CN115611867B - (1, 1-Trichloro-2) carbamate derivative and preparation method and application thereof - Google Patents

Abstract

The present invention relates to (1,1,1-trichloro-2) carbamate derivatives, and the present invention also relates to their preparation methods and related medical uses. To achieve the above object, the present invention provides a (1,1,1-trichloro-2) carbamate derivative, the general structural formula of which is shown in formula (I): Compared with the prior art, the tumor inhibitory activity of the target compound of the present invention is greatly enhanced compared with apcin, and it shows good inhibitory effect in multiple tumor cell lines; it also has a good microtubule aggregation inhibitory effect, opening up a new way to find a dual-function cancer treatment method targeting the APC pathway and microtubule inhibition.

Description

(1,1,1-Trichloro-2-carbamate derivatives and preparation methods and applications thereof

Technical Field

The present invention relates to (1,1,1-trichloro-2)carbamate derivatives, and also relates to their preparation methods and related medical uses.

Background Art

Malignant tumors are one of the main causes of death in most countries in the world today, and they pose a serious threat to human health. Surveys show that the incidence of malignant tumors is increasing year by year. With the rapid development of molecular biology, scientists have a deeper and more comprehensive understanding of the signaling pathways and targets related to the occurrence and development of malignant tumors, and have achieved important results in designing anti-tumor drugs based on these signaling pathways and targets. As the view that "malignant tumors are a type of cell cycle disease" has been widely recognized, research on anti-tumor drugs targeting cell cycle regulation has become a hot topic.

Cell cycle dysregulation is a common feature of human cancer, and the accumulation of mutations in tumor cells can lead to abnormal proliferation, genomic instability, and chromosomal instability. Based on this phenomenon, many drugs have been developed to inhibit different cell cycle stages for cancer treatment. Inhibiting the initial stage of the cell cycle may produce living quiescent cells, while targeting mitosis has several possibilities for killing cancer cells. Targeting mitosis is a long-tested approach, and new drugs targeting mitotic spindle microtubule elements and non-microtubule effectors of mitosis are currently being widely studied. Among them, drugs that affect the mitotic spindle are recognized components of many cancer treatment regimens.

Normally, the cell mitosis process is subject to complex and delicate regulation, and ultimately the replicated genome is divided into two identical daughter cells. Mitosis is divided into five different phases: prophase, prometaphase, metaphase, anaphase, and telophase. Prophase transitions from G2 to mitosis, accompanied by the condensation of chromosomes and the disintegration of the nuclear envelope. In prometaphase, the chromosome centromeres attach to the spindle microtubules through a search and capture mechanism. In metaphase, the chromosomes converge on the equatorial plate, equidistant from the centrosome. The centromere microtubules shorten in anaphase, separating the sister chromatids and moving them to opposite poles, which is called separation. Finally, the cell is divided into two during telophase and cytokinesis, forming two daughter cells (Figure 1). A large number of small molecule compounds have been designed and studied for proteins related to the mitotic phase.

Anti-microtubule drugs are the first treatment method targeting mitosis. Microtubules play an important role in maintaining cell morphology, cell division, signal transduction, and material transport. Microtubules polymerize into spindles in the early stage of mitosis, and the spindles pull chromosomes to move toward the two poles in mitosis and enter two cells to complete cell proliferation. Because microtubules play an extremely important role in cell division, they have become one of the important targets of anticancer drugs, and microtubule inhibitors have been clinically proven to have significant therapeutic effects on a variety of malignant tumors. Microtubule polymerizers (including paclitaxel and docetaxel) and microtubule depolymerizers (including vinorelbine) target primary tubulin, destroy the kinetic stability of microtubule polymerization-depolymerization, thereby blocking the cell cycle in the mitotic phase and inducing tumor cell apoptosis. However, these drugs not only act on proliferating tumor cells, but also show obvious side effects on normal cells without abnormal proliferation. In addition, the expression of multidrug resistance (MDR) proteins and tubulin isotypes (such as tubulin mutations) is also related to microtubule inhibitory drugs.

Therefore, the identification of novel mitotic drug targets other than tubulin has attracted widespread attention recently. In recent years, several mitotic targets have been explored, including drugs targeting microtubules, kinases, motor proteins, and multiprotein complexes. Among them, the ubiquitin-proteasome (UPS) pathway-mediated substrate ubiquitination and its subsequent orderly degradation process runs through the entire cell's plasma membrane system and is necessary for cells to undergo mitosis throughout the cell cycle. Moreover, it is of great significance in the treatment of various diseases caused by ubiquitin system disorders, especially malignant tumors. In general, the ubiquitination process requires the joint participation of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). In this process, ubiquitin (a highly conserved 76-residue protein) is first connected to the ubiquitin-activating enzyme (E1) in an ATP-powered reaction. The activated ubiquitin is then transferred to a small ubiquitin-conjugating enzyme (E2) to form a thioester-linked E2-ubiquitin intermediate (E2~Ub). E2, acting alone or in conjunction with an E3 ubiquitin ligase, conjugates ubiquitin to the ε-amino group of a lysine residue in the substrate protein, forming an isopeptide bond. Strict control of these seemingly simple sequential actions of E1-E2-E3 allows for accurate and appropriately timed ubiquitination/proteolysis.

The specificity of protein degradation by the ubiquitination system is determined by the ubiquitin ligase E3. Among them, the anaphase promoting complex (APC) is the largest E3 ubiquitin ligase, which is composed of at least 14 subunits and a co-activator. After activation by cyclin 20 (Cdc20) or Cdc20 homolog 1 (Cdh1), two different E3 ubiquitin ligase complexes APC ^Cdc20 or APC ^Cdh1 are formed (Figure 2). APC ^Cdc20 destroys key regulators in the cell growth cycle during the transition from metaphase to anaphase and regulates the cell cycle process, while APC ^Cdh1 plays an important role mainly in the G1 phase.

More and more studies have shown that Cdc20 promotes the occurrence and development of tumors. High expression of Cdc20 has been found in many tumors, and its expression level is closely related to the pathological degree and prognosis of tumor patients. The higher the expression level of Cdc20, the greater the risk. In short, the expression level of Cdc20 is an effective prognostic indicator for tumor patients, providing a basis for Cdc20 as a new target for the development of anti-tumor drugs. In contrast, another co-activator of APC/C, Cdh1, is considered a tumor suppressor.

At present, with the deepening of research on Cdc20, more and more APC ^Cdc20 substrates have been discovered (Table 1). Some substrates are directly or indirectly involved in the cell division process. Cdc20 can regulate the ubiquitination or degradation process of these substrates, thereby regulating the cell cycle process. Therefore, the discovery of more APC ^Cdc20 substrates will help to understand the molecular mechanism of Cdc20 in regulating cell cycle progression.

Table 1 APCCdc20 downstream substrates and their functions

Using compounds to inhibit the Cdc20 pathway is a new idea for treating tumors. Cdc20 targeted inhibitors can directly act on Cdc20 and inhibit the activity of the APC ^Cdc20 complex. Apcin is the only reported Cdc20-specific inhibitor. It acts on the WD40 region at the N-terminus of Cdc20, blocking the binding of Cdc20 to downstream substrates and inhibiting the ubiquitination and degradation of downstream substrates (Figure 3). In addition, many compounds have been reported to indirectly affect the Cdc20 pathway and exert anti-tumor effects. In addition, the active ingredients in some natural products also have the function of regulating the Cdc20 pathway (Table 2)

Table 2 Structure and function of some Cdc20 inhibitors

Premature exit from mitosis by mitotic slippage is considered one of the major mechanisms by which cells develop resistance to antimitotic drugs. Both mechanisms of action of microtubule inhibitors involve drug binding to vinca, taxane, or colchicine sites, resulting in unattached centromeres that activate the SAC, and, as a result, mitotic cell death or mitotic slippage is observed.

In the normal cell cycle, mitotic exit is driven by APC/C–Cdc20-dependent degradation of cyclin B1. Inhibition of mitotic progression triggers the SAC and inhibits APC/C–Cdc20. However, this inhibitory effect is transient and slow, and cyclin B1 may be gradually degraded despite failure to pass the mitotic checkpoint. Cells that escape mitotic cell death can die in subsequent cell cycle stages, arrest in the tetraploid state, or undergo several rounds of division. Therefore, it is foreseeable that completely blocking mitotic exit may be an effective strategy to induce mitotic cell death. Inhibition of cyclin B1 degradation in vivo by genetic ablation of Cdc20 has been shown to be more effective in killing tumor cells than conventional compounds targeting spindle assembly. On the other hand, classical microtubule-inhibiting antimitotic drugs (vincristine or paclitaxel) induce only partial responses in aggressive tumors, while Cdc20 ablation leads to complete mitotic arrest, resulting in massive apoptotic cell death and complete tumor elimination in vivo. Meanwhile, the study showed that in vitro, tumor cells without Cdc20 were unable to escape metaphase arrest and died by mitotic cell death within 6-30 hours. Importantly, mitotic exit inhibition also affected pRb-null, p53-null or SAC-deficient cells, indicating its broad utility and complementing the poor efficacy of microtubule inhibitors in cells with SAC inactivation. Overall, these results suggest that targeting Cdc20 to inhibit Cyclin B1 degradation may be very effective in killing tumor cells.

Therefore, designing small molecule inhibitors that simultaneously target Cdc20, which inhibits mitotic exit, and tubulin, which activates the SAC, is an effective approach to trigger mitotic cell death. The purpose of mitotic exit inhibitors is to prevent the consumption of cyclin B, which causes cells to be arrested in mitosis and die. A preclinical study demonstrated that knocking out Cdc20 was more effective in killing resistant cells compared to paclitaxel treatment. Another study combined the APC inhibitor proTAME with two antimitotic drugs, paclitaxel or MLN-8054, resulting in activation of apoptosis in cancer cells. By reviewing clinical trials of antimitotic agents, it is clear that better cancer treatment effects can be achieved when dual-target inhibitors are designed.

Current anti-mitotic strategies focus on SAC activation, and the main cancer treatment approach is to use microtubule drugs to activate the SAC and induce apoptosis, but their effectiveness is limited by background Cyclin B1 degradation and mitotic slippage due to residual APC/C-Cdc20 activity. Cancer cells, in particular, display high rates of mitotic slippage due to mutations that disrupt the SAC, making treatments unsuccessful. Therefore, compounds targeting factors downstream of the checkpoint, such as Cdc20, are more effective in killing cells, especially in cells that are prone to slippage and resistant to apoptosis, compared with other mitotic inhibitors, including MIA and kinesin inhibitors.

Apcin is the only reported CDC20-specific inhibitor so far. Its anti-tumor activity is weak and cannot be applied clinically. This may be related to the extensive protein-protein interaction between CDC20 and its substrates.

Currently, there are no reports of anti-tumor compounds with dual functions targeting CDC20 and microtubule network. This "dual-function strategy" can reduce intranuclear replication and prevent background Cyclin B1 proteolysis, thereby enhancing the blocking effect of mitosis, while effectively preventing the occurrence of mitotic slippage, which can increase the sensitivity of cancer cells to drugs and open up new avenues for the development of cancer treatments based on microtubule inhibitors.

Summary of the invention

The present invention aims to provide a novel (1,1,1-trichloro-2)carbamate derivative, a preparation method of the compound and its application as a possible anti-liver cancer drug.

To achieve the above object, the technical solution of the present invention is:

A (1,1,1-trichloro-2-carbamate derivative, the general structural formula of which is shown in formula (I-III):

n is 0-1;

X is O;

Wherein, _R1 is selected from hydrogen, aromatic ring, substituted aromatic ring, aromatic heterocycle, substituted aromatic heterocycle, aliphatic heterocycle, substituted aliphatic heterocycle, C1-C4 open chain amine, C1-C6 straight chain or branched alkoxy, C1-C6 straight chain or branched alkane, C3-C6 aliphatic ring and substituted C3-C6 aliphatic ring, aliphatic lenalidomide substituted C1-C8 straight chain or branched alkyl, lenalidomide substituted C1-C8 straight chain or branched alkoxyalkyl.

In formula I, _R2 , _R3 , and _R4 are independently selected from hydrogen, amino, hydroxyl, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 alkylamino, and the phenyl portion of a quinoline ring; Y is selected from N and CH; when Y is N, R2, R3, and R4 are not simultaneously hydrogen;

In the structural formula II-III, Y is selected from N, CH; R ₅ and R ₆ are independently selected from amino, H, halogen, C ₁ -C ₈ alkyl, C ₁ -C ₈ alkoxy, C ₁ -C ₈ haloalkyl, C ₁ -C ₈ alkylamino,

In a preferred embodiment, the aromatic ring is selected from benzene, naphthalene and anthracene; the aromatic heterocycle is selected from pyrrole, furan, thiophene, imidazole, thiazole, oxazole, pyrazole, isoxazole, thiadiazole, oxadiazole, triazole, tetrazole, pyridine, pyrimidine, pyrazine, pyridazine, purine, quinoline, isoquinoline, indole, acridine and carbazole; the aliphatic heterocycle is selected from pyrrolidine, piperazine, piperidine, morpholine and tetrahydrofuran; and the substituents in the substituted aromatic ring, substituted aromatic heterocycle and substituted aliphatic heterocycle are selected from C1-C6 straight chain or branched alkoxy, C1-C6 straight chain or branched alkyl, C1-C6 hydroxyalkyl, hydroxyl, amino, nitro, haloalkyl and halogen.

In a preferred embodiment, the substituents in the substituted aromatic ring, substituted aromatic heterocycle and substituted aliphatic heterocycle are selected from methyl, ethyl, propyl, isopropyl, methoxy, hydroxyethyl, hydroxyl, amino, nitro, trifluoromethyl and halogen.

In a preferred embodiment, the (1,1,1-trichloro-2-carbamate derivative has a general structural formula as shown in Formula I:

Where n is 1;

X is O;

_R1 is selected from hydrogen, C1-C4 straight or branched alkoxy, C1-C4 straight or branched alkane, C3-C6 aliphatic ring and substituted C3-C6 aliphatic ring, benzene ring, substituted benzene ring, aromatic heterocycle, substituted aromatic heterocycle, aliphatic heterocycle, substituted aliphatic heterocycle;

The aromatic heterocycle is selected from pyrrole, imidazole, thiazole, oxazole, pyrazole, pyridine, pyrimidine, and indole;

The aliphatic heterocycle is selected from pyrrolidine, piperazine, piperidine, and morpholine;

The substituents in the substituted aromatic ring, substituted aromatic heterocycle, and substituted aliphatic heterocycle are selected from methyl, ethyl, methoxy, hydroxyethyl, amino, nitro, trifluoromethyl, and halogen;

_R2 , _R3 , _R4 are selected from amino, H, =O, halogen, _C1 - _C3 alkyl, _C1 - _C3 alkoxy, phenyl part of quinoline ring; Y is selected from N, CH; _R2 , _R3 , _R4 are not hydrogen at the same time.

In a preferred embodiment, the (1,1,1-trichloro-2-carbamate derivative has a general structural formula as shown in Formula II:

Where n is 0-1;

X is O;

_R1 is selected from one of C1-C4 straight chain or branched alkoxy, C1-C4 straight chain or branched alkane, C3-C6 aliphatic ring and substituted C3-C6 aliphatic ring benzene ring, substituted benzene ring, aromatic heterocycle, substituted aromatic heterocycle, aliphatic heterocycle and substituted aliphatic heterocycle.

The aromatic heterocycle is selected from pyrrole, imidazole, thiazole, oxazole, pyrazole, pyridine, pyrimidine, and indole;

The aliphatic heterocycle is selected from pyrrolidine, piperazine, piperidine, and morpholine;

The substituents in the substituted aromatic ring, substituted aromatic heterocycle, and substituted aliphatic heterocycle are selected from methyl, ethyl, methoxy, hydroxyethyl, amino, nitro, trifluoromethyl, and halogen;

_R5 and _R6 are selected from amino, H, halogen, _C1 - _C7 alkyl, _C1 - _C7 alkoxy, _C1 - _C7 haloalkyl, _C1 - _C7 alkylamino,

Y is N, CH.

In a preferred embodiment, the (1,1,1-trichloro-2-carbamate derivative has a general structural formula as shown in Formula III:

Where n is 1;

X is O;

_R1 is selected from one of C1-C4 straight chain or branched alkoxy, C1-C4 straight chain or branched alkane, C3-C6 aliphatic ring and substituted C3-C6 aliphatic ring benzene ring, substituted benzene ring, aromatic heterocycle, substituted aromatic heterocycle, aliphatic heterocycle and substituted aliphatic heterocycle.

The aromatic heterocycle is selected from pyrrole, imidazole, thiazole, oxazole, pyrazole, pyridine, pyrimidine, and indole;

The aliphatic heterocycle is selected from pyrrolidine, piperazine, piperidine, and morpholine;

The substituents in the substituted aromatic ring, substituted aromatic heterocycle, and substituted aliphatic heterocycle are selected from methyl, ethyl, methoxy, hydroxyethyl, amino, nitro, trifluoromethyl, and halogen;

_R5 and _R6 are selected from amino, H, halogen, Y is N, CH.

In a preferred embodiment, the (1,1,1-trichloro-2-carbamate derivative has the following structural formula:

The present invention also provides a method for synthesizing the compounds of formula (I), formula (II) and formula (III), and the synthetic routes are as follows:

The synthesis steps of the (1,1,1-trichloro-2-carbamate derivatives include:

1. Reacting a hydroxy-substituted compound with p-nitrophenyl chloroformate in an anhydrous aprotic solvent to obtain an intermediate;

2. The intermediate reacts with aqueous ammonia in a mixed solvent of halogenated alkane and methanol to obtain a carbamate product;

3. The carbamate product is refluxed with chloral hydrate at 80-100°C to obtain the (1,1,1-trichloro-2)carbamate part;

4. The (1,1,1-trichloro-2-carbamate part is first chlorinated with thionyl chloride and then reacted with amine to obtain the target compound.

Preferably, the molar feed ratio of the reaction in step 1 is hydroxy-substituted compound: p-nitrophenyl chloroformate: chloral hydrate = 1: (1.2-1.5): (8-12).

Preferably, step 1 is reacted for 4-12 h to obtain an intermediate.

Preferably, a weak base may be added to catalyze the reaction in step 1 to accelerate the reaction process.

Preferably, the weak base is any one of triethylamine and diisopropylethylamine or a combination thereof, and the molar feed ratio is: hydroxyl substituted compound: base = 1:1.2-1:1.5.

Preferably, the anhydrous aprotic solvent is dichloromethane.

Preferably, in the mixed solvent of halogenated alkane and methanol in step 2, the mass ratio of halogenated alkane to methanol is 1:1-5:1.

Preferably, the concentration of aqueous ammonia in step 2 is 10-35%.

Preferably, the molar feed ratio of step 3 reaction is: carbamate: chloral hydrate = 1:8-1:12

Preferably, the reflux time in step 3 is 6-48h.

Preferably, the reaction temperature in step 3 is 100°C.

Preferably, step 4 is reacted at a temperature of rt-50°C.

Preferably, in step 4, the (1,1,1-trichloro-2-carbamate portion (1 equivalent) is first dissolved in an anhydrous aprotic solvent, chlorinated with thionyl chloride (5 equivalents), and then subjected to a substitution reaction with a raw material amine (1.2 equivalents) in an anhydrous dichloromethane: anhydrous acetonitrile (1:1) mixed solvent to generate the corresponding target compound.

The present invention also provides the use of the above-mentioned (1,1,1-trichloro-2-carbamate derivatives in the preparation of drugs for treating breast cancer, melanoma, lung adenocarcinoma, hepatocellular carcinoma, cervical cancer and ovarian cancer.

The present invention is further explained below:

The present invention is based on the structure of the cdc20 specific inhibitor apcin, and introduces different substituent groups on its pyrimidine ring to obtain a variety of derivatives (structural core I). It is found that after the introduction of chlorine atoms, the compound has more interaction sites with cdc20, the affinity is enhanced, and its inhibitory effect on tumor cells is greatly enhanced.

After the pyrimidine in the apcin structure is replaced by purine (structural core II) or benzopyrazole (structural core III), the inhibitory effect on tumor cells is further enhanced. On the basis of maintaining a certain inhibitory effect on cdc20, the introduction of purine or benzopyrazole enhances the interaction between the compound and tubulin dimers, resulting in microtubule aggregation inhibition. The compound thus has a dual-function anti-tumor effect and is expected to become a new anti-tumor lead compound.

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

1. The present invention enriches the structure of cdc20 inhibitors based on apcin and obtains three structural mother cores;

2. The tumor inhibitory activity of the target compound of the present invention is much stronger than that of apcin, and it shows good inhibitory effect in multiple tumor cell lines;

3. Through the study of the anti-tumor activity mechanism of the compound, the present invention found that in addition to the original cdc20 inhibitory activity of apcin, the new compound also has a good microtubule aggregation inhibitory effect, which opens up a new way to find a dual-function cancer treatment method targeting the APC pathway and microtubule inhibition.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a simplified diagram of the mitotic process and related target proteins and complexes;

Figure 2 shows that the APC/C RING E3 ubiquitin ligase binds to its co-activator Cdc20/Cdh1 to promote substrate ubiquitination;

Figure 3 shows the D-box binding pocket of the small molecule compound Apcin binding to the side of the WD40 region (left) and the small molecule compound TAME binding to APC3 by simulating the isoleucine-arginine (IR) tail of Cdc20 and Cdh1, interfering with the IR tail binding site;

FIG4 is the immunofluorescence experimental results of drug-treated HepG2 cell lines;

FIG. 5 is the result of apoptosis experiment of HepG2 cell line treated with drugs (fluorescence image of compound promoting cell apoptosis).

DETAILED DESCRIPTION

The present invention will be described in detail below in conjunction with the embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

Example 1

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4-methylpyrimidin-2-)amino)-ethyl)-carbamate (5b)

Step 1

Synthesis of 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethylcarbamate

Weigh 30.0g of metronidazole (0.175mol) and put it into a 500mL eggplant-shaped bottle, then add 300mL of anhydrous dichloromethane to dissolve, 30mL of anhydrous triethylamine (0.210mol) in turn, stir evenly, add 42.5g of p-nitrophenyl chloroformate (0.210mol) in an ice bath, and finally add 30mL of anhydrous dichloromethane to rinse the bottle mouth. Move to room temperature and stir to react overnight. TLC monitoring shows that the metronidazole reaction is basically complete, and stop the reaction. At 10°C, add 62mL of ammonia water (28%) slowly to the reaction solution using a constant pressure dropping funnel, react for 3h, and the reaction is complete. The reaction solution was filtered to obtain 58.3 g of solid, which was dissolved with 200 mL of sodium hydroxide solution (1 mol/L), extracted three times with 200 mL of dichloromethane, and the organic layers were combined, concentrated under reduced pressure, and dried to obtain 18.6 g of yellow crystalline compound 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethylcarbamate. Two-step yield: 50%, melting point: 151.5-153.1°C.

Step 2

Synthesis of 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate

Weigh 77.0g of chloral hydrate (0.467mol) and put it into a 100mL eggplant-shaped bottle, place it in an oil bath and heat it. After the chloral hydrate melts, slowly add 10.0g of the compound 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethylcarbamate (0.047mol), stir and react at 100°C. After reacting for 48h, stop the reaction. Cool the reaction solution to room temperature, add 50mL of ethyl acetate, ultrasonically turn it into a turbid liquid, and then filter it to obtain 11.6g of white compound. Yield: 68%, melting point: 168.7～169.7°C.

Step 3

Using the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-methyl-2-aminopyrimidine as raw materials, compound 5b was obtained.

The specific operation is: weigh 0.50g of the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate (1.38mmol) and put it into a 50mL eggplant-shaped bottle, then measure 30mL of anhydrous dichloromethane and add it to the reaction bottle, add 1 drop of triethylamine, stir evenly, and slowly add 0.50mL of thionyl chloride (6.91mmol) with a constant pressure dropping funnel under an ice bath. After the addition is complete, transfer to an oil bath pot, reflux reaction at 40°C under nitrogen protection overnight to stop the reaction. After the reaction solution is cooled to room temperature, it is concentrated under reduced pressure, 20mL of anhydrous dichloromethane is added to fully dissolve, and concentrated under reduced pressure, and repeated 3 times to remove the residual thionyl chloride to obtain a yellow solid. The obtained yellow solid was dissolved in 15 mL of anhydrous dichloromethane and 15 mL of anhydrous acetonitrile, and another raw material amine (1.66 mmol) was added to the reaction solution. After 24 hours, 10 mL of methanol was added and stirred for 30 minutes to quench the reaction. The reaction solution was concentrated under reduced pressure and dissolved in 50 mL of dichloromethane, washed 3 times with 50 mL of saturated brine, and concentrated under reduced pressure to obtain a crude product. After the crude product was dissolved in 20 mL of methanol, it was mixed with 80-100 mesh silica gel, loaded with 200-300 mesh silica gel, and purified by column chromatography. The elution system used dichloromethane and methanol solution, and the target compound was obtained after separation and purification.

The target compound is a white solid, yield: 21.42%, melting point: 209.0-209.9°C, HPLC: 97.19%. 1HNMR (500MHz, DMSO-d6): δ8.27 (d, J = 5.0 Hz, 1H), 8.00 (s, 1H), 7.97 (d, J = 8.8 Hz, 1H), 6.97 (d, J = 9.5 Hz, 1H), 6.73 (d, J = 5.0 Hz, 1H), 6.60 (t, J = 9.2 Hz, 1H), 4.56-4.46 (m, 3H), 4.35 (dd, J = 10.7, 5.7 Hz, 1H), 2.39 (s, 3H), 2.31 (s, 3H).13C NMR (500MHz, DMSO-d6): δ160.69,155.24,152.06,138.88,133.53,112.85,102.88,70.12,63.46,45.90,24.10,14.37.HRMS(ESI)m/z calcd for[C14H16Cl3N7O4+H ]+:452.0329,found:452.0401.

Example 2

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4-methoxypyrimidin-2-)amino)-ethyl)-carbamate (5c)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-methoxy-2-aminopyrimidine were used as raw materials to obtain compound 5c. Pale yellow solid, yield: 32.66%, melting point: 81.6-83.3°C, HPLC: 98.63%. 1H NMR (500MHz, DMSO-d6): δ8.14(d,J＝5.7Hz,1H),8.01(d,J＝16.7Hz,2H),7.05(s,1H),6.57(d,J＝13.3Hz,1H),6.28(d,J＝5.7Hz,1H),4.51(dd,J＝17.4,4.3Hz ,3H),4.38–4.33(m,1H),3.85(s,3H),2.39(s,3H).13C NMR (500MHz, DMSO-d6): δ170.22,160.87,158.59,155.27,152.06,138.88,133.51,102.59,99.44,70.35,63.48,53.74,45.89,14.36.HRMS(ESI)m/z calcd for[C 14H16Cl3N7O5+H]+:468.0278,found:468.0370.

Example 3

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((6-oxo-1,6-dihydropyrimidin-2-)amino)ethyl)carbamic acid (5d)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and isocytosine were used as raw materials to obtain compound 5d. Pale yellow solid, yield: 16.24%, melting point: 192.3-193.2°C, HPLC: 98.39%. 1H NMR (500MHz, DMSO-d6): δ11.14(s,1H),8.71(d,J＝8.4Hz,1H),8.01(s,1H),7.64(d,J＝6.1Hz,1H),7.11(d,J＝9.3Hz,1H),6.49(t,J＝9.1Hz,1H),5.72(d, J＝6.1Hz,1H),4.55–4.48(m,3H),4.37(dd,J＝7.5,5.0Hz,1H),2.41(s,3H).13C NMR (500MHz, DMSO-d6): δ162.08,155.74,155.43,153.95,152.10,138.89,133.56,105.94,101.66,69.29,63.28,46.00,14.41.HRMS(ESI)m/z calcd for[C13H14 Cl3N7O5+H]+:454.0122,found:454.0200.

Example 4

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4-aminopyrimidin-2-)amino)-ethyl)-carbamate (5e)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2,4-diaminopyrimidine were used as raw materials to obtain compound 5e. Pale yellow solid, yield: 28.69%, melting point: 104.2-105.0°C, HPLC: 98.55%. 1H NMR (500MHz, DMSO-d6): δ8.02(s,1H),7.95(d,J＝8.5Hz,1H),7.74(d,J＝5.3Hz,1H),6.64(s,2H),6.53(t,J＝9.0Hz,1H),6.15(s,1H),5.87(d,J＝5.1Hz,1 H),4.56-4.46(m,3H),4.32(d,J＝11.4Hz,1H),2.40(s,3H).13C NMR (500MHz, DMSO-d6): δ164.54,160.64,156.04,155.13,152.07,138.88,133.52,103.38,97.97,69.98,63.36,45.92,14.37.HRMS(ESI)m/z calcd for[C13H15Cl 3N8O4+H]+:453.0282,found:453.0360.

Example 5

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4-chloropyrimidin-2-)amino)-ethyl)-carbamate (5f)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-chloro-2-aminopyrimidine were used as raw materials to obtain compound 5f. White solid, yield: 20.18%, melting point: 182.3-183.4°C, HPLC: 98.75%. 1H NMR (500MHz, DMSO-d6): δ8.40(d,J＝4.8Hz,1H),8.06(d,J＝8.4Hz,1H),7.99(s,1H),7.87(s,1H),6.98(d,J＝5.0Hz,1H),6.55(s,1H),4.56–4.47(m,3H), 4.40–4.35(m,1H),2.41(s,3H).13C NMR(500MHz,DMSO-d6): δ161.18,155.35,152.08,138.90,133.51,112.61,102.13,70.30,63.51,45.88,14.41.HRMS(ESI )m/z calcd for[C13H13Cl4N7O4+H]+:471.9783,found:471.9864.

Example 6

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4-trifluoromethylpyrimidine-2-)amino)-ethyl)-carbamate (5 g)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-trifluoromethyl-2-aminopyrimidine were used as raw materials to obtain compound 5g. Yellow oil, yield: 5.26%, HPLC: 97.81%. 1H NMR (500MHz, DMSO-d6): δ8.79(d,J＝4.4Hz,1H),8.12(s,1H),8.03(d,J＝8.6Hz,1H),7.98(s,1H),7.28(d,J＝4.9Hz,1H),6.61(s,1H),4.57–4.46(m,3H) ,4.40–4.34(m,1H),2.40(s,3H).HRMS(ESI)m/z calcd for[C14H13Cl3F3N7O4+H]+:506.0047,found:506.0140.

Example 7

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((pyrimidine-4-)amino)-ethyl)-carbamate (6a)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-aminopyrimidine were used as raw materials to obtain compound 6a. Yellow solid, yield: 32.24%, melting point: 179.4-180.2°C, HPLC: 99.79%. 1H NMR (500MHz, DMSO-d6): δ8.59–8.48(m,2H),8.21(d,J＝5.9Hz,1H),8.04–7.94(m,2H),6.88(d,J＝5.9Hz,1H),6.77(t,J＝8.6Hz,1H),4.50(d,J＝11.0Hz,3 H),4.35(dd,J＝10.7,4.5Hz,1H),2.39(s,3H).13C NMR (500MHz, DMSO-d6): δ161.10,158.30,155.81,155.59,152.14,138.87,133.56,107.09,101.98,68.80,63.24,46.04,14.41.HRMS(ESI)m/z calcd for[C13H14 Cl3N7O4+H]+:438.0173,found:438.0264.

Example 8

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((quinoline-2-)amino)-ethyl)-carbamate (6b)

Steps 1 and 2 are the same as in Example 1.

According to the synthesis method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-aminoquinoline were used as raw materials to obtain compound 6b. Dark yellow solid, yield: 25.62%, melting point: 119.9-121.0°C, HPLC: 99.75%. 1H NMR (500MHz, DMSO-d6): δ8.35(d,J＝8.8Hz,1H),8.03–7.98(m,2H),7.71(d,J＝7.9Hz,1H),7.59–7.54(m,2H),7.49(d,J＝9.2Hz,1H),7.27(t,J＝7.2Hz,1H ),7.13(d,J＝8.9Hz,1H),6.94(t,J＝9.0Hz,1H),4.51(dd,J＝11.7,4.8Hz,3H),4.35–4.30(m,1H),2.34(s,3H).13C NMR (500MHz, DMSO-d6): δ155.57,155.16,152.16,147.19,138.82,137.80,133.55,129.84,127.96,126.72,124.23,123.01,113.30,102.69,69.54,63 .17,46.07,14.38.HRMS(ESI)m/z calcd for[C18H17Cl3N6O4+H]+:487.0377,found:487.0463.

Example 9

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((isoquinolin-3-yl)amino-)ethyl)-carbamate (6c)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 3-aminoisoquinoline were used as raw materials to obtain compound 6c. Yellow solid, yield: 28.32%, melting point: 122.6-124.2°C, HPLC: 99.50%. 1H NMR (500MHz, DMSO-d6): δ8.96(s,1H),8.27(d,J＝8.7Hz,1H),7.99(s,1H),7.90(d,J＝8.2Hz,1H),7.64(d,J＝8.3Hz,1H),7.58–7.54(m,1H),7.32–7.28( m,1H),7.05(s,1H),6.81–6.72(m,2H),4.49(d,J＝11.5Hz,3H),4.33(d,J＝5.3Hz,1H),2.34(s,3H).13C NMR (500MHz, DMSO-d6): δ155.57,152.99,152.12,151.48,138.86,138.56,133.53,131.03,128.23,125.27,124.18,123.73,103.23,100.17,70.84,63 .23,46.02,14.36.HRMS(ESI)m/z calcd for[C18H17Cl3N6O4+H]+:487.0377,found:487.0469.

Example 10

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((isoquinolin-1-yl)amino)-ethyl)-carbamate (6d)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 1-aminoisoquinoline were used as raw materials to obtain compound 6d. White solid, yield: 27.43%, melting point: 113.5-114.8°C, HPLC: 97.92%. 1H NMR (500MHz, DMSO-d6): δ8.06(d,J＝8.4Hz,1H),8.02–7.94(m,2H),7.84(d,J＝8.0Hz,1H),7.78(d,J＝8.7Hz,1H),7.73(t,J＝7.5Hz,1H),7.64(t,J＝7.6Hz, 1H),7.35(d,J＝8.8Hz,1H),7.18(d,J＝5.7Hz,1H),7.07(t,J＝8.7Hz,1H),4.49(dd,J＝21.6,11.7Hz,3H),4.37–4.31(m,1H),2.36(s,3H).13C NMR (500MHz, DMSO-d6): δ155.11,152.99,151.98,140.88,138.89,137.32,133.50,130.92,127.44,127.06,122.66,117.67,113.11,103.34,69.26,63 .53,45.81,14.34.HRMS(ESI)m/z calcd for[C18H17Cl3N6O4+H]+:487.0377,found:487.0463.

Embodiment 11

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(1H-benzo[d]imidazol-1-yl)-2,2,2-trichloroethyl)-carbamate (6e)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and benzimidazole were used as raw materials to obtain compound 6e. White solid, yield: 22.13%, melting point: 123.6-124.9°C, HPLC: 97.97%. 1H NMR (500MHz, DMSO-d6): δ9.68(d,J＝9.8Hz,1H),8.63(s,1H),8.00(d,J＝6.1Hz,1H),7.96(t,J＝11.1Hz,1H),7.71(t,J＝7.8Hz,1H),7.33(t,J＝7.4Hz,1H), 7.27(t,J＝7.4Hz,1H),6.88(t,J＝14.6Hz,1H),4.60–4.51(m,3H),4.40(dt,J＝8.5,3.8Hz,1H),2.38(s,3H).13C NMR (500MHz, DMSO-d6): δ155.67,152.01,142.82,142.73,138.91,134.14,133.57,123.96,123.03,120.18,112.05,99.86,73.06,63.74,45.86,14.34 .HRMS(ESI)m/z calcd for[C16H15Cl3N6O4+H]+:461.0220,found:461.0127.

Example 12

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-(6-(2,2,2-trichloro-1-((2-(2-methyl-5-nitro-1H-imidazol-1-yl)-ethoxycarbonylamino-)-ethyl)-amino)-9H-purin-9-yl-ethyl)-carbamate (6f)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and adenine were used as raw materials to obtain compound 6f. Dark yellow solid, yield: 10.56%, melting point: 173.8-175.3°C, HPLC: 97.12%. 1H NMR (500MHz, DMSO-d6): δ9.63(d,J＝9.3Hz,1H),8.62(s,1H),8.48(s,1H),7.97(s,2H),7.87(d,J＝21.2Hz,2H),6.95(d,J＝10.0Hz,1H),6.90(s,1H),4. 58–4.34(m,8H),2.39(s,3H),2.37(s,3H).HRMS(ESI)m/zcalcd for[C23H23Cl6N13O8+H]+:819.9924,found:820.0001.

Example 13

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4,6-dichloropyrimidin-2-)amino)-ethyl)-carbamate (7a)

Steps 1 and 2 are the same as in Example 1.

According to the synthesis method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-amino-4,6-dichloropyrimidine were used as raw materials to obtain compound 7a. Pale yellow solid, yield: 46.23%, melting point: 2175.5-176.3°C, HPLC: 99.73%. 1H NMR (500MHz, DMSO-d6): δ8.56(d,J＝9.2Hz,1H),8.17(d,J＝9.0Hz,1H),7.98(s,1H),7.21(s,1H),6.45(t,J＝9.1Hz,1H),4.55–4.48(m,3H),4.38(dt,J＝ 9.5,4.4Hz,1H),2.42(s,3H).13C NMR(500MHz,DMSO-d6):δ160.73,155.42,152.12,138.90,133.50,111.40,101.52,70.47,63.53,45.90,14.45.HRMS(ESI)m/ zcalcd for[C13H12Cl5N7O4+H]+:505.9393,found:505.9472.

Embodiment 14

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((4,6-dichloro-5-methylpyrimidin-2-)amino)-ethyl)-carbamate (7b)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-amino-4,6-dichloro-5-methylpyrimidine were used as raw materials to obtain compound 7b. White solid, yield: 46.66%, melting point: 140.3-142.1°C, HPLC: 99.82%. 1H NMR (400MHz, DMSO-d6): δ8.24(d,J＝9.3Hz,1H),8.15(d,J＝9.0Hz,1H),7.99(s,1H),6.40(t,J＝9.1Hz,1H),4.56–4.46(m,3H),4.37(dt,J＝9.3,3.9Hz,1H) ,2.42(s,3H),2.26(s,3H).13C NMR(400MHz,DMSO-d6):δ158.23,155.43,152.15,138.90,133.55,117.65,101.74,70.55,63.51,45.92,15.41,14.47.HRMS( ESI)m/z calcd for[C14H14Cl5N7O4+H]+:519.9550,found:519.9629.

Embodiment 15

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((5-amino-4,6-dichloropyrimidine-2-)amino)-ethyl)-carbamate (7c)

Steps 1 and 2 are the same as in Example 1.

According to the synthesis method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2,5-diamino-4,6-dichloropyrimidine were used as raw materials to obtain compound 7c. Yellow-brown solid, yield: 27.68%, melting point: 92.9-93.7°C, HPLC: 99.73%. 1H NMR (500MHz, DMSO-d6): δ8.00(d,J＝8.9Hz,1H),7.98(s,1H),7.21(d,J＝9.5Hz,1H),6.26(t,J＝9.2Hz,1H),5.16(s,2H),4.56–4.45(m,3H),4.37(dt,J＝9 .6,4.6Hz,1H),2.40(s,3H).13C NMR(500MHz,DMSO-d6): δ155.34,152.07,151.11,145.55,138.88,133.49,129.80,102.40,71.00,63.41,45.92,14.40.HRMS (ESI)m/z calcd for[C13H13Cl5N8O4+H]+:520.9502,found:520.9581.

Example 16

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-((2-amino-4,6-dichloropyrimidine-5-yl)amino)-2,2,2-trichloroethyl)-carbamate (7d)

Steps 1 and 2 are the same as in Example 1.

According to the synthesis method of step 3 of Example 1, 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2,5-diamino-4,6-dichloropyrimidine were used as raw materials to obtain compound 7d. White solid, yield: 20.18%, melting point: 125.1-125.7°C, HPLC: 99.14%. 1H NMR (500MHz, DMSO-d6): δ8.35(s,1H),8.34(d,J＝9.5Hz,1H),8.01(s,1H),7.76–7.74(m,1H),7.26(s,2H),5.58(t,J＝10.0Hz,1H),4.61(d,J＝10.6Hz,1 H),4.47(d,J＝11.9Hz,3H),4.42–4.35(m,1H),2.40(s,3H).13C NMR (500MHz, DMSO-d6): δ158.85,156.40,155.83,152.17,138.83,133.53,121.90,102.23,75.27,63.21,46.09,14.45.HRMS(ESI)m/z calcd for[C13H13Cl5N8O4+ H]+:520.9502,found:520.9574.

Embodiment 17

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((6-chloro-9H-purin-9-yl)-ethyl)-carbamate (8a)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 6-chloropurine were used as raw materials to obtain compound 8a. Pale yellow solid, yield: 21.32%, melting point: 101.4-101.8°C, HPLC: 96.82%. 1H NMR (500MHz, DMSO-d6): δ9.76(d,J＝10.0Hz,1H),8.98(s,1H),8.93(s,1H),7.91(s,1H),7.00(d,J＝10.0Hz,1H),4.57–4.45(m,4H),2.37(s,3H).13C NMR( 500MHz, DMSO-d6): δ155.56,153.11,152.38,151.86,150.38,144.65,138.98,133.45,130.31,98.77,71.56,63.87,45.75,14.35.HRMS(ESI)m/zcalcd for[C14H12Cl4N8O4+H]+:496.9736,found:496.9782.

Embodiment 18

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((6-(N,N-dimethylamino)-9H-purin-9-yl)-ethyl)-carbamate (8b)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 6-dimethylaminopurine were used as raw materials to obtain compound 8b. White solid, yield: 20.23%, melting point: 113.6-115.2°C, HPLC: 98.22%. 1H NMR (500MHz, CDCl3): δ8.51(s,1H),8.31(s,1H),7.96(s,1H),7.94(s,1H),6.65(d,J＝9.7Hz,1H),4.64–4.58(m,3H),4.54–4.49(m,1H),3.58(s,6H) ,2.47(s,3H).13C NMR(500MHz,DMSO-d6): δ155.52,154.74,153.01,151.93,151.01,138.94,137.06,133.50,118.26,99.65,70.73,63.80,45.81,14.32. HRMS(ESI)m/z calcd for[C16H18Cl3N9O4+H]+:506.0547,found:506.0638.

Embodiment 19

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(6-benzamido-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8c)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and N6-benzoyl adenine were used as raw materials to obtain compound 8c. White solid, yield: 22.65%, melting point: 183.7-184.9°C, HPLC: 97.27%. 1H NMR (500MHz, DMSO-d6): δ11.35(s,1H),9.71(d,J＝9.9Hz,1H),8.85(s,1H),8.78(s,1H),8.06(d,J＝7.4Hz,2H),7.97(s,1H),7.66(t,J＝7.2Hz,1H),7.5 7(t,J＝7.3Hz,2H),7.05(d,J＝9.9Hz,1H),4.58(d,J＝11.0Hz,3H),4.45(s,1H),2.38(s,3H).13C NMR (500MHz, DMSO-d6): δ166.08,155.59,152.94,152.91,151.92,151.30,141.70,138.97,133.61,133.49,133.05,129.01,128.97,124.43,99.26,71 .09,63.89,45.78,14.33.HRMS(ESI)m/z calcd for[C21H18Cl3N9O5+H]+:582.0496,found:582.0577.

Embodiment 20

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(6-benzylamino-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8d)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 6-benzylaminopurine were used as raw materials to obtain compound 8d. White solid, yield: 23.21%, melting point: 121.0-121.8°C, HPLC: 98.42%. 1H NMR (500MHz, DMSO-d6): δ9.60(d,J＝10.1Hz,1H),8.60(s,1H),8.48(s,1H),8.29(s,1H),7.98(s,1H),7.35(d,J＝7.4Hz,2H),7.29(t,J＝7.5Hz,2H),7.2 1(t,J＝7.2Hz,1H),6.92(d,J＝10.1Hz,1H),4.72(s,2H),4.56(t,J＝9.3Hz,3H),4.42(dd,J＝8.8,5.6Hz,1H),2.38(s,3H).13C NMR (500MHz, DMSO-d6): δ155.52,154.90,153.76,151.93,149.63,140.31,138.94,138.30,133.50,128.68,127.68,127.13,118.14,99.55,70.82,63. 81,45.80,43.45,14.33.HRMS(ESI)m/z calcdfor[C21H20Cl3N9O4+H]+:568.0704,found:568.0795.

Embodiment 21

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(6-amino-2-chloro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8e)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-chloroadenine were used as raw materials to obtain compound 8e, a white solid, yield: 20.18%, melting point: 196.5-197.6°C, HPLC: 98.62%. 1H NMR (500MHz, DMSO-d6): δ9.61(d,J＝10.0Hz,1H),8.45(s,1H),7.99(d,J＝20.3Hz,3H),6.73(d,J＝9.8Hz,1H),4.57(d,J＝9.3Hz,3H),4.44(d,J＝8.1Hz,1H) ,2.38(s,3H).13C NMR(500MHz,DMSO-d6): δ157.40,155.54,154.23,151.91,151.23,138.96,138.72,133.47,116.94,99.23,70.90,63.85,45.79,14.32. HRMS(ESI)m/z calcd for[C14H13Cl4N9O4+H]+:511.9845,found:511.9915.

Embodiment 22

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-((2,6-diamino)-9H-purin-9-yl)-ethyl)-carbamate (8f)

Steps 1 and 2 are the same as in Example 1.

According to the synthesis method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2,6-diaminopurine were used as raw materials to obtain compound 8f. Pale yellow solid, yield: 25.36%, melting point: 165.8-166.6°C, HPLC: 98.68%. 1H NMR (400MHz, DMSO): δ9.47(d,J＝10.2Hz,1H),8.03(d,J＝7.8Hz,2H),6.85(s,2H),6.73(d,J＝10.1Hz,1H),6.07(s,2H),4.58(ddd,J＝16.2,9.9,5.1Hz,3H) ,4.39(dd,J＝7.6,4.6Hz,1H),2.40(s,3H).13C NMR (400MHz, DMSO): δ161.21,156.74,155.56,152.60,152.00,138.97,134.48,133.53,111.95,100.10,70.22,63.64,45.88,14.35.HRMS(ESI)m/z calcd for[C1 4H15Cl3N10O4+H]+:493.0343,found:493.0416.

Embodiment 23

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8 g)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-fluoroadenine were used as raw materials to obtain compound 8g. White solid, yield: 19.26%, melting point: 168.1-169.6°C, HPLC: 96.92%. 1H NMR (500MHz, DMSO-d6): δ9.60 (d, J=10.0Hz, 1H), 8.42 (s, 1H), 8.00 (d, J=31.1Hz, 3H), 6.70 (d, J=10.0Hz, 1H), 4.57 (d, J=10.1Hz, 3H), 4.44 (d, J=7.5Hz, 1H), 2.38 (s, 3H).13C NMR(500MHz,DMSO-d6)δ160.25,158.62,158.40,158.23,155.54,151.91,151.71,151.55,138.96,138.61,133.46,116.28,99.26,71.02,63.85,45.79 ,14.30.HRMS(ESI)m/z calcd for[C14H13Cl3FN9O4+H]+:496.0140,found:496.0222.

Embodiment 24

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(2-amino-6-chloro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8h)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-amino-6-chloropurine were used as raw materials to obtain compound 8h. White solid, yield: 22.16%, melting point: 140.4-140.9°C, HPLC: 97.69%. 1H NMR (500MHz, DMSO-d6): δ9.61(d,J＝9.9Hz,1H),8.40(s,1H),7.96(s,1H),7.26(s,2H),6.78(d,J＝10.0Hz,1H),4.53(t,J＝35.8Hz,4H),2.38(s,3H).13C N MR (500MHz, DMSO-d6): δ160.75,155.58,154.62,151.90,150.53,139.79,139.01,133.45,122.32,99.33,70.70,63.74,45.83,14.33.HRMS(ESI)m/z calcd for[C14H13Cl4N9O4+H]+:511.9845,found:511.9923.

Embodiment 25

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-(6-chloro-2-fluoro-9H-purin-9-yl)-ethyl)-carbamate (8i)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-fluoro-6-chloropurine were used as raw materials to obtain compound 8i as a white solid with a yield of 23.16%, a melting point of 171.3-172.9°C, and a HPLC of 98.85%. 1H NMR(500MHz,DMSO-d6): δ9.80(d,J＝9.7Hz,1H),8.97(s,1H),7.92(s,1H),6.82(d,J＝10.0Hz,1H),4.60–4.47(m,4H),2.38(s,3H).13C NMR(500MHz,DMSO-d6): δ157.93,156.21,155.56,154.42,154.28,152.12,151.98,151.87,145.47 ,138.99,133.39,129.60,98.42,71.92,63.92,45.76,14.36.HRMS(ESI)m/ zcalcd for[C14H11Cl4N8O4+H]+:514.9641,found:514.9718.

Embodiment 26

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(2-amino-6-benzyloxy-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8j)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and O-6-benzylguanine were used as raw materials to obtain compound 8j, a white solid, with a yield of 24.53%, a melting point of 171.6-172.8°C, and a HPLC of 96.77%. 1H NMR (500MHz, DMSO-d6): δ9.54(d,J＝10.1Hz,1H),8.17(s,1H),8.00(s,1H),7.52(d,J＝7.3Hz,2H),7.40(t,J＝7.3Hz,2H),7.38–7.34(m,1H),6.79(d,J＝ 7.3Hz,3H),5.50(s,2H),4.56(t,J＝10.3Hz,3H),4.41(d,J＝10.3Hz,1H),2.39(s,3H).13C NMR (500MHz, DMSO-d6): δ160.69,155.57,155.03,151.95,138.98,136.91,136.74,133.50,129.04,128.89,128.59,112.66,99.77,70.47,67.61,63.6 8,45.86,14.33.HRMS(ESI)m/z calcd for[C21H20Cl3N9O5+H]+:584.0653,found:584.0735.

Embodiment 27

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-(2,6-dichloro-9H-purin-9-yl)-ethyl)-carbamate (8k)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2,6-dichloropurine were used as raw materials to obtain compound 8k. White solid, yield: 19.33%, melting point: 129.9-131.7°C, HPLC: 99.76%. 1H NMR (500MHz, DMSO-d6): δ9.77 (d, J = 9.8Hz, 1H), 9.01 (d, J = 21.3Hz, 1H), 7.91 (s, 1H), 6.86 (d, J = 9.8Hz, 1H), 4.59–4.46 (m, 4H), 2.37 (s, 3H). 13C NMR (500MHz, DMSO-d6): δ155.56,153.83,152.55,151.87,151.22,145.42,139.01,133.38,130.10,98.46,71.80,63.92,45.76,14.37.HRMS(ESI)m/z calcdfor[C14H11Cl5N8O4+H]+:530.9346,found:530.9421.

Embodiment 28

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(2-acetyl-amino-6-oxo-3,6-dihydro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8l)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl (2,2,2-trichloro-1-hydroxyethyl)carbamate and N-2-acetylguanine were used as raw materials to obtain compound 81. Pale yellow solid, yield: 22.16%, melting point: 197.5-198.3°C, HPLC: 99.67%. 1H NMR (400MHz, DMSO-d6): δ12.13 (s, 1H), 11.90 (s, 1H), 9.67 (d, J = 9.1 Hz, 1H), 8.29 (s, 1H), 7.99 (s, 1H), 6.76 (d, J = 9.8 Hz, 1H), 4.50 (d, J = 54.2 Hz, 4H), 2.38 (s, 3H), 2.19 (s, 3H).13C NMR (500MHz, DMSO-d6): δ174.19,155.64,155.20,151.91,149.53,149.04,139.04,136.98,133.50,119.25,99.23,70.80,63.69,45.89,24.24,14.32. HRMS(ESI)m/z calcd for[C16H16Cl3N9O6+H]+:536.0289,found:536.0362.

Embodiment 29

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(6-amino-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (8m)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and adenine were used as raw materials to obtain compound 8m, a white solid, yield: 12.36%, melting point: 187.1-187.9°C, HPLC: 98.68%. ¹ H NMR (400MHz, DMSO-d ₆ ): δ9.59 (d, J = 10.1 Hz, 1H), 8.46 (s, 1H), 8.21 (s, 1H), 7.98 (s, 1H), 7.48 (s, 2H), 6.89 (d, J = 10.1Hz, 1H), 4.61-4.52 (m, 3H), 4.47 -4.39(m,1H),2.38(s,3H). ¹³ C NMR(101MHz,DMSO-d ₆ ): δ156.60,155.57,153.74,151.98,150.22,138.96,138.31,133.47,117.59,99.66,70.90,63.84,45.90,14.51.HRMS(ESI)m/z calcd for[C ₁₄ H ₁₄ Cl ₃ N ₉ O ₄ +H] ⁺ :478.0234,found:478.0313.

Embodiment 30

Benzyl-(1-(6-amino-2-chloro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9a)

Using benzyl carbamate as the starting material, the preparation method is the same as step 2 of Example 1. The crude intermediate (2,2,2-trichloro-1-hydroxyethyl) benzyl carbamate is obtained and directly used for the next step reaction.

According to the synthesis method of step 3 of Example 1, the intermediate (2,2,2-trichloro-1-hydroxyethyl) benzyl carbamate and 2-chloroadenine were used as raw materials to obtain compound 9a, a white solid, yield: 21.32%, melting point: 198.6-199.9°C, HPLC: 98.49%. 1H NMR (400MHz, DMSO-d6): δ9.69(d,J＝10.1Hz,1H),8.49(s,1H),8.05(s,2H),7.37(dd,J＝15.1,7.4Hz,5H),6.87(d,J＝10.2Hz,1H),5.21–5.12(m,2H).13C NMR (400MHz, DMSO-d6): δ157.42,155.85,154.31,151.25,138.78,136.18,128.95,128.86,128.80,116.92,99.48,70.91,67.70.HRMS(ESI)m/z calcd for[C15H12Cl4N6O2+H]+:448.9776,found:448.9853.

Embodiment 31

(1-Methyl-5-nitro-1H-imidazol-2-yl)-methyl-(1-(6-amino-2-chloro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9b)

Using ronidazole as the starting material, the preparation method is the same as step 2 of Example 1. The crude intermediate (1-methyl-5-nitro-1H-imidazolin-2-yl)methyl (2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate (1-methyl-5-nitro-1H-imidazolin-2-yl)methyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-chloroadenine were used as raw materials to obtain compound 9b, a white solid, yield: 23.16%, melting point: 185.8-186.3°C, HPLC: 98.24%. 1H NMR (500MHz, DMSO-d6): δ9.87(d,J＝10.0Hz,1H),8.48(s,1H),8.06(d,J＝12.8Hz,3H),6.83(d,J＝10.0Hz,1H),5.32(q,J＝13.6Hz,2H),3.91(s,3H).13C NMR( 500MHz, DMSO-d6): δ157.42,155.27,154.30,151.26,147.57,139.93,138.75,132.24,116.90,99.37,70.93,59.34,33.98.HRMS(ESI)m/z calcdfor[C13H11Cl4N9O4+H]+:497.9688,found:497.9766.

Embodiment 32

Ethyl-(1-(6-amino-2-chloro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9c)

Using ethyl carbamate as the starting material, the preparation method is the same as step 2 of Example 1. The crude intermediate (2,2,2-trichloro-1-hydroxyethyl) ethyl carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate (2,2,2-trichloro-1-hydroxyethyl) ethyl carbamate and 2-chloroadenine were used as raw materials to obtain compound 9c, a white solid, yield: 19.89%, melting point: 219.5-220.4°C, HPLC: 98.67%. 1H NMR (400MHz, DMSO-d6): δ9.53(d,J＝10.2Hz,1H),8.51(s,1H),8.04(s,2H),6.83(d,J＝10.2Hz,1H),4.14(ddd,J＝10.7,7.0,3.7Hz,2H),1.22(t,J＝7.1Hz,3 H).13C NMR (400MHz, DMSO-d6): δ157.42,155.92,154.28,151.25,138.86,116.91,99.57,70.85,62.25,14.77.HRMS(ESI)m/z calcd for[C10H10Cl4N6O2+H]+:386.9616,found:386.96970.

Embodiment 33

(1-Methyl-5-nitro-1H-imidazol-2-yl)-methyl-(1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9d)

Using ronidazole as the starting material, the preparation method is the same as step 2 of Example 1. The crude intermediate (1-methyl-5-nitro-1H-imidazolin-2-yl)methyl (2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate (1-methyl-5-nitro-1H-imidazolin-2-yl)methyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-fluoroadenine were used as raw materials to obtain compound 9d. White solid, yield: 21.63%, melting point: 205.8-206.9°C, HPLC: 99.00%. 1H NMR (500MHz, DMSO-d6): δ9.85 (d, J=9.9Hz, 1H), 8.45 (s, 1H), 8.04 (d, J=24.3Hz, 3H), 6.80 (d, J=9.9Hz, 1H), 5.33 (q, J=13.6Hz, 2H), 3.92 (s, 3H).13C NMR (500MHz, DMSO-d6): δ160.29,158.66,158.41,158.24,155.26,151.73,151.57,147.58,139.91,138.62,132.22,116.24,102.69,99.37,84.10,71. 03,59.33,33.96.HRMS(ESI)m/z calcd for[C13H11Cl4N9O4+H]+:481.9984,found:482.0055.

Embodiment 34

Ethyl-(1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9e)

Using ethyl carbamate as the starting material, the preparation method is the same as step 2 of Example 1. The crude intermediate (2,2,2-trichloro-1-hydroxyethyl) ethyl carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate (2,2,2-trichloro-1-hydroxyethyl) ethyl carbamate and 2-fluoroadenine were used as raw materials to obtain compound 9e, a white solid, yield: 18.21%, melting point: 208.3-209.8°C, HPLC: 99.12%. 1H NMR (400MHz, DMSO-d6): δ9.50(d,J＝10.1Hz,1H),8.48(s,1H),8.05(d,J＝45.7Hz,2H),6.80(d,J＝10.2Hz,1H),4.14(dd,J＝13.3,6.4Hz,2H),1.21(t,J＝7.0 Hz,3H).13C NMR(400MHz,DMSO-d6): δ160.29,158.65,158.41,158.24,155.91,151.73,151.57,138.73,116.29,116.26,99.60,70.96,62.21,14.75.HRMS(ESI )m/z calcd for[C10H10Cl4N6O2+H]+:370.9915,found:370.9988.

Embodiment 35

2-N-Morpholinoethyl-(1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9f)

Using hydroxyethylmorpholine as the starting material, the preparation method is the same as step 2 of Example 1. The intermediate 2-morpholinoethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate crude product is obtained and directly used for the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-N-morpholinoethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-fluoroadenine were used as raw materials to obtain compound 9f. Pale yellow solid, yield: 10.42%, HPLC: 96.96%. ¹ H NMR (500 MHz, DMSO-d ₆ ): δ9.68 (d, J=9.1 Hz, 1H), 8.55 (s, 1H), 8.03 (d, J=38.0 Hz, 2H), 6.79 (d, J=10.0 Hz, 1H), 4.21 (s, 2H), 3.03 (d, J=7.3 Hz, 4H), 2.60 (s, 2H), 2.46 (s, 4H). ¹³ C NMR (126 MHz, DMSO-d ₆ ): δ158.32(d,J＝5.9Hz),155.78(s),138.96(s),130.10(s),102.96(s),99.51(s),83.94(s),71.03(s),70.20(s),65.83(s),65.54(s),56.68(d, J＝5.6Hz),53.32(s).HRMS(ESI)m/z calcd for[C ₁₄ H ₁₈ Cl ₃ FN ₇ O ₃ +H] ⁺ :456.0515, found:456.0517.

Embodiment 36

2-N-Morpholinoethyl-(2,2,2-trichloro-1-(2,6-diamino-9H-purin-9-yl)-ethyl)-carbamate (9 g)

Using hydroxyethylmorpholine as the starting material, the preparation method is the same as step 2 of Example 1. The intermediate 2-morpholinoethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate crude product is obtained and directly used for the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-morpholinoethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-aminoadenine were used as raw materials to obtain compound 9g. White solid, yield: 10.07%, HPLC: 97.8%. 1H NMR (500MHz, DMSO-d6): δ9.44 (d, J＝10.1Hz, 1H), 8.11 (s, 1H), 6.90-6.80 (m, 3H), 6.08 (s, 2H), 4.18 (s, 2H), 3.51 (s, 4H), 2.51 (s, 2H), 2.38 (s, 4H). NMR HRMS (ESI) m/zcalcd for [C14H19Cl3N8O3+H]+: 453.0718, found: 453.0724.

Embodiment 37

2-(Pyridin-2-yl)-ethyl-(1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9h)

Using 2-hydroxyethylpyridine as the starting material, the preparation method is the same as that of Example 2. The crude intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-fluoroadenine were used as raw materials to obtain compound 9h. White solid, yield: 26.58%, HPLC: 97.28%. 1H NMR (500MHz, DMSO-d6) δ9.78 (d, J = 9.7 Hz, 1H), 8.75 (s, 1H), 8.55 (s, 1H), 8.37 (s, 1H), 8.07-7.92 (m, 3H), 7.81 (s, 1H), 6.70 (d, J = 9.9 Hz, 1H), 4.51 (d, J = 6.9 Hz, 2H), 3.39 (s, 2H).13C NMR(126MHz, DMSO-d6)δ160.22(s),158.58(s),158.33(s),155.69(s),154.21(s),142.99(s),139.02(d,J=3.0Hz),127.59(s),125.26(s),116.20(s), 99.37(s),70.98(s),64.02(s),33.43(s).HRMS(ESI)m/z calcd for[C15H13Cl3FN7O2+H]+:448.0253,found:448.0257.

Embodiment 38

2-(Pyridin-2-yl)-ethyl-(2,2,2-trichloro-1-(2,6-diamino-9H-purin-9-yl)-ethyl)-carbamate (9i)

Using 2-hydroxyethylpyridine as the starting material, the preparation method is the same as that of Example 2. The crude intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 2-aminoadenine were used as raw materials to obtain compound 9i. White solid, yield: 27.38%, HPLC: 95.32%. 1H NMR (500MHz, DMSO) δ9.34 (d, J = 10.1 Hz, 1H), 8.48 (d, J = 3.9 Hz, 1H), 8.07 (s, 1H), 7.69 (t, J = 7.1 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.24-7.19 (m, 1H), 6.91-6.77 (m, 3H), 6.08 (s, 2H), 4.52-4.43 (m, 2H), 3.08 (t, J = 6.5 Hz, 2H).13C NMR(101MHz,DMSO)δ161.23(s),158.02(s),156.75(s),152.59(s),149.56(s),137.00(s),134.68(s),123.90(s),122.23(s),111.95(s),100.45(s), 70.19(s),64.90(s),60.23(s),37.14(s).HRMS(ESI)m/z calcd for[C15H15Cl3N8O2+H]+:445.0456,found:445.0460.

Embodiment 39

Benzyl-(1-(6-amino-2-fluoro-9H-purin-9-yl)-2,2,2-trichloroethyl)-carbamate (9j)

Using benzyl carbamate as the starting material, the preparation method is the same as step 2 of Example 1. The crude intermediate (2,2,2-trichloro-1-hydroxyethyl) benzyl carbamate is obtained and directly used for the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate (2,2,2-trichloro-1-hydroxyethyl) benzyl carbamate and 2-fluoroadenine were used as raw materials to obtain compound 9j, a white solid, yield: 19.82%, melting point: 234.8-235.7°C, HPLC: 96.24%. 1H NMR (500MHz, DMSO-d6): δ9.67(d,J＝10.1Hz,1H),8.46(s,1H),8.06(d,J＝47.0Hz,2H),7.46-7.31(m,5H),6.82(d,J＝10.2Hz,1H),5.16(q,J＝12.2Hz,2H) .13CNMR (126MHz, DMSO-d6): δ160.30,158.67,158.42,158.25,155.85,151.72,151.56,138.65,136.20,128.94,128.82,116.29,99.53,71.01,67.68. HRMS(ESI)m/z calcd for[C15H12Cl4N6O2+H]+:433.0071,found:433.0146.

Embodiment 40

2-N-Morpholinoethyl-(1-(4-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2,2-trichloroethyl)-carbamate (10a)

Hydroxyethylmorpholine was used as the starting material and the preparation method was the same as step 2 of Example 1. The crude intermediate 2-morpholinoethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate was obtained and directly used for the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-morpholinoethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-aminopyrazolo[3,4-d]pyrimidine were used as raw materials to obtain compound 10a, a white solid, yield: 19.56%, HPLC: 95.17%. 1H NMR (500MHz, DMSO) δ9.27(d,J＝7.9Hz,1H),8.38(s,1H),8.22(d,J＝23.0Hz,2H),7.79(s,1H),7.15(d,J＝9.7Hz,1H),4.14(s,2H),3.48(s,4H),2.51(s, 2H),2.37(s,4H).13C NMR(126MHz,DMSO)δ158.49(s),157.03(s),155.34(s),135.09(s),99.91(s),99.65(s),71.72(s),66.48(s),62.90(s),57.14(s) ),53.79(s).HRMS(ESI)m/zcalcd for[C14H18Cl3N7O3+H]+:438.0604,found:438.0610.

Embodiment 41

2-(Pyridin-1-yl)-ethyl-(2,2,2-trichloro-1-(4-(2,2,2-trichloro-1-((2-(2-pyridin-2-yl)-ethoxycarbonylamino-)-ethyl)-amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl-ethyl)-carbamate (10b)

Using 2-hydroxyethylpyridine as the starting material, the preparation method is the same as that of Example 2. The crude intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-aminopyrazolo[3,4-d]pyrimidine were used as raw materials to obtain compound 10b, a white solid, yield: 9.84%, HPLC: 97.73%. 1H NMR (500MHz, DMSO) δ9.34(d,J＝9.2Hz,1H),9.06(d,J＝7.8Hz,1H),8.61(s,1H),8.51(s,1H),8.48–8.43(m,2H),8.39–8.31(m,1H),7.66(s,2H),7.29(d ,J＝7.3Hz,2H),7.19(d,J＝4.4Hz,4H),4.42(dd,J＝8.0,5.7Hz,4H),3.04(d,J＝7.7Hz,4H).13C NMR (126MHz, DMSO) δ158.09 (d, J = 12.8Hz), 156.30 (s), 155.89 (s), 155.31 (s), 149.51 (d, J = 5.0Hz), 136.89 (s), 134.91 (s), 123.91 (d, J = 6.1Hz), 122.14 (s) ,101.65(s),100.39(s),99.39(s),71.92(s),68.88(s),64.88(s),64.50(s),37.27(d,J＝11.4Hz).HRMS(ESI)m/z calcd for[C25H23Cl6N9O4+H]+:724.0077 found :724.0083.

Embodiment 42

2-(Pyridin-2-yl)-ethyl-(2,2,2-trichloro-1-(4-hydroxy-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-ethyl)-carbamate (10c)

Using 2-hydroxyethylpyridine as the starting material, the preparation method is the same as that of Example 2. The crude intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthesis method of step 3 of Example 1, the intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-hydroxypyrazolo[3,4-d]pyrimidine were used as raw materials to obtain compound 10c, a light yellow solid, with a yield of 7.22% and an HPLC result of 95.06%. 1H NMR (500MHz, DMSO) δ12.49(s,1H),9.41(d,J＝9.6Hz,1H),8.46(s,1H),8.28(s,1H),8.22(s,1H),7.68(t,J＝6.7Hz,1H),7.31(d,J＝7.0Hz,1H),7.25–7. 19(m,1H),7.12(d,J＝9.8Hz,1H),4.42(dd,J＝13.4,6.5Hz,2H),3.05(t,J＝6.3Hz,2H).13C NMR(101MHz,DMSO)δ157.99(s),157.44(s),154.15(s),149.73(s),149.45(s),137.04(s),136.81(s),124.05(s),122.17(s),116.17(s),106.07(s), 99.15(s),72.11(s),64.92(s),37.14(s)HRMS(ESI)m/z calculated for[C15H13Cl3N6O3+H]+:431.0187,found:431.0193

Embodiment 43

2-(Pyridin-1-yl)-ethyl-(2,2,2-trichloro-1-(4-(2,2,2-trichloro-1-(2-(2-pyridin-2-yl)-ethoxycarbonylamino-)-ethoxy)-1H-pyrazolo[3,4-d]pyrimidin-1-yl-ethyl)-carbamate (10d)

Using 2-hydroxyethylpyridine as the starting material, the preparation method is the same as that of Example 2. The crude intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate is obtained and directly used in the next step reaction.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(pyridin-2-yl)ethyl-(2,2,2-trichloro-1-hydroxyethyl)carbamate and 4-hydroxypyrazolo[3,4-d]pyrimidine were used as raw materials to obtain compound 10d, a light yellow solid, with a yield of 7.22% and an HPLC result of 96.23%. 1H NMR (500MHz, DMSO) δ9.48 (s, 1H), 9.36 (d, J = 9.4Hz, 1H), 9.14 (s, 1H), 8.65 (s, 1H), 8.46 (s, 2H), 7.68 (s, 2H), 7.39 (d, J = 10.0Hz, 1H), 7.30 (d, J = 6.7Hz, 2H),7.20(s,2H),6.91(d,J＝14.1Hz,1H),4.48(dd,J＝15.2,7.7Hz,4H),3.15–3.01(m,4H).

Embodiment 44

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(1-(6-amino-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-2,2,2-trichloroethyl)-carbamate (10e)

Steps 1 and 2 are the same as in Example 1.

According to the synthetic method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 6-aminopyrazolo[3,4-d]pyrimidine were used as raw materials to obtain compound 10e. Pale yellow solid, yield: 14.65%, HPLC: 98.62%. 1H NMR (500MHz, DMSO) δ9.63(d,J＝10.9Hz,1H),8.53(s,1H),8.05(d,J＝47.0Hz,3H),7.73(s,1H),6.81(d,J＝9.8Hz,1H),4.19(d,J＝5.7Hz,4H),2.37(s,3H) .HRMS(ESI)m/z calcd for[C14H14Cl3N9O4+H]+:478.0302,found:478.0309.

Embodiment 45

2-(2-Methyl-5-nitro-1H-imidazol-1-yl)-ethyl-(2,2,2-trichloro-1-(6-chloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-ethyl)-carbamate (10f)

Steps 1 and 2 are the same as in Example 1.

According to the synthesis method of step 3 of Example 1, the intermediate 2-(2-methyl-5-nitro-1H-imidazolin-1-yl)ethyl(2,2,2-trichloro-1-hydroxyethyl)carbamate and 6-chloropyrazolo[3,4-d]pyrimidine were used as raw materials to obtain the hydrochloride of compound 10f. Pale yellow solid, yield: 9.08%, HPLC: 95.06%. 1H NMR (500MHz, DMSO) δ11.89(d,J＝3.4Hz,1H),9.64(d,J＝9.5Hz,1H),8.98(s,1H),8.01(d,J＝3.7Hz,1H),7.99(s,1H),6.89(d,J＝10.0Hz,1H),4.54(s,4H) ,2.40(s,3H).HRMS(ESI)m/z calcd for[C14H12Cl4N8O4+H]+:496.9803,found:497.0073.

Embodiment 46

Cancer cell proliferation inhibition assay

In order to verify the anticancer activity of the designed synthetic compounds, this project designed two parts of activity tests. First, the MTT method was used to screen the proliferation inhibition activity of all compounds in six cell lines: breast cancer MCF-7, melanoma A375, lung adenocarcinoma A549, hepatocellular carcinoma HepG2, cervical cancer Hela, and ovarian cancer Carvo-3. The positive control drug selected was Apcin, a specific inhibitor of Cdc20.

The principle of the MTT method is that the succinate dehydrogenase in the mitochondria of living cells can reduce exogenous tetrazolium bromide (MTT) to insoluble blue-purple crystals and deposit them in the cells, while dead cells do not have this function. DMSO can dissolve the purple crystals in the cells, and the absorbance value detected at a wavelength of 490nm using an enzyme-linked immunosorbent assay can reflect the number of living cells.

The final _IC50 value was obtained by three parallel experiments using the MTT method described above.

(1) Experimental Materials

a. Cell lines: MCF-7, A375, A549, HepG2, Hela, Carvo-3 cell lines

b. Reagents and instruments: Methyl tetrazolium (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; newborn calf serum, fetal bovine serum, trypsin, RPMI-1640, DMEM and penicillin-streptomycin were purchased from Gibco; other reagents were purchased from Sigma-Aldrich unless otherwise specified; _CO2 incubator (Thermo), microplate reader (Thermo) and fluorescence inverted microscope (Olympus).

(2) Experimental content

a. Take cells with a live cell ratio of more than 90% for the experiment (MTT method). Collect cells in the logarithmic phase, adjust the cell suspension concentration to 4-5×10 ⁴ cells/mL, add 100 μL to each well, and plate the cells to be tested to a density of 4000-5000/well.

b. Incubate at 37°C with 5% CO ₂ until the cell monolayer covers the bottom of the well (96-well flat-bottom plate), add drugs in different concentration gradients to make the final concentration of the compound in the wells 300, 100, 30, 10, 3, 1, 0.3, 0.1, 0.03, 0 μM/mL, 100 μL per well, and make 3 replicate wells at the same time to compare the differences between groups.

c. Place in a 5% CO ₂ , 37°C constant temperature incubator for 48 hours and observe under an inverted microscope.

d. Add 10 μL of MTT solution (5 mg/mL, i.e., 0.5% MTT) to each well and continue culturing for 4 h.

e.Terminate the culture after 4 hours and carefully remove the culture medium in the wells.

f. Add 150 μL DMSO to each well and shake at low speed for 10 min on a shaker to fully dissolve the crystals. Measure the absorbance of each well at a wavelength of 490 nm using an enzyme-linked immunosorbent assay and calculate the inhibition rate.

g. Set up zero adjustment wells (culture medium, MTT, DMSO) and control wells (cells, drug dissolution medium of the same concentration, culture medium, MTT, DMSO) at the same time.

(3) Data processing

Each concentration gradient was repeated three times, and the SPSS13.0 software was used to fit the curve with concentration as the horizontal axis and inhibition rate as the vertical axis to calculate the inhibition rate (IC ₅₀ value) of the compound. The absorbance OD value of each well was measured at a detection wavelength of 490nm on an ELISA instrument, and the cell growth inhibition rate was calculated as follows:

Table 1: Inhibition data of the tested compounds on cancer cell lines

The results show:

Compared with the specific Cdc20 inhibitor Apcin, the anticancer activity of most of the obtained target compounds was improved. Compounds containing dichloro-substituted pyrimidines (7a-7d) and compounds containing 2-fluoroadenine side chains (8g, 9d, 9e, 9f, 9h) showed the most effective antiproliferative activity. In order to further confirm the targeting of the compounds, 7d and 9f with the best activity were selected to continue the cell cycle and apoptosis studies designed by the present invention.

Embodiment 47

Human hepatocellular carcinoma cell (Hep-G2) cell cycle experiment

(1) Experimental Materials

a. Cell line: Human hepatocellular carcinoma cell HepG2

b. Reagents and instruments: The multifunctional imaging system was purchased from Biotek; the phosphorylated histone H3 antibody was purchased from CST; the manufacturers of PBS, trypsin digestion solution, DMSO, fetal bovine serum, DMEM, 96-well cell culture plates, T25 cell culture flasks, CO2 cell culture incubator, low-speed centrifuge, cell counter, and biological clean bench were the same as those used in the cancer cell proliferation inhibition experiment.

(2) Experimental content

a. Take HepG2 cells growing in the logarithmic phase and wash them three times with 1×PBS, 2 minutes each time; add trypsin to the culture flask/culture dish for digestion and digest in a _CO2 incubator for 2 minutes. Add medium (DMEM) to the culture flask/culture dish to terminate digestion, and use a sterile pipette to blow the cells until the cells are in a single suspension state;

b. Count the cells using a cell counting plate and plate the cells.

c. Apply drugs and incubate in a constant temperature box for 2 days.

d. Aspirate the medium from each well, add 100 μl of 4% paraformaldehyde to each well, and incubate at room temperature for 15 min.

e. Aspirate and rinse three times with PBS.

f. Add 0.1 ml of permeabilization solution to each well and incubate at room temperature for 15 minutes.

g. Aspirate the permeate and rinse three times with PBS.

h. Add 100 μl of blocking buffer to each well and incubate at room temperature for 15 min.

i. Aspirate the blocking buffer, add 50 μl of primary antibody solution to each well, and incubate at 4°C overnight.

j. Aspirate the primary antibody solution and rinse three times with PBS, add 50 μl of secondary antibody solution to each well and incubate at room temperature for 60 min (protected from light).

k. Aspirate the secondary antibody solution and rinse three times with PBS, add 100 μl PBS to each well and proceed to imaging.

As shown in Figure 4, the immunofluorescence experiment results of HepG2 cell lines treated with drugs. Drug group: compound Apcin (100 μM), 7d (30 μM), 9f (0.3 μM). Drug treatment time: 48 h.

Compared with the solvent control group in HepG2 cells, 30 μM compound 7d and 0.3 μM compound 9f induced an increase in phospho-histone H3-positive cells, from 3.8% to 6.9% and 10.0%, respectively. Compared with the DMSO control, 100 μM positive compound Apcin led to an increase of about 2.2 times in the number of PHH3-positive cells. Compared with the positive control drug Apcin, compounds 7d and 9f showed stronger efficacy in blocking cell mitotic exit, and compound 9f was the most effective mitotic blocker, showing a strong M phase arrest effect at 0.3 μM.

Embodiment 48

Human hepatocellular carcinoma cell (Hep-G2) apoptosis experiment

(1) Experimental Materials

a. Cell line: Human hepatocellular carcinoma cell HepG2

b. Reagents and instruments: The multifunctional imaging system was purchased from Biotek; the Annexin-FITC/PI apoptosis kit was purchased from Solebao; the manufacturers of PBS, trypsin digestion solution, DMSO, fetal bovine serum, RPMI 1640, 24-well cell culture plates, T25 cell culture flasks, CO2 cell culture incubator, low-speed centrifuge, cell counter, and biological clean bench were the same as those used in the cancer cell proliferation inhibition experiment.

(2) Experimental procedures

a. Take HepG2 cells in logarithmic phase, wash them 3 times with 1×PBS, 2 minutes each time, add trypsin to the culture flask/dish for digestion, and digest in _CO2 incubator for 2 minutes. Add medium (DMEM) to the culture flask/dish to stop digestion, blow the cells with a sterile pipette until the cells are in a single suspension state. Count them with a cell counting plate and plate them.

b. Add drugs and incubate at 37°C overnight.

c. Dilute 3 ml of Binding Buffer (10×) to 30 ml with 27 ml of deionized water, using 3 ml each time.

d. Add 2 μl ANNEXIN V-FITC and stain for 10 minutes at room temperature, away from light. It is also advisable to use a shaker or shake manually several times.

e. Add 5 μl PI and incubate for 5 min at room temperature in the dark.

f. Aspirate the staining solution, add PBS to rinse, and use for imaging.

As shown in Figure 5, the results of the apoptosis experiment of HepG2 cell lines treated with drugs. Drug group: compound Apcin (100μM), 9f (0.3μM); drug treatment time: 24h. When the drug was treated for 24h, high concentrations of Apcin had a significant effect on promoting cell apoptosis, and most cells were in the early apoptosis stage, but there were also late apoptotic cells. High concentrations of 9f had a significant effect on promoting cell apoptosis, and most cells were in the early apoptosis stage. Compound 7d (30μM) also had a pro-apoptotic effect.

The above mechanism studies preliminarily show that compounds 9f and 7d can significantly inhibit the proliferation of cancer cells. At the same time, studies have shown that the compounds designed in this project inhibit the proliferation of cancer cells by blocking the cell cycle and promoting cell apoptosis.

The contents explained in the above embodiments should be understood as these embodiments are only used to more clearly illustrate the present invention, and are not used to limit the scope of the present invention. After reading the present invention, various equivalent forms of modifications to the present invention by those skilled in the art all fall within the scope defined by the claims attached to this application.

Claims (2)

1. A (1, 1-trichloro-2) carbamate derivative, characterized by the following structural formula:

2. use of a (1, 1-trichloro-2) carbamate derivative according to claim 1 for the preparation of a medicament for the treatment of lung adenocarcinoma a549, hepatocellular carcinoma HepG2 and ovarian cancer Carvo-3.