Cdc25 phosphatase degradation agent based on quinolinedione skeleton as well as preparation method and application of Cdc25 phosphatase degradation agent

Abstract

The invention discloses a Cdc25 phosphatase degradation agent based on a quinolinedione skeleton as well as a preparation method and application of the Cdc25 phosphatase degradation agent, and belongs to the field of medicinal chemistry. The degradation agent has a general formula structure as shown in a formula (I), recognizes Cdc25 phosphatase through a quinolinedione derivative, is coupled with an E3 ubiquitin ligase ligand through a connecting chain, and induces ubiquitination of target protein and proteasome dependent degradation. The compound can obviously reduce the level of Cdc25 phosphatase in cells, has obvious anti-tumor activity and can be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and biomedicine, specifically relating to a protein hydrolysis-targeting chimeric (PROTAC) molecule based on a quinolinidone backbone, which can be used as a Cdc25 phosphatase degrader, and also relates to its preparation method and its application in the preparation of antitumor drugs. Background Technology

[0002] Malignant tumors pose a serious threat to human health, and their treatment methods include surgery, radiotherapy, chemotherapy, and targeted therapy. Among these, molecular targeted therapy, as the core of precision medicine, has made progress in targeting specific oncogenic proteins, but it still often faces challenges such as acquired resistance and off-target toxicity. There is an urgent need to develop novel targeted drugs that combine high efficacy, high selectivity, and a broad therapeutic window.

[0003] The activity of cyclin-dependent kinases (CDKs) is precisely regulated by their phosphorylation state. The Cdc25 phosphatase family (including Cdc25A, B, and C isoforms) activates CDKs through dephosphorylation and is a key positive regulator driving cell cycle progression. Cdc25 phosphatases are frequently overexpressed in various human tumors and are associated with tumor progression and poor prognosis, thus being considered highly promising anti-tumor targets. However, due to the flat and highly conserved active sites of Cdc25 phosphatases, developing potent and selective traditional small molecule inhibitors presents significant challenges, making them typical "drug-difficult" targets.

[0004] Proteolytic targeting chimeras (PROTACs) offer a novel strategy for targeting these "drug-resistant" proteins. PROTAC molecules induce ubiquitination of the target protein by simultaneously binding to both the target protein and an E3 ubiquitin ligase, leading to its degradation by the proteasome. Compared to inhibitors that only block protein function, degradative agents can directly eliminate the target protein, potentially offering advantages such as longer duration of action, overcoming drug resistance, and higher selectivity. Currently, there are no publicly reported PROTAC degradative agents targeting Cdc25 phosphatase.

[0005] Therefore, developing a PROTAC molecule that can efficiently and selectively degrade Cdc25 phosphatase is of great significance for opening up new avenues for anti-tumor therapy. Summary of the Invention

[0006] The primary objective of this invention is to provide a Cdc25 phosphatase degrader based on a quinolinidone skeleton.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned degradation agent.

[0008] Another object of the present invention is to elucidate the bioactivity of the above-mentioned degrading agent in degrading Cdc25 phosphatase and in antitumor activity, and to provide its use in the preparation of antitumor drugs.

[0009] This invention provides the structure of a Cdc25 phosphatase degrader based on a quinolinidone skeleton and its preparation method. This invention also provides some activity screening results of the above compound and its uses.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] I. Cdc25 phosphatase degrading agents based on the quinolinidone skeleton

[0012] This invention provides a Cdc25 phosphatase degrading agent based on a quinolinidone skeleton having the structure shown in general formula (I).

[0013]

[0014] In general formula (I):

[0015] X is either C or N;

[0016] Y is either C or N, and there is exactly one X and Y containing N;

[0017] The linker is selected from one of the following groups:

[0018]

[0019] The R group represents the E3 ubiquitin ligase ligand moiety and is selected from one of the following groups:

[0020]

[0021] According to a preferred embodiment of the present invention, the compounds are compounds A1-A18 and B1-B16 having specific structural formulas, the structures of which are shown below:

[0022]

[0023] II. Preparation method of Cdc25 phosphatase degrading agent based on quinolinidone skeleton

[0024] A method for preparing a Cdc25 phosphatase degrading agent based on a quinolinidone skeleton, the synthetic route is as follows:

[0025] (1) Synthesis of intermediate 2a-2i, the synthetic route is as follows:

[0026]

[0027] Reagents and conditions: (i) H2N–(CH2) with different structures n –COOH, n=2-7,9,10 or NH2-PEG2-COOH, DMSO, DIEA, 110°C, 6h;

[0028] Where R1 is , ;

[0029] (2) The synthetic route for the target compounds A1-A9 and B1-B9 is as follows:

[0030]

[0031] Reagents and conditions: (i) 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester, DIEA, CH2Cl2, 6 h; (ii) 4 mol hydrochloric acid, 1,4-dioxane solution, 4 h; (iii) intermediate 2a-2i, EDCI, HOBt, DIEA, CH2Cl2, 12 h.

[0032] Where L-1 is , ;

[0033] (3) The synthesis route of intermediate 18a-18g is as follows:

[0034]

[0035] Reagents and conditions: (i) 4-methylthiazole, [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium(II) chloride, potassium acetate, N,N-dimethylacetamide, 90°C, 18h; (ii) 4 mol hydrochloric acid, ethyl acetate, rt, 4h; (iii) HATU, DIEA, N,N-dimethylformamide, rt, 12h; (iv) LiOH, MeOH: THF: H2O = 5: 4: 3 (volume ratio), rt.

[0036] Where R2 is , R3 is , ;

[0037] (4) The synthetic route for the target compounds B1-B16 is as follows:

[0038]

[0039] Reagents and conditions: (i) Intermediate 18a-18h, triethylamine, CH2Cl2, rt, 6 h;

[0040] Among them, L-2 is , .

[0041] III. Application of Cdc25 phosphatase degraders based on quinolinidone skeleton

[0042] This invention provides the use of the above-mentioned Cdc25 phosphatase degrading agent based on a quinolinedione skeleton in the preparation of antitumor drugs. Experiments have demonstrated that the Cdc25 phosphatase degrading agent based on a quinolinedione skeleton of this invention can be used as an antitumor drug targeting Cdc25.

[0043] As shown in Table 1, 5-fluorouracil (5-FU), compound NSC663285, and lead compound NSC663284 were selected as positive controls. The in vitro antitumor activity of the synthesized target compounds A1-A18 and B1-B16 was evaluated, and the in vitro cytotoxicity of the drugs was determined by the CCK-8 assay.

[0044] As shown in Table 2, 5-FU, compound NSC663285, and lead compound NSC663284 were selected as positive controls. The in vitro antitumor activity of the synthesized target compounds A2, A4, A7, A8, B6, and B14 was evaluated, and the in vitro cytotoxicity of the drugs was determined by the CCK-8 assay.

[0045] As shown in Table 3, the in vitro selectivity index (SI; SI = CC) was used to evaluate the synthesized target compounds A2, A4, A7, A8, B6, and B14. 50 / IC 50 )evaluate.

[0046] like Figure 1 As shown, the in vitro antitumor mechanism of the representative compound B6 was evaluated.

[0047] This invention discloses a Cdc25 phosphatase degrading agent based on a quinolinedione skeleton, its preparation method, antitumor activity test results, and its first application as a Cdc25 phosphatase degrading agent. Experiments have shown that the Cdc25 phosphatase degrading agent based on a quinolinedione skeleton can be used as an antitumor drug targeting Cdc25. Attached Figure Description

[0048] Figure 1 Research on the mechanism by which compound B6 induces the degradation of the target protein Cdc25.

[0049] The study included: (a) time-dependent degradation; (b) concentration-dependent degradation and its effect on downstream cyclins; (c) determination of degradation efficiency (DC50); (d) validation of concentration-dependent degradation; and (e, f) the effects of lysosomal inhibitor (CQ) and proteasome inhibitor (MG-132) treatments, as well as control compounds (NSC663285, NSC663284), on B6 degradation. Internal control: GAPDH.

[0050] The technical solution of the present invention will be further described in detail below through embodiments. Detailed Implementation

[0051] The technical solution of the present invention will be described below through embodiments. To make the objectives, technical solutions, and advantages of this application clearer, the present invention will be described clearly and completely below in conjunction with embodiments. The following description is intended to further elaborate on the content of the present invention; unless otherwise specified, the meanings of the technical terms used are consistent with the common understanding of those skilled in the art.

[0052] The compounds of this invention can be synthesized using commercially available reagents according to the methods described below, or prepared using conventional methods and other reagents in the art. Compounds of formulas A1-A18 or B1-B16 can be prepared by a variety of methods, including standard chemical synthetic routes. Representative general synthetic strategies are listed below, and the relevant compounds can be obtained using known organic synthetic techniques. When referring to the examples below, those skilled in the art can combine conventional group substitutions to obtain similar derivatives without departing from the spirit of the invention. For sensitive or reactive groups, protecting groups can be used according to conventional chemical principles, and their operation and deprotection follow standard organic synthetic methods (TW Greene & PGM Wuts, Protective Groups in Organic Synthesis, 3rd ed., Wiley, 1999), and removed at appropriate stages in a manner well known in the art. The selection of synthetic methods, reaction conditions, and the sequence of steps should be adapted to the preparation of the target compound.

[0053] Those skilled in the art can identify the stereocenters that may be present in compounds of formulas A1-A18 or B1-B16. Therefore, this invention covers all possible stereoisomers, including racemic mixtures and single enantiomers. If the target compound is a single enantiomer, it can be obtained through stereospecific synthesis or by resolving the final product or intermediate. The resolving of the starting materials, intermediates, or final product can be performed using methods known in the art (see EL Eliel, SH Wilen & LN Mander, Stereochemistry of Organic Compounds, Wiley-Interscience, 1994).

[0054] Example 1. Preparation of the compound

[0055] A method for synthesizing Cdc25 phosphatase PROTAC degrading agents based on a quinolinidone skeleton includes:

[0056] (1) Synthesis of intermediate 2a-2i, the synthetic route is as follows:

[0057]

[0058] 1.0 eq. of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione 1a was added to 12 mL of DMSO, followed by DIEA (4.0 eq.) and H2N–(CH2) of different structures. n –COOH or NH2-PEG2-COOH (1.2 eq.), heated at 110 ℃ and stirred for 6 h. After the reaction was completed by TLC monitoring, an equal volume of 1 mol / L hydrochloric acid was added, and the mixture was extracted with ethyl acetate (15 mL × 4). The upper organic phases were combined and collected, and then washed with saturated sodium chloride solution (15 mL × 3). The mixture was dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation under reduced pressure. The crude product was mixed with 100-200 mesh silica gel and separated by column chromatography to obtain intermediates 2a-2i.

[0059] (2) The synthetic route for the target compounds A1-A9 and B1-B9 is as follows:

[0060]

[0061] Intermediate 3a (0.83 g, 3.64 mmol, 1.0 eq.) and DIEA (1.00 g, 7.25 mmol, 2.00 eq.) were added to 20 mL of dichloromethane, followed by tert-butyl 4-(2-aminoethyl)piperazine-1-carboxylate (1.00 g, 4.36 mmol, 1.2 eq.). The reaction was carried out at room temperature for 6 h. After the reaction was monitored by TLC, water was added to quench the reaction, and the mixture was extracted with dichloromethane (15 mL × 3). The lower organic phases were combined and collected. The organic phase was then washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and dichloromethane was removed by rotary evaporation under reduced pressure. The crude product was then separated by column chromatography (dichloromethane → methanol:dichloromethane = 1:100 → 1:50) with silica gel mixed with the solution to obtain the crude product. Intermediates 4a and 5a were obtained by preparative thin-layer chromatography (methanol: dichloromethane = 1:100 → 1:60) or by preparative HPLC (water: acetonitrile = 70:30 → water: acetonitrile = 10:90, 20 min).

[0062] Intermediate 4a or 5a (0.5 g, 1.19 mmol) was added to a 25 mL round-bottom flask, followed by 6 mL of a 4 M dioxane hydrochloride solution. The mixture was reacted at room temperature for 4 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure. The pH of the reaction solution was adjusted to approximately 7 using saturated sodium bicarbonate solution. Then, n-butanol (10 mL × 3) was added for extraction. The upper organic phases were combined, the n-butanol was removed by rotary evaporation, and the mixture was dissolved in tetrahydrofuran. The mixture was filtered, and the filtrate was removed by rotary evaporation under reduced pressure to obtain intermediate 6a or 7a.

[0063] Intermediate 2a-2i (1.1 eq.) was dissolved in 15 mL of dichloromethane, and EDCI (1.5 eq.) and HOBt (0.15 eq.) were added. The mixture was activated in an ice bath for 30 min. After removing the ice bath, DIEA (0.5 eq.) and intermediate 6a or 7a (1.0 eq.) were added, and the mixture was reacted at room temperature for 12 h. After the reaction was completed as monitored by TLC, the reaction was quenched with 15 mL of saturated sodium bicarbonate solution, and the mixture was extracted with dichloromethane (15 mL × 3). The organic phases were combined and collected. The organic phases were then washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and dichloromethane was removed by rotary evaporation under reduced pressure. The crude product was mixed with 100-200 mesh silica gel and separated into target compounds A1-A18 by column chromatography.

[0064] The spectral data of compounds A1-A18 are as follows:

[0065] A1: Reddish-brown solid, yield 46%, Mp: 134-136 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.89 (dd, J = 4.6, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.9,1.7 Hz, 1H, Ph-H), 7.79 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.59 (dd, J = 8.6,7.1 Hz, 1H, Ph-H), 7.48 (s, 1H, NH), 7.14 (d, J = 8.6 Hz, 1H, Ph-H), 7.03 (d,J = 7.0 Hz, 1H, Ph-H), 6.76 (t, J = 6.3 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4Hz, 1H, CH), 3.85 (d, J = 6.3 Hz, 2H, CH2), 3.52 (d, J = 6.1 Hz, 6H, CH2×3), 2.91 – 2.83 (m, 1H, CH), 2.63 (t, J = 6.4 Hz, 3H, CH2, CH), 2.56 (s, 3H,CH2, CH), 2.38 (s, 2H, CH2), 2.35 (s, 2H, CH2), 2.06 – 1.98 (m, 1H, CH). ESI-MS: m / z 648.32 (M + H) + . C 31 H 30 ClN7O7[647.19].

[0066] A2: Reddish-brown solid, yield 25%, Mp: 112-114 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.95 (dd, J = 4.7, 1.8 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.59 (dd, J = 8.4,7.2 Hz, 1H, Ph-H), 7.37 (s, 1H, NH), 7.14 (d, J = 8.6 Hz, 1H, Ph-H), 7.03 (d,J = 7.0 Hz, 1H, Ph-H), 6.76 (t, J = 6.3 Hz, 1H, NH), 5.05 (dd, J = 12.7, 5.4Hz, 1H, CH), 3.85 (d, J = 6.3 Hz, 2H, CH2), 3.53 (d, J = 6.2 Hz, 6H, CH2×3), 2.92 – 2.83 (m, 1H, CH), 2.65 (q, J = 7.3 Hz, 3H, CH × 3), 2.60 – 2.54 (m, 3H, CH × 3), 2.41 (s, 2H, CH2), 2.35 (d, J = 13.9 Hz, 2H, CH2), 2.07 –1.98 (m, 1H, CH). ESI-MS: m / z 648.13 (M + H) + . C 31 H 30 ClN7O7[647.19].

[0067] A3: Reddish-brown solid, yield 40%, Mp: 180-182 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.90 (dd, J = 4.6, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz,1H, Ph-H), 7.48 (s, 1H, NH), 7.16 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d, J = 7.0Hz, 1H, Ph-H), 6.67 (t, J = 6.2 Hz, 1H, NH), 5.06 (dd, J = 12.9, 5.4 Hz, 1H,CH), 3.85 (d, J = 7.3 Hz, 2H, CH2), 3.35 – 3.24 (m, 6H, CH2× 3), 2.95 – 2.82(m, 1H, CH), 2.59 (d, J = 18.0 Hz, 4H, CH2× 2), 2.37 (t, J = 7.9 Hz, 6H,CH2× 3), 2.03 (d, J = 13.1 Hz, 1H, CH), 1.80 – 1.73 (m, 2H, CH2). 13 C NMR(100 MHz, DMSO-d6) δ 178.62, 173.33, 170.61, 170.57, 169.28, 167.79, 153.37,146.84, 136.71, 134.21, 132.70, 128.86, 117.73, 110.85, 109.52, 55.40, 52.98,52.56, 48.98, 45.18, 42.00, 41.45, 31.45, 29.80, 24.68, 22.63. ESI-MS: m / z666.22 (M + H) + . C 32 H 32 ClN7O7[661.21].

[0068] A4: Reddish-brown solid, yield 21%, Mp: 150-152 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.95 (dd, J = 4.6, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.7,1.7 Hz, 1H, Ph-H), 7.75 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz,1H, Ph-H), 7.36 (s, 1H, NH), 7.17 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d, J = 7.0Hz, 1H, Ph-H), 6.67 (t, J = 6.1 Hz, 1H), 5.06 (dd, J = 12.8, 5.4 Hz, 1H, CH), 3.85 (d, J = 6.4 Hz, 2H, CH2), 3.41 (s, 2H, CH2), 3.31 (d, J = 6.6 Hz, 4H, CH2× 2), 2.95 – 2.84 (m, 1H, CH), 2.66 – 2.53 (m, 4H, CH2× 2), 2.43 – 2.35 (m, 6H, CH2× 3), 2.04 (dd, J = 12.9, 6.7 Hz, 1H, CH), 1.78 (t, J = 7.3 Hz, 2H, CH2). 13 C NMR (100 MHz, DMSO-d6) δ 180.36, 173.34, 170.60, 169.28, 167.79,154.97, 148.16, 146.84, 136.71, 134.90, 132.70, 127.37, 117.73, 110.85,109.52, 57.42, 55.40, 53.02, 52.56, 48.98, 45.32, 42.01, 41.57, 31.45, 29.81,24.68, 22.63. ESI-MS: m / z 666.10 (M + H) + . C 32 H 32 ClN7O7[661.21].

[0069] A5: Reddish-brown solid, yield 47%, Mp: 142-144 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.8, 4.6 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz,1H, Ph-H), 7.47 (s, 1H, NH), 7.10 (d, J = 8.6 Hz, 1H, CH), 7.02 (d, J = 7.0Hz, 1H, CH), 6.57 (t, J = 6.0 Hz, 1H, NH), 5.06 (dd, J = 12.9, 5.4 Hz, 1H,CH), 3.85 (d, J = 6.1 Hz, 2H, CH2), 3.31 (d, J = 7.2 Hz, 4H, CH2× 2), 3.17(d, J = 4.1 Hz, 2H, CH2), 2.88 (t, J = 8.8 Hz, 1H, CH), 2.58 (t, J = 10.7 Hz, 4H, CH2× 2), 2.42 – 2.30 (m, 6H, CH2× 3), 2.03 (d, J = 13.1 Hz, 1H, CH), 1.55 (d, J = 9.6 Hz, 6H, CH2× 3). 13 C NMR (100 MHz, DMSO-d6) δ 178.62,173.32, 170.83, 170.60, 167.78, 153.36, 146.85, 136.73, 134.21, 132.66,128.85, 117.72, 110.85, 109.45, 53.10, 52.61, 49.07, 48.99, 45.25, 42.59,41.99, 41.35, 36.93, 32.29, 31.44, 28.72, 28.50, 22.63, 22.52. ESI-MS: m / z 676.25 (M + H) + . C 33 H 34 ClN7O7[675.22].

[0070] A6: Reddish-brown solid, yield 30%, Mp: 121-123 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (dd, J = 8.5,7.1 Hz, 1H, Ph-H), 7.47 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d,J = 7.0 Hz, 1H, Ph-H), 6.54 (t, J = 5.9 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4Hz, 1H, CH), 3.85 (q, J = 6.2 Hz, 2H, CH2), 3.32 – 3.17 (m, 6H, CH2× 3), 3.00 – 2.83 (m, 1H, CH), 2.66 – 2.54 (m, 4H, CH2), 2.42 – 2.31 (m, 6H, CH2×3), 2.07 – 1.98 (m, 1H, CH), 1.65 – 1.50 (m, 4H, CH2× 2). 13 C NMR (100 MHz, DMSO-d6) δ 178.62, 173.33, 170.93, 170.61, 169.40, 167.78, 153.36, 146.87,136.77, 134.21, 132.65, 128.86, 117.66, 110.85, 109.41, 53.11, 52.61, 48.99,45.27, 42.20, 41.33, 32.56, 31.44, 28.99, 26.52, 24.99, 22.62. ESI-MS: m / z676.13 (M + H) + . C 33 H 34 ClN7O7[675.22].

[0071] A7: Reddish-brown solid, yield 50%, Mp: 165-167 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (dd, J = 8.5,7.1 Hz, 1H, Ph-H), 7.47 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d,J = 7.0 Hz, 1H, Ph-H), 6.54 (t, J = 5.9 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4Hz, 1H, CH), 3.85 (q, J = 6.2 Hz, 2H, CH2), 3.35 – 3.23 (m, 6H, CH2× 3),2.93 – 2.83 (m, 1H, CH), 2.66 – 2.53 (m, 4H, CH2× 2), 2.40 (d, J = 4.6 Hz,2H, CH2), 2.34 (d, J = 4.7 Hz, 2H, CH2), 2.28 (t, J = 7.4 Hz, 2H, CH2), 2.06 –2.00 (m, 1H, CH), 1.58 (t, J = 7.4 Hz, 2H, CH2), 1.50 (q, J = 7.5 Hz, 2H,CH2), 1.38 – 1.31 (m, 2H, CH2). 13 C NMR (100 MHz, DMSO-d6) δ 178.62, 173.33,170.93, 170.61, 169.40, 167.78, 153.36, 146.87, 136.77, 134.21, 132.65,128.86, 117.66, 110.85, 109.41, 53.11, 52.61, 48.99, 45.27, 42.20, 41.33,32.56, 31.44, 28.99, 26.52, 24.99, 22.62. ESI-MS: m / z 690.24 (M + H) + .C 34 H 36 ClN7O7[689.24].

[0072] A8: Reddish-brown solid, yield 24%, Mp: 136-138 ℃. 1 H NMR (400 MHz, DMSO-d6) δ11.13 (s, 1H, NH), 8.95 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.9,1.8 Hz, 1H, Ph-H), 7.75 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (dd, J = 8.6,7.0 Hz, 1H, Ph-H), 7.39 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d,J = 7.0 Hz, 1H, Ph-H), 6.54 (t, J = 5.9 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4Hz, 1H, CH), 3.85 (d, J = 6.3 Hz, 2H, CH2), 3.29 (q, J = 6.7 Hz, 6H, CH2×3), 2.94 – 2.84 (m, 1H, CH), 2.73 – 2.53 (m, 4H, CH2× 2), 2.41 (s, 2H, CH2), 2.36 (s, 2H, CH2), 2.29 (t, J = 7.4 Hz, 2H, CH2), 2.06 – 2.00 (m, 1H, CH), 1.62 – 1.55 (m, 2H, CH2), 1.51 (t, J = 7.7 Hz, 2H, CH2), 1.35 (d, J = 7.1 Hz, 2H, CH2). 13 C NMR (100 MHz, DMSO-d6) δ 173.34, 170.95, 170.60, 154.97, 146.87,136.78, 132.64, 127.41, 110.84, 53.16, 48.99, 42.20, 41.45, 32.57, 31.44,29.00, 26.52, 24.99. ESI-MS: m / z 690.09 (M + H) + . C 34 H 36 ClN7O7[689.24].

[0073] A9: Reddish-brown solid, yield 41%, M.p.: 155 - 157 °C. 1 H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.9,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.6 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz,1H, Ph-H), 7.48 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d, J = 7.0Hz, 1H, Ph-H), 6.55 (t, J = 6.0 Hz, 1H, NH), 5.06 (dd, J = 12.8, 5.4 Hz, 1H,CH), 3.85 (d, J = 6.4 Hz, 2H, CH2), 3.35 (s, 2H, CH2), 3.29 (q, J = 7.2 Hz,4H, CH2× 2), 2.94 – 2.83 (m, 1H, CH), 2.59 (d, J = 17.1 Hz, 4H, CH2× 2),2.39 (s, 2H, CH2), 2.34 (s, 2H, CH2), 2.26 (t, J = 7.3 Hz, 2H, CH2), 2.07 –1.99 (m, 1H, CH), 1.60 – 1.53 (m, 2H, CH2), 1.50 – 1.43 (m, 2H, CH2), 1.32 (s,4H, CH2× 2). 13 C NMR (100 MHz, DMSO-d6) δ 178.62, 173.33, 170.99, 170.61,169.41, 167.78, 153.36, 146.88, 136.77, 134.21, 132.65, 128.85, 117.66,110.85, 109.43, 55.39, 53.11, 52.61, 48.99, 45.27, 42.24, 41.32, 32.57,31.44, 29.04, 28.92, 26.60, 25.20, 22.62. ESI-MS: m / z 704.28 (M + H) + .C 35 H38 ClN7O7 [703.25].

[0074] A10: Reddish-brown solid, yield 28%, M.p.: 132 - 134 °C. 1 H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.95 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8, 1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (t, 1H, Ph-H), 7.36 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d, J = 7.0 Hz, 1H, Ph-H), 6.54 (t, J = 6.0 Hz, 1H, NH), 5.05 (dd, J = 12.9, 5.4 Hz, 1H, CH), 3.85 (q, J = 6.3 Hz, 2H, CH2), 3.29 (q, J = 6.7 Hz, 2H, CH2), 2.94 – 2.83 (m, 1H, CH), 2.58 (dd, J = 10.7, 4.5 Hz, 4H, CH2× 2), 2.41 (t, J = 4.8 Hz, 2H, CH2), 2.36 (t, J = 5.0 Hz, 2H, CH2), 2.27 (t, J = 7.4 Hz, 2H, CH2), 2.07 – 1.99 (m, 1H, CH), 1.55 (d, J = 7.2 Hz, 2H, CH2), 1.47 (q, J = 7.0 Hz, 2H, CH2), 1.32 (s, 4H, CH2× 2). 13C NMR (100 MHz, DMSO-d6) δ 180.36, 173.33,171.01, 170.61, 169.41, 167.78, 154.97, 146.88, 136.77, 134.91, 132.65,127.37, 117.66, 110.85, 109.43, 57.44, 53.17, 52.60, 48.99, 45.42, 42.24,41.45, 40.40, 32.57, 31.44, 29.04, 28.93, 26.61, 25.20, 22.62. ESI-MS: m / z704.11 (M + H) + . C 35 H 38 ClN7O7[703.25].

[0075] A11: Reddish-brown solid, yield 36%, Mp: 135-137 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.11 (s, 1H, NH), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.9,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (dd, J = 8.6,7.1 Hz, 1H, Ph-H), 7.46 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d,J = 7.0 Hz, 1H, Ph-H), 6.53 (t, J = 5.9 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4Hz, 1H, CH), 3.89 – 3.83 (m, 2H, CH2), 3.29 (q, J = 6.8 Hz, 6H, CH2× 3),2.92 – 2.84 (m, 1H, CH), 2.58 (q, J = 11.6 Hz, 4H, CH2), 2.40 (s, 2H, CH2),2.34 (d, J = 5.6 Hz, 2H, CH2), 2.25 (t, J = 7.4 Hz, 2H, CH2), 2.06 – 2.00 (m,1H, CH), 1.56 (d, J = 7.2 Hz, 2H, CH2), 1.46 (t, J = 7.2 Hz, 2H, CH2), 1.31(s, 6H, CH2× 3). 13 C NMR (100 MHz, DMSO-d6) δ 178.61, 173.32, 171.04, 170.59,169.43, 167.78, 153.36, 146.88, 136.76, 134.20, 132.63, 128.86, 117.65,110.86, 109.43, 57.72, 53.11, 52.61, 49.00, 45.27, 42.27, 41.49, 41.31,32.63, 31.45, 29.16, 29.11, 29.04, 26.70, 25.21, 22.62. ESI-MS: m / z 718.29 (M+ H) + . C 36 H 40 ClN7O7[717.27].

[0076] A12: A reddish-brown solid with a yield of 26%, M.p.: 105 - 107 °C. 1 H NMR (400 MHz, DMSO-d6) δ11.11 (s, 1H, NH), 8.95 (dd, J = 4.8, 1.8 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 7.9, 4.6 Hz, 1H, Ph-H), 7.58 (t, J = 7.9 Hz,1H, Ph-H), 7.36 (s, 1H, NH), 7.09 (d, J = 8.7 Hz, 1H, Ph-H), 7.02 (d, J = 7.3Hz, 1H, Ph-H), 6.53 (s, 1H, NH), 5.05 (dd, J = 12.8, 5.4 Hz, 1H, CH), 3.86(d, J = 6.3 Hz, 2H, CH2), 3.29 (d, J = 6.6 Hz, 6H, CH2× 3), 2.91 (d, J =15.9 Hz, 1H, CH), 2.59 (dd, J = 11.0, 4.9 Hz, 4H, CH2× 2), 2.42 (s, 2H,CH2), 2.37 (d, J = 4.9 Hz, 2H, CH2), 2.30 – 2.25 (m, 2H, CH2), 2.03 (d, J =12.7 Hz, 1H, CH), 1.57 (s, 2H, CH2), 1.47 (s, 2H, CH2), 1.31 (s, 6H, CH2×3). 13 C NMR (100 MHz, DMSO-d6) δ 180.36, 173.33, 171.05, 170.60, 169.42,167.78, 154.97, 146.88, 136.77, 134.91, 132.64, 127.38, 117.67, 110.86,109.43, 57.43, 53.17, 52.60, 49.00, 45.42, 42.27, 41.44, 32.63, 31.45, 29.17,29.10, 29.04, 26.70, 25.21, 22.62. ESI-MS: m / z 718.25 (M + H) + . C36 H 40 ClN7O7 [717.27].

[0077] A13: Reddish-brown solid, yield 35%, M.p.: 153 - 155 °C. 1 H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8, 1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.57 (dd, J = 8.6, 7.0 Hz, 1H, Ph-H), 7.48 (s, 1H, NH), 7.08 (d, J = 8.6 Hz, 1H, Ph-H), 7.01 (d, J = 7.0 Hz, 1H, Ph-H), 6.53 (t, J = 6.0 Hz, 1H, NH), 5.06 (dd, J = 12.9, 5.4 Hz, 1H, CH), 3.85 (q, J = 6.2 Hz, 2H, CH2), 3.35 (s, 4H, CH2×2), 3.27 (t, J = 6.6 Hz, 2H, CH2), 2.94 – 2.84 (m, 1H, CH), 2.59 (d, J = 17.2 Hz, 4H, CH2×2), 2.40 (d, J = 4.9 Hz, 2H, CH2), 2.34 (d, J = 4.9 Hz, 2H, CH2), 2.24 (t, J = 7.4 Hz, 2H, CH2), 2.07 – 2.00 (m, 1H, CH), 1.56 (d, J = 7.2 Hz, 2H, CH2), 1.45 (d, J = 8.6 Hz, 2H, CH2), 1.30 (s, 2H, CH2), 1.28 – 1.20 (m, 8H, CH2×4). 13CNMR (100 MHz, DMSO-d6) δ 178.62, 173.30, 171.02, 170.58, 167.77, 153.35,146.89, 136.75, 134.20, 132.65, 128.85, 117.64, 110.84, 109.45, 57.76, 53.13,52.63, 49.00, 45.30, 42.29, 41.53, 41.32, 32.66, 31.45, 29.37, 29.27, 29.18,29.13, 26.77, 25.27, 22.62. ESI-MS: m / z 746.30 (M + H) + . C 38 H 44 ClN7O7[745.30].

[0078] A14: Reddish-brown solid, yield 25%, Mp: 128-120 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.13 (s, 1H, NH), 8.95 (dd, J = 4.8, 1.7 Hz, 1H, Ph-H), 8.32 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 7.8, 4.7 Hz, 1H, Ph-H), 7.57 (t, J = 8.6,7.0 Hz, 1H, Ph-H), 7.37 (s, 1H, NH), 7.09 (d, J = 8.6 Hz, 1H, Ph-H), 7.01 (d,J = 7.0 Hz, 1H, Ph-H), 6.53 (t, J = 5.9 Hz, 1H, NH), 5.07 – 5.03 (m, 1H, CH),3.85 (d, J = 5.8 Hz, 2H, CH2), 3.28 (q, J = 6.7 Hz, 6H, CH2× 3), 2.91 – 2.85(m, 1H, CH), 2.62 – 2.56 (m, 4H, CH2× 2), 2.42 (d, J = 4.7 Hz, 2H, CH2),2.36 (t, J = 5.1 Hz, 2H, CH2), 2.27 (d, J = 7.4 Hz, 2H, CH2), 2.05 – 2.01 (m,1H, CH), 1.55 (d, J = 7.0 Hz, 2H, CH2), 1.45 (d, J = 7.2 Hz, 2H, CH2), 1.26 –1.22 (m, 8H, CH2× 4). 13 C NMR (100 MHz, DMSO-d6) δ 180.35, 173.33, 171.09,170.59, 169.42, 167.78, 154.96, 146.88, 136.77, 134.91, 132.63, 127.37,117.65, 110.85, 109.42, 57.43, 53.17, 52.60, 48.99, 45.44, 42.28, 41.45,32.66, 31.44, 29.36, 29.25, 29.18, 29.12, 26.75, 25.26, 22.62. ESI-MS: m / z746.19 (M + H) + . C 38 H 44ClN7O7[745.30].

[0079] A15: Reddish-brown solid, yield 38%, Mp: 182-184 ℃. 1 H NMR (400 MHz, DMSO-d6) δ11.10 (s, 1H, NH), 8.90 (dd, J = 4.6, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz,1H, Ph-H), 7.46 (s, 1H, NH), 7.08 (d, J = 8.6 Hz, 1H, Ph-H), 7.02 (d, J = 7.0Hz, 1H, Ph-H), 6.51 (t, J = 5.8 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4 Hz, 1H,CH), 3.86 (q, J = 6.2 Hz, 2H, CH2), 3.28 (q, J = 6.9 Hz, 4H, CH2× 2), 2.93 –2.85 (m, 1H, CH), 2.60 (d, J = 16.7 Hz, 4H, CH2× 2), 2.40 (s, 2H, CH2), 2.38– 2.32 (m, 2H, CH2), 2.24 (t, J = 7.5 Hz, 2H, CH2), 2.07 – 2.00 (m, 1H, CH), 1.56 (d, J = 7.5 Hz, 2H, CH2), 1.44 (s, 2H, CH2), 1.31 (s, 2H, CH2), 1.28 –1.20 (m, 10H, CH2× 5). 13C NMR (100 MHz, DMSO-d6) δ 178.61, 173.33, 171.06,170.60, 169.41, 167.78, 153.36, 146.87, 136.76, 134.21, 132.63, 128.86,117.65, 110.85, 109.41, 53.12, 52.62, 48.99, 45.29, 42.28, 40.54, 40.17,32.66, 31.44, 29.41, 29.36, 29.30, 29.20, 29.12, 26.78, 25.27, 22.62. ESI-MS: m / z 760.30 (M + H) + . C 39 H 46 ClN7O7[759.31].

[0080] A16: Reddish-brown solid, yield 25%, Mp: 158-160 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.12 (s, 1H, NH), 8.95 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.33 (dd, J = 7.8,1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.57 (dd, J = 8.5,7.1 Hz, 1H, Ph-H), 7.36 (s, 1H, NH), 7.08 (d, J = 8.6 Hz, 1H, Ph-H), 7.01 (d,J = 7.0 Hz, 1H, Ph-H), 6.53 (t, J = 5.9 Hz, 1H, NH), 5.05 (dd, J = 12.8, 5.4Hz, 1H, CH), 3.85 (q, J = 6.2 Hz, 2H, CH2), 3.37 (s, 4H, CH2× 2), 3.28 (d, J= 6.7 Hz, 2H, CH2), 2.93 – 2.84 (m, 1H, CH), 2.59 (dd, J = 11.3, 5.1 Hz, 4H,CH2× 2), 2.42 (t, J = 4.9 Hz, 2H, CH2), 2.36 (t, J = 5.0 Hz, 2H, CH2), 2.26(t, J = 7.5 Hz, 2H, CH2), 2.06 – 2.00 (m, 1H, CH), 1.60 – 1.52 (m, 2H, CH2),1.45 (s, 2H, CH2), 1.33 – 1.29 (m, 2H, CH2), 1.28 – 1.20 (m, 10H, CH2 × 5). 13C NMR (100 MHz, DMSO) δ 180.36, 173.32, 171.05, 170.59, 169.42, 167.78,154.97, 146.88, 136.76, 134.90, 132.64, 127.37, 117.65, 110.85, 109.43,57.44, 53.17, 52.62, 48.99, 45.43, 42.28, 41.44, 32.66, 31.45, 29.42, 29.37,29.31, 29.21, 29.12, 26.78, 25.27, 22.62. ESI-MS: m / z 760.15 (M + H) + .C 39 H 46 ClN7O7[759.31].

[0081] A17: Reddish-brown solid, yield 47%, Mp: 143-145 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.11 (s, 1H, NH), 8.90 (dd, J = 4.6, 1.8 Hz, 1H, Ph-H), 8.32 (dd, J = 7.8,1.8 Hz, 1H, Ph-H), 7.79 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (t, J = 7.8 Hz,1H, Ph-H), 7.45 (s, 1H, NH), 7.13 (d, J = 8.6 Hz, 1H, Ph-H), 7.04 (d, J = 7.0Hz, 1H, Ph-H), 6.57 (t, J = 5.8 Hz, 1H, NH), 5.06 (dd, J = 13.0, 5.3 Hz, 1H,CH), 3.84 (q, J = 6.1 Hz, 2H, CH2), 3.66 (t, J = 6.5 Hz, 2H, CH2), 3.59 (t, J= 5.4 Hz, 2H, CH2), 3.46 (t, J = 5.4 Hz, 2H, CH2), 3.37 (s, 4H, CH2× 2), 2.93 – 2.84 (m, 1H, CH), 2.60 – 2.52 (m, 6H, CH2× 3), 2.39 (d, J = 5.7 Hz, 2H, CH2), 2.37 – 2.30 (m, 2H, CH2), 2.10 – 2.01 (m, 1H, CH). 13 C NMR (100 MHz, DMSO-d6) δ 178.62, 173.31, 170.57, 169.39, 169.04, 167.75, 153.37, 146.85,136.73, 134.21, 132.55, 128.85, 117.90, 111.16, 109.68, 69.10, 67.14, 53.02,52.54, 49.01, 45.34, 42.11, 41.36, 33.13, 31.45, 22.62. ESI-MS: m / z 692.28 (M+ H) + . C 33 H 34 ClN7O8[691.22].

[0082] A18: Reddish-brown solid, yield 33%, Mp: 127-129 ℃. 1H NMR (400 MHz, DMSO-d6) δ11.11 (s, 1H, NH), 8.95 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.32 (dd, J = 7.9,1.7 Hz, 1H, Ph-H), 7.74 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.58 (dd, J = 8.6,7.1 Hz, 1H, Ph-H), 7.33 (s, 1H, NH), 7.13 (d, J = 8.6 Hz, 1H, Ph-H), 7.04 (d,J = 7.0 Hz, 1H, Ph-H), 6.58 (t, J = 5.8 Hz, 1H, NH), 5.05 (dd, J = 12.9, 5.4Hz, 1H, CH), 3.84 (q, J = 6.2 Hz, 2H, CH2), 3.66 (t, J = 6.5 Hz, 2H, CH2),3.59 (t, J = 5.4 Hz, 2H, CH2), 3.46 (dd, J = 7.3, 3.6 Hz, 2H, CH2), 3.42 –3.38 (m, 4H, CH2× 2), 2.93 – 2.84 (m, 1H, CH), 2.56 (t, J = 6.5 Hz, 6H, CH2× 3), 2.41 (d, J = 4.6 Hz, 2H, CH2), 2.37 (d, J = 5.7 Hz, 2H, CH2), 2.10 –2.00 (m, 1H, CH). 13 C NMR (100 MHz, DMSO-d6) δ 180.36, 173.31, 170.56, 169.40,169.09, 167.75, 154.98, 146.85, 136.73, 134.91, 132.55, 127.37, 117.91,111.17, 109.69, 69.11, 67.15, 57.39, 53.04, 52.54, 49.01, 45.48, 42.12,41.48, 33.15, 31.45, 22.63. ESI-MS: m / z 692.25 (M + H) + . C 33 H 34 ClN7O8[691.22].

[0083] (3) The synthesis route of intermediate 18a-18g is as follows:

[0084]

[0085] [(S)-1-(4-bromophenyl)ethyl] tert-butyl carbamate 8a (1.0 g, 3.33 mmol, 1.0 eq.), 4-methylthiazole (0.67 g, 6.76 mmol, 2.0 eq.), and potassium acetate (0.65 g, 6.62 mmol, 2.0 eq.) were added to 20 mL of N,N-dimethylacetamide (DMA), followed by palladium acetate (0.08 g, 0.36 mmol, 0.1 eq.). The reaction was carried out under N2 protection at 90 °C for 18 h. After the reaction was completed under TLC monitoring, the mixture was allowed to return to room temperature. The mixture was filtered through diatomaceous earth and extracted with ethyl acetate (15 mL × 3). The upper organic phases were combined and collected, then washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to remove ethyl acetate. The crude product was then separated by column chromatography (ethyl acetate: petroleum ether = 1:4) with 100-200 mesh silica gel to obtain intermediate 9a. 9a: white solid, yield 32%, MP: 120-122 ℃. 1 H NMR(400 MHz, DMSO-d6) δ 8.95 (s, 1H, thiazole-H), 7.41 (d, J = 8.2 Hz, 3H, Ph-H×2, NH), 7.35 (d, J = 8.1 Hz, 2H, Ph-H×2), 4.62 (t, J = 7.6 Hz, 1H, CH), 2.42 (s, 3H, CH3), 1.34 (s, 9H, CH3× 3), 1.29 (d, J = 7.0 Hz, 3H, CH3). ESI-MS: m / z 319.17 (M + H) + . C 17 H 22 N2O2S [318.14].

[0086] Intermediate 9a (0.5 g, 1.57 mmol) was added to a 25 mL round-bottom flask, followed by 6 mL of ethyl acetate solution containing 4 M hydrochloric acid. The reaction was carried out at room temperature for 3 h. After the reaction was completed as monitored by TLC, the solvent was removed by rotary evaporation under reduced pressure to give a yellow solid 10a. 10a: yellow solid, yield 42%, Mp: 135-137 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.98 (s, 1H,thiazole-H), 7.47 (d, J = 8.3 Hz, 2H, Ph-H × 2), 7.42 (d, J = 2.2 Hz, 2H,Ph-H × 2), 7.41 – 7.34 (m, 2H, NH2), 4.02 (d, J = 6.6 Hz, 1H, CH), 2.46 (s,3H, CH3), 1.27 (s, 3H, CH3). ESI-MS: m / z 218.92 (M + H) + . C 12 H 14 N2S [218.09].

[0087] N-Boc-L-tert-leucine 11a (1.5 g, 6.49 mmol, 1.0 eq.) was added to 15 mL of DMF, followed by HATU (3.7 g, 9.73 mmol, 1.5 eq.), and the mixture was activated in an ice bath for 30 min. The ice bath was removed, and DIEA (3.4 g, 26.3 mmol, 4.0 eq.) and trans-4-hydroxy-L-proline methyl ester hydrochloride 12a (1.41 g, 7.76 mmol, 1.2 eq.) were added. The mixture was reacted overnight at room temperature. After the reaction was complete as monitored by TLC, the reaction was quenched with 15 mL of saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate (15 mL × 3). The organic phases were combined and collected. The organic phase was then washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and the ethyl acetate was removed by rotary evaporation under reduced pressure to obtain crude product 13a. 13a (1.0 eq.) was then dissolved in 24 mL of a mixed solvent (MeOH:THF:H2O = 5:4:3), and lithium hydroxide (3.0 eq.) was added. The mixture was stirred at room temperature for 8 h. After the reaction was complete, the solvent was removed by rotary evaporation under reduced pressure, and the pH was adjusted to 2-3 with 1 M hydrochloric acid, precipitating a white solid, 14a. 14a: white solid, yield 34%, Mp: 112-114 ℃. 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H, COOH), 6.49 (d, J= 9.4 Hz, 1H, NH), 5.20 (s, 1H, OH), 4.33 (s, 1H, CH), 4.27 (t, J = 8.3 Hz,1H, CH), 4.16 (d, J = 9.4 Hz, 1H, CH), 3.66 (d, J = 10.7 Hz, 1H, CH), 3.59 (d, J = 10.6 Hz, 1H, CH), 2.14 – 2.08 (m, 1H, CH), 1.89 (td, J = 8.9, 4.5 Hz,1H, CH), 1.38 (s, 9H, CH3× 3), 0.94 (s, 9H, CH3× 3). ESI-MS: m / z 344.92 (M+ H) + . C 16 H 28 N2O6[344.19].

[0088] Intermediate 14a (0.55 g, 1.60 mmol, 1.2 eq.) and HATU (0.91 g, 2.39 mmol, 1.5 eq.) were added to 15 mL of DMF and activated in an ice bath for 30 min. The ice bath was removed, and DIEA (0.82 g, 6.34 mmol, 4.0 eq.) and intermediate 10a (0.41 g, 1.61 mmol, 1.0 eq.) were added. The reaction was allowed to proceed overnight at room temperature. After the reaction was complete as monitored by TLC, the reaction was quenched with 15 mL of saturated sodium bicarbonate solution, and extracted with ethyl acetate (15 mL × 3). The upper organic phases were combined and collected. The organic phase was washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and ethyl acetate was removed by rotary evaporation under reduced pressure. The crude product was mixed with 100-200 mesh silica gel and separated by column chromatography (methanol:dichloromethane = 1:60) to obtain intermediate 15a. 15a: White solid, yield 56%, Mp: 127-129 ℃. 1H NMR (400 MHz, DMSO-d6) δ 8.99 (s, 1H,thiazole-H), 8.43 (d, J = 7.7 Hz, 1H, NH), 7.44 (d, J = 8.3 Hz, 2H, Ph-H ×2), 7.37 (d, J = 8.3 Hz, 2H, Ph-H × 2), 6.45 (d, J = 9.3 Hz, 1H, NH), 5.14(d, J = 3.5 Hz, 1H, OH), 4.90 (t, J = 7.2 Hz, 1H, CH), 4.45 (t, J = 8.2 Hz,1H, CH), 4.28 (s, 1H, CH), 4.14 (d, J = 9.2 Hz, 1H, CH), 3.58 (d, J = 4.5 Hz,2H, CH2), 2.46 (s, 3H, CH3), 2.06 – 2.00 (m, 1H, CH), 1.82 – 1.72 (m, 1H, CH),1.38 (d, J = 3.0 Hz, 12H, CH3× 4), 0.93 (s, 9H, CH3× 3). ESI-MS: m / z544.95 (M + H) + , 567.22 (M + Na) + . C 28 H 40 N4O5S [544.27].

[0089] Intermediate 15a (1 g, 1.84 mmol) was added to a 25 mL round-bottom flask, followed by 6 mL of ethyl acetate solution containing 4 M hydrochloric acid. The reaction was carried out at room temperature for 3 h. After the reaction was completed by TLC monitoring, the solvent was removed by rotary evaporation under reduced pressure to obtain a colorless oily substance 16a. Different alkane carboxylic acid derivatives containing tert-butyloxycarbonyl groups (1.2 eq.) were dissolved in 15 mL DMF, and HATU (1.5 eq.) was added. The mixture was activated in an ice bath for 30 min. The ice bath was removed, and DIEA (3.0 eq.) and intermediate 16a (1.0 eq.) were added. The reaction was carried out at room temperature overnight. After the reaction was completed by TLC monitoring, the reaction was quenched with 15 mL of saturated sodium bicarbonate solution, and the mixture was extracted with ethyl acetate (15 mL × 3). The organic phases were combined and collected. The organic phase was washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and ethyl acetate was removed by rotary evaporation under reduced pressure. The crude product was mixed with 100-200 mesh silica gel and separated into target compounds 17a-17h by column chromatography. The Boc group was removed in ethyl acetate solution of 4 M hydrochloric acid and the intermediate 18a-18h was obtained by the operation described above.

[0090] 18a: Yellow-green oily substance, yield 33%, Mp: 139-141 ℃. 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.44 (d, J = 7.7 Hz, 1H, NH), 7.90 (d, J = 9.1 Hz,1H, NH), 7.44 (d, J = 8.4 Hz, 2H, CH2), 7.38 (d, J = 8.3 Hz, 2H, CH2), 6.71(t, J = 5.6 Hz, 1H, NH), 5.18 (d, J = 3.6 Hz, 1H, OH), 4.95 – 4.88 (m, 1H,CH), 4.51 (d, J = 9.3 Hz, 1H, CH), 4.44 (t, J = 8.1 Hz, 1H, CH), 4.28 (t, J =3.6 Hz, 1H, CH), 3.59 (d, J = 3.3 Hz, 2H, CH2), 3.11 (d, J = 3.9 Hz, 2H, CH2),2.46 (s, 3H, CH3), 2.42 – 2.37 (m, 1H, CH), 2.33 – 2.28 (m, 1H, CH), 2.03(td, J = 8.4, 4.0 Hz, 1H, CH), 1.79 (ddd, J = 12.9, 8.5, 4.6 Hz, 1H, CH),1.39 (s, 3H, CH3), 1.37 (s, 9H, CH3× 3), 0.94 (s, 9H, CH3× 3). 13 C NMR (100MHz, DMSO-d6) δ 171.10, 170.74, 169.89, 155.84, 151.98, 148.20, 145.17,131.59, 130.13, 129.28, 126.85, 78.02, 69.22, 59.02, 56.92, 56.73, 53.72,48.18, 42.01, 38.19, 37.30, 36.35, 35.70, 28.70, 26.90, 22.93, 18.41, 17.15,16.46, 12.65. ESI-MS: m / z 616.42 (M + H) + . C 31 H 45 N5O6S [615.31].

[0091] 18b: Yellow-green oily substance, yield 66%, Mp: 164-166 ℃. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.43 (d, J = 7.9 Hz, 1H, NH), 7.87 (d, J = 9.3 Hz,1H, NH), 7.44 (d, J = 8.4 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.3 Hz, 2H, Ph-H× 2), 6.83 (t, J = 5.7 Hz, 1H, NH), 5.15 (d, J = 3.5 Hz, 1H, OH), 4.91 (q, J= 7.1 Hz, 1H, CH), 4.51 (d, J = 9.3 Hz, 1H, CH), 4.43 (t, J = 8.1 Hz, 1H,CH), 4.28 (s, 1H, CH), 3.61 (d, J = 3.9 Hz, 2H, CH2), 3.13 (dd, J = 7.5, 3.8Hz, 2H, CH2), 2.46 (s, 3H, CH3), 2.26 – 2.19 (m, 1H, CH), 2.14 – 2.08 (m, 1H,CH), 1.58 (q, J = 6.7 Hz, 2H, CH2), 1.42 – 1.35 (m, 14H, CH3× 3, CH2 × 2), 0.94 (s, 9H, CH3× 3). ESI-MS: m / z 630.29 (M + H) + . C 32 H 47 N5O6S [629.32].

[0092] (4) The synthetic route for the target compounds B1-B16 is as follows:

[0093]

[0094] Intermediate 3a (1.0 eq.) and triethylamine (1.0 eq.) were added to 20 mL of dichloromethane, followed by intermediate 18a for 18 h. The reaction was allowed to proceed at room temperature for 6 h. After the reaction was complete as monitored by TLC, the reaction was quenched with water, extracted with dichloromethane (15 mL × 3), and the organic phases were combined and collected. The organic phase was then washed with saturated sodium chloride solution (15 mL × 3), dried over anhydrous magnesium sulfate, filtered, and dichloromethane was removed by rotary evaporation under reduced pressure. The crude product was then separated by column chromatography (dichloromethane → methanol:dichloromethane = 1:100 → 1:50) with silica gel mixed with the solution. The crude product was then purified by preparative thin-layer chromatography or preparative HPLC to obtain the target compounds B1 to B16.

[0095] B1: Reddish-brown solid, yield 31%, Mp: 165-167 ℃. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.91 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.41 (d, J =7.9 Hz, 1H, NH), 8.33 (dd, J = 7.9, 1.7 Hz, 1H, Ph-H), 8.11 (d, J = 9.2 Hz, 1H, NH), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.56 (s, 1H, NH), 7.44 (d, J =8.2 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.2 Hz, 2H, Ph-H × 2), 5.16 (d, J = 3.5Hz, 1H, OH), 4.91 (t, J = 7.2 Hz, 1H, CH), 4.54 (d, J = 9.3 Hz, 1H, CH), 4.42 (d, J = 8.0 Hz, 1H, CH), 4.28 (s, 1H, CH), 4.01 – 3.95 (m, 2H, CH2), 3.60 (d,J = 7.0 Hz, 2H, CH2), 2.75 – 2.64 (m, 1H, CH), 2.58 (t, J = 7.0 Hz, 1H, CH), 2.46 (s, 3H, CH3), 2.03 (s, 1H, CH), 1.83 – 1.75 (m, 1H, CH), 1.37 (d, J =6.9 Hz, 3H, CH3), 0.92 (s, 9H, CH3× 3).13 C NMR (100 MHz, DMSO-d6) δ 178.74,171.08, 170.09, 169.77, 153.40, 151.97, 148.21, 145.11, 134.20, 131.59,130.15, 129.29, 128.88, 126.85, 69.24, 59.04, 57.03, 56.93, 56.77, 48.18,38.16, 36.56, 36.06, 35.80, 35.69, 34.22, 26.86, 22.89, 16.44. ESI-MS: m / z707.03 (M + H) + , 729.41 (M + Na) + . C 35 H 39 ClN6O6S [706.23].

[0096] B2: Reddish-brown solid, yield 52%, Mp: 115-117 ℃. 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.95 (d, J = 5.3 Hz, 1H, Ph-H), 8.43 (d, J = 7.8Hz, 1H, NH), 8.33 (d, J = 7.9 Hz, 1H, Ph-H), 8.12 (d, J = 9.2 Hz, 1H, NH),7.75 (dd, J = 8.0, 5.3 Hz, 1H, Ph-H), 7.51 – 7.46 (m, 1H, NH), 7.44 (d, J =8.0 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.0 Hz, 2H, Ph-H × 2), 5.17 (d, J = 3.4Hz, 1H, OH), 4.91 (t, J = 8.0 Hz, 1H, CH), 4.53 (d, J = 9.2 Hz, 1H, CH), 4.43(t, J = 8.2 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.96 (s, 2H, CH2), 3.59 (dd, J =13.4, 7.3 Hz, 2H, CH2), 2.68 (q, J = 7.6 Hz, 1H, CH), 2.46 (s, 3H, CH3), 2.01(d, J = 10.3 Hz, 1H, CH), 1.78 (s, 1H, CH), 1.37 (d, J = 6.9 Hz, 3H, CH3),1.23 (s, 1H, CH), 0.92 (s, 9H, CH3× 3). 13 C NMR (100 MHz, DMSO-d6) δ 169.74,152.00, 148.22, 145.13, 134.93, 131.60, 130.15, 129.30, 126.86, 69.23, 59.03,48.17, 38.19, 35.81, 26.86, 22.92, 16.46. ESI-MS: m / z 707.01 (M + H) + , 729.39(M + Na) + . C 35 H 39 ClN6O6S [706.23].

[0097] B3: Reddish-brown solid, yield 25%, M.p.: 165 - 167 °C. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.39 (d, J =7.8 Hz, 1H, NH), 8.33 (dd, J = 7.9, 1.7 Hz, 1H, Ph-H), 7.94 (d, J = 9.3 Hz,1H, NH), 7.80 (dd, J = 7.8, 4.6 Hz, 1H, Ph-H), 7.72 (s, 1H, NH), 7.44 (d, J =8.3 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.3 Hz, 2H, Ph-H × 2), 5.11 (d, J = 3.6Hz, 1H, OH), 4.93 (q, J = 7.1 Hz, 1H,CH), 4.53 (d, J = 9.3 Hz, 1H, CH), 4.42(t, J = 8.0 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.75 (q, J = 6.8 Hz, 2H, CH2),3.64 – 3.56 (m, 2H, CH2), 2.46 (s, 3H, CH3), 2.34 (q, J = 7.2 Hz, 1H, CH),2.22 (dt, J = 14.5, 7.3 Hz, 1H, CH), 2.01 (t, J = 10.4 Hz, 1H, CH), 1.85 (dd,J = 11.0, 5.9 Hz, 2H, CH2), 1.79 (td, J = 8.5, 4.3 Hz, 1H, CH), 1.37 (d, J =7.0 Hz, 3H, CH3), 0.93 (s, 9H, CH3× 3). 13C NMR (100 MHz, DMSO-d6) δ 172.08,171.08, 169.98, 153.33, 151.97, 148.22, 145.13, 131.59, 130.16, 129.30,128.88, 126.85, 69.22, 59.01, 56.91, 56.73, 48.16, 38.18, 35.71, 32.65,26.92, 22.92, 16.46. ESI-MS: m / z 721.02 (M + H) + . C 36 H 41 ClN6O6S [720.25].

[0098] B4: Reddish-brown solid, yield 48%, Mp: 132-134 ℃. 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.95 (dd, J = 4.8, 1.8 Hz, 1H, Ph-H), 8.39 (d, J =7.8 Hz, 1H, NH), 8.32 (dd, J = 7.9, 1.8 Hz, 1H, Ph-H), 7.94 (d, J = 9.2 Hz,1H, NH), 7.74 (dd, J = 7.9, 4.6 Hz, 1H, Ph-H), 7.62 (s, 1H, NH), 7.44 (d, J =8.0 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.1 Hz, 2H, Ph-H × 2), 5.12 (d, J = 3.5Hz, 1H, OH), 4.92 (t, J = 7.3 Hz, 1H, CH), 4.53 (d, J = 9.2 Hz, 1H, CH), 4.43(t, J = 8.0 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.74 (q, J = 6.8 Hz, 2H, CH2),3.64 – 3.56 (m, 2H, CH2), 2.46 (s, 3H, CH3), 2.34 (q, J = 7.4 Hz, 1H, CH),2.23 (q, J = 7.1 Hz, 1H, CH), 2.02 (t, J = 11.1 Hz, 1H, CH), 1.89 – 1.78 (m,3H, CH2, CH), 1.37 (d, J = 7.0 Hz, 3H, CH3), 0.94 (s, 9H, CH3× 3). 13 C NMR(100 MHz, DMSO-d6) δ 180.46, 172.09, 171.08, 169.98, 154.94, 151.97, 148.22,145.12, 134.89, 131.58, 130.16, 129.30, 127.30, 126.85, 69.22, 59.02, 56.92,48.17, 38.19, 35.72, 32.66, 26.93, 22.91, 16.46. ESI-MS: m / z 721.09 (M + H) + ,743.45 (M + Na) + . C 36 H41 ClN6O6S [720.25].

[0099] B5: Reddish-brown solid, yield 58%, M.p.: 118 - 120 °C. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.90 (dd, J = 4.6, 1.7 Hz, 1H, Ph-H), 8.43 – 8.40(m, 1H, NH), 8.33 (dd, J = 7.9, 1.7 Hz, 1H, Ph-H), 7.88 – 7.85 (m, 1H, NH),7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.77 (s, 1H, NH), 7.44 (d, J = 8.0 Hz,2H, Ph-H × 2), 7.39 (d, J = 8.0 Hz, 2H, Ph-H × 2), 5.14 (t, J = 3.2 Hz, 1H,OH), 4.92 (t, J = 7.2 Hz, 1H, CH), 4.53 – 4.50 (m, 1H, CH), .41 (t, J = 4.1Hz, 1H, CH), 4.28 (s, 1H, CH), 3.73 (q, J = 7.0 Hz, 2H, CH2), 3.60 (d, J =5.1 Hz, 2H, CH2), 2.46 (s, 3H, CH3), 2.32 – 2.27 (m, 1H, CH), 2.16 (d, J = 6.3Hz, 1H, CH), 2.01 (s, 1H, CH), 1.79 (d, J = 8.3 Hz, 1H), CH, 1.62 – 1.57 (m,2H, CH2), 1.47 (d, J = 5.7 Hz, 2H, CH2), 1.39 – 1.36 (m, 3H, CH3), 0.92 (s,9H, CH3× 3). 13C NMR (100 MHz, DMSO-d6) δ 178.85, 172.38, 172.29, 171.10,170.03, 153.31, 151.99, 148.22, 145.13, 134.22, 131.59, 130.15, 129.30,128.90, 126.85, 69.23, 59.01, 56.81, 56.74, 48.17, 38.19, 35.69, 35.12,31.04, 28.42, 26.90, 23.22, 23.07, 22.92, 16.45. ESI-MS: m / z 735.02 (M + H) + 757.42 (M + Na) + . C 37 H 43 ClN6O6S [734.27].

[0100] B6: Reddish-brown solid, yield 32%, Mp: 129-131 ℃. 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.95 (dd, J = 4.8, 1.8 Hz, 1H, Ph-H), 8.41 (d, J =7.8 Hz, 1H, NH), 8.32 (dd, J = 7.9, 1.8 Hz, 1H, Ph-H), 7.87 (d, J = 9.3 Hz,1H, NH), 7.74 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.64 (s, 1H, NH), 7.44 (d, J =8.0 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.0 Hz, 2H, Ph-H × 2), 5.14 (d, J = 3.5Hz, 1H, OH), 4.91 (t, J = 7.2 Hz, 1H, CH), 4.51 (d, J = 9.3 Hz, 1H, CH), 4.41(t, J = 8.1 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.72 (t, J = 6.8 Hz, 2H, CH2),3.60 (d, J = 5.5 Hz, 2H, CH2), 2.46 (s, 3H, CH3), 2.33 – 2.26 (m, 1H, CH),2.16 (dd, J = 14.3, 7.4 Hz, 1H, CH), 2.00 (d, J = 9.6 Hz, 1H, CH), 1.78 (t, J= 8.4 Hz, 1H, CH), 1.64 – 1.53 (m, 4H, CH2× 2), 1.37 (d, J = 7.0 Hz, 3H,CH3), 0.92 (s, 9H, CH3× 3). 13 C NMR (100 MHz, DMSO-d6) δ 180.52, 172.38,171.09, 170.02, 151.99, 148.22, 145.13, 134.90, 131.59, 130.15, 129.30,127.30, 126.85, 69.22, 59.01, 56.81, 48.17, 38.19, 35.70, 35.11, 31.03,26.90, 23.08, 22.92, 16.46. ESI-MS: m / z 735.10 (M + H) +, 757.49 (M + Na) + .C 37 H 43 ClN6O6S [734.27].

[0101] B7: Reddish-brown solid, yield 32%, M.p.: 116 - 118 °C. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.90 (dd, J = 4.6, 1.7 Hz, 1H, Ph-H), 8.43 (d, J =7.8 Hz, 1H, NH), 8.33 (dd, J = 7.9, 1.7 Hz, 1H), 7.84 (d, J = 9.3 Hz, 1H,NH), 7.79 (dd, J = 7.9, 4.6 Hz, 1H, Ph-H), 7.74 (s, 1H, NH), 7.44 (d, J = 8.0Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.0 Hz, 2H, Ph-H × 2), 5.16 (d, J = 3.4 Hz,1H, OH), 4.93 – 4.88 (m, 1H, CH), 4.51 (d, J = 9.3 Hz, 1H, CH), 4.42 (t, J =8.0 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.72 (d, J = 7.3 Hz, 2H, CH2), 3.59 (s,2H, CH2), 2.46 (s, 3H, CH3), 2.30 – 2.24 (m, 1H, CH), 2.15 – 2.09 (m, 1H, CH),2.00 (d, J = 9.8 Hz, 1H, CH), 1.78 (s, 1H, CH), 1.61 (d, J = 7.1 Hz, 2H,CH2), 1.49 (d, J = 14.6 Hz, 2H, CH2), 1.37 (d, J = 6.9 Hz, 3H, CH3), 1.29 (t,J = 7.6 Hz, 2H, CH2), 0.91 (s, 9H, CH3× 3). ESI-MS: m / z 749.05 (M + H) + ,771.42 (M + Na) + . C 38 H45 ClN6O6S [748.28].

[0102] B8: Reddish-brown solid, yield 55%, M.p.: 145 - 147 °C. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.95 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.41 (d, J =7.7 Hz, 1H, NH), 8.33 (dd, J = 7.8, 1.7 Hz, 1H, Ph-H), 7.84 (d, J = 9.4 Hz,1H, NH), 7.74 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.69 – 7.55 (m, 1H, NH), 7.44(d, J = 8.0 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.1 Hz, 2H, Ph-H × 2), 5.12 (s,1H, OH), 4.91 (t, J = 7.3 Hz, 1H, CH), 4.51 (d, J = 9.3 Hz, 1H, CH), 4.41 (t,J = 8.1 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.71 (d, J = 7.0 Hz, 2H, CH2), 3.60(d, J = 4.2 Hz, 2H, CH2), 2.46 (s, 3H, CH3), 2.28 (dd, J = 14.3, 7.3 Hz, 1H,CH), 2.15 – 2.10 (m, 1H, CH), 2.01 (s, 1H, CH), 1.79 (td, J = 8.7, 4.3 Hz,1H), 1.62 (t, J = 7.3 Hz, 2H, CH2), 1.49 (dd, J = 12.1, 5.6 Hz, 2H, CH2), 1.37(d, J = 6.9 Hz, 3H, CH3), 1.32 – 1.27 (m, 2H, CH2), 0.91 (s, 9H, CH3× 3). 13CNMR (100 MHz, DMSO-d6) δ 172.45, 171.10, 170.03, 152.00, 148.21, 145.14,134.92, 131.60, 130.14, 129.30, 127.29, 126.85, 69.22, 59.00, 56.78, 48.17,38.19, 35.67, 35.29, 31.10, 26.89, 26.25, 25.64, 22.92, 16.45. ESI-MS: m / z749.08 (M + H) + , 771.45 (M + Na) + . C 38 H 45 ClN6O6S [748.28].

[0103] B9: Reddish-brown solid, yield 37%, Mp: 123-125 ℃. 1H NMR (400 MHz, DMSO-d6) δ9.06 (s, 1H, thiazole-H), 8.90 (dd, J = 4.8, 1.7 Hz, 1H, Ph-H), 8.41 (d, J =7.8 Hz, 1H, NH), 8.33 (dd, J = 7.9, 1.7 Hz, 1H, Ph-H), 7.82 (d, J = 9.2 Hz,1H, NH), 7.73 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.61 (s, 1H, NH), 7.45 (d, J =8.1 Hz, 2H, Ph-H × 2), 7.39 (d, J = 8.1 Hz, 2H, Ph-H × 2), 4.92 (t, J = 7.2Hz, 1H, CH), 4.52 (dd, J = 9.4, 1.7 Hz, 1H, CH), 4.42 (t, J = 8.1 Hz, 1H,CH), 4.28 (s, 1H, CH), 3.72 (d, J = 7.2 Hz, 2H, CH2× 2), 3.61 (d, J = 3.8Hz, 2H, CH2), 3.17 (s, 1H, CH), 2.46 (s, 3H, CH3), 2.28 – 2.21 (m, 1H, CH),2.12 (q, J = 7.0 Hz, 1H, CH), 2.00 (d, J = 9.7 Hz, 1H, CH), 1.82 – 1.75 (m,1H, CH), 1.54 – 1.42 (m, 4H, CH2× 2), 1.38 (d, J = 6.9 Hz, 3H, CH3), 1.30 –1.25 (m, 4H, CH2× 2), 0.94 (s, 9H, CH3× 3). 13 C NMR (100 MHz, DMSO-d6) δ172.49, 171.11, 170.05, 152.25, 147.82, 145.28, 131.82, 129.96, 129.31,126.87, 69.22, 59.00, 56.80, 48.18, 38.20, 35.66, 35.26, 31.24, 28.71, 28.58,26.91, 26.38, 25.78, 22.93, 16.28. ESI-MS: m / z 763.04 (M + H)+ . C 39 H 47 ClN6O6S [762.30].

[0104] B10: Reddish-brown solid, yield 53%, M.p.: 156 - 158 °C. 1 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.94 (dd, J = 4.8, 1.7 Hz, 1H, Ph-H), 8.40 (d, J =7.8 Hz, 1H, NH), 8.32 (dd, J = 7.9, 1.7 Hz, 1H, Ph-H), 7.82 (d, J = 9.2 Hz,1H, NH), 7.73 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.61 (s, 1H, NH), 7.44 (d, J =8.3 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.4 Hz, 2H, Ph-H × 2), 5.12 (s, 1H,OH), 4.92 (t, J = 7.2 Hz, 1H, CH), 4.52 (d, J = 9.4 Hz, 1H, CH), 4.42 (t, J =8.1 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.70 (q, J = 7.1 Hz, 2H, CH2), 3.60 (s,2H, CH2), 2.46 (s, 3H, CH3), 2.26 (m, 1H, CH), 2.12 (q, J = 6.9 Hz, 1H, CH),2.00 (d, J = 9.9 Hz, 1H, CH), 1.79 (td, J = 8.5, 4.3 Hz, 1H), 1.60 (t, J =7.4 Hz, 2H, CH2), 1.51 – 1.44 (m, 2H, CH2), 1.37 (d, J = 7.0 Hz, 3H, CH3),1.28 (d, J = 8.1 Hz, 4H, CH2× 2), 0.93 (s, 9H, CH3× 3). 13C NMR (100 MHz, DMSO-d6) δ 172.47, 171.10, 170.04, 152.00, 148.21, 145.15, 134.91, 131.59,130.14, 129.30, 127.29, 126.85, 69.22, 59.00, 56.77, 48.16, 38.20, 35.67,28.84, 26.91, 26.35, 25.82, 22.94, 16.46. ESI-MS: m / z 763.07 (M + H) + .C 39 H 47 ClN6O6S [762.30].

[0105] B11: Reddish-brown solid, yield 40%, Mp: 122-124 ℃. 11H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.90 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.41 (d, J =7.7 Hz, 1H, NH), 8.32 (dd, J = 7.8, 1.7 Hz, 1H, Ph-H), 7.84 – 7.81 (m, 1H,NH), 7.80 (dd, J = 7.8, 4.7 Hz, 1H, Ph-H), 7.71 (s, 1H, NH), 7.44 (d, J = 8.0Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.0 Hz, 2H, Ph-H × 2), 5.13 (s, 1H, OH),4.93 (d, J = 7.2 Hz, 1H, CH), 4.50 (d, J = 7.3 Hz, 1H, CH), 4.44 – 4.40 (m,1H, CH), 4.28 (s, 1H, CH), 3.71 (d, J = 7.0 Hz, 1H, CH), 3.61 (s, 2H, CH2),3.16 (d, J = 6.5 Hz, 1H, CH), 2.46 (s, 3H, CH3), 2.24 (d, J = 7.2 Hz, 1H,CH), 2.09 (d, J = 7.0 Hz, 1H, CH), 2.00 (d, J = 9.4 Hz, 1H, CH), 1.81 – 1.77(m, 1H, CH), 1.49 – 1.42 (m, 4H, CH2× 2), 1.38 (s, 3H, CH3), 1.24 (s, 6H,CH2× 3), 0.92 (s, 9H, CH3 × 3). ESI-MS: m / z 777.21 (M + H) + . C 40 H 49 ClN6O6S [776.31].

[0106] B12: Reddish brown solid, yield 36%, M.p.: 155 - 157 °C. 11H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.95 (dd, J = 4.7, 1.6 Hz, 1H, Ph-H), 8.40 (d, J =7.8 Hz, 1H, NH), 8.33 (dd, J = 7.8, 1.6 Hz, 1H, Ph-H), 7.86 – 7.82 (m, 1H,NH), 7.80 (dd, J = 7.8, 4.7 Hz, 1H, Ph-H), 7.73 (s, 1H, NH), 7.43 (d, J = 8.1Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.1 Hz, 2H, Ph-H × 2), 5.12 (s, 1H, OH),4.93 (d, J = 7.2 Hz, 1H, CH), 4.50 (d, J = 7.3 Hz, 1H, CH), 4.44 – 4.40 (m,1H, CH), 4.28 (s, 1H, CH), 3.71 (d, J = 7.1 Hz, 1H, CH), 3.61 (s, 2H, CH2),3.16 (d, J = 6.5 Hz, 1H, CH), 2.46 (s, 3H, CH3), 2.24 (d, J = 7.2 Hz, 1H,CH), 2.09 (d, J = 7.0 Hz, 1H, CH), 2.00 (d, J = 9.4 Hz, 1H, CH), 1.82 – 1.75(m, 1H, CH), 1.51 – 1.44 (m, 4H, CH2× 2), 1.38 (s, 3H, CH3), 1.24 (s, 6H,CH2× 3), 0.92 (s, 9H, CH3 × 3). ESI-MS: m / z 777.40 (M + H) + . C 40 H 49 ClN6O6S [776.31].

[0107] B13: Reddish-brown solid, yield 38%, M.p.: 112 - 114 °C. 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.90 (dd, J = 4.7, 1.6 Hz, 1H, Ph-H), 8.40 (d, J =7.8 Hz, 1H, NH), 8.33 (dd, J = 7.8, 1.6 Hz, 1H, Ph-H), 7.85 – 7.82 (m, 1H,NH), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.72 (s, 1H, NH), 7.43 (d, J = 8.1Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.1 Hz, 2H, Ph-H × 2), 5.12 (d, J = 3.5 Hz,1H, OH), 4.93 (d, J = 7.2 Hz, 1H, CH), 4.52 (d, J = 7.3 Hz, 1H, CH), 4.42 (s,1H, CH), 4.28 (s, 1H, CH), 3.72 (d, J = 7.3 Hz, 1H, CH), 3.60 (s, 2H, CH2),3.16 (d, J = 6.5 Hz, 1H, CH), 2.46 (s, 3H, CH3), 2.24 (d, J = 7.2 Hz, 1H,CH), 2.09 (d, J = 7.0 Hz, 1H, CH), 2.00 (d, J = 9.4 Hz, 1H, CH), 1.81 – 1.77(m, 1H, CH), 1.49 – 1.42 (m, 4H, CH2× 2), 1.38 (s, 3H, CH3), 1.24 (s, 8H,CH2× 4), 0.93 (s, 9H, CH3× 3). 13 C NMR (100 MHz, DMSO-d6) δ 172.53, 171.10,170.06, 153.31, 151.97, 148.22, 145.14, 131.59, 130.15, 129.29, 126.85,69.22, 59.00, 56.79, 48.17, 38.20, 35.66, 35.34, 29.14, 28.64, 26.90, 26.59,25.87, 22.91, 16.52, 16.46. ESI-MS: m / z 790.98 (M + H) +, 813.83 (M + Na) + .C 41 H 51 ClN6O6S [790.33].

[0108] B14: Reddish-brown solid, yield 45%, M.p.: 169 - 171 °C. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.94 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.41 (d, J =7.8 Hz, 1H, NH), 8.32 (dd, J = 7.8, 1.7 Hz, 1H, Ph-H), 7.82 (d, J = 9.2 Hz,1H, NH), 7.73 (dd, J = 7.8, 4.7 Hz, 1H, Ph-H), 7.63 (s, 1H, NH), 7.44 (d, J =8.1 Hz, 2H, Ph-H × 2), 7.38 (d, J = 8.1 Hz, 2H, Ph-H × 2), 5.13 (d, J = 3.5Hz, 1H, OH), 4.91 (t, J = 7.2 Hz, 1H, CH), 4.51 (d, J = 9.3 Hz, 1H, CH), 4.40(d, J = 8.1 Hz, 1H, CH), 4.28 (s, 1H, CH), 3.73 – 3.68 (m, 2H, CH2), 3.60 (s,2H, CH2), 2.46 (s, 3H, CH3), 2.28 – 2.22 (m, 1H, CH), 2.13 – 2.08 (m, 1H, CH),2.01 (s, 1H, CH), 1.81 – 1.76 (m, 1H, CH), 1.61 (s, 2H, CH2), 1.46 (d, J =7.2 Hz, 2H, CH2), 1.37 (d, J = 6.9 Hz, 3H, CH3), 1.32 – 1.21 (m, 8H, CH2×4), 0.93 (s, 9H, CH3 × 3). ESI-MS: m / z 791.01 (M + H) + , 813.52 (M + Na) + .C 41 H 51 ClN6O6S [790.33].

[0109] B15: Reddish-brown solid, yield 51%, Mp: 154-156 ℃. 1 H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.95 (d, J = 4.9 Hz, 1H, Ph-H), 8.44 (d, J = 7.7Hz, 1H, NH), 8.34 (d, J = 7.9 Hz, 1H, Ph-H), 7.96 (d, J = 9.0 Hz, 1H, NH),7.80 (dd, J = 8.0, 4.6 Hz, 1H, Ph-H), 7.43 (d, J = 8.0 Hz, 2H, Ph-H × 2),7.37 (d, J = 8.0 Hz, 2H, Ph-H × 2), 5.15 (s, 1H, OH), 4.91 (s, 1H, CH), 4.51(d, J = 9.3 Hz, 1H, CH), 4.43 (s, 1H, CH), 4.28 (s, 1H, CH), 3.78 (d, J =11.9 Hz, 2H, CH2), 3.60 (s, 3H, CH3), 2.46 (s, 3H, CH3), 2.01 (s, 2H, CH2), 1.83 – 1.74 (m, 4H, CH2× 2), 1.37 (d, J = 7.1 Hz, 3H, CH3), 0.94 (s, 9H, CH3× 3). ESI-MS: m / z 747.10 (M + H) + . C 38 H 43 ClN6O6S [746.27].

[0110] B16: Reddish-brown solid, yield 38%, Mp: 132-134 ℃. 1H NMR (400 MHz, DMSO-d6) δ8.99 (s, 1H, thiazole-H), 8.96 (dd, J = 4.7, 1.7 Hz, 1H, Ph-H), 8.42 (d, J =7.8 Hz, 1H, NH), 8.35 (dd, J = 7.9, 1.7 Hz, 1H, Ph-H), 7.88 (d, J = 9.1 Hz,1H, NH), 7.80 (dd, J = 7.9, 4.7 Hz, 1H, Ph-H), 7.44 (d, J = 8.0 Hz, 2H, Ph-H× 2), 7.37 (d, J = 8.1 Hz, 2H, Ph-H × 2), 5.15 (d, J = 3.5 Hz, 1H, OH),4.91 (m, 1H, CH), 4.49 (d, J = 9.2 Hz, 1H, CH), 4.41 (d, J = 8.2 Hz, 1H, CH),4.29 (s, 1H, CH), 3.89 (d, J = 12.6 Hz, 1H, CH), 3.82 (d, J = 13.2 Hz, 1H,CH), 3.59 (d, J = 9.7 Hz, 3H, CH3), 2.45 (s, 3H, CH3), 2.00 (d, J = 10.4 Hz,1H, CH), 1.87 (s, 1H, CH), 1.78 (s, 3H, CH3), 1.65 (s, 1H, CH), 1.37 (d, J =7.0 Hz, 3H, CH3), 0.96 (s, 2H, CH2), 0.92 (s, 9H, CH3 × 3). ESI-MS: m / z747.25 (M + H) + . C 38 H 43 ClN6O6S [746.27].

[0111] Example 2. Screening and evaluation of the in vitro antitumor activity of the target compound

[0112] To evaluate the antiproliferative activity of the synthesized Cdc25 degraders, their inhibitory rates against six different cancer cell lines (A549, HepG2, DU-145, Mia-PaCa-2, HCT15, and MDA-MB-231) and their cytotoxicity against normal human embryonic kidney cells HEK-293T were determined. After treating cells with the compounds at a concentration of 2 μM for 48 h, their proliferation inhibition rate was detected using the CCK-8 assay, with 5-fluorouracil (5-FU), NSC663284, and NSC663285 serving as controls. Table 1 summarizes the antiproliferative activity and cytotoxicity data of each compound at 2 μM. The results showed that most compounds exhibited significant inhibitory activity against A549, HepG2, DU-145, Mia-PaCa-2, and HCT-15 at this concentration, but had no significant effect on MDA-MB-231.

[0113]

[0114] Table 1. Antitumor cell proliferation inhibition rate of PROTAC series compounds 1 (%)

[0115] 1 The experimental results are expressed as mean ± SD (n ≥ 3).

[0116] To determine the half-maximal inhibitory concentration (IC50) of the preferred compound 50 Compounds A2, A4, A7, A8, B6, and B14, which exhibited significant inhibitory activity against the proliferation of sensitive cell lines and low cytotoxicity at 2 μM, were selected for assay. The results (Table 2) show that most compounds showed significant IC50 activity against the six tumor cell lines. 50 The antiproliferative activity of the compound reached submicromolar levels, showing superior activity compared to the positive control drug pentafluorouracil and the lead compound NSC663284. However, some compounds exhibited high cytotoxicity. Therefore, the selectivity index (SI; SI = CC) was further calculated. 50 / IC 50 Data (Table 3) show that these compounds exhibited the strongest inhibitory effect on the HCT-15 cell line, and also achieved the best SI value in this cell line, superior to 5-FU and NSC663284. Among them, compound B6 inhibited the IC50 of HCT-15 cells. 50 The value was 0.35 ± 0.11 µM, and the selectivity index (SI) was 14.9.

[0117] Table 2. Antitumor cell proliferation activity of active compounds

[0118]

[0119] 1 IC 50 / 1 CC 50 values ​​are expressed as the mean ± SD (n ≥ 3)

[0120] Table 3 SI Index of Active Compounds 1

[0121]

[0122] 1 SI = CC 50 / IC 50

[0123] Example 3. Evaluation of the in vitro antitumor mechanism of the target compound

[0124] Based on the screening results of the antiproliferative activity of the target compounds, the representative compound B6 was selected for Western blot experiments to evaluate its degradation effects on Cdc25A, Cdc25B, and Cdc25C, and to further explore its antitumor molecular mechanism. Since HCT-15 cells are sensitive to the target compounds, this cell line was used to detect the compounds' degradation ability on Cdc25 phosphatase. Using GAPDH as an internal control, the protein expression levels were quantitatively analyzed using ImageJ software.

[0125] The results showed that in the time-dependent degradation experiment (see...) Figure 1 a) Compound B6 at 5 μM degraded Cdc25 phosphatase 3 h after administration, with significant degradation after 6 h, and the degradation effect increased with time. The results indicate that compound B6 can degrade Cdc25A, Cdc25B, and Cdc25C in HCT-15 cells in a time-dependent manner. In concentration-dependent experiments (see...) Figure 1 (b and 1d), compound B6, when used to treat HCT-15 cells at concentrations ranging from 0.1 to 5 μM, showed increased degradation activity in Cdc25A, Cdc25B, and Cdc25C DCs with increasing concentration. 50 The values ​​were 0.97 μM, 2.02 μM, and 4.67 μM, respectively (see...). Figure 1 c).

[0126] To verify whether the experiment relied on the ubiquitin-proteasome pathway, the proteasome inhibitor MG-132 was added. The results showed (see...). Figure 1e) In the blank control group, MG-132 had no significant effect on Cdc25 phosphatase expression. However, in the drug-treated group, the degradation effect of B6 on Cdc25A and Cdc25C was reversed, and the degradation effect on Cdc25B was also weakened, indicating that its degradation mechanism is proteasome-dependent. (Competitive experiment) Figure 1 f) further indicates that the degradation of Cdc25B by the representative compound B6 is dependent on the ubiquitin-proteasome pathway. Furthermore, compound B6 can upregulate the expression of p-CDK1 and p-CDK2 (see f) Figure 1 (b) suggests that it may weaken the dephosphorylation function of Cdc25 phosphatase by degrading it.

[0127] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although they have been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions without departing from the spirit and scope of the present invention.

Claims

1. A Cdc25 phosphatase degrading agent based on a quinolinidone skeleton, characterized in that, It has the structure shown in general formula (I): In general formula (I): X is either C or N; Y is either C or N, and there is exactly one X and Y containing N; The linker is selected from one of the following groups: The R group represents the E3 ubiquitin ligase ligand moiety and is selected from one of the following groups: 。 2. The Cdc25 phosphatase degrading agent based on a quinolinidone skeleton as described in claim 1, characterized in that, It is one of the following compounds: 。 3. The method for preparing the Cdc25 phosphatase degrading agent based on the quinolinidone skeleton as described in claim 2, characterized in that, The synthesis route is as follows: (1) Synthesis of intermediate 2a-2i, the synthetic route is as follows: Reagents and conditions: (i) H2N–(CH2) with different structures n –COOH, n=2-7,9,10 or NH2-PEG2-COOH, DMSO, DIEA, 110°C, 6h; Where R1 is , ; (2) The synthetic route for the target compounds A1-A9 and B1-B9 is as follows: Reagents and conditions: (i) 4-(2-aminoethyl)piperazine-1-carboxylic acid tert-butyl ester, DIEA, CH2Cl2, 6 h; (ii) 4 mol hydrochloric acid, 1,4-dioxane solution, 4 h; (iii) intermediate 2a-2i, EDCI, HOBt, DIEA, CH2Cl2, 12 h. Where L-1 is , ; (3) The synthesis route of intermediate 18a-18g is as follows: Reagents and conditions: (i) 4-methylthiazole, [1,1'-bis(di-tert-butylphosphine)ferrocene]palladium(II) chloride, potassium acetate, N,N-dimethylacetamide, 90°C, 18h; (ii) 4 mol hydrochloric acid, ethyl acetate, rt, 4h; (iii) HATU, DIEA, N,N-dimethylformamide, rt, 12h; (iv) LiOH, MeOH: THF: H2O = 5: 4: 3 (volume ratio), rt. Where R2 is , R3 is , ; (4) The synthesis route for target compounds B1-B16 is as follows: Reagents and conditions: (i) Intermediate 18a-18h, triethylamine, CH2Cl2, rt, 6 h; Among them, L-2 is , .

4. The use of the Cdc25 phosphatase degrader based on the quinolinidone skeleton as described in any one of claims 1-2 in the preparation of antitumor drugs.

5. The application as described in claim 4, wherein the antitumor drug is an antitumor drug targeting Cdc25.

6. An antitumor pharmaceutical composition comprising a Cdc25 phosphatase degrader based on a quinolinidone backbone as described in any one of claims 1-2 and one or more pharmaceutically acceptable carriers.

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