CN101007794A - N,N'-disubstituted piperazine derivative, and its preparation method and medicinal composition and uses - Google Patents

Abstract

The invention relates to the fields of drug design, medicinal chemistry and pharmacology. The present invention uses computer virtual screening based on the three-dimensional structure of potassium ion channel crystals to seek molecules that can stimulate or inhibit the activity of potassium ion channels, and derivates the screened compounds to provide a structural formula with potassium ion channel inhibitory activity as shown in the figure below The shown N, N'-disubstituted piperazine derivatives and their pharmaceutically acceptable salts, the present invention also relates to the preparation method of N, N'-disubstituted piperazine derivatives and their pharmaceutical composition and use. Such compounds can be used as potassium ion channel agonists or potassium ion channel inhibitors, and selectively move K ⁺ into or out of cells by stimulating or inhibiting the activity of potassium ion channels, so the compounds and pharmaceutical compositions of the present invention can be used for effective treatment of and / or drugs for the prevention of cardiovascular and cerebrovascular diseases closely related to the physiology, pathology and pharmacology of the cardiovascular system.

Description

N, N'-disubstituted piperazine derivatives, preparation method, pharmaceutical composition and use thereof

technical field

The present invention relates to the fields of drug design, medicinal chemistry and pharmacology. More specifically, the present invention uses computer virtual screening based on the three-dimensional structure of potassium ion channel crystals to seek molecules that can stimulate or inhibit the activity of potassium ion channels, and the screened Derivatized compound molecules, thereby providing novel N, N'-disubstituted piperazine derivatives with potassium ion channel inhibitory activity, the present invention also relates to the preparation method of N, N'-disubstituted piperazine derivatives And its pharmaceutical composition and application.

Background technique

Potassium ion channel is a special membrane protein that exists in all cells and has high selectivity and high permeability to potassium ions. Potassium ion channels are involved in many important cellular functions, such as controlling the current excitability of cells and excitation/response coupling, etc. In this way, potassium ion channels regulate the current pattern of nerves and hearts, release neurotransmitters, and control muscle contraction and the secretion of hormones; in non-current excitatory cells, potassium ion channels reflect their physiological functions by regulating the conduction pathway of single channels. Compared with other types of ion channels, there are many subtypes of potassium ion channels. If the potassium ion channel gene is mutated, it will lead to the occurrence of some cardiovascular and cerebrovascular diseases and nervous system diseases, such as arrhythmia, long QT syndrome (long QT syndrome), short-term movement disorder (episodic Ataxia), muscular atrophy (myotonic muscular dystrophy) ) and epilepsy. Potassium ion channels can be further divided into different types such as voltage-dependent, ligand (or receptor)-dependent, ATP-sensitive, calcium-activated and sodium-activated. Dozens of subtypes of voltage-dependent potassium channels alone have been cloned. The structures of the remaining types of potassium channels are not yet fully understood. They are all closely related to the physiology, pathology and pharmacology of the cardiovascular system.

Potassium ion (K ⁺ ) channels can selectively move K ⁺ into or out of cells with changes in membrane potential, or with activation of anions and/or ligands. The main role of K ⁺ channels is to maintain the resting membrane potential; another role is to repolarize action potentials in sensitive cells. Potassium channels represent different classes of ion channel proteins, and several toxins have now been elucidated to act primarily by blocking one or more specific K ⁺ channels. Cardiac cells are characterized by a remarkable diversity of different subtypes of K ⁺ channels. During an action potential, several types of K ⁺ channels can be opened due to the depolarization of the membrane, and the sum of the ion currents carried by these different channels will cause the repolarization of the membrane to reach the resting potential. Transient outward K ⁺ channels, one of these types of channels, are capable of generating a rapid (within 1-10 ms) initiation of a current of ions (I ₁₀ ) followed by a rapid (10-200 ms) decay (inactivation) upon membrane depolarization . I ₁₀ plays the most prominent role in the initial repolarization of the cardiac action potential and is blocked by several nonspecific pharmaceutical compounds such as aminopyridines and Tedisamil, a class III antiarrhythmic agent. Blocking I ₁₀ results in prolongation of the cardiac action potential. Prolongation of the cardiac action potential duration is thought to be a mechanism for the suppression of remobilized atrial and ventricular cardiosomal disturbances. Currently available Class III antiarrhythmic compounds, most of which act by blocking a single subtype of K ⁺ channels known as fast-type delayed rectifier K ⁺ channels.

At the amino acid sequence level, the homology between different subfamilies is about 40%, and the homology between the same subfamilies is about 70%. The Kv channel is composed of four α subunits forming an ion pore region, and each α subunit is composed of six highly conserved transmembrane segments (S1-S6) and C-terminal and N-terminal of different lengths inside and outside the cell. The S4 fragment has high homology with the voltage-gated sodium ion channel. It has both hydrophilic and lipophilic properties, and every three residues have a positively charged amino acid, which is a voltage-gated potassium ion channel. main components of activation. Terminal amino acids are thought to be involved in voltage-dependent inactivation. The connection between the S5 and S6 fragments penetrates deep into the membrane and participates in the formation of the ion pore region, which is called the H5 region or pore region in potassium ion channels. The structure of the pore region is highly conserved in potassium ion selective channels, especially the sequence (T/SXXTXGY(F)G) is regarded as the characteristic sequence of potassium ion selectivity. Removing G or Y has a great impact on ion selectivity, such as the selectivity of Shaker channel will be completely lost. Heginbotham et al. 2 mutated the residues in the Shaker channel pore area one by one, and found that 4 residues are very critical, they are TVGG, which are located at the 1, 5, 6 and 8 positions of the characteristic sequence, and the mutation of these residues will lead to Channels lose selectivity.

In April 1998, Doyle et al. published the crystal structure of the first ion-selective channel in Science. The resolution of the crystal structure was 3.2 Å, which was derived from the potassium ion channel of the bacteria Streptomyces lividans. KcsA has only two transmembrane structures connected by the pore region, but its amino acid sequence homology is closer to the voltage-gated potassium channel than the Kir channel (also two transmembrane structures). In the pore region, KcsA The sequence of is almost identical to the Kv channel. The crystal structure shows that the KcsA channel is a tetramer, and four identical α subunits are symmetrically distributed around the central pore region. Two transmembrane alpha helices (S1 and S2) are connected. The channel is shaped like an inverted cone. TVGYG residues form a narrow selective filter funnel, under which there is a cavity with a diameter of about 10 Ȧ, the carbonyl of the main chain points to the hole, and the side chain is away from the hole area, that is, the dipole moment of the helix is concentrated here. The channel length of the water-containing pore area is about 18 Ȧ, and the total length of the ion channel pore area is about 45 Ȧ. In 2005, Stephen et al. published the crystal structure of the potassium ion channel of the mammalian voltage-dependent Shaker family in Science. The resolution of the crystal structure is 2.9 Ȧ. This structure includes the complete transmembrane pore region and the voltage-sensitive region VS site, The middle T1 domain and the β subunit involved in redox reactions. But the amino acid residues in the selectivity filter region outside the mouth of these crystal structures are highly conserved. This provides the possibility to use computer to screen the active effective molecules of potassium ion channel.

The present invention selects the potassium ion channel as the target, and uses the means of computer-aided drug design to carry out virtual screening on the compounds in the commercially available ACD (http://www.mdl.com/) library. Better compounds are chemically synthesized and derivatized, as well as tested for biological activity. Biological activity test proves that the compound we obtained has good inhibitory activity on potassium ion channel. ACD library is the abbreviation of Available Chemicals Directory, which means a dictionary of chemicals that can be purchased at present. MDL company has close relationship with nearly a thousand chemical pharmaceutical companies in the world, constantly updates the database, and grasps the recent products, prices and packaging of various chemical pharmaceutical companies through the network. So far, the ACD library has contained information on nearly 1,300,000 compounds, and each compound has commonly used physical and chemical properties data, structure, price, packaging, manufacturer and contact information (address, telephone, fax, or email, etc.) ( http://www.mdl.com/products/pdfs/acd_ds.pdf), so ACD is the information source for various research institutes and pharmaceutical companies to find and order raw materials.

Contents of the invention

The purpose of the present invention is to use computer virtual screening based on the three-dimensional structure of the potassium ion channel crystal to seek molecules that can stimulate or inhibit the activity of the potassium ion channel, thereby providing a novel N, N'-disubstituted piperazine with potassium ion channel inhibitory activity kind of derivatives.

Another object of the present invention is to provide a preparation method of N, N'-disubstituted piperazine derivatives.

Another object of the present invention is to provide a pharmaceutical composition containing the above compound that can effectively treat or prevent cardiovascular system diseases closely related to potassium ion channels and related complications.

Another object of the present invention is to provide the use of the above compounds and pharmaceutical compositions in the preparation of medicines for treating or preventing cardiovascular system diseases closely related to potassium ion channels and related complications.

The invention uses computer virtual screening based on the three-dimensional structure of the potassium ion channel crystal to seek effective molecules with potassium ion channel activity. In April 1998, Doyle published the first crystal structure of the ion-selective channel of the bacterium Streptomyces lividans in Science. The inventor of the present invention utilizes the crystal structure of the potassium ion channel, and information on the action sites of toxins such as quaternary ammonium ion (QA), 4-aminoquinoline, scorpion venom, snake venom, and bee venom on the potassium ion channel, and selects the potassium ion channel The two active sites were searched in three-dimensional databases using the molecular docking program DOCK4.0 to find possible inhibitors. The small molecule three-dimensional structure database searched by the present inventor is ACD-3D small molecule database, which has about 250,000 compounds. Due to the high-throughput screening technology for new potassium ion channel inhibitors, there are still some difficulties, and the role of virtual screening is particularly significant. Using this method, the inventors performed virtual screening, sorting and scoring on the ACD database, and selected 22 potential ligands. Electrophysiological analysis showed that 6 compounds had high inhibitory activity, which was 300-1000 times that of TEA (tetraethylamine). Bioanalytical results showed that these compounds were _IK- specific blockers. Among them, the N, N'-disubstituted piperazine compounds have different degrees of inhibitory effects on I _K and I _A , and their IC ₅₀ are shown in Table 2. Among them, 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine has high selectivity in extracellular inhibition, and its inhibition on _IK is 16.3 times that of _IA ( _IC50 ratio is 16.3). The present inventors derivatized 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine to obtain a series of derivatives with better agonist or inhibitory activity on potassium ion channels.

The present invention provides novel N, N'-disubstituted piperazine derivatives represented by the following structural formula I or pharmaceutically acceptable salts thereof:

in:

n=0-1 integer;

R ₁ and R ₂ each independently represent hydrogen, halogen, C1-C6 linear or branched saturated or unsaturated hydrocarbon group, C3-C7 cycloalkyl group, benzyl, aromatic group or 5-7 membered heterocyclic group;

The halogen is fluorine, chlorine, bromine or iodine;

The aryl group can be phenyl, substituted phenyl, naphthyl or biphenyl; wherein the substituted phenyl group can include 1 to 4 substituents, which can be selected from halogen, C1-C6 straight chain Or branched hydrocarbon group, cyano group, nitro group, amino group, hydroxyl group, hydroxymethyl group, trifluoromethyl group, trifluoromethoxy group, carboxyl group, C1-C4 alkoxy group, mercapto group and C1-C4 acyl group;

The 5-7 membered heterocyclic group may contain 1-3 heteroatoms selected from oxygen, sulfur or nitrogen, and/or be combined by phenyl in structural formula I, and/or contain one or more heteroatoms selected from Halogen, C1-C6 straight chain or branched chain hydrocarbon group, cyano group, nitro group, amino group, hydroxyl group, hydroxymethyl group, trifluoromethyl group, trifluoromethoxy group, carboxyl group, C1-C4 alkoxy group, mercapto group, C1- Substituents for C4 acyl and aryl groups.

The pharmaceutically acceptable salts of N, N'-disubstituted piperazine derivatives provided by the present invention can be specifically listed with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, Salts formed with organic acids such as malic acid, tartaric acid or citric acid or acidic amino acids such as aspartic acid and glutamic acid after forming esters with inorganic bases, such as sodium, potassium, calcium, aluminum salts and ammonium salts; or with organic bases Formed salts, such as methylamine salt, ethylamine salt, ethanolamine salt, etc.; or hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, Salts of inorganic acids such as nitric acid and phosphoric acid, or salts with organic acids such as formic acid, acetic acid, picric acid, methanesulfonic acid, and ethanesulfonic acid.

A preferred embodiment of N, N'-disubstituted piperazine derivatives of structural formula I of the present invention or a pharmaceutically acceptable salt thereof is 1-substituted benzyl-4-[2-(4-substituted-benzenesulfonate Acyl)-ethyl]-piperazine compounds or pharmaceutically acceptable salts thereof:

where n=1;

R ₁ is hydrogen or fluorine;

_R2 is as defined above.

Another preferred embodiment of N, N'-disubstituted piperazine derivatives of structural formula I of the present invention or a pharmaceutically acceptable salt thereof is 1-substituted benzyl-4-[2-(4-substituted-benzene Sulfonyl)-ethyl]-piperazine compounds or pharmaceutically acceptable salts thereof:

where n=1;

_R1 is as defined above;

R ₂ is hydrogen or chlorine.

Another preferred embodiment of N, N'-disubstituted piperazine derivatives of structural formula I of the present invention or pharmaceutically acceptable salts thereof is 1-(benzo[1,3]dioxolane-5- Methylene)-4-[2-(4-substituted-benzenesulfonyl)-ethyl]-piperazine compounds or their pharmaceutically acceptable salts:

where n=1;

R ₁ is [1,3]dioxolane combined with benzo;

_R2 is as defined above.

The fourth preferred embodiment of N, N'-disubstituted piperazine derivatives of structural formula I of the present invention or a pharmaceutically acceptable salt thereof is 1-substituted phenyl-4-[2-(4-substituted- Benzenesulfonyl)-ethyl]-piperazine compounds or their pharmaceutically acceptable salts:

where n=0;

R ₁ and R ₂ are as defined above.

The present invention provides a method for preparing N, N'-disubstituted piperazine derivatives of structural formula I. The steps of the method are as follows:

(1) In an organic solvent, under the action of a base, piperazine monohydrochloride reacts with a compound of structural formula a to obtain a compound of structural formula b;

Wherein said organic solvent is tetrahydrofuran, ether, dimethylformamide, ethylene glycol dimethyl ether, ethylene glycol diethyl ether, dioxane, ethanol, methanol or ethyl acetate; said base is an organic base Or inorganic base, wherein organic base is pyridine, triethylamine, 4-dimethylaminopyridine (DMAP) or diisopropylethylamine, and inorganic base is sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide; R ₁ is as defined above; X is Cl or Br.

(2) the compound of structural formula c reacts with 2-chloroethyl acetate to obtain the compound of structural formula d;

(3) the compound of structural formula d obtains the compound of structural formula e through hydrolysis, chlorination;

(4) The compound of structural formula b obtained in the above step (1) is reacted with the compound of structural formula e obtained in step (3), and the N, N'-disubstituted piperazine derivatives represented by structural formula I of the present invention are obtained after separation.

In the preparation method provided by the present invention, the used raw material piperazine monohydrochloride can be obtained by reacting piperazine with excess hydrogen chloride to obtain piperazine dihydrochloride, and then react with an equivalent amount of piperazine to obtain piperazine monohydrochloride ; The halides of structural formula a are commercially available.

The preparation method of N,N'-disubstituted piperazine derivatives provided by the invention has the advantages of mild reaction conditions, abundant and easy-to-obtain raw materials, simple operation and post-treatment, and the like.

The pharmaceutically acceptable salts of the N,N'-disubstituted piperazine derivatives of the present invention can be prepared by conventional pharmaceutical salt-forming methods.

The N, N'-disubstituted piperazine derivatives of the present invention can effectively treat and prevent cardiovascular system diseases and diseases closely related to potassium ion channels by activating/inhibiting channel activity in computer virtual screening and potassium ion channel binding experiments. Its related complications, such as hypertension, coronary heart disease, myocardial infarction and other cardiovascular and cerebrovascular diseases. In the model of the interaction between N, N'-disubstituted piperazine derivatives and potassium ion channels, it was observed that all compounds had an effect on the model in the concentration range of 10 ^-7 -10 ^-5 M.

The N, N'-disubstituted piperazine derivatives of the present invention have very low toxicity.

The pharmaceutical composition of the present invention contains a therapeutically effective amount of N,N'-disubstituted piperazine derivatives of the above structural formula I or pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable carriers.

The compound and pharmaceutical composition of the present invention can be used to prepare medicines for treating or preventing cardiovascular and cerebrovascular diseases caused by potassium ion channel regulation.

The compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions or aerosols, etc., and can be present in suitable solid or liquid carriers or Diluent and appropriate sterile equipment for injection or infusion. The pharmaceutical composition may further contain smelling agents, flavoring agents and the like.

The ideal ratio of the pharmaceutical composition provided by the present invention is that the compound of formula I as the active ingredient accounts for 65%-99.5% of the total weight ratio, and the rest is 0.5-35% of the total weight ratio, or better 1-20% , or preferably 1-10% pharmaceutically acceptable carrier, diluent or solution or saline solution.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the field of pharmacy.

The compounds and pharmaceutical compositions of the present invention can be clinically used in mammals, including humans and animals, and can be administered through oral, nasal, dermal, pulmonary, or gastrointestinal routes. Oral administration is most preferred. The best preferred daily dose is 0.01-200 mg/kg body weight, taken once, or 0.01-100 mg/kg body weight in divided doses. Regardless of the method of administration, the optimal dosage for an individual should depend on the specific treatment. Usually, start with a small dose and gradually increase the dose until you find the most suitable dose.

Description of drawings

Figure 1 shows the first 300 compounds with lower binding energies, where A represents the binding energies of these 300 compounds calculated by DOCK 4.0; B represents the binding energies (BE ₁ ) and Sybyl of the 300 compounds calculated by DOCK Binding energy (BE ₂ ).

Figure 2 illustrates the effect of extracellular application of 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine (Ia) on rat hippocampal neurons.

Figure 3 illustrates the effect of intracellular administration of 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine (Ia) on delayed rectifier potassium channels in rat hippocampal neurons.

Detailed ways

The invention is further illustrated in the following examples. These examples serve only to illustrate the invention, but do not limit the invention in any way. All parameters in the examples, as well as the rest of the description, are based on mass unless otherwise stated.

Example 1

Computer Virtual Screening :

In April 1998, Doyle published the crystal structure of the first ion-selective channel in Science, which was derived from the potassium ion channel of the bacterium Streptomyces lividans. Using the crystal structure of the potassium channel, as well as the information on the action sites of quaternary ammonium ion (QA), 4-aminoquinoline, and toxins such as scorpion venom, snake venom, and honeybee venom on the potassium channel, two active sites of the potassium channel were selected, and the molecular The docking program DOCK4.0 carried out a three-dimensional database search on them to find possible blockers. The small molecule three-dimensional structure database searched is the ACD-3D small molecule database, which has about 250,000 compounds.

experimental method:

(1) Structure processing of small molecule compounds

Before the three-dimensional structure of small molecules can be used for molecular docking, it must go through a series of data format conversions. First, output the three-dimensional structure in the database in MDL/RDF format, then convert the corresponding RDF file into MDL/SDF format, and finally convert the SDF file into SYBYL/MOL2 data format (DOCK4.0 comes with MDL/SDF file conversion into a program in SYBYL/MOL2 format). Finally, use the SPL program in the molecular simulation software SYBYL to carry out hydrogenation and charge processing on the small molecule database MOL2. The charges are Gasteiger-Marsili charges.

(2) Macromolecular structure processing

Checks for missing residues, deletes ligand and all water molecules in the receptor molecule, adds perhydrogen and Kollman All type charges to the molecule.

(3) Description of the active site

Considering the action site of the potassium channel inhibitor, the amino acid residues within 8 Ȧ around the TEA binding site Tyr82 were defined as the active pocket region of the outer mouth; the other binding pocket of TEA was calculated using the AutoMS program to calculate the molecular surface of the active site of the potassium channel, The sphgen program is then used to generate shape ball sets describing the active sites, and the molecular docking program uses these ball sets to speed up the matching of small molecules and macromolecular active sites during docking.

(4) Calculation grid file

Use the Grid program to calculate the grid file of the electrostatic interaction and van der Waals interaction energy in the box.

(5) Molecular docking

Use DOCK4.0 to search the three-dimensional database to conduct a rough screening of 250,000 compounds in the ACD library, the parameters used are (10, 10), and then select the best 5000 molecules in the docking results, using the parameters (25, 25) These molecules were searched more carefully, and finally 300 molecules with higher binding energy were selected for further analysis.

(6) Docking structure processing

Use the Docking module in SYBYL to optimize the results of DOCK4.0, and keep the macromolecular structure unchanged during the optimization process. Finally, the binding energy of the complex is calculated according to the following formula:

E _binding =E _RL -E _R -E _L

where _ER is the energy of the complex, _ER is the energy of the receptor, and _EL is the energy of the ligand. The calculation adopts Tripos force field, GASTHUCK charge, and optimizes the energy gradient RMS to be less than 0.5kcal/mol·A.

Experimental results:

The interaction force between the selected 300 compounds and the potassium ion channel, the binding energy calculated by DOCK is from -38.33 to -22.76kcal/mol, and the binding energy calculated by Sybyl is from -178.85 to -72.15kcal/mol (as shown in 1, Figure 1 shows the first 300 compounds with lower binding energy, where A is the binding energy of these 300 compounds calculated by DOCK4.0 (■: total binding energy; ●: van der Waals interaction; ▲: electrostatic force); B represents the binding energy (BE ₁ ) calculated by DOCK and the binding energy (BE ₂ ) calculated by Sybyl of 300 compounds (▲: compound 1-6; ■: compound 7-22; ◆: compound 23- 300.)), the data of the two have great consistency. Moreover, parameters such as shape matching, H-bond formation potential with potassium ion channels, and drug-like properties were also considered, and 22 compounds (numbered: 01-22) were selected for further analysis. The binding free energies of 22 compounds and potassium ion channels were calculated by AutoDock3.0. The various bonding properties of the 22 compounds are shown in Table 1.

Table 1: Binding energy (kcal/mole), rank and XlogP values of 22 compounds selected from 300 candidate compounds:

compound BE ₁ ^a R ₁ ^a BE ₂ ^b R ₂ ^b BE ₃ ^c R ₃ ^c XlogP 01 -35.16 4 -75.437 225 -16.33 16 1.29 02 -35.16 4 -78.822 191 -17.29 12 1.04 03 -34.32 7 -96.668 80 -15.23 17 2.59 04 -35.50 2 -75.069 231 -25.57 9 3.31 05 -38.33 1 -100.505 67 -17.00 13 2.00 06 -29.51 51 -132.34 12 -9.81 20 1.87 07 -33.85 9 -82.245 167 -16.57 14 0.35 08 -29.10 57 -84.331 154 -45.68 3 -2.09 09 -32.82 13 -175.019 2 -26.52 7 4.42 10 -35.40 3 -92.112 104 -45.95 2 0.51 11 -28.67 64 -91.259 110 6.64 twenty two -0.89 12 -29.06 57 -75.32 227 -6.14 twenty one 5.05 13 -30.49 33 -81.235 176 -46.50 1 -1.13 14 -32.96 11 -103.654 59 -27.32 6 0.15 15 -32.04 17 -82.745 163 -39.4 5 3.89 16 -32.91 12 -91.974 107 -26.33 8 2.69

17 -33.12 10 -83.070 161 -13.74 18 0.96 18 -28.67 64 -83.269 160 -22.71 11 -0.12 19 -32.21 15 -87.947 131 -42.21 4 -0.19 20 -33.90 9 -77.120 205 -16.34 15 -0.49 twenty one -34.46 6 -87.807 132 -11.31 19 -1.82 twenty two -28.51 68 -100.82 64 -25.57 10 0.19

^a Binding energy (BE ₁ ) and ordering (R ₁ ) calculated using DOCK

^b Binding energy (BE ₂ ) and ordering (R ₂ ) calculated using Sybyl

^c Binding energy (BE ₃ ) and ordering (R ₃ ) calculated using AutoDock

Example 2

Preparation of 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine (Ia)

At normal temperature, feed freshly prepared dry HCl gas into a 200ml three-necked flask containing 8.6g (0.1mol) anhydrous piperazine dissolved in 50ml THF. Under vigorous stirring, react for 6h, remove HCl gas, add 8.6g (0.1mol) of anhydrous piperazine, react at room temperature for 2h, then reflux for 6h under vigorous stirring. Cool, filter and wash the filter cake with cold THF. After drying, 22.4 g of white crystals of piperazine monohydrochloride were obtained (92.3% yield).

Put 4.86g (0.02mol) of piperazine monohydrochloride and 20ml of THF into a 100ml eggplant-shaped bottle. At 0-5°C, 2.28 g (0.018 mol) benzyl chloride dissolved in 20 ml THF was added dropwise, and the drop was completed after 1 h. Reacted overnight at room temperature, refluxed for 2 hours, and evaporated the solvent. Then, add 30ml of water, adjust the pH=8-9 with saturated potassium carbonate solution, extract with dichloromethane (20ml×3), combine the organic layers and dry with _MgSO4 , filter, and concentrate the filtrate to obtain N-benzylpiperazine 2.15 g of pale yellow oil (67.9% yield).

Add 0.01mol (1.125g) of 2-chloroethyl acetate and 0.01mol (1.99g) of sodium p-chlorobenzenesulfinate into 20ml of ethylene glycol dimethyl ether, reflux for 36 hours, cool, add 20ml of water, and use Extract with dichloromethane (20ml×3), combine the organic layers and use _MgSO4Dry , filter, evaporate most of the solvent, silica gel column separation (dichloromethane: ethyl acetate: petroleum ether=1: 1: 20, volume ratio) 1.12 g of white crystals of 2-(4-chloro-benzenesulfonyl)ethyl acetate were obtained (yield 44.4%).

Add 0.01mol (2.52g) of 2-(4-chloro-benzenesulfonyl)ethyl acetate to 20ml of 75% ethanol, add 20ml of 40% sodium hydroxide, and reflux for 8 hours. Part of the solvent was evaporated, then extracted with dichloromethane (20ml×3), the organic layers were combined and dried with _MgSO4 , filtered, and the filtrate was concentrated to give 2-(4-chlorobenzenesulfonyl) ethanol gray oil: 1.82g (product rate of 82.4%).

Dissolve 1.0 mmol (0.221 g) of 2-(4-chlorobenzenesulfonyl)ethanol in 5 ml of dichloromethane, add 0.5 g of phosphorus pentachloride, react in an ice-water bath for 2 hours, and react overnight at room temperature. 10ml of ice water was added dropwise within 1 hour, then extracted with dichloromethane (20ml×3), the organic layer was dried with MgSO , filtered, and separated _by column chromatography (dichloromethane: ethyl acetate: petroleum ether=1:1: 20, volume ratio), to obtain 1-chloro-4-(2-chloro-ethanesulfonyl)-benzene off-white liquid: 0.21 g (87.5% yield).

Add 1mmol (0.238g) 1-chloro-4-(2-chloro-ethanesulfonyl)-benzene, 1mmol (0.176g) N-benzylpiperazine, 1.5mmol (0.207g) K ₂ CO to a 50ml reaction flask _3. 10mg I ₂ and 20mlTHF, reflux reaction for 3 hours; then stop the reaction, evaporate the solvent, add 20ml water, extract with dichloromethane (20ml×3), combine the organic layers and use MgSO ₄ Dry, filter, and after the filtrate is concentrated, Silica gel column separation (dichloromethane: ethyl acetate: petroleum ether = 1:1:4, volume ratio) to obtain 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]- 0.312 g of white crystals of piperazine (82.3% yield).

IR (KBr): 3448, 1579, 1479 cm ^-1 . ¹ H NMR (CDCl ₃ ): 2.62(t, 8H); 2.55(t, 2H); 3.75(t, 2H); 3.84(s, 2H); 7.25(m, 5H); 7.50(m, 2H); 7.83 (m, 2H). HRMS (SCI) m/z calcd for M ⁺ , 378.1169; found, 378.1182.

Example 3

Preparation of 1-(4-fluorophenyl)-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine (Ib)

Replace 1-benzylpiperazine with 1-(4-fluorophenyl)-piperazine, operate as described in Example 2, all the other required raw materials, reagents and preparation methods are the same as Example 2, to obtain the target compound 1-( 4-fluorophenyl)-4-[2-(4-chloro-phenylsulfonyl)-ethyl]-piperazine.

IR (KBr): 1625 cm ^-1 . ¹ HNMR (CDCl ₃ ) ppm: 2.55(t, 4H); 2.85(t, 2H); 2.98(t, 4H); 3.35(t, 2H); 6.82(m, 2H); 6.94(m, 2H) ; 7.54(m, 2H); 7.87(m, 2H). HRMS (SCI) m/z calcd for M ⁺ , 382.0918; found, 382.0922.

Example 4

Preparation of 1-(2-fluorophenyl)-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine (Ic)

Replace 1-benzylpiperazine with 1-(2-fluorophenyl)-piperazine, operate as described in Example 2, all the other required raw materials, reagents and preparation methods are the same as Example 2, to obtain target compound 1-( 2-fluorophenyl)-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine.

¹ HNMR (CDCl ₃ ) ppm: 2.55(t, 4H); 2.85(t, 2H); 2.98(t, 4H); 3.35(t, 2H); 6.90(m, 2H); 7.02(m, 2H); 7.54 (m, 2H); 7.87 (m, 2H). HRMS (SCI) m/zcalcd for M+, 382.0918; found, 382.0925.

Example 5

1-(Benzo[1,3]dioxolan-5-ylmethyl)-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-

Preparation of piperazine (Id)

Use benzo[1,3]dioxolan-5-ylmethyl bromide instead of benzyl chloride, operate as described in Example 2, and the rest of the required raw materials, reagents and preparation methods are the same as in Example 2 to obtain the target compound 1 -(Benzo[1,3]dioxolan-5-ylmethyl)-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine.

IR (KBr): 1580 cm ^-1 . ¹ H NMR (CDCl ₃ ): 2.56(t, 8H); 2.85(t, 2H); 3.85(t, 2H); 3.98(s, 2H); 5.88(s, 2H); ); 7.50-7.83 (m, 4H). HRMS (SCI) m/z calcd for M ⁺ , 422.1067; found, 422.1067.

Example 6

Preparation of 1-(diphenylmethyl)-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine (Ie)

Replace benzyl chloride with diphenylmethyl bromide, operate as described in Example 2, and all the other required raw materials, reagents and preparation methods are the same as in Example 2 to obtain the target compound 1-(diphenylmethyl)-4-[ 2-(4-Chloro-benzenesulfonyl)-ethyl]-piperazine.

IR (KBr): 1625 cm ^-1 . ¹ H NMR (CDCl ₃ ): 2.68(t, 8H); 2.55(t, 2H); 3.75(t, 2H); 5.15(s, 1H); 7.05-7.14(m, 10H); ); 7.88 (m, 2H). HRMS (SCI) m/z calcd for M ⁺ , 454.1482; found, 454.1491.

Example 7

Preparation of 1-phenyl-4-(2-benzenesulfonyl-ethyl)-piperazine (If)

Sodium benzenesulfinate is used to replace sodium p-chlorobenzenesulfinate, 1-phenylpiperazine is used to replace 1-benzylpiperazine, and the operation described in Example 2 is followed, and the rest of the required raw materials, reagents and preparation methods are implemented in the same way

Example 2, the target compound 1-phenyl-4-(2-benzenesulfonyl-ethyl)-piperazine was obtained.

IR (KSr): 1605 cm ^-1 . ¹ HNMR (CDCl ₃ ) ppm: 2.55 (t, 4H,); 2.85 (t, 2H); 2.98 (t, 4H); 3.35 (t, 2H); 6.82-7.14 (m, 5H); 2H); 7.87 (m, 2H). HRMS (SCI) m/z calcd for M ⁺ , 364.1012; found, 364.1018.

Example 8

Preparation of 1-(4-fluorobenzyl)-4-(2-benzenesulfonyl-ethyl)-piperazine (Ig)

Replace sodium p-chlorobenzenesulfinate with sodium benzenesulfinate, replace 1-benzylpiperazine with 1-(4-fluorobenzyl)piperazine, operate as described in Example 2, and the rest of the required raw materials and reagents And the preparation method is the same as in Example 2 to obtain the target compound 1-(4-fluorobenzyl)-4-(2-benzenesulfonyl-ethyl)-piperazine.

IR(KBr): 1588cm ^-1 ; ¹ HNMR(CDCl ₃ )ppm: 2.55(t, 4H,); 2.85(t, 2H); 2.98(t, 4H); 3.35(t, 2H); 2H); 6.82 (m, 2H); 6.94 (m, 2H); 7.54 (m, 2H); 7.87 (m, 2H); HRMS (SCI) m/z calcd for M ⁺ , 396.1075; found, 396.1081.

Example 9

Preparation of 1-(2-fluorobenzyl)-4-(2-benzenesulfonyl-ethyl)-piperazine (Ih)

Replace sodium p-chlorobenzenesulfinate with sodium benzenesulfinate, replace 1-benzylpiperazine with 1-(2-fluorobenzyl)piperazine, operate as described in Example 2, and the rest of the required raw materials and reagents And the preparation method is the same as in Example 2 to obtain the target compound 1-(2-fluorobenzyl)-4-(2-benzenesulfonyl-ethyl)-piperazine.

IR(KBr): 1622cm ^-1 ; ¹ HNMR(CDCl ₃ )ppm: 2.55(t, 4H); 2.85(t, 2H); 2.98(t, 4H); 3.35(t, 2H); 5.26(s, 2H ); 6.90(m, 2H); 7.02(m, 2H); 7.54(m, 2H); 7.87(m, 2H) HRMS(SCI) m/z calcd for M ⁺ , 396.1075; found, 396.1077

Example 10

Preparation of 1-[2-(Benzo[1,3]dioxolane-5-sulfonyl)-ethyl]-4-phenyl-piperazine (Ii)

Replace 1-benzylpiperazine with 1-phenylpiperazine, replace sodium p-chlorobenzenesulfinate with sodium benzo[1,3]dioxolane-5-sulfinate, as described in Example 2 Operation, all the other required raw materials, reagents and preparation methods are the same as in Example 2, to obtain the target compound 1-[2-(benzo[1,3]dioxolane-5-sulfonyl)-ethyl]-4-benzene base-piperazine.

Test Example

Biological activity test:

Experiment Name: Acute Isolation and Whole-Cell Patch Clamp Recording of Pyramidal Cells in the CA1 Region of the Rat Hippocampus

Experimental method: 5-9 days old Spragur-Dawley newborn rats were taken, and the whole brain was taken out after decapitation, placed in artificial cerebrospinal fluid (ACSF ^* ) at 0°C, and thin slices of brain with a thickness of about 400-500 μm were cut with a vibrating microtome. The part of the hippocampus was removed and transferred into standard extracellular solution (ES ^* ) containing 1 mg/ml proteaseXIII and incubated at 32°C for 8 minutes. After taking it out, rinse it with fresh external fluid for 2-3 times, transfer the hippocampus into the external fluid containing trypsin inhibitor 1mg/ml and bovine serum albumin 1mg/ml to terminate the enzymatic hydrolysis, and continue oxygenating and standing still for later use. The CAI area was isolated under a dissecting microscope, and the tissue block was gently blown with three pipettes with decreasing caliber (2-0.5 mm), the supernatant containing hippocampal pyramidal cells was transferred to a culture dish, most of the suspension was sucked off and removed. Replace with new standard extracellular fluid and start recording.

表示。The experiment used the Axopatch 200A amplifier to record the current response in the whole-cell patch clamp mode, and the room temperature was 21-23°C. The inner diameter of the tip of the electrode is 1-2μm, and the impedance is 3-5MΩ after filling the inner liquid. The clamping voltage command and the recording of the membrane current are controlled by pClamp 6.2 software through the DigiData1200A A/D conversion interface. The resting clamping voltage is -50mv, the sampling frequency is 10-40KHz, and the filtering frequency is 1-10KHz. The series resistance compensation is generally 75-85%, and the capacitance compensation is 60-80%. The leakage current and residual capacitance current are eliminated by the P/4 method. Two kinds of potassium currents with different kinetic characteristics can be recorded in acutely isolated rat hippocampal CA1 pyramidal cells: the fast-inactivating instantaneous potassium current (I _A ) and the very slowly decaying delayed outward rectifying potassium current (I _K ). In the experiment, the voltage method was used to separate the total potassium current and I _K : the clamping voltage was first supercharged from -50mv to -110mv, and then depolarized to +60mv with a voltage step of 10mv, and the mixed total of I _A and I _K could be recorded. current; when the clamping voltage is inserted into an intermediate voltage of -50mv and 50ms after the hyperpolarization of -110mv, the rapidly decaying I _A component can be completely inactivated, and this has just separated the I _K component; the total current minus I _K is to get I _A ² . The drugs used were formulated in external fluid containing 1 μM TTX to block sodium channels. The drug delivery system is a Bio-Logic RSC-100 18-tube rapid drug delivery device. The means and standard errors of the data obtained using

express.

Compounds to be tested: Ia-Ii

Experimental results:

(1) As shown in Figure 2, the effect of extracellular application of 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine on rat hippocampal neurons. The left column of Figure A shows the separation of I _K and I _A : the upper raw current map is the total potassium current IT, the middle is I _K , and the lower is the current obtained by subtracting I _K from IT, which is I _A . The graphs in the right column are IT, I _K and I _A after administration, respectively. The voltage used to activate IT was hyperpolarized to -110mV followed by depolarization to +60mV for 400ms; the voltage used to activate _IK was similar to IT, but with a brief 50ms depolarization of -50mV before depolarization cation voltage. Panel B is a concentration-effect curve for the inhibition of _IK and _IA by 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine. I _A is the peak value, and I _K is the measured value 300ms after depolarization. (n=5～12).

(2) As shown in Figure 3, intracellular use of 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine on the delayed rectification of potassium ions in rat hippocampal neurons The role of the channel. In the figure, the ordinate is the relative value of _IK , and the abscissa is the time of intracellular administration. The concentration of the compound used in the cell inhibits _IK outside the cell by about 75%.

The biological activity test shows that the N,N'-disubstituted piperazine derivatives in the examples have different degrees of inhibitory effects on I _K and I _A , and their IC ₅₀ are shown in Table 2. Among them, 1-benzyl-4-[2-(4-chloro-benzenesulfonyl)-ethyl]-piperazine has the highest selectivity for extracellular inhibition, and its inhibition on I _K is 16.3 times that of I _A ( _IC50 ratio is 16.3).

Table 2:

compound _IC50 value (μM) IC ₅₀ (I _A /I _K ) ratio no I _K I _A Ia 60±7 980±150 16.3 6 Ib 18±3 124±9 6.9 6 Ic 56±7 800±10 14.3 6 Id 2.1±0.5 28±4 13.3 6 Ie 67±9 737±48 11 6 If 524±14 972±34 1.9 6

Ig 83±8 244±26 2.9 6 Ih 80±7 800±10 10.0 6 Ii 21±0.5 128±4 6.10 6

n is the number of neurons tested

Claims (12)

1, the N that has following structural formula I, N '-disubstituted piperazine derivative or its pharmacy acceptable salt:

Wherein:

The integer of n=0-1;

R ₁And R ₂Saturated or unsaturated alkyl, C3-C7 cyclic hydrocarbon radical, benzyl, aromatic base or the 5-7 unit heterocyclic radical of representing hydrogen, halogen, C1-C6 straight or branched independently of one another;

Wherein said halogen is fluorine, chlorine, bromine or iodine;

Described aromatic base is phenyl, substituted-phenyl, naphthyl or xenyl; Wherein said substituted-phenyl comprises 1～4 substituting group, and this substituting group is selected among halogen, C1-C6 straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxyl group, sulfydryl and the C1-C4 acyl group;

The first heterocyclic radical of described 5-7 contains 1-3 heteroatoms that is selected from oxygen, sulphur or nitrogen; and/or by the phenyl among the structural formula I and close, and/or contain one or more substituting groups that are selected from halogen, C1-C6 straight or branched alkyl, cyano group, nitro, amino, hydroxyl, methylol, trifluoromethyl, trifluoromethoxy, carboxyl, C1-C4 alkoxyl group, sulfydryl, C1-C4 acyl group and aromatic base.

2, N according to claim 1, N '-disubstituted piperazine derivative or its pharmacy acceptable salt is characterized in that, described N, N '-disubstituted piperazine derivative is 1-substituted benzyl-4-(2-(4-replacement-benzenesulfonyl)-ethyl)-piperazine compounds, wherein, and n=1; R ₁Be hydrogen or fluorine.

3, N according to claim 1, N '-disubstituted piperazine derivative or its pharmacy acceptable salt is characterized in that, described N, N '-disubstituted piperazine derivative is 1-substituted benzyl-4-(2-(4-replacement-benzenesulfonyl)-ethyl)-piperazine compounds, wherein, and n=1; R ₂Be hydrogen or chlorine.

4, N according to claim 1; N '-disubstituted piperazine derivative or its pharmacy acceptable salt; it is characterized in that; described N; N '-disubstituted piperazine derivative is 1-(benzo [1,3] dioxolane-5-methylene radical)-4-(2-(4-replacement-benzenesulfonyl)-ethyl)-piperazine compounds, wherein; n=1, R ₁Be [1, the 3] dioxolane that closes with benzo.

5, N according to claim 1, N '-disubstituted piperazine derivative or its pharmacy acceptable salt is characterized in that; described N; N '-disubstituted piperazine derivative is 1-substituted-phenyl-4-(2-(4-replacement-benzenesulfonyl)-ethyl)-piperazine compounds, wherein, and n=0.

6, according to each described N of claim 1～5, N '-disubstituted piperazine derivative or its pharmacy acceptable salt, it is characterized in that described pharmacy acceptable salt is to form behind the ester sodium salt, sylvite, calcium salt, aluminium salt or the ammonium salt that forms with mineral alkali with propionic acid, oxalic acid, propanedioic acid, succsinic acid, fumaric acid, toxilic acid, lactic acid, oxysuccinic acid, tartrate or citric acid organic acid or aspartic acid, L-glutamic acid acidic amino acid again; Or the methylamine salt, ethylamine salt or the ethanolamine salt that form with organic bases; Or form hydrochloride, hydrobromate, hydrofluoride, vitriol, nitrate, phosphoric acid salt, formate, acetate, picrate, mesylate or esilate behind the ester with Methionin, arginine, ornithine basic aminoacids.

7, the described N of a kind of claim 1, the preparation method of N '-disubstituted piperazine derivative, this method comprises:

(1) in organic solvent, under the effect of alkali, the compound of piperazine mono-hydrochloric salts and structural formula a reacts, and obtains the compound of structural formula b;

Wherein said organic solvent is tetrahydrofuran (THF), ether, dimethyl formamide, glycol dimethyl ether, ethylene glycol diethyl ether, dioxane, ethanol, methyl alcohol or ethyl acetate; Described alkali is organic bases or mineral alkali, and wherein organic bases is pyridine, triethylamine, 4-dimethylamino pyridine (DMAP) or diisopropylethylamine, and mineral alkali is yellow soda ash, salt of wormwood, sodium hydroxide or potassium hydroxide; R ₁Definition according to claim 1; X is Cl or Br;

(2) compound of structural formula c and acetate 2-chloroethene ester react the compound of structural formula d, R wherein ₂Definition according to claim 1;

(3) compound of structural formula d gets the compound of structural formula e through hydrolysis, chlorination;

(4) compound of the compound of the structural formula b that obtains of above-mentioned steps (1) and the structural formula e that step (3) obtains reaction obtains the N that structural formula I represents, N '-disubstituted piperazine derivative after the separation.

8, a kind of pharmaceutical composition is characterized in that, said composition contains the described N of the claim 1 for the treatment of significant quantity, N '-disubstituted piperazine derivative or its pharmacy acceptable salt.

9, pharmaceutical composition according to claim 8 is characterized in that, described N, and N '-disubstituted piperazine derivative or its pharmacy acceptable salt account for 65%～99.5% of this pharmaceutical composition gross weight ratio.

10, according to Claim 8 or 9 described pharmaceutical compositions, it is characterized in that said composition further comprises one or more pharmaceutically acceptable carriers, odorant agent, flavouring agent, vehicle or diluent.

11, the described N of claim 1, N '-disubstituted piperazine derivative or its pharmacy acceptable salt purposes aspect the medicine of preparation treatment or prevention cardiovascular system diseases and related complication thereof.

12, the purposes of the described pharmaceutical composition of claim 8 aspect the medicine of preparation treatment or prevention cardiovascular system diseases and related complication thereof.

Images