Piperazine derivative as well as preparation method and application thereof

Abstract

The invention relates to a piperazine derivative as well as a preparation method and application thereof. The structure of the piperazine derivative is a compound as shown in a formula (I) and a stereoisomer, a tautomer or pharmaceutically acceptable salt thereof, wherein in the formula (I), a ring A is selected from aryl R7 or heteroaryl R8; r1 is selected from hydrogen, C1-4 alkyl, cycloalkyl R9, aryl R10, heteroaryl R11 or heterocyclic group R12; r2 is selected from hydrogen, halogen, nitryl, cyano, C1-4 alkyl or C1-4 alkoxy; and R3 is selected from an aryl group R13, a heteroaryl group R14, a cycloalkyl group R15 or a heterocyclic group R16. The invention also provides a preparation method of the piperazine derivative provided by the invention. The invention also provides an application of the piperazine derivative provided by the invention in preparation of drugs for resisting eimeria and / or toxoplasma gondii. The problems that toxoplasma gondii resisting drugs are deficient, and existing coccidiosis resisting drugs are high in drug resistance are solved. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a piperazine derivative and a preparation method and application thereof. Background Art

[0002] In the field of public health and animal husbandry, parasitic diseases, especially those caused by Toxoplasma gondii and Eimeria, pose a severe challenge. As a common zoonotic parasite, Toxoplasma gondii poses a great threat to pregnant humans and animals and people with low immunity. Currently, treatments such as pyrimethamine + sulfadiazine, trimethoprim + sulfamethoxazole, and spiramycin are widely used in clinical practice. Although they can control the clinical symptoms of toxoplasmosis to a certain extent, they have no effect on latent infection. And they are accompanied by significant side effects, such as easily causing allergies in patients, suppressing patients' bone marrow, and generally poor tolerance. Therefore, the development of a new anti-Toxoplasma drug that can treat the latent infection stage and significantly reduce toxic side effects is of great significance to ensuring human public health safety and the healthy development of animal husbandry.

[0003] Chicken coccidiosis is a parasitic protozoan disease caused by Eimeria coccidia characterized by severe intestinal damage. It has become a major disease in the poultry industry. This disease not only significantly reduces the growth performance and feed conversion rate of broiler chickens, but also easily causes diarrhea and even death, causing more than 10 billion US dollars in economic losses to the global poultry industry each year. At present, the prevention and control of Eimeria coccidiosis mainly rely on anticoccidial drugs and live attenuated oocyst vaccines for chicken coccidiosis. However, due to the abuse and inefficient use of drugs, clinical strains have generally developed resistance to a variety of existing drugs, and the existing live oocyst vaccines are also insufficient in their protective efficacy when facing specific coccidia species such as Eimeria toxicans. This situation urgently requires the development of new, safer and more efficient prevention and control methods. Summary of the invention

[0004] The purpose of the present invention is to provide a piperazine derivative and a preparation method and application thereof, so as to solve the problems of shortage of anti-Toxoplasma drugs and strong drug resistance of existing anticoccidial drugs.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0006] A piperazine derivative, the structure of which is a compound represented by formula (I) and its stereoisomers, tautomers or pharmaceutically acceptable salts thereof:

[0007]

[0008] In formula (I),

[0009] Ring A is selected from aromatic R 7or heteroaryl R 8 ;

[0010] R 1 Selected from hydrogen, C 1-4 Alkyl, cycloalkyl R 9 , aryl R 10 , heteroaryl R 11 or heterocyclic group R 12 ;

[0011] R 2 Selected from hydrogen, halogen, nitro, cyano, C 1-4 Alkyl or C 1-4 Alkoxy;

[0012] R 3 Selected from aryl R 13 , heteroaryl R 14 , cycloalkyl R 15 or heterocyclic group R 16 .

[0013] Preferably, the aromatic group R 7 or heteroaryl R 8 There are 0 to 5 substituents R 4 ; The substituent R 4 is selected from hydrogen, halogen, cyano, hydroxy, amino, nitro, trifluoromethyl, trifluoromethoxy, C 1-4 Alkyl or C 1-4 Alkoxy.

[0014] According to the above technical means, the piperazine derivatives of the present invention have a skeleton of quinazolinone and its analogs, and have the advantages of good water solubility, low cytotoxicity, and high in vitro and in vivo anticoccidial and anti-Toxoplasma activities through experimental determination, thus solving the problems of strong drug resistance of existing anticoccidial drugs and the shortage of anti-Toxoplasma drugs.

[0015] Preferably, the C 1-4 Alkyl, cycloalkyl R 9 , aryl R 10 , heteroaryl R 11 , heterocyclic group R 12 There is at least one substituent R 5 ; The substituent R 5 Selected from aryl R 17 , halogenated aryl, cycloalkyl aryl, halogenated alkyl aryl, halogenated alkoxy aryl, heterocyclic group R 18 , halogenated heterocyclic group, heteroaryl R 19, halogenated heteroaryl, halogenated alkyl heteroaryl, halogenated alkoxy heteroaryl, alkyl formyl, aryl formyl, halogenated aryl formyl, heteroaryl formyl, halogenated heteroaryl formyl, halogenated arylsulfonyl, hydroxyalkyl, hydroxyalkynyl, amino, aminoalkyl, hydrogen, halogen, carboxyl, ester, cyano, C 1-4 Alkyl or C 1-4 Alkoxy.

[0016] Preferably, the aromatic group R 13 , heteroaryl R 14 , cycloalkyl R 15 , heterocyclic group R 16 There is at least one substituent R 6 ; The substituent R 6 Selected from aryl R 20 , halogenated aryl, cycloalkyl aryl, halogenated alkyl aryl, halogenated alkoxy aryl, heterocyclic group R 21 , halogenated heterocyclic group, heteroaryl R 22 , halogenated heteroaryl, halogenated alkyl heteroaryl, halogenated alkoxy heteroaryl, alkyl formyl, aryl formyl, halogenated aryl formyl, heteroaryl formyl, halogenated heteroaryl formyl, halogenated arylsulfonyl, hydroxyalkyl, hydroxyalkynyl, amino, aminoalkyl, hydrogen, halogen, carboxyl, ester, cyano, C 1-4 Alkyl or C 1-4 Alkoxy.

[0017] Preferably, the pharmaceutically acceptable salt is a salt formed by an acid and a nitrogen having a lone pair of electrons in compound (I).

[0018] Preferably, the acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, trifluoroacetic acid, tartaric acid, lactic acid, maleic acid, fumaric acid, malic acid or citric acid.

[0019] Preferably, the cycloalkyl group R 9 and cycloalkyl R 15 is a monocyclic or bicyclic hydrocarbon group having 3 to 10 ring carbon atoms, i.e., C 3-10 Cycloalkyl, the bicyclic hydrocarbon group has a cycloalkyl portion consisting of two saturated carbon rings having two atoms in common.

[0020] Preferably, the aromatic group R 7 , aryl R 10 , aryl R 13 , aryl R 17 and aromatic R 20 It is a monocyclic, bicyclic or tricyclic carbon ring system, at least one of which is aromatic, and the whole is C with 6 to 14 ring members. 6-14 -Aryl.

[0021] Preferably, the heteroaryl group R 8, heteroaryl R 11 , heteroaryl R 14 , heteroaryl R 19 and heteroaryl R 22 It is a monocyclic, bicyclic or tricyclic ring system of at least one valence, wherein at least one ring is aromatic, contains 1 to 4 heteroatoms, and the whole is a C ring containing 5 to 12 ring members. 5-11 -heteroaryl, the heteroatom being selected from O, S or N.

[0022] Preferably, the heterocyclic group R 12 , heterocyclic group R 16 , heterocyclic group R 18 and heterocyclic group R 21 C with 3 to 12 ring atoms 2-11 -heterocyclyl, and is a saturated or partially saturated monocyclic or bicyclic heterocycle, at least one of the ring atoms is selected from the heteroatom in the group consisting of N, O and S, and the remaining ring atoms are selected from carbon; a bicyclic heterocyclyl refers to a heterocycle consisting of two rings having two common ring atoms, and the bridge separating the two rings is a single bond, a chain of at least one ring atom, or a spirocycle where the two rings are connected via one common ring atom.

[0023] Preferably, the C 1-4 The alkyl group refers to a saturated straight-chain or branched hydrocarbon group having 1 to 4 carbon atoms.

[0024] Preferably, the C 1-4 The alkoxy group refers to an alkoxy group having 1 to 4 carbon atoms in total and connected to the rest of the molecule through O.

[0025] Preferably, the aromatic group R 7 , aryl R 10 , aryl R 13 , aryl R 17 and aromatic R 20 C selected from 6 to 12 ring members 6-12 -Aryl.

[0026] Preferably, the aromatic group R 7 , aryl R 10 , aryl R 13 , aryl R 17 and aromatic R 20 C selected from 6 to 10 ring members 6-12 -Aryl.

[0027] Preferably, the heteroaryl group R 8 , heteroaryl R 11 , heteroaryl R 14 , heteroaryl R 19 and heteroaryl R 22 C selected from 5 to 10 ring members 5-9-heteroaryl.

[0028] Preferably, the heterocyclic group R 12 , heterocyclic group R 16 , heterocyclic group R 18 and heterocyclic group R 21 C selected from 3 to 12 ring atoms 3-8 -heterocyclic group.

[0029] Preferably, the aromatic group R 7 , aryl R 10 , aryl R 13 , aryl R 17 and aromatic R 20 is selected from phenyl, naphthyl, indanyl or anthracenyl.

[0030] Preferably, the heteroaryl group R 8 , heteroaryl R 11 , heteroaryl R 14 , heteroaryl R 19 and heteroaryl R 22 is selected from pyridyl, pyrimidinyl, pyrrolyl, thienyl, furanyl, indolyl or quinolinyl.

[0031] Preferably, the heterocyclic group R 12 , heterocyclic group R 16 , heterocyclic group R 18 and heterocyclic group R 21 is selected from azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, 1,6-diazaspiro[3.4]octane, 1,7-diazaspiro[3.5]nonane, 2-azaspiro[3.3]heptane, 2-oxa-7-azaspiro[3.5]nonane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2,7-diazaspiro[3.5]nonane, 2,6-diazaspiro[3.5]nonane, 7-oxa-2-azaspiro[3.5]nonane, morpholinyl or thiomorpholinyl.

[0032] Preferably, the cycloalkyl group R 9 and cycloalkyl R 15 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

[0033] Preferably, the C 1-4 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.

[0034] Preferably, the C 1-4 The alkoxy group is selected from methoxy, ethoxy, propoxy or tert-butoxy.

[0035] Preferably, the compound of formula (I) is selected from any one of the following compounds ((Ia), (Ib), (Ic), (Id), (Ie)):

[0036]

[0037] Among them, the substituent R 6 Selected from aryl R 20 , halogenated aryl, cycloalkyl aryl, halogenated alkyl aryl, halogenated alkoxy aryl, heterocyclic group R 21 , halogenated heterocyclic group, heteroaryl R 22 , halogenated heteroaryl, halogenated alkyl heteroaryl, halogenated alkoxy heteroaryl, alkyl formyl, aryl formyl, halogenated aryl formyl, heteroaryl formyl, halogenated heteroaryl formyl, halogenated arylsulfonyl, hydroxyalkyl, hydroxyalkynyl, amino, aminoalkyl, hydrogen, halogen, carboxyl, ester, cyano, C 1-4 Alkyl or C 1-4 Alkoxy;

[0038] The aryl group R 20 is selected from phenyl, naphthyl, indanyl or anthracenyl;

[0039] The heterocyclic group R 21 is selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, 1,6-diazaspiro[3.4]octane, 1,7-diazaspiro[3.5]nonane, 2-azaspiro[3.3]heptane, 2-oxa-7-azaspiro[3.5]nonane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2,7-diazaspiro[3.5]nonane, 2,6-diazaspiro[3.5]nonane, 7-oxa-2-azaspiro[3.5]nonane, morpholinyl or thiomorpholinyl;

[0040] The heteroaryl group R 22 is selected from pyridyl, pyrimidinyl, pyrrolyl, thienyl, furanyl, indolyl or quinolyl;

[0041] The C 1-4 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl;

[0042] The C 1-4 The alkoxy group is selected from methoxy, ethoxy, propoxy or tert-butoxy.

[0043] Preferably, the compound described in formula (I) is selected from any one of the following compounds 1 to 25:

[0044]

[0045] The present invention also provides a method for preparing the piperazine derivatives of the present invention, comprising the following steps:

[0046]

[0047] The compound represented by formula (II) is subjected to a substitution reaction to obtain a compound represented by formula (III);

[0048] The compound represented by formula (III) is hydrolyzed and then reacted with an amine compound to obtain a compound represented by formula (I).

[0049] Among them, A, R 1 , R 2 and R 3 Same definition as above.

[0050] Preferably, the method for preparing the piperazine derivative comprises the following steps:

[0051] Dissolve the compound represented by formula (II) and a base in an organic solvent, add an alcohol compound or a phenol compound, stir and react at room temperature, monitor the reaction with a thin layer plate, add water after the reaction is completed, extract with an organic solvent, combine the organic phases, concentrate, and chromatograph to obtain the compound represented by formula (III);

[0052] The compound represented by formula (III) and a base are dissolved in a mixed solvent and stirred at room temperature. After the hydrolysis is completed, an acid is added to adjust the pH value. The precipitated product is evaporated to dryness and directly dissolved in an organic solvent. Then, a condensing agent, an acid binding agent and an amine compound are added and stirred for reaction. After the reaction is completed, water is added and extracted with an organic solvent. The organic phases are combined, concentrated, and chromatographed to obtain the compound represented by formula (I);

[0053] The mixed solvent is a mixture of water and an organic solvent.

[0054] Preferably, the organic solvent is selected from at least one of pyridine, chloroform, dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, chlorobenzene, toluene, acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide.

[0055] Preferably, the base is selected from at least one of sodium hydroxide, potassium hydroxide, cesium carbonate, potassium carbonate, sodium bicarbonate, sodium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and 2,6-lutidine.

[0056] Preferably, the condensing agent is selected from at least one of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) uronium hexafluorophosphate (HATU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N,N′-carbonyldiimidazole (CDI), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and tripyrrolidinophosphonium bromide hexafluorophosphate (PyBrOP).

[0057] Preferably, the acid binding agent is selected from at least one of ammonia water, methylamine hydrochloride, ethylamine, isopropylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, azetidine, tetrahydropyrrole, piperidine hydrochloride, dimethylamine, diethylamine, aniline, benzylamine, 3,4-methylenedioxyaniline, 6-aminobenzothiazole, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and 2,6-lutidine.

[0058] Preferably, the alcohol compound is selected from at least one of 4-hydroxy-pyridine, 4-hydroxypyrimidine, 4-hydroxy-piperidine, N-methyl-4-hydroxypiperidine and cyclohexanol.

[0059] Preferably, the phenolic compound is selected from phenol.

[0060] Preferably, the amine compound is selected from at least one of ammonia, methylamine hydrochloride, dimethylamine, ethylamine, isopropylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, azetidine, tetrahydropyrrole, piperidine hydrochloride, diethylamine, aniline, benzylamine, 3,4-methylenedioxyaniline and 6-aminobenzothiazole;

[0061] Preferably, the acid is selected from at least one of hydrochloric acid, sulfuric acid and phosphoric acid.

[0062] The present invention also provides a use of the piperazine derivative of the present invention in the preparation of an anti-Eimeria drug;

[0063] The invention also provides an application of the piperazine derivatives of the invention in preparing drugs against Toxoplasma gondii.

[0064] Beneficial effects of the present invention:

[0065] The piperazine derivatives of the present invention have a skeleton of quinazolinone and its analogs, and have the advantages of good water solubility, low cytotoxicity, and high in vitro anti-coccidial and anti-toxoplasma activities, while solving the problems of lack of clinical drugs for treating toxoplasmosis and very serious drug resistance of coccidia.

[0066] The preparation method of the piperazine derivative provided by the present invention adopts a molecular hybridization strategy and is synthesized by a nucleophilic substitution reaction. The synthesis process has the advantages of simple and mild conditions, readily available synthetic raw materials, and low production cost. The piperazine derivative is suitable for development into anticoccidial and anti-toxoplasmic drugs and has potential new drug development value in the field of pharmaceutical chemistry technology.

[0067] The piperazine derivatives provided by the present invention have shown high inhibitory activity in vitro against Toxoplasma gondii and coccidiostat, and have the advantage of low toxicity to host cells, thus proving that the piperazine derivatives have potential application prospects in drugs with anticoccidiostat and anti-Toxoplasma gondii activities and have value for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Figure 1 is the mass spectrum of compound 1;

[0069] Figure 2 is the mass spectrum of compound 2;

[0070] Figure 3 is the mass spectrum of compound 3;

[0071] Figure 4 is the mass spectrum of compound 4;

[0072] Figure 5 is the mass spectrum of compound 5;

[0073] Figure 6 is the mass spectrum of compound 14;

[0074] Figure 7 is the mass spectrum of compound 15. DETAILED DESCRIPTION

[0075] The following will describe the embodiments of the present invention with reference to the accompanying drawings and preferred embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be understood that the preferred embodiments are only for illustrating the present invention, not for limiting the scope of protection of the present invention.

[0076] If no specific techniques or conditions are specified in the specific embodiments, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0077] Example 1

[0078] A method for preparing a piperazine derivative as shown in compound 1,

[0079] The synthesis process of compound 1 is as follows:

[0080]

[0081] The steps of the preparation method include:

[0082] Step 1: Place methyl 2-aminobenzoate (compound 1-1) (15.1 g, 100 mmol) in a flask, add ethanol (200 mL) and triethylamine (15.2 g, 150 mmol); add 4-fluorophenyl isothiocyanate (18.4 g, 120 mmol) to the above solution at 0°C; reflux the reaction solution overnight; when the raw material is no longer reduced as monitored by the thin layer plate, terminate the reaction and cool in an ice bath; filter the precipitated solid under reduced pressure, and wash the filter cake with anhydrous ethanol 3 times (40 mL×3) to obtain a white solid compound 1-2 (3-(4-fluorophenyl)-2-sulfoxy-2,3-dihydroquinazolin-4(1H)-one) (yield 19.8 g, yield 71%). LC-MS (m / z): 273.1 [M+H] + .

[0083] Step 2: Compound 1-2 (16.3 g, 60 mmol) was placed in a flask, and chloroform (200 mL) was added; sulfonyl chloride (16.2 g, 120 mmol) was slowly added under stirring at 0°C; the reaction solution was stirred under reflux for 16 hours, and the reaction was terminated after the reaction raw material was no longer reduced as monitored by the thin layer chromatography plate, the solid was filtered under reduced pressure, the filter cake was washed with chloroform 3 times (50 mL×3), and the filtrate was collected; the filtrate was washed with water 3 times (50 mL×3), and evaporated to dryness under reduced pressure; the crude product was purified by chromatography on a silica gel column, and eluted with petroleum ether / ethyl acetate (v / v=10 / 1 to 1 / 1) as the eluent to obtain a light yellow powder 1-3 (2-chloro-3-(4-fluorophenyl)quinazolin-4(3H)-one (compound 1-3)) (yield 9.37 g, yield 57%). LC-MS (m / z): 275.1 [M+H] + .

[0084] Step 3: Compound 1-3 (8.2 g, 30 mmol) was added to a round-bottom flask containing anhydrous ethanol (150 mL), and triethylamine (4.5 g, 45 mmol) and 1-Boc-piperazine (6.7 g, 36 mmol) were added at room temperature. After the addition was completed, the reaction solution was reacted under reflux for 16 hours; after the raw material no longer disappeared as monitored by the thin layer plate, the reaction was cooled to room temperature, the precipitated solid was filtered under reduced pressure, and the filter cake was washed with anhydrous ethanol 3 times (30 mL × 3); evaporated to dryness under reduced pressure, and the crude product was recrystallized with isopropanol to obtain an off-white solid compound 1-4 (4-(3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)piperazine-1-carboxylic acid tert-butyl ester) (yield 8.14 g, yield 64%). LC-MS (m / z): 425.1 [M+H] + .

[0085] Step 4: Add compound 1-4 (8.0 g, 18.9 mmol) to an eggplant-shaped flask containing N,N-dimethylformamide (100 mL), add cesium carbonate (12.3 g, 37.8 mmol) and phenol (1.8 g, 18.9 mmol) successively, and stir at room temperature overnight; after the reaction is completed, add water (100 mL), extract with ethyl acetate 3 times (60 mL×3), wash the combined organic phase with saturated brine (50 mL×3), dry with anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure; the crude product is recrystallized from ethanol to obtain a light yellow solid compound 1-5 (4-(4-oxo-3-(4-phenoxyphenyl)-3,4-dihydroquinazolin-2-yl)piperazine-1-carboxylic acid tert-butyl ester) (yield 5.27 g, yield 56%). LC-MS (m / z): 499.1 [M+H] + .

[0086] Step 5: Add compound 1-5 (4.5 g, 9.0 mmol) into a round-bottom flask containing dichloromethane (60 mL), stir evenly, then add trifluoroacetic acid (10.3 g, 90 mmol); stir the reaction solution at room temperature overnight, and evaporate the solvent under reduced pressure after the disappearance of the starting material monitored by TLC plate. The crude product is directly used in the next step reaction. The crude product was dissolved in methanol (50 mL), potassium carbonate (2.5 g, 18 mmol) and ethyl bromoacetate (1.5 g, 9.0 mmol) were added successively, and stirred at room temperature for 16 hours; after the raw material was no longer disappeared as monitored by thin layer plate, methanol was evaporated under reduced pressure, ethyl acetate (80 mL) was added to the residue, and the mixture was washed with water 3 times (30 mL × 3), and the organic phase was washed with saturated brine (30 mL × 3); after drying over anhydrous sodium sulfate, the mixture was filtered, and the filtrate was concentrated under reduced pressure; the crude product was purified by chromatography on a silica gel column, eluted with dichloromethane / methanol (v / v = 50 / 1 to 30 / 1), and the eluent was concentrated to obtain a light yellow solid compound 1-6 (ethyl 2-(4-(4-oxo-3-(4-phenoxyphenyl)-3,4-dihydroquinazolin-2-yl)piperazin-1-yl)acetate) (yield 2.1 g, yield 48%). LC-MS (m / z): 485.1 [M+H] + .

[0087] Step 6: Compound 1-6 (2.0 g, 4.1 mmol) was added to a round-bottom flask containing methanol (20 mL) and water (10 mL). After stirring evenly, 2 mol / L sodium hydroxide (8.2 mL) was added and stirred at room temperature overnight. After the raw material disappeared as monitored by thin layer plate, the pH value was adjusted to 7-8 with 3 mol / L hydrochloric acid. The precipitate was filtered and the filter cake was washed with water 3 times (30 mL × 3). The filter cake was evaporated under reduced pressure and the crude product was directly used for the next step reaction. The crude product was dissolved in N, N-dimethylformamide (30 mL), and a condensation agent HATU (2.3 g ,6.1mmol), ammonia water (1.4g, 41mmol) (ammonia water is used as both an acid-binding agent and an amine compound), react at room temperature for 6 hours. After the reaction is completed, water (50mL) is added and stirred for 10 minutes, then extracted with ethyl acetate 3 times (30mL×3). The combined organic phase is washed with saturated brine (30mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure; the crude product is purified by silica gel column chromatography using dichloromethane / methanol (v / v=25 / 1) as the elution solvent to obtain the product compound 1 (yield 0.54g, yield 29%). LC-MS (m / z): 456.1[M+H] + . 1 H NMR (400 MHz, DMSO-d 6)δ7.90(d,J=8.0Hz,1H),7.66(d,J=8.0Hz,1H),7.59(t,J=8.0Hz,1H),7.36(t,J=8.0Hz,2H),7.29(d,J=12.0Hz,1H),7.27(s,2H), 7.12(dt,J=20.0Hz,J=8.0Hz,2H),7.00(d,J=12.0Hz,2H),6.96(d,J=8.0Hz,2H),3.70(s,4H),3.19(s,2H),2.61(t,J=4.0Hz,4H).

[0088] Examples 2 to 13 are respectively methods for preparing the piperazine derivatives represented by Compounds 2 to 13.

[0089] Among them, the preparation steps and conditions of Compounds 2 to 13 are the same as those of Compound 1, except for the different amine compounds in Step 6 shown in Table 1, and the amine compounds in Examples 2 to 13 are also used as acid binding agents. The names and structural formulas of the synthesized Compounds 2 to 13 are shown in Table 1.

[0090] Table 1 Names and structural formulas of compounds 2 to 13

[0091]

[0092]

[0093]

[0094]

[0095] Embodiments 18 to 20 are methods for preparing the piperazine derivatives represented by compounds 18 to 20.

[0096] The preparation steps and conditions of Compounds 18 to 20 are the same as those of Compound 5, except for the o-amino aromatic acid ester compound in Step 1. The names and structural formulas of the synthesized Compounds 18 to 20 are shown in Table 2.

[0097] Table 2 Names and structural formulas of compounds 18 to 20

[0098]

[0099] Embodiment 21

[0100] A method for preparing a piperazine derivative as shown in compound 21,

[0101] Among them, the synthetic route of compound 21 is as follows:

[0102]

[0103] Step 1: Dissolve compound 1-4 (8.5 g, 20 mmol) in 1,4-dioxane (80 mL), add 4 mol / L hydrochloric acid / 1,4-dioxane solution (20 mL) dropwise at room temperature, react at room temperature for 6 hours, evaporate the solvent under reduced pressure after the reaction, and use the crude product directly in the next step. The above crude product solution was dissolved in methanol (80 mL), potassium carbonate (4.1 g, 30 mmol) and ethyl bromoacetate (3.34 g, 20 mmol) were added successively, and stirred at room temperature for 16 hours; after the raw material was no longer disappeared as monitored by thin layer plate, methanol was evaporated under reduced pressure, ethyl acetate (80 mL) was added to the residue, and the mixture was washed with water 3 times (30 mL × 3), and the organic phase was washed with saturated brine (30 mL × 3); after drying over anhydrous sodium sulfate, the mixture was filtered, and the filtrate was concentrated under reduced pressure; the crude product was purified by chromatography on a silica gel column, eluted with dichloromethane / methanol (v / v = 50 / 1 to 30 / 1), and the eluent was concentrated to obtain a light yellow solid compound 1-7 (ethyl 2-(4-(3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)piperazin-1-yl)acetate) (yield 5.1 g, yield 62%). LC-MS (m / z): 412.1 [M+H] + .

[0104] Step 2: Compound 1-7 (4.93 g, 12 mmol) was added to a round-bottom flask containing methanol (40 mL) and water (20 mL), stirred evenly, and then 2 mol / L sodium hydroxide (24 mL) was added, and stirred overnight at room temperature; after the raw material disappeared as monitored by thin layer plate, the pH value was adjusted to 7-8 with 3 mol / L hydrochloric acid, the precipitate was filtered, the filter cake was washed with water 3 times (30 mL × 3), the filter cake was evaporated under reduced pressure, and the crude product was directly used for the next step reaction; the crude product was dissolved in N, N-dimethylformamide (100 mL), and a condensation agent HATU (6.8 g, 18 mmol) and cyclopropylamine (1.37 g, 24 mmol) (wherein , cyclopropylamine is used as an acid-binding agent and an amine compound at the same time), react at room temperature for 6 hours. After the reaction is completed, water (100 mL) is added and stirred for 10 minutes, then extracted with ethyl acetate 3 times (60 mL×3). The combined organic phase is washed with saturated brine (60 mL×3), dried over anhydrous sodium sulfate, filtered, and the filtrate is concentrated under reduced pressure; the crude product is purified by silica gel column chromatography using dichloromethane / methanol (v / v=25 / 1) as the elution solvent to obtain the product compound 1-8 (N-cyclopropyl-2-(4-(3-(4-fluorophenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)piperazine-1-yl)acetamide) (yield 2.68 g, yield 53%). LC-MS (m / z): 422.1 [M+H] + .

[0105] Step 3: Add compound 1-8 (0.42 g, 1 mmol) to an eggplant-shaped flask containing N,N-dimethylformamide (10 mL), add sodium hydrogen (0.05 g, 2 mmol) and 4-hydroxy-pyridine (0.11 g, 1.2 mmol) in an ice bath, and stir at room temperature overnight; after the reaction, add water (10 mL), extract with ethyl acetate 3 times (15 mL × 3), wash the combined organic phase with saturated brine (20 mL × 3), dry over anhydrous sodium sulfate, filter, and concentrate the filtrate under reduced pressure; the crude product is recrystallized from ethanol to obtain a light yellow solid compound 21 (N-cyclopropyl-2-(4-(4-oxo-3-(4-(pyridin-4-yloxy)phenyl)-3,4-dihydroquinazolin-2-yl)piperazin-1-yl)acetamide) (yield 0.24 g, yield 48%). LC-MS (m / z): 497.1 [M+H] + .

[0106] Examples 22 to 25 are respectively the preparation methods of the piperazine derivatives represented by Compounds 22 to 25.

[0107] The preparation steps and conditions of Compounds 22 to 25 are the same as those of Compound 21, except for the alcohol derivative in Step 3. The names and structural formulas of the synthesized Compounds 22 to 25 are shown in Table 3.

[0108] Table 3 Names and structural formulas of compounds 22 to 25

[0109]

[0110] Detection and analysis

[0111] 1. Mass spectrometry

[0112] The compounds obtained in Example 1, Example 2, Example 3, Example 4, Example 5, Example 14 and Example 15 were subjected to mass spectrometry analysis. The results are as follows: Figures 1 to 7 shown.

[0113] 2. Activity test on inhibition of Toxoplasma gondii (T.gondii) and human foreskin fibroblasts (HFF)

[0114] The piperazine derivatives (Compounds 1 to 25) prepared in Examples 1 to 25 were tested for their inhibitory activity against Toxoplasma gondii (T. gondii) and human foreskin fibroblasts (HFF) using the CCK-8 method, and then the absorbance at 490 nm was rapidly measured using an ELISA instrument, and the IC 50 The specific results are shown in Table 4.

[0115] Table 4 Inhibitory activity of compounds against Toxoplasma gondii (T.gondii) and human foreskin fibroblasts (HFF)

[0116]

[0117]

[0118] From the analysis in Table 4, it can be seen that the piperazine derivatives of the present invention have good activity in inhibiting Toxoplasma gondii, among which the in vitro inhibitory activity of compound 5 is comparable to that of the marketed drug pyrimethamine. The in vitro cytotoxicity of this class of compounds is greater than 100 μM, which is significantly better than the marketed drugs pyrimethamine and sulfadiazine.

[0119] 3. In vitro anticoccidial activity assay

[0120] MDBK cells were infected with sporulated oocysts of E. tenella to establish an in vitro infection model, and then the in vitro anti-coccidial activities of the piperazine derivatives (Compounds 1 to 25) prepared in Examples 1 to 25 were investigated. The inhibition of the growth and development of coccidia by the compounds was detected by RT-qPCR, the toxicity of the compounds to MDBK cells was tested by CCK-8 method, and the IC 50 The specific results are shown in Table 5.

[0121] Table 5 Inhibitory activity of compounds against coccidia

[0122]

[0123]

[0124] As shown in Table 5, the piperazine derivatives of the present invention have a strong inhibitory effect on Eimeria tenella. The IC 50 The value is comparable to that of the marketed drug sulfamethoxazole, and the IC value of MDBK inhibition is small. 50 The values ​​were all greater than 100 μM, significantly better than the marketed drugs diclazuril and sulfamethazine.

[0125] It can be seen from Table 4 and Table 5 that the piperazine derivatives of the present invention have good inhibitory activity against Toxoplasma gondii and Eimeria tenella, have low toxicity to host cells, and have potential development value.

[0126] In summary, the piperazine derivatives provided by the present invention have a quinazolinone and its analogue skeleton, and have the advantages of good water solubility, low cytotoxicity, and high in vitro anti-Toxoplasma and anti-coccidial activities.

[0127] The invention provides a method for preparing a piperazine derivative, which is synthesized through the steps of ring closure, nucleophilic substitution, hydrolysis, condensation reaction and the like, and the synthesis process has the advantages of simple and mild conditions and low production cost, and is suitable for developing anti-toxoplasma and anticoccidial drugs, and has potential new drug development value in the field of pharmaceutical chemistry technology.

[0128] The in vitro anti-Toxoplasma and anticoccidial activity tests of the piperazine derivatives provided by the present invention show that most of them exhibit high inhibitory activity and have the advantage of low toxicity to host cells, thereby proving that the piperazine derivatives have potential application prospects in anti-Toxoplasma and anticoccidial active drugs.

[0129] The above embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Any equivalent substitution or change made by a person skilled in the art based on the present invention is within the protection scope of the present invention.

Claims

1. A piperazine derivative, characterized in that: The structure is a compound shown in formula (I) and its stereoisomers, tautomers or pharmaceutically acceptable salts thereof: In formula (I), Ring A is selected from aryl R7 or heteroaryl R8; R1 is selected from hydrogen, C 1-4 Alkyl, cycloalkyl R9, aryl R 10 , heteroaryl R 11 or heterocyclic group R 12 ; R2 is selected from hydrogen, halogen, nitro, cyano, C 1-4 Alkyl or C 1-4 Alkoxy; R3 is selected from aromatic R 13 , heteroaryl R 14 , cycloalkyl R 15 or heterocyclic group R 16 .

2. The piperazine derivative according to claim 1, characterized in that The aryl R7 or heteroaryl R8 has 0 to 5 substituents R4; the substituents R4 are selected from hydrogen, halogen, cyano, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, C 1-4 Alkyl or C 1-4 Alkoxy; and / or, the C 1-4 Alkyl, cycloalkyl R9, aryl R 10 , heteroaryl R 11 , heterocyclic group R 12 There is at least one substituent R5; the substituent R5 is selected from aromatic R 17 , halogenated aryl, cycloalkyl aryl, halogenated alkyl aryl, halogenated alkoxy aryl, heterocyclic group R 18 , halogenated heterocyclic group, heteroaryl R 19 , halogenated heteroaryl, halogenated alkyl heteroaryl, halogenated alkoxy heteroaryl, alkyl formyl, aryl formyl, halogenated aryl formyl, heteroaryl formyl, halogenated heteroaryl formyl, halogenated arylsulfonyl, hydroxyalkyl, hydroxyalkynyl, amino, aminoalkyl, hydrogen, halogen, carboxyl, ester, cyano, C 1-4 Alkyl or C 1-4 Alkoxy; and / or, the aromatic group R 13 , heteroaryl R 14 , cycloalkyl R 15 , heterocyclic group R 16 There is at least one substituent R6; the substituent R6 is selected from aromatic R 20 , halogenated aryl, cycloalkyl aryl, halogenated alkyl aryl, halogenated alkoxy aryl, heterocyclic group R 21 , halogenated heterocyclic group, heteroaryl R 22 , halogenated heteroaryl, halogenated alkyl heteroaryl, halogenated alkoxy heteroaryl, alkyl formyl, aryl formyl, halogenated aryl formyl, heteroaryl formyl, halogenated heteroaryl formyl, halogenated arylsulfonyl, hydroxyalkyl, hydroxyalkynyl, amino, aminoalkyl, hydrogen, halogen, carboxyl, ester, cyano, C 1-4 Alkyl or C 1-4 Alkoxy; And / or, the pharmaceutically acceptable salt is a salt formed by an acid and a nitrogen having a lone pair of electrons in compound (I); The acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, trifluoroacetic acid, tartaric acid, lactic acid, maleic acid, fumaric acid, malic acid or citric acid.

3. The piperazine derivative according to claim 2, characterized in that The cycloalkyl group R9 and the cycloalkyl group R 15 is a monocyclic or bicyclic hydrocarbon group having 3 to 10 ring carbon atoms, i.e., C 3-10 Cycloalkyl, the bicyclic hydrocarbon group has a cycloalkyl portion consisting of two saturated carbon rings with two common atoms; And / or, the aryl R7, aryl R 10 , aryl R 13 , aryl R 17 and aryl R 20 It is a monocyclic, bicyclic or tricyclic carbon ring system, at least one of which is aromatic, and the whole is C with 6 to 14 ring members. 6-14 - aryl; and / or, the heteroaryl R8, heteroaryl R 11 , heteroaryl R 14 , heteroaryl R 19 and heteroaryl R 22 It is a monocyclic, bicyclic or tricyclic ring system of at least one valence, wherein at least one ring is aromatic, contains 1 to 4 heteroatoms, and the whole is a C ring containing 5 to 12 ring members. 5-11 - heteroaryl, wherein the heteroatom is selected from O, S or N; And / or, the heterocyclic group R 12 , heterocyclic group R 16 , heterocyclic group R 18 and heterocyclic group R 21 C with 3 to 12 ring atoms 2-11 -heterocyclyl, and is a saturated or partially saturated monocyclic or bicyclic heterocycle, at least one of the ring atoms is selected from the heteroatom in the group consisting of N, O and S, and the remaining ring atoms are selected from carbon; a bicyclic heterocyclyl refers to a heterocycle consisting of two rings having two common ring atoms, the bridge separating the two rings being a single bond, a chain of at least one ring atom, or a spirocycle where the two rings are connected via one common ring atom; and / or, the C 1-4 Alkyl refers to a saturated straight-chain or branched hydrocarbon group having 1 to 4 carbon atoms; and / or, the C 1-4 The alkoxy group refers to an alkoxy group having 1 to 4 carbon atoms in total and connected to the rest of the molecule through O.

4. The piperazine derivative according to claim 2, characterized in that The aryl group R7, aryl group R 10 , aryl R 13 , aryl R 17 and aryl R 20 is selected from phenyl, naphthyl, indanyl or anthracenyl; and / or, the heteroaryl R8, heteroaryl R 11 , heteroaryl R 14 , heteroaryl R 19 and heteroaryl R 22 is selected from pyridyl, pyrimidinyl, pyrrolyl, thienyl, furanyl, indolyl or quinolyl; And / or, the heterocyclic group R 12 , heterocyclic group R 16 , heterocyclic group R 18 and heterocyclic group R 21 is selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, 1,6-diazaspiro[3.4]octane, 1,7-diazaspiro[3.5]nonane, 2-azaspiro[3.3]heptane, 2-oxa-7-azaspiro[3.5]nonane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2,7-diazaspiro[3.5]nonane, 2,6-diazaspiro[3.5]nonane, 7-oxa-2-azaspiro[3.5]nonane, morpholinyl or thiomorpholinyl; and / or, the cycloalkyl group R9 and the cycloalkyl group R 15 is selected from cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; and / or, the C 1-4 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; and / or, the C 1-4 The alkoxy group is selected from methoxy, ethoxy, propoxy or tert-butoxy.

5. The piperazine derivative according to claim 1, characterized in that The compound described in formula (I) is selected from any one of the following compounds: Wherein the substituent R6 is selected from the group consisting of aromatic R 20 , halogenated aryl, cycloalkyl aryl, halogenated alkyl aryl, halogenated alkoxy aryl, heterocyclic group R 21 , halogenated heterocyclic group, heteroaryl R 22 , halogenated heteroaryl, halogenated alkyl heteroaryl, halogenated alkoxy heteroaryl, alkyl formyl, aryl formyl, halogenated aryl formyl, heteroaryl formyl, halogenated heteroaryl formyl, halogenated arylsulfonyl, hydroxyalkyl, hydroxyalkynyl, amino, aminoalkyl, hydrogen, halogen, carboxyl, ester, cyano, C 1-4 Alkyl or C 1-4 Alkoxy; The aryl group R 20 is selected from phenyl, naphthyl, indanyl or anthracenyl; The heterocyclic group R 21 is selected from the group consisting of azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, 1,6-diazaspiro[3.4]octane, 1,7-diazaspiro[3.5]nonane, 2-azaspiro[3.3]heptane, 2-oxa-7-azaspiro[3.5]nonane, 2-oxa-6-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 2,7-diazaspiro[3.5]nonane, 2,6-diazaspiro[3.5]nonane, 7-oxa-2-azaspiro[3.5]nonane, morpholinyl or thiomorpholinyl; The heteroaryl group R 22 is selected from pyridyl, pyrimidinyl, pyrrolyl, thienyl, furanyl, indolyl or quinolyl; The C 1-4 The alkyl group is selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl; The C 1-4 The alkoxy group is selected from methoxy, ethoxy, propoxy or tert-butoxy.

6. The piperazine derivative according to claim 1, characterized in that The compound described in formula (I) is selected from any one of the following compounds:

7. A method for preparing a piperazine derivative according to any one of claims 1 to 6, characterized in that: The following steps are involved: The compound represented by formula (II) is subjected to a substitution reaction to obtain a compound represented by formula (III); The compound represented by formula (III) is hydrolyzed and then reacted with an amine compound to obtain a compound represented by formula (I).

8. The method for preparing a piperazine derivative according to claim 7, characterized in that: The following steps are involved: Dissolving the compound represented by formula (II) and a base in an organic solvent, adding an alcohol compound or a phenol compound, stirring to react, adding water after the reaction, extracting with an organic solvent, combining the organic phases, concentrating, and chromatography to obtain the compound represented by formula (III); The compound represented by formula (III) and a base are dissolved in a mixed solvent and stirred. After the hydrolysis is completed, an acid is added to adjust the pH value. The precipitated product is evaporated to dryness and directly dissolved in an organic solvent. Then, a condensing agent, an acid binding agent and an amine compound are added and stirred for reaction. After the reaction is completed, water is added and extracted with an organic solvent. The organic phases are combined, concentrated, and chromatographed to obtain the compound represented by formula (I); The mixed solvent is a mixture of water and an organic solvent.

9. The method for preparing a piperazine derivative according to claim 8, characterized in that: The organic solvent is selected from at least one of pyridine, chloroform, dichloromethane, ethyl acetate, tetrahydrofuran, 1,4-dioxane, chlorobenzene, toluene, acetonitrile, N,N-dimethylformamide and dimethyl sulfoxide; and / or, the base is selected from at least one of sodium hydroxide, potassium hydroxide, cesium carbonate, potassium carbonate, sodium bicarbonate, sodium carbonate, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and 2,6-lutidine; and / or, the condensing agent is selected from at least one of N,N,N′,N′-tetramethyl-O-(7-azabenzotriazole-1-yl) uronium hexafluorophosphate (HATU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), N,N′-carbonyldiimidazole (CDI), benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU), benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) and tripyrrolidinylphosphonium bromide hexafluorophosphate (PyBrOP); and / or, the acid-binding agent is selected from at least one of ammonia water, methylamine hydrochloride, ethylamine, isopropylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, azetidine, tetrahydropyrrole, piperidine hydrochloride, dimethylamine, diethylamine, aniline, benzylamine, 3,4-methylenedioxyaniline, 6-aminobenzothiazole, triethylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine and 2,6-lutidine; And / or, the alcohol compound is at least one selected from 4-hydroxy-pyridine, 4-hydroxypyrimidine, 4-hydroxy-piperidine, N-methyl-4-hydroxypiperidine and cyclohexanol; and / or, the phenolic compound is selected from phenol; and / or, the amine compound is selected from at least one of ammonia, methylamine hydrochloride, dimethylamine, ethylamine, isopropylamine, cyclopropylamine, cyclobutylamine, cyclopentylamine, cyclohexylamine, azetidine, tetrahydropyrrole, piperidine hydrochloride, diethylamine, aniline, benzylamine, 3,4-methylenedioxyaniline and 6-aminobenzothiazole; And / or, the acid is selected from at least one of hydrochloric acid, sulfuric acid and phosphoric acid.

10. Use of the piperazine derivative according to any one of claims 1 to 6, characterized in that: Use of the piperazine derivatives in preparing drugs against Eimeria; And / or, use of the piperazine derivative in preparing drugs against Toxoplasma gondii.

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