Bladder cancer cell inhibitor taking TNKS2 protein as target as well as preparation method and application of bladder cancer cell inhibitor

Abstract

The invention relates to a bladder cancer cell inhibitor taking TNKS2 protein as a target as well as a preparation method and application thereof, the bladder cancer cell inhibitor is a 1, 3, 5-triazine compound, the structural formula of the bladder cancer cell inhibitor is shown in the specification, and the preparation method comprises the following steps: taking 2, 4, 6-trichloro-1, 3, 5-triazine as a raw material, under an alkaline condition, dissolving the 2, 4, 6-trichloro-1, 3, 5-triazine in dichloromethane, stirring in an ice bath, filtering, washing, and drying to obtain the bladder cancer cell inhibitor taking TNKS2 protein as a target. Then adding N-(2-aminoethyl) morpholine, continuously stirring to react, and filtering separated solids after the reaction is finished to obtain a compound 1; dissolving the compound 1 and 2-(2-pyridyl) ethylamine in methanol, reacting at room temperature, separating out a white solid, and filtering to obtain a compound 2; the bladder cancer cell inhibitor is obtained by taking tetra (triphenyl) phosphine palladium as a catalyst, enabling a compound 2 to react with a phenylboronic acid derivative under an alkaline condition and in a nitrogen atmosphere, and performing post-treatment after the reaction is finished. Compared with the prior art, the product prepared by the invention has better bladder cancer cell selectivity and inhibitory activity.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a bladder cancer cell inhibitor with TNKS2 protein as a target point and a preparation method and application thereof. BACKGROUND

[0002] Bladder cancer is a common malignant tumor of the urinary system with a high incidence rate worldwide. According to the global cancer statistics in 2020, the newly diagnosed bladder cancer cases in men accounted for 4.4% of all malignant tumors, ranking 6th in the incidence of malignant tumors. In China, bladder cancer ranks 13th in the incidence spectrum of malignant tumors, with a crude incidence rate of 5.80 / 10 million, a Chinese standard incidence rate of 3.60 / 10 million and a world standard incidence rate of 3.57 / 10 million. It is worth noting that the incidence rate in men is 3.8 times that in women, and the incidence rate in urban areas is higher than that in rural areas, and the incidence rate in eastern regions is higher than that in central and western regions. At the same time, data shows that the incidence rate of bladder cancer is showing a trend of youth.

[0003] In recent years, the role of TNKS2 protein in the molecular regulation process of bladder cancer has been gradually discovered. TNKS2 (Tankyrase 2) protein plays an important role in the occurrence of bladder cancer. It mainly promotes tumor development by regulating the Wnt / β-catenin signaling pathway and affecting telomere stability. Specifically, TNKS2 can modify Axin protein through PARylation, promote its degradation, inhibit the degradation of β-catenin, and enhance the activity of the Wnt signaling pathway. The abnormal activation of this signaling pathway plays a key role in the occurrence of bladder cancer, which can lead to uncontrolled cell proliferation. In addition, TNKS2 is also related to telomere maintenance. It regulates the stability of telomeres by interacting with TRF1, helps tumor cells maintain telomere length, and thus escapes cell senescence and apoptosis caused by telomere shortening. This mechanism may be particularly important in bladder cancer cells, because telomere stabilization is one of the important ways for tumor cells to gain immortality. Studies have shown that TNKS2 is abnormally elevated in various cancers, including bladder cancer. Its high expression in bladder cancer is closely related to the malignancy and invasion ability of tumors, and TNKS2 is a potential therapeutic target. By inhibiting the activity of TNKS2, it is expected to restore the normal regulation of the Wnt signaling pathway and weaken the telomere maintenance ability of tumor cells, thereby inhibiting the occurrence and development of bladder cancer.

[0004] In recent years, progress has been made in the study of target inhibitors for TNKS2. For example, XAV939 is a known TNKS inhibitor that inhibits the activity of the Wnt / β-catenin signaling pathway by stabilizing Axin protein, thereby inhibiting the proliferation of tumor cells. In bladder cancer cells, TNKS2 inhibitors can act through a similar mechanism, reducing β-catenin levels and inhibiting tumor growth. In addition, the new inhibitor RK-287107 showed inhibition of the Wnt signaling pathway in vitro and animal models, and significantly inhibited tumor growth. These studies provide a theoretical basis for TNKS2 as a target for bladder cancer treatment, and are expected to bring new treatment options for bladder cancer patients. SUMMARY

[0005] The purpose of the present application is to provide a bladder cancer cell inhibitor targeting TNKS2 protein and its preparation method and application, which has good bladder cancer cell inhibitory activity.

[0006] The purpose of the present application can be achieved by the following technical solutions:

[0007] In one aspect, the present application provides a bladder cancer cell inhibitor targeting TNKS2 protein, which is a 1,3,5-triazine compound, and its structural formula is:

[0008]

[0009] wherein R 1 ~R 5 each independently selected from any one of hydrogen atom, halogen atom, alkyl group, hydroxyl group, ester group, naphthyl group, diphenylamino group, trifluoromethyl group, aldehyde group, ketone group, alkoxy group, nitro group, amino group, carboxyl group, and methylenedioxy group.

[0010] Preferably, R 1 ~R 5 each independently selected from any one of hydroxyl group, alkoxy group and ester group.

[0011] Preferably, the structural formula of the bladder cancer cell inhibitor comprises any one of the following:

[0012]

[0013]

[0014] Preferably, the structural formula of the bladder cancer cell inhibitor is:

[0015]

[0016] Secondly, the present invention provides a method for preparing a bladder cancer cell inhibitor targeting the TNKS2 protein, comprising the following steps:

[0017] S1: Using 2,4,6-trichloro-1,3,5-triazine as a raw material, it was dissolved in dichloromethane under alkaline conditions and stirred in an ice bath. Then N-(2-aminoethyl)morpholine was added and the reaction was continued with stirring. After the reaction was completed, the precipitated solid was filtered to obtain compound 1.

[0018] S2: Compound 1 was dissolved in methanol and reacted with 2-(2-pyridyl)ethylamine at room temperature under alkaline conditions. A white solid precipitated and was obtained by filtration to give compound 2.

[0019] S3: Using tetra(triphenyl)phosphine palladium as a catalyst, compound 2 is reacted with a phenylboronic acid derivative under alkaline conditions and a nitrogen atmosphere. After the reaction is completed, post-treatment is performed to obtain the bladder cancer cell inhibitor.

[0020] Preferably, the structural formula of compound 1 is:

[0021]

[0022] Preferably, the structural formula of compound 2 is:

[0023]

[0024] Preferably, in steps S1, S2, and S3, the alkaline conditions are provided by adding an alkali, which includes any one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, and triethylamine.

[0025] Preferably, in step S1, the molar ratio of 2,4,6-trichloro-1,3,5-triazine, N-(2-aminoethyl)morpholine, and the base is 1:1:1 to 1:2:2.

[0026] Preferably, in step S1, the temperature of the ice bath is -10 to 10°C, the stirring time in the ice bath is 20 to 40 minutes, and the stirring speed is 200 to 600 rpm.

[0027] Preferably, in step S1, the reaction time for continued stirring is 1-6 hours, and the stirring speed is 200-600 rpm.

[0028] Preferably, in step S2, the molar ratio of compound 1, 2-(2-pyridyl)ethylamine, and base is 1:1:1 to 1:2:2, the room temperature is 0-25°C, and the reaction time is 1-6 hours.

[0029] Preferably, in step S3, the molar ratio of compound 2, phenylboronic acid derivative, base, and tetra(triphenyl)phosphine palladium is 1:1:2:0.05-1:1.5:3:0.1.

[0030] Preferably, in step S3, the reaction temperature of compound 2 with the phenylboronic acid derivative is 80-110°C, and the reaction time is 1-6 hours.

[0031] More preferably, in step S3, the reaction temperature of compound 2 with the phenylboronic acid derivative is 80°C, and the reaction time is 1-4 hours.

[0032] Preferably, in step S3, the chemical formula of the phenylboronic acid derivative is:

[0033]

[0034] Among them, R 1 ~R 5 Each group is independently selected from any one of the following: hydrogen atom, halogen atom, alkyl group, hydroxyl group, ester group, naphthyl group, diphenylamino group, trifluoromethyl group, aldehyde group, ketone group, alkoxy group, nitro group, amino group, carboxyl group, and methylenedioxy group.

[0035] Preferably, in step S3, the post-processing includes column chromatography.

[0036] Preferably, in step S3, the eluent used for column chromatography is a mixture of ethyl acetate and petroleum ether.

[0037] More preferably, in step S3, the volume ratio of ethyl acetate to petroleum ether is 1:20-1:5.

[0038] Preferably, the method for preparing the bladder cancer cell inhibitor targeting TNKS2 protein includes the following steps:

[0039] This method employs a multi-step synthetic reaction. Using 2,4,6-trichloro-1,3,5-triazine as a starting material, 2,4,6-trichloro-1,3,5-triazine is dissolved in dichloromethane. An equimolar amount of solid sodium hydroxide is added, and the mixture is stirred in an ice bath for 30 min. An equimolar amount of N-(2-aminoethyl)morpholine is then added, and the mixture is stirred for 1-6 h. The precipitated solid is filtered to obtain compound 1. In the second step, compound 1 is dissolved in methanol with 2-(2-pyridyl)ethylamine. The reaction is carried out under alkaline conditions at room temperature for 1-6 h, precipitating a white solid, which is then filtered to obtain compound 2. In the third step, compound 2 reacts with a series of different phenylboronic acids using tetra(triphenyl)phosphine palladium as a catalyst. The reaction is carried out under alkaline conditions in a nitrogen atmosphere at 80-110 °C for 1-4 h to obtain the bladder cancer cell inhibitor.

[0040] Preferably, the synthetic route of the bladder cancer cell inhibitor targeting the TNKS2 protein is as follows:

[0041]

[0042] Preferably, the bladder cancer cell inhibitor targeting the TNKS2 protein is obtained through screening using computer-aided drug design methods, specifically including the following steps:

[0043] (1) Identifying core targets and constructing a virtual screening system

[0044] TNKS2 (PDB ID: 4HYF) was identified as the key target. This protein plays an important regulatory role in the Wnt / β-catenin signaling pathway and is a core target for drug design. Based on this, a screening database and validation system were constructed.

[0045] Approximately 400,000 small molecule compounds were obtained from the ZINC database. The Lipinski Rule of Five was used for preliminary screening to exclude compounds that did not meet the basic properties of drug molecules, thus narrowing down the screening scope.

[0046] Using the Surflex-Dock module of Sybyl-X2.1 software, the filtered compounds were molecularly docked with TNKS2 to screen out candidate molecules with strong binding ability to the target site.

[0047] Verification via reconnection Confirm the reliability of the docking method to ensure the accuracy of subsequent screening results.

[0048] (2) Screen and analyze lead compounds to identify structural advantages.

[0049] From the molecular docking results, ZINC11726230 was selected as the most promising lead compound, with a binding free energy (-160.167 kJ / mol) superior to known inhibitors XAV-939 and G007-LK. At the same time, computer simulation analysis of the binding mode of this compound with TNKS2 revealed the core structural advantage: its benzene ring can penetrate deep into the adenosine binding pocket of TNKS2 to form a key hydrophobic interaction. This binding characteristic provides the core basis for subsequent structural optimization.

[0050] (3) Structure-based drug design (SBDD) for the targeted design of 1,3,5-triazine derivatives

[0051] Based on the binding mode of the lead compound and combined with the structural characteristics of known active molecules, targeted structural optimization and derivation design are carried out:

[0052] Based on the structural advantages of G007-LK, the derivatives were constructed using "1,3,5-triazine" as the parent core. The core optimization direction was to modify the benzene ring of the lead compound. The influence of different modifications on the binding ability was predicted by computer simulation. Finally, 14 derivatives (compounds 4-1 to 4-14) were designed to enhance the binding affinity of the compounds to TNKS2.

[0053] Simultaneously, the feasibility of synthesis was evaluated using a computer: a concise three-step synthetic route was designed using 2,4,6-trichloro-1,3,5-triazine as the starting material. By changing different phenylboronic acid derivatives, structural diversity was achieved, which not only facilitates the rapid construction of compound libraries, but also provides diverse molecules for subsequent structure-activity relationship (SAR) studies.

[0054] (4) Molecular dynamics (MD) simulations confirmed the preferred derivative structure.

[0055] Computer-level verification of the activity and stability of the designed derivatives:

[0056] The long-term stability of the preferred compound bound to TNKS2 was evaluated by performing 50 ns MD simulation.

[0057] Binding free energy was calculated using the MM / PBSA method (Molecular Mechanics-Poisson Bolzmann Surface Area), confirming that the binding affinity of derivatives such as compound 4-2 is superior to that of the lead compound.

[0058] Further analysis of the binding mode revealed that compound 4-2 can form more hydrogen bonds with amino acid residues of TNKS2, which further explains its excellent activity and ultimately determined the structure of the target 1,3,5-triazine compound.

[0059] Thirdly, the present invention also provides the application of the bladder cancer cell inhibitor targeting the TNKS2 protein described above in the field of preparing anti-bladder cancer drugs.

[0060] Compared with the inhibitor XAV939, the 1,3,5-triazine bladder cancer cell inhibitors provided in this invention (represented by compound 4-2) exhibit significant advantages in synthesis, structure, and overall performance. In terms of synthesis methods, this inhibitor possesses the core advantages of a simple route and ease of industrialization: it uses commercially available and low-cost 2,4,6-trichloro-1,3,5-triazine as a starting material, ensuring readily available and cost-effective raw materials; key reaction steps can be carried out only under ice bath or room temperature conditions, and the final coupling reaction conditions are mature and reliable, resulting in a safer and lower-energy-consumption synthesis process with significantly improved operational simplicity; the target product can be obtained through three steps, with compound 4-2 achieving a yield as high as 69%, demonstrating excellent potential for process scale-up; simultaneously, by replacing the readily available phenylboronic acid reagent, a diverse compound library can be rapidly constructed, greatly facilitating structure-activity relationship studies and lead compound optimization, exhibiting excellent derivatization capabilities and structural diversity.

[0061] In terms of structural design, the 1,3,5-triazine bladder cancer cell inhibitors provided by this invention exhibit novel, rational, and highly targeted characteristics: their core chemical skeleton is completely different from XAV939, belonging to a novel chemical entity; based on the concept of computer-aided drug design, this invention precisely optimizes the active pocket of the TNKS2 protein, and the morpholine and pyridine groups contained in the screened 1,3,5-triazine compounds can significantly enhance the binding ability with target proteins and cell permeability, and the design logic is both scientific and targeted; in addition, the introduction of variable aryl(R) provides ample space for molecular structure regulation, and the activity and selectivity of the molecules can be optimized through fine adjustment to further enhance the targeting effect.

[0062] In terms of overall performance, this inhibitor also exhibits the advantages of reliable activity and outstanding potential: the IC50 of the prepared compound 4-2... 50 The effective value was 9.049 μM, comparable to 8.889 μM for XAV939, demonstrating an inhibitory effect on bladder cancer cells of equivalent strength to the positive control XAV939. The highly significant P-values ​​(e.g., 0.00508 for compound 4-2, 0.00014 for compound 4-5, and 0.00004 for compound 4-11) fully demonstrate the high reliability of its inhibitory effect, with a clear mechanism of action, indicating it is not a coincidence. Excellent binding free energy (ΔG) data (e.g., -64.896 kcal / mol for compound 4-2) indicate that this series of compounds has a strong affinity for the TNKS2 target protein, laying a solid foundation for subsequent performance optimization. Simultaneously, the advantageous 1,3,5-triazine skeleton provides a good drug-like basis, facilitating further optimization of pharmacokinetic properties and highlighting its potential for drug development.

[0063] In summary, this invention represents a breakthrough in terms of synthetic feasibility, structural novelty, and reliability of biological data. It provides a TNKS2 inhibitor solution that is easier to produce, structurally novel, and exhibits highly significant inhibitory effects.

[0064] Compared with the prior art, the present invention has the following beneficial effects:

[0065] (1) The present invention provides a bladder cancer cell inhibitor of 1,3,5-triazine compound targeting TNKS2 protein, which has good bladder cancer cell inhibitory activity.

[0066] (2) Among the 1,3,5-triazine compounds prepared in this invention as bladder cancer cell inhibitors, compound 4-2 has an IC50 value of [missing information]. 50 The value was 9.049 μM, while the IC50 of the positive control XAV939 was... 50 With a concentration of 8.889 μM, compound 4-2 exhibits inhibitory activity comparable to the positive control XAV939, achieving an equivalent inhibitory effect on bladder cancer cells.

[0067] (3) Based on the computer-aided drug design method, this invention screened and designed a series of derivatives with 1,3,5-triazine as the backbone. The synthesized 1,3,5-triazine compounds have a better binding mode with the TNKS2 protein pocket, thereby improving the inhibitory activity of the compounds. Through bladder cancer cell inhibitory activity test and calculation, it was shown that the data of 9 compounds were statistically significant. Among them, the statistical significance of compounds 4-1 (p value 0.00508), 4-4 (p value 0.00014), and 4-10 (p value 0.00004) was particularly prominent, indicating that the inhibitory effect of the 1,3,5-triazine compounds prepared by this invention as bladder cancer cell inhibitors is highly reliable.

[0068] (4) Compared with XAV939, the 1,3,5-triazine compound bladder cancer cell inhibitor of the present invention has significant advantages in synthesis. It can be prepared by using low-cost and readily available raw materials as starting materials and through three mild reactions. Not only is the product yield high, but the preparation process is also safe, energy-saving and easy to operate, with outstanding industrialization potential. Detailed Implementation

[0069] This embodiment is implemented based on the technical solution of the present invention, and provides detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiment.

[0070] A bladder cancer cell inhibitor targeting the TNKS2 protein is a 1,3,5-triazine compound with the following structural formula:

[0071]

[0072] Among them, R 1 ~R 5 Each group is independently selected from any one of the following: hydrogen atom, halogen atom, alkyl group, hydroxyl group, ester group, naphthyl group, diphenylamino group, trifluoromethyl group, aldehyde group, ketone group, alkoxy group, nitro group, amino group, carboxyl group, and methylenedioxy group.

[0073] Its preparation method includes the following steps:

[0074] S1: Using 2,4,6-trichloro-1,3,5-triazine as a raw material, it was dissolved in dichloromethane under alkaline conditions and stirred in an ice bath. Then N-(2-aminoethyl)morpholine was added and the reaction was continued with stirring. After the reaction was completed, the precipitated solid was filtered to obtain compound 1.

[0075] S2: Compound 1 was dissolved in methanol and reacted with 2-(2-pyridyl)ethylamine at room temperature under alkaline conditions. A white solid precipitated and was obtained by filtration to give compound 2.

[0076] S3: Using tetra(triphenyl)phosphine palladium as a catalyst, compound 2 is reacted with a phenylboronic acid derivative under alkaline conditions and a nitrogen atmosphere. After the reaction is completed, post-treatment is performed to obtain the bladder cancer cell inhibitor.

[0077] The embodiments of the present invention will be described in detail below.

[0078] Example 1: Preparation of Compounds 4-14

[0079]

[0080] (1) In the first step reaction, 1 mmol of 2,4,6-trichloro-1,3,5-triazine was weighed, dissolved in dichloromethane, 1 mmol of sodium hydroxide was added, and the mixture was stirred in an ice bath for 30 min. Then, 1.0 mmol of N-(2-aminoethyl)morpholine was added, and the mixture was stirred for 2-4 h. The precipitated solid was filtered to obtain compound 1.

[0081] (2) In the second step of the reaction, 1 mmol of compound 1 and 1 mmol of 2-(2-pyridyl)ethylamine were dissolved in methanol and reacted at room temperature for 2-4 h. A white solid was precipitated and filtered to obtain compound 2.

[0082] (3) In the third step, 1 mmol of compound 2 and 1.1 mmol of 2-methylphenylboronic acid were dissolved in tetrahydrofuran, and 0.1 mmol of tetra(triphenyl)phosphine palladium and 1 mmol of NaOH were added. The reaction was carried out at 80 °C for 2 h under a nitrogen atmosphere. The product was purified by column chromatography to obtain N2-(2-morpholinoethyl)-N4-(2-(pyridin-2-yl)ethyl)-6-(o-tolyl)-1,3,5-triazine-2,4-diamine. The yield of the target compound 4-14 as a reddish-brown solid product was 55%.

[0083] Compound 4-14 was characterized by NMR and mass spectrometry, and the data are as follows:

[0084] 1 H NMR(401MHz,Chloroform-d)δ8.50(d,J=5.5Hz,1H),7.80–7.47(m,2H),7.21(dd,J=13.6,7.7Hz,3H),7.07(t,J=6.9Hz,2H),6.14(dd,J= 160.5,66.9Hz,2H),3.92–3.70(m,2H),3.65(t,J=4.7Hz,4H),3.53–3.32(m,2H),3.04–2.91(m,2H),2.56(s,2H),2.43(d,J=13.8Hz,7H).

[0085] 13 C NMR (101MHz, CDCl3) δ165.74,162.28,159.46,149.38,136.42,128.96,125.75,123.44,121.45,66.95,57.23,53.46,40.04,37.09,20.42.

[0086] HRMS(ESI)calcd for C 23 H 29 N7O[M+H] + :420.250635;Found:420.251427. The above analysis results show that the obtained product is the expected product.

[0087] Example 2 Preparation of compound 4-1

[0088]

[0089] Compound 4-1 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0090] 4-1 Yield of yellowish-brown solid product: 69%

[0091] 1 H NMR(401MHz,Chloroform-d)δ8.51(dd,J=13.9,5.4Hz,2H),8.29(d,J=57.5Hz,1H),7.56(d,J=7.9Hz,2H),7.41(p,J=8.7,7.8Hz,2H),7.10(d,J=7.0Hz,2H), 6.64–6.31(m,1H),6.30–6.05(m,1H),3.87–3.81(m,2H),3.66(s,4H),3.47(p ,J=8.7,7.4Hz,2H),3.02(t,J=7.0Hz,2H),2.53(d,J=6.4Hz,2H),2.47(s,4H).

[0092] 13 C NMR (101MHz, CDCl3) δ166.24,162.68,162.58,157.20,156.38,149.42,136.53,1 31.29,128.25,121.63,121.51,66.94,66.91,56.86,53.38,40.23,37.66,37.10.

[0093] HRMS(ESI)calcd for C 22 H 27 N7O[M+H] + :406.234985; Found:406.235334.

[0094] Example 3 Preparation of compound 4-2

[0095]

[0096] Compound 4-2 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0097] 4-2 Yellow oily product yield: 70%

[0098] 1H NMR(401MHz,Chloroform-d)δ8.56(d,J=5.0Hz,1H),8.08(d,J=41.0Hz,2H),7.60(t,J=7.6Hz,1H),7.16(dt,J=13.2,8.2Hz,3H),5.77 (t,J=39.1Hz,2H),4.11(q,J=7.1Hz,2H),3.92(d,J=33.0Hz,2H),3.72(s,4H),3.09(t,J=6.6Hz,2H),2.63–2.54(m,2H),2.49(s,4H).

[0099] 13 C NMR (101MHz, CDCl3) δ167.08,161.70,160.46,155.71,153.05,151.24,149.48,13 6.58,128.39,123.51,121.59,116.78,115.64,66.98,60.45,53.45,40.21,37.72.

[0100] HRMS(ESI)calcd for C 24 H 25 F2N7O[M+H] + :442.216141; Found:442.216902.

[0101] Example 4 Preparation of compound 4-3

[0102]

[0103] Compound 4-3 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0104] 4-3 Yield of yellowish-brown solid product: 66%

[0105] 1 H NMR(401MHz,Chloroform-d)δ8.55(d,J=4.9Hz,1H),8.44–8.11(m,2H),7.59(t,J=7.7Hz,1H),7.38(t,J=7.0Hz,2H),7.22–7. 09(m,2H),5.97–5.58(m,2H),3.88(t,J=28.6Hz,2H),3.46(s,3H),3.09(d,J=7.6Hz,2H),2.53(d,J=31.2Hz,7H),2.17(s,2H).

[0106] 13 C NMR (101MHz, CDCl3) δ166.42,149.43,137.57,129.10,127.83,125.11,123.54,122.26,121.59,63.72,56.07,53.46,50.30,39.76,37.85.

[0107] HRMS(ESI)calcd for C 22 H 26 N7OCL[M+H] + :440.196013; Found:440.195801.

[0108] Example 5 Preparation of compound 4-4

[0109]

[0110] Compound 4-4 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0111] 4-4 Yield of yellowish-brown solid product: 55%

[0112] 1 H NMR (401MHz, DMSO-d6) δ8.50(d,J=4.4Hz,1H),7.82–7.67(m,3H),7.63(d,J=7.7Hz,1H),7.62–7.48(m,2H),7.25(dt,J=18. 1,6.0Hz,2H),6.72(d,J=7.8Hz,1H),3.58(tq,J=12.7,6.2,5.0Hz,8H),2.97(d,J=7.6Hz,1H),2.42(dt,J=21.1,6.3Hz,7H).

[0113] 13 C NMR (101MHz, DMSO) δ165.83,159.50,159.45,149.58,149.53,136.94,136.41,123.73,123.63,122.03,114.8 7,66.86,66.67,57.78,57.44,53.76,40.75,40.54,40.33,40.12,39.91,39.71,39.50,37.84,37.46,37.26.

[0114] HRMS(ESI)calcd for C 22 H 27 N7O2[M+H]+ :422.229900; Found:422.230164.

[0115] Example 6 Preparation of compounds 4-5

[0116]

[0117] Compounds 4-5 were synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0118] 4-5 Yellowish-brown solid product yield: 73%

[0119] 1 H NMR(401MHz,Chloroform-d)δ8.56(d,J=4.9Hz,1H),8.37–8.05(m,2H),7.58(t,J=7.7Hz,1H),7.28(t,J=7.6Hz,4H),7.14(d,J=14.4Hz,6H),7.08(d,J=7 .5Hz,4H),6.17–5.62(m,2H),3.92(d,J=30.4Hz,2H),3.72(s,4H),3.54(dd, J=56.1,14.0Hz,2H),3.09(t,J=7.2Hz,2H),2.62–2.55(m,2H),2.49(s,4H).

[0120] 13 C NMR(101MHz,Chloroform-d)δ166.07,159.65,154.68,149.44,147.28,136.48,132.34 ,129.40,125.17,123.64,123.50,121.81,121.49,66.99,57.50,53.51,40.27,37.79.

[0121] HRMS(ESI)calcd for C 34 H 36 N8O[M+H] + :573.309464; Found:573.307990.

[0122] Example 7 Preparation of compounds 4-6

[0123]

[0124] Compounds 4-6 were synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0125] 4-6. Yield of dark brown solid product: 55%

[0126] 1 H NMR(401MHz,Chloroform-d)δ8.54(d,J=4.9Hz,1H),8.34(s,1H),8.22(s,1H) ,7.58(td,J=7.8,1.9Hz,1H),7.19–7.08(m,2H),6.92(d,J=8.3Hz,2H),5.84(s ,1H),5.66(s,1H),3.96(s,1H),3.84(s,4H),3.74–3.70(m,4H),3.67(d,J=7. 0Hz, 2H), 3.56 (s, 1H), 3.12–3.04 (m, 2H), 2.58 (d, J = 10.5Hz, 2H), 2.48 (s, 3H).

[0127] 13 C NMR (101MHz, CDCl3) δ166.78,163.30,149.42,136.54,130.94,129.73,129.38,123.54,121 .53,113.51,77.42,77.11,76.79,66.97,58.31,55.38,53.47,40.24,39.00,38.16,28.96.

[0128] HRMS(ESI)calcd for C 23 H 29 N7O2[M+H] + :436.245550; Found:436.245470.

[0129] Example 8 Preparation of compounds 4-7

[0130]

[0131] Compounds 4-7 were synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0132] 4-7 Yield of reddish-brown solid product: 66%

[0133] 1H NMR(401MHz,Chloroform-d)δ8.53(d,J=4.9Hz,2H),8.45–8.39(m,1H),8.29(d,J=7.8Hz,1H),8 .05(d,J=8.1Hz,3H),7.56(t,J=7.6Hz,2H),7.11(dd,J=12.9,7.0Hz,3H),5.95(d,J=15.7Hz,1H) ,5.79(s,1H),5.27(s,1H),3.96(d,J=6.6Hz,1H),3.91(s,6H),3.80(d,J=5.3Hz,1H),3.70(s,6 H),3.67–3.63(m,2H),3.55(s,1H),3.50–3.41(m,1H),3.11–3.03(m,3H),2.56(d,J=9.1Hz,3H).

[0134] 13 C NMR (101MHz, CDCl3) δ167.89,166.21,159.43,150.26,137.32,133.14,129.4 4,128.21,123.48,121.53,67.88,58.13,54.06,51.23,41.35,38.34,37.11.

[0135] HRMS(ESI)calcd for C 24 H 29 N7O3[M+H] + :442.216132; Found:442.216141.

[0136] Example 9 Preparation of compounds 4-8

[0137]

[0138] Compounds 4-8 were synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0139] 4-8 Yield of reddish-brown solid product: 76%

[0140] 1H NMR(401MHz,Chloroform-d)δ8.52(d,J=5.0Hz,2H),7.58(t,J=7.9Hz,3H),7.12(dd,J=7.9,4.3Hz,5H),6.31–5.88(m,3H),3.93–3.83(m,3H),3.82(s,1H) ),3.80–3.69(m,5H),3.68(d,J=4.8Hz,6H),3.51(t,J=5.8Hz,3H),3.39(q,J =5.7Hz,1H),3.04(d,J=6.3Hz,3H),2.55(t,J=6.1Hz,3H),2.48–2.43(m,6H).

[0141] 13 C NMR (101MHz, CDCl3) δ164.19,161.13,159.83,157.41,150.74,147.49,135.19,124.04,122.13,66.27,56.75,52.59,41.46,33.13.

[0142] HRMS(ESI)calcd for C 22 H 25 N7OF2[M+H] + :442.216141; Found:442.216013.

[0143] Example 10 Preparation of compounds 4-9

[0144]

[0145] Compounds 4-9 were synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0146] 4-9 Yellowish-brown solid product yield: 67%

[0147] 1H NMR(401MHz,Chloroform-d)δ9.30–9.06(m,1H),8.71–8.49(m,2H),8.35–8.26(m,1H),7.63–7.54(m,2H),7.22–7.12(m,2H),5.86(t,J=35.8Hz,2 H),3.92(ddd,J=32.9,16.5,6.4Hz,2H),3.75(t,J=4.8Hz,4H),3.67–3.4 0(m,2H),3.19–3.04(m,2H),2.94(s,1H),2.87(s,1H),2.67–2.53(m,4H).

[0148] 13 C NMR (101MHz, CDCl3) δ166.26,161.31,160.17,159.37,149.39,148.38,139.18,136.64,13 3.90,129.10,125.63,125.51,123.51,123.28,121.62,66.92,57.31,53.49,53.36,40.22,37.08

[0149] HRMS(ESI)calcd for C 22 H 26 N8O3[M+H] + :451.220063; Found:451.219918.

[0150] Example 11 Preparation of compounds 4-10

[0151]

[0152] Compound 4-10 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0153] 4-10 Dark brown solid product yield: 78%

[0154] 1H NMR(401MHz,Chloroform-d)δ8.63(d,J=85.9Hz,2H),8.13–7.79(m,3H),7.52(dq,J=23.3,7.8Hz,4H),7.10(t,J=8.3Hz,2H),5. 94(d,J=150.7Hz,2H),3.87(d,J=42.6Hz,2H),3.68(s,4H),3.46(d,J=38.4Hz,2H),3.20–2.92(m,2H),2.52(s,2H),2.44(s,4H).

[0155] 13 C NMR(101MHz,Chloroform-d)δ165.89,161.31,159.49,156.92,149.47,136.45,135.76,134.0 5,130.39,128.37,126.35,125.88,125.15,121.51,66.95,57.22,53.44,42.15,40.06,37.12.

[0156] HRMS(ESI)calcd for C 26 H 29 N7O[M+H] + :456.250635; Found:456.251598.

[0157] Example 12 Preparation of Compounds 4-11

[0158]

[0159] Compound 4-11 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0160] 4-11 Yield of dark brown solid product: 80%

[0161] 1H NMR(401MHz,Chloroform-d)δ8.88(d,J=44.1Hz,1H),8.55(dd,J=21.2,4.8Hz,2H),8.41(d,J=46.2Hz,1H) ,7.96(s,1H),7.88(d,J=9.3Hz,2H),7.58(d,J=7.9Hz,2H),7.51(d,J=7.2Hz,2H),7.13(q,J=6.2Hz,2H),6 .00–5.65(m,2H),4.08(d,J=18.6Hz,1H),3.92–3.80(m,2H),3.74(s,4H),3.64(d,J=30.2Hz,2H),3.55–3. 34(m,1H),3.14(d,J=16.0Hz,2H),3.03(t,J=6.4Hz,1H),2.61(s,2H),2.52(s,2H),2.43(d,J=13.2Hz,1H).

[0162] 13 C NMR (101MHz, CDCl3) δ166.63,165.02,160.09,148.60,136.94,134.73,133.62,131.65,130.97,129.2 7,128.80,127.16,126.13,125.63,123.50,121.59,121.49,66.97,57.41,53.49,39.84,37.71,37.04.

[0163] HRMS(ESI)calcd for C 26 H 29 N7O[M+H] + :456.250635; Found:456.250822.

[0164] Example 13 Preparation of compounds 4-12

[0165]

[0166] Compound 4-12 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0167] 4-12 Yellowish-brown solid product yield: 68%

[0168] 1H NMR(401MHz,Chloroform-d)δ8.75–8.10(m,2H),7.63(dt,J=28.4,7.7Hz,3H),7.14(dt,J=14.6,8.4Hz,2H),6.28–5.66(m,2H),4.04–3.8 3(m,2H),3.70(dt,J=18.5,4.5Hz,6H),3.62–3.32(m,2H),3.07(dt,J=25.6,6.6Hz,2H),2.62–2.53(m,2H),2.48(dd,J=13.7,6.0Hz,4H).

[0169] 13 C NMR (101MHz, CDCl3) δ166.23,163.13,159.73,153.29,150.28,136.55,129.3 3,126.15,123.49,121.65,67.69,54.12,53.35,51.91,40.89,38.70,37.70.

[0170] HRMS(ESI)calcd for C 24 H 25 N7O3[M+H] + :450.224814; Found:450.225404.

[0171] Example 14 Preparation of compounds 4-13

[0172]

[0173] Compound 4-13 was synthesized using different phenylboronic acid derivatives according to the method in Example 1. The NMR and mass spectrometry results are as follows:

[0174] 4-13 Yellowish-brown solid product yield: 77%

[0175] 1 H NMR (401MHz, Chloroform-d) δ8.54(d,J=4.9Hz,1H),7.87(t,J=51.5Hz,2H),7.58(t,J=7.6Hz,1H),7.19–7.08(m,2H),6.84(s,1H),5.73(d,J= 43.4Hz,2H),3.88(t,J=22.6Hz,2H),3.73–3.69(m,4H),3.51(d,J=45.1Hz,2H),3.08(t,J=6.5Hz,2H),2.57(q,J=6.6,6.0Hz,2H),2.48(s,4H).

[0176] 13 C NMR (101MHz, CDCl3) δ166.26,165.57,164.64,159.51,152.68,149.43,137.21,13 2.24,128.46,124.24,122.60,121.53,67.65,57.26,53.48,40.97,39.74,37.60.

[0177] HRMS(ESI)calcd for C 23 H 26 N7O[M+H] + :474.222370; Found:472.222894.

[0178] The obtained compounds underwent initial screening. In this stage, all candidate compounds were dissolved in DMSO solution and their activity was evaluated at a concentration of 10 μM. Based on the OD value determination results, combined with statistical analysis (P < 0.05) and structural characterization, five potentially active compounds (4-1, 4-2, 4-5, 4-9, and 4-10) and the positive control XAV-939 were ultimately selected for further studies. To accurately assess the compound activity, concentration gradient experiments were performed on the screened compounds, and the IC50 values ​​were calculated. The results are shown in Table 2.

[0179] This invention uses the CCK8 method to test the inhibitory effect of the target compound on the proliferation of bladder cancer cells. The test method is as follows:

[0180] (1) Prepare 100 μL of cell suspension in a 96-well plate. Pre-incubate the plate in an incubator for 24 hours (37°C, 5% CO2).

[0181] (2) Add 10 μL of blank concentration, 2.5 μM / L, 5 μM / L, 10 μM / L, 20 μM / L and 40 μM / L of the test substance to the culture plate.

[0182] (3) Incubate the culture plate in an incubator for 24 hours.

[0183] (4) Add 10 μL of CCK solution to each well.

[0184] (5) Incubate the culture plate in an incubator for 4 hours.

[0185] (6) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0186] Table 1: Chemical structure, p-value, and binding free energy of the compounds

[0187]

[0188]

[0189]

[0190] Table 2: Chemical structures and IC50 values ​​of the five compounds and the positive control XAV-939 50 value

[0191]

[0192]

[0193] Experimental results show that compound 4-2 prepared in this invention exhibits the most significant inhibitory effect, with an IC50 value of [missing value]. 50 The value was similar to that of the positive control XAV-939, indicating that it has similar biological activity.

Claims

1. A bladder cancer cell inhibitor targeting the TNKS2 protein, characterized in that, The bladder cancer cell inhibitor is a 1,3,5-triazine compound with the following structural formula: Among them, R 1 ~R 5 Each group is independently selected from any one of the following: hydrogen atom, halogen atom, alkyl group, hydroxyl group, ester group, naphthyl group, diphenylamino group, trifluoromethyl group, aldehyde group, ketone group, alkoxy group, nitro group, amino group, carboxyl group, and methylenedioxy group.

2. The bladder cancer cell inhibitor targeting TNKS2 protein according to claim 1, characterized in that, The structural formula of the bladder cancer cell inhibitor includes any of the following:

3. A method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to any one of claims 1-2, characterized in that, Includes the following steps: S1: Using 2,4,6-trichloro-1,3,5-triazine as a raw material, it was dissolved in dichloromethane under alkaline conditions and stirred in an ice bath. Then N-(2-aminoethyl)morpholine was added and the reaction was continued with stirring. After the reaction was completed, the precipitated solid was filtered to obtain compound 1. S2: Compound 1 was dissolved in methanol and reacted with 2-(2-pyridyl)ethylamine at room temperature under alkaline conditions. A white solid precipitated and was filtered to obtain compound 2. S3: Using tetra(triphenyl)phosphine palladium as a catalyst, compound 2 is reacted with a phenylboronic acid derivative under alkaline conditions and a nitrogen atmosphere. After the reaction is completed, post-treatment is performed to obtain the bladder cancer cell inhibitor.

4. The method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to claim 3, characterized in that, In steps S1, S2, and S3, the alkaline conditions are provided by adding an alkali, which includes any one of sodium hydroxide, potassium hydroxide, sodium bicarbonate, sodium carbonate, potassium bicarbonate, potassium carbonate, and triethylamine.

5. The method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to claim 3, characterized in that, In step S1, the molar ratio of 2,4,6-trichloro-1,3,5-triazine, N-(2-aminoethyl)morpholine, and the base is 1:1:1 to 1:2:2; the temperature of the ice bath is -10 to 10°C; the stirring time in the ice bath is 20 to 40 minutes; the stirring speed is 200 to 600 rpm; and the reaction is continued for 1 to 6 hours at a stirring speed of 200 to 600 rpm.

6. The method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to claim 3, characterized in that, In step S2, the molar ratio of compound 1, 2-(2-pyridyl)ethylamine, and base is 1:1:1 to 1:2:2, the room temperature is 0-25℃, and the reaction time is 1-6h.

7. The method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to claim 3, characterized in that, In step S3, the molar ratio of compound 2, phenylboronic acid derivative, base, and tetra(triphenyl)phosphine palladium is 1:1:2:0.05-1:1.5:3:0.1, the reaction temperature of compound 2 and phenylboronic acid derivative is 80-110℃, and the reaction time is 1-6h.

8. The method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to claim 3, characterized in that, In step S3, the chemical formula of the phenylboronic acid derivative is: Among them, R 1 ~R 5 Each group is independently selected from any one of the following: hydrogen atom, halogen atom, alkyl group, hydroxyl group, ester group, naphthyl group, diphenylamino group, trifluoromethyl group, aldehyde group, ketone group, alkoxy group, nitro group, amino group, carboxyl group, and methylenedioxy group.

9. The method for preparing a bladder cancer cell inhibitor targeting TNKS2 protein according to claim 3, characterized in that, In step S3, the post-processing includes column chromatography, and the eluent used is a mixture of ethyl acetate and petroleum ether, wherein the volume ratio of ethyl acetate to petroleum ether is 1:20-1:

5.

10. The use of a bladder cancer cell inhibitor targeting the TNKS2 protein as described in any one of claims 1-2 in the field of preparing anti-bladder cancer drugs.

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