A 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidin-5(4H)-one compound, and a preparation method and use thereof

Abstract

The application discloses a 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-ketone compound and a preparation method and application thereof. The anti-convulsion activity of the compound is evaluated by adopting a subcutaneous pentylenetetrazol (Sc-PTZ) and a mouse maximum electroshock (MES) model, and the neurotoxicity of the compound is evaluated by adopting a rotating rod method; experimental data show that the target compound with the structure of formula I provided by the application has clear and strong anti-convulsion activity and extremely low neurotoxicity, and the conclusion proves that the compound with the structure of formula I has the possibility of being developed into a new type of epilepsy treatment drug with low neurotoxicity.

Description

Technical Field

[0001] This invention relates to a 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound, its preparation method, and its uses. Background Technology

[0002] Epilepsy is a clinical syndrome caused by recurrent abnormal electrical discharges in the brain's nerve cells, leading to brain dysfunction. It typically begins in childhood and persists throughout life. It affects approximately 1% of the global population, with about 10 million people in my country and approximately 400,000 new cases each year. Epilepsy not only causes suffering for patients and their families but also places a significant burden on society. Currently, epilepsy treatment primarily relies on antiepileptic drugs. Although antiepileptic drugs have evolved from first-generation to third-generation drugs over the past few decades, currently used antiepileptic drugs only control seizures in about 70% of patients. Furthermore, many patients experience neurogenic side effects associated with existing drugs, including sedation and cognitive impairment. Therefore, developing novel structural molecules with low neurotoxicity and antiepileptic activity remains an urgent need for epilepsy treatment. Our team's authorized Chinese patent (CN201611115587.6) reports the preparation of [1,2,4]-triazolo[1,5-a]pyrimidine-7-one heterocyclic compounds and their potential applications in epilepsy treatment. Further in-depth research based on this patent led to the filing of a Chinese patent (CN201910571753.0) and the publication of research papers in the *European Journal of Medicinal Chemistry*, 2020, 185, 11824; and *Bioorganic Chemistry*, 2022, 19, 105565. While these compounds exhibit some anticonvulsant activity, they still suffer from significant side effects such as sedation and motor incoordination. Summary of the Invention

[0003] To address the sedative and ataxia-like toxic side effects of existing [1,2,4]-triazolo[1,5-a]pyrimidine-7-one heterocyclic compounds, this invention provides a novel 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound and its preparation method. This compound exhibits good antiepileptic activity and virtually no sedative or ataxia-like side effects.

[0004] To solve the above problems, the present invention adopts the following technical solution:

[0005] The present invention provides a novel 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound, characterized in that the compound and its pharmaceutically acceptable salt have the structure shown in formula (I).

[0006]

[0007] Secondly, the present invention also provides a pharmaceutical composition containing the above-mentioned 4-butyl-2,7-bis(2-fluorophenyl)-triazolidine-5(4H)-one compound, wherein the compound (I) is the active ingredient, and simultaneously contains one or more pharmaceutical carriers or one or more known pharmaceutical diluents.

[0008] Furthermore, the present invention provides the use of the above-described 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one or pharmaceutical composition in the treatment and / or prevention of various epileptic seizures, neurological diseases, etc. in animals (including humans).

[0009] This invention provides a method for preparing the above-mentioned 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound, comprising the following steps:

[0010] S1: Malonate derivative 1 and derivative 2 cyclize under the catalysis of an acidic catalyst to generate derivative 3.

[0011] S2: Derivative 3 undergoes a substitution reaction with halobutane, n-butylmethane sulfonate or n-butyl-p-toluene sulfonate under the action of a base to generate 4-butyl-2,7-bis(2-fluorophenyl)triazolopyrimidine-5(4H)-one (I).

[0012] Furthermore, in the cyclization reaction in step S1, the acidic catalyst used is selected from formic acid, acetic acid, etc., with acetic acid being preferred.

[0013] In the substitution reaction in step S2, the base used is selected from cesium carbonate, sodium carbonate, potassium carbonate, sodium tert-butoxide, potassium tert-butoxide, sodium amino, potassium amino, potassium hydroxide, sodium hydride, etc., with sodium hydride being preferred.

[0014] The solvent used in the condensation reaction in step S2 is selected from aprotic solvents such as DMF, THF, CH3CN, and DMSO, with DMF and THF being preferred.

[0015] The chemical reaction formula for preparing the 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound provided by this invention is as follows:

[0016]

[0017] The present invention provides a 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound, namely the compound of formula (I) or a pharmaceutical composition, which has excellent therapeutic effects in treating and / or preventing various epileptic seizures, neurological diseases, etc. in animals (including humans).

[0018] The 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound provided by this invention contains the essential elements required for drug production in its structure: a hydrogen-bonded domain (HBD), a hydrophobic domain (A), and an electron-donating moiety (D). Compound I is obtained by introducing a specific o-fluorophenyl group and a suitable length of n-butyl carbon chain into the 2,7 positions and nitrogen atom of the triazolopyrimidine-5(4H)-one skeleton. This specific combination ensures that the obtained molecule interacts well with the target to produce specific activity, while also possessing blood-brain barrier crossing ability and good pharmacokinetic properties to ensure efficient transport to the target in vivo. Compound I exhibits good anti-epileptic activity in mouse models and shows no central nervous system-related toxic side effects such as sedation or motor instability at saturated solubility.

[0019] Its anticonvulsant activity was determined using subcutaneous pentylenetetrazol (Sc-PTZ) and a mouse model of maximum electrical seizures (MES), and its neurotoxicity was determined using the rotating bar method. Pharmacological data showed that this compound had ED. 50 The value was 9.17 mg / kg, TD 50 With a concentration >813.40 mg / kg, the anticonvulsant activity is significantly superior (and its toxicity is significantly lower) than the positive control drugs phenytoin sodium, valproate, and carbamazepine, and the compound does not exhibit central nervous system-related neurotoxicity such as sedation or motor incoordination at maximum solubility. Based on the above technical solutions, the 4-butyl-2,7-bis(2-fluorophenyl)-triazolidine-5(4H)-one compound provided by this invention has good clinical application prospects in the treatment and / or prevention of various epileptic seizures and neurological diseases in animals (including humans). Detailed Implementation

[0020] This invention discloses the bioactivity of the compound 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one and its preparation method. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0021] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0022] All the raw material compounds described in this invention can be purchased commercially, and the compound shown in formula (I) can be synthesized using conventional compound synthesis methods according to the following synthetic route.

[0023] Example 1

[0024] o-Fluorobenzaldehyde, dimethyl malonate (1-1.5 equivalents), piperidine (0.02-0.1 equivalents), acetic acid (0.1-0.5 equivalents), and toluene solvent were added to a reaction flask, and the mixture was refluxed for 5-6 hours. After the reaction was completed, the product was concentrated under reduced pressure to obtain derivative 1. Derivative 1 and 3-amino-1,2,4-triazole derivative 2 were refluxed in glacial acetic acid for 20 hours, and then separated to obtain pure derivative 3 in 42% yield.

[0025]

[0026] Example 2

[0027] Sodium hydrogen (1.5-3 equivalents) was added to derivative 3 and solvent N,N-dimethylformamide. After stirring at room temperature for 15-30 minutes, bromobutane, butyl methanesulfonate, or butyl p-toluenesulfonate was added. The reaction solution was reacted at 80°C for 5-6 hours and then separated to obtain a white solid 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound (I) with a yield of 75-78%.

[0028] Using the same alkylation method, I analogs without carbon chains were prepared (their structures are shown in Table 2).

[0029]

[0030] The structure of the obtained compound was determined by 1 Confirmed by 1H NMR and ESI-MS, the 1H NMR and mass spectrometry data are shown in Table 1.

[0031] Table 1: NMR and mass spectrometry characterization data of compound I

[0032]

[0033] Example 3

[0034] The antiepileptic activity and toxicity of 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one and its series derivatives prepared in Example 2 were tested. PTZ and MES mouse models are the most commonly used animal models to evaluate the antiepileptic activity of the compounds.

[0035] 3.1 Animal preparation and compound formulation

[0036] Animal husbandry: Kunming white mice (20±2g each), half male and half female, were used. Before the experiment, the male and female mice were housed in separate cages with free access to food and water. The room was well-ventilated, kept at a constant temperature (22-25℃), and the relative humidity was controlled (around 50%). Normal room lighting was provided (12 hours of bright light, 12 hours of dark light). Mice were allowed free access to food particles (4% mineral mixture, 10% corn oil, 20% sucrose, 0.2% cellulose, 10.5% casein (95% pure), 54.3% starch, and water) to help them quickly become familiar with the experimental environment. Animal husbandry followed optimal standards to avoid unnecessary interference with the animals. The screening dose for the initial screening of the compounds' antiepileptic activity was 30mg / kg. All compounds and positive control drugs were dissolved in dimethyl sulfoxide (DMSO) during preparation, with an injection volume of 0.1mL / 20g. In the Sc-PTZ model, the screening dose of pentylenetetrazol was 85 mg / kg, dissolved in 0.9% physiological saline, and the injection volume was 0.2 mL / 20 g.

[0037] 3.2 Subcutaneous pentylenetetrazol (Sc-PTZ) test

[0038] The ability of the test compound to block pentylenetetrazole-induced clonic seizures or tonic seizures induced by electric shock can be interpreted as a measure of anticonvulsant efficacy. Mice were injected intraperitoneally with a prepared compound solution (30 mg / kg), followed 30 minutes later by subcutaneous injection of pentylenetetrazole (85 mg / kg). Mice were observed for the next 30 minutes, with the number of clonic seizures (ranging from violent shaking or trembling of the limbs) and the number of tonic seizures (limbs pulling or pushing towards the body or stiffly, usually with maximum extension of the hind legs) used as criteria for determining the duration of the seizure.

[0039] 3.3 Maximum Electroconvulsive Seizure (MES) Experiment

[0040] The JTC-1 type convulsion and pain sensory stimulator was used for stimulation output. The voltage was set to 80V, with a single stimulus of 0.3s. The ears and jaws of the mice were moistened with 0.9% saline solution and clamped with alligator clips. The mice were suspended and relaxed before stimulation was administered. Mice exhibiting hind leg rigidity were suitable for the experiment. The test compound was administered intraperitoneally at a dose of 30mg / kg. The absence of significant hind limb rigidity and extension indicated that the test compound possessed anti-maximal electroconvulsive activity.

[0041] The measured data are shown in Table 2, which lists the activity evaluation results of this type of compound with different alkyl chain lengths in Sc-PTZ and MES animal models.

[0042] Table 2: Preliminary activity evaluation of compounds obtained from different alkyl groups in Sc-PTZ and MES models.

[0043]

[0044]

[0045] As can be seen from the initial screening results of antiepileptic drugs in Table 2, compounds with n-butyl groups attached to the N atom exhibited the best antiepileptic activity in the PTZ mouse model. Shortening or increasing the carbon chain length led to a significant decrease in activity, and the activity changes were not obvious.

[0046] 3.4 Quantitative testing of activity and toxicity

[0047] Anticonvulsant activity at half the effective dose (ED) 50 Neurotoxicity is indicated by the median toxic dose (TD). 50 )express.

[0048] Compounds that showed 3 / 3 protection in mice during the initial screening for antiepileptic activity were subjected to quantitative activity testing. The quantitative neurotoxicity of the active compounds was determined using a rotundus fatigue tester (6 r / min). Before the test, mice were trained to remain on the rotundus for one minute. The trained mice were then randomly divided into groups of five. Thirty minutes after intraperitoneal injection of the target compound, the mice were placed on the rotundus; those that could remain on the rotundus for one minute without falling were considered effective. Both the quantitative activity test and the neurotoxicity test used a modified Kohl's method.

[0049] The protection index is considered an indicator of the safety and tolerability between the dose of an anticonvulsant drug and the dose of a compound that produces acute adverse reactions (such as sedation, motor incoordination, ataxia, or other neurotoxic manifestations); a higher value indicates greater safety of the compound. This is achieved by measuring the TD (total dose) in the trial. 50 Value divided by the corresponding ED 50 The value is used to calculate the protection index of the compound under study.

[0050] The compounds that showed 3 / 3 protection in mice in the initial screening were subjected to activity quantification, i.e., the median effective dose (ED50) test. 50 The neurotoxicity of the active compound, i.e., the median toxic dose (TD), was determined using a rotator-type fatigue analyzer. 50 The test data are shown in Table 3, which lists the activity and neurotoxicity test results of the target compound, the compound in the comparative document, and the positive control drug.

[0051] Table 3. Efficacy of target compound, compound in comparative file, and positive control drug. 50 TD 50 and PI value

[0052]

[0053] The comparison of activity and neurotoxicity results presented in Table 3 shows that, in the PTZ mouse model, the target compound exhibited better antiepileptic activity than the compound in the comparative file and the positive control. In addition to its good activity, the outstanding advantage of the target compound is its significantly lower neurotoxicity compared to the compound in the comparative file and the positive control. It showed no neurotoxicity at saturated solubility, and its protection index in animals was greater than 88.7, far exceeding that of the compound and the positive control reported in the comparative file. Its excellent antiepileptic activity and extremely low neurotoxicity make it a promising candidate for clinical application.

[0054] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidin-5(4H)-one compound characterized by, The compound and its pharmaceutically acceptable salt have the structure shown in formula (I), 2. A pharmaceutical composition, characterized by, The compound of claim 1 is used as an active ingredient, and one or more pharmaceutically acceptable carriers or one or more known pharmaceutically acceptable diluents are contained.

3. Use of the compound of claim 1 or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating or preventing convulsion or epilepsy.

4. A process for the preparation of 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidin-5(4H)-one (I) according to claim 1, characterized in that, The method comprises the following steps: S1: malonic acid ester derivative 1 is cyclized with compound 2 under the catalysis of an acidic catalyst to generate compound 3; S2: compound 3 is subjected to substitution reaction with n-butyl methane sulfonate or n-butyl p-toluenesulfonate under the action of a base to generate 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-ketone (I); 。 5. The preparation method as described in claim 4, characterized in that, In the cyclization reaction in step S1, the acidic catalyst used is selected from one of formic acid and acetic acid.

6. The method of claim 4, wherein the step of forming the first and second layers is performed by a method comprising: In the substitution reaction in step S2, the base used is selected from one of cesium carbonate, sodium carbonate, potassium carbonate, sodium tert-butoxide, potassium tert-butoxide, sodium amide, potassium amide, potassium hydroxide and sodium hydride.

7. The method of claim 4, wherein the step of forming the first and second layers is performed by a method comprising: In the substitution reaction in step S2, the solvent used is selected from one of polar aprotic solvents DMF, THF, CH3CN and DMSO.

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