CN116120383A

CN116120383A - Compound for inhibiting RNA virus and preparation method thereof

Info

Publication number: CN116120383A
Application number: CN202310052531.4A
Authority: CN
Inventors: 沈振波; 曾皓宇
Original assignee: Guangdong Papien Biotechnology Co ltd
Current assignee: Guangdong Papien Biotechnology Co ltd
Priority date: 2023-02-02
Filing date: 2023-02-02
Publication date: 2023-05-16

Abstract

The invention provides a compound for inhibiting RNA virus and a preparation method thereof, wherein the compound is a nucleotide inhibitor, has obvious inhibition effect on various viruses, and particularly aims at inhibiting influenza virus, new coronavirus and hepatitis C virus.

Description

Compound for inhibiting RNA virus and preparation method thereof

Technical Field

The invention belongs to the field of chemical synthesis, and in particular relates to a compound for inhibiting RNA viruses, a preparation method thereof and application of the compound in preventing and/or treating influenza viruses, new coronaviruses and hepatitis C viruses.

Background

Viruses are the most common infectious disease pathogen, are continuously occurring in human history, and severely threaten human health. Viruses are classified from genetic sources, and are largely classified into RNA viruses and DNA viruses. According to the infection route, it can be classified into respiratory viruses, enteroviruses, etc. The molecular mechanism of viral genome replication is complex involving a variety of viral and host elements, with RNA-dependent RNA polymerase (RNA dependent RNA polymerase, rdRp) being a class of polymerases that synthesize complementary RNA strands using single-stranded RNA as a template, and being a core component of RNA virus replication. RdRp is a multidomain viral transferase and all RNA viruses, except for the retrovirus family, encode RdRp. Despite the significant differences in RdRp sequences between different virus species, the three-dimensional structure of their core catalytic domains is highly conserved, making it a major target for the development of broad-spectrum antiviral drugs.

Inhibitors against RdRp can be divided into 4 classes: nucleoside (nucleotide) analogs, non-nucleoside (nucleotide) analogs, inhibitors of protein-protein interactions, and targeted covalent inhibitors. Currently, FDA approves the clinically useful anti-RdRp drugs such as ribavirin, fepira Weiba lo Sha Weizhi, sofosbuvir, rad Wei Heda zebuvir, and the like.

Influenza virus (IFV) is an enveloped virus with a segmented negative-strand RNA genome, belonging to the orthomyxoviridae family. Viruses can be classified into three types, A, B and C, according to their antigenic differences in nucleoprotein and matrix protein. Influenza a is the primary pathogen in most influenza cases and is therefore of greatest concern. When influenza virus invades host cells, it first binds sialic acid residues on host cell receptors through the top of HA on the envelope, and receptor-mediated endocytosis enters into the capsule, and then forms further vesicles, which bind lysosome-activated uncoating, and NP complex (RNP) in the viral core is released into the cytoplasm, and enters into the nucleus through the nuclear pores for replication and expression. The replication generates a large amount of RNP, and then the RNP enters cytoplasm for packaging, and the nucleocapsid RNP is combined with MI protein, and then the MI protein is fused with a double-layer lipid membrane and interacts with cytoplasmic tail of virus membrane glycoprotein to initiate release of virus, and then other host cells are infected.

The novel coronaviruses are large, enveloped positive-strand RNA viruses. The coronavirus genome encodes several structural and non-structural proteins, the non-structural proteins (Nsps) facilitating viral replication and transcription. The membrane (M), envelope (E) and spike protein (S) constitute structural proteins and are associated with the envelope. Spike proteins enter cells by binding to angiotensin converting enzyme 2 (ACE-2). Regarding SARS-CoV-2, its life cycle begins with the activation of S protein by cellular serine protease (TMPRSS 2) and trypsin-like protease from the respiratory tract (TMPRSS 11D) to bind to ACE2 receptor enzyme (TMPRSS 11D) to activate S protein to bind to ACE2 receptor. Upon binding of the virus to the ACE2 receptor, conformational changes occur in the spike protein allowing for endosomal fusion between the viral envelope and host cell membrane. SARS-CoV-2 then releases the RNA into the cytoplasm, which translates and replicates to form a transcriptional and replicative complex. Next, the positive RNA strand is translated into a negative strand template, so that new genomic and subgenomic mRNAs can be synthesized. These mRNAs are translated and transcribed to produce structural and accessory proteins. Viral proteins and RNA genomes are put together in endoplasmic reticulum and golgi apparatus virions, and finally transported by vesicles and released from the cell host to infect new cells.

The Hepatitis C Virus (HCV) genome is similar in structural and phenotypic characteristics to human flaviviruses and pestiviruses, and therefore is classified as a single-stranded positive-strand RNA virus in the genus hepatitis virus of the flaviviridae family. The HCV genome encodes a 3010 amino acid polyprotein precursor, flanked by 5 'and 3' non-coding regions, which are critical for viral replication and translation. Translation of the HCV protein starts with the 5-UTR IRES, and the polyprotein is processed by cellular proteases to produce structural proteins (Core, envelope E1, envelope E2) that form viral particles, and by viral proteases to produce non-structural proteins (p 7, NS2, NS3, NS4A, NS4B, NS a and NS 5B). 7. The nonstructural proteins are the main targets of the current Direct-acting antiviral drug (Direct-actingAntiviral Agents, DAA) treatment, and perform multiple functions to replicate the virus in host cells.

Specifically, the RdRp inhibitor has an inhibiting effect on one or more viruses of influenza virus, novel coronavirus, hepatitis C virus and the like, but due to the characteristics of high infectivity, easy variation and the like of the viruses, the existing virus inhibitor generates certain drug resistance, and the general structural characteristics of the RdRp of RNA viruses and the research and development strategy of the inhibitor are known, so that the RdRp inhibitor has positive significance for research and development of broad-spectrum antiviral drugs. The invention designs and synthesizes a new compound for inhibiting RNA virus, explores the action and effect of the compound on influenza virus, novel coronavirus and hepatitis C virus, and has great practical significance for improving the drug resistance of the existing virus inhibitor and developing novel multifunctional antiviral drugs.

Disclosure of Invention

In order to solve the problems and the defects in the prior art, the invention provides a novel compound which is a nucleotide inhibitor and can play a role in preventing/treating influenza virus, novel coronavirus and hepatitis C virus.

According to a first aspect of the present invention there is provided a compound of formula I:

（I）。

according to another aspect of the present invention there is provided a process for the synthesis of the above compound of formula I, characterized by the following synthetic route:

；

wherein Bn on compounds 11-17 represents benzyl, TMS on compounds 14-15 represents trimethylsilyl, and Boc on compound 20 and compound 2 represents t-butoxycarbonyl.

Preferably, the synthetic route is as follows:

。

in the above synthetic route, the english abbreviations represent the following meanings: bnBr, benzyl bromide; DMF, dimethylformamide; etOH, ethanol; DMP, dess-martin periodate; DCM, dichloromethane; nBuLi, n-butyllithium; THF, tetrahydrofuran; TMS, tetramethyl silicon; DMAP, 4-dimethylaminopyridine; -TMS, -trimethylsilyl; bu (Bu) ₃ SnH, tri-n-butyltin hydride; AIBN, azobisisobutyronitrile; TBAF; tetrabutylammonium fluoride; acOH; acetic acid; BCl (binary coded decimal) ₃ Boron trichloride; -Boc, t-butoxycarbonyl; the availability of EDCI,carbodiimide hydrochloride; TEA, triethanolamine.

Further, the specific procedure for converting compound 10 to compound 11 is as follows: mixing a compound 10, benzyl bromide and DMF, cooling to-5 ℃, adding sodium hydride in batches, and slowly heating to room temperature for reaction for 1.5-3 hours; slowly adding water to quench reaction, and sequentially extracting, washing, drying and passing through a column to obtain the compound 11.

Further, the specific procedure for converting compound 11 to compound 12 is as follows: compound 11 was mixed with a 95% ethanol solution of KOH and reacted under reflux overnight; cooling to room temperature, and sequentially extracting, washing, drying and passing through a column to obtain a compound 12; wherein, the 95% ethanol solution refers to ethanol with the volume ratio of 95%.

Further, the specific procedure for the conversion of compound 12 to compound 13 is as follows: compound 12, dess-martin periodate, and dichloromethane were mixed and reacted overnight at room temperature in an ice bath; and then sequentially extracting, washing, drying and passing through a column to obtain the compound 13.

Further, the specific procedure for the conversion of compound 13 to compound 14 is as follows: mixing trimethylsilyl acetylene, n-butyllithium and THF, stirring at-73 to-83 ℃ for 0.5-1 h, dropwise adding a THF solution of a compound 13 into the mixture, reacting for 0.5-1 h, and then heating to 0 ℃ for reacting for 0.5-1.5 h; quenching reaction by saturated ammonium chloride, and sequentially extracting, washing, drying and passing through a column to obtain the compound 14.

Further, the specific procedure for converting compound 14 to compound 15 is as follows:

mixing a compound 14, oxalyl chloride monomethyl ester, DMAP and DCM, and reacting for 0.5-1.5 h at room temperature; adding water to quench the reaction, and then sequentially extracting, washing and drying the reaction product to obtain the compound 15.

Further, the specific procedure for the conversion of compound 15 to compound 16 is as follows: mixing a compound 15, AIBN, tri-n-butyl tin hydride and toluene, and refluxing at 105-115 ℃ for 1.5-3 hours; the reaction product was then concentrated under reduced pressure and passed through a column to give compound 16.

Further, the specific procedure for the conversion of compound 16 to compound 17 is as follows: mixing the compound 16, TBAF, acetic acid and THF, and stirring for 2-3 hours at room temperature; and then sequentially extracting, washing, drying and passing through a column to obtain the compound 17.

Further, the specific procedure for the conversion of compound 17 to compound 18 is as follows: compound 17, boron trichloride, dichloromethane were mixed, ice-bath was slowly stirred to room temperature overnight; the reaction product is extracted, washed, dried and passed through column to obtain compound 18.

Further, the specific procedure for the conversion of compound 18 to compound of formula I is as follows: mixing compound 18, compound 23, magnesium chloride and acetonitrile in 1ml, heating to 45-55 ℃ for reaction for 5-15 min, and then dropwise adding DIPEA (N, N-diisopropylethylamine) into the mixture, and reacting overnight; cooling to room temperature, extracting, washing, drying and passing through a column sequentially to obtain the compound shown in the formula I.

Further, the specific procedure for the conversion of compound 19 to compound 20 is as follows: mixing isobutanol, N-t-butoxycarbonyl-L-alanine, EDCI, DMAP and dichloromethane, and stirring for 2-4 hours at room temperature; the reaction product was then sequentially extracted, washed, dried, and subjected to column chromatography under reduced pressure to give compound 20.

Further, the specific procedure for the conversion of compound 20 to compound 21 is as follows: mixing the compound 20 with 1, 4-dioxane in an ice bath, then dropwise adding a 1, 4-dioxane solution of hydrogen chloride, and naturally heating to room temperature to react overnight after the dropwise adding is finished; then the reaction product is concentrated under reduced pressure and dried in turn to obtain the compound 21.

Further, the specific procedure for the conversion of compound 21 to compound 23 is as follows: mixing the compound 21 with DCM, cooling to-83 to-73 ℃, adding phenyl dichlorophosphate into the mixture, then dropwise adding TEA, stirring the mixture for 10-20 min at-83 to-73 ℃ after the dropwise adding, naturally heating to room temperature, and stirring for 1-3 h; then cooling the reaction system to-25-15 ℃, adding TEA into the reaction system, slowly adding a methylene dichloride solution of p-nitrophenol, and heating to room temperature after the dripping is completed and stirring overnight; adding a proper amount of water for quenching reaction; and then sequentially extracting, washing, drying, decompressing and concentrating the reaction product and passing through a column to obtain the compound 23.

According to a third aspect of the present invention there is provided the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis and/or treatment of influenza virus.

According to a fourth aspect of the present invention there is provided the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis and/or treatment of novel coronaviruses.

According to a fifth aspect of the present invention there is provided the use of a compound of formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis and/or treatment of hepatitis c virus.

The invention has the following beneficial effects:

(1) The compound of the present invention or a pharmaceutically acceptable salt thereof is an RNA polymerase inhibitor which, upon entry into cells infected with a virus, binds to the active site of RNA polymerase (RdRP) in the form of an active triphosphate, prevents further expansion of the RNA strand and terminates RNA replication.

(2) Experiments prove that the compound or the pharmaceutically acceptable salt thereof can inhibit various RNA viruses, has excellent pharmaceutical effects, and is particularly used for inhibiting influenza viruses, new coronaviruses and hepatitis C viruses.

(3) The nucleotide inhibitor has the advantages of simple synthesis method, mild synthesis condition and high feasibility.

Drawings

FIG. 1 is a mass spectrum of the compound of the present invention of formula I.

FIG. 2 is a hydrogen spectrum of the compound of the present invention of formula I.

FIG. 3 is a carbon spectrum of the compound of the present invention of formula I.

Detailed Description

In order that those skilled in the art will better understand the present invention, a technical solution of the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments.

Example 1: synthesis of Compounds of formula I

Synthesis of Compound 11

Dehydrated uridine (

compound

10,3g,1 eq), benzyl bromide (5 g,2.2 eq) and 30ml DMF were mixed, cooled to 0℃and sodium hydride (60%, 1.4g,2.6 eq) was added in portions and allowed to react slowly at room temperature for 2h. Post-treatment: slowly adding water for quenching reaction, sequentially extracting the reaction product by ethyl acetate, washing by brine, drying by anhydrous sodium sulfate, and passing through a column to obtain a compound 11,3.6g, wherein the yield is: 66%.

Synthesis of Compound 12

Compound 11 (100 mg,1 eq) and 0.1mol/L KOH 95% ethanol solution 2ml were mixed and reacted under reflux overnight. Post-treatment: cooling to room temperature, sequentially extracting the reaction product with ethyl acetate, washing with brine, drying with anhydrous sodium sulfate, and passing through a column to obtain a compound 12, 54mg, and the yield: 51%.

Synthesis of Compound 13

Compound 12 (54 mg,1 eq), dess-martin periodate (170 mg,3 eq) and 5ml dichloromethane were mixed and reacted overnight at room temperature in an ice bath. Post-treatment: the reaction product was sequentially subjected to dichloromethane extraction, brine washing, drying over anhydrous sodium sulfate, and column chromatography to give compound 13, 40mg, yield: 75%.

Synthesis of Compound 14

Trimethylsilylacetylene (70 mg,3 eq), n-butyllithium (1.6 mol/L,0.45ml,3 eq) and 1ml THF were mixed, stirred at-78℃for 0.5h, a THF solution of compound 13 (100 mg,1 eq) was added dropwise, reacted for 0.5h, and heated to 0℃for 1h. Post-treatment: the reaction product was sequentially subjected to ethyl acetate extraction, brine washing, anhydrous sodium sulfate drying, and column chromatography to give compound 14, 35mg, yield: 40%.

Synthesis of Compound 15

Compound 14 (100 mg,1 eq), oxalyl chloride monomethyl ester (46 mg,2 eq), DMAP (48 mg,2 eq) and 2ml DCM were mixed and reacted at room temperature for 1h. Post-treatment: the reaction product was quenched with water, extracted with DCM, washed three times with water, washed twice with brine, and concentrated by organic phase drying to give crude compound 15, 110mg, yield: 95%.

Synthesis of Compound 16

Compound 15 (110 mg,1 eq), 4mg AIBN, tri-n-butyltin hydride (221 mg,4 eq) and 5ml toluene were mixed and refluxed at 110℃for 2h. Post-treatment: the reaction product was concentrated under reduced pressure and passed through a column to give compound 16, 56mg, yield: 57%.

Synthesis of Compound 17

Compound 16 (112 mg,1 eq), TBAF (2 eq), acetic acid (1 eq), 1ml thf were mixed and stirred at room temperature for 2h. Post-treatment: the reaction product was sequentially subjected to ethyl acetate extraction, brine washing, drying over anhydrous sodium sulfate, and column chromatography to give compound 17, 93mg, yield: 98%.

Synthesis of Compound 18

Compound 17 (80 mg,1 eq), boron trichloride (2.5 eq), dichloromethane were mixed, and the ice bath was stirred slowly to room temperature overnight. Post-treatment: the reaction product was quenched with water, and then extracted with dichloromethane, washed with brine, dried over anhydrous sodium sulfate, and passed through a column to give compound 18, 30mg, yield: 60%.

Synthesis of Compound 20

Isobutanol, N-t-butoxycarbonyl-L-alanine, EDCI (1.1 eq) and DMAP (0.1 eq) were dissolved in 40ml of methylene chloride solution with stirring and stirred at room temperature for 3 hours. Post-treatment: adding a proper amount of water and methylene dichloride extraction liquid into the reaction product, sequentially washing with brine, drying with anhydrous sodium sulfate, decompressing and passing through a column to obtain a compound 20,4.1g, and obtaining the yield: 57.6%.

Synthesis of Compound 21

Compound 20 (4.1 g,1 eq) was dissolved in 30ml dioxane with stirring in an ice bath, hydrogen chloride dioxane solution (1 mol/L,3 eq) was added dropwise thereto, and the mixture was allowed to spontaneously warm to room temperature after completion of the addition, and reacted overnight. Post-treatment: the reaction product was concentrated and dried under reduced pressure to give compound 21,3.0g, yield: 97.1%.

Synthesis of Compound 23

Compound 21 (3 g,1 eq) was dissolved in 40ml DCM, cooled to-78 ℃, phenyl dichlorophosphate (1 eq) was added, and TEA (2 eq) was added dropwise; stirring at-78deg.C for 15min after dripping, naturally heating to room temperature, and stirring for 2 hr; then the reaction system was cooled to-20 ℃, TEA (1.15 eq) was added, and then a solution of p-nitrophenol (0.9 eq) in dichloromethane was slowly added; after completion of the dropwise addition, the mixture was warmed to room temperature and stirred overnight. Post-treatment: adding a proper amount of water for quenching reaction, and sequentially carrying out dichloromethane extraction and liquid separation, brine washing, anhydrous sodium sulfate drying, and reduced pressure concentration and column passing on the reaction product to obtain a compound 23,3.8g.

Synthesis of Compound of formula I (PPN-301)

Compound 18 (30 mg,1 eq), fragment compound 23 (1.2 eq), magnesium chloride (1 eq) and acetonitrile 1ml were mixed, heated to 50 ℃ and reacted for 10min, DIPEA (2.5 eq) was added dropwise and reacted overnight. Post-treatment: cooling to room temperature, adding water and ethyl acetate into the reaction product for extraction, washing with saturated sodium bicarbonate and brine, drying with anhydrous sodium sulfate, and passing through a column to obtain a compound PPN-301, 25mg, yield: 39%.

Referring to FIGS. 1-3, the mass spectrum, hydrogen spectrum and carbon spectrum of the compound of formula I are shown. Wherein, mass spectrum data is: MS (ESI) m/z calculated for C ₂₄ H ₃₀ N ₃ O ₉ P[M+H] ⁺ 536.1, found 536.1; the hydrogen spectrum data are: ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.39 (s, 1H), 7.60 (d,J= 8.0 Hz, 1H), 7.45 – 7.33 (m, 2H), 7.23 –7.16 (m, 3H), 6.22 (d,J= 7.6 Hz, 1H), 6.16 – 6.03 (m, 2H), 5.52 (d,J= 8.0 Hz, 1H), 4.45 – 4.07 (m, 3H), 4.01 – 3.72 (m, 4H), 3.56 – 3.40 (m, 1H),3.21 (d,J= 2.4 Hz, 1H), 1.93 – 1.76 (m, 1H), 1.24 (d,J= 7.2Hz, 3H), 0.86 (d,J=6.7 Hz, 6H); the carbon spectrum data are: ¹³ C NMR (125MHz, DMSO-d ₆ ) δ173.61, 163.42, 151.08, 150.66, 141.29, 130.09(2C), 125.02, 120.48(2C), 101.59,84.11, 82.86, 79.56, 77.26, 74.29, 70.68, 65.03, 50.34, 43.05, 27.69, 20.25,19.21(2C)。

example 2: cytotoxicity detection protocol

Vero E6 cells were inoculated into 96 well cell culture plates and cultured overnight for experiments when the confluence reached 80-90%. Different doses of the drugs to be tested PPN301, PPN311, PPN316, PPN317 and (final concentrations of 1.0mM, 100. Mu.M, 10. Mu.M, 1.0. Mu.M, 0.1. Mu.M, 10 nM) were added to each well, an equal volume of medium was added to the control wells, cell viability was detected by CCK-8 after 72h incubation, and the effect of the compounds on cell growth was evaluated by calculation of CC 50. Experimental data on Vero E6 cytotoxicity are shown in table 1.

TABLE 1 experimental data on cytotoxicity of Vero E6

Final concentration of sample (μM)	PPN301 cell viability.+ -. SD (%)	PPN311 cell viability.+ -. SD (%)	PPN316 cell viability.+ -. SD (%)	PN317 cell viability.+ -. SD (%)
					1000	2.89±0.51	2.31±1.00	1.80±0.38	2.12±0.26
100	78.48±4.07	64.63±8.09	24.71±4.18	45.78±5.19
					10	95.14±7.04	87.14±11.54	54.34±6.35	68.34±8.37
1	98.43±6.80	81.70±14.49	67.28±10.69	76.98±11.23
					0.1	94.06±11.27	84.34±17.33	74.92±4.14	85.44±5.49
0.01	95.22±4.74	80.79±12.68	85.87±12.52	89.42±4.37

The structural formulas of PPN316, PPN317, PPN311 in table 1 are as follows:

，PPN311；

，PPN316；

，PPN317。

example 3: new coronavirus (SARS-CoV-2) inhibition assay

Vero E6 cells were plated in 96-well cell plates, 1.6X104 cells/well, D10 medium at 37℃with 5% CO ₂ Culturing overnight. The cell culture supernatants were aspirated and the drugs to be tested were added at different concentrations per well, each with 6 concentration gradients (100. Mu.M, 33.3. Mu.M, 11.1. Mu.M, 3.7. Mu.M, 1.2. Mu.M, 0.4. Mu.M), 4 replicate wells per gradient, 37℃and 5% CO ₂ Incubate in incubator for 1h. An equal volume of virus dilution supernatant (moi=0.1) was then added per well, with vehicle control, negative control without drug and virus, positive control being set. 37 ℃,5% CO ₂ After 1h of infection in the incubator, the virus-test drug mixed medium was aspirated, and after washing with 1 XPBS, the medium was changed to a medium containing only the test drug to continue the culture, 6 concentration gradients (100. Mu.M, 33.3. Mu.M, 11.1. Mu.M, 3.7. Mu.M, 1.2. Mu.M, 0.4. Mu.M) were set, 4 replicate wells per gradient, and positive, vehicle and negative controls were set. 37 ℃,5% CO ₂ After culturing for 24 hours, the following steps are sequentially carried out:

(1) cell supernatant was collected, viral RNA was extracted, used for Real-time PCR virus quantification, and the inhibition ratio of the drug to viral replication was calculated.

(2) The culture supernatant was discarded from the cell plate, and 4% PFA 200. Mu.L/well was added thereto, and the mixture was fixed at room temperature for more than 3 hours. After Triton punching and membrane rupture sealing, SARS-N Rabbit monoclonal antibody acts as a primary antibody for 1h, A488 anti-Rabbit IgG acts as a secondary antibody for 1h, and plates are washed and fluorescence observation is carried out.

The results of the inhibition test data (24 h) of the replication of the new coronavirus in Vero E6 cells are shown in table 2.

TABLE 2 inhibition of SARS-CoV-2 replication in VeroE6 cells experimental data (24 h)

Final concentration of sample (μM)	PPN301 inhibition ± SD (%)	PPN311 inhibition ± SD (%)	PPN316 inhibition ± SD (%)	PPN317 inhibition ± SD (%)
					100	99.03±0.03	59.92±2.83	19.65±0.06	25.14±1.31
33.3	91.46±0.28	53.13±9.32	67.21±0.72	50.35±4.36
					11.1	80.50±2.67	35.09±3.42	16.79±0.75	28.68±2.19
3.7	16.93±8.49	32.84±1.68	26.65±1.78	26.71±1.65
					1.2	29.47±17.23	25.58±3.78	11.11±1.37	16.42±2.49
0.4	40.63±5.13	10.52±12.03	21.84±0.32	17.38±4.29

Example 4: toxicity eliminating pharmacodynamic detection for mice

A K18-hACE2 mouse model is built, and 5-14 weeks old K18-hACE2 mice (Ji kang) are adopted, wherein the weight of the mice is 20-30 g, 8 mice in each group are randomly divided, and a normal control group, a SARS-CoV infection group, a PPN301 group and a PPN303 group are adopted. New coronavirus (Delta) nasal drops infect K18-hACE2 transgenic mice at a preliminary dose of 105PFU. 2 hours after infection, the test subjects were given by gavage to the drug group mice for 5 consecutive days, 1 time/day. Daily changes in body weight were recorded after infection and animal death was recorded within 5 days of infection, and the results are shown in table 3.

TABLE 3 survival of mice

Days (days)	Normal control group	SARS-CoV-2 infection group	PPN303	PPN311	PPN316	PPN317
								1	8	8	8	8	8	8
2	8	5	7	4	5	5
							3	8	2	6	1	3	2
4	8	0	5	0	1	1
							5	8	0	5	0	0	0

Example 5: inhibition effect on influenza virus

Virus infectivity assay (determination of TCID 50)

Taking out virus liquid, dissolving, continuously performing 10-fold gradient dilution with serum-free double-antibody-free culture medium, diluting to 101-109 (dilution degree can be increased or decreased according to the situation), inoculating virus of each dilution into corresponding host cell, wherein influenza virus H1N1, H3N2 into MDCK (NBL-2) cells, arranging 6-8 multiple holes for each dilution, respectively inoculating virus liquid of different concentrations into corresponding 96-well plates growing into single-layer cells, each hole having 100 μL, placing into 5% CO ₂ Adsorbing the supernatant in a constant temperature incubator at 35 ℃ for 1-2h, and washing with PBS for 1-2 times; adding 100 μl of cell maintenance solution per well, continuously culturing while setting 6-8 normal cell control groups, and placing 5% CO at 35deg.C and 37deg.C respectively ₂ Culturing in incubator, observing virus growth every day, observing cell morphology change under inverted microscope, recording characteristic Cytopathic (CPE) hole number, recording result after 3-7d, counting pathological change holes when cytopathic rate reaches 50% or above, and counting non-pathological change holes when cytopathic rate is less than 50%.

The extent of lesions (CPE) in cells was recorded on a 6-level scale:

-: the cells grow normally without lesions;

and (3) the following steps: cytopathy is less than 10% of the whole monolayer;

+: cytopathy is less than 25% of the whole monolayer;

++: cytopathy is less than 50% of the whole monolayer;

+++: cytopathy is less than 75% of the whole monolayer;

++++: cytopathy accounts for more than 75% of the whole monolayer of cells.

The result judging and calculating method comprises the following steps: the half-infectious amount TCID50 of the virus was calculated according to the Reed-Muench method.

Proportional Distance (PD) = (percent of lesions above 50% to 50%)/(percent of lesions above 50% to percent of lesions below 50%); wherein the culture holes with cytopathic rate up to 50% and above are pathological holes, and the culture holes with cytopathic rate less than 50% are non-pathological holes.

In vitro antiviral assay

(1) Experimental method

In vitro antiviral assays were performed after 96 well plate cells had grown into monolayers. The virus attack amount is 10-100 TCID50. After virus adsorbed on monolayer cells for 1-2h, washing off virus liquid, adding 100 mu L of liquid medicine and 100 mu L of maintaining liquid into each hole, and setting virus control and blank cell control. CO at a corresponding temperature ₂ Culturing in incubator, observing the lesions under inverted microscope every day, continuously observing for several days, and recording CPE of each hole. When the virus is in the control group the lesions reach "++++", and when the normal cell control group has no lesion, and determining the optimal time point for CCK-8 detection, performing CCK-8 detection, and measuring the absorbance value at the wavelength of 450nm of the microplate reader. The experiment was repeated 2 times and the average of the results of 3 experiments was calculated.

(2) The 50% effective concentration (IC 50) and Therapeutic Index (TI) of the drug were calculated according to the Reed-Muench method. Viral inhibition = (drug group OD 450-virus control group OD 450)/(normal cell control group OD 450-virus control group OD 450). ti=tc50/IC 50.

TABLE 4 inhibition of H1N1 Virus

Names of Compounds	Concentration of	H1N1 virus inhibition rate
			PPN301
	10 μM	71.24
			PPN311	10 μM	50.61
PPN316	10 μM	48.38
			PPN317	10 μM	49.77

TABLE 5 inhibition of H3N2 Virus

Names of Compounds	Concentration of	H3N2 virus inhibition rate
			PPN301
	10 μM	40.86
			PPN311	10 μM	20.16
PPN316	10 μM	25.79
			PPN317	10 μM	22.56

According to an experiment for inhibiting influenza virus, the compound PPN301 provided by the invention can effectively inhibit influenza virus H1N1 and H3N2 relative to other compounds PPN311, PPN316 and PPN317, and the compound or pharmaceutically acceptable salt thereof provided by the invention can be used for preparing medicaments for treating influenza virus infection.

Example 6: inhibiting effect on hepatitis C virus

The basic procedure of example 6 is the same as in example 5, except that the virus is replaced with hepatitis C virus. Wherein the hepatitis C virus is adsorbed at 37 ℃ for 1-2 hours, and the supernatant is discarded. The inhibitory effect of each compound on hepatitis c virus is shown in table 6 below.

TABLE 6 inhibitory effect on hepatitis C Virus

Names of Compounds	Concentration of	Hepatitis C Virus inhibition Rate
			PPN301
	10 μM	60.46
			PPN311	10 μM	40.61
PPN316	10 μM	48.19
			PPN317	10 μM	44.13

According to the experiment of inhibiting the hepatitis C virus, the compound PPN301 provided by the invention can effectively inhibit the hepatitis C virus relative to other compounds PPN311, PPN316 and PPN317, and the compound or pharmaceutically acceptable salt or ester thereof provided by the invention can be used for preparing medicaments for treating hepatitis C virus infection.

The above embodiments are only for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention, but these modifications or substitutions are all within the scope of the present invention.

Claims

1. A compound having the structure shown in formula I:

（I）。

2. a method of synthesizing the compound of claim 1, wherein the synthetic route is as follows:

；

wherein Bn on compounds 11-17 represents benzyl, TMS on compounds 14-16 represents trimethylsilyl, and Boc on compound 20 and compound 2 represents t-butoxycarbonyl.

3. The method of claim 2, wherein the synthesis path is as follows:

。

4. a method according to claim 3, wherein the conversion of compound 10 to compound 11 is performed as follows:

mixing a compound 10, benzyl bromide and DMF, cooling to-5 ℃, adding sodium hydride in batches, and slowly heating to room temperature for reaction for 1.5-3 hours; slowly adding water to quench reaction, and sequentially extracting, washing, drying and passing through a column to obtain the compound 11.

5. A method according to claim 3, wherein the conversion of compound 11 to compound 12 is performed as follows:

compound 11 was mixed with a 95% ethanol solution of KOH and reacted under reflux overnight; cooling to room temperature, and sequentially extracting, washing, drying and passing through a column to obtain a compound 12; wherein, the 95% ethanol solution refers to ethanol with the volume ratio of 95%.

6. A method according to claim 3, wherein the conversion of compound 12 to compound 13 is performed as follows:

compound 12, dess-martin periodate, and dichloromethane were mixed and reacted overnight at room temperature in an ice bath; and then sequentially extracting, washing, drying and passing through a column to obtain the compound 13.

7. A method according to claim 3, wherein the conversion of compound 13 to compound 14 is performed as follows:

mixing trimethylsilyl acetylene, n-butyllithium and THF, stirring at-73 to-83 ℃ for 0.5-1 h, dropwise adding a THF solution of a compound 13 into the mixture, reacting for 0.5-1 h, and then heating to 0 ℃ for reacting for 0.5-1.5 h; quenching reaction by saturated ammonium chloride, and sequentially extracting, washing, drying and passing through a column to obtain the compound 14.

8. A method according to claim 3, wherein the conversion of compound 14 to compound 15 is performed as follows:

9. A method according to claim 3, wherein the conversion of compound 15 to compound 16 is performed as follows:

mixing a compound 15, AIBN, tri-n-butyl tin hydride and toluene, and refluxing at 105-115 ℃ for 1.5-3 hours; the reaction product was then concentrated under reduced pressure and passed through a column to give compound 16.

10. A method according to claim 3, wherein the conversion of compound 16 to compound 17 is performed as follows:

mixing the compound 16, TBAF, acetic acid and THF, and stirring for 2-3 hours at room temperature; and then sequentially extracting, washing, drying and passing through a column to obtain the compound 17.

11. A method according to claim 3, wherein the conversion of compound 17 to compound 18 is performed as follows:

compound 17, boron trichloride, dichloromethane were mixed, ice-bath was slowly stirred to room temperature overnight; the reaction product is extracted, washed, dried and passed through column to obtain compound 18.

12. A process according to claim 3, wherein the conversion of compound 18 to compound of formula I is carried out as follows:

mixing compound 18, compound 23, magnesium chloride and acetonitrile in 1ml, heating to 45-55 ℃ for reaction for 5-15 min, and then dropwise adding DIPEA into the mixture for reaction overnight; cooling to room temperature, extracting, washing, drying and passing through a column sequentially to obtain the compound shown in the formula I.

13. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis and/or treatment of influenza virus.

14. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis and/or treatment of novel coronaviruses.

15. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis and/or treatment of hepatitis c virus.