CN111606891B

CN111606891B - (1,1,1-trichloro-2) carbamate derivative and preparation method and application thereof

Info

Publication number: CN111606891B
Application number: CN202010377518.2A
Authority: CN
Inventors: 陈卓; 胡高云; 李乾斌; 黄攀
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2020-04-15
Filing date: 2020-05-07
Publication date: 2023-01-17
Anticipated expiration: 2040-05-07
Also published as: CN115611867A; CN111606891A

Abstract

The invention relates to (1,1,1-trichloro-2) carbamate derivatives, a preparation method thereof and related medical application. In order to achieve the purpose, the invention provides a (1,1,1-trichloro-2) carbamate derivative, which has a structural general formula shown in formula (I-III):

Description

(1,1,1-trichloro-2) carbamate derivative and preparation method and application thereof

Technical Field

The invention relates to (1,1,1-trichloro-2) carbamate derivatives, a preparation method thereof and related medical application.

Background

Malignant tumors are one of the main causes of death in most countries in the world today, and seriously threaten human health. The investigation shows that the incidence rate of malignant tumor is in a trend of increasing year by year. With the rapid development of molecular biology, scientists have more deep and comprehensive knowledge on the related signal pathways and targets for the generation and development of malignant tumors, and have achieved important results in designing anti-tumor drugs based on the signal pathways and targets. With the widely accepted view that "malignant tumor is a kind of cell cycle disease", the research of anti-tumor drugs aiming at cell cycle regulation becomes a hot spot.

Cell cycle disorders are a common feature of human cancers, and mutations accumulated 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. Inhibition of the initial stages of the cell cycle may give rise to quiescent cells in life, whereas targeting mitosis has several possibilities to kill cancer cells. Targeting mitosis is a long-established approach, and currently widely studied are novel drugs targeting mitotic spindle microtubule elements and targeting mitotic non-microtubule effectors, with drugs affecting the mitotic spindle being well-recognized components of many cancer treatment regimens.

In general, the cellular mitotic process is subject to complex and delicate regulation and ultimately divides the replicated genome into two identical daughter cells. Mitosis is divided into five distinct phases: prophase, prometaphase, metaphase, anaphase and telophase. The prophase transitions from G2 to mitosis, with the onset of chromosome condensation and nuclear envelope breakdown. In prometaphase, chromosomal centromeres are attached to spindle microtubules by search and capture mechanisms. In metaphase, chromosomes converge on the equatorial plate equidistant from the centrosome. At a later stage, the centromere microtubules become shortened, and sister chromatids are separated and moved to the opposite polarity, which is called segregation. Finally, the cell is bisected during telophase and cytokinesis, forming two daughter cells (fig. 1). A large number of small molecule compounds have been designed and studied against mitosis-related proteins.

Anti-microtubule drugs are the first therapeutic approach to mitosis, and microtubules play an important role in maintaining cell morphology, cell division, signal transduction, and substance transport. Microtubules are polymerized into spindles in the mitotic prophase, and the spindles pull chromosomes to move to the two poles in mitosis to enter two self cells to complete cell proliferation. Microtubules play an extremely important role in cell division, and are now one of the important targets of anticancer drugs, and microtubule inhibitors have been clinically proven to have significant efficacy against a variety of malignant tumors. Microtubule polymerization agents (including paclitaxel and docetaxel) and microtubule depolymerization agents (including vinorelbine) target primary tubulin, disrupting the kinetic stability of microtubule polymerization-depolymerization, thereby blocking the cell cycle in mitotic phase and inducing apoptosis of tumors. However, these drugs not only act on proliferating tumor cells, but also exhibit significant side effects on non-abnormally proliferating normal cells. In addition, the expression of multidrug resistance (MDR) proteins and tubulin isoforms (e.g., mutations in tubulin) has also been associated with microtubule inhibiting drugs.

Thus, the identification of novel mitotic drug targets other than tubulin has recently received much attention. In recent years, several mitotic targets have been explored, including drugs that target microtubules, kinases, motor proteins, and multi-protein complexes. Among them, ubiquitin-proteasome (UPS) pathway-mediated ubiquitination of substrates and their subsequent ordered degradation process, which are carried out throughout the plasma membrane system of cells, are essential for cells to undergo mitosis throughout the cell cycle, and have important significance in treating various diseases caused by disorders of the ubiquitin system, especially malignant tumors. In general, the ubiquitination process requires the co-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 linked to ubiquitin activating enzyme (E1) in an ATP-powered reaction. The activated ubiquitin is then transferred to a small ubiquitin-binding enzyme (E2) to form a thioester-linked E2-ubiquitin intermediate (E2. About. Ub). E2 acts alone or in combination with E3 ubiquitin ligase to conjugate ubiquitin to the epsilon-amino group of lysine residues in substrate proteins to form isopeptide bonds. Tight control of these seemingly simple sequential actions of E1-E2-E3 can achieve accurate and proper timing of ubiquitination/proteolysis.

While the specificity of the ubiquitination system to degrade proteins is determined by ubiquitin ligase E3. Wherein, the cell division later-stage Promoting Complex (APC) is the largest E3 ubiquitin ligase, consists of at least 14 subunits and a coactivator, and forms two different E3 ubiquitin ligase complexes APC after being activated by cell division cyclin 20 (Cdc 20) or Cdc20 homolog 1 (Cdh 1) ^Cdc20 Or APC ^Cdh1 (FIG. 2), APC ^Cdc20 The transition phase from metaphase to anaphase of mitosis disrupts key regulators in the cell growth cycle, regulates cell cycle progression, and APC ^Cdh1 Plays an important role mainly in the G1 phase.

More and more researches show that Cdc20 promotes the occurrence and development of tumors, a phenomenon of Cdc20 high expression is found in many tumors, the expression degree is closely related to the pathological degree and prognosis of tumor patients, and the higher the expression level of Cdc20 is, the higher the risk is. In conclusion, the expression level of Cdc20 is an effective prognostic index for tumor patients, and provides a basis for Cdc20 as a novel antitumor drug development target. In contrast, cdh1, another co-activator of APC/C, is considered to be a tumor suppressor.

At present, with the research on Cdc20 being deepened, more and more APC are available ^Cdc20 Substrates were found (Table 1), some of which are involved directly or indirectly in cell division processes, cdc20 regulating ubiquitination or degradation processes of these substrates and thus regulating cell cycle progression, and thus, more APC were found ^Cdc20 The substrate helps to understand the molecular mechanism by which Cdc20 regulates cell cycle progression.

TABLE 1 APCCdc20 downstream substrates and their function

The use of compounds to inhibit the Cdc20 pathway is a new approach to the treatment of tumors. The Cdc20 targeted inhibitor can directly act on Cdc20 and inhibit APC ^Cdc20 The activity of the complex. Apcin is the only reported specific inhibitor of Cdc20 at present, and acts on WD40 region at the N-terminal of Cdc20, blocks Cdc20 from binding with downstream substrates, and inhibits ubiquitination and degradation processes of downstream substrates (fig. 3). In addition, many compounds are reported to indirectly affect the Cdc20 pathway and exert antitumor effects. In addition, the active ingredients in some natural products also have the function of regulating Cdc20 pathway (Table 2)

TABLE 2 partial Cdc20 inhibitor Structure and function

Premature withdrawal of cells from mitosis by mitotic slippage is considered to be one of the major mechanisms by which cells develop resistance to antimitotic drugs. Both mechanisms of action of microtubule inhibitors activate the SAC by drug binding to the vinca, taxane or colchicine sites, producing unattached centromeres, and thus, mitotic cell death or mitotic glide is observed.

Whereas in the normal cell cycle, mitotic withdrawal is driven by APC/C-Cdc20 dependent cyclin B1 degradation. Inhibition of the mitotic process triggers SAC and inhibits APC/C-Cdc20. However, this inhibition is transient and slow, and cyclin B1 may gradually degrade despite the failure of the mitotic checkpoint. Cells that escape mitotic cell death can die at a later stage of the cell cycle, either halting at the tetraploid state or undergoing several rounds of division. Thus, it is envisioned that complete blocking of mitotic withdrawal may be an effective strategy for inducing mitotic cell death. Inhibition of cyclin B1 degradation in vivo by gene ablation of Cdc20 has been shown to be more effective in killing tumor cells than compounds assembled with traditional targeting spindles. On the other hand, classical microtubule-inhibiting antimitotic drugs (vincristine or paclitaxel) induce only partial responses in aggressive tumors, and Cdc20 ablation can lead to complete arrest of the division phase, leading to massive apoptotic cell death and complete elimination of the tumor in vivo. Meanwhile, studies have shown that in vitro, cdc 20-free tumor cells fail to pendulate metaphase arrest and die of mitotic cell death within 6-30 hours. Importantly, mitotic withdrawal inhibition also affected pRb-null, p53-null or SAC-deficient cells, suggesting a broad utility, making up for the poor therapeutic effect of microtubule inhibitors on SAC-inactive cells. Overall, these results indicate that targeting Cdc20 to inhibit Cyclin B1 degradation may be very effective in killing tumor cells.

Therefore, the design of small molecule inhibitors that simultaneously target Cdc20, which inhibits mitotic withdrawal, and tubulin, which activates SAC, is an effective method of triggering mitotic cell death. The purpose of mitotic withdrawal inhibitors is to prevent the depletion of cyclin B, thereby causing the cell to be arrested in mitosis and die. One preclinical study demonstrated that knockout of Cdc20 is more effective at killing resistant cells than paclitaxel treatment. Another study used the APC inhibitor proTAME in combination with either paclitaxel or MLN-8054, two anti-mitotic drugs, resulting in activation of cancer cell apoptosis. By reviewing clinical trials of antimitotic agents, it is evident that better cancer treatment results can be obtained when designed dual-target inhibitors are employed.

Current anti-mitotic strategies focus on SAC activation, and the main cancer treatment is activation of SAC and induction of apoptosis using microtubule drugs, but their effectiveness is limited by background Cyclin B1 degradation and mitotic slippage due to residual APC/C-Cdc20 activity. Cancer cells in particular, show a high rate of mitotic glide due to mutations that disrupt SAC, making treatment ineffective. Thus, compounds directed against factors downstream of the checkpoint such as Cdc20 are more effective in killing cells, particularly in glide-prone and anti-apoptotic cells, than other mitotic inhibitors (including MIA and kinesin inhibitors).

Apcin, the only reported CDC20 specific inhibitor at present, has weak antitumor activity, has no application and clinical application, and may be related to the protein-protein interaction of CDC20 and a wider range of substrates.

At present, no anti-tumor compound with double functions of CDC20 and a microtubule network is reported, the 'double-function strategy' can reduce the endonuclear replication and prevent the proteolysis of background Cyclin B1, thereby enhancing the mitosis retardation effect, effectively preventing the occurrence of mitosis slippage, improving the sensitivity of cancer cells to drugs and opening up a new way for developing a cancer treatment method based on a microtubule inhibitor.

Disclosure of Invention

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

In order to achieve the purpose, the technical scheme of the invention is as follows:

a (1,1,1-trichloro-2) carbamate derivative has a structural general formula shown in formula (I-III):

n is 0 to 1;

x is O;

wherein R is ₁ Selected from hydrogen, aromatic ring, substituted aromatic ring, aromatic heterocycle, substituted aromatic heterocycle, aliphatic heterocycle, substituted aliphatic heterocycle, C1-C4 open chain amino, C1-C6 straight chain or branched chain alkoxy, C1-C6 straight chain or branched chain alkane, C3-C6 aliphatic heterocycleA ring and one of a substituted C3-C6 aliphatic ring, a fatty lenalidomide substituted C1-C8 straight chain or branched chain alkyl group and a lenalidomide substituted C1-C8 straight chain or branched chain alkoxy alkyl group.

In the structural formula I, R ₂ ，R ₃ ，R ₄ Independently selected from hydrogen, amino, hydroxy, halogen, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 alkylamino, the phenyl moiety of a quinoline ring; y is selected from N, CH; when Y is N, R2, R3 and R4 are not hydrogen at the same time;

in the structural formula II-III, Y is selected from N and CH; r ₅ And R ₆ Independently selected from amino, H, halogen, C ₁ -C ₈ Alkyl radical, C ₁ -C ₈ Alkoxy radical, C ₁ -C ₈ Haloalkyl, C ₁ -C ₈ An alkylamino group,

Preferably, the aromatic ring is selected from the group consisting of 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 heterocyclic ring is selected from pyrrolidine, piperazine, piperidine, morpholine and tetrahydrofuran; the substituent in the substituted aromatic ring, the substituted aromatic heterocyclic ring and the substituted aliphatic heterocyclic ring is 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.

Preferably, 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 scheme, the (1,1,1-trichloro-2) carbamate derivative has a structural general formula shown in formula I:

wherein n is 1;

x is O;

R ₁ one of hydrogen, C1-C4 straight chain or branched chain alkoxy, C1-C4 straight chain or branched chain alkane, C3-C6 fat ring and substituted C3-C6 fat ring, benzene ring, substituted benzene ring, aromatic heterocyclic ring, substituted aromatic heterocyclic ring, aliphatic heterocyclic ring and substituted aliphatic heterocyclic ring is selected;

the aromatic heterocyclic ring is selected from pyrrole, imidazole, thiazole, oxazole, pyrazole, pyridine, pyrimidine and indole;

the aliphatic heterocyclic ring is selected from pyrrolidine, piperazine, piperidine and morpholine;

the substituent in the substituted aromatic ring, the substituted aromatic heterocyclic ring and the substituted aliphatic heterocyclic ring is selected from methyl, ethyl, methoxy, hydroxyethyl, amino, nitro, trifluoromethyl and halogen;

R ₂ ，R ₃ ，R ₄ selected from amino, H, = O, halogen, C ₁ -C ₃ Alkyl radical, C ₁ -C ₃ Alkoxy, the phenyl portion of the quinoline ring; y is selected from N, CH; r ₂ ，R ₃ ，R ₄ Not hydrogen at the same time.

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

wherein n is 0-1;

x is O;

R ₁ is selected from one of C1-C4 straight chain or branched chain alkoxy, C1-C4 straight chain or branched chain alkane, C3-C6 aliphatic ring and substituted C3-C6 aliphatic ring benzene ring, substituted benzene ring, aromatic heterocyclic ring, substituted aromatic heterocyclic ring, aliphatic heterocyclic ring and substituted aliphatic heterocyclic ring.

R ₅ and R ₆ Selected from amino, H, halogen, C ₁ -C ₇ Alkyl radical, C ₁ -C ₇ Alkoxy radical, C ₁ -C ₇ Haloalkyl, C ₁ -C ₇ An alkylamino group,

Y is N, CH.

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

wherein n is 1;

x is O;

R ₅ and R ₆ 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 invention also provides a synthetic method of the compounds shown in the formula (I), the formula (II) and the formula (III), and the synthetic route is as follows:

the synthesis steps of the (1,1,1-trichloro-2) carbamate derivative comprise:

1. reacting a hydroxyl substituted compound with chloroformic acid p-nitrophenyl ester in an anhydrous aprotic solvent to obtain an intermediate;

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

3. refluxing the carbamate product and chloral hydrate at 80-100 deg.C to obtain (1,1,1-trichloro-2) carbamate part;

4. the (1,1,1-trichloro-2) carbamate moiety is chlorinated with thionyl chloride and then reacted with an amine to give the target compound.

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

Preferably, the reaction in step 1 is carried out for 4-12h to obtain an intermediate.

Preferably, the reaction in the step 1 can be accelerated by adding weak base for catalysis.

Preferably, the weak base is any one or combination of triethylamine and diisopropylethylamine, and the molar charge ratio is as follows: hydroxy-substituted compound base = 1.2-1.

Preferably, the anhydrous aprotic solvent is dichloromethane.

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

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

Preferably, the reaction molar charge ratio of the step 3 is as follows: carbamate chloral hydrate =1:8-1

Preferably, the refluxing time of the step 3 is 6-48h.

Preferably, the reaction temperature in step 3 is 100 ℃.

Preferably, step 4 is carried out at a temperature rt-50 ℃.

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

The invention also provides application of the (1,1,1-trichloro-2) carbamate derivative in preparation of medicines for treating breast cancer, melanoma, lung adenocarcinoma, hepatocellular carcinoma, cervical cancer and ovarian cancer.

The invention is further explained below:

according to the invention, based on the structure of cdc20 specific inhibitor apcin, different substituent groups are introduced on the pyrimidine ring of the cdc20 specific inhibitor apcin to obtain a plurality of derivatives (structural parent nucleus I), and after chlorine atoms are introduced, the compounds and the cdc20 have more interaction sites and enhanced affinity, so that the inhibition effect of the compounds on tumor cells is greatly enhanced.

After pyrimidine in the apcin structure is replaced by purine (structure mother nucleus II) or benzopyrazole (structure mother nucleus III), the inhibition effect on tumor cells is further enhanced. On the basis of keeping a certain cdc20 inhibition effect, the interaction between the compound and tubulin dimer is enhanced due to introduction of purine or benzopyrazole, so that the microtubule aggregation inhibition effect is generated. The compound has double-function anti-tumor effect and is expected to become a novel anti-tumor lead compound.

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

1. the invention enriches the structure of the cdc20 inhibitor on the basis of apcin, and obtains three structure mother nuclei;

2. compared with apcin, the tumor inhibition activity of the target compound is greatly enhanced, and the target compound has good inhibition effect in a plurality of tumor cell strains;

3. according to the invention, through the research on the anti-tumor activity mechanism of the compound, the novel compound has the original cdc20 inhibition activity of apcin and also has a better microtubule aggregation inhibition effect, and a novel way is developed for searching a cancer treatment method aiming at the APC pathway and microtubule inhibition dual functions.

Drawings

Figure 1 is a diagrammatic view of mitotic progression and associated target proteins and complexes;

FIG. 2 shows that APC/C RING E3 ubiquitin ligase combines with its co-activator Cdc20/Cdh1 to promote substrate ubiquitination;

FIG. 3 is a graph showing that small molecule compound Apcin binds to the D-box binding pocket (left) on the side of the WD40 region and that small molecule compound TAME interferes with APC3 binding by mimicking the isoleucine-arginine (IR) tail of Cdc20 and Cdh 1;

FIG. 4 shows the results of immunofluorescence experiments with drug-treated HepG2 cell lines;

FIG. 5 shows the results of apoptosis experiments (fluorescence of compound pro-apoptosis) for drug-treated HepG2 cell lines.

Detailed Description

The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments of the present invention may 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 (5 b)

Step 1

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

Weighing 30.0g of metronidazole (0.175 mol) and placing the metronidazole into a 500mL eggplant-shaped bottle, then sequentially adding 300mL of anhydrous dichloromethane for dissolution and 30mL of anhydrous triethylamine (0.210 mol), stirring uniformly, adding 42.5g of chloroformic acid p-nitrophenyl ester (0.210 mol) under the ice bath condition, and finally supplementing 30mL of anhydrous dichloromethane for rinsing the bottle mouth. The mixture is moved to room temperature and stirred for overnight, and the metronidazole reaction is monitored by TLC to be basically completed, and the reaction is stopped. 62mL of ammonia (28%) was slowly added to the reaction solution at 10 ℃ via a constant pressure dropping funnel, and the reaction was completed for 3 hours. The reaction solution was filtrated to obtain 58.3g of a solid, and the obtained solid was dissolved in 200mL of a sodium hydroxide solution (1 mol/L), then extracted 3 times with 200mL of dichloromethane, and the organic layers were combined, concentrated under reduced pressure, and dried to obtain 18.6g of a yellow crystalline compound, 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethylcarbamate. The two-step yield: 50%, melting point: 151.5-153.1 ℃.

Step 2

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

77.0g of chloral hydrate (0.467 mol) is weighed and put into a 100mL eggplant-shaped bottle, the bottle is placed on an oil bath pot for heating, after the chloral hydrate is melted, 10.0g of compound 2- (2-methyl-5-nitro-1H-imidazoline-1-yl) ethyl carbamate (0.047 mol) is slowly added, the reaction is stirred at 100 ℃, and the reaction is stopped after 48 hours of reaction. The reaction solution was cooled to room temperature, 50mL of ethyl acetate was added thereto, and the mixture was sonicated to obtain a cloudy solution which was then subjected to suction filtration to obtain 11.6g of a white compound. Yield: 68%, melting point: 168.7-169.7 ℃.

Step 3

The intermediate 2- (2-methyl-5-nitro-1H-imidazoline-1-yl) ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 4-methyl-2-aminopyrimidine are used as raw materials to obtain a compound 5b.

The specific operation is as follows: weighing 0.50g of intermediate 2- (2-methyl-5-nitro-1H-imidazoline-1-yl) ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate (1.38 mmol) and placing the intermediate into a 50mL eggplant-shaped bottle, then weighing 30mL of anhydrous dichloromethane and adding the anhydrous dichloromethane into the reaction bottle, dropwise adding 1 drop of triethylamine, stirring uniformly, slowly dropwise adding 0.50mL of thionyl chloride (6.91 mmol) by using a constant-pressure dropping funnel under an ice bath, transferring the mixture into an oil bath after dropwise adding, carrying out reflux reaction at 40 ℃ under the protection of nitrogen and standing overnight, and stopping the reaction. After the reaction solution was cooled to room temperature, the reaction solution was concentrated under reduced pressure, and 20mL of anhydrous dichloromethane was added to dissolve the reaction solution sufficiently, and the concentration under reduced pressure was repeated 3 times to remove the remaining thionyl chloride to obtain a yellow solid. The resulting yellow solid was dissolved in 15mL of anhydrous dichloromethane and 15mL of anhydrous acetonitrile, and another starting amine (1.66 mmol) was added to the reaction solution, after 24h 10mL of methanol was added and the reaction was quenched by stirring for 30 min. The reaction solution was concentrated under reduced pressure, dissolved in 50mL of dichloromethane, washed with 50mL of saturated brine for 3 times, and concentrated under reduced pressure to give a crude product. Dissolving the crude product with 20mL of methanol, mixing with 80-100 mesh silica gel, loading the silica gel into a column with 200-300 mesh, purifying by column chromatography, and separating and purifying by using dichloromethane and methanol solution in an elution system to obtain the target compound.

Target compound was white solid, yield: 21.42%, melting point: 209.0-209.9 ℃, HPLC:97.19%.1H NMR (500 MHz, DMSO-d 6): delta 8.27 (d, J =5.0Hz, 1H), 8.00 (s, 1H), 7.97 (d, J =8.8Hz, 1H), 6.97 (d, J =9.5Hz, 1H), 6.73 (d, J =5.0Hz, 1H), 6.60 (t, J =9.2Hz, 1H), 4.56-4.46 (m, 3H), 4.35 (dd, J =10.7,5.7Hz, 1H), 2.39 (s, 3H), 2.31 (s, 3H). 13C NMR (500MHz, DMSO-d6 delta 36 zxft 3236, 102.88,70.12,63.46,45.90,24.10,14.37 HRMS (ESI) m/z cad for [ 14H 3H ] +/-3719H 16H, 3763H, 3762 + 3719H).

Example 2

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

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

Compound 5c was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 4-methoxy-2-aminopyrimidine. Light yellow solid, yield: 32.66%, melting point: 81.6-83.3 ℃, HPLC:98.63 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.14 (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 delta): 170.22,160.87,158.59,155.27,152.06,138.88,133.51,102.59,99.44,70.35,63.48,53.74,45.89, 14.36.MS (ESI) m/z calcd for [ C14H16Cl3N7O5+ H ] +:468.0278, und.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 (5 d)

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

Compound 5d was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and isocytosine by the synthetic method of step 3 of example 1. Light yellow solid, yield: 16.24%, melting point: 192.3 to 193.2 ℃, HPLC:98.39%.1H NMR (500MHz, DMSO-d 6): delta 11.14 (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 (500 MHz, DMSO-d6 delta 162.08,155.74,155.43,153.95,152.10,138.89,133.56,105.94,101.66,69.29,63.28,46.00, 14.41.HRMS (ESI) m/z cad for [ C13H14Cl3N7O5+ H ] +, 454.0202.454, 0.0120.

Example 4

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

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

Compound 5e was obtained from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2,4-diaminopyrimidine by the synthetic method of step 3 of example 1. Light yellow solid, yield: 28.69%, melting point: 104.2-105.0 ℃, HPLC:98.55 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.02 (s, 1H), 7.95 (d, J =8.5 Hz, 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, 1H), 4.56-4.46 (m, 3H), 4.32 (d, J =11.4Hz, 1H), 2.40 (s, 3H). 13C NMR (500MHz, DMSO-d 6): delta 164.54, 160.64,156.04,155.13,152.07,138.88,133.52,103.38,97.97,69.98,63.36,45.92, ESI 14.37.028 (19) m/C [ 13H ] H + 13H [ 13H ] H + 13H [ 453H ] H + 13H ] H.

Example 5

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

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

Compound 5f was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 4-chloro-2-aminopyrimidine. White solid, yield: 20.18%, melting point: 182.3-183.4 ℃, HPLC:98.75 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.40 (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-d 6): delta 161.18,155.35,152.08,138.90, 133.51,112.61,102.13,70.30,63.51,45.88,14.41.HRMS (ESI) m/z calcd for [ C13H13Cl4N7O4+ H ] + (471.9783, 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.

Compound 5g was obtained by following the synthesis procedure of step 3 of example 1, starting from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 4-trifluoromethyl-2-aminopyrimidine. Yellow oil, yield: 5.26%, HPLC:97.81 percent. 1H NMR (500MHz, DMSO-d 6) Δ 8.79 (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.

Example 7

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

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

Compound 6a was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 4-aminopyrimidine by the synthetic method of step 3 of example 1. Yellow solid, yield: 32.24%, melting point: 179.4-180.2 ℃, HPLC:99.79% of the total weight of the composition. 1H NMR (500MHz, DMSO-d 6): delta 8.59-8.48 (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, 3H), 4.35 (dd, J =10.7,4.5Hz, 1H), 2.39 (s, 3H), 13C NMR (500MHz, DMSO-d 6): 161.10,158.30, 155.81,155.59,152.14,138.87,133.56,107.09,101.98,68.80,63.24,46.04,14.41 HRMS (ESI) for m/z calc [ 13H 3H ] 3+ 7H, 0264H, 3H + 0264H).

Example 8

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

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

Compound 6b was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2-aminoquinoline. Dark yellow solid, yield: 25.62%, melting point: 119.9-121.0 ℃, HPLC:99.75 percent. 1H NMR (500MHz, DMSO-d 6): δ 8.35 (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-d 6) δ 5363 zxv5363, 129.84,127.96,126.72,124.23,123.01,113.30,102.69, 63.63, 63, 18C NMR (500mhz, dmso-d6 δ 5363, 18C × 18Cl, 18H) for N + 18H + 18Cl 19, 18H + 18H.

Example 9

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

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

Compound 6c was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 3-aminoisoquinoline. Yellow solid, yield: 28.32%, melting point: 122.6-124.2 ℃, HPLC:99.50 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.96 (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-d 6): delta 155.57,152.99,152.12,151.48,138.86,138.56,133.53,131.03,128.23, 125.27,124.18,123.73,103.23,100.17,70.84,63.23,46.02, 18C NMR (500MHz, 17H), 18H + 18H [ 18H ]:17H ].

Example 10

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

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

Compound 6d was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 1-aminoisoquinoline. White solid, yield: 27.43%, melting point: 113.5-114.8 ℃, HPLC:97.92 percent. 1H NMR (500mhz, dmso-d 6): δ 8.06 (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 5003 (NMR (500mhz, dmso-d6 δ 3238, 38 × 38, 18 × 19H), 18 × 18Cl [ 18Cl ] 19, 18 × 17Cl ].

Example 11

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

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

Compound 6e was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and benzimidazole as starting materials according to the synthesis procedure of step 3 of example 1. White solid, yield: 22.13%, melting point: 123.6-124.9 ℃, HPLC:97.97%.1H NMR (500MHz, DMSO-d 6): δ 9.68 (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-d 6) δ 155.67,152.01,142.82,142.73,138.91,134.14,133.57,123.96, 123.03,120.18,112.05,99.86,73.06,63.74,45.86, 14.34.34 g (ms), 15.461 [ + 15H + hrc ] 15H + 3H).

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 (6 f)

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

Compound 6f was obtained according to the synthesis method of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and adenine. Dark yellow solid, yield: 10.56%, melting point: 173.8-175.3 ℃, HPLC:97.12 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.63 (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/z cad 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 (7 a)

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

Compound 7a was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2-amino-4,6 dichloropyrimidine by the synthetic method of step 3 of example 1. Light yellow solid, yield: 46.23%, melting point: 2175.5-176.3 ℃, HPLC:99.73 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.56 (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-d 6): delta 160.73,155.42,152.12,138.90, 133.50,111.40,101.52,70.47,63.53,45.90,14.45 HRMS (ESI) m/z calcd for [ C13H12Cl5N7O4+ H ] +: 505.93505.9493, fo72).

Example 14

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (2,2,2-trichloro-1- ((4,6-dichloro-5-methylpyrimidin-2-) amino) -ethyl) -carbamic acid ester (7 b)

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

Compound 7b was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 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. White solid, yield: 46.66%, melting point: 140.3-142.1 ℃, HPLC:99.82 percent. 1H NMR (400MHz, DMSO-d 6): δ 8.24 (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-d 6): δ 158.23,155.43,152.15,138.90, 133.55,117.65,101.74,70.55,63.51,45.92,15.41,14.47.HRMS (ESI) m/z calcd for [ C14H14Cl5N7O4+ H ] +, 519.50, 519.29.

Example 15

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (2,2,2-trichloro-1- ((5-amino-4,6-dichloropyrimidin-2-) amino) -ethyl) -carbamic acid ester (7 c)

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

Compound 7c was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2,5-diamino-4,6 dichloropyrimidine. Tan solid, yield: 27.68%, melting point: 92.9-93.7 ℃, HPLC:99.73 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.00 (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-d 6): delta 155.34,152.07,151.11, 145.55,138.88,133.49,129.80,102.40,71.00,63.41,45.92,14.40 HRMS (ESI) m/z calc for [ C13H13Cl5N8O4+ H ] +, 520.95520.958, 520.958, 520.9581H).

Example 16

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (1- ((2-amino-4,6-dichloropyrimidin-5-) amino) -2,2,2-trichloroethyl) -carbamic acid ester (7 d)

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

Compound 7d was obtained according to the synthesis method of step 3 of example 1 using 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2,5-diamino-4,6 dichloropyrimidine as starting materials. White solid, yield: 20.18%, melting point: 125.1-125.7 ℃, HPLC:99.14 percent. 1H NMR (500MHz, DMSO-d 6): delta 8.35 (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, 1H), 4.47 (d, J =11.9Hz, 3H), 4.42-4.35 (m, 1H), 2.40 (s, 3H), 13C NMR (500MHz, DMSO-d6 delta): 8978 zft 8978, 156.40,155.83,152.17,138.83,133.53, 156.90, 102.23, 121.27, 63.21,46.09, 14.45.ESI) m/z for [ C.958H ] + 958H ] C [ C.95520H ] C [ C.3H ].

Example 17

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

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

Compound 8a was obtained by the synthetic method of step 3 of example 1 using intermediate 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 6-chloropurine as starting materials. Pale yellow solid, yield: 21.32%, melting point: 101.4-101.8 ℃, HPLC:96.82 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.76 (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-d 6): delta 155.56,153.11,152.38,151.86,150.38,144.65,138.98,133.45,130.31,98.77, 71.56,63.87,45.75,14.35 HRMS (ESI) m/z calcd for [ C14H12Cl4N8O4+ H ] +:496.9736, found.

Example 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 (8 b)

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

Compound 8b was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 6-dimethylaminopurine. White solid, yield: 20.23%, melting point: 113.6-115.2 ℃, HPLC:98.22 percent. 1H NMR (500MHz, CDCl3): delta 8.51 (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-d 6): delta 155.52,154.74,153.01,151.93,151.01,138.94,137.06,133.50, 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.

Example 19

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (1- (6-benzamido-9H-purin-9-yl) -2,2,2-trichloroethyl) -carbamic acid ester (8 c)

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

Compound 8c was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and N6-benzoyladenine. White solid, yield: 22.65%, melting point: 183.7-184.9 ℃, HPLC:97.27%.1H NMR (500MHz, DMSO-d 6): δ 11.35 (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), 7.57 (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 (500 mhz, dmso-d 6): δ 5363 zft 5363, 133.05,129.01,128.97,124.43,99.26,71.09,63.89,45.78, 14.33.esims (ESI) m/3 Cl [ 9Cl 3Cl ] for 3H + 3Cl 3H).

Example 20

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

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

Compound 8d was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 6-benzylaminopurine. White solid, yield: 23.21%, melting point: 121.0-121.8 ℃, HPLC:98.42 percent. 1H NMR (500MHz, DMSO-d 6): δ 9.60 (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), 7.21 (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-d 6): delta 155.52,154.90,153.76,151.93,149.63,140.31, 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 calcd for [ C21H20Cl3N9O4+ H ] +:568.0704, found 568.0795.

Example 21

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (1- (6-amino-2-chloro-9H-purin-9-yl) -2,2,2-trichloroethyl) -carbamic acid ester (8 e)

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

Compound 8e was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2-chloroadenine. White solid, yield: 20.18%, melting point: 196.5-197.6 ℃, HPLC:98.62 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.61 (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-d 6): delta 157.40,155.54,154.23,151.91,151.23,138.96,138.72, 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.

Example 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 (8 f)

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

Compound 8f was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2,6-diaminopurine by the synthetic method of step 3 of example 1. Light yellow solid, yield: 25.36%, melting point: 165.8-166.6 ℃, HPLC:98.68 percent. 1H NMR (400MHz, DMSO): delta 9.47 (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): delta 161.21,156.74,155.56, 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 cad for [ C14H15Cl 3H 041 + 3H 3+ 493, 493H + 43H).

Example 23

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (1- (6-amino-2-fluoro-9H-purin-9-yl) -2,2,2-trichloroethyl) -carbamic acid ester (8 g)

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

According to the synthetic method of step 3 of example 1, using intermediate 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-fluoroadenine as starting materials, 8g of compound was obtained. White solid, yield: 19.26%, melting point: 168.1-169.6 ℃, HPLC:96.92 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.60 (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-d 6) delta 160.25,158.62,158.40,158.23,155.54,151.91,151.71, 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, und 2 FO).

Example 24

2- (2-methyl-5-nitro-1H-imidazol-1-yl) -ethyl- (1- (2-amino-6-chloro-9H-purin-9-yl) -2,2,2-trichloroethyl) -carbamic acid ester (8H)

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

Compound 8H was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediate 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2-amino-6-chloropurine. White solid, yield: 22.16%, melting point: 140.4-140.9 ℃, HPLC:97.69 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.61 (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 NMR (500MHz, DMSO-d 6): delta 160.75,155.58,154.62,151.90,150.53,139.79,139.01,133.45,122.32, 99.33,70.70,63.74,45.83,14.33 HRMS (ESI) m/z calcd for [ C14H13Cl4N9O4+ H ] +:511.9845, found: 511.9923.

Example 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 (8 i)

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

Compound 8i was obtained according to the synthesis method of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2-fluoro-6-chloropurine. White solid, yield: 23.16%, melting point: 171.3-172.9 ℃, HPLC:98.85 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.80 (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 (500 MHz, DMSO-d 6): delta 157.93,156.21,155.56,154.42,154.28,152.12,151.98,151.87,145.47,138.99,133.39, 129.60,98.42,71.92,63.92,45.76,14.36 HRMS (ESI) m/z calc for [ C14H11Cl4N8O4+ H ] +:514.9641, found:514.9718.

Example 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 (8 j)

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

Compound 8j was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and O-6-benzylguanine. White solid, yield: 24.53%, melting point: 171.6-172.8 ℃, HPLC:96.77 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.54 (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 (500 MHz, DMSO-d 6): delta 8978 zft 8978, 128.59,112.66,99.77,70.47,67.61,63.68,45.86, ESI 14.33.0621 Cl/584 nF [ 3H ] + 3H ].

Practice of example 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 (8 k)

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

Compound 8k was obtained from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 2,6-dichloropurine as starting materials according to the synthesis method of step 3 of example 1. White solid, yield: 19.33%, melting point: 129.9-131.7 ℃, HPLC:99.76 percent. 1H NMR (500MHz, DMSO-d 6): delta 9.77 (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-d 6): delta 8978 xft 8978, 71.80,63.92,45.76,14.37 HRMS (ESI) m/z cald for [ C14H11Cl5N8O4+ H ]: 530.9346, found 530.9421.

Example 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 (8 l)

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

According to the synthesis method of step 3 of example 1, 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 8l. Pale yellow solid, yield: 22.16%, melting point: 197.5-198.3 ℃, HPLC:99.67 percent. 1H NMR (400MHz, DMSO-d 6): δ 12.13 (s, 1H), 11.90 (s, 1H), 9.67 (d, J =9.1Hz, 1H), 8.29 (s, 1H), 7.99 (s, 1H), 6.76 (d, J =9.8Hz, 1H), 4.50 (d, J =54.2Hz, 4H), 2.38 (s, 3H), 2.19 (s, 3H). 13C NMR (500DMSO-d 6): MHz 174.19,155.64,155.20,151.91,149.53,149.04, 139.04,136.98,133.50,119.25,99.23,70.80,63.69,45.89,24.24,14.32 HRMS (ESI) m/z cad for [ C16H16Cl 3N: 9O6+ H ] + -536.0289, 536, 62 fof.

Example 29

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

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

Compound 8m was obtained according to the synthesis method of step 3 of example 1, starting from intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and adenine. White solid, yield: 12.36 percent of the total weight of the steel, melting point: 187.1-187.9 ℃, HPLC:98.68 percent. ¹ H NMR(400MHz,DMSO-d ₆ ):δ9.59(d,J＝10.1Hz,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.

Example 30

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

The preparation method is the same as that of step 2 in example 1, taking benzyl carbamate as a starting material. The crude product of the intermediate (2,2,2-trichloro-1-hydroxyethyl) benzyl carbamate is obtained and then directly put into the next reaction.

Compound 9a was obtained according to the synthesis procedure of step 3 of example 1, starting from benzyl carbamate and 2-chloroadenine, intermediates (2,2,2-trichloro-1-hydroxyethyl). White solid, yield: 21.32%, melting point: 198.6-199.9 ℃, HPLC:98.49 percent. 1H NMR (400MHz, DMSO-d 6): δ 9.69 (d, J =10.1Hz, 1H), 8.49 (s, 1H), 8.05 (s, 2H), 7.37 (dd, J =15.1,7.4 Hz, 5H), 6.87 (d, J =10.2Hz, 1H), 5.21-5.12 (m, 2H), 13C NMR (400MHz, DMSO-d 6): δ 157.42, 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, unford 448.9853.

Example 31

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

The preparation method is the same as that of step 2 of example 1, with the use of the metronidazole as a starting material. To obtain the crude product of the intermediate (1-methyl-5-nitro-1H-imidazoline-2-yl) methyl (2,2,2-trichloro-1-ethoxyl) carbamate, and directly putting the crude product into the next reaction.

Compound 9b was obtained according to the synthesis procedure of step 3 of example 1, starting from intermediates (1-methyl-5-nitro-1H-imidazolin-2-yl) methyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-chloroadenine. White solid, yield: 23.16%, melting point: 185.8-186.3 ℃, HPLC:98.24%.1H NMR (500MHz, DMSO-d 6): delta 9.87 (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 (500 MHz, DMSO-d 6): delta 157.42,155.27,154.30,151.26,147.57,139.93,138.75,132.24, 116.90,99.37,70.93,59.34,33.98 HRMS (ESI) m/z calcd for [ C13H11Cl4N9O4+ H ] +: 497.3288, found: 62 zxft 3262.

Example 32

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

The procedure is as in example 1, step 2, using ethyl carbamate as the starting material. The intermediate (2,2,2-trichloro-1-ethoxyl) ethyl carbamate crude product is obtained and then directly put into the next reaction.

Compound 9c was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediates (2,2,2-trichloro-1-hydroxyethyl) ethyl carbamate and 2-chloroadenine. White solid, yield: 19.89%, melting point: 219.5-220.4 ℃, HPLC:98.67%.1H NMR (400MHz, DMSO-d 6): δ 9.53 (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, 3H), 13C NMR (400MHz, DMSO-d 6): δ 157.42,155.92,154.28,151.25,138.86,116.91,99.57,70.85,62.25,14.77 HRMS (ESI) m/z calcd for [ C10H10Cl4N6O2+ H ] +:386.9616, found.

Example 33

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

The preparation method is the same as that of step 2 of example 1, with the use of the metronidazole as a starting material. The intermediate (1-methyl-5-nitro-1H-imidazoline-2-yl) methyl (2,2,2-trichloro-1-ethoxyl) carbamate crude product is obtained and then directly put into the next reaction.

Compound 9d was obtained according to the synthesis method of step 3 of example 1, starting from intermediate (1-methyl-5-nitro-1H-imidazolin-2-yl) methyl (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-fluoroadenine. White solid, yield: 21.63%, melting point: 205.8-206.9 ℃, HPLC:99.00 percent. 1H NMR (500MHz, DMSO-d 6): δ 9.85 (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 (500 MHz, DMSO-d 6): δ 160.29,158.66,158.41,158.24,155.26,151.73,151.57,147.58,139.91,138.62, 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.

Example 34

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

Compound 9e was obtained according to the synthesis method of step 3 of example 1, starting from the intermediates (2,2,2-trichloro-1-hydroxyethyl) ethyl carbamate and 2-fluoroadenine. White solid, yield: 18.21%, melting point: 208.3-209.8 ℃, HPLC:99.12 percent. 1H NMR (400MHz, DMSO-d 6): δ 9.50 (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 =7.0Hz, 3H), 13C NMR (400MHz, DMSO-d 6): δ 160.29,158.65,158.41,158.24,155.91,151.73,151.57,138.73,116.29,116.26,99.60,70.96,62.21, 14.75 HRMS (ESI) m/z calcd for [ C10H10Cl4N6O2+ H ] +:370.9915, found 370.9988.

Example 35

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

The preparation method is the same as that of the step 2 of the example 1 by taking the hydroxyethyl morpholine as a starting material. Obtaining the crude product of the intermediate 2-morpholine ethyl- (2,2,2-trichloro-1-ethoxyl) carbamate and directly putting the crude product into the next reaction.

Compound 9f was obtained from the intermediate 2-N-morpholinoethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-fluoroadenine by the synthetic method of step 3 of example 1. Pale yellow solid, yield: 10.42%, HPLC:96.96 percent. ¹ H NMR(500 MHz,DMSO-d ₆ ):δ9.68(d,J＝9.1Hz,1H),8.55(s,1H),8.03(d,J＝38.0Hz,2H),6.79(d,J＝10.0Hz, 1H),4.21(s,2H),3.03(d,J＝7.3Hz,4H),2.60(s,2H),2.46(s,4H). ¹³ C NMR(126MHz,DMSO-d ₆ ):δ 158.32(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.

Example 36

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

The preparation method is the same as that of the step 2 of the example 1 by taking the hydroxyethyl morpholine as a starting material. The intermediate 2-morpholine ethyl- (2,2,2-trichloro-1-ethoxyl) carbamate crude product is obtained and then directly put into the next reaction.

Compound 9g was obtained according to the synthesis method of step 3 of example 1, starting from intermediate 2-morpholinoethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-aminoadenine. White solid, yield: 10.07%, HPLC:97.8 percent. 1H NMR (500 MHz, DMSO-d 6). Delta.9.44 (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/z calcd for [ C14H19Cl3N8O3+ H ] +: 3238 zx3238, found:453.0724.

Example 37

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

The preparation method is the same as the step 2 of the example by using 2-hydroxyethyl pyridine as a starting material. The intermediate 2- (pyridine-2-yl) ethyl- (2, 2,2-trichloro-1-ethoxyl) carbamate crude product is obtained and then directly put into the next reaction.

Compound 9h was obtained according to the synthetic method of step 3 of example 1, starting from intermediate 2- (pyridin-2-yl) ethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-fluoroadenine. White solid, yield: 26.58%, HPLC:97.28%.1H NMR (500MHz, DMSO-d 6) delta 9.78 (d, J =9.7Hz, 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 =9.9Hz, 1H), 4.51 (d, J =6.9Hz, 2H), 3.39 (s, 2H). 13C NMR (126 MHz, DMSO-d 6) delta 160.22(s), 158.58(s), 158.33(s), 155.69(s), 154.21(s), 142.99(s), 139.02 (d, J =3.0 Hz), 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.

Example 38

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

Compound 9i was obtained according to the synthesis method of step 3 of example 1, starting from intermediate 2- (pyridin-2-yl) ethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 2-aminoadenine. White solid, yield: 27.38%, HPLC:95.32 percent. 1H NMR (500MHz, DMSO). Delta.9.34 (d, J =10.1Hz, 1H), 8.48 (d, J =3.9Hz, 1H), 8.07 (s, 1H), 7.69 (t, J =7.1Hz, 1H), 7.29 (d, J =7.6Hz, 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 =6.5Hz, 2H), 13C NMR (101MHz, DMSO). Delta. 161.23(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.

Example 39

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

Compound 9j was obtained according to the synthesis procedure of step 3 of example 1, starting from benzyl carbamate and 2-fluoroadenine, the intermediates (2,2,2-trichloro-1-hydroxyethyl). White solid, yield: 19.82%, melting point: 234.8-235.7 ℃, HPLC:96.24%.1H NMR (500MHz, DMSO-d 6): δ 9.67 (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), 13C NMR (126mhz, dmso-d 6): δ 160.30, 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.

Example 40

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

The preparation method is the same as the step 2 of the example by taking hydroxyethyl morpholine as a starting material. Obtaining the crude product of the intermediate 2-morpholinoethyl- (2,2,2-trichloro-1-ethoxyl) carbamate and directly putting the crude product into the next reaction.

Compound 10a was obtained from the intermediates 2-morpholinoethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 4-aminopyrazolo [3,4-d ] pyrimidine according to the synthesis method of step 3 of example 1. White solid, yield: 19.56%, HPLC:95.17 percent. 1H NMR (500mhz, dmso) δ 9.27 (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) δ 158.49(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.esis, ms (H) 18H + hrc Cl [ 14Cl 3/3 Cl ]: 438.0604, found.

EXAMPLE 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 (10 b)

Compound 10b was obtained according to the synthetic method of step 3 of example 1, starting from intermediate 2- (pyridin-2-yl) ethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 4-aminopyrazolo [3,4-d ] pyrimidine. White solid, yield: 9.84%, HPLC:97.73 percent. 1H NMR (500MHz, DMSO) delta 9.34 (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) delta 158.09 (d, J =12.8 Hz), 156.30(s), 4924 zxft 24 (4924), 155.31(s), 149.51 (d, J =5.0 Hz), 136.89(s), 134.91(s), 123.91 (d, J =6.1 Hz), 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 =11.4 Hz). HRMS (ESI) m/z calcd for [ C25H23Cl6N9O4+ H ] +:724.0077, found.

Example 42

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

The preparation method is the same as the step 2 of the example by using 2-hydroxyethyl pyridine as a starting material. The intermediate 2- (pyridine-2-yl) ethyl- (2,2,2-trichloro-1-ethoxyl) carbamate crude product is obtained and then directly put into the next reaction.

Compound 10c was obtained according to the synthesis procedure of step 3 of example 1, starting from the intermediate 2- (pyridin-2-yl) ethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 4-hydroxypyrazolo [3,4-d ] pyrimidine. Pale yellow solid, yield: 7.22%, HPLC:95.06 percent. 1H NMR (500MHz, DMSO). Delta.12.49 (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). Delta.8652 zxft 2(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 calcaulded for [ C15H13Cl3N6O3+ H ] +:431.0187, found 431.0193

Example 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-pyrazoio [3,4-d ] pyrimidin-1-yl-ethyl) -carbamate (10 d)

Compound 10d was obtained from the intermediate 2- (pyridin-2-yl) ethyl- (2,2,2-trichloro-1-hydroxyethyl) carbamate and 4-hydroxypyrazolo [3,4-d ] pyrimidine, following the synthetic method of step 3 of example 1. Pale yellow solid, yield: 7.22%, HPLC: 96.23 percent. 1H NMR (500mhz, dmso) δ 9.48 (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 =14.1 hz, 1h), 4.48 (dd, J =15.2,7.7hz, 4h), 3.15-3.01 (m, 4H).

Example 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 (10 e)

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

Compound 10e was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 6-aminopyrazolo [3,4-d ] pyrimidine, according to the synthesis method of step 3 of example 1. Light yellow solid, yield: 14.65%, HPLC:98.62 percent. 1H NMR (500mhz, dmso) δ 9.63 (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.

Example 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 (10 f)

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

The hydrochloride of compound 10f was obtained from the intermediates 2- (2-methyl-5-nitro-1H-imidazolin-1-yl) ethyl (2,2, 2-trichloro-1-hydroxyethyl) carbamate and 6-chloropyrazolo [3,4-d ] pyrimidine, following the synthesis procedure of step 3 of example 1. Pale yellow solid, yield: 9.08%, HPLC:95.06 percent. 1H NMR (500mhz, dmso) δ 11.89 (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 cad for [ C14H12Cl4N8O4+ H ] +:496.9803, found 497.0073.

Example 46

Experiment for inhibition of cancer cell proliferation

To validate the anticancer activity of the designed synthetic compounds, the subject designed two major activity tests. Firstly, MTT method is used to screen proliferation inhibition activity of breast cancer MCF-7, melanoma A375, lung adenocarcinoma A549, hepatocellular carcinoma HepG2, cervical carcinoma Hela and ovarian cancer Carvo-3,6 cell lines on all compounds, and the selected positive contrast drug is Cdc20 specific inhibitor Apcin.

The principle of the MTT method is that succinate dehydrogenase in mitochondria of living cells can reduce exogenous tetrazolium bromide (MTT) into difficultly soluble blue-purple crystals and deposit the crystals in the cells, but dead cells do not have the function. DMSO can dissolve purple crystal in cells, and enzyme linked immunity detector can detect absorbance at 490nm wavelength to reflect viable cell number.

The final IC was obtained by the MTT method described above, three replicates ₅₀ The value is obtained.

(1) Experimental Material

a. Cell lines: MCF-7, A375, A549, hepG2, hela, carvo-3 cell lines

b. Reagents and instrumentation: tetramethylazo salts (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich; newborn bovine serum, fetal bovine serum, pancreatin, RPMI-1640, DMEM and penicillin-streptomycin were purchased from Gibco; other reagents were purchased from Sigma-Aldrich, inc. without specific reference; CO2 ₂ Incubator (Thermo), enzyme-linked immunosorbent assay (Thermo)) And fluorescence inverted microscope (Olympus).

(2) Contents of the experiment

a. The experiment (MTT method) was carried out using cells having a viable cell ratio of 90% or more. Collecting logarithmic phase cells, adjusting cell suspension concentration to 4-5 × 10 ⁴ Adding 100 mu L of the test cells per mL of the test cells per well, and plating to adjust the density of the test cells to 4000-5000 per well.

b.5％CO ₂ Incubate at 37 ℃ until the cell monolayer is spread to the bottom of the well (96-well flat bottom plate), add different concentration gradients of drug to give final concentrations of 300, 100, 30,10, 3,1, 0.3, 0.1, 0.03, 0 μ M/mL compound in the well of 100 μ L per well, and make 3 replicates simultaneously for comparison of group differences.

c. Put into 5% of CO ₂ Incubation in a constant temperature incubator at 37 ℃ for 48h, and observation under an inverted microscope.

d. Add 10. Mu.L of MTT solution (5 mg/mL, i.e., 0.5% MTT) per well and continue the incubation for 4h.

After e.4h, the culture was terminated and the culture medium in the wells was carefully aspirated.

f. Add 150. Mu.L DMSO into each well, and shake on a shaker for 10min at low speed to dissolve the crystals sufficiently. And measuring the absorbance of each hole at 490nm of detection wavelength of an enzyme-linked immunosorbent assay (ELISA) detector, and calculating the inhibition rate.

g. The zero-setting wells (culture medium, MTT, DMSO) and the control wells (cells, drug dissolution medium of the same concentration, culture solution, MTT, DMSO) are also set.

(3) Data processing

Repeating three multiple wells per concentration gradient, fitting a curve with the concentration as abscissa and the inhibition rate as ordinate using SPSS13.0 software, and calculating the inhibition rate (IC) of the compound ₅₀ Value). Measuring the absorbance OD value of each hole at the detection wavelength of 490nm on an enzyme-labeling instrument, and calculating the cell growth inhibition rate according to the following formula:

table 1: inhibition data of tested compounds on cancer cell lines

The results show that:

compared with a specific Cdc20 inhibitor Apcin, the anticancer activity of most of the obtained target compounds is improved. Compounds containing dichloro-substituted pyrimidines (7 a-7 d) and 2-fluoroadenine side chains (8 g, 9d, 9e, 9f, 9 h) showed the most potent antiproliferative activity. To further confirm targeting of the compounds, the cell cycle and apoptosis studies designed by the present invention were continued with the selection of the most active 7d and 9f.

Example 47

Human hepatoma cell (Hep-G2) cycle experiments

(1) Experimental Material

a. Cell lines: human liver cancer cell HepG2

b. Reagents and instrumentation: multifunctional imaging systems were purchased from Biotek corporation; phosphorylated histone H3 antibody purchased from CST; the manufacturers of PBS, pancreatin digestive juice, DMSO, fetal calf serum, DMEM, 96-well cell culture plates, T25 cell culture bottles, CO2 cell culture boxes, low-speed centrifuges, cell counters and biological clean benches are the same as those in the cancer cell proliferation inhibition experiment.

(2) Content of the experiment

a. Taking HepG2 cells growing in logarithmic phase, washing with 1 × PBS for 3 times, 2min each time; adding pancreatin and CO into culture bottle/culture dish ₂ Digesting in incubator for 2min. Adding a culture medium (DMEM) into the culture bottle/culture dish to stop digestion, and blowing and beating the cells by using a sterilizing pipette until the cells are in a single suspension state;

b. counting with a cell counting plate and plating.

c. And (5) applying the medicine, and incubating in a constant temperature box for 2d.

d. The medium was aspirated from each well, 100. Mu.l of 4% paraformaldehyde was added to each well, and incubation was performed at room temperature for 15min.

e. Aspirate and rinse three times with PBS.

f. 0.1ml of permeate was added to each well and incubated for 15min at room temperature.

g. The permeate was aspirated and washed three times with PBS.

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

i. Blocking buffer was aspirated, 50. Mu.l of primary antibody solution was added to each well, and incubation was performed overnight at 4 ℃.

j. The primary antibody solution was aspirated and washed three times with PBS, and 50. Mu.l of secondary antibody solution was added to each well and incubated at room temperature for 60min (protected from light).

k. The secondary antibody solution was aspirated and washed three times with PBS, and 100. Mu.l of PBS was added to each well for imaging.

FIG. 4 shows the results of immunofluorescence experiments with drug-treated HepG2 cell lines. Drug group: the compound Apcin (100. Mu.M), 7d (30. Mu.M), 9f (0.3. Mu.M). The treatment time of the medicine is as follows: and (5) 48h.

Compared to the solvent control in HepG2 cells, 30 μ M of compound 7d and 0.3 μ M of compound 9f induced an increase in phosphohistone H3 positive cells, i.e. from 3.8% to 6.9% and 10.0%, respectively. Whereas 100 μ M of the positive compound Apcin resulted in an approximately 2.2-fold increase in PHH 3-positive cell number compared to the DMSO control. Compounds 7d and 9f showed greater potency in arresting mitotic withdrawal in cells compared to the positive control, apcin, while compound 9f was the most potent mitotic blocker, exhibiting strong M-phase arrest at 0.3 μ M.

Example 48

Apoptosis test of human hepatoma cells (Hep-G2)

(1) Experimental materials

a. Cell lines: human liver cancer cell HepG2

b. Reagents and instrumentation: multifunctional imaging systems were purchased from Biotek corporation; annexin-FITC/PI apoptosis kit purchased from Solebao; the manufacturers of PBS, pancreatin digest, DMSO, fetal calf serum, RPMI 1640, 24-well cell culture plates, T25 cell culture bottles, CO2 cell culture boxes, low-speed centrifuges, cell counters, and biological clean benches were the same as those used in the cancer cell proliferation inhibition experiments.

(2) Experimental procedure

a. Taking HepG2 cells growing in log phase, washing with 1 × PBS for 3 times (2 min each time), adding pancreatin and CO into culture bottle/culture dish ₂ Digesting in incubator for 2min. Medium (DMEM) was added to the flask/dish to stop digestion and the cells were pipetted with a sterile pipette until the cells were in a single suspension. Counting with a cell counting plate and plating.

b. The drugs were added and incubated overnight at 37 ℃.

c. 3ml Binding Buffer (10X) to 30ml 3ml each time was diluted with 27ml deionized water.

d. Add 2. Mu.l ANNEXIN V-FITC stain for 10min at room temperature, protected from light, and shake several times, preferably with a shaker, or manually.

e. Add 5. Mu.l PI, room temperature, protected from light and incubate for 5min.

f. The staining solution was aspirated and washed with PBS for imaging.

FIG. 5 shows the apoptosis test results of drug-treated HepG2 cell lines. Drug group: compound Apcin (100 μ M), 9f (0.3 μ M); the treatment time of the medicine is as follows: and (5) 24h. High concentrations of Apcin had a significant pro-apoptotic effect when the drug was treated for 24h, with most cells in the early stage, but late-withered cells were also present. Whereas a high concentration of 9f had a significant pro-apoptotic effect and most cells were in the early-wither stage. Compound 7d (30. Mu.M) also had a pro-apoptotic effect.

The above mechanism studies preliminarily show that the compounds 9f and 7d can significantly inhibit cancer cell proliferation, and at the same time, the studies show that the compounds designed by the present subject exert the effect of inhibiting cancer cell proliferation by retarding the cell cycle and promoting apoptosis.

The foregoing examples are set forth to provide a clear illustration of the invention and are not to be construed as limiting the scope thereof, which is defined in the claims appended hereto.

Claims

1. A (1,1,1-trichloro-2) carbamate derivative is characterized in that the structural general formula is shown as formula (II-III):

n is 0 to 1;

x is O;

wherein R1 is selected from hydrogen, benzene ring, pyridine and morpholine;

y is selected from N, CH;

in the formula II, R ₅ And R ₆ Selected from amino, H, halogen, C1-C7 alkyl, C1-C7 alkoxy, C1-C7 haloalkyl, C1-C7 alkylamino,

In the formula III, R ₅ And R ₆ Selected from amino, H, halogen,

2. The (1,1,1-trichloro-2) carbamate derivative is characterized in that the structural general formula of the (1,1,1-trichloro-2) carbamate derivative is shown as the formula II:

wherein n is 0-1;

x is O;

r1 is selected from benzene ring, pyridine and morpholine;

R ₅ and R ₆ Selected from amino, H, halogen, C1-C7 alkyl, C1-C7 alkoxy, C1-C7 haloalkyl, C1-C7 alkylamino,

Y is N, CH.

3. The (1,1,1-trichloro-2) carbamate derivative is characterized in that the structural general formula of the (1,1,1-trichloro-2) carbamate derivative is shown as the formula III:

wherein n is 1;

x is O;

r1 is selected from benzene ring, pyridine and morpholine;

R ₅ and R ₆ Selected from amino, H, halogen,

Y is N.

4. A (1,1,1-trichloro-2) amino methyl ester derivative is characterized by the following structural formula:

5. a process for the preparation of the (1,1,1-trichloro-2) carbamate derivative according to any one of claims 1 to 4, comprising the steps of:

s1, reacting a hydroxyl substituted compound with chloroformic acid p-nitrophenyl ester in anhydrous dichloromethane to obtain an intermediate;

s2, reacting the intermediate with ammonia water in a mixed solvent of halogenated alkane and methanol to obtain a carbamate product;

s3, refluxing the carbamate product and chloral hydrate at 80-100 ℃ to obtain a (1,1,1-trichloro-2) carbamate part;

s4, (1,1,1-trichloro-2) carbamate part is firstly chlorinated with thionyl chloride, and then reacts with amine to obtain a target compound.

6. The preparation method according to claim 5, wherein the step S1 reaction is catalyzed by a weak base to accelerate the reaction process, and the weak base is any one of triethylamine, diisopropyldiethylamine or a combination thereof.

7. The method according to claim 5, wherein the step S2 is carried out by mixing halogenated alkane with methanol in a solvent mixture of halogenated alkane: the mass of the methanol is 1:1-5:1.

8. The use of the (1,1,1-trichloro-2) carbamate derivative of any one of claims 1 to 4 in the preparation of a medicament for the treatment of lung adenocarcinoma a549, hepatocellular carcinoma HepG2, and ovarian cancer Caov-3.