KR101984129B1 - Novel 1,2,4-Triazole Compound And Pharmaceutical Composition For Inhibiting Urease Comprising The Same - Google Patents

Abstract

The present invention provides a 1,2,4-triazole compound represented by formula 1 or a pharmaceutically acceptable salt thereof: In formula 1, R is a methoxy group, a fluoro or bromo group, Y is a thiophenyl group, a pyroyl group, an ethyl phenyl group, a benzyl group or a fluorophenyl group, and n is 0 or 1. The 1,2,4-triazole compound of the present invention has a form of Schiff base and can keep being stabilized by thione-thiol tautomerism. The compounds of the present invention have activity for inhibiting urease to be used for treating or preventing gastrointestinal disorders such as gastritis, a stomach cancer, stomach ulcer and the like caused by Helicobacter, and kidney stones, and maintain nitrogen element of soil to be used as a fertilizing composition for enhancing fertility of soil.

Description

TECHNICAL FIELD The present invention relates to a 1,2,4-triazole compound, and a novel pharmaceutical composition for inhibiting urease comprising the 1,2,4-triazole compound.

The present invention relates to a novel 1,2,4-triazole compound and a pharmaceutical composition containing it as an active ingredient, and more specifically to a 1,2,4-triazole-based Schiff base compound represented by the following formula ≪ RTI ID = 0.0 > and / or < / RTI >

(Wherein R is methoxy, fluoro or bromo group, Y is thiophenyl, pyrroyl, ethylphenyl, benzyl or fluorophenyl group, and n is 0 or 1).

The development of new therapeutic agents using natural or non-toxic precursors is one of the basic goals of pharmaceutical chemistry for the design and development of new drugs. The 5-membered heteroaromatic ring is an important building block of a broad range of biologically active molecules and has become an important structural motif in drug discovery. Similarly, the 1,2,4-triazole-containing compounds can be used in combination with anticancer, antiinflammatory, antidiabetic, anti-malarial, proliferative inhibitors, cell division inhibitors, anti-vascular, anticonvulsant, antifungal, and urease, And exhibit various biological actions including the same enzyme inhibition potential. Changes in the chemical structure of substituted 1,2,4-triazoles lead to other biological effects, as they strongly alter their activity and affect cell-tissue interactions.

The Schiff base refers to a compound represented by RCH = NR '(wherein R and R' are aryl or alkyl groups) and is a chemical compound such as a chelating agent, a stabilizer, a biologically active agent, Have been widely used in many fields of Various heterocycle-derived Schiff bases have been reported to have various biological activities. In addition, Schiff bases containing 1,2,4-triazole nuclei have attracted great interest in chemistry and biology for many years due to their ease of synthesis and extensive biological activity including antineoplastic, antiviral, antibacterial and corrosion inhibitors.

Biochem. Biophys. Res. Commun. 319 (2004) 1053-1063 reports a series of 25 1,2,4-triazole-3-thione compounds f indicating the possibility of urease inhibition. Arch. Pharm. Chem. Life Sci. 346 (2013) 423-446 summarized the library of triazole-based compounds g-m as potent urease inhibitors. Recently, Eur. J. Med. Chem. 67 (2013) 230-242 reported microwaves that helped in the synthesis of relatively complex triazole compounds n as urease inhibitors (see FIG. 1).

The superior urease inhibition potential is described by Eur. J. Med. Chem. 45 (2010) 5200-5207, or their Schiff bases, Arch. Pharm. Chem. Life Sci. Was observed by the designed Schiff base of 1,2,4-triazole, which is not directly linked by the imine linkage with the triazole skeleton (e. G. P) reported in EP-A-347 (2014) 387-397 .

Arch. Pharm. Chem. Life Sci. 346 (2013) 423-446. Eur. J. Med. Chem. 84 (2014) 639-650 Eur. J. Med. Chem. 67 (2013) 230-242. Eur. J. Med. Chem. 45 (2010) 5200-5207. Arch. Pharm. Chem. Life Sci. 347 (2014) 387-397.

The present inventors have developed novel 1,2,4-triazole-Schiff base compounds having urease inhibitory activity based on information on 1,2,4-triazole compounds that have been studied. In addition, the present inventors analyzed the urease inhibitory effect of the Schiff base compound and developed a pharmaceutical composition and a fertilizer composition for treating or preventing stomach diseases and kidney stones using the same.

The present invention provides a 1,2,4-triazole compound or a pharmaceutically acceptable salt thereof,

[Chemical Formula 1]

(Wherein R is methoxy, fluoro or bromo group, Y is thiophenyl, pyrroyl, ethylphenyl, benzyl or fluorophenyl group, and n is 0 or 1).

In one embodiment, the compound is a 1,2,4-triazole compound wherein R is a bromo or methoxy group, Y is ethylphenyl, thiophenyl or benzyl, n is 0, or a pharmaceutically acceptable salt thereof, Acceptable salt.

In one embodiment, the compound is (7a) (E) -3- (2-fluorophenyl) -4 - [(thiophen-2- ylmethylene) amino] -1H-1,2,4-triazole -5 (4H) -thione; (7b) (E) -3- (2-bromophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7c) (E) -3- (2-Methoxyphenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (4H) -lH-pyrrolo [2,3-d] pyrimidin-2-yl] - thion; (7E) -4 - [{(lH-pyrrol-2-yl) methylene} amino] -3- (2- methoxyphenyl) - thion; (7f) (E) -3- (3-Fluorophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7 g) (E) -3- (3-Bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7h) (E) -3- (3-methoxyphenyl) -4 - [(2-phenylethylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7i) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-fluorobenzyl) -1H-1,2,4-triazole-5 (4H) -thione; (7j) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-methoxyphenyl) -1H-1,2,4-triazole-5 (4H) -thione; And (7k) (E) -3- (4-methoxybenzyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole- Or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition for inhibiting urease comprising the above compound as an active ingredient.

In one embodiment, the pharmaceutical composition is for the treatment or prevention of one or more gastrointestinal disorders selected from the group consisting of acute gastritis, chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer, gastric lymphoma, caused by Helicobacter pylori Can be used.

In one embodiment, the pharmaceutical composition may be used for the prevention or treatment of kidney stones.

In one embodiment, the pharmaceutical composition may comprise from 0.001% to 90% by weight of the compound, based on the total weight of the composition.

The present invention also provides a fertilizer composition for enhancing soil soil strength comprising the above compound as an active ingredient.

The present invention also provides a process for the preparation of said compounds comprising the steps of: (i) reacting an aralkanoic acid (1) with, in the presence of 1,2-dichloroethane as a solvent and phosphorus oxychloride as a chlorinating agent, To synthesize an aralkanoic acid chloride (2); (Ii) The aralkanoic acid chloride (2) obtained in the above step (i) is dissolved in acetonitrile, which is then added dropwise to a solution containing hydrazine hydrate, TEA and acetonitrile and refluxed to obtain aralkanoic acid hydrazide 3); (Iii) adding potassium hydroxide, methanol and carbon disulfide to the aralkanoic acid hydrazide (3) obtained in the step (ii) to produce carbodithioate (5); (Iv) adding hydrazine hydrate and purified water to the carbodithioate (5) produced in the step (iii), refluxing for 10 to 12 hours overnight, and then neutralizing with hydrochloric acid to produce the triazole compound (6) ; And (v) dissolving the triazole compound (6) and the aldehyde produced in the step (iv) in methanol to prepare the 1,2,4-triazole compound of the present invention.

The novel 1,2,4-triazole compounds of the present invention have a Schiff base form and can be maintained in a stable state by thion-thiol tautomerization. The 1,2,4-triazole compounds have a urease inhibitory activity and have a therapeutic or preventive effect on gastrointestinal diseases such as gastritis, gastric cancer, gastric ulcer, and kidney stones caused by Helicobacter pylori, and urea, To ammonia, thereby enhancing the ground's intelligence.

FIG. 1 shows the molecular structure of conventional 1,2,4-triazole-based urease inhibiting compounds.
Fig. 2 shows the formulas of the novel 1,2,4-triazole compounds 7a to 7k of the present invention.
Figure 3 shows a single crystal X-ray structure of compound 7k, created by an atom-numbering scheme.
4 is a packing diagram for compound 7k in a crystal lattice, and the dotted line represents intermolecular hydrogen bonding of compound 7k.
Figure 5 shows a double reciprocal line double reciprocal plot for urease inhibition in the presence of compound 7b: (a) when using urea as the substrate, the concentrations of 7b were 0, 13.7, 27.5 and 55 mM , And the urea concentrations were 3.12, 6.25, 12.5, 25, 50 and 100 mM, respectively. The illustrations are for the determination of the inhibition constant, Figure 5b shows the slope versus inhibitor (7b) concentration, (7b) concentration. Straight lines were drawn using linear least squares.
Figure 6 shows the overlap of ligands within the active site. (a) shows the overlay of all the minimum energy docking pores inside the pocket, the ligand is shown in cyan, the major residue in green, and the backbone receptor in the form of a cartoon and a ribbon. (b) shows an enlarged view of an enzyme active site prepared to have a surface.
7 shows two-dimensionally an aspect in which compounds 7b, 7g and 7h, which are the ligands with the highest inhibitory activity, interact with major residues at the active site of the urease enzyme, wherein (a) represents compound 7b, ) Represents 7 g of the compound, and (c) represents the compound 7h.

The present invention provides a 1,2,4-triazole compound or a pharmaceutically acceptable salt thereof,

[Chemical Formula 1]

(Wherein R is a methoxy, fluoro or bromo group, Y is thiophenyl, pyrroyl, ethylphenyl, benzyl or fluorophenyl group, and n is 0 or 1).

In one embodiment, the compound is a compound wherein R is a bromo or methoxy group, preferably a bromo group; Y is an ethylphenyl, thiophenyl or benzyl group, preferably an ethylphenyl group; or a 1,2,4-triazole compound wherein n is 0, or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound is (7a) (E) -3- (2-fluorophenyl) -4 - [(thiophen-2- ylmethylene) amino] -1H-1,2,4-triazole -5 (4H) -thione; (7b) (E) -3- (2-bromophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7c) (E) -3- (2-Methoxyphenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (4H) -lH-pyrrolo [2,3-d] pyrimidin-2-yl] - thion; (7E) -4 - [{(lH-pyrrol-2-yl) methylene} amino] -3- (2- methoxyphenyl) - thion; (7f) (E) -3- (3-Fluorophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7 g) (E) -3- (3-Bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7h) (E) -3- (3-methoxyphenyl) -4 - [(2-phenylethylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione; (7i) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-fluorobenzyl) -1H-1,2,4-triazole-5 (4H) -thione; (7j) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-methoxyphenyl) -1H-1,2,4-triazole-5 (4H) -thione; And (7k) (E) -3- (4-methoxybenzyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole- , Or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound is preferably (7b) (E) -3- (2-bromophenyl) -4 - [(thiophen-2- ylmethylene) amino] (3-bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole (4H) -thione and (7h) (E) -3- (3-methoxyphenyl) -4 - [(2- phenylethylidene) amino] Sol-5 (4H) -thione, or a pharmaceutically acceptable salt thereof.

In one embodiment, the compound is more preferably (7 g) (E) -3- (3-bromophenyl) -4 - [(2- phenylpropylidene) amino] Triazol-5 (4H) -thione, or a pharmaceutically acceptable salt thereof.

In one embodiment, the 1,2,4-triazole compound may be in Schiff base form.

In one embodiment, the 1,2,4-triazole compound is capable of acting as a thione-thiol tautomer.

The present invention also provides a pharmaceutical composition for inhibiting urease comprising the above compound as an active ingredient.

In one embodiment, the pharmaceutical composition may be used for the treatment or prevention of one or more gastric diseases selected from the group consisting of acute gastritis, chronic gastritis, gastric ulcer, duodenal ulcer, gastric cancer and gastric lymphoma caused by Helicobacter pylori.

Helicobacter pylori secretes a strong urease, urease, and hydrolyzes one molecule of urea (H ₂ NCONH ₂ ) in the gastric juice to form two molecules of ammonia (NH ₃ ). Ammonia produced by Helicobacter pylorease increases the pH in the gastric juice and damages the gastric mucous layer. In addition, ammonia itself inhibits the oxygen consumption of the gastric mucosal layer cells and ATP production of mitochondria, and ultimately ammonia forms monochloroamine to generate reactive oxygen species, Inflammation, and even DNA damage, which is known to promote cancer development. Therefore, the urease inhibitor can be effectively used for the prevention or treatment of gastric diseases caused by Helicobacter pylori.

In one embodiment, the pharmaceutical composition may be used for the prevention or treatment of kidney stones.

Urease inhibitors can be used to dissolve kidney stones containing struvite stones, prevent the production of new stones, and treat urinary tract infections.

In one embodiment, the pharmaceutical composition may include the compound in an amount of 0.001 to 90% by weight based on the total weight of the composition, but may be appropriately adjusted depending on the symptom of the disease, progression, have.

In one embodiment, the pharmaceutical composition may further comprise a pharmaceutically acceptable diluent or carrier.

The pharmaceutically acceptable carrier may be a carbohydrate compound (for example, lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, etc.) commonly used in pharmaceuticals, acacia rubber, calcium phosphate, (Such as corn oil, cottonseed oil, soybean oil, olive oil, coconut oil, cottonseed oil, corn oil, cottonseed oil, ), Polyethylene glycol, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components.

The present invention can also provide a fertilizer composition for enhancing soil soil strength comprising the above-mentioned compound as an active ingredient.

The hydrolysis of urea by urease in the soil follows the same formula as (NH ₂ ) CO + 2H ₂ 0 ⇒ 2NH ₄ HCO ₃ ⇒ 2NH ₃ + H ₂ 0 + CO ₂ , pH rise and nitrogen loss. Therefore, the compounds of the present invention having an urease inhibiting effect can help increase the soil strength by preventing soil pH increase and nitrogen loss after using urea fertilizer.

The present invention also relates to a process for the production of (1) a process for the production of (2) a process for the production of (2) a process for the production of an organoaluminum compound, comprising the steps of (i) reacting an aralkanoic acid (1) under reflux in the presence of 1,2-dichloroethane as a solvent and phosphorus oxychloride as a chlorinating agent; (Ii) The aralkanoic acid chloride (2) obtained in the above step (i) is dissolved in acetonitrile, which is then added dropwise to a solution containing hydrazine hydrate, TEA and acetonitrile and refluxed to obtain aralkanoic acid hydrazide 3); (Iii) adding potassium hydroxide, methanol and carbon disulfide to the aralkanoic acid hydrazide (3) obtained in the step (ii) to produce carbodithioate (5); (Iv) adding hydrazine hydrate and purified water to the carbodithioate (5) produced in the step (iii), refluxing for 10 to 12 hours overnight, and then neutralizing with hydrochloric acid to produce the triazole compound (6) ; And (v) dissolving the triazole compound (6) and the aldehyde produced in the step (iv) in methanol to prepare the 1,2,4-triazole compound of the present invention. (See Scheme 1).

Hereinafter, the present invention will be described in more detail with reference to Examples. The present embodiments are illustrative of the present invention and are not intended to limit the scope of the present invention.

<Examples>

[ Example  One: New  1,2,4- Triazole  Synthesis of compounds 7a to 7k]

Substrates and reagents

The phenyl, aryl and aralkylcarboxylic acids were purchased from Alfa Aesar and Aldrich. In this experiment, ethanol, methanol, chloroform, water (Samchun Chemical), H ₂ SO ₄ and HCl (Jin Chemical & Pharmaceutical Co. Ltd.) were used. The urease (EC 3.5.1.5), thiourea, EDTA, sodium nitroprusside and active chloride from Jack bean were purchased from Sigma-Aldrich and all reagents used for biological analysis were analytical grade.

The synthesis route of the 1,2,4-triazole compounds 7a to 7k of the present invention

The following Reaction Scheme 1 schematically shows the synthesis route of the 1,2,4-triazole compounds 7a to 7k of the present invention.

[Reaction Scheme 1]

Synthesis conditions of the compound 7a-7k: (i) POCl ₃ , ClCH ₂ CH ₂ Cl, reflux 3 hours; (Ii) hydrazine hydrate, TEA, MeCN, reflux for 3 hours; (Iv) CS ₂ , KOH, methanol, stirring, 0 ° C, 1 hour; (v) hydrazine hydrate (80%), water, reflux, 10-12 hours; (Vi) different substituted triazole compounds, substituted aldehydes, methanol, microwave pulses (10 min).

First, in the presence of 12 ml of 1,2-dichloroethane as a solvent and 0.4 ml of phosphorus oxychloride as a chlorinating agent, 1 mmol of aralkanoic acid (1) was reacted under reflux for 3 hours. The reaction solution was cooled to room temperature and the solvent was removed under reduced pressure to give the aralkanoic acid chloride (2) which was used in the next step without further purification.

The obtained aralkanoic acid chloride (2) was dissolved in 80 ml of acetonitrile, and then added dropwise to a solution containing 1 mmol of hydrazine hydrate, 0.5 ml of TEA and 20 ml of acetonitrile, and the solution was stirred at room temperature for 3 minutes while monitoring by thin layer chromatography (TLC) Lt; / RTI &gt; After all of the starting material was consumed, the reaction mixture was cooled to room temperature, and the solvent was evaporated under reduced pressure to obtain crude substituted aromatic acid hydrazide (3) as a white solid. This was purified by column chromatography and crystallized with methanol.

The substituted aromatic acid hydrazide (3) (0.125 mol, 1 eq) was added to potassium hydroxide (0.125 mol, 1 eq) and cooled in ice. Carbon disulfide (0.125 mol, 1 eq) was added thereto with constant stirring for 1 hour to form a solid product of the potassium salt of hydrazine carbodithioate (5). The solid product was filtered, washed with cold diethyl ether, dried and used in the next step without further purification.

The potassium salt of the hydrazine carbodithioate (5) was added to deionized water (50 ml), followed by addition of hydrazine hydrate (0.250 mol) and refluxing overnight. The reaction mixture turned yellow-green with the emission of hydrogen sulfide and eventually became homogeneous. This was then poured into ice and neutralized with hydrochloric acid to give a white precipitate of the substituted triazole compound (6) which was filtered and washed with cold water and crystallized in methanol.

A solution prepared by dissolving the substituted triazole compound (6) (0.125 mol, 1 eq) in methanol (20 mL) and the different substituted aldehydes (0.125 mol, 1 eq) in methanol (20 mL) was mixed , The reaction was monitored by TLC for 2 minutes at regular intervals while being exposed to microwave for 10 minutes. Upon complete consumption of the starting material, the excess solvent was evaporated under reduced pressure and the resulting powder was crystallized from methanol. The product was purified by column chromatography using a n-hexane: ethyl acetate solvent system and then crystallized with methanol to give Schiff base compounds 7a to 7k of the present invention.

Monitoring synthetic products

The reaction course was monitored by TLC analysis and R _ζ The values were determined using a pre-coated silica gel aluminum plate (Kieselgel 60 F ₂₅₄ ) (Merck). TLC was visualized under UV lamp (VL-4LC). The melting point was measured with a Fisher Scientific Melting Point measuring device and used without correction.

[ Example  2: spectroscopic analysis of synthetic products]

The FTIR spectrum was recorded on a KBr pellet by an FTIR-8400S spectrometer (Shimadzu). ^{The 1} H and ¹³ C NMR spectra were recorded on a Bruker Avance 400 MHz spectrometer and the chemical shifts were recorded in the downstream δ values (ppm) from the internal tetramethylsilane of the designated organic solution. Peak multiplicity is represented by s singlet, d singular, and m singular. Single crystal data were obtained using a Bruker SMART APEX II X-ray diffractometer CCD with a Mo X-ray tube at room temperature and a graphite monochromator.

(7a) (E) -3- (2- Fluorophenyl ) -4 - [(thiophene-2- Ylmethylene ) Amino] -1H-1,2,4- Triazole -5 (4H) -thione

Light pink; Yield 83%; R _f : 0.54 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, aromatic protons), 7.38-7.29 (m, 2H, m, 1H, aromatic protons), 7.79-7.77 (m, , Aromatic protons), 7.27 to 7.26 (m, 1H, aromatic protons), 7.25 to 7.13 (m, 1H, aromatic protons); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 162.1, 159.6, 157.7, 149.5, 136.8, 136.2, 133.4, 131.9, 129.7, 128.9, 124.9, 122.3, 115.8.

(7b) (E) -3- (2- Bromophenyl ) -4 - [(thiophene-2- Ylmethylene ) Amino] -1H-1,2,4- Triazole -5 (4H) - Tion

Light gray; Yield: 84%; R _f : 0.54 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, aromatic protomer), 7.47-7.27 (m, 2H, m, 1H, aromatic protons), 7.57-7.49 (m, 1H, aromatic protons) , Aromatic protons), 7.27-7.20 (m, 2H, aromatic protons); ¹³ C NMR (100 MHz, DMSO-d ₆ )? 162.7, 162.0, 157.4, 151.4, 150.2, 136.9, 136.1, 135.3, 134.9, 133.3, 131.8, 129.0, 120.5.

(7c) (E) -3- (2-methoxyphenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-

Light pink; Yield 86%; R _f : 0.53 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, aromatic protons), 7.28-7.18 (m, 3H), 13.71 (s, 1H, NH), 10.16 , aromatic protons), 6.96 ~ 6.84 (m, 2H, aromatic protons), 3.73 (s, 3H, OCH 3); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 161.9, 158.6, 157.3, 151.0, 136.9, 136.1, 133.3, 130.5, 129.0, 128.7, 127.2, 114.3, 55.5.

(4H) -lH-pyrrolo [2,3-d] pyrimidin-2-yl] - Tion

Off white; Yield 86%; R _f : 0.53 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ (M, 2H, aromatic protons), 7.49-7.45 (m, 3H, aromatic protons), 7.29-7.17 (m, 1H, , Aromatic protons), 6.82-6.80 (m, 1H, aromatic protons), 6.24-6.22 (m, 1H, aromatic protons), 5.55 (s, 1H, NH); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 161.8, 156.4, 150.0, 135.1, 132.0, 131.8, 131.5, 125.5, 120.4, 120.2, 109.9.

(7E) -4 - [{(lH-pyrrol-2-yl) methylene} amino] -3- (2- methoxyphenyl) - Tion

Ivory; Yield 83%; R _f : 0.52 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 2H, aromatic protons), 7.27-7.11 (m, 3H, aromatic protons), 6.86-3.81 (m, 1H, , aromatic protons), 6.29 ~ 6.22 (m, 1H, aromatic proton), 5.59 (s, 1H, NH), 3.81 (s, 3H, OCH 3); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 162.0, 159.6, 156.4, 149.4, 132.0, 129.7, 125.5, 124.9, 122.5, 120.2, 115.8, 109.9, 55.5.

(7f) (E) -3- (3-fluorophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-

Brown; Yield 81%; R _f : 0.56 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, NH), 10.14 (s, 1H, imine proton), 7.86-7.50 (m, 3H, aromatic protomer), 7.46-7.11 (m, 5H, aromatic protomer), 2.99-2.90 , Aliphatic protons), 1.94 (d, 3H, J = 6.4, aliphatic protons); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 166.6, 151.4, 135.3, 131.8, 131.6, 125.5, 120.4, 114.4, 30.09, 18.4.

(7 g) (E) -3- (3-bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-

Antique white; Yield 82%; R _f : 0.56 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, aromatic protomer), 7.78-7.72 (m, 1H, aromatic protomer), 7.57-7.44 (m, 3H) , Aromatic protons), 7.32-7.20 (m, 4H, aromatic protons), 2.85-2.72 (m, 1H, aliphatic protons), 1.24 (d, 3H, aliphatic protons); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 162.0, 157.4, 151.4, 150.2, 136.9, 136.1, 135.3, 134.9, 133.3, 131.7, 129.0, 120.5, 30.5, 19.4.

(7h) (E) -3- (3- Methoxyphenyl ) -4 - [(2- Phenylethylidene ) Amino] -1H-1,2,4- Triazole -5 (4H) - Tion

purple; Yield 81%; R _f : 0.52 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, aromatic protons), 7.28-7.18 (m, 5H), 13.73 (s, 1H, NH), 10.16 , aromatic protons), 6.90 ~ 6.84 (m, 2H, aromatic protons), 4.02 (s, 2H, aliphatic), 3.96 (s, 3H, OCH 3); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 161.9, 158.6, 157.3, 152.1, 138.9, 136.9, 136.1, 134.2, 133.3, 131.4, 130.5, 129.0, 127.7, 114.3, 55.5, 30.3.

(4i?) - thione (7i) (Z) -4 - [(2- fluorobenzylidene) amino] -3- (4-fluorobenzyl)

cream color; Yield 83%; R _f : 0.54 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ (M, 2H, aromatic protons), 7.39-7.22 (m, 2H, aromatic protons), 7.31-7.27 (m, 2H, , aromatic protons), 7.22 ~ 7.11 (m, 2H, aromatic protons), 4.10 (s, 2H, aliphatic CH _2); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 163.3, 163.0, 162.4, 162.0, 159.6, 149.8, 131.9, 131.6, 129.8, 129.2, 124.9, 116.9, 115.8, 30.1.

(7j) (Z) -4 - [(2-fluorobenzyliden) amino] -3- (4- methoxyphenyl)

Light purple; Yield 81%; R _f : 0.51 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H), 7.99-7.91 (m, 2H, aromatic protons), 7.51-7.44 (m, 2H, aromatic protons), 7.22-7.13 (m, 2H, , aromatic protons), 6.89 ~ 6.80 (m, 2H, aromatic protons), 3.71 (s, 3H, OCH 3); ^{13 C NMR (100 MHz, DMSO} -d 6) δ 162.2, 160.8, 158.6, 152.1, 151.1, 131.6, 131.1, 130.4, 129.3, 127.7, 127.3, 116.9, 114.4, 55.5.

(7) (E) -3- (4-methoxybenzyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-

yellow; Yield 89%; R _f : 0.52 (chloroform: methanol, 9: 1); ^{1 H NMR (400 MHz, DMSO} -d 6) δ 1H, aromatic protons), 7.38-7.29 (m, 2H, m, 1H, aromatic protons), 7.79-7.77 (m, 1H, aromatic protons) , aromatic protons), 7.27 ~ 7.26 (m, 1H, aromatic proton), 7.26 ~ 7.13 (m, 2H, aromatic protons), 4.18 (s, 3H, OCH 3), 3.40 (s, 2H, aliphatic, CH ₂₎ ; ^{13 C NMR (100 MHz, DMSO} -d 6) δ 162.1, 159.6, 157.7, 149.5, 136.8, 136.2, 133.4, 131.9, 129.7, 128.9, 124.9, 122.3, 115.8, 55.5.

Structural analysis and correction

Data collection: APEX II (Bruker, 2005); Cell purification: SAINT (Bruker, 2005); Data organization: SAINT (Bruker, 2005); Structural Analysis Program: SHELXS97 (Sheldrick, 2008); Structural Correction Program: SHELXL97 (Sheldrick, 2008); Molecular Graphics: SHELXTL (Sheldrick, 2008); Calculations of molecular graphics and materials were performed by Ortep-3 (Farrugia, 2012) and Win GX publishing routines (Farrugia, 2012) for Windows. Crystallographic data are deposited with the supplementary publication number CCDC-1033154 at the Cambridge Crystallographic Data Center.

<Experimental Example>

[ Experimental Example  1: pharmacological analysis]

Preparation of receptor

A molecular operating environment (MOE 2014) by the Chemical Computing Group (CCG) was used in the present invention. Protein preparation steps include 3D protonation, energy minimization, and active site identification. Proteins were minimized and 3-D protonated using MOE's structural preparation module. Since the protein does not have a co-crystallized ligand, the MOE site finder module was used to identify the active site, and to find the active site, the two nickel ion binding chains were selected. The pocket was found to be a deep cleft containing both nickel ions and hydrophobic and hydrophilic amino acid residues in the interior.

Preparation of ligand

Ligand files for molecular docking studies were prepared in MOE-2014 and then energy optimization was performed at the standard MMFF 94 force field level using a 0.0001 kcal / mol energy gradient convergence criterion. Optimized geometries were stored in a molecular database (mdb) file for further study.

Urease inhibition assay protocol

Urease activity was determined by measuring the amount of ammonia produced by the indophenol method. That is, the assay was carried out by adding 10 ml of urease enzyme (5 U / ml) and 10 ml of the compound sample of the present invention to 40 ml of buffer (100 mM urea, 0.01 MK ₂ HPO ₄ , 1 mM EDTA and 0.01 M LiCl ₂ , pH 8.2) The mixture was incubated in a 96-well plate at 37 DEG C for 30 minutes. To each of the wells was added a phenol reagent (1%, phenol w / v and 0.005%, w / v sodium nitroprusside) and 40 ml of an alkaline reagent (0.5%, w / v NaOH and 0.1% Respectively. Absorbance at 625 nm was measured after 30 minutes using a microplate reader (OPTI _Max , Tunable). All reactions were performed three times, and thiourea was used as a standard inhibitor of urease.

Kinetic analysis for urease inhibition assay

The inhibition type was determined using a line Weber-Burk plot of 1 / urea versus 1 / absorbance. The urease inhibition rate was determined by varying the urea concentration in the presence of the test compound (7b) at different concentrations (0, 13.7, 27.5 and 55 mM). On the other hand EI dissociation constant (K _i) is determined by a secondary re-plot of the slope against inhibitor concentration, ESI dissociation constant (K _{i `)} is the X-axis crossing of the secondary re-plot of 1 / V versus inhibitor concentration (line Section of the Weber-Burk plot). That is, the same as that described in the urease inhibition assay protocol, except that the urea concentration was changed from 3.12 mM to 100 mM. Urease activity was determined by measuring the ammonia production rate using the indophenol method described above. The results (change in absorbance per minute) were processed using SoftMaxPro software.

Study of molecular docking for urease

The optimized ligand was docked with soybean urease (PDB code: 3AL4) protein using the MOE-Dock program. A total of 30 independent docking runs were performed using the MOE docking simulation program. All docked structures were analyzed and the structure with the highest point for each compound was selected for further study. 2D ligand-protein interactions were visualized using a MOE ligand interaction program. We created 30 structures for each compound using two algorithms with rescoring capabilities: the default MOE docking parameters Triangle Matcher, London dG and GB vi / WSA dG. As a result of the docking run, the mdb output file was generated containing all the docking results and several structures of the ligand.

[Results and discussion]

Synthesis of Target Compounds 7a-7k

Conversion of the substituted carboxylic acid (1) to the substituted acid chloride (2) was found by the disappearance of the gentle signal in the range of 3400-2500 cm ^-1 due to the acidic hydroxyl group in the FT-IR spectral data. In addition, new signals of 3346, 3296 and 3156 cm ^-1 due to the primary and secondary amino groups of the acid hydrazide appeared in the IR spectrum by the formation of the substituted acid hydrazide (3). On the other hand, the carbonyl stretching vibration 1634cm ^-1 Slight change in at 1733cm ^-1, indicating that a successful transition to a substituted acid chloride disubstituted aromatic acid hydrazide 3. ^{In the 1} H NMR spectrum, the appearance of broad monomers due to primary and secondary amino groups at 9.23 ppm and 4.25 ppm, respectively, further confirms the successful synthesis of the aromatic acid hydrazide (3).

The formation of the substituted triazole compound (6) was analyzed by FT-IR, ¹ H NMR and ¹³ C NMR spectral data. In the FT-IR spectrum, the appearance of a relatively broad peak in the range of 3280, 3169 cm ^-1 for the NH ₂ stretching vibration, and a new signal at 1321 cm ^-1 for the C = S stretching vibration, ) To the substituted triazole compound (6). Further, in the ¹ H NMR, the formation of the substituted triazole compound (6) was confirmed by the specific down-field signal at 13.57 ppm due to secondary amino proton resolution of the triazole ring. The condensation of the different substituted aldehydes with the substituted triazole compound (6) was confirmed by ¹ H NMR and ¹³ C NMR spectral analysis. Schiff base formation was confirmed by slight down-field shift of the NH signal from 13.57 ppm to 13.88 ppm and complete disappearance of the NH ₂ signal at 5.60 ppm.

On the other hand, the presence of the down-field single line in the range of 10.03 to 10.18 ppm by the immune proton and further the appearance of additional signals in both the carbon and proton NMR spectra, 7k, respectively.

Compounds 7a to 7k Tion - Thiol Tautomer

Compounds 7a-7k of the present invention have a hydrogen atom that is liable to 1,2,4-triazole nucleus, which hydrogen atom is on a ring nitrogen atom, or a sulfur atom bonded to a 5-membered ring structure . Due to this nature of the hydrogen atom, it is theoretically possible to have two tautomeric stereostructures as in the following scheme.

[Reaction Scheme 2]

In the FT-IR spectral analysis, compounds 7a-7k clearly showed signs of thionotomer structure because there was no signal in the range of 2500-2400 cm ^-1 due to the terminal thiol (-SH) stretching vibration.

In order to analyze the main isomer of the Schiff base, single crystal X-ray diffraction analysis was performed on Compound 7k in the above compound to obtain single crystal data as shown in Table 1 below. As a result, it was found that the compound 7k mainly exists as a thione tautomer structure represented by the following formula (2).

X-ray diffraction analysis data on compound 7k division Experimental value division Experimental value Empirical formula C ₁₅ H ₁₄ N ₄ OS ₂ Cell formula unit, z 4 Chemical food 330.42 Density (calculated value) 1.374 mg / m ³ Temperature 296 (2) K Extinction coefficient 0.340 m ² / M Crystal system Monoclinic system F (000) 688 Space group P 21 / c Minimum theta value 2.37˚ a 12.330 (8) Å Maximum theta value 28.01 b 7.540 (5) Å Color yellow c 18.134 (12) Å Maximum crystal size 0.20 mm ³ alpha 90 (1) ° Intermediate crystal size 0.18 mm ³ beta 108.713 (9) Minimum crystal size 0.15 mm ³ gamma 90 (1) ° Diffraction wavelength 0.71073 nm
Cell volume 1596.9 (18) Å ³
Calibration method Full-matrix least squares for F ²

The packing diagram of compound 7k shows that a molecular chain parallel to the short x-axis is formed through a typical inversion-symmetric H bonding system (Fig. 4). The main structure of the NH-C = S tautomer was confirmed by the relative size of the molecules involved and the direct localization and free correction of the NH 3 hydrogen. In the crystal structure, intermolecular N-H ... S hydrogen bonding is believed to have the effect of stabilizing the crystal by linking 7k molecules of the compound to an endless chain (Fig. 4). The bonding length (Å) and the bonding angle (°) of the hydrogen bond of the compound 7k are shown in Table 2 below.

The bonding length (Å) and bonding angle (°) of the hydrogen bond of the compound 7k D-H ... A d (D-H) &lt; RTI ID = 0.0 &gt; [A] d (H ... A) [A] d (D ... A) [A] <(DHA) [°] N (3) -H (3) S (13) # 1 0.85 (2) 2.48 (2) 3.323 (2) 170.8 (19)

Symmetric transformations used to generate equivalent atoms: # 1-x, -y + 1, -z

Geometrical structure of compound molecules

The crystals for X-ray diffraction analysis were obtained by slow evaporation of a 7 k methanol solution at room temperature and the single crystal data were analyzed using a Bruker SMART APEX II X-ray tube equipped with a molybdenum (Mo) X-ray tube, a graphite monochromator and a CCD detector, Ray diffractometer using 100 (1) K (Table 1). The H atom of the NH group was freely calibrated and the methyl group was corrected to an ideal functional group that is rotatable but not tip; Other H atoms were included using a riding group. The final wR2 was 0.1038 and 0.1111 for all reflections and the R1 values are typically 0.0410 and 0.0488. Experimental structural parameters including bond length and bond angle are summarized in the following Tables 3 and 4, and thus the atomic numbered structure is shown in FIG.

Briefly, in compound 7k, the N (1) -C (5) and N (1) -C (2) bond lengths are 1.378 (2) and 1.3811 , Suggesting that electron clouds are delocalized within the triazole ring. On the other hand, the bond length of C (2) -S (13) is 1.6795 (18) Å, which indicates that the bond length of C-SH is 1.80 Å and C = S characteristics. This fact is a strong indication that the molecule is involved in thion-thiol tautomerization, but that the main structure of compound 7k is a thione structure.

The bond length [Å] in compound 7k Combination Coupling length [Å] Combination Coupling length [Å] N (1) -C (5) 1.378 (2) C (12) -H (12) 0.9300 N (1) -C (2) 1.3811 (19) C (14) -C (15) 1.520 (2) N (1) -N (6) 1.4037 (18) C (14) -H (14A) 0.9700 C (2) -N (3) 1.338 (2) C (14) -H (14B) 0.9700 C (2) -S (13) 1.6795 (18) C (15) -C (16) 1.386 (2) N (3) -N (4) 1.377 (2) C (15) -C (20) 1.391 (2) N (3) -H (3) 0.85 (2) C (16) -C (17) 1.387 (3) N (4) -C (5) 1.299 (2) C (16) -H (16) 0.9300 C (5) -C (14) 1.496 (2) C (17) -C (18) 1.386 (3) N (6) -C (7) 1.281 (2) C (17) -H (17) 0.9300 C (7) -C (8) 1.439 (2) C (18) -O (21) 1.362 (2) C (7) -H (7) 0.9300 C (18) -C (19) 1.391 (3) C (8) -C (12) 1.370 (2) C (19) -C (20) 1.376 (3) C (8) -S (9) 1.7242 (17) C (19) -H (19) 0.9300 S (9) -C (10) 1.703 (2) C (20) -H (20) 0.9300 C (10) -C (11) 1.345 (3) O (21) -C (22) 1.412 (3) C (10) -H (10) 0.9300 C (22) -H (22A) 0.9600 C (11) -C (12) 1.415 (3) C (22) -H (22B) 0.9600 C (11) -H (11) 0.9300 C (22) -H (22C) 0.9600

The remaining bond length of the triazole skeleton is N (4) -C (5); 1.299 (2), C (2) -N (3); 1.338 (2), N (3) -N (4); 1.377 (2) and N (3) -H (3); 0.85 (2) A. The bond length value for N (6) -C (7) is 1.281 (2), while the bond length for N (1) -N (6) shows a single bond characteristic corresponding to 1.4037 Which is a double bond structure, which is connected through the formation of an imine bond between the triazole skeleton and the side chain-bonded functional group. In the same way, the combined lengths of C (8) -S (9) and S (9) -C (10) are 1.7242 (17) and 1.703 (2), respectively, .

On the other hand, the binding length to the residue of the remaining thiophene skeleton, i.e. C (8) -C (12); 1.370 (2), C (10) -C (11); 1.345 (3), C (11) -C (12); 1.415 (3) showed a slight deviation from the standard bond length for single and double bonds, which is due to the fragmentation of the electron cloud within the thiophene moiety. The bond length of the remaining molecules was within the normal range as shown in Table 3 above.

The bonding angle [deg.] In the compound 7k [ Combination Coupling angle [°] Combination Coupling angle [°] C (5) -N (1) -C (2) 108.62 (12) C (5) -C (14) -H (14A) 109.3 C (5) -N (1) -N (6) 122.02 (12) C (15) -C (14) -H (14A) 109.3 C (2) -N (1) -N (6) 128.33 (13) C (5) -C (14) -H (14B) 109.3 N (3) -C (2) -N (1) 102.57 (13) C (15) -C (14) -H (14B) 109.3 N (3) -C (2) -S (13) 128.50 (12) H (14A) -C (14) -H (14B) 108.0 N (1) -C (2) -S (13) 128.92 (11) C (16) -C (15) -C (20) 117.75 (16) C (2) -N (3) -N (4) 114.00 (14) C (16) -C (15) -C (14) 121.11 (15) C (2) -N (3) -H (3) 126.2 (14) C (20) -C (15) -C (14) 121.09 (15) N (4) -N (3) -H (3) 119.7 (14) C (15) -C (16) -C (17) 122.07 (16) C (5) -N (4) -N (3) 104.23 (13) C (15) -C (16) -H (16) 119.0 N (4) -C (5) -N (1) 110.55 (13) C (17) -C (16) -H (16) 119.0 N (4) -C (5) -C (14) 125.40 (15) C (18) -C (17) -C (16) 119.17 (16) N (1) -C (5) -C (14) 124.04 (14) C (18) -C (17) -H (17) 120.4 C (7) -N (6) -N (1) 114.93 (12) C (16) -C (17) -H (17) 120.4 N (6) -C (7) -C (8) 119.99 (13) O (21) -C (18) -C (17) 124.93 (17) N (6) -C (7) -H (7) 120.0 O (21) -C (18) -C (19) 115.55 (16) C (8) -C (7) -H (7) 120.0 C (17) -C (18) -C (19) 119.52 (17) C (12) -C (8) -C (7) 126.60 (14) C (20) -C (19) -C (18) 120.45 (17) C (12) -C (8) -S (9) 110.87 (12) C (20) -C (19) -H (19) 119.8 C (7) -C (8) -S (9) 122.53 (12) C (18) -C (19) -H (19) 119.8 C (10) -S (9) -C (8) 91.57 (9) C (19) -C (20) -C (15) 121.05 (16) C (11) -C (10) -S (9) 112.46 (14) C (19) -C (20) -H (20) 119.5 C (11) -C (10) -H (10) 123.8 C (15) -C (20) -H (20) 119.5 S (9) -C (10) -H (10) 123.8 C (18) -O (21) -C (22) 118.44 (17) C (10) -C (11) -C (12) 112.68 (16) O (21) -C (22) -H (22A) 109.5 C (10) -C (11) -H (11) 123.7 O (21) -C (22) -H (22B) 109.5 C (12) -C (11) -H (11) 123.7 H (22A) -C (22) -H (22B) 109.5 C (8) -C (12) -C (11) 112.42 (15) O (21) -C (22) -H (22C) 109.5 C (8) -C (12) -H (12) 123.8 H (22A) -C (22) -H (22C) 109.5 C (11) -C (12) -H (12) 123.8 H (22B) -C (22) -H (22C) 109.5 C (5) -C (14) -C (15) 111.56 (13) H (22B) -C (22) -H (22C) 109.5

The binding angle of the molecule 7k was within the normal range. O (21) -C (22) -H (22A), O (21) -C (22) -H (22B) 21) -C (22) -H (22C), H (22A) -C (22) -H (22C) The coupling angle was 109.5 °, indicating that it was a tetrahedron.

All bonding angles of the benzene ring were within the normal range, and the bonding angle of the triazole ring showed slight deviation because of the strain of the 5-member ring structure. The coupling angle of C (5) -N (1) -C (2) showed a higher distortion of 108.62 (12) close to the tetrahedral structure, ) Was smaller at 102.57 (13). The internal bond angle of the triazole nucleus is C (2) -N (3) -N (4); 114.00 (14) and C (5) -N (4) -N (3); 104.23 (13), which indicates the external coupling angle to the triazole nucleus, C (5) -N (1) -N (6); 122.02 (12), C (2) -N (1) -N (6); 128.33 (13), N (3) -C (2) -S (13); 128.50 (12), N (1) -C (2) -S (13); 128.92 (11), C (2) -N (3) - H (3); 126.2 (14), N (4) - N (3) - H (3); 119.7 (14), N (4) -C (5) -C (14); 125.40 (15) and N (1) -C (5) -C (14); 124.04 (14).

The coupling angle of C (7) -N (6) -N (1) is 114.93 (12) which is slightly smaller than triangular structure. On the other hand, since the thiophene skeleton has ring strain, a very small bonding angle of 91.57 (9) was observed in C (10) -S (9) -C (8).

Similarly, the shortening of the internal coupling angle was observed for the thiophene nucleus. C (11) -C (10) -S (9); 112.46 (14), C (8) -C (12) -C (11); 112.42 (15) and C (11) -C (10) -S (9); 112.46 (14). On the other hand, the external coupling angle is C (11) -C (10) -H (10); 123.8, S (9) -C (10) -H (10); 123.8, C (10) -C (11) -H (11); 123.7, C (12) -C (11) -H (11); 123.7, C (8) -C (12) -H (12); 123.8, C (11) -C (12) -H (12); 123.8 and C (7) -C (8) -S (9); 122.53 (12), which is close to the coupling angle of the triangular structure. The binding angles for the remaining molecules were within the normal range, as shown in Table 4 above.

[ Experimental Example  2: Biological evaluation]

The urease inhibitory activity of the compound 7a-7k

The synthesized Compound 7a-7k (see Chemical Formula 1) was evaluated for urease inhibitory activity using thiourea (IC ₅₀ value 20.9 ± 0.92 mM) as a reference inhibitor. All IC ₅₀ values were recorded three times and the data are expressed as mean ± SEM (mean standard error, n = 3).

[Chemical Formula 1]

Results of urease inhibition test on Compound 7a-7k
Comparative Example
R
Y
n Urease inhibition test
(IC ₅₀ ± ^a SEM μM) 7a 2-F 2-thiophenyl 0 42.59 + - 4.35 7b 2-Br 2-thiophenyl 0 17.02 + - 4.63 7c 2-OCH ₃ 2-thiophenyl 0 39.30 ± 5.03 7d 2-F 2- 0 186.8 ± 16.95 7e 2-OCH ₃ 2- 0 35.71 ± 6.28 7f 3-F 2-ethylphenyl 0 77.34 + - 7.92 7g 3-Br 2-ethylphenyl 0 8.02 ± 0.59 7h _3-OCH 3 benzyl 0 20.29 + - 2.76 7i 4-F 2-fluorophenyl One 159.15 + 21.38 7j 4-OCH ₃ 2-fluorophenyl 0 25.45 ± 3.09 7k 4-OCH ₃ 2-thiophenyl One 52.54 + - 3.21 ^b thiourea ^b 20.9 ± 0.92

^a SEM = standard error of mean; These values are expressed as mean ± SEM of three individual measurement results.

^b urea inhibition test standard.

As shown in Table 5, compounds 7g and 7b had the IC ₅₀ values of 8.02 ± 0.59 and 17.02 ± 4.63 mM, respectively, and were most potent against thiourea as a reference inhibitor. Among them, compound 7b was used for kinetic analysis for tracking the mechanism of action. Compound 7b exhibited a mixed inhibition pattern with EI and ESI dissociation constants of 3 and 42 mM, respectively, in a line Weber-Burk plot.

Further, the compounds 7g and 7b have 2-ethylphenyl and 2-thiophenyl groups as substituents Y and 3-bromo and 2-bromo groups as substituents R, respectively. There was no extra carbon between the triazole skeleton and the side-bonded aryl groups.

Compound 7h has an IC ₅₀ value of 20.29 ± 2.76 mM, indicating the possibility of urease inhibition similar to that of the reference thiourea. Compound 7h has a 3-OCH ₃ group as a substituent R and a benzyl group as a substituent Y. From this, it was found that the urease inhibition activity was reduced by substituting the bromo group at the 3-position of the aryl ring with another substance.

On the other hand, compound 7j, 7e, 7c and 7a are brought to each 25.45 ± 3.09, 35.71 ± 6.28, 39.30 ± 5.03 and 42.59 ± 4.35 mM of IC ₅₀ values, showed a moderate urease inhibitory activity. In both of these compounds, there was no substitution of the bromo group of the side chain-bonded aryl ring. The activity of the remaining compounds ranged from 77.34 占 7.92 to 186.8 占 .95 mM.

Compound 7d had an IC ₅₀ value of 186.8 ± 16.95 mM, which was the lowest among the compounds. This compound has a fluoro group at the 2-position of the aryl ring and a 2-pyrroyl group as the substituent Y.

As for the relationship between the inhibition of the substituents and the overall structural activity of the synthesized compound, the 2-ethylphenyl group bonded through the imine formation with the central triazole nucleus, as well as the bromo substituent at the 3 and 2 positions of the aryl ring, Thereby imparting strong inhibitory properties to the compound. Methoxy substitution on the side chain bonded aryl ring leads to modest inhibition while a strong electron withdrawing fluorine substituent on the side chain bonded aryl ring has been shown to induce minimal inhibitory activity.

On the other hand, as the substituent Y bonded to the central triazole skeleton through imine bond, the 2-ethylphenyl group gives the urease inhibition property, but the 2-pyrroyl group seems to decrease the inhibitory activity of the compound.

Inhibition Mechanism of Compound 7b

Kinetic studies for urease inhibition assays were performed by a 1 / [S] versus 1 / V line Weber-Burk plot in the presence of different concentrations of inhibitor 7b (see FIG. 5).

In addition, the EI dissociation constants (K _i ) and the ESI dissociation constants (K _{i `} ) were determined to obtain the path by the second re-plot. When increasing the concentration of the substrate (urea), a linear series intersecting within the second quadrant was obtained.

As the concentration of compound (7b) increased, V _max decreased as K _m increased. From this, it can be seen that the K _i and K _{i `} values of the compound (7b) are 3 and 42 mM, respectively (FIGS. 5B and 5C), and the compound 7b is an inhibitor of the mixed type for urease there was.

Study of molecular docking for urease

Molecular docking of the 7a-7k series on the crystal structure of urease from soybean koji was carried out using the MOE program. The most advantageous docking structure of all compounds was superimposed within the cavity of the active site, which has both hydrophobic and hydrophilic amino acids. The hydrophobic portion comprises Leu 523, Ala 636, Ala 440, Met 637 and Met 588 while the hydrophilic portion comprises Arg 439, His 492, Asp 494, His 519, His 593, Glu 493 and Arg 609 amino acids. It has been observed that two Ni ions play an important role by bridgeing the amino acid residues within the active site.

Thirty structures of the structural ligands were all docked in the pocket, despite the differences in binding energy level and interaction with the amino acid residues within the active site. The docked poses were prioritized based on the combination of free energy and ligand interaction. MOE ranked the docked structures on the basis of the bond energy (London dG) and the bond free energy in the "S" region. Detailed docking results are summarized in Table 6, which is based on the two-dimensional interaction of the most preferred docking structure. All of the preferred docking structures were inside the cavity as shown in FIG.

The most preferred binding mode of the docking structure showed that the ligands 7b, 7g and 7h were tightly immobilized in the cavity of the active site via hydrogen bonding. Very significant hydrogen bond interactions with Met 637, Arg 439 and Met 588 were observed. Ni ions seem to play an important role in the orientation of residues near the active site through chelation.

Figure 7 shows the three-dimensional interactions of the most important inhibitors, compounds 7b, 7g and 7h. These ligands were hydrogen bonded to the active site residues and tightly immobilized on the side chain and backbone of the urease enzyme.

The phenyl ring was found to be involved in the arene-H interaction in the case of the most active ligand. Only ligand 7c showed arene-arene stacking. All inhibitors exhibited the binding free energy of a narrow range of S regions (-5.0312 to -6.0760 kcal / mol) and the binding energy of London dG of the Escore-1 region (-8.5129 to -9.7104 kcal / mol).

Compounds that do not exhibit significant interactions such as compounds 7i and 7d have been found to be inert in experimental screening. Compounds 7b, 7g and 7h showed significant interactions with the amino acid residues of the active site and found that they exhibit much better activity than the reference drug in experimental screening. This may be due to the strong binding interaction of a number of these ligands with the amino acid residues of soybean curd urease compared to the reference drug thiourea. The docking modes of the most potent compounds 7b, 7g and 7h inhibited the catalytic activity of urease by tightly immobilizing through strong hydrogen bonding, Pi-H interaction and very strong polar interaction of the active site.

Considering the specificity of the active site residues in urease catalysis, some of the 7a-7k compounds make strong contact with the side chains of the urease residues, which results in some of the 7a-7k compounds Arg 439, Ala 636, Ala 440, Met 637, His 592, Asp 494, Arg 439, Met 588 and Met 637 residues.

Nickel ions not only directly participate in the structure of the active site but also play an important role in positioning other major residues of the active site to catalyze through various interactions such as hydrogen bonding and chelation .

compound

IC ₅₀
(μM) Bonding energy
(kcal / mol) Hydrogen bond Areen-H
Interaction Arend-Arlen
Hydrophobic interaction
Action
polarity
Mutual
Action
S London dG Interaction residue Street
(A) Interaction residue Street
(A) Interaction residue Street
(A)
7a
42.59
-5.2136
-9.0120
-
-
-
-
-
- Ala636
Ala440
Ala436 Gln635
Arg439
7b
17.02
-10.1855
-8.5129
Arg439
3.6
Ala440
4.36
- Ala636
Ala440
Met637 Arg439
His593
Gln635
7c
39.30
-5.1051
-8.6355
Arg439
4.26
Arg439
3.54
His539
3.99 Ala636
Ala440
Ala436
Met637 His593
Asp494
Arg439
His594
7d
186.8
-5.0632
-8.8014
-
-
-
-
-
- Ala636
Ala440
Ala436 Arg439
Gln635

7e
35.71
-5.4208
-8.8897
-
His594
4.71
-
- Ala636
Ala440
Met637 Arg439
Gln635
His594
7f
77.34
-5.5278
-8.6227
Arg439
2.86
Arg439
3.98
-
- Ala636
Ala440
Met637 His593
Asp494
Arg439
Gln635

7g

8.02

-5.6639

-9.2664
Met637
CME92
Arg439
4.15
3.42
2.92
Arg439
Arg609
2.42
4.86


-

-
Ala636
Ala440
Met637 His593
Asp494
Arg439
Gln635
Asp494
7h
20.29
-6.0760
-9.7104 Arg609
Arg609
Met637 4.4
4.11
4.07
His593
3.93
-
-
Ala440 His593
Asp494
Arg439
Gln635
7i
159.15
-5.1402
-9.0249
-
-
-
-
-
- Ala636
Ala440
Met637 His594
Arg439
Gln635
7j
25.45
-5.2134
-9.5137 Arg609
His593
4.2
3.95
-
-
-
- Ala636
Ala440 His593
Asp494
Arg439
7k
52.54
-5.1353
-9.1899
Arg439
2.91
Arg439
-
- Ala636
Ala440
Met637 His593
Arg439
Gln635 Thiourea
20.9
-2.3530
-5.9201 Asp494
Arg609 3.15
3.02
3.68
-
-
-
-
Ala440
His593

conclusion

The synthesized Schiff base compound 7a-7k of the present invention was analyzed for its structure by FT-IR, ¹ H NMR, ¹³ C NMR and single crystal X-ray diffraction analysis. Among them, Compound 7k has a predominant thione structure in the thione-thiol tautomer structure according to the X-ray single crystal structure. As a result of the bioactivity and kinetics analysis of the synthesized compounds, the Schiff base compounds 7a-7k of the present invention showed a considerable possibility as a urease inhibitor.

Compounds 7g and 7b in the above compounds exhibited excellent urease inhibitory activity even when compared with thiourea (IC ₅₀ 20.9 ± 0.92 mM), which is a reference material, with IC ₅₀ values of 8.02 ± 0.59 mM and 17.02 ± 4.63 mM, respectively. Compound 7b had EI and ESI dissociation constants of 3 and 42 mM, respectively, and showed a mixed inhibition pattern as a result of analysis of the reaction rate through a line weber-buck plot. The compound 7h having the highest urease inhibitory activity as the third of the above compounds has an IC ₅₀ value of 20.29 ± 2.76 mM, which is similar to the inhibitory activity of thiourea as a reference substance. On the other hand, compound 7j, 7e, 7c and 7a are brought to each 25.45 ± 3.09 mM, 35.71 ± 6.28 mM, 39.30 ± 5.03 mM and 42.59 ± 4.35 mM in the IC ₅₀ value and to exhibit a suitable urease inhibitory activity. The 1,2,4-triazole-Schiff base compound of the present invention showed superior urease inhibitory activity as compared with the conventional triazole-based molecule or Schiff base compound which was not directly imine-coupled with the triazole skeleton. Further, as a result of examining the urease inhibition method by the above-mentioned compounds 7a-7k, it can be seen that the compounds 7b, 7g and 7h are competitive inhibitors for targeting the amino acid of the urease active site.

Claims (9)

delete delete delete delete Acute gastritis, chronic gastritis, gastric ulcer, duodenal ulcer caused by Helicobacter pylori comprising as active ingredients a compound selected from the group consisting of the following 1,2,4-triazole compounds, or a pharmaceutically acceptable salt thereof: , Gastric cancer, and gastric lymphoma. The pharmaceutical composition for treating or preventing at least one gastric disease selected from the group consisting of:
(7a) (E) -3- (2-Fluorophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7b) (E) -3- (2-bromophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7c) (E) -3- (2-Methoxyphenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(4H) -lH-pyrrolo [2,3-d] pyrimidin-2-yl] - thion;
(7E) -4 - [{(lH-pyrrol-2-yl) methylene} amino] -3- (2- methoxyphenyl) - thion;
(7f) (E) -3- (3-Fluorophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7 g) (E) -3- (3-Bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7h) (E) -3- (3-methoxyphenyl) -4 - [(2-phenylethylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7i) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-fluorobenzyl) -1H-1,2,4-triazole-5 (4H) -thione;
(7j) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-methoxyphenyl) -1H-1,2,4-triazole-5 (4H) -thione; And
(7k) (E) -3- (4-methoxybenzyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazol-5 (4H) -thione. A pharmaceutical composition for the prevention or treatment of kidney stone comprising, as an active ingredient, a compound selected from the group consisting of the following 1,2,4-triazole compounds, or a pharmaceutically acceptable salt thereof:
(7a) (E) -3- (2-Fluorophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7b) (E) -3- (2-bromophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7c) (E) -3- (2-Methoxyphenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(4H) -lH-pyrrolo [2,3-d] pyrimidin-2-yl] - thion;
(7E) -4 - [{(lH-pyrrol-2-yl) methylene} amino] -3- (2- methoxyphenyl) - thion;
(7f) (E) -3- (3-Fluorophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7 g) (E) -3- (3-Bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7h) (E) -3- (3-methoxyphenyl) -4 - [(2-phenylethylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7i) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-fluorobenzyl) -1H-1,2,4-triazole-5 (4H) -thione;
(7j) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-methoxyphenyl) -1H-1,2,4-triazole-5 (4H) -thione; And
(7k) (E) -3- (4-methoxybenzyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazol-5 (4H) -thione. delete A fertilizer composition for enhancing soil soil strength comprising a compound selected from the group consisting of the following 1,2,4-triazole compounds or a pharmaceutically acceptable salt thereof as an active ingredient:
(7a) (E) -3- (2-Fluorophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7b) (E) -3- (2-bromophenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7c) (E) -3- (2-Methoxyphenyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(4H) -lH-pyrrolo [2,3-d] pyrimidin-2-yl] - thion;
(7E) -4 - [{(lH-pyrrol-2-yl) methylene} amino] -3- (2- methoxyphenyl) - thion;
(7f) (E) -3- (3-Fluorophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7 g) (E) -3- (3-Bromophenyl) -4 - [(2-phenylpropylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7h) (E) -3- (3-methoxyphenyl) -4 - [(2-phenylethylidene) amino] -1H-1,2,4-triazole-5 (4H) -thione;
(7i) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-fluorobenzyl) -1H-1,2,4-triazole-5 (4H) -thione;
(7j) (Z) -4 - [(2-fluorobenzylidene) amino] -3- (4-methoxyphenyl) -1H-1,2,4-triazole-5 (4H) -thione; And
(7k) (E) -3- (4-methoxybenzyl) -4 - [(thiophen-2-ylmethylene) amino] -1H-1,2,4-triazol-5 (4H) -thione. delete

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