Application of isoindolinone compounds in the preparation of antiviral drugs

Abstract

The present invention discloses the application of a class of isoindolinone compounds in the preparation of antiviral drugs. The isoindolinone compounds have the structure shown in the following general formula I. Wherein, R1 is an amino group or a substituted amino group or H; R2 is an amino group or H; R3 is a straight chain or branched chain alkyl group containing 1-6 carbons, a benzyl group, a phenyl group or a substituted phenyl group; R4 or R5 is a straight chain or branched chain alkyl group containing 1-6 carbons or H, which are the same or different; or R3, R4, R5 and the connected carbon atoms together constitute a substituted or unsubstituted cycloalkyl group containing 5-10 carbons, and the cycloalkyl group includes a monocyclic alkyl group or a polycyclic alkyl group. Experiments have shown that the compounds not only have significant antiviral activity, but also have the advantages of low cytotoxicity and high selectivity index. The present invention provides a new technical means for antiviral treatment, especially for anti-EV71 enterovirus treatment.

Description

Technical Field

[0001] The present invention relates to a new type of isoindolinone compound and a preparation method thereof, and also relates to the application of the compound in antiviral drugs, in particular to the application of anti-enteroidal EV71 virus drugs. The present invention belongs to the field of medical technology. Background Art

[0002] At present, there are about 100 known viruses that can infect humans. Although most viruses are self-limiting except for highly pathogenic viruses such as HIV, HBV or HCV, these viruses still pose serious harm to high-risk groups such as the elderly, children and pregnant women. Moreover, with the increase in international travel, as an infectious pathogen, once a cluster outbreak occurs, viral infection will inevitably bring huge challenges to the global public health and medical system. At present, there are only more than 80 antiviral drugs approved in the world, and about half of the antiviral drugs are developed for the treatment of AIDS and viral hepatitis, which means that most viral infections are in short supply or have no controllable drugs. EV71 is highly contagious and has caused many outbreaks and epidemics around the world, especially in the Asia-Pacific region. In recent years, there have been reports of EV71 infection in my country. EV71 infection can be asymptomatic or manifest as a self-limiting influenza-like illness. EV71 is an important pathogen that causes hand, foot and mouth disease in children. The susceptible population is mainly infants and children, especially those under 5 years old. The disease usually manifests as rashes and herpes on the hands, feet, mouth and other parts of the body, as well as systemic fever. Some children with severe HFMD will develop aseptic meningitis, brainstem encephalitis and other nervous system-related diseases, which may lead to death. The inactivated EV71 vaccine launched in my country at the end of 2015 has a preventive and protective effect against HFMD caused by EV71 infection, but it is currently not a vaccine in the immunization program, so the vaccination rate for infants and young children across the country is not high. In addition, a small number of vaccine recipients have experienced side effects such as erythema, rash, dizziness, and fever. There is also a phenomenon of developing encephalitis after vaccination with EV71 vaccine ( Human vaccines & immunotherapeutics , 2021, 17 (7): 2097-2100). Currently, most anti-EV71 drugs are still in the preclinical research stage, such as viral capsid protein VP1 inhibitors (Pleconaril and Vapendavir) or inhibitors targeting different non-structural proteins of EV71 (Quercetin, Metrifudil, Rupintrivir, DC07090). The clinical treatment of hand, foot and mouth disease mainly relies on symptomatic treatment with intravenous ribavirin, and the treatment effect is not satisfactory ( Journal of medical virology , 2019, 91 (2): 201-207). Therefore, there is no specific anti-EV71 drug available clinically.

[0003] Isoindol-1-one is a class of chemical skeletons with simple structures. Its derivatives have been reported to have a variety of biological activities such as anti-tumor, anti-bacterial, anti-inflammatory, and anti-angiogenesis, but antiviral activity has rarely been reported. Some researchers have found that isoindolinone derivatives have inhibitory activity against tobacco mosaic virus (TMV) ( Archives of pharmacal research , 2022, 45 (8): 572-583). The inventors previously confirmed that N-phenyl substituted isoindolinone compounds represented by LJ0306 have significant inhibitory activity against human EV71 virus (Molecules. 2019, 24(5), E985). In further studies, the inventors found that when the phenyl group is replaced by a saturated ring structure, it is beneficial to improve the activity and reduce cytotoxicity. The ability of N-alkyl isoindolinone derivatives to cross the cell membrane in vitro is also significantly improved compared with N-phenyl substituted isoindolinone. The compounds listed below are EV71 inhibitors reported in the literature.

[0004]

[0005] The inventors of the present invention have discovered a new class of antiviral compounds and their pharmaceutical salts by conducting medicinal chemistry research on substituted isoindolinone compounds. The research found that this class of compounds not only has significant antiviral activity, but also has the advantages of low cytotoxicity and high selectivity index, and has the potential for better oral absorption. Summary of the invention

[0006] The purpose of the present invention is to provide a novel antiviral compound and its pharmaceutically acceptable salt for use in the preparation of antiviral drugs.

[0007] In order to achieve the above object, the present invention adopts the following technical means:

[0008] The present invention provides an application of an isoindolinone compound or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug. The isoindolinone compound has a structure shown in the following general formula I:

[0009] ;

[0010] in:

[0011] R1 is amino or substituted amino or H; when R1 is substituted amino, the substituent is selected from a straight chain or branched alkyl or acyl group containing 1 to 6 carbon atoms; R2 is amino or H;

[0012] R3 is a straight chain or branched chain alkyl group containing 1 to 6 carbon atoms, benzyl, phenyl or substituted phenyl, wherein the substituent is selected from a straight chain or branched chain alkyl group containing 1 to 6 carbon atoms, halogen, and a straight chain or branched chain alkyl group containing 1 to 6 carbon atoms substituted with halogen;

[0013] R4 or R5 is a straight or branched alkyl group containing 1 to 6 carbon atoms or H, which may be the same or different;

[0014] Or R3, R4, R5 and the carbon atoms to which they are connected together form a substituted or unsubstituted cycloalkyl group containing 5-10 carbon atoms, wherein the cycloalkyl group includes a monocyclic alkyl group or a polycyclic alkyl group.

[0015] Among them, preferably, in the general formula I:

[0016] R1 is amino or H;

[0017] R2 is amino or H; R1 and R2 are not amino at the same time;

[0018] R3 is methyl, ethyl, isobutyl, phenyl, benzyl or halogen-substituted phenyl;

[0019] R4 and R5 are methyl or H, which are the same or different;

[0020] Or R3, R4, R5 and the carbon atom to which they are connected together form cyclohexyl, cyclopentyl, cycloheptyl, 4-oxocyclohexyl or adamantyl.

[0021] Wherein, preferably, the isoindolinone compound is:

[0022] 2-propylisoindol-1-one;

[0023] 2-(sec-butyl)isoindol-1-one;

[0024] 2-(4-chlorobenzyl)isoindol-1-one;

[0025] 2-Phenylethylisoindol-1-one;

[0026] 2-neopentylisoindol-1-one;

[0027] 2-Cyclohexylisoindol-1-one;

[0028] 2-Cyclopentylisoindol-1-one;

[0029] 2-Cycloheptylisoindol-1-one;

[0030] 2-(1-adamantyl)isoindol-1-one;

[0031] 2-(4-Oxocyclohexyl)isoindol-1-one;

[0032] 6-amino-2-cyclohexylisoindol-1-one;

[0033] 7-amino-2-cyclohexylisoindol-1-one.

[0034] Preferably, the medicine contains a therapeutically effective amount of one or more compounds of the present invention or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.

[0035] Preferably, the pharmaceutically acceptable salt refers to the product generated by the isoindolinone compound and acid, including inorganic acid salts and organic acid salts of isoindolinone compounds.

[0036] Among them, preferably, the inorganic acid salt of the isoindolinone compound is the hydrochloride, phosphate or sulfate of the isoindolinone compound; the organic acid salt of the isoindolinone compound is the acetate, lactate, succinate, fumarate, maleate, citrate, benzoate, methanesulfonate or p-toluenesulfonate of the isoindolinone compound.

[0037] Among them, preferably, the virus is an enterovirus.

[0038] Among them, preferably, the enterovirus is EV71 enterovirus.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention has discovered a new class of antiviral compounds and their pharmaceutical salts through the medicinal chemistry research on substituted isoindolinone compounds. The research found that the compounds not only have significant antiviral activity, but also have the advantages of low cytotoxicity and high selectivity index. The invention provides an effective technical means for antiviral treatment, especially for anti-EV71 enterovirus treatment. DETAILED DESCRIPTION

[0041] Unless otherwise defined, the terms used in this document have the following meanings:

[0042] "Substitution" may be, but is not limited to, substitution by one or more substituents selected from the group consisting of: halogen, alkoxy, hydroxy, alkyl, amino, alkylamino, aminoalkyl and phenyl. For example, "substituted benzene ring" may be, but is not limited to, a benzene ring monosubstituted with a substituent of: alkyl, alkoxy, amino, trifluoromethyl, halogen or a benzene ring polysubstituted with these substituents at different positions.

[0043] "Alkyl" can be, but is not limited to, a straight or branched chain alkyl group having 1 to 6 carbon atoms, more preferably a C1-C4 lower alkyl group, for example, methyl, ethyl, isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, etc.

[0044] "Alkoxy" can be but is not limited to an alkoxy group having 1-6 carbon atoms, more preferably a lower alkoxy group having C1-C4, for example, methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, isohexoxy, etc.

[0045] "Halo" or "halogen" may be fluorine, chlorine, bromine or iodine, more preferably bromine or iodine.

[0046] "Aminoalkyl" may be, but is not limited to, an alkyl group having 1 to 6 carbon atoms substituted with an amino group, for example, aminomethyl, aminoethyl, aminoisopropyl, amino-n-propyl, amino-n-butyl, aminoisobutyl, amino-sec-butyl, amino-tert-butyl, amino-n-pentyl, aminoisopentyl, amino-n-hexyl, aminoisohexyl, etc. More preferably, it is an amino C1-C4 alkyl group.

[0047] "Amino", "alkylamino" and "dialkylamino" refer to -NH2, -NHR and -NR2, respectively, with R being an alkyl group as defined above. The two alkyl groups attached to the nitrogen atom in the dialkyl moiety may be the same or different.

[0048] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0049] Example 1: Synthesis of 2-cyclohexylisoindol-1-one

[0050] 229 mg of methyl 2-bromomethylbenzoate was dissolved in 4 mL of acetonitrile, and 276 mg of potassium carbonate and 171 μL of cyclohexylamine were added under stirring, and the mixture was reacted at room temperature for 2 hours. After the reaction was completed by TLC monitoring, 10 mL of ethyl acetate and 5 mL of distilled water were added to the reaction system for extraction, and the organic phase was washed with saturated sodium chloride solution, dried with an appropriate amount of anhydrous magnesium sulfate, filtered, and separated by column chromatography (petroleum ether: ethyl acetate = 7:3) to obtain the product 2-cyclohexylisoindole-1-one (yield 76.1%), mass spectrometry (ESI + ): m / z=216.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.69 (d, J = 7.5 Hz, 1H), 7.60(d, J= 4.2 Hz, 2H), 7.49 (dd, J = 7.9, 4.0 Hz, 1H), 4.45 (s, 2H), 4.02 (t, J = 11.9 Hz, 1H), 1.78 (dd, J = 28.9, 11.9 Hz, 4H), 1.67 (d, J = 13.3 Hz, 1H),1.54 (q, J = 12.3, 10.7 Hz, 2H), 1.39 (q, J = 13.0 Hz, 2H), 1.16 (q, J = 12.9Hz, 1H).

[0051] The following compounds were obtained in a manner similar to Example 1:

[0052] 2-Propylisoindol-1-one, mass spectrometry (ESI + ): m / z=176.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6)δ 7.67 (d, J = 7.5 Hz, 1H), 7.59 (d, J = 4.0 Hz, 2H), 7.48 (dd, J = 7.8, 3.9Hz, 1H), 4.46 (s, 2H), 3.48 (t, J = 7.2 Hz, 2H), 1.62 (q, J = 7.3 Hz, 2H),0.87 (t, J = 7.4 Hz, 3H).

[0053] 2-(Sec-butyl)isoindol-1-one, mass spectrometry (ESI + ): m / z=190.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.91 – 7.18 (m, 4H), 4.38 (q, J = 17.7 Hz, 2H), 4.20 (s, 1H), 1.59 (s, 2H), 1.21 (s, 3H), 0.79 (s, 3H).

[0054] 2-(4-Chlorobenzyl)isoindol-1-one, mass spectrometry (ESI + ): m / z=258.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.75 (d, J = 7.5 Hz, 1H), 7.66 – 7.48 (m, 3H), 7.42 (d, J = 8.2Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 4.74 (s, 2H), 4.39 (s, 2H).

[0055] 2-Phenylethylisoindol-1-one, mass spectrometry (ESI + ): m / z=238.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.66 (d, J = 7.5 Hz, 1H), 7.57 (td, J = 8.0, 6.4 Hz, 2H), 7.47 (td, J =7.2, 1.7 Hz, 1H), 7.35 – 7.13 (m, 5H), 4.39 (s, 2H), 3.78 (t, J = 7.4 Hz,2H), 2.93 (t, J = 7.4 Hz, 2H).

[0056] 2-Neopentylisoindol-1-one, mass spectrometry (ESI + ): m / z=204.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.69 (d, J = 7.5 Hz, 1H), 7.60 (d, J = 6.8 Hz, 2H), 7.53 – 7.47 (m,1H), 4.60 (s, 2H), 3.36 (s, 2H), 0.97 (s, 9H).

[0057] 2-Cyclopentylisoindol-1-one, mass spectrometry (ESI +): m / z=202.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.66 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 4.2 Hz, 2H), 7.47 (dt, J = 8.0,4.1 Hz, 1H), 4.58 (m, 1H), 4.46 (s, 2H), 1.92 – 1.82 (m, 2H), 1.79 – 1.53 (m, 6H).

[0058] 2-Cycloheptylisoindol-1-one, mass spectrometry (ESI + ): m / z=230.2(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.66 (d, J = 7.6 Hz, 1H), 7.58 (d, J = 4.1 Hz, 2H), 7.51 – 7.44 (m,1H), 4.45 (s, 2H), 4.21 (m, 1H), 1.89 – 1.43 (m, 12H).

[0059] 2-(1-adamantyl)isoindol-1-one, mass spectrometry (ESI + ): m / z=268.2(M+H) + , 1 H NMR (500 MHz, CDCl3) δ 7.79 (d, J = 6.7 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.46 – 7.38 (m,2H), 4.47 (s, 2H), 2.31 (s, 6H), 2.17 (s, 3H), 1.75 (q, J = 11.8 Hz, 6H).

[0060] 2-(4-Oxocyclohexyl)isoindol-1-one, mass spectrometry (ESI + ): m / z=230.1(M+H) + , 1H NMR (500MHz, CDCl3) δ 7.91 – 7.83 (m, 1H), 7.56 (td, J = 7.4, 1.2 Hz, 1H), 7.52 –7.44 (m, 2H), 4.80 (m, J = 12.1, 3.8 Hz, 1H), 4.37 (s, 2H), 2.63 (td, J =14.4, 5.9 Hz, 2H), 2.51 (m, J = 15.2, 4.8, 2.3 Hz, 2H), 2.22 (m, J = 12.8,6.1, 3.2 Hz, 2H), 2.02 (m, J = 12.9, 4.4 Hz, 2H).

[0061] Example 2: Synthesis of 6-amino-2-cyclohexylisoindol-1-one

[0062] Using 2-bromomethyl-5-nitrobenzoic acid methyl ester (804 mg, 2.93 mmol) and cyclohexylamine as raw materials, the intermediate 6-nitro-2-cyclohexylisoindolin-1-one (500 mg, yellow-white solid) was obtained according to the synthetic method of compound 1, with a yield of 65.6%. Then the intermediate was dissolved in MeOH (10 mL), the air in the bottle was replaced by N2, 10% Pd / C (100.2 mg, 0.05 mmol) was added and stirred evenly, and medium-pressure hydrogenation reduction reaction was carried out for 2 hours. The reaction solution was filtered, concentrated and dried to obtain a yellow-white solid product. Mass spectrum (ESI + ): m / z=231.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.19(d, J = 8.1 Hz, 1H), 6.84 (d, J = 1.7 Hz, 1H), 6.79 (dd, J = 8.0, 1.8 Hz,1H), 5.42 (s, 2H), 4.22 (s, 2H), 3.95 (t, J = 11.8 Hz, 1H), 1.78 (d, J = 12.9Hz, 2H), 1.66 (dd, J= 30.1, 11.9 Hz, 3H), 1.54 – 1.42 (m, 2H), 1.34 (q, J =12.9 Hz, 2H), 1.13 (q, J = 12.8 Hz, 1H).

[0063] Example 3: Synthesis of 7-amino-2-cyclohexylisoindol-1-one

[0064] Using 2-bromomethyl-6-nitrobenzoic acid methyl ester and cyclohexylamine as raw materials, a white solid product was obtained according to a synthetic method similar to the above 6-amino-2-cyclohexylisoindol-1-one. Mass Spectrum (ESI + ): m / z=231.1(M+H) + , 1 H NMR (500 MHz, DMSO- d 6) δ 7.17 (t, J = 7.7 Hz, 1H), 6.61 (d, J = 7.3 Hz, 1H), 6.54(d, J = 8.1 Hz, 1H), 6.02 (s, 2H), 4.28 (s, 2H), 3.90 (t, J = 11.9 Hz, 1H),1.79 (d, J = 13.2 Hz, 2H), 1.67 (dd, J = 27.5, 12.2 Hz, 3H), 1.55 – 1.44 (m,2H), 1.35 (q, J = 13.0 Hz, 2H), 1.13 (q, J = 12.9 Hz, 1H).

[0065] Example 4: Determination of anti-EV71 activity of the compounds of the present invention

[0066] 3×10 5 Vero cells were seeded in 96-well culture plates at a density of 10 / mL and infected with enterovirus 71 type 10 after 24 hours. -4 After 2 hours of adsorption, the virus solution was discarded, and maintenance solution containing samples of different dilutions and positive control drugs was added. At the same time, cell control wells and virus control wells were set up, and cultured at 5% CO2 and 37°C. When the cytopathic effect (CPE) of the virus control group reached 4+, the cytopathic effect (CPE) of each group was observed, and the half inhibitory concentration (IC) of the sample to the virus was calculated using the Reed-Muench method. 50). The results of the anti-EV71 activity test of the synthesized compounds in the present invention in cell culture are shown in Table 1.

[0067]

[0068]

[0069] *Inhibitory concentration of the compound on EV71 virus, + indicates IC 50 Values ​​above 100 μM, ++ indicates IC 50 Values ​​are in the range of 10-100 μM, +++ indicates IC 50 Values ​​are in the range of 1-10 μM, ++++ indicates IC 50 The value is below 1μM. #: The molecular weight of all compounds in the table retains three significant figures and is rounded off.

[0070] Example 5: Evaluation of compound cell membrane permeability

[0071] Caco-2 cells were cultured at 1×10 5 / cm 2 Inoculated on a 24-well polycarbonate membrane. After 21 days of culture, the cells were washed with HBSS solution to remove interfering substances on the cell surface. After pre-incubation at 37°C for 0.5 h, the test compound (50 μM) was added to the A side of the polycarbonate membrane. 50 μL was sampled on the opposite side 120 min after addition, and LC-MS was used to detect the permeability of the compound from the A side to the B side of the polycarbonate membrane. The experiment set the hypotonic positive drug group enalapril (100 μM) group as a negative control, with 3 wells in each group. The results are shown in Table 2.

[0072] .

Claims

1. Use of an isoindolinone compound or a pharmaceutically acceptable salt thereof in the preparation of an anti-enterovirus EV71 drug, characterized in that: The isoindolinone compound has a structure shown in the following general formula I: in: R1 is amino or H; R2 is amino or H; R1 and R2 will not be amino groups at the same time; R3 is methyl, ethyl, isobutyl, phenyl, benzyl or halogen-substituted phenyl; R4 and R5 are methyl or H, which are the same or different; Or R3, R4, R5 and the carbon atom to which they are connected together form cyclohexyl, cyclopentyl, cycloheptyl, 4-oxocyclohexyl or adamantyl.

2. The use according to claim 1, characterized in that The isoindolinone compound is: 2-propylisoindol-1-one; 2-(sec-butyl)isoindol-1-one; 2-(4-Chlorobenzyl)isoindol-1-one; 2-Phenylethylisoindol-1-one; 2-neopentylisoindol-1-one; 2-Cyclohexylisoindol-1-one; 2-Cyclopentylisoindol-1-one; 2-Cycloheptylisoindol-1-one; 2-(1-adamantyl)isoindol-1-one; 2-(4-Oxocyclohexyl)isoindol-1-one; 6-amino-2-cyclohexylisoindol-1-one; 7-amino-2-cyclohexylisoindol-1-one.

3. The use according to claim 1 or 2, characterized in that: The medicine contains a therapeutically effective amount of one or more compounds as claimed in claim 1 or 2 or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.

4. The use according to claim 1, characterized in that The pharmaceutically acceptable salts refer to products generated by the isoindolinone compounds and acids, including inorganic acid salts and organic acid salts of isoindolinone compounds.

5. The use according to claim 4, characterized in that The inorganic acid salt of the isoindolinone compound is the hydrochloride, phosphate or sulfate of the isoindolinone compound; the organic acid salt of the isoindolinone compound is the acetate, lactate, succinate, fumarate, maleate, citrate, benzoate, methanesulfonate or p-toluenesulfonate of the isoindolinone compound.

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