Application of benzoylaniline compound in preparation of broad-spectrum antiviral drugs

Abstract

The invention belongs to the field of antiviral compounds, and discloses an application of a benzoylaniline compound in preparation of broad-spectrum antiviral drugs. The benzoylaniline compound has a general formula as shown in a formula I, wherein R1, R2, R3, R4 and R5 are at least one of H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl and nitryl; wherein H on the C1-4 alkyl group and H on the C1-4 alkoxy group are optionally substituted by halogen atoms, and the condition is that at least two of R1, R2, R3, R4 and R5 are halogens, but not more than 4; at least one of R1, R2, R3, R4 and R5 is F, but not more than 2. The compound disclosed by the invention has a relatively good in-vitro antiviral effect, and the compound BAB162 is wide in antiviral spectrum and has a remarkable inhibition effect on various viruses at low concentration. The BAB162 shows an obvious treatment effect in a piglet viral diarrhea infection model and a mouse acute hepatitis virus infection model.

Description

Technical Field

[0001] This invention belongs to the field of antiviral compounds, and particularly relates to the application of benzoylaniline compounds in the preparation of broad-spectrum antiviral drugs. Background Technology

[0002] Infectious diseases are a major cause of high global morbidity and mortality, with viral diseases accounting for more than half. Currently, viral diseases are spreading particularly rapidly, and the risk of emerging novel viruses is constantly increasing. In livestock farming, the prevention and control of viral diseases faces a severe challenge, with an increasing number of zoonotic diseases and frequent cross-species transmission. New and old viruses emerge alternately. While vaccine control is an important strategy for viral diseases, it easily accelerates the development of drug resistance, often leaving a situation where no effective drugs are available during outbreaks of new viruses.

[0003] Small molecule antiviral compounds are a major strategy for effectively combating viral infections. Totally synthesized compounds, due to their stability, simple processes, rapid synthesis, and ease of modification, overcome the limitations of natural product structures and long development cycles, providing lead compounds for subsequent antiviral compound development. In recent years, novel antiviral compounds developed based on total synthesis strategies have received significant attention in the field of drug discovery. The total synthesis of over 20 natural products has successfully solved the construction of all representative skeletons in xanthane-type sesquiterpenoid natural products, providing a material basis for the subsequent discovery of antiviral lead compounds (Angew Chem Int Ed Engl. 2017, 56, 51). Synthesizing compounds with structures similar to natural products but with low isolation yields and difficult structural modification is also a major advantage of total synthesis. For example, a concise and efficient chemical synthesis route for prostaglandin was developed using an oxidative dearomatization strategy, providing a solution for the efficient elimination of HIV latent in immune cells (Chem. 2018, 4, 12). The FDA approval of simeprevir and vaniprevir for hepatitis C in 2014 represents an excellent example of chemically synthesized drugs entering clinical trials, providing targeted therapies for the treatment of chronic hepatitis C infection (J. Med. Chem. 2014, 57, 1673; J. Med. Chem. 2010, 53, 2443). These studies demonstrate that chemical total synthesis can effectively enrich the candidate library of antiviral drugs, yielding more novel antiviral compounds with different or multiple targets, while also enriching structure-activity relationship analysis, further promoting the development of subsequent antiviral drugs.

[0004] Existing benzoylaniline derivatives struggle to balance activity and toxicity. For example, their backbones often contain potentially toxic groups (such as nitro groups), and due to their host pathway-targeting mechanisms, they frequently exhibit high host cell toxicity (Med Res Rev. 2023, 43:897-931). Furthermore, the development of existing derivatives is largely limited to efficacy evaluation against single or a few specific viruses, and even minor chemical modifications often lead to drastic changes in the antiviral spectrum. There is a lack of systematic optimization strategies for the antiviral properties of this core component. Summary of the Invention

[0005] To address the limitations of existing technologies in benzoylaniline derivatives regarding their limited antiviral spectrum and the difficulty in balancing toxicity and activity, this invention provides an application of benzoylaniline compounds in the preparation of broad-spectrum antiviral drugs. By modifying functional groups, compounds with different activities are obtained, and the compound BAB162, exhibiting the best efficacy, is screened. The compound provided by this invention has a broad antiviral spectrum, good antiviral effect, and significant in vivo therapeutic efficacy.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] The application of benzoylaniline compounds in the preparation of broad-spectrum antiviral drugs, which have the general formula shown in Formula I:

[0008] ;

[0009] R1, R2, R3, R4, and R5 are selected from at least one of H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl, and nitro; wherein the H on the C1-4 alkyl or C1-4 alkoxy is optionally replaced by a halogen atom, provided that at least two of R1, R2, R3, R4, and R5 are halogens, but no more than four.

[0010] Furthermore, at least one of R1, R2, R3, R4, and R5 is F, but no more than two.

[0011] The above-mentioned benzoylaniline compound is at least one of the following compounds:

[0012] .

[0013] Further, preferably, the above-mentioned benzoylaniline compound is .

[0014] Furthermore, the aforementioned benzoylaniline compounds can also exist in the form of their pharmaceutically acceptable salts.

[0015] A broad-spectrum antiviral drug comprising the above-described benzoylaniline compound or a pharmaceutically acceptable salt of a benzoylaniline compound.

[0016] Furthermore, the antiviral drugs mentioned above target at least one virus from the families Coronaviridae, Arteritis Viridae, Orthomyxoviridae, Reoviridae, and Double-stranded RNA Viridae. Specifically, the Coronaviridae family includes porcine epidemic diarrhea virus or mouse hepatitis virus; the Arteritis Viridae family includes porcine reproductive and respiratory syndrome virus; the Orthomyxoviridae family includes influenza virus; the Reoviridae family includes avian reovirus; and the Double-stranded RNA Viridae family includes infectious bursal disease virus.

[0017] The beneficial effects of this invention are:

[0018] The compounds provided by this invention exhibit good in vitro antiviral effects. Among them, the preferred compound BAB162 has a broad antiviral spectrum and shows significant inhibitory effects against a variety of viruses, including those from the Coronaviridae, Arterioviridae, Orthomyxoviridae, and Reoviridae families, even at low concentrations. BAB162 also demonstrated significant therapeutic effects in both a swine viral diarrhea infection model and a mouse acute hepatitis virus infection model. This invention enriches the antiviral drug molecular library and provides a new approach for the prevention and treatment of viral infections. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The 1H NMR spectrum (solvent DMSO) of compound BAB162 prepared in Example 1.

[0021] Figure 2 The carbon NMR spectrum of compound BAB162 prepared in Example 1.

[0022] Figure 3 The image shows a high-resolution mass spectrum in positive ion mode of compound BAB162 prepared in Example 1.

[0023] Figure 4 The high-resolution mass spectrum of compound BAB162 prepared in Example 1 is shown in negative ion mode.

[0024] Figure 5 The viral load of enteroviruses in PEDV-infected piglets under the action of compound BAB162.

[0025] Figure 6 The viral load in the livers of MHV mice infected with compound BAB162 is shown. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Porcine epidemic diarrhea virus (PEDV)-HB2020, LW / L, and ZJ08, and influenza virus (IAV) H3N3 are strains preserved in our laboratory; PEDV-JMS is preserved in the research group of Professor Liu Pinghuang at China Agricultural University; mouse hepatitis virus (MHV)-A59 was kindly donated by Professor Peng Guiqing of Huazhong Agricultural University; mink parvovirus (MEV)-SD7 was kindly donated by Professor Xie Zhijing of Shandong Agricultural University; porcine reproductive and respiratory syndrome virus (PRRSV)-40 and porcine rotavirus standard strain PoRV OSU were kindly donated by Associate Professor Wang Yongqiang of China Agricultural University; Newcastle disease virus (NDV)-SG10 is preserved in the research group of Professor Zhang Guozhong of China Agricultural University; vaccinia virus (VACV) is preserved in the research group of Professor Peng Chen of China Agricultural University; avian reovirus (ARV)-S1133 and infectious bursal disease virus (IBDV)-Lx are preserved in the research group of Associate Professor Wang Yongqiang of China Agricultural University. Except for PEDV-LW / L and PEDV-ZJ08, which are vaccine strains, all other strains are clinical strains.

[0028] This invention also provides a method for preparing compound BAB162, comprising the following steps:

[0029] (S1) Under an inert gas atmosphere, 1,2-difluoro-3-chlorobenzene was reacted in the presence of organolithium and organic amine at a controlled temperature of -80°C to -60°C for 2-4 h. The reaction solution was poured into dry ice and heated to room temperature and stirred for 12-24 h to obtain the intermediate product Cpd1.

[0030] (S2) Intermediate Cpd1 reacts with tert-butanol in the presence of an azide compound to give intermediate Cpd2;

[0031] (S3) The intermediate product Cpd2 was dissolved in ethyl acetate, and saturated hydrochloric acid ethyl acetate was added and stirred. A white solid precipitated to obtain the intermediate product Cpd3.

[0032] (S4) 5-chlorosalicylic acid reacts with oxalyl chloride to give an acyl chloride intermediate;

[0033] (S5) The intermediate product Cpd3 and the acyl chloride intermediate were reacted at 60°C to 80°C for 15-20 h in the presence of potassium iodide. After the solid was precipitated, it was processed to obtain the compound BAB162 of formula (I).

[0034] Example 1

[0035] The preparation method of the benzoylaniline compound BAB162 in this embodiment is as follows:

[0036] .

[0037] The specific steps are as follows:

[0038] (S1) Tetrahydrofuran was added to a three-necked flask and stirred to displace nitrogen. The temperature was lowered to -75°C, and 29.8 g of butyllithium (1 eq) was added dropwise under controlled temperature. Then, 47.1 g of diisopropylamine (1 eq) was added dropwise under controlled temperature, and the mixture was stirred for 10 min. Next, 100 g of 1,2-difluoro-3-chlorobenzene (1 eq) was added dropwise. After the addition was completed, the reaction mixture was kept at this temperature for 2 h. The reaction mixture was poured into dry ice and allowed to warm naturally to room temperature overnight with stirring. The reaction mixture was concentrated to remove tetrahydrofuran, and 100 mL of water and 200 mL of ethyl acetate were added. The pH was adjusted to 3. The mixture was separated, and the organic phase was concentrated to obtain the crude product. The crude product was then slurried with n-hexane and filtered to obtain 72 g of the intermediate product Cpd1.

[0039] (S2) In a single-necked flask, add 72 g (1 eq) of Cpd1 and dissolve it evenly in 720 mL of dioxane. Then, add 350 mL of tert-butanol, 112 g (4 eq) of triethylamine, and 115 g of diphenyl azidophosphate (1.5 eq) sequentially. After adding nitrogen, purge the mixture three times and react overnight. Pour the reaction solution into ice, concentrate and extract with ethyl acetate, and stir the organic phase (petroleum ether:ethyl acetate = 20:1) to obtain 72 g of the intermediate product Cpd2.

[0040] (S3) Add 72 g of Cpd2 to a single-necked flask, dissolve it in 720 mL of ethyl acetate, then add 720 mL of saturated ethyl hydrochloride (HCl / EA, 1 g / 4 mL) and stir overnight. A white solid gradually precipitates out. The solid obtained after filtration is the intermediate product Cpd3.

[0041] (S4) Dissolve 100 g of salicylic acid in 1 L of dichloromethane, add 8 mL of DMF, add 1.2 eq of oxaloyl chloride under ice bath conditions, react at room temperature for 2 h, the reaction becomes clear, and after the reaction is completed, evaporate to dryness to obtain the acyl chloride compound for later use.

[0042] (S5) In a single-necked flask, add 50 g (1 eq) of Cpd3 and stir with acetonitrile until homogeneous. Add potassium iodide (62 g (2 eq)) and then slowly add 43 g (dissolved in acetonitrile) (1.2 eq) of the acyl chloride compound prepared in step (S4). React at 80°C for 16 h. Let stand overnight until the solid precipitates. Filter the solid and dissolve the filter cake in ethyl acetate. Wash twice with water, dry the organic phase, and then evaporate to dryness. Pulp the solid with dichloromethane and filter to obtain product BAB162.

[0043] Figure 1 This is the 1H NMR spectrum of compound BAB162 obtained in Example 1 (solvent: deuterated dimethyl sulfoxide). 1 ¹H NMR (600 MHz, DMSO-d⁶) δ 12.24 (s, 1H), 10.78 (s, 1H), 8.08–8.05 (m, 1H), 7.90 (d, J = 2.4 Hz, 1H), 7.48 (dd, J = 8.8, 2.4 Hz, 1H), 7.44–7.41 (m, 1H), 7.07 (d, J = 8.8 Hz, 1H), a total of 7 hydrogen signals. Figure 2 This is the carbon NMR spectrum of compound BAB162 obtained in Example 1. 13 C NMR (150 MHz, DMSO-d6) δ 163.49, 156.04, 146.62 (dd, J = 244.5, 13.5 Hz), 142.59 (dd, J = 247.5, 13.5 Hz), 134.03, 129.96, 127.58 (d, J = 7.5 Hz), 125.39 (d, J = 4.5 Hz), 123.88, 119.59 (d, J = 12.0 Hz), 118.47, 118.45, 116.03 (d, J = 15.0 Hz), a total of 13 carbon signals. Figure 3 This is a high-resolution mass spectrum in positive ion mode of compound BAB162 obtained in Example 1, with the measured accurate mass-to-charge ratio [M+H]. + It is 317.9889. Figure 4 This is a high-resolution mass spectrum of compound BAB162 obtained in Example 1 in negative ion mode, with the measured accurate mass-to-charge ratio [MH]. - The value is 315.9732. (Passed) Figures 1-4 The structure of compound BAB162 obtained in the preparation example can be determined.

[0044] Implementation Results Example

[0045] (1) Antiviral activity of the compound against coronavirus

[0046] The antiviral activity of BAB025, BAB027, BAB031, BAB120, and BAB162 against alpha coronavirus PEDV-HB2020 was tested (n=3). The results are shown in Table 1 below.

[0047] Table 1. Antiviral activity of representative compounds against coronavirus PEDV

[0048]

[0049] As shown in Table 1, BAB162 exhibits the best antiviral activity, IC50. 50 The concentration was 0.028 μM. Other compounds, BAB025, BAB027, BAB031, and BAB120, also showed varying degrees of antiviral activity.

[0050] (2) Determination of the antiviral spectrum of compound BAB162

[0051] The benzoyl aniline compound BAB162 was subjected to IC50 reaction using the conventional MTT method. 50 The measurements were performed using GraphPad Prism 9.0 for nonlinear fitting.

[0052] After cell digestion and dispersion, the cells were seeded at an appropriate density into 96-well plates and placed in a 37°C, 5% CO2 cell culture incubator for static culture to grow into a monolayer of cells. The test compound was serially diluted 2-fold using DMEM, and the virus was simultaneously diluted 2-fold with virus maintenance solution to achieve an appropriate multiplicity of infection. 100 μL / well was added to each well, with three replicates for each compound concentration. Positive control wells, negative control wells, and blank control wells were also included. The cells were incubated at 37°C, 5% CO2 for 24–72 h, depending on the CPE level in the positive control wells. Detection was performed when the endpoint was reached. The culture medium was discarded, and the cells were washed twice with sterile PBS buffer. 100 μL of 1 mg / mL MTT working solution was added to each well, and the cells were incubated at 37°C, 5% CO2 for another 4 h. 150 μL of 100 mg / mL SDS-HCl solution was added to each well, and the cells were incubated at room temperature for 24 h until the precipitated formazan was completely dissolved. The absorbance of each well at 570 nm (reference wavelength 630 nm) was measured using a microplate reader. The viral inhibition rate was calculated using the following formula: Inhibition (%) = [(Asv - Av) / (Ac - Av)] × 100%, where Asv represents the experimental wells, Av represents the positive control wells, and Ac represents the negative control wells. The results are shown in Table 2 below.

[0053] Table 2 Antiviral spectrum determination

[0054]

[0055] As shown in Table 2, the compounds of Formula I prepared in this invention have a broad antiviral spectrum. Compound BAB162 showed inhibitory effects on members of the Coronaviridae (PEDV, MHV), PRRSV, IAV, ARV, and IBDV families. Among them, it had high SI values ​​of 66 and >100 against PEDV and MHV, respectively.

[0056] (3) The efficacy of compound BAB162 in a model of viral diarrhea in piglets

[0057] Four-day-old English Large White piglets, weighing between 1.5 and 2.0 kg, were used. After acclimatization, they were randomly divided into four groups of five piglets each. Each piglet was orally infected with 1 mL of the virulent strain PEDV-JMS (10... 3 TCID 50 / head). One day before viral infection, piglets were orally administered BAB162 at doses of 10 mg / kg bw and 20 mg / kg bw, respectively. The infection status of piglets was recorded within 7 days after infection. Figure 5 This refers to the viral load in the susceptible intestinal segments and contents of piglets under the influence of BAB162. In a piglet viral diarrhea model, the viral load decreased under the influence of BAB162 at both 10 mg / kg bw and 20 mg / kg bw, and the inhibitory effect was significantly stronger than that in the saline group.

[0058] (4) The therapeutic effect of compound BAB162 in a mouse model of viral hepatitis infection.

[0059] Five- to six-week-old female BALB / c mice, weighing 14–16 g, were used. After acclimatization, they were randomly divided into six groups of 12 mice each. Each mouse was intraperitoneally infected with 100 μL of MHV-A59 (10⁸ TCID₅₀ / mouse). 0.5 h post-infection, mice were intraperitoneally injected with the positive control drug remdesivir at doses of 5 mg / kg bw, 15 mg / kg bw, 25 mg / kg bw, and 40 mg / kg bw. The infection status of the mice was recorded over 7 days post-infection (dpi). Figure 6This describes the change in viral load in the liver of mice infected with BAB162. In a mouse model of viral hepatitis infection, the viral load in the liver was significantly reduced within 7 days after treatment with BAB162 at doses of 15 mg / kg bw and 25 mg / kg bw, in a concentration-dependent manner. Furthermore, there was no significant difference in treatment efficacy between 25 mg / kg bw BAB162 and 40 mg / kg bwremdesivir.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of benzoylaniline compounds in the preparation of broad-spectrum antiviral drugs, characterized in that, The benzoyl aniline compound has the general formula shown in Formula I: ； R1, R2, R3, R4, and R5 are selected from at least one of H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl, and nitro; wherein the H on the C1-4 alkyl or C1-4 alkoxy is optionally replaced by a halogen atom, provided that at least two of R1, R2, R3, R4, and R5 are halogens, but no more than four; and at least one of R1, R2, R3, R4, and R5 is F, but no more than two.

2. The use of the benzoylaniline compound according to claim 1 in the preparation of broad-spectrum antiviral drugs, characterized in that, The benzoyl aniline compound is at least one of the following compounds: 。 3. The application of the benzoylaniline compound according to claim 2 in the preparation of broad-spectrum antiviral drugs, characterized in that, The benzoylaniline compound is .

4. A broad-spectrum antiviral drug, characterized in that, The broad-spectrum antiviral drug comprises benzoylaniline compounds or pharmaceutically acceptable salts of benzoylaniline compounds; wherein the benzoylaniline compounds have the general formula shown in Formula I: ； R1, R2, R3, R4, and R5 are selected from at least one of H, C1-4 alkyl, C1-4 alkoxy, halogen, hydroxyl, and nitro; wherein the H on the C1-4 alkyl or C1-4 alkoxy is optionally replaced by a halogen atom, provided that at least two of R1, R2, R3, R4, and R5 are halogens, but no more than four; and at least one of R1, R2, R3, R4, and R5 is F, but no more than two.

5. The broad-spectrum antiviral drug according to claim 4, characterized in that, The antiviral drug targets at least one virus from the families Coronaviridae, Arterioviridae, Orthomyxoviridae, Reoviridae, and Double-stranded RNA Viridae.

6. The broad-spectrum antiviral drug according to claim 5, characterized in that, The coronaviruses mentioned are selected from porcine epidemic diarrhea virus or mouse hepatitis virus.

7. The broad-spectrum antiviral drug according to claim 5, characterized in that, The arteritis virus family mentioned is porcine reproductive and respiratory syndrome virus.

8. The broad-spectrum antiviral drug according to claim 5, characterized in that, The Orthomyxoviridae family mentioned refers to influenza viruses.

9. The broad-spectrum antiviral drug according to claim 5, characterized in that, The reovirus family mentioned refers to avian reoviruses.

10. The broad-spectrum antiviral drug according to claim 5, characterized in that, The double-stranded RNA virus family mentioned is infectious bursal disease virus.

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