Thiophene compounds, their preparation and use as enterovirus inhibitors
By synthesizing thiophene compounds, the lack of enterovirus inhibitors in existing technologies has been solved, achieving effective inhibition of viruses such as EV71 with less cytotoxicity, and providing a new treatment approach.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-02-26
- Publication Date
- 2026-03-31
AI Technical Summary
Currently, there is a lack of effective enterovirus inhibitors, especially for EV71 enterovirus, resulting in a lack of treatment options for diseases such as hand-foot-mouth disease. Furthermore, existing drugs lack good inhibitory activity and have low cytotoxicity.
Thiophene compounds were designed and synthesized, and novel compounds were formed by linking amide groups with para-substituted phenyl groups and 3,5-substituted benzyl groups. These compounds exhibited good inhibitory activity against various enteroviruses such as EV71 and low cytotoxicity.
The compound exhibited good inhibitory activity against enteroviruses such as EV71, EVD68, CVA16, CVB1, and Echov-6, with EC50 ranging from 0.1 to 5.0 μM and CC50 greater than 50 μM, significantly improving the survival rate of mice infected with enteroviruses.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of medicinal chemistry, specifically relating to a thiophene compound, its preparation, and its application as an enterovirus inhibitor. Background Technology
[0002] Enteroviruses (EVs) are a class of pathogens that invade and multiply within intestinal cells, inducing various types of inflammation in human intestinal epithelial cells. In 1970, the International Committee on Virus Nomenclature classified them as belonging to the genus Enterovirus within the family Picornaviridae, and named them according to their ordinal numbers, such as enterovirus types 68, 69, 70, 71, and 72.
[0003] in,
[0004] Enterovirus 71 (EV71) is a non-enveloped single-stranded RNA virus that was first isolated from the feces of an infant with encephalitis in 1969 and is the main causative agent of hand-foot-mouth disease.
[0005] This virus is an acute infectious disease that primarily causes fever and rashes and blisters on the hands, feet, and mouth in infants and young children. Furthermore, it can also lead to serious neurological disorders such as encephalitis and meningitis, posing a serious threat to the life and health of newborns.
[0006] EV71 infection is a global infectious disease, with outbreaks and epidemics reported in most parts of the world, drawing widespread attention and vigilance from countries around the globe. Unfortunately, however, there is still a lack of effective drugs to treat hand-foot-and-mouth disease. Therefore, the design and synthesis of a series of novel and effective EV71 inhibitors is particularly urgent.
[0007] In view of this, the present invention is hereby proposed. Summary of the Invention
[0008] To address the problems and / or shortcomings of existing technologies, the present invention aims to provide a thiophene compound, its preparation, and its application as an enterovirus inhibitor. This type of compound comprises a thiophene compound linked to a para-substituted phenyl group and a benzyl group substituted at the 3,5-position via an amide group. Its novel and unique structure exhibits good inhibitory activity against various enteroviruses such as EV71 and EVD68, with relatively low cytotoxicity, providing a new potential option for pharmaceutical treatment of enterovirus infections.
[0009] This invention provides a compound of Formula I or a pharmaceutically acceptable salt, crystal form, or solvate thereof;
[0010]
[0011] R1, R2, R3, and R4 are independently selected from halogens, C1-C4 alkyl groups, or halogen-substituted C1-C4 alkyl groups.
[0012] Furthermore,
[0013] In any of the above technical solutions (compound or its salt, crystal form, solvate), R1 is a halogen; R2 is a halogen or a C1-C4 alkyl group; R3 and R4 are independently selected from halogens, C1-C4 alkyl groups or halogen-substituted C1-C4 alkyl groups; preferably, the halogen is selected from fluorine, chlorine or bromine, and / or the C1-C4 alkyl group is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.
[0014] Furthermore,
[0015] In any of the above technical solutions (compound or its salt, crystal form, solvate), R1 is fluorine, chlorine or bromine; R2 is chlorine or methyl; R3 and R4 are independently selected from fluorine, chlorine, bromine, methyl, tert-butyl, fluorine-substituted methyl or fluorine-substituted tert-butyl.
[0016] Furthermore,
[0017] In any of the above technical solutions (compound or its salt, crystal form, solvate), the compound represented by Formula I is:
[0018] The present invention also provides a method for preparing the compound represented by Formula I, which includes the following steps:
[0019] i
[0020]
[0021] In the presence of a haloalkanes solvent and an organic amine, compound 5 undergoes a condensation reaction with compound 4 to remove HCl, producing the compound shown in formula I.
[0022] ii、
[0023]
[0024] Compound 1 and Compound 2 undergo a condensation reaction in an alcohol solvent to remove H2O, generating Compound 3, which is then reduced by MBH4 to generate Compound 4.
[0025] Among them, R1, R2, R3, and R4 are defined as described in any of the preceding items, and M is an alkali metal.
[0026] Furthermore,
[0027] In any of the above technical solutions (the preparation method of the compound shown in Formula I), in step i,
[0028] The molar ratio between compound 4 and compound 5 is 1:0.9 to 1.5, preferably 1:1.0 to 1.2;
[0029] And / or,
[0030] The amount of alcohol solvent used per gram of compound 4 is 10-80 mL, preferably 45-60 mL;
[0031] And / or,
[0032] The molar ratio between compound 4 and the organic amine is 1:0.9 to 1.5, preferably 1:1.0 to 1.4;
[0033] And / or,
[0034] The reaction temperature for the condensation and removal of HCl is 10–40°C, preferably 10–30°C;
[0035] And / or,
[0036] The reaction time for condensation to remove HCl is 3 to 24 hours, preferably 5 to 12 hours.
[0037] Furthermore,
[0038] In any of the above technical solutions (the method for preparing the compound shown in Formula I), in step ii,
[0039] The molar ratio between compound 1 and compound 2 is 1:0.9 to 1.5, preferably 1:0.9 to 1.2;
[0040] And / or,
[0041] The amount of alcohol solvent used per gram of compound 1 is 10-80 mL, preferably 45-60 mL;
[0042] And / or,
[0043] The molar ratio between compound 1 and MBH4 is 1:0.9 to 1.5, preferably 1:1.2 to 1.5;
[0044] And / or,
[0045] The reaction temperature for condensation to remove H2O is 10–40°C, preferably 10–30°C;
[0046] And / or,
[0047] The reaction time for condensation to remove H2O is 3 to 24 hours, preferably 5 to 20 hours;
[0048] And / or,
[0049] The reaction temperature for MBH4 reduction is 10–40°C, preferably 10–30°C;
[0050] And / or,
[0051] The reaction time for MBH4 reduction is 1 to 12 hours, preferably 2.5 to 5 hours.
[0052] Furthermore,
[0053] In any of the above technical solutions (the method for preparing the compound shown in Formula I),
[0054] The halocarbon solvent mentioned is dichloromethane;
[0055] And / or,
[0056] The organic amine mentioned is triethylamine;
[0057] And / or,
[0058] The alcohol solvent is methanol or ethanol;
[0059] And / or,
[0060] The MBH4 mentioned is sodium borohydride or potassium borohydride.
[0061] also,
[0062] The present invention also provides the use of the compound represented by Formula I above, or a pharmaceutically acceptable salt, crystal form, or solvate thereof, in the preparation of enterovirus inhibitors; for example, the enterovirus is EV71, EVD68, CVA16, CVB1, or Echov-6.
[0063] The present invention also provides a pharmaceutical composition for treating and / or preventing enterovirus infections:
[0064] It is a preparation made with the compound of Formula I as described in any of the preceding items, or its pharmaceutically acceptable salt, crystal form, or solvate, as the active ingredient, plus commonly used pharmaceutical excipients; for example, the enterovirus infection disease mentioned is hand-foot-mouth disease.
[0065] The compounds of this invention have a fundamental core component: a thiophene group linked to a para-substituted phenyl group and a 3,5-substituted benzyl group, resulting in a novel and unique structure. In vitro cell experiments show that the compounds of this invention exhibit good inhibitory activity against enteroviruses such as EV71, EVD68, CVA16, CVB1, and Echov-6. 50 Between 0.1 and 5.0 μM, and with relatively low cytotoxicity, CC 50Greater than 50 μM. In vivo bioactivity assays showed that the compounds of this invention can effectively improve the survival rate of mice infected with enteroviruses, further demonstrating the inhibitory effect and / or significant therapeutic effect of the compounds on enteroviruses, providing a new potential option for pharmaceutical treatment of enterovirus infections.
[0066] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention. Detailed Implementation
[0067] The present invention will now be clearly and completely described in conjunction with specific embodiments. Those skilled in the art will understand that the embodiments described below are some, but not all, embodiments of the present invention, and are only used to illustrate the present invention, and should not be regarded as a limitation on the scope of protection of the present invention.
[0068] In this invention, unless otherwise specified, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.
[0069] Regarding the definitions of terms used in this invention, unless otherwise stated, the initial definitions provided herein apply to the term throughout the text; for terms not specifically defined herein, the meanings that a person skilled in the art would give them should be given based on the disclosure and / or context.
[0070] C a ~C b Alkyl refers to any alkyl group containing a to b carbon atoms. For example, C1 to C3 alkyl refers to alkyl groups containing 1 to 3 carbon atoms, such as methyl, ethyl, propyl, etc.
[0071] The term "pharmaceutically acceptable" generally refers to a drug that is chemically or physically compatible with other components that make up a drug dosage form and physiologically compatible with receptors.
[0072] The term "salt" refers to the above-mentioned compound or its isomers, which form acidic and / or basic salts with inorganic and / or organic acids and bases. It also includes zwitterionic salts and quaternary ammonium salts, such as alkyl ammonium salts.
[0073] The term "solvate" refers to a compound formed by one or more solvent molecules with the compound of this invention. Solvents that form solvates include, but are not limited to, water, methanol, ethanol, aminoethanol, isopropanol, dimethyl sulfoxide, acetic acid, ethyl acetate, etc.
[0074] Example 1
[0075] 1. Preparation of compound B
[0076]
[0077] 12 g (approximately 0.11 mol) of p-methylaniline and 19.5 g (approximately 0.11 mol) of 3,5-dichlorobenzaldehyde were added to 650 mL of ethanol and stirred at room temperature for 15 h (condensation reaction) to generate compound A (intermediate). Without separation, 5.8 g (approximately 0.15 mol) of sodium borohydride was added in batches or gradually, and the reaction was continued at room temperature for 3 h (reduction reaction) to generate compound B. The mixture was concentrated, the solid precipitated was filtered, and recrystallized from ethanol or ethanol-petroleum ether (volume ratio 2:1). After drying, compound B was given as a white or pale yellow solid with a yield of 65.8%.
[0078] Compound B: C 14 H 13 Cl2N, m / z: 265.0422; melting point: 176~178℃.
[0079] NMR data for compound B: 1 H NMR (DMSO-d6): δ7.58(s,H),7.35(s,H),7.31(d,2H),7.09(d,2H),6.47(d,2H),4.32(d,2H),2.32(m,3H); 13 C NMR (DMSO-d6): δ146.3, 144.4, 135.5, 129.6, 128.4, 127.2, 113.4, 47.0, 21.3.
[0080] 2. Preparation of compound C
[0081]
[0082] 20 g (approximately 0.11 mol) of 5-chloro-2-thiophenecarboxyl chloride, 27 g (approximately 0.1 mol) of compound B, and 12.5 g (approximately 0.12 mol) of triethylamine were added to 1500 mL of dichloromethane. The mixture was stirred at 15 °C for 8 h. After the reaction was completed by HPLC or TLC, the mixture was concentrated and purified by silica gel column chromatography (mobile phase: petroleum ether-ethyl acetate = 1:2, v / v) to give compound C, a white or pale yellow solid, with a yield of 45.3%.
[0083] Compound C:C 19 H 14 Cl3NOS, m / z: 408.9857.
[0084] NMR data for compound C: 1H NMR (DMSO-d6): δ8.08(s,H),7.60(s,H),7.32(m,4H),7.14(d,2H),5.40(d,2H),2.31(m,3H); 13 C NMR (DMSO-d6): δ163.7,144.4,140.7,138.3,136.8,136.1,135.9,135.5,133.4,129.8,129.2,128.4,127.2,48.4,21.3.
[0085] Example 2
[0086] 1. Preparation of compound D
[0087]
[0088] Compound D was prepared from p-chloroaniline and 3,5-dimethylbenzaldehyde using a method similar to that in Example 1, with a yield of 64.5%. Compound D: C 15 H 16 ClN, m / z: 245.0965.
[0089] 2. Preparation of compound E
[0090]
[0091] Compound E was prepared from 5-chloro-2-thiophenecarboxyl chloride and compound D using a method similar to that in Example 1, with a yield of 39.8%. Compound E: C 20 H 17 Cl2NOS, m / z: 389.0406.
[0092] Example 3
[0093]
[0094] Following a method similar to that in Example 1, compound F was prepared from p-chloroaniline and 3,5-dichlorobenzaldehyde as raw materials; then, compound G was prepared from 5-chloro-2-thiophenecarboxyl chloride and compound F as raw materials.
[0095] Example 4
[0096]
[0097] Following a method similar to that in Example 1, compound H was prepared from p-chloroaniline and 3,5-bis(trifluoromethyl)benzaldehyde; then, compound I was prepared from 5-chloro-2-thiophenecarboxyl chloride and compound H.
[0098] Example 5
[0099]
[0100] Following a method similar to that in Example 1, compound J was prepared from p-methylaniline and 3,5-bis(tert-butyl)benzaldehyde; then, compound K was prepared from 5-bromo-2-thiophenecarboxyl chloride and compound J.
[0101] Example 6
[0102]
[0103] Following a method similar to that in Example 1, compound L was prepared from p-chloroaniline and 3-chloro-5-(trifluoromethyl)benzaldehyde; then, compound M was prepared from 5-bromo-2-thiophenecarboxyl chloride and compound L.
[0104] Example 7
[0105]
[0106] Following a method similar to that in Example 1, compound N was prepared from p-chloroaniline and 3-bromo-5-chlorobenzaldehyde; then, compound O was prepared from 5-bromo-2-thiophenecarboxyl chloride and compound N.
[0107] Example 8
[0108] 1. Cytotoxicity test
[0109] The Cell Counting Kit-8 (CCK-8) is a rapid and highly sensitive assay kit widely used for detecting cell proliferation and cytotoxicity. Its working principle is as follows: in the presence of an electron-coupling reagent, it is reduced by mitochondrial dehydrogenases to generate a highly water-soluble, orange-yellow formazan product. The intensity of the color is directly proportional to cell proliferation and inversely proportional to cytotoxicity. The OD value is measured using a microplate reader to indirectly reflect the number of viable cells.
[0110] During the experiment, different dilutions of the drug were added to 96-well plates filled with RD cells after 24 hours. After incubation at 37°C for 8–24 hours, 20 μL of MTS / PMS mixed solution was added, and the plates were incubated at 37°C for another 4 hours. The OD value at 450 nm was then measured using a microplate reader.
[0111] The inhibition rate (%) of the compound = [1 - (EN) / (PN)] × 100, where "E" represents the OD value of the experimental group, "P" represents the OD value of the positive control group, and "N" represents the OD value of the negative control group. (Using CC...) 50(half-cytotoxic concentration) is used as an indicator for evaluating the cytotoxicity of compounds.
[0112] 2. Anti-EV71 activity test
[0113] After EV71 virus infects Vero, RD and other cells, it causes cytopathic effects after a certain period of time. The degree of cytopathic effects can reflect the inhibitory level of the medicinal compound on the virus.
[0114] In the experiment, a mixture of EV71 virus and different dilutions of the drug was added to 96-well plates filled with RD cells, with an area ratio of 80% and 8 replicates per dilution. The plates were incubated at 37°C for 24–48 hours. After the virus control group cells showed complete cytopathic effect, 20 μL of MTS / PMS mixed solution was added, and the plates were incubated at 37°C for another 4 hours. The OD value at 490 nm was then measured using a microplate reader.
[0115] The inhibition rate (%) of the compound = [1 - (EN) / (PN)] × 100, where "E" represents the OD value of the experimental group, "P" represents the OD value of the positive control group, and "N" represents the OD value of the negative control group. Using EC 50 (half-maximal effective concentration) is used as an indicator for evaluating antiviral activity.
[0116] Using enviroxime as a control, cytotoxicity tests and anti-EV71 activity tests were conducted on compounds C, E, G, I, K, M, and O prepared in the aforementioned examples, and the selectivity index (SI) of the compounds was calculated. The results are shown in Table 1.
[0117] CC 50 The half-maximal cytotoxicity (MCC) concentration; a higher value indicates lower cytotoxicity.
[0118] EC 50 The half-maximal effective concentration (WMC) indicates that the lower the value, the better the inhibitory effect on the virus.
[0119] SI: for CC 50 With EC 50 The higher the ratio, the higher the safety and / or likelihood of the drug.
[0120] Table 1. Cytotoxicity and anti-EV71 activity results of the compounds synthesized in this invention.
[0121] test subjects <![CDATA[CC 50 (μM)]]> <![CDATA[EC 50 (μM)]]> SI Compound C 158.470 0.204 776.8 Compound E 134.126 0.225 596.1 Compound G 125.395 0.308 407.1 Compound I 147.802 0.257 575.1 compound K 160.228 0.120 1335.2 Compound M 152.304 0.262 581.3 Compound O 130.496 0.285 457.9 Envelopime 28.0 0.140 200
[0122] also,
[0123] The study also found that the aforementioned compounds of the present invention also exhibit good inhibitory activity against enteroviruses such as EVD68, CVA16, CVB1, and Echov-6. 50 Between 0.1 and 5.0 μM, CC 50 Greater than 50 μM.
[0124] Example 9
[0125] In vivo bioactivity testing:
[0126] Experimental groups: blank control group, virus control group, compound C treatment group, compound E treatment group, compound K treatment group, and enrofloxacin treatment group. EV-A71 GZ-CII was administered at a rate of 1.2 × 10⁻⁶. 8 Mice were infected with PFU via intraperitoneal injection at a lethal dose. Ten mice were used in each group, and the average of two administrations was taken. Six hours before challenge, compounds C, E, and K were administered orally at doses of 0.05 mg / kg / day and 0.2 mg / kg / day, respectively, while enviroxime was administered orally at a dose of 0.2 mg / kg. Administration was twice daily for 14 consecutive days. Mouse weight changes, morbidity, and mortality were monitored daily. Results are shown in Table 2.
[0127] Table 2. Results of in vivo anti-EV71 virus activity of the compounds synthesized in this invention.
[0128]
[0129] The experimental results showed that all mice in the virus control group died. The survival rates of mice were 55% and 100% when compound C was administered at doses of 0.05 mg / kg / d and 0.2 mg / kg / d, respectively; the survival rates of mice were 50% and 100% when compound E was administered at doses of 0.05 mg / kg / d and 0.2 mg / kg / d, respectively; and the survival rates of mice were 60% and 100% when compound K was administered at doses of 0.05 mg / kg / d and 0.2 mg / kg / d, respectively. All of these results were superior to those of enviroxime.
Claims
1. A compound represented by Formula I or a pharmaceutically acceptable salt thereof; wherein, R1, R2, R3 and R4 are as defined in claim 1, and M is an alkali metal. characterized in that The compound is selected from the following structural formula:
2. A process for the preparation of a compound of formula I, characterized in that, It comprises the following steps: i、 Compound 5 is condensed with compound 4 in a halogenated hydrocarbon solvent and in the presence of an organic amine to remove HCl, thereby generating a compound represented by Formula I; ii, Compound 1 is condensed with compound 2 in an alcohol solvent to remove H2O, thereby generating compound 3, which is then reduced by MBH4 to generate compound 4; wherein, R1, R2, R3 and R4 are as defined in claim 1, and M is an alkali metal.
3. The preparation method according to claim 2, characterized in that, In step i, the molar ratio between compound 4 and compound 5 is 1:0.9-1.5; and / or, the amount of alcohol solvent corresponding to each gram of compound 4 is 10-80 mL; and / or, the molar ratio between compound 4 and the organic amine is 1:0.9-1.5; and / or, the reaction temperature for removing HCl is 10-40℃; and / or, the reaction time for removing HCl is 3-24 h.
4. The production method according to claim 3, characterized by, In step i, the molar ratio between compound 4 and compound 5 is 1:1.0-1.2; and / or, the amount of alcohol solvent corresponding to each gram of compound 4 is 45-60 mL; and / or, the molar ratio between compound 4 and the organic amine is 1:1.0-1.4; and / or, the reaction temperature for removing HCl is 10-30℃; and / or, the reaction time for removing HCl is 5-12 h.
5. The preparation method according to claim 2, characterized in that, In step ii, the molar ratio between compound 1 and compound 2 is 1:0.9-1.5; and / or, the amount of alcohol solvent corresponding to each gram of compound 1 is 10-80 mL; and / or, the molar ratio between compound 1 and MBH4 is 1:0.9-1.5; and / or, the reaction temperature for removing H2O is 10-40℃; and / or, the reaction time for removing H2O is 3-24 h; and / or, the reaction temperature for MBH4 reduction is 10-40℃; and / or, the reaction time for MBH4 reduction is 1-12 h.
6. The production method according to claim 5, wherein In step ii, the molar ratio between compound 1 and compound 2 is 1:0.9-1.2; and / or, the amount of alcohol solvent corresponding to each gram of compound 1 is 45-60 mL; and / or, the molar ratio between compound 1 and MBH4 is 1:1.2-1.5; and / or, the reaction temperature for removing H2O is 10-30℃; and / or, the reaction time for removing H2O is 5-20 h; and / or, the reaction temperature for MBH4 reduction is 10-30℃; and / or, the reaction time for MBH4 reduction is 2.5-5 h.
7. The preparation method according to any one of claims 2-6, characterized in that, the halogenated hydrocarbon solvent is dichloromethane; and / or, the organic amine is triethylamine; and / or, the alcohol solvent is methanol or ethanol; and / or, the MBH4 is sodium borohydride or potassium borohydride.
8. Use of the compound represented by Formula I or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an enterovirus inhibitor.
9. Use of a compound of Formula I according to claim 8 or a pharmaceutically acceptable salt thereof for the preparation of an enterovirus inhibitor, characterized in that, The enterovirus is EV71, EVD68, CVA16, CVB1 or Echov-6.
10. A pharmaceutical composition for treating and / or preventing an enterovirus infection disease, characterized by, It is a preparation of the compound shown in formula I of claim 1 or its pharmaceutically acceptable salt as active ingredient, plus the usual adjuvant of pharmacy.
11. The pharmaceutical composition for treating and / or preventing an enterovirus infection disease according to claim 10, characterized in that, The enterovirus infection disease is hand-foot-mouth disease.