were added. The mixture was stirred at 80°C overnight. After completion of the reaction, the product was purified by silica gel column chromatography. The product was dissolved in DCM (20 mL) and TFA (20 mL), stirred for 30 minutes, and the solvent was evaporated and dried to afford compound 10-c as a white powder (1.35 g, 95% yield).

[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). 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 sequentially. The mixture was stirred at room temperature overnight. The product was purified by reverse-phase column chromatography to obtain the TFA salt of compound 10 (AKT-MeTAG Ctr, 1.71 g, 69% yield) as a white solid.

[0141] To a solution of compound 10 (50 mg, 0.068 mmol) in CH2Cl2 (2 mL) was added TFA (2 mL) and stirred at room temperature for 32 h. After the starting material was consumed, the volatiles were removed in vacuo. Purification on a silica gel column afforded AKT-MeTAG 1N (38 mg, 88%), designated 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 reaction mixture was stirred under H2 at room temperature for 2 h. After the starting material was consumed, the reaction mixture was filtered and the filtrate was collected and evaporated to give 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 product 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. Purification by silica gel column gave AKT-MeTAG 1 (28 mg, 34% over three steps) 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),3.31(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,1H),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] Following the same procedure as AKT-MeTAG 1, TFA (2 ml) was added to a solution of compound 9 (63 mg, 0.11 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 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 completion of the reaction, 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. 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. Purification by silica gel column gave AKT-MeTAG 2 (59 mg, 54% over three steps) 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 hours. After completion of the reaction, the product 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%), which was 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 Figure 10 The synthesis of p300-MeTAG 1-4 was carried out by the following route:

[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 dried over Na2SO4, filtered, concentrated, and then dissolved in MeOH / H2O (v / v, 1 / 1, 10 mL). 2M NaOH (3 mL) was added and the reaction was continued at 50°C for 2 h. The reaction solution was cooled to room temperature, the pH was adjusted to 2-3 with HCl, and the mixture was extracted with ethyl acetate. The organic phase was collected to obtain intermediate compound 12 for the next reaction.

[0155] Pd(dppf)Cl2 (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 aqueous NaHCO3 solution (100 mL) was added to the mixture. The mixture was then degassed by evacuating and flushing with nitrogen (repeated three 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). MgSO4 and activated carbon were added to the separated organic phase, 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 product crystallization. The supernatant was pipetted and the process was repeated. Finally, the obtained solid was dried under vacuum to obtain product 13-b as a light beige solid (8.57 g, yield 73%).

[0157] To a solution of compound 13-b (1.18 g, 5.00 mmol) and DIPEA (CAS No. 7087-68-5, 1.05 mL, 6.00 mmol) in tetrahydrofuran (THF, 25 mL) was added 4-(2-Aminoethyl)morpholine (CAS No. 2038-03-1, 0.787 mL, 6.00 mmol). The mixture was stirred at room temperature for 16 hours, followed by the addition of 4-(2-Aminoethyl)morpholine (0.262 mL, 2.00 mmol) and continued stirring for 5 hours. After completion of the reaction, the mixture was partitioned between ethyl acetate (20 mL) and water (20 mL). The separated organic phase was washed with water (20 mL) and saturated brine (20 mL), dried over anhydrous MgSO4, and the solvent was evaporated to afford product 13-c as an orange solid (1.63 g, 94% yield).

[0158] A 1.0 M aqueous Na2S2O4 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), dried over anhydrous MgSO4, and the solvent was evaporated to obtain 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, basified with 0.1 M aq. NaOH, and extracted with ethyl acetate. The organic phase was collected and washed with brine. The mixture was dried over Na2SO4, filtered, and separated on a silica gel column to yield compound 14 (70 mg, 48%), designated 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 a microwave at 150° C. for 10 minutes.

[0164] The reaction mixture was basified by adding 1M NaOH solution and then extracted with ethyl acetate (3 mL). The organic phase was washed with water (3 mL) and saturated brine (3 mL), filtered through a hydrophobic filter, and the solvent was evaporated with nitrogen. The crude product was purified by silica gel column chromatography to obtain compound 15 as a light yellow gum (yield 27 mg, 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), and HATU (104 mg, 0.27 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After completion of the reaction, 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 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), and HATU (104 mg, 0.27 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After completion of the reaction, 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 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), and HATU (100 mg, 0.263 mmol) were stirred in DMF (1 mL) at room temperature for 2 h and monitored by TLC. After completion of the reaction, 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 on a silica gel column afforded compound 18 (100 mg, 60%), designated 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 starting 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 p300-MeTAG 1 (17 mg, 20% over three steps), which was 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 steps for synthesizing p300-MeTAG 2 were the same as those for synthesizing p300-MeTAG 1. Compound 16 (98 mg, 0.12 mmol) was dissolved in CH2Cl2 (2 ml) and TFA (2 ml) was added. 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 completion of the reaction, the mixture 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 2 (43 mg, 38% over three steps) and 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 completion of the reaction, 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% over 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 were 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) and stirred at room temperature for 2 h. After completion of the reaction, 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 4 (32 mg, 23% over 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 concentration of one to one 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 for predicting the preferred orientation of one molecule (ligand) when binding to another molecule (receptor, such as a protein or enzyme). In this study, we employed a semi-flexible docking approach to generate stable complexes. To predict plausible ternary complexes, we divided the small molecule into three parts: two ligands that bind to the target protein and PRMT5, and a linker. We docked the ligands to the corresponding target proteins and then constructed bound conformations of the two proteins and the small molecule based on the docking site or active region of the protein. First, we downloaded the crystal structure of the target protein from the Protein Data Bank (www.rcsb.org). We then prepared the protein using AutoDock 1.5.2 software. During preparation, we removed water molecules, added polar hydrogen atoms, and calculated Gasteiger charges. We then generated a local charge distribution on the protein and defined AutoDock atom types. To understand the torsion angles and rotatable bonds of the molecule, we used the Torsion Tree menu in ADT to view and customize them. Next, we set parameters in the Grid module. We defined the docking region as a 40×40×40 box to encompass the entire active site of the protein molecule. All other parameters remained at their default values. For small molecules, we used AutoDock software for preparation. We also used AutoDock Vina software for molecular docking. Finally, we used Discover Studio software to construct an 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] To further investigate the interactions and stability of the ternary complex, molecular dynamics (MD) simulations were performed using GROMACS2021 software for 30 nanoseconds. The AMBER99SB-ILDN force field was used to establish the protein topology file, and the small molecule ligand topology file was generated using sobtop software and the GAFF force field. A truncated cubic TIP3P solvent box was added at a distance of 1 nm from the system, and Na / Cl was added to balance the charge. Energy minimization was then performed using 2500 steps of the steepest descent method and 2500 steps of the conjugate gradient method, respectively. While maintaining the system temperature at 298.15 K, 100 picosecond NVT ensemble simulations and 100 picosecond NPT equilibrium simulations were performed. Finally, under periodic boundary conditions, dynamic simulations of 30 or 50 nanoseconds were performed, and the PME method was used to calculate the long-range electrostatic interactions. The non-bonded cutoff distance was set to 1 nm, the system pressure was 101.325 kPa, the integration time step was 2 fs, and the trajectory was saved every 100 picoseconds. Through molecular dynamics simulations, the binding conformations of the two proteins and molecules were obtained, providing 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 understand 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] 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 supplemented with 10% fetal bovine serum. All cells were maintained at 37°C and 5% CO2.

[0196] Transfections were performed using lipofectamine 2000 transfection reagent purchased from Life Technologies according to 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 generated by transfecting the indicated cells with lentiviral vectors carrying PRMT5 shRNA.

[0197] 2. Cell proliferation ability detection experiment

[0198] Cell Counting Kit-8 assay to analyze the effects of drugs on cell proliferation

[0199] The Cell Counting Kit-8, also known as CCK-8, is a rapid, highly sensitive assay based on WST-8 and widely used for detecting cell proliferation and cytotoxicity. WST-8 is a compound similar to MTT. In the presence of an electron coupling reagent, it is reduced by certain dehydrogenases within mitochondria to produce an orange-yellow compound. The color darkens with increasing cell proliferation, while the color lighter with increasing cytotoxicity. For the same cell population, the color intensity is linearly correlated with cell number.

[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 drug was plated at a density of 100 μL / well in a 96-well plate. Serial two-fold dilutions were made 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 indicated time, followed by treatment with CCK-8 reagent (NCM Biotech, China) for 4 hours. The absorbance was read at 450 nM and 690 nm.

[0201] A=OD450-OD690.

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

[0203] See the results Figure 4 A.

[0204] 3. Immunoprecipitation Assay and Immunoblotting Analysis

[0205] To investigate whether the three MeTAC molecules mentioned in this invention can exert their proximity-induced effects in cells, and whether MeTAC function is affected in the presence of PRMT5 inhibitors or under conditions of PRMT5 knockdown, immunoprecipitation and immunoblotting analysis were performed. The specific experimental procedures were as follows:

[0206] 1) Cell preparation

[0207] 293T, VCaP, and MCF7 cells were cultured at 3 × 10 4Cells were seeded at a density of 100 μL / well at 400 μL / well in a 6-well plate, and 2 ml of cell culture was added to each well. After 24 h of adherent culture, the cells were treated with various drugs. After the specified treatment time, the cells were lysed with 100 μL / well of IP buffer containing protease inhibitors (Sigma-Aldrich; Merck KGaA, Missouri, USA) and phosphatase inhibitors (Sigma-Aldrich; Merck, Inc., Missouri, USA). A portion was aliquoted as WCL, and the total protein content in each sample was quantified using a BCA quantification kit. The sample volume was adjusted to ensure consistent protein concentration in each sample.

[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 hours. Subsequently, centrifuge at 13,000 rpm for 10 minutes, wash the beads three times with IP buffer, add loading buffer, and incubate at 100°C for 10 minutes to completely denature the proteins.

[0209] 3) Separate the samples from the different groups by SDS-PAGE. Prepare a 10% polyacrylamide separating gel containing SDS and a 5% polyacrylamide stacking gel. Add the prepared samples and an equal volume of prestained protein sample to the sample wells for electrophoresis separation. Electrophoresis conditions are as follows: Set the voltage to 70V and separate for approximately 15 minutes, until the bromophenol blue reaches the end of the separating gel. Then adjust the voltage to 120V and separate for approximately 60 minutes, until the bromophenol blue reaches approximately 1 cm from the end of the separating gel, at which point the electrophoresis is stopped.

[0210] 4) Transfer the protein sample to a membrane. All Western blot experiments in this article use PVDF membranes. Place the following on a membrane transfer apparatus: three layers of filter paper, PVDF membrane, gel, and three layers of filter paper. Set the transfer current to 1 mA and the transfer time to 1 hour.

[0211] 5) Blocking: Immerse the transferred PVDF membrane in a blocking solution containing 5% BSA and incubate at room temperature for 1 hour to remove the effects of nonspecific adsorption.

[0212] 6) Primary Antibody Incubation: Prepare different antibody dilutions as needed and incubate overnight at 4°C to allow the antibody to recognize the specific antigen.

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

[0214] 8) Color development. Prepare a color development solution, soak the PVDF membrane completely with secondary antibody, and analyze the color using a chemiluminescence analyzer. All immunoblotting experiments were repeated at least three times with similar results. Semi-quantitative analysis of immunoblot bands was performed using ImageJ software (NIH).

[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 immobilized with PRMT5 antibody, and then incubated with HRP-labeled detection antibody at 37°C for 1 hour. Then, 3,3',5,5'tetramethylbenzidine (TMB) was used as the detection system (450 nm) to detect whether the level of PRMT5 was inhibited.

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

[0219] See the 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) to form a cell suspension, and counted;

[0223] (2) Cell seeding: 400–1,000 cells / well (depending on cell growth, 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 individual clones exceeds 50. Change the medium every 3 days and observe the cell status;

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

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

[0227] (6) Wash the cells several times with PBS, dry them, and take pictures with a digital camera (take pictures 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 mark horizontal lines on the back of a 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 approximately 5×10 5 C4-2 cells, whichever can be fully confluent overnight;

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

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

[0233] (5) Cell culture and observation: Place cells in a 37°C, 5% CO2 incubator and treat them for a specific time according to specific groups and concentrations; take samples at 0, 6, 12, 24, 48, 72, and 96 hours, and 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 intercellular distance 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 Detection of Apoptosis

[0237] (1) Cell inoculation and treatment: 1×10 cells were inoculated into each experimental group in a 6-well culture plate. 5C4-2 cells / well were allowed to adhere for 24 hours, and then treated with drugs according to the specific group concentration 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 the flow tube, and add 5 μl of Annexin V-Alexa Fluor to the single positive tube. TM Add 488 or 10 μl of PI propidium iodide dye and mix gently.

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

[0242] (6) Incubate at room temperature in the dark for 10–20 min, add 400 μl of 1× Binding Buffer, then place in an ice bath and analyze on a flow cytometer. Select the following channels:

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

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

[0245] The results are as follows Figure 4 As shown in D, it was demonstrated that AR-MeTAG 1 could induce apoptosis in C4-2 cells.

[0246] Example 4: Mass spectrometry analysis

[0247] Arginine methylation sites were identified using nano-LC-MS / MS equipped with electrospray ionization, based on previously reported analyses. 293T cells were stably transfected with 10 μg of 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 pellet pulled down from anti-HA agarose beads from 293T cells was first dissolved in denaturing buffer (8 M urea and 0.1 M Tris-HCl, pH 8.5). The pellet was then centrifuged at 14,000 g for 20 min at 4°C. Subsequently, the urea in the protein mixture was replaced with 50 mM NH₄HCO₃, and the mixture was centrifuged at 14,000 g for 20 min at 20°C, repeated three times. Proteins were then digested overnight with sequencing-grade modified trypsin (Promega) at a 50:1 protease ratio at 37°C.

[0248] To map 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 were: 5-28% Solution B (50 min), 28-90% Solution B (2 min), and 90% Solution B (10 min). The flow rate was 200 nL / min, the spray voltage was 2.0 kV, and the heated capillary temperature was 275°C. Mass spectrometry / mass spectrometry experiments were performed in an Orbitrap using high energy collisional 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 at 5000 and the normalized collision energy was set at 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 Figure 3, treatment with P300-MeTAG 1 resulted in methylation of 12 arginine residues of P300.

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

Claims

1. A proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation, characterized in that: The target protein ligand is connected to the PRMT5 ligand through a linker; 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 P300 protein; 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): Formula (1); linker is ; When the target protein ligand is the pan-AKT inhibitor AZD5363, the structural formula of the MeTAG is shown in formula (2): Formula (2), n=1; linker is ; When the target protein ligand is a bromodomain targeting P300 protein, the structural formula of the MeTAG is shown in formula (3): Formula (3); linker is .

2. The use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 1 in the preparation of a drug for treating prostate cancer, characterized in that: The structural formula of the MeTAG is shown in formula (1).

3. The use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 2 in the preparation of a drug for treating prostate cancer, 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.

4. Use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 1 in the preparation of a preparation promoting cell growth, characterized in that: The structural formula of the MeTAG is shown in formula (2).

5. The use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 4 in the preparation of a preparation promoting cell growth, characterized in that: The cells include MCF7, H9C2, HDF and HUVEC.

6. Use of the proximity-induced small molecule chimera platform MeTAG targeting protein arginine methylation according to claim 1 in the preparation of a P300 protein activator, characterized in that: The structural formula of the MeTAG is shown in formula (3).

7. 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 6, characterized in that: The MeTAG was used to prepare a preparation for activating P300 protein in HCT116 cells.

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