阅读说明：本技术 一种不会引起心率过缓的核苷酸类化合物 (Nucleotide compound without causing bradycardia ) 是由 沈振波 于 2021-02-07 设计创作，主要内容包括：本发明涉及一种核苷酸结构的化合物,该核苷酸类化合物可以用于抑制RNA病毒,且由于该化合物在特定位点采取了特定构型,该化合物和胺碘酮共同作用时不存在引起心律过缓的副作用。(The invention relates to a compound with a nucleotide structure, which can be used for inhibiting RNA virus, and because the compound adopts a specific configuration at a specific site, the compound and amiodarone do not have the side effect of causing bradyarrhythmia when acting together.)

1. A compound based on a nucleotide structure, wherein the structure of the compound is shown as formula I or a pharmaceutically acceptable salt or ester of formula I:

wherein:

R₁、R₂independently is hydrogen, methyl, halogen, hydroxy, alkoxy, alkynyl or cyano, and R₁And R₂Cannot be simultaneously hydrogen;

R₃is hydroxy, alkoxy or C₁-C₈An alkyl group;

connection R₁、R₂The carbon atom of the group is in the S configuration.

2. The compound of claim 1, wherein: r₃Is 2-ethyl-butyl.

3. The compound of claim 1, wherein: the compound is selected from at least one of the compounds shown in the following structures:

4. use of a compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt or ester thereof, in the manufacture of a medicament for the treatment and/or prophylaxis of RNA viruses.

5. The use of claim 4, wherein the RNA virus is COVID-19.

6. The use of claim 4, wherein the RNA virus is an influenza virus.

7. The use of claim 4, wherein the influenza virus is H1N 1.

Technical Field

The present invention relates to compounds based on nucleotide structures and to the use of such compounds based on nucleotide structures for inhibiting the replication of RNA viruses.

Background

Viral infection is the major infectious disease in the world at present, accounts for more than three quarters of the infectious disease, and seriously threatens the health and life of human beings.

Among antiviral drugs currently on the market, nucleosides account for more than half of them, and have a very important position in antiviral treatment. Nucleoside drugs, as inhibitors of viral polymerase or reverse transcriptase, undergo stepwise phosphorylation after entering cells and are converted into nucleoside triphosphate analogs, which exert antiviral effects. However, the clinical application of nucleoside drugs is greatly limited due to the defects of high toxicity, short half-life in vivo, easy induction of drug-resistant strains and the like.

Amiodarone (amiodarone) has been used as an antiarrhythmic drug for over 30 years, and 1/3, which accounts for the prescription of antiarrhythmic drugs, has become an indispensable member in antiarrhythmic drug therapy. The amiodarone pharmacokinetics is characterized by slow absorption and long half-life, and the elimination half-life can be as long as more than 60 days after long-term oral treatment.

The FDA in the united states updated the instruction information for hepatitis c antiviral drugs ledipasvir/sofosbuvir (harvoni) sofosbuvir (sovaldi), all of which are nucleoside compounds, and there were many clinical reports that patients suffered from bradycardia leading to death after combining these drugs with amiodarone.

It is particularly noted that amiodarone has a long half-life and that many patients or medical staff are likely to have a bradycardia when undergoing antiviral therapy by ignoring the presence of amiodarone.

Due to the existence of chiral environment in organisms, different optical isomers of the same drug molecule enter human bodies, and the chiral receptors, enzymes, carriers and the like in the bodies are treated as completely different molecules, so that the drug effects are often remarkably different. For example, the R-isomer of thalidomide has sedative effect, but the S-isomer and metabolite thereof have strong embryotoxic and teratogenic effects, which once caused thousands of cases of short limb teratogenesis. Ketamine is a commonly used barbiturate intravenous anesthetic, which has no respiratory depression but has adverse effects. At present, the medicine is still administrated in a racemic form, but the side effect is large, and after operation, the diseases generate hallucinations, unstable mood and mental disorder. Studies have shown that intoxication is mainly caused by the dextro-isomer, while side effects are mainly caused by the levo-isomer.

Disclosure of Invention

The invention provides a compound based on a nucleotide structure, which does not cause the side effect of bradyarrhythmia when being used together with antiarrhythmic drug amiodarone.

The scheme of the invention is as follows:

a compound based on a nucleotide structure, wherein the structure of the compound is shown as formula I or a pharmaceutically acceptable salt or ester of formula I:

wherein:

R₁、R₂independently is hydrogen, methyl, halogen, hydroxy, alkoxy, alkynyl or cyano, and R₁And R₂Cannot be simultaneously hydrogen;

R₃is hydroxy, alkoxy or C₁-C₈An alkyl group;

connection R₁、R₂The carbon atom of the group is in the S configuration.

As a further improvement of the above technical scheme, R₃Is 2-ethyl-butyl.

As a further improvement of the above technical solution, the compound is selected from at least one of the compounds shown in the following structures:

in another aspect, the invention also provides the use of the compound or the pharmaceutically acceptable salt or ester thereof in the preparation of a medicament for the treatment and/or prevention of RNA viruses.

As a further improvement of the technical proposal, the RNA virus is COVID-19.

As a further improvement of the above technical solution, wherein the RNA virus is an influenza virus.

As a further improvement of the above technical solution, wherein the influenza virus is H1N 1.

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

1. the compound or the pharmaceutically acceptable salt or ester thereof can not cause the side effect of bradycardia when being used with the antiarrhythmic medicament amiodarone, and the antiviral medicaments such as sofosbuvir and the like can cause the side effect of bradycardia when being used with the amiodarone.

2. The nucleotide analogs of the present invention have a phosphate group that blocks action, are also substituted at specific sites, have good cell permeability, enable entry into infected eukaryotic cells, are prodrugs, are converted to the active triphosphate form by cellular enzymes, the activated drug binds to the active site of RNA polymerase (RdRP) and is then incorporated into RNA, inhibits further extension of the RNA strand and terminates RNA replication due to the modification at the 2' position, and compete with natural ribonucleotides as RNA polymerase inhibitors. The compound or the pharmaceutically acceptable salt or ester thereof has good inhibitory effect on new coronavirus and influenza.

Drawings

Figure 1 shows the effect on heart rate shared by different configurational compounds and amiodarone.

Detailed Description

In order to more fully understand the technical contents of the present invention, the technical solutions of the present invention will be further described and illustrated with reference to the following specific embodiments.

Example 1: combination of Compound 1 with amiodarone

IPS induces generation of cardiomyocytes, and the cardiomyocytes are evenly paved on a 24-well plate.

2. The cardiomyocyte spontaneous action potentials were recorded using PatchMaste software and HEKA EPC-10 amplifier, whole cell recording mode, 10kHz sampling frequency.

3. The flow rate of the gravity perfusion system is adjusted to 5mL/min, and the temperature control device is adjusted to continuously keep the temperature in the recording tank at 35-36 ℃.

4. Taking a climbing sheet, covering a recording groove on an objective table with extracellular fluid, drawing an electrode with the water resistance value of 6-8M omega, filling electrode fluid with the height of 1-2cm, fixing the electrode on a micro-distance manipulator, finding out cells with clear cell outlines under a microscope, moving the electrode by using the micro-distance manipulator to enable the electrode to slowly approach the cells, pressing down 0.2M omega and sealing by negative pressure after the electrode approaches the cells, giving short-forcing negative pressure again when the sealing resistance is approximately equal to or more than 1G omega, removing all compensation by Setup after the sealing peak shape changes into a membrane breaking peak shape and the resistance value is stabilized at G omega 3-5min, selecting a recording mode of C-clamp, removing stimulation current (I is equal to 0pA), and recording the spontaneous action potential of the myocardial cells by perfusion administration. Before cell perfusion administration, an extracellular fluid containing 0.1% DMSO is perfused for 5min, after ensuring that the action potential distribution frequency is stable in the perfusion administration process, the drugs with the final concentration of 3 mu M compound 1 or compound 1 x and 0.3 mu M Amiodarone (Amiodarone) are respectively perfused and administered to the extracellular fluid, and the change condition of the action potential of the cells before and after administration is recorded.

The extracellular fluid was as shown in Table 1 below (the pH of the extracellular fluid was adjusted to about 7.4 with NaOH or HCl).

The intracellular fluid was as shown in Table 2 below (the pH of the intracellular fluid was adjusted to about 7.4 with KOH and HCl).

TABLE 1

TABLE 2

The structural formula of compound 1 is as follows:

the structural formula of the compound 1 is as follows, and the structural formula is different from the compound 1 in that the configuration of a C atom connected with a cyano group is different, the C atom is in an R configuration, and the S configuration is at the position 1 of the compound.

As shown in fig. 1, by analyzing the action potential of each group of cardiomyocytes, there was no significant change in the action potential of the cardiomyocytes after perfusion of compound 1 and compound 1 alone. However, after the compound 1 and amiodarone were used together, the action potential of the cardiomyocytes was significantly prolonged in action time, while the action potential of the cardiomyocytes was not significantly changed after the compound 1 and amiodarone were used together. Therefore, compared with the R configuration, the S-type nucleotide compound does not cause the side effect of bradyarrhythmia when being used together with the antiarrhythmic medicament amiodarone.

Example 2: inhibitory Effect of Compounds 1 to 3 on New coronavirus

The following experiments were conducted by the Guangzhou customs technology center BSL-3 laboratory.

Day 0 cell plating

96-well cell plating of Vero E6 cells, 1.6X 10⁴cells/well, D10 Medium at 37 ℃ with 5% CO₂The culture was carried out overnight.

Day 1 antiviral experiment

1. Drug dilution: the drug was stored at a concentration of 10mM, and diluted with DMEM medium to 10. mu.M, 5. mu.M, 1. mu.M action concentration group and 20. mu.M, 10. mu.M, 2. mu.M action concentration group, respectively.

2. Pretreatment: the experiment is respectively provided with 4 groups of experiments including a cell control group, a virus infection group, a drug virus action group and a drug control group, and each drug is provided with 3 compound wells. Cell culture supernatant was aspirated before Vero E6 cells were infected with virus, and culture medium at concentrations of 10. mu.M, 5. mu.M, and 1. mu.M was added to drug-virus-affected group and drug-control group at 100. mu.L/well. 37 ℃ and 5% CO₂And culturing for 2 h.

3. The antiviral effect is as follows: pretreatment ofAll the supernatant was then aspirated in the BSL-3 laboratory, and the 2019-nCoV (COVID-19) virus was diluted in D2 medium to give an MOI of 0.05 and a volume of 25. mu.L/well. The diluted virus 25. mu.L/well was mixed with the drug 20. mu.M, 10. mu.M, 2. mu.M working concentration group (25. mu.L/well) to form a drug-virus working group of 50. mu.L/well. The remaining groups were mixed with the corresponding D2 medium instead of drugs or viruses. Incubating the mixed culture medium with cells at 37 deg.C and 5% CO₂And culturing for 1 h.

4. Cell culture: after mixing the incubated cells, the supernatant was discarded, and 10. mu.M, 5. mu.M, and 1. mu.M drug medium (D2 as a diluent) was added to each drug-virus-affected group at 100. mu.L/well. The other groups were supplemented with the corresponding D2 or drug media 100. mu.L/well at 37 ℃ with 5% CO₂And culturing for 24 h.

Day 2IFA detection

1. Fixing a cell plate: in the BSL-3 laboratory, the culture supernatant was discarded from the cell plate and 4% PFA 200. mu.L/well was added and fixed at room temperature for more than 3 h.

IFA detection: after Triton punching and membrane breaking and sealing, SARS-N Rabbit monoclonal antibody acts for 1h as a primary antibody, A488anti-Rabbit IgG acts for 1h as a secondary antibody, and the plate is washed and observed by fluorescence.

The results of the experiment are shown in table 3 below:

TABLE 3

Name of Compound	Concentration of	Inhibitory ratio (%) of New coronavirus
			Compound 1	10μM	24.22％
Compound 2	10μM	10.80％
			Compound 3	10μM	19.58％

According to inhibition experiments on new coronavirus, the compounds 1-3 can also effectively inhibit the new coronavirus, and the compounds or pharmaceutically acceptable salts or esters thereof can be used for preparing a medicine for treating the infection of the new coronavirus.

Example 2

Inhibitory Effect of Compounds 1 to 3 on influenza Virus

Virus infectivity assay (assay of TCID 50)

Taking out virus solution, dissolving, and continuously diluting with serum-free and double-antibody-free culture medium by 10 times gradient, wherein the dilution can be 10¹～10⁹(can increase or reduce the dilution according to the situation) inoculating the virus of each dilution into corresponding host cells, wherein the influenza virus H1N1 is inoculated into MDCK (NBL-2) cells, each dilution is provided with 6-8 multiple wells, virus liquid with different concentrations is respectively inoculated into corresponding 96-well plates growing into monolayer cells, each well is 100 mu L, the 96-well plates are placed in a 5% CO2 constant temperature incubator, the influenza virus (H1N1) is adsorbed at 35 ℃ for 1-2H, then supernatant is discarded, and the cells are washed 1-2 times by PBS; adding 100 μ L of cell maintenance liquid per well, continuously culturing, simultaneously setting 6-8 normal cell control groups, culturing in 35 deg.C and 37 deg.C 5% CO2 incubator, observing virus growth condition every day, observing cell morphological change under inverted microscope and recording number of wells with characteristic cytopathic effect (CPE), recording result after 3-7d, wherein culture wells with cell morbidity rate of 50% or above are considered as diseased wells, and culture wells with cell morbidity rate of less than 50% are considered as non-diseased wells.

The degree of cytopathic effect (CPE) was recorded according to grade 6 criteria:

-: the cells grow normally and no disease is generated;

+ -: cytopathic effects are less than 10% of the whole monolayer of cells;

+: cytopathic effects are less than 25% of the whole monolayer of cells;

++: cytopathic effects are less than 50% of the whole monolayer of cells;

+++: cytopathic effects are less than 75% of the whole monolayer of cells;

++++: cytopathic effects account for more than 75% of the entire monolayer of cells.

And a result judgment and calculation method comprises the following steps:

half of the infected amount of the virus, TCID50, was calculated according to the Reed-Muench method.

Proportional Distance (PD) ═ (percentage of lesions above 50% — 50%)/(percentage of lesions above 50% — percentage of lesions below 50%)

Wherein the culture wells with a cytopathic rate of 50% or more are considered as diseased wells, and the culture wells with a cytopathic rate of less than 50% are considered as non-diseased wells.

In vitro antiviral assay

(1) Experimental methods

In vitro antiviral assays were performed after the 96-well plate cells were grown into monolayers. The virus attack amount is 10-100 TCID 50. After the virus adsorbs monolayer cells for 1-2h, washing off virus liquid, respectively adding 100 μ L of liquid medicine and 100 μ L of maintenance liquid into each well, and setting virus control and blank cell control. CO at the corresponding temperature₂Incubate in incubator, observe the lesions under inverted microscope every day for several consecutive days, and record CPE for each well. When the cytopathic effect of the virus control group reaches "++++" and the normal cell control group has no pathological changes, the optimum time point of CCK-8 detection is determined, CCK-8 detection is carried out, and the absorbance value (OD value) is measured at the wavelength of 450nm of an enzyme labeling instrument. The experiment was repeated 2 times and the average of the results of 3 experiments was calculated.

(2) The calculation method comprises the following steps:

the drug 50% effective concentration (IC50) and Therapeutic Index (TI) were calculated according to the Reed-Muench method.

The virus inhibition rate was (drug group OD 450-virus control group OD 450)/(normal cell control group OD 450-virus control group OD 450).

TI＝TC50/IC50。

The results of the experiment are shown in table 4 below:

TABLE 4

Name of Compound	Concentration of	H1N1 Virus inhibitory Rate (%)
			Compound 1	10μM	28.53％
Compound 2	10μM	9.88％
			Compound 3	10μM	13.76％

According to the inhibition experiment of the compound on the influenza virus, the compound 1-3 can effectively inhibit the H1N1 virus, and the compound or the pharmaceutically acceptable salt or ester thereof can be used for preparing the medicine for treating the influenza virus infection.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, so that any simple modification, equivalent change and modification made to the above embodiment according to the technical spirit of the present invention are within the scope of the technical solution of the present invention.