CN114044757A - Triazole-4- (N-substituted formamide) -5-triazene compound and preparation method and application thereof - Google Patents

Triazole-4- (N-substituted formamide) -5-triazene compound and preparation method and application thereof Download PDF

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CN114044757A
CN114044757A CN202111003569.XA CN202111003569A CN114044757A CN 114044757 A CN114044757 A CN 114044757A CN 202111003569 A CN202111003569 A CN 202111003569A CN 114044757 A CN114044757 A CN 114044757A
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substituted
triazole
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triazene
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CN114044757B (en
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崔孙良
周显晶
曾林伟
陈忠
汪仪
徐层林
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Zhejiang University ZJU
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    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
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Abstract

The invention discloses a 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound and a preparation method and application thereof. Particularly, the compound has a structure similar to that of rufinamide, has obvious electrophysiological activity and can reduce Na ion channelv1.1 half of the inactivation voltage, thereby shortening the Na ion channelv1.1 open time. Part of the compounds show better anti-epileptic effect than rufinamide on animal epilepsy models, such as a pentylenetetrazol acute seizure model (PTZ) and a maximal electric shock seizure Model (MES).

Description

Triazole-4- (N-substituted formamide) -5-triazene compound and preparation method and application thereof
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a preparation method of a 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound, which is used for a sodium ion channel Nav1.1 half the activation voltage and half the deactivation voltage were tested.
Background
Triazole compounds are a class of substances with biological activity or medicinal value. The drug rufinamide on the market has a triazole parent nucleus structure and is used for the adjuvant therapy of the epilepsy Lennox-Gastaut syndrome (LGS). Particularly, the compound has a structure similar to that of rufinamide, has obvious electrophysiological activity and can reduce Na ion channelv1.1 half of the inactivation voltage, thereby shortening the Na ion channelv1.1 open time. Part of the compounds show better anti-epileptic effect than rufinamide on animal epilepsy models, such as a pentylenetetrazol acute seizure model (PTZ) and a maximal electric shock seizure Model (MES).
Figure BDA0003236445320000011
Disclosure of Invention
The invention provides a preparation method of a 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound and evaluation of biological activity of a sodium ion channel Nav1.1. Meanwhile, part of compounds have sodium ion channels Na on animal epilepsy models and cellular levelsv1.1 half the activation voltage and half the deactivation voltage have better biological activity than rufinamide.
The 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound has a structure shown in a formula (I):
Figure BDA0003236445320000012
in the formula (I), R1Is substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted C2～C4Alkyl, wherein the substituent on the phenyl is halogen or C1～C6Alkyl, wherein the substituent on the benzyl is halogen or C1～C6Alkyl radical, said C2～C4The substituent on the alkyl group is one or more fluorine substituted phenyl groups;
R2is hydrogen, substituted or unsubstituted C1～C8Alkyl, phenyl, benzyl, said C1～C8The substituent on the alkyl is methoxyacyl;
R3and R4Independently selected from the group consisting of C1～C4Alkyl, or R3、R4To the connection R3And R4Form a five-or six-membered ring containing N, C and O, which may be substituted with an acyl group.
Cell and animal experiments show that the structure has better biological activity than rufinamide.
Preferably, R is1 Is 2, 6-difluorobenzyl, 2, 6-difluorophenyl, 2- (2, 6-difluorophenyl) ethyl, 2, 4-difluorobenzyl, 2-fluorobenzyl, 4-fluorobenzyl, phenyl, benzyl, 2-chlorobenzyl, 4-methylbenzyl and n-butyl; r2Hydrogen, methyl, cyclopropyl, phenyl, benzyl; r3And R4Independently selected from methyl, isopropyl or R3、R4To the connection R3And R4The N of (A) forms pyrrolidine, piperidine, morpholine, N-formyl piperazine.
Preferably, the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound is selected from one of the following specific compounds:
Figure BDA0003236445320000021
Figure BDA0003236445320000031
Figure BDA0003236445320000041
Figure BDA0003236445320000051
the invention also provides a preparation method of the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound, which is characterized by comprising the following steps:
step 1: the secondary amine and n-butyl lithium generate lithium amide at the temperature of minus 78 ℃, and the newly prepared lithium amide THF solution is introduced into the atmosphere of nitrous oxide gas for reaction for 4 hours;
step 2: during the above reaction, 2- (2-propargyloxy) tetrahydropyran was reacted with ethyl magnesium bromide reagent for 2 h. Adding the prepared 1- (2-tetrahydropyranyloxy) propynylmagnesium bromide into the reaction in the step 1, and reacting at 50 ℃ overnight to obtain an intermediate 1;
and step 3: 1 mol% of intermediate 1 [ Ir (cod) Cl]2Generating an intermediate 2 with alkyl azide under a catalyst;
and 4, step 4: intermediate 2 was reacted under acidic conditions with MeOH/HCl (pH 2-3) to give intermediate 3. Intermediate 3 was dissolved in PBS (pH 6.8)/acetonitrile (v/v 1:1) to give intermediate 4 at 3eq NaClO2/10 mol% TEMPO at 40 ℃;
and 5: and reacting the intermediate 4 with equivalent CDI at room temperature for 30min, and then reacting with organic amine at room temperature to generate the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound.
The structure of the intermediate 1 is shown as a formula (II):
Figure BDA0003236445320000061
the structure of the intermediate 2 is shown as the formula (III):
Figure BDA0003236445320000062
the structure of the intermediate 3 is shown as a formula (IV)
Figure BDA0003236445320000063
The structure of the intermediate 4 is shown as a formula (V)
Figure BDA0003236445320000064
Wherein R is1、R2、R3And R4As defined above.
During the reaction, the secondary amine reacts with N-butyllithium to form aminolithium, which is in N2Reacting for a period of time in an atmosphere of O, and then reacting with 1- (2-tetrahydropyranyloxy) propynylmagnesium bromide to generate a triazene derivative intermediate 1. Triazene intermediate 1 in [ Ir (cod) Cl]2Generating a triazole derivative intermediate 2 with alkyl azide under the action of a catalyst. Removing THP protecting group from triazole derivative intermediate 2 under acidic condition to obtain intermediate 3, and oxidizing hydroxyl in intermediate into acid under NaClO2/10 mol% TEMPO system to obtain intermediate 4. Finally, the carboxyl group of the intermediate 4 is activated by CDI, and then is subjected to condensation reaction with amine to generate the target compound.
Preferably, the iridium catalyst is [ Ir (cod) Cl]2The dosage of (CAS: 12112-67-3) is 1-2% of the molar weight of the triazene compound, and the iridium catalyst has good applicability to the substrate in the invention and high catalytic efficiency.
Preferably, the 1- (2-tetrahydropyranyloxy) propynylmagnesium bromide is obtained by reacting tetrahydro-2- (2-propynyloxy) -2H-pyran with ethylmagnesium bromide in an amount of 1.5eq based on the secondary amine substrate.
Preferably, the hydroxyl group in the intermediate 3 is oxidized into the intermediate 4, and the optimal conditions are as follows: NaClO2 is 3eq, 10 mol% TEMPO catalytic amount, reaction solvent is PBS/acetonitrile 1:1 mixed solvent, wherein PBS buffer solution pH is 6.8, 40 ℃.
Preferably, R is1 Is 2, 6-difluorobenzyl, 2, 6-difluorophenyl, 2- (2, 6-difluorophenyl) ethyl, 2, 4-difluorobenzyl, 2-fluorobenzyl, 4-fluorobenzyl, phenyl, benzyl, 2-chlorobenzyl, 4-methylbenzyl or n-butyl.
Preferably, R is2Hydrogen, methyl, cyclopropyl, phenyl, benzyl.
Preferably, R is3And R4Independently selected from methyl, isopropyl or R3、R4To the connection R3And R4The N of (A) forms pyrrolidine, piperidine, morpholine, N-formyl piperazine.
Preferably, the triazene intermediate 1 compounds can be prepared by conventional methods, specifically in the literature (Angew. chem. int. Ed.2015,54, 302-305).
Preferably, the compound can obviously reduce Na ion channelv1.1 half of the inactivation voltage, thereby shortening the Na ion channelv1.1 open time.
Preferably, the compound shows better anti-epileptic effect than rufinamide on animal models, such as a pentylenetetrazol acute epileptic seizure model (PTZ) and a maximal electric shock epileptic seizure Model (MES).
Drawings
FIG. 1 is a diagram of the product obtained in example 11HNMR spectrogram;
FIG. 2 shows the product obtained in example 113CNMR spectrogram;
FIG. 3 shows the product obtained in example 21HNMR spectrogram;
FIG. 4 shows the product obtained in example 213CNMR spectrogram;
FIG. 5 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-1 as measured in example 23;
FIG. 6 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-2 as measured in example 23;
FIG. 7 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-3 as measured in example 23;
FIG. 8 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-4 as measured in example 23;
FIG. 9 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-5 as measured in example 23;
FIG. 10 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-6 as measured in example 23;
FIG. 11 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-7 as measured in example 23;
FIG. 12 is a graph of Nav1.1 sodium channel current activation and deactivation before and after administration of Compound Z-8 as measured in example 23;
FIG. 13 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-9 as measured in example 23;
FIG. 14 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-10 as measured in example 23;
FIG. 15 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-10 as measured in example 23;
FIG. 16 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-12 as measured in example 23;
FIG. 17 is a graph of Nav1.1 sodium channel current activation and deactivation before and after administration of Compound Z-13 as measured in example 23;
FIG. 18 is a graph showing the current activation and inactivation curves of Nav1.1 sodium channel before and after administration of Compound Z-14 as measured in example 23;
FIG. 19 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-15 as measured in example 23;
FIG. 20 is a graph showing the current activation and deactivation of Nav1.1 sodium channels before and after administration of Compound Z-16 as measured in example 23;
FIG. 21 shows the effect of some of the compounds of example 24 on the acute seizure model (PTZ) of pentylenetetrazol:
FIG. 22 shows the effect of some of the compounds of example 24 on the maximal electroshock seizure Model (MES).
Detailed Description
The present invention will be described in detail with reference to the following specific examples, which are only for explaining the technical solutions of the present invention and do not limit the present invention in any way.
The 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound used in the invention can be prepared by the following method:
Figure BDA0003236445320000091
example 1
Figure BDA0003236445320000092
Step 1: 15mmol diisopropylamine was added to a two-necked flask under argon and 15mL dry THF was added. 6.6mL of n-butyl (2.5M, 16.5mmol) were slowly added dropwise at-78 deg.C, and the reaction was continued for 30min after the addition was complete, followed by bringing to room temperature. Introducing the newly prepared lithium diisopropylamide THF solution into N2O gas atmosphere, adding 15mL of anhydrous THF, and carrying out rt reaction for 4 h;
step 2: during the above reaction, 2.8g of 2- (2-propargyloxy) tetrahydropyran (20mmol) were added to a two-necked flask under argon and 20mL of anhydrous THF were added. Slowly dripping 20mL of CH3MgBr reagent (1M) at room temperature, and reacting for 2h at 50 ℃ after dripping is finished;
and step 3: after the reaction in the step 1 is finished, adding N2And replacing O gas with argon protection. Then the step 2 reaction solution was transferred to the step 1 reaction system through a syringe and reacted at 50 ℃ overnight. After the reaction, 30mL of water was added for quenching, the mixture was filtered through celite, extracted three times with ethyl acetate, washed once with saturated NaCl, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. Performing column chromatography separation and purification by using neutral Al2O3 as a stationary phase and a petroleum ether/ethyl acetate system as an eluent to obtain 2.5g of a white solid intermediate 1 with a yield of 62%;
and 4, step 4: in a 25mL single-neck reaction flask, 1g of intermediate 1(3.7mmol), 633mg of 2- (azidomethyl) -1, 3-difluorobenzene (3.7mmol), 25mg of [ Ir (cod) Cl ]2 catalyst (1 mol%, 0.037mmol) were added. 10mL of methylene chloride was added and the reaction was carried out at room temperature. After the reaction is finished, silica gel is used as a stationary phase, and a petroleum ether/ethyl acetate system is used as an eluent to carry out column chromatography separation and purification. 1.1g of intermediate 2 was obtained as a white solid in a yield of 68%.
And 5: in a 50mL single-neck flask, 1.1g of intermediate 2 was added, 10mL of MeOH was added, and the reaction solution was adjusted to pH 2 to 3 with hydrochloric acid and reacted at room temperature. After the reaction, methanol in the reaction solution was removed under reduced pressure to obtain intermediate 3. Intermediate 3 was dissolved in 10mL of PBS (pH 6.8)/10mL of acetonitrile (v/v 1:1), and 680mg of NaClO2, 40mg of TEMPO, and 1 to 2 drops of 6 to 14% NaClO solution were added at room temperature, and reacted at 40 ℃. After the reaction was completed, the reaction solution was quenched with NaClO2 remaining in the NaSO3 solution, and then made alkaline with 1N NaOH solution. And (3) decompressing and spinning out acetonitrile in the reaction liquid, extracting with diethyl ether for three times, discarding an organic layer and reserving an aqueous layer. Adjusting the water layer to be acidic by hydrochloric acid, separating out a large amount of white solid, filtering and drying to obtain 560mg of a white solid intermediate 4 with the yield of two steps of 61%;
step 6: in a 25mL single-necked flask, 560mg (1.5mmol) of intermediate 4 and 290mg of CDI (1.8mmol) were added, and 5mL of acetonitrile was added to react at room temperature for 30 min. After TLC detection of the intermediate 4, 0.5mL of 28-30% ammonia water was added and the reaction was continued at room temperature. After the reaction, acetonitrile in the reaction solution was removed by vacuum distillation. The crude product was dissolved in 15mL of dichloromethane, washed three times with 1M HCl, once with saturated NaCl, dried over anhydrous Na2SO4, and the solvent was removed under reduced pressure. The desired product was obtained by column chromatography using dichloromethane/methanol eluent, 420mg of white solid, yield 76%.
The physical properties and spectrum data of the product are as follows: a white solid; mp: 166.0-166.2 ℃;1H NMR(400MHz,CDCl3)δ7.34-7.26(m,1H),6.92-6.86(m,2H),5.64(s,2H),5.32-5.26(m,1H),4.20-4.10(m,1H),1.39(d,J＝6.8Hz,3H),1.29(d,J＝6.8Hz,3H)；13C NMR(101MHz,CDCl3)δ161.48(dd,J＝251.6,7.2Hz),161.15(s),146.79(s),130.86(t,J＝10.3Hz),125.53(s),111.73-111.49(m),110.87(t,J＝18.4Hz),51.01(s),48.49(s),39.77(s),23.09(s),18.79(s)；HRMS(EI)(m/z):calcd for 366.1854(M+),366.1854；found,366.1851。
of the product1The HNMR spectrum is shown in figure 1,13the CNMR spectrum is shown in FIG. 2.
Example 2
Figure BDA0003236445320000111
Step 1: 20mmol dimethylamine was added to a two-necked flask under argon and 15mL anhydrous THF was added. 8.8mL of n-butyl (2.5M, 22mmol) were slowly added dropwise at-78 deg.C, and the reaction was continued for 30min after the addition was complete, followed by moving to room temperature. The freshly prepared lithium dimethylamide THF solution is passed over N2In the atmosphere of O gas, 20mL of anhydrous THF is added, and rt reaction is carried out for 4 h;
step 2: during the above reaction, 4.2g of 2- (2-propargyloxy) tetrahydropyran (30mmol) were added to a two-necked flask under argon and 30mL of anhydrous THF were added. Slowly dropwise adding 30mL of CH at room temperature3MgBr reagent (1M), reacting for 2h at 50 ℃ after the dropwise addition is finished; '
And step 3: after the reaction in the step 1 is finished, adding N2And replacing O gas with argon protection. Then the step 2 reaction solution was transferred to the step 1 reaction system through a syringe and reacted at 50 ℃ overnight. After the reaction is finished, adding 50mL of water for quenching, filtering by using kieselguhr, extracting by using ethyl acetate for three times, washing by using saturated NaCl once and using anhydrous Na2SO4Drying and removing the solvent under reduced pressure. Using neutral Al2Separating and purifying by column chromatography with O3 as stationary phase and petroleum ether/ethyl acetate system as eluent to obtain 780mg oily liquid intermediate 1 with yield of 18.5%;
and 4, step 4: into a 25mL single-neck reaction flask, 300mg of intermediate 1(1.4mmol), 260mg of 2- (azidoethyl) -1, 3-difluorobenzene (1.4mmol), and 9.5mg of [ Ir (cod) Cl were charged]2Catalyst (1 mol%, 0.014 mmol). 5mL of methylene chloride was added and the reaction was carried out at room temperature. After the reaction is finished, silica gel is used as a stationary phase, and a petroleum ether/ethyl acetate system is used as an eluent to carry out column chromatography separation and purification. 450mg of intermediate 2 as a red thick solid are obtained in 82% yield.
And 5: in a 25mL single-neck flask, 450mg of intermediate 2 was added, 5mL of MeOH was added, and the reaction solution was adjusted to pH 2 to 3 with hydrochloric acid and reacted at room temperature. After the reaction, methanol in the reaction solution was removed under reduced pressure to obtain intermediate 3. Intermediate 3 was dissolved in 5mL PBS (pH 6.8)/5mL acetonitrile (v/v 1:1), and 311mg NaClO was added at room temperature2、18mgTEMPO, 1-2 drops of 6-14% NaClO solution, and reacting at 40 ℃. After the reaction is finished, NaSO is used3Solution quenching of residual NaClO in the reaction solution2The reaction solution was then made alkaline with 1N NaOH solution. And (3) decompressing and spinning out acetonitrile in the reaction liquid, extracting with diethyl ether for three times, discarding an organic layer and reserving an aqueous layer. Adjusting the water layer to be acidic by hydrochloric acid, separating out a large amount of white solid, filtering and drying to obtain 210mg of white solid intermediate 4, wherein the yield of the two steps is 56%;
step 6: in a 25mL single-necked flask, 97mg (0.3mmol) of intermediate 4 and 54mg of CDI (0.33mmol) were added, and 3mL of acetonitrile was added to react at room temperature for 30 min. After TLC detection of the intermediate 4, 0.25mL of 28-30% ammonia water was added and the reaction was continued at room temperature. After the reaction, acetonitrile in the reaction solution was removed by vacuum distillation. The crude product was dissolved in 15mL of dichloromethane, washed three times with 1M HCl, once with saturated NaCl, anhydrous Na2SO4Drying and removing the solvent under reduced pressure. The desired product was obtained by column chromatography using dichloromethane/methanol eluent in the form of a white solid (80 mg, 83% yield).
The physical properties and spectrum data of the product are as follows: a white solid; mp: 191.5 to 192.0 ℃;1H NMR(500MHz,CDCl3)δ7.21-7.15(m,2H),7.06-6.77(m,2H)5.52(s,1H),4.88-4.38(m,1H),3.60(d,J＝4.7Hz,3H),3.38-3.17(m,5H)；13C NMR(126MHz,CDCl3)δ162.75,161.80(dd,J＝247.9,8.2Hz),145.33,129.58,128.93-128.71(m),113.09(t,J＝18.8Hz),111.30(dd,J＝20.2,5.8Hz),47.14,44.02,36.61,23.20；HRMS(EI)(m/z):calcd for C13H16F2N7O(M+),324.1384；found,324.1378。
of the product1The HNMR spectrum is shown in FIG. 3,13the CNMR spectrum is shown in FIG. 4.
Examples 3 to 22
Figure BDA0003236445320000121
Examples 3-22 were prepared in the same manner as in examples 1 and 2, except that R was1、R2、R3Fragment substratesThe resulting product structures are shown in Table 1, substituting other substrate materials of similar structure.
TABLE 1 product structures of examples 3-22
Figure BDA0003236445320000122
Figure BDA0003236445320000131
Figure BDA0003236445320000141
Figure BDA0003236445320000151
Example 231-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene Compound sodium ion channel Nav1.1 biological Activity
2.1 test Compound information
Table 2 test compound information
Figure BDA0003236445320000161
Figure BDA0003236445320000171
Figure BDA0003236445320000181
2.2 solvent
Name: DMSO (dimethyl sulfoxide)
The source is as follows: sigma purchase, item number: 276855 500mL
Molecular weight: 78.13
Storage conditions are as follows: sealing at normal temperature and storing in dark
2.3 cellular information
Species of species&Strain: Nav1.1-Flp-InTM T-RExTM293 cell line
(stably express Na)v1.1 Flp-In of channelsTM T-RExTM293 cell)
The source is as follows: internal construction
The culture conditions are as follows: 5% CO2Incubator at 37 ℃
Freezing and storing conditions: liquid nitrogen
2.4 solution and reagent information
Extracellular fluid formulation (mM): 140NaCl, 5KCl, 1CaCl2、1.25MgCl210HEPES, 10Glucose, pH adjusted to 7.4 with NaOH.
Intracellular fluid formulation (mM): 130CsF, 10NaCl, 10EGTA, 10HEPES, pH adjusted to 7.2 with CsOH.
Notes on abbreviations
HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid, N- (2-hydroxyethyl) piperazine-N' - (2-ethanesulfonic acid)
EGTA: ethylene glycol bis (2-aminoethyl ether) tetraacetic acid
Figure BDA0003236445320000182
Figure BDA0003236445320000191
2.5 Experimental apparatus
Patch clamp amplifier (Axopatch 200B, Axon, USA)
Digital-to-analog converter (DigiData 1550B, Axon, USA)
Inverted microscope (IX51, Olympus, Japan)
Rapid drug delivery system (RSC-200, Bio-Logic, France)
Micromanipulator (MX7600R, Syskiyou, USA)
Electrode drawing instrument (P-97, Sutter, USA)
Glass electrode (BF150-86-10, Sutter, USA)
Shockproof platform and screen (63-534, TMC, USA)
Data acquisition and analysis software (pClamp 11, Axon, USA)
Carbon dioxide incubator (HERACell 150i, Thermo, USA)
Biological safety cabinet (MODEL 1384, Thermo, USA)
Water purifier (Milli Q, Millipore, USA)
2.6 Experimental methods
1. Preparation of Compounds
Compounds were made up in DMSO as 100mM stock solutions and stored at-20 ℃. On the day of the assay, compound DMSO stock solutions were thawed at room temperature and then diluted with extracellular fluid to the final assay concentration for the desired assay.
2. Cell culture and processing
Stably express Nav1.1 Flp-InTMT-RExTM293 cells were cultured in 35mm diameter cell culture dishes at 37 ℃ in 5% CO2The culture box is used for culturing, subculture is carried out according to the proportion of 1:4 every 48 hours, and the formula of a culture medium is as follows: 90% DMEM (Invitrogen), 10% fetal bovine serum (Gibco) and 50g/mL Hygromycin B (Invitrogen). On the day of the experiment, the cell culture was aspirated, rinsed once with extracellular fluid, and digested for 1 minute at room temperature with 0.25% Trypsin-EDTA (Invitrogen) solution. The digestion solution was aspirated, resuspended in extracellular fluid and the cells transferred to a petri dish for electrophysiological recording.
3. Electrophysiological recording procedure
Stably express Nav1.1 Flp-In of sodium channelTMT-RExTM293 cells, Na recording by Whole cell patch clamp technique at room temperaturev1.1 sodium channel current. The glass microelectrode is formed by drawing a glass electrode blank (BF150-86-10, Sutter) by a drawing instrument, the tip resistance after the liquid in the electrode is poured is about 2-5M omega, and the glass microelectrode can be connected to a patch clamp amplifier after being inserted into an amplifier probe. The clamped voltage and data recording is controlled and recorded by a pClamp 11 software through a computer, the sampling frequency is 20kHz, and the filtering frequency is 2 kHz. After obtaining a whole-cell record, cell clampingAt-120 mV, the channel activation process was recorded by giving 20ms stimulation of different depolarization voltages (from-120 mV to +60mV, 10mV apart for each voltage step) and calculating the half activation voltage (V1/2, the voltage at which channel activation reaches 50% of maximum conductance). Na (Na)v1.1 sodium channel inactivation Process sodium channel currents at different conditioned voltages were induced by applying different conditioned voltages from-120 mV to +50mV (10 mV per voltage step interval, 200ms duration) followed by applying a further voltage stimulus of-20 mV, 20ms, to record the channel inactivation process and calculate the half inactivation voltage (V)1/2I.e., the voltage at which channel deactivation reaches 50% of maximum conductance). The channel activation and inactivation process was again recorded 1 minute after compound administration and V was calculated1/2At least 3 cells (n.gtoreq.3) were tested.
4. Data processing
The data analysis was performed using pClamp 11, GraphPad Prism 5 and Excel software. The voltage-dependent activation and deactivation curves were fitted by the Boltzmann equation to calculate V1/2：
Y＝Bottom+(Top-Bottom)/(1+exp((V1/2-X)/Slope))
Where X represents the depolarization voltage, Y represents the corresponding conductance at the depolarization voltage, and Bottom and Top are the minimum and maximum conductance values, respectively.
5. Quality control
The experimental data in the report meet the following quality control indicators:
whole cell sealing impedance >1G omega
The series resistance is compensated by more than 80% if being more than 10M omega
No obvious spontaneous decay or increase of the current before administration
6. Test results
Na before and after administration of the Compoundv1.1 Δ V of channel Current activation (activation) and deactivation (inactivation)1/2And Δ V1/2。
TABLE 3 Pre-and post-administration of Compounds Nav1.1 channel Δ V1/2And Δ V1/2
Figure BDA0003236445320000211
Nav1.1 sodium channel current activation and inactivation curves before and after test compound administration
Figure BDA0003236445320000221
Figure BDA0003236445320000231
Figure BDA0003236445320000241
Example 241-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene Compounds animal experiments
3.1 Pentrazole acute seizure model (PTZ)
The effect of the preferred compounds Z-2, Z-3, Z-6 on the acute seizure model of Pentylenetetrazol (PTZ): adult male ICR mice, weighing 25-30g, were used. Mice were randomly grouped by weight: solvent group, Rufinamide positive control group and different compound (dosage is 50mg/kg) groups, and each group contains 5-6 ICR male mice. In the experiment, solvents or drugs are respectively administered by intragastric administration, 100mg/kg PTZ is injected in an abdominal cavity after 1h, and then the observation is immediately started and the seizure condition of the mice is recorded, wherein the observation time is 30 minutes. The behavioral evaluation grading standard is as follows: level 0: no obvious reaction; level 1: dull loss of spirit, twitching of ears and face; and 2, stage: the whole body is twitched without lifting forelimbs; and 3, level: one or both forelimbs are raised; 4, level: one side of the body is down with convulsion; and 5, stage: the back falls on the ground, or the whole body tonic clonic attack; and 6, level: death after rigidity. The rating of seizures in each mouse was recorded (panel a), from the start of dosing to the level 2 seizure potential (panel B), level 4 seizure potential (panel C) and level 6 seizure potential (panel D).
3.2 maximal electroshock epileptic seizure Model (MES)
Preferred effects of compounds Z-2, Z-3, Z-6 on the model of maximal electroshock seizures: adult male ICR mice, weighing 25-30g, were used. Mice were randomly grouped by weight: solvent group, Rufinamide positive control group and different compound (dosage is 50mg/kg) groups, and each group contains 5-6 ICR male mice. In the experiment, the stomach is respectively infused with solvent or medicine, after 1h, the stimulation clips of the maximum electroshock instrument are soaked by normal saline, the auricles of the two ears of the mouse are respectively clipped, and the stimulation is given with electrical stimulation, the stimulation frequency is 50Hz, the stimulation duration is 0.2s, and the stimulation current intensity is 25 mA. Mouse behavioral rating: level 1: running; and 2, stage: strengthening forelimbs; and 3, level: the hind limbs were strong and straight. The maximal seizure rating (panel a), the time to reach the tertiary seizure potential (panel B), and the duration of tonic-tonic clonic seizures (panel C) were recorded for the mice in this model.

Claims (9)

1.1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compounds, which are characterized by having a structure shown in a formula (I):
Figure FDA0003236445310000011
in the formula (I), R1Is substituted or unsubstituted phenyl, substituted or unsubstituted benzyl, substituted or unsubstituted C2～C4Alkyl, wherein the substituent on the phenyl is halogen or C1～C6Alkyl, wherein the substituent on the benzyl is halogen or C1～C6Alkyl radical, said C2～C4The substituent on the alkyl group is one or more fluorine substituted phenyl groups;
R2is hydrogen, substituted or unsubstituted C1～C8Alkyl, phenyl, benzyl, said C1～C8The substituent on the alkyl is methoxyacyl;
R3and R4Independently selected from the group consisting of C1～C4Alkyl, or R3、R4To the connection R3And R4Form a five-or six-membered ring which contains N, C and O and which may be substituted by an acyl group。
2. The 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound of claim 1, wherein R is1Is 2, 6-difluorobenzyl, 2, 6-difluorophenyl, 2- (2, 6-difluorophenyl) ethyl, 2, 4-difluorobenzyl, 2-fluorobenzyl, 4-fluorobenzyl, phenyl, benzyl, 2-chlorobenzyl, 4-methylbenzyl and n-butyl; r2Hydrogen, methyl, cyclopropyl, phenyl, benzyl; r3And R4Independently selected from methyl, isopropyl or R3、R4To the connection R3And R4The N of (A) forms pyrrolidine, piperidine, morpholine, N-formyl piperazine.
3. The 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound of claim 1, which is selected from one of the following specific compounds:
Figure FDA0003236445310000012
Figure FDA0003236445310000021
Figure FDA0003236445310000031
Figure FDA0003236445310000041
Figure FDA0003236445310000051
4. a preparation method of the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound according to any one of claims 1 to 3, which is characterized by comprising the following steps:
step 1: the secondary amine and n-butyl lithium generate a lithium amide THF solution at the temperature of-70-80 ℃, and the newly prepared lithium amide THF solution is introduced into a nitrous oxide gas atmosphere for reaction for 3-5 h;
step 2: during the reaction period, reacting 2- (2-propargyloxy) tetrahydropyran with an ethyl magnesium bromide reagent for 1-3 h to prepare 1- (2-tetrahydropyranyloxy) propyne magnesium bromide; adding the prepared 1- (2-tetrahydropyranyloxy) propyne magnesium bromide into the reaction in the step (1) for reacting overnight at 40-60 ℃ to obtain an intermediate 1;
and step 3: intermediate 1 in [ Ir (cod) Cl]2Generating an intermediate 2 with alkyl azide under a catalyst;
and 4, step 4: the intermediate 2 is put into a hydrogen chloride methanol solution to generate an intermediate 3; intermediate 3 was dissolved in PBS in acetonitrile and then washed with NaClO2Generating an intermediate 4 under the action of TEMPO;
the pH value of the hydrogen chloride methanol solution is 2-3;
and 5: the intermediate 4 reacts with CDI at room temperature for 30min, and then reacts with organic amine at room temperature to generate a 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound;
the structure of the intermediate 1 is shown as a formula (II):
Figure FDA0003236445310000052
the structure of the intermediate 2 is shown as the formula (III):
Figure FDA0003236445310000053
the structure of the intermediate 3 is shown as a formula (IV)
Figure FDA0003236445310000061
The structure of the intermediate 4 is shown as a formula (V)
Figure FDA0003236445310000062
Wherein R is1、R2、R3And R4Is as defined in any one of claims 1 to 3.
5. The method for preparing 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compounds according to claim 4, wherein the iridium catalyst [ Ir (cod) Cl]2The dosage of the triazine compound is 1-2% of the molar weight of the triazine compound.
6. The preparation method of the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound as claimed in claim 4, wherein the 1- (2-tetrahydropyranyloxy) propiolic magnesium bromide is obtained by reacting tetrahydro-2- (2-propianyloxy) -2H-pyran with ethyl magnesium bromide, and the dosage of the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound is 1-2 eq of a secondary amine substrate.
7. The preparation method of the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound according to claim 4, wherein the condition for oxidizing the hydroxyl in the intermediate 3 into the intermediate 4 is as follows: NaClO23eq, 10 mol% TEMPO catalytic amount, reaction solvent PBS/acetonitrile 1:1 mixed solvent, PBS buffer pH 6.8, 40 ℃.
8. The application of the 1-substituted-1H-1, 2, 3-triazole-4- (N-substituted formamide) -5-triazene compound as claimed in any one of claims 1 to 3 in preparation of antiepileptic drugs.
9. The 1-substituted ketone as defined in claim 9The application of the 1H-1,2, 3-triazole-4- (N-substituted formamide) -5-triazene compound in antiepileptic drugs is characterized in that the antiepileptic drugs are used for reducing Na of a sodium ion channelv1.1 half of the inactivation voltage, thereby shortening the Na ion channelv1.1 open time.
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GB1403050A (en) * 1971-11-19 1975-08-13 Chinoin Gyogyszer Es Vegyeszet Triazene derivatives and process for the preparation thereof
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US4049639A (en) * 1971-11-19 1977-09-20 Erzsebet Kovacsovics 5-Amido imido triazene triazoles and pyrazoles with photographic and antimycotic utility
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