4-butyl-2, 7-bis (2-fluorophenyl)-triazolopyrimidine-5 (4H)-ketone compound as well as preparation method and application thereof

Abstract

The invention discloses a 4-butyl-2, 7-bis (2-fluorophenyl)-triazolopyrimidine-5 (4H)-ketone compound as well as a preparation method and application of the 4-butyl-2, 7-bis (2-fluorophenyl)-triazolopyrimidine-5 (4H)-ketone compound. The anti-convulsion activity of the compound is evaluated by adopting subcutaneous pentyltetrazole (Sc-PTZ) and a mouse maximum electroconvulsion experiment (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 as shown in the formula I shows clear and relatively strong anti-convulsion activity and extremely low neurotoxicity, and the conclusion proves that the compound with the structure as shown in the formula I has the possibility of being developed into a novel epilepsy treatment medicine with low neurotoxicity. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound and a preparation method and use thereof Background Art

[0002] Epilepsy is a clinical syndrome in which repeated abnormal discharges of brain nerve cells lead to brain dysfunction, which usually begins in childhood and lasts a lifetime. It affects about 1% of the world's population, and there are about 10 million epilepsy patients in my country, with about 400,000 new cases each year. Epilepsy not only causes pain to patients and their families, but also brings a huge burden to society. At present, the treatment of epilepsy mainly relies on the use of anti-epileptic drugs. Although anti-epileptic drugs have developed from the first generation to the third generation in the past few decades, the anti-epileptic drugs currently used in clinical practice can only control epileptic seizures in about 70% of patients. In addition, many patients will experience neurogenic side effects associated with existing drugs, including sedation and cognitive dysfunction. Therefore, the development of new structural molecules with low neurotoxicity and anti-epileptic activity is still an urgent need for the treatment of epilepsy. The Chinese patent (CN201611115587.6) authorized by our team reports the preparation of [1,2,4]-triazolo[1,5-a]pyrimidine-7-one heterocyclic compounds and their potential application in the treatment of epilepsy; based on further in-depth research on the content of this patent, a Chinese patent (CN201910571753.0) was applied for and a research paper was published in European Journal of Medicinal Chemistry, 2020, 185, 11824; Bioorganic Chemistry, 2022, 19, 105565. Although these compounds show certain anticonvulsant activity, they still have the disadvantage of large side effects such as sedation and movement disorders. Summary of the invention

[0003] In order to solve the toxic side effects such as sedation and movement disorder of [1,2,4]-triazolo[1,5-a]pyrimidin-7-one heterocyclic compounds in the prior art, the present invention provides a novel 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidin-5(4H)-one compound and a preparation method thereof. The compound has good anti-epileptic activity and almost no sedative and movement disorder side effects.

[0004] In order to solve the above problems, the present invention adopts the following technical solutions:

[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 a 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)-triazolopyrimidine-5(4H)-one compound, which has the compound (I) as the active ingredient and contains one or / and multiple pharmaceutical carriers or one or / and multiple known pharmaceutical diluents.

[0008] Again, the present invention provides the use of the above-mentioned 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one or pharmaceutical composition in treating and / or preventing various epileptic seizures, nervous system diseases, etc. in animals (including humans).

[0009] The present 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 are cyclized to generate derivative 3 under the catalysis of an acidic catalyst.

[0011] S2: Derivative 3 undergoes a substitution reaction with halogenated n-butane, n-butyl methane sulfonate or n-butyl p-toluene sulfonate in the presence of a base to generate 4-butyl-2,7-bis(2-fluorophenyl)triazolopyrimidin-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., among which acetic acid is 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 amide, potassium amide, potassium hydroxide, sodium hydride, etc., among which sodium hydride is preferred.

[0014] The solvent used for the condensation reaction in step S2 is selected from aprotic solvents such as DMF, THF, CH3CN, DMSO, etc., among which DMF and THF are preferred.

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

[0016]

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

[0018] The 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound provided by the present invention contains the basic elements required for drugs in terms of structural characteristics: hydrogen bonding domain (HBD), hydrophobic domain (A) and electron donor part (D). A specific o-fluorophenyl group and a n-butyl carbon chain of suitable length are introduced into the 2,7 positions and nitrogen atom of the triazolopyrimidine-5(4H)-one skeleton to obtain compound I. These specific combinations ensure that the obtained molecule has good interaction with the target to produce specific activity, and at the same time have the ability to pass the blood-brain barrier and good pharmacokinetic properties to ensure its efficient transport to the target in the body; the obtained compound I has good anti-epileptic activity in a mouse model and does not show any sedation, movement disorders and other central nervous system-related toxic side effects at saturated solubility.

[0019] Its anticonvulsant activity was determined by subcutaneous pentylenetetrazol (Sc-PTZ) and the maximal electroconvulsive mouse model (MES), and its neurotoxicity was determined by the rotarod method. 50 The value is 9.17mg / kg, TD 50 The value is >813.40 mg / kg, and the anticonvulsant activity is significantly better than (and the toxicity is significantly lower than) the positive control drugs phenytoin sodium, valproate and carbamazepine, and the compound does not show central nervous system-related neurotoxicity such as sedation and movement disorders at the maximum solubility. From the above technical scheme, it can be seen that the 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compound provided by the present invention has good clinical application prospects in the treatment and / or prevention of various epileptic seizures, nervous system diseases, etc. in animals (including humans). DETAILED DESCRIPTION

[0020] The present invention discloses the biological activity of 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one compounds and methods for preparing the same. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0021] In order to enable those skilled in the art to better understand the present invention, the present invention is further described in detail below in conjunction with specific implementation methods.

[0022] The raw material compounds described in the present invention can be purchased through commercial channels, and the compound represented by formula (I) can be synthesized according to the following synthetic route using conventional compound synthesis methods.

[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 solvent toluene were added to the reaction flask and refluxed for 5-6 hours. After the reaction was completed, the derivative 1 was obtained by concentration under reduced pressure. After the derivative 1 and the 3-amino-1,2,4-triazole derivative 2 were refluxed in glacial acetic acid for 20 hours, the pure product of the derivative 3 was obtained by separation and treatment, and the yield was 42%.

[0025]

[0026] Example 2

[0027] Sodium hydrogen (1.5-3 equivalents) is added to the derivative 3 and the solvent N,N-dimethylformamide, and the mixture is stirred at room temperature for 15-30 minutes, followed by the addition of butyl bromide or butyl methanesulfonate or butyl p-toluenesulfonate. The reaction solution is 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, analogs of I without carbon chain were prepared (their structures are shown in Table 2).

[0029]

[0030] The obtained compound structure 1 HNMR and ESI-MS confirmed, and its H NMR spectrum and mass spectrum 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 tests were performed on 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one and its derivatives prepared in Example 2. PTZ and MES mouse models are the most commonly used animal models to evaluate the antiepileptic activity of compounds.

[0035] 3.1 Animal preparation and compound preparation

[0036] Animal husbandry: 20±2g Kunming white mice were selected, half male and half female. Before the experiment, the male and female experimental mice were kept in cages with free access to food and water. The room was well ventilated, the temperature was constant (22-25℃), the relative humidity in the room was controlled (about 50%), and the light in the room was normal (12 hours of bright light, 12 hours of shade). Mice were allowed to freely contact food particles (4% mineral mixture, 10% corn oil, 20% sucrose, 0.2% cellulose, 10.5% casein (95% pure), 54.3% starch and water) to enable mice to become familiar with the experimental environment as soon as possible. Animal husbandry followed the best standard rules to avoid unnecessary interference with animals. When the anti-epileptic activity of the compound was initially screened, the screening dose was 30mg / kg. During the preparation process, all compounds and positive control drugs were dissolved in dimethyl sulfoxide (DMSO) and the injection volume was 0.1mL / 20g. In the Sc-PTZ model, the screening dose of pentylenetetrazol was 85 mg / kg, dissolved in 0.9% saline, and the injection volume was 0.2 mL / 20 g.

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

[0038] The ability of the test compound to block PTZ-induced clonic seizures or to block electric shock-induced tonic seizures can be interpreted as a measure of anticonvulsant efficacy. The prepared compound solution was injected intraperitoneally into mice (30 mg / kg), and PTZ (85 mg / kg) was injected subcutaneously 30 minutes later. The mice were observed for the next 30 minutes, and the number of clonic seizures (ranging from violent shaking or vibration of the limbs) and tonic seizures (pulling or pushing the limbs toward the body or rigidly, usually with maximum extension of the hind legs) was used as the basis for judging the current epileptic seizure.

[0039] 3.3 Maximum electroconvulsive (MES) experiment

[0040] The JTC-1 type convulsion and pain experimental AC stimulator was used for stimulation output, the voltage was set to 80V, and the single stimulation was 0.3s. The ears and jaws of the experimental mice were moistened with 0.9% saline and clamped with crocodile clips. The experimental mice were suspended and relaxed before being stimulated. The experimental mice with hind leg stiffness can be used for the experiment. The test compound was intraperitoneally administered at a dose of 30mg / kg. No obvious hind limb stiffness and extension indicated that the test compound had anti-maximal electroconvulsive activity.

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

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

[0043]

[0044]

[0045] From the preliminary anti-epileptic screening results in Table 2, it can be seen that the compounds with n-butyl groups attached to the N atom showed the best anti-epileptic activity in the PTZ mouse model. Shortening or increasing the carbon chain length resulted in a significant decrease in activity and there was no obvious pattern in the activity changes.

[0046] 3.4 Quantitative activity and toxicity testing

[0047] The anticonvulsant activity was measured as the median effective dose (ED 50 ) and neurotoxicity is expressed as median toxic dose (TD 50 )express.

[0048] The compounds that protected 3 / 3 of the mice in the initial screening of anti-epileptic activity were subjected to quantitative activity tests. The quantitative neurotoxicity of the active compounds was determined by a rotating rod fatigue instrument (6r / min). Before the test, the experimental mice were trained to stay on the rolling rod for 1 minute, and then the trained experimental mice were randomly divided into groups of 5 per group. After 30 minutes of intraperitoneal injection of the target compound, the experimental mice were placed on the rolling rod. Those that could stay on the rolling rod for one minute without falling were considered effective. The modified Coulthard method was used for both the quantitative activity test and the neurotoxicity test.

[0049] The protection index is considered to be an indicator of the safety and tolerability between the dose of the anticonvulsant drug and the dose of the compound that produces acute adverse reactions (such as sedation, motor coordination disorders, ataxia or other neurotoxic manifestations). The larger the value, the higher the safety of the compound. 50 Divide the value by the corresponding ED 50 The calculated result is the protection index of the compound under study.

[0050] The compounds that protected 3 / 3 of the mice in the initial screening were tested for activity quantification, i.e., the half effective dose ED 50 The neurotoxicity of the active compound was determined by the rotarod fatigue test, i.e., the median toxic dose TD 50 The measurement 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 ED of target compounds, compounds in comparative documents and positive control drugs 50 , TD 50 and PI value

[0052]

[0053] From the comparison of activity and neurotoxicity results presented in Table 3, it can be seen that in the PTZ mouse model, the target compound exhibits better anti-epileptic activity than the comparative document compound and the positive control drug. In addition to good activity, the outstanding advantage of the target compound is that its neurotoxicity is much lower than that of the compounds and positive control drugs in the comparative documents. It does not show any neurotoxicity at saturated solubility, and its protection index for animals is greater than 88.7, which is much higher than that of the compounds and positive control drugs reported in the comparative documents. Its good anti-epileptic activity and extremely low neurotoxicity give it a good prospect for clinical application.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as 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 in that The compound according to claim 1 is used as an active ingredient, and contains one or / and multiple pharmaceutical carriers or one or / and multiple known pharmaceutical diluents.

3. Use of the compound according to claim 1 or the pharmaceutical composition according to claim 2 in a drug for treating or preventing convulsions or epilepsy.

4. A method for preparing 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidin-5(4H)-one (I) according to claim 1, characterized in that: The steps include: S1: Malonate derivative 1 and derivative 2 are cyclized to generate derivative 3 under the catalysis of an acidic catalyst. S2: Derivative 3 undergoes a substitution reaction with halogenated n-butane, n-butyl methane sulfonate or n-butyl p-toluene sulfonate in the presence of a base to generate 4-butyl-2,7-bis(2-fluorophenyl)-triazolopyrimidine-5(4H)-one (I).

5. The preparation method as claimed 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 preparation method as claimed in claim 4, characterized in that: 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 preparation method as claimed in claim 4, characterized in that: In the substitution reaction in step S2, the solvent used is selected from one of the polar aprotic solvents DMF, THF, CH3CN, and DMSO.

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