CN104825456B - A kind of new opplication of the excellent body of urea hydrolytic velocity inhibitor - Google Patents
A kind of new opplication of the excellent body of urea hydrolytic velocity inhibitor Download PDFInfo
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Abstract
The invention discloses a kind of new opplication of the excellent body of urea hydrolytic velocity inhibitor.The structural formula of the urea hydrolytic velocity inhibitor is shown in formula I.Present invention application red blood cell model discrimination is inhibited the compound of urea hydrolytic velocity, test result indicate that, such compound（Such as excellent body）The erythrocyte membrane of urea hydrolytic velocity UT B mediations can be suppressed to the penetrating of urea, and its effect is in dose-dependence；Excellent body is acted on mdck cell no cytotoxicity in effective dosage ranges, illustrates that the effect of excellent body suppression cell-permeant urea is unrelated with its cytotoxicity；Excellent body gradually strengthens the inhibitory action of urea hydrolytic velocity UT B；Excellent body is reversible to the inhibitory action of UT B；Results from vivo experiments shows that excellent body can significantly increase rat urine amount；Reduce the concentration of urea in rat urine；And its osmotic pressure is reduced, show that excellent body generates urea selective diuresis in vivo.
Description
Technical field
The present invention relates to a kind of new opplication of the excellent body of urea hydrolytic velocity inhibitor.
Background technology
1. diuretics is applied and research and development focus at present
Diuretics acts on kidney, can increase the discharge of water.Clinically it is mainly used in treating the oedema that a variety of causes draws,
Can be used to treat some non-edema diseases, such as be can be used alone as a line medicine or use treatment blood high with other drugs compatibility
Pressure, reduces the incidence and case fatality rate of cardiovascular and cerebrovascular disease.Conventional diuretics is broadly divided into three classes at present：High-effect diuresis
Medicine, middle efficiency diuretics, low ceiling diuretic.High ceiling diuretic and middle efficiency diuretics are mainly respectively by specific suppression
Medullary loop ascending branch Na processed+/K+/2Cl-Co-transport and distal convoluted tubule Na+/Cl-Co-transport, suppresses the reabsorption of NaCl, drop
The urine concentrating power of low kidney, discharge is a large amount of close to isotonic urine.But long-term use these diuretics can cause electrolyte disorderly
Unrest, such as Diagnostic value, low blood sodium, hypomagnesemia etc..The low ceiling diuretic of clinical practice is mainly some isokalaemic diuretics, by
Concetrated pipe and distal convoluted tubule antagonism aldosterone, the row's of showing sodium protect the effect of potassium, and long-term use can cause hyperkalemia etc. bad anti-
Answer [Mann SJ.The silent epidemic of thiazide-induced hyponatremia, J
ClinHypertens,2008,10:477-84].Therefore, find and exploitation does not cause the new diuretics of electrolyte disturbance to be profit
Urinate the focus of drug development.Urea hydrolytic velocity（UT）Play extremely important, selectivity knockout in urinary concentrating mechanism
Urea passage can block urea cycle path in kidney, reduce urine concentrating capacity, not influence Na+、K+、Cl-In the case of excretion,
Produce urea selective diuresis.Urea hydrolytic velocity inhibitor can not appreciably affect body electrolyte as diuretics
In the case of balance, lower permeable pressure head in the kidney that urea cycle is set up in kidney, so as to produce diuresis, be adapted to hypertension etc.
Chronic disease patient's long-term use.Therefore, researching and developing new diuretics as drug target using urea hydrolytic velocity will give hypertension
And the patient of the cardiovascular and cerebrovascular disease that occurs together brings glad tidings.
2. urea cycle process in urinary concentrating mechanism and kidney
The crude urine that normal person is formed daily there are about 180 liters, and the whole urine volume of actual discharge daily only has 1.5 liters or so.Urine
Element is the most abundant solute of content in urine, accounts for 40~50% of solute total amount in urine, and urea concentration may be up to Plasma Urea in urine
More than 100 times [Yang B and Bankir L.Renal handling of urea in transgenic mice of concentration
lacking the urea transporter UT-B,Am J Physiol Renal Physiol,2005,288:F881-
F896].Urea be participate in urinary concentrating mechanism major solute, its with urea cycle mechanism in kidney, by counter-current multiplication and adverse current
Exchange process, concentration is gradually increased by the inside myeloid tissue of medulla externa, and sodium chloride forms the osmotic pressure of cortex renis to kidney medulla together
Gradient, so that kidney effectively can be such that water and some solutes are effectively received by resorption by concentrated urine.Urea cycle in kidney
Mechanism is specifically included：（1）Concetrated pipe, to the reabsorption of water and to the not penetrating of urea, causes urea in collection under pitressin regulation and control
Close the concentration of pipe inner height；（2）Inner medullary collecting duct end makes the urea of high concentration penetrate into internal medullary mass the infiltrative increase of urea
Interstitial tissue；（3）Medullary substance urea passes through the straight vessels ascending branch of internal medullary mass constantly by blood band to Renal Cortex, further through straight
Thin vessels descending branch and medullary loop descending branch thin segment particular section are taken back medullary substance again to the penetrating of urea, thus maintain from cortex renis to
The Urea Gradient and osmotic pressure gradient of kidney medulla（See Fig. 2）, this process is in urinary concentrating mechanism with very important effect
[Sands JM.Renal urea transporters,CurrOpinNephrolHypertens,2004,13:525-532],
In addition to the straight vessels ascending branch endothelial cell of internal medullary mass is with the penetrating urea of micropore mode, each part mentioned above to the permeability of urea by
Urea passage（Urea transporter, UT）Mediation [Smith CP and Rousselet G.Facilitative Urea
transporters,J MembrancBiol,2001,183:1-14]。
Urea passage is the membrane channel protein of specific penetrating urea.7 members are cloned at present, has been belonging respectively to
Two subfamilies of UT-A and UT-B, UT-A subfamilies include 6 members（UT-A1 to UT-A6）By same gene（Slc14a2）Through
Different promoters regulate and control and [Bagnasco SM.Gene structure of urea produced by post transcription cleavage
transporters,Am J Physiol,2003,284:F3–F10;Shayakul C and Hediger
MA.TheSLC14gene family of urea transporters,Pfluegers Arch,2004447:603-609],
UT-B subfamily only one of which members UT-B.There are expression of 5 urea hydrolytic velocities in kidney different parts, UT-A1, UT-A3
And UT-A4（UT-A4 is only expressed in rat）In the expression of kidney concetrated pipe epithelial cell, UT-A2 is in kidney medullary loop descending branch thin segment table
Reach, UT-A5, UT-A6 are expressed in testis, colon respectively.UT-B is by another gene（Slc14a1）Expression, is positioned at kidney straight
Thin vessels descending branch endothelial cell, red blood cell and multiple histoorgans.In UT-A1, UT-A2, UT-A3, UT-A4 and UT-B mediation kidney
The urea permeability of urea cycle corresponding site, plays an important role during urea cycle in kidney, participates in urinary concentrating mechanism.
3. urea channel functional knocks out and can produce urea selective diuresis and reduce blood pressure
Using urea channel gene knock-out mice model [Yang B, Bankir L, Gillepsie A.Urea-
selective concentrating defect in transgenic mice lacking urea transporter
UT-B,J BiolChem,2002,277:10633-10637] the renal physiology result of study that carries out shows, missing UT-B's
Mice displayed no goes out developmental anomaly.UT-B is knocked out does not influence glomerular filtration rate(GFR, kidney weight and in urine beyond urea
Other major solutes（Na+、K+、Cl-）Clearance rate.But its urine concentrating capacity there occurs substantially change：Urine volume increases, urine infiltration
Pressure drop is low, urine urea and plasma wrea concentration proportion are only the 50% of wild-type mice.Test result indicate that, UT-B is directly small in kidney
The urea transhipment of blood vessel mediation accounts for 1/3rd [Bankir L, the Chen K and Yang that kidney always urinates concentrating capacity
B.Renal handling of urea in transgenic mice lacking the urea transporter UT-
B,Am J Physiol,2004,286:F144-F151].Under basal conditions, urine is concentrated UT-Al/UT-A3 Gene-Deficient Mices
Ability drops to the 35% of wild-type mice urine concentrating capacity, and its urine volume is higher than wild-type mice 3 times.And taken the photograph in strict control
After entering liquid 5 days, their osmotic pressure of urine is not improved.UT-A1/UT-A3 knock out mice urea is in kidney internal medullary mass
Accumulation also substantially reduces (being the 1/3 of normal level) [Fenton RA, Chou CL, Stewart GS.Urinary
concentrating defect in mice with selective deletion of phloretin-sensitive
urea transporters in the renal collecting duct,ProcNatlAcadSci,2004,101:7469-
7474;Fenton,R.A.,Flynn A,Shodeinde A.Renal phenotype of UT-A urea transporter
knockout mice,J Am SocNephrol.2005,16:1583–1592].Therefore, selectivity knocks out UT-B or UT-A1/
UT-A3 can block urea cycle path in kidney, reduce urine concentrating capacity, not influence Na+、K+、Cl-In the case of, produce urea
Selective diuresis.We recent research result show, are detected with Mouse tail artery non-invasive blood pressure under normal physiological condition
Method measures mouse blood pressure, the systolic pressure of UT-B knock-out mices（systolic）, diastolic pressure (diastolic) and mean blood pressure
(mean) it is significantly lower than wild-type mice.According to above result of study, it is proposed that urea hydrolytic velocity inhibitor can be researched and developed
Scientific hypothesis as diuretics.Advantage of the urea hydrolytic velocity inhibitor as diuretics is not influence fluid electrolyte to put down
Weighing apparatus, is adapted to hypertension chronic diseases patient's long-term use.
The content of the invention
It is an object of the invention to provide a kind of new opplication of the excellent body of urea hydrolytic velocity inhibitor.
The invention provides the new opplication of compound shown in Formulas I, specially：With compound shown in Formulas I or its pharmaceutically may be used
The salt or soluble compound of receiving are following any one product of active component：
1）Urea hydrolytic velocity inhibitor；
2）The medicine of diuresis；
3）Instrument medicine for studying urea hydrolytic velocity；
In the Formulas I, R1And R2It is identical or different, it is selected from any one in following radicals：
Halogen, phenyl, first naphthyl, benzyl, halogen substitution phenyl,2,3- dihydrobenzos [b]
[1,4] dioxin -6- amidos, the benzyl containing substituted base, the anilino- containing substituted base, the furyl containing substituted base, contain
The thiadiazolyl group of substituted base, the thiazolyl containing substituted base, the pyridine radicals containing substituted base, C1-C6 containing substituted base
Alkyl,Benzothiazole -2- amidos, quinary heterocyclic radical and hexa-member heterocycle base；
Wherein, the benzyl containing substituted base, the anilino- containing substituted base, the furyl containing substituted base, contain
Replace thiadiazolyl group, the thiazolyl containing substituted base, the pyridine radicals containing substituted base, the alkane of the C1-C6 containing substituted base of base
In base, substitution base is selected from phenyl, methyl, ethyl, amino, halogen, group-4 ethyl formate, dioxanes base, methoxyphenyl, piperidines
Base, methylamino, methoxyl group, isopropyl, hydroxyl, carbonyl, acetyl group, acetamido, methyl formate base, group-4 ethyl formate and second
At least one of acetoacetic ester base.
Additionally, with compound shown in above-mentioned Formulas I or its pharmaceutically acceptable salt or soluble compound prepare it is following
Application in any one product, falls within protection scope of the present invention：
1）Urea hydrolytic velocity inhibitor；
2）The medicine of diuresis；
3）Instrument medicine for studying urea hydrolytic velocity.
In the Formulas I, R1Selected from least one of following radicals：
R2Any one selected from following radicals：
Hydroxy benzenes amido,4- Ethyl formates anilino-,4- (4- methoxyphenyls) thiazole -2- amidos,2,3- dihydrobenzenes
And [b] [1,4] dioxin -6- amidos,2-（Piperidin-1-yl）Anilino-,Furans -2- first
Base amido,2- amino -4- methyl -5- Ethyl formates thiazolyl,2- methyl thiazoliums
Diazole 5- amino,The amino of 2- ethyls thiadiazoles -5,2,5- dimethoxyanilines
Base,The aminopyridine base of 2- methyl -6,2- isopropyl -5- chiral lactones base,3- hydroxy benzenes amido,Acetylbenzene amido,3- acetamidos
Anilino-,Methyl formate thiazole -2- the amidos of 4- methyl -5,4,5- dimethyl thiophenes
Azoles -2- amidos,4- ethyl acetate thiazole -2- amidos andBenzo thiophene
Azoles -2- amidos.
Specifically, compound shown in the Formulas I is compound shown in Formulas I -1, namely " excellent body "：
In the urea hydrolytic velocity inhibitor, urea hydrolytic velocity is UT-B and/or UT-A.
The preparation method of compound is as shown in Figure 2 shown in above-mentioned formula I.
Because the protein structure of UT-B has been elucidated with [Levin EJ, Cao Y and Zhou
M.Structureandpermeationmechanismof a mammalian urea transporter.ProcNatlAcad
Sci,2012,109:11194-9.Levin EJ,Quick M,and Zhou M.Crystal structure of a
bacterial homologue of the kidney urea transporter.Nature,2009,462,757-761],
For screening urea channel inhibitor theoretical foundation has been established with its inhibitory action mechanism is studied.The early-stage Study result table of inventor
Bright, the erythrocyte membrane urea permeability of UT-B mediations is 50 times of [Yang B, and of bilayer lipid membrane urea permeability
Verkman AS.Analysis of double knockout mice lacking aquaporin-1and urea
transporter UT-B.Evidence for UT-B-facilitated water transport in
erythrocytes,J BiolChem,2002,277:36782-36786], UT-B removes the also penetrating water of penetrating urea and urea class
Like thing acetamide [Zhao D, Sonawane ND, Levin MH, and Yang B.Comparative transport
efficiencies of urea analogues through urea transporter UT-B,BiochimBiophys
Acta,2007,1768:1815-1821].According to erythrocyte membrane high level expression aquaporin AQP1（The penetrating water of specificity
Memebrane protein）With the characteristic of urea hydrolytic velocity UT-B, the seminar where inventor establishes the high pass of urea channel inhibitor
Amount screening model [Marc H.Levin, Ricardo de la Fuente, and A.S.Verkman.Urearetics:a
small molecule screen yields nanomolar potency inhibitors of urea transporter
UT-B,The FASEB J,2007,21:551-563], see that red blood cell is placed on 1.25M acetamides (the penetrating acetyl of UT-B by Fig. 1
The speed of amine is slower than penetrating urea, is suitable to measurement permeability inhibitory action) in normal saline solution, it is contained within red blood cell highly concentrated
The acetamide of degree, then red blood cell is quickly put into isotonic physiological saline.The hyperosmosis that intracellular high concentration acetamide is produced
By water by AQP1 rapid transports to intracellular, acetamide is gone out in the presence of extracellular concentration difference by UT-B rapid transports in the cell
Cell, makes intra-erythrocyte osmotic pressure reach balance rapidly, and cell volume change is not obvious.If urea passage is blocked, carefully
Intracellular acetamide is unable to rapid transport and goes out cell, and water is entered thin by caused intraor extracellular permeable pressure head by AQP1 rapid transports
Born of the same parents, so as to cause erythrocyte volume to increase rapidly so that rupture.EF discharges the amount of hemoglobin, can be used as compound
To the evaluation index of urea hydrolytic velocity UT-B inhibitory activity.ELIASA surveys 710nm absorbances, can calculate erythrocytolysis
Rate.
The present invention is using urea channel inhibitor screening model by high throughput screening system from the small molecule chemical combination for synthesizing
The clue compound with urea passage UT-B inhibitory activity is screened in thing storehouse.From 650 derivatives of clue compound（Chemistry
Structure is shown in Table 1）In sift out the best candidate compound of dose-effect relationship " excellent body ", its structure is shown in formula I -1.Excellent body and its part derive
Thing has the activity of the stronger suppression penetrating urea of UT-B and UT-A, and the half that it suppresses to urea passage UT-B in vitro is effective
Dosage is below micromolar levels, and no obvious cytotoxicity.Experiment in vivo finds：Giving excellent body in vivo can dramatically increase greatly
Mouse urine volume, reduces urine urea level, while reducing osmotic pressure of urine, shows with good urea selective diuresis.
Brief description of the drawings
Fig. 1 is red blood cell urea channel inhibitor screening model.
Fig. 2 is cleavage rate of the excellent body to red blood cell.
Fig. 3 is that red blood cell high flux screening model screening conditions optimize figure.
Fig. 4 is the CDCC of excellent body.
Fig. 5 is inhibitory action of the excellent body to the penetrating urea of mdck cell UT-A.
Fig. 6 is the Time-activity-curve of the excellent body diuresis of rat skin lower injection.
Fig. 7 is influence figure of the excellent body to various parameters（Average value+standard deviation, n=6）.
Fig. 8 is the influence figure of excellent body and Hydrochioro to myeloid tissue in kidney（Average value+standard deviation, n=6）.
Fig. 9 is influence figure of the excellent body to various parameters（Average value+standard deviation, n=6）.
Specific embodiment
With reference to specific embodiment, the present invention is further elaborated, but the present invention is not limited to following examples.Under
Experimental technique described in embodiment is stated, unless otherwise specified, conventional method is；The reagent and biomaterial, such as without special
Illustrate, commercially obtain.
Embodiment 1, the screening of UT-B inhibitor and pharmacodynamic evaluation
1st, screening test method
1）Blood is taken, 15ml graduated centrifuge tubes are placed in（It is suspended from the PBS containing liquaemin）In, centrifugation, 3000r/min,
10min, abandons supernatant；
2）The PBS with blood equivalent is added, is centrifuged, 3000r/min, 10min abandon supernatant；
3）The cell suspension that red blood cell to specific volume is 2% is diluted with the hypertonic PBS containing 1.25M acetamides；
4）Red cell suspension is placed in incubation at room temperature 2h makes intraor extracellular acetamide concentration balance, is regularly mixed with pipettor
Close；
5）Take the 99 above-mentioned red cell suspensions of μ l to be placed in 96 hole each holes of round bottom microwell plate, be subsequently adding 1 μ l testing compounds
（Such as excellent body）, mix, it is incubated at room temperature 6min（Final concentration of 20 μM of testing compound, DMSO final concentration of 1%）；
6）The flat black wall microwell plate in 96 holes separately is taken, the 180 isotonic PBS of μ l (containing 1%DMSO) are added per hole；
7）Take above-mentioned steps 5）The μ l of red cell suspension 20, are rapidly added in 96 orifice plates, quick to mix；
8）In 5min absorbance, wavelength 710nm are surveyed with ELIASA；
9）Every piece of microwell plate is all provided with Positive control wells（Non-specific UT-B inhibitor phloretin）, negative control hole
（PBS）.
Calculate erythrocytolysis rate：
The dissolution rate percentage computing formula of red blood cell, wherein AtestIt is the absorbance of instrument connection, AnegIt is negative control
The absorbance in hole, AposIt is the absorbance of Positive control wells.Erythrocyte splitting rate, extinction are calculated by surveying absorbing wavelength 710
Angle value stabilization, absorbance is unchanged within least one hour, as a result sees Fig. 2.
2nd, red blood cell high flux screening model screening conditions optimization
With the acetamide of various concentrations（0-3.0M）Red blood cell is incubated, the absorbance of 710nm is surveyed, amount effect curve is drawn,
When acetamide concentration is 1.1-1.25M, positive controls（phloretin）Absorbance difference with solvent control group is maximum,
Therefore the acetamide of selection 1.1-1.25M carries out subsequent experimental, as a result sees Fig. 3.
3rd, urea channel inhibitor clue compound is found
Be increase find urea passage clue compound chance, this project team first according to UT-B protein conformations,
Computer simulation screening is carried out to the compound with Formulas I female ring structure in computer chemical simulation method, 2319 changes are selected
Compound, DMSO is dissolved in by above-claimed cpd, and 1mM concentration application liquid is diluted in 96 hole microwell plates as screening compounds storehouse.
Take people, rabbit（Japanese white big ear rabbit）, rat（SD rats）, mouse（C57 mouse）Four red blood cells of kind, with
Red blood cell urea channel inhibitor screening model, the preliminary screening of urea channel inhibitor is carried out to above-mentioned screening compounds storehouse,
Screening compounds concentration is 10M, repeats screening once, determines clue compound.
4th, clue compound specificity suppresses urea passage
In order to determine clue compound action specificity, red blood cell is balanced with isotonic PBS or 1.25M acetamides PBS respectively,
Clue compound（10μM）After incubation, in fast transfer to isotonic PBS, erythrocyte splitting rate is detected.Result is：Use isotonic PBS
The red blood cell of incubation, has no that red blood cell is substantially cracked, and the red blood cell being incubated with 1.25M acetamides PBS, erythrocyte splitting.Table
The rupture of bright red blood cell be clue compound specificity suppress urea hydrolytic velocity urea permeability caused by.
5. optimal clue compound is determined
Based on the parent nucleus of the clue compound structure for being obtained, replace the replacement chemical constitution analog of base,
Postsearch screening small molecule libraries are set up, using above-mentioned model and method screening and determination activity, dose-effect experiment result is obtained（Table
1）（Note：IC in table 150It is concentration when HRBC's dissolution rate is 50%）.
Through com-parison and analysis, compound shown in the Formulas I -1 for having better inhibition effect to four kinds is selected as preferredization
Compound, is named as excellent body, and excellent body is shown in Table 1 with the chemical structural formula of other compounds.
6. excellent body compound is without obvious cytotoxicity
In order to study the cytotoxicity of above-claimed cpd, using CCK-8 kits（Colleague's chemistry institute）, complete
Mdck cell toxicity test, is as a result shown in Fig. 4, shows the excellent body of compound without notable CDCC.
This research CCK-8 kit detection compound cytotoxicities：By the mdck cell suspension inoculation of exponential phase
In 96 well culture plates（1×104Individual cells/well/100 μ l）, give 100 μ l per hole and contain 10% hyclone, 100U/ml moulds
The DMEM nutrient solutions of element and 100 μ g/ml streptomysins, put 37 DEG C, cultivate in 5%CO2 incubators.When cell 70%-80% is merged,
Serum starvation synchronizing of 12h.Then 100 μ l are given per hole and contains various concentrations（0.128,0.64,3.2,16 with 80 μM）
The DMEM nutrient solutions of compound, cultivate 12h.10 μ l CCK-8 detection liquid is given per hole, 37 DEG C of lucifuges are incubated 1h, detect 470nm
OD values.Blank well is set simultaneously（Culture medium, CCK-8）, control wells（Cell, the dissolving medium of same concentrations compound, culture
Base, CCK-8）, every group sets 3 multiple holes.Calculate cell survival rate：
Cell survival rate（%）=[（OD experimental port-OD blank wells）/（OD control wells-OD blank wells）]×100%
Wherein, OD refers to the absorbance in each hole.
7. excellent body specificity suppresses urea passage UT-A
To determine inhibitory action of the excellent body to UT-A, the mdck cell of stabilization expression UT-A1 is cultivated in Transwell
Into close monolayer, UT-A protein deliveries to cytoplasma membrane are stimulated with forskolin, 15min is incubated with excellent body, will
Nutrient solution below Transwell changes the nutrient solution containing 15mM urea into, is cultivated on special time detection Transwell
Urea concentration in liquid, evaluates inhibitory action of the excellent body to UT-A urea permeabilities.
Test result indicate that, excellent body significantly inhibits the urea permeability of UT-A1 mediations（Fig. 5 A）, its inhibitory activity with it is positive
Control compound non-specificity urea channel inhibitor phloretin inhibition strengths are identical（Fig. 5 B）, point out excellent body to UT-B and
UT-A has identical inhibitory activity.
8. excellent body has diuresis
From 8 week old male SD rats, body weight 180-200g.Adaptation environment on the 2nd in metabolic cage is previously placed in, observation is freely
Whether urine volume is stablized under the conditions of drinking water diet.Two hours urines are collected in morning 8-10 points on the 3rd, then the agent of hypodermic injection difference
Measure excellent body（3.125th, 12.5,50,100mg/kg body weight）, hereafter collect every 2 hours urines, altogether collect administration after eight it is small
When.The urine of every 2 hours before and after administration is each sufficiently mixed, measurement urine volume, osmotic pressure of urine value（Uosm）, urine urea
(Uurea) level.
Influence of the excellent body to rat urine volume is shown in（Fig. 6 A）, the change after administration over time, rat unit time urination amount
Gradually increase, after being administered 2~4 hours, unit interval urination amount reaches top, and plateau continues 2~4 hours, starts afterwards
Decline.After administration the urine volume of 6~8 hours with compare and still dramatically increase before administration（P<0.05）.
Excellent body on rat osmotic pressure of urine and the Time-activity-curve of urine urea concentration influence respectively such as（Fig. 6 B）With（Fig. 6 C）It is shown,
Change after administration over time, osmotic pressure of urine and urine urea concentration are gradually reduced, and the lowest point was reached at 2~4 hours, are then returned
Rise, infiltration voltage levels change always with urea concentration, and simply numerical value is bigger, show that the change of osmotic pressure of urine is main by urea in urine
Caused by change in concentration, the two Changing Pattern changes with urine volume conversely, prompting urea selective diuresis.
Once, twenty-four-hour urine liquid is collected in continuous injection 5 times to the excellent bodies of every 6 hours hypodermic injection 50mg/kg, is measured 24 hours
Urine volume, osmotic pressure of urine (Uosm), urine urea (Uurea), infiltration solute excretion, urea excretion and non-uremic solutes excretion.
Test result indicate that, the excellent body of 50mg/kg dosage substantially increases urine volume（Fig. 7 A）, reduce osmotic pressure of urine（Fig. 7 B）With
Urine urea concentration（Fig. 7 C）, and solute excretion is permeated to the unit interval（Fig. 7 D）, urea excretion（Fig. 7 E）It is molten with non-urea
Matter excretion（Fig. 7 F）Have not significant impact.By contrast, positive control medicine Hydrochioro（HCTZ）In diuresis simultaneously,
Substantially increase the excretion of infiltration solute（Fig. 7 D）, and it is mainly due to non-uremic solutes（Mainly sodium, potassium, chlorion）Row
Discharge increase causes（Fig. 7 F）.Urea excretion rate before and after administration is similar, shows that the diuresis that excellent body is produced are by excellent body shadow
Ring caused by the caused urine concentrating capacity reduction of urea cycle in kidney, it does not cause obvious sodium, potassium, chlorion to be lost.
To the osmotic pressure of myeloid tissue in the kidney of above-mentioned experimental rat（Fig. 8 A）, urea level（Fig. 8 B）With non-uremic solutes
Level（Fig. 8 C）Knowable to being detected, excellent body causes in kidney substantially to be reduced with tissue infiltration pressure and urea level, and internally with
The non-uremic solutes of tissue have not significant impact.By contrast, Hydrochioro does not change the osmotic pressure of myeloid tissue, urea in kidney
With non-urea level.
These experimental results point out excellent body by blocking urea cycle in kidney, change the urea concentration of myeloid tissue in kidney,
Make, from cortex renis to the reduction of the osmotic pressure gradient of kidney medulla tissue, diuresis to be produced by reducing urine concentrating capacity.
Prohibit water 18 hours to rat, then the excellent bodies of every 6 hours hypodermic injection 50mg/kg, urine was collected at the 24th hour, survey
Osmotic pressure of urine, urine urea and the non-uremic solutes level of urine, even if finding in the case where water is prohibited, the urine of the rat of the excellent body of injection
Osmotic pressure（Uosm, Fig. 9 A）, urine urea (Uurea, Fig. 9 B) and level still is below control rats, rather than uremic solutes
（Unon-urea solutes, Fig. 9 C）Level does not have significant change, it was demonstrated that excellent body still has stronger in the case of maximum urine concentrating capacity
Diuresis, and the diuresis of excellent body do not influence the non-uremic solutes such as sodium, potassium, chlorine.
Blood biochemistry Testing index shows, after excellent body is acted on 24 hours, blood Na+、K+、Cl-, urea, creatinine, blood fat do not send out
Raw to change, by contrast, Hydrochioro causes blood Na+、K+、Cl-Level reduction, urea level increases, cholesterolemia, glycerine three
Ester and low-density lipoprotein are raised.Excellent body is pointed out not cause electrolyte to be lost and metabolic disorder.
The structural formula and IC of the compound of the screening of table 150Value
Claims (5)
1. compound shown in Formulas I or its pharmaceutically acceptable salt or soluble compound are in following any one product are prepared
Application：
1) urea hydrolytic velocity inhibitor；
2) medicine of diuresis；
3) for studying the instrument medicine of urea hydrolytic velocity；
In the Formulas I, R1And R2It is identical or different, it is selected from any one in following radicals：
Halogen, phenyl, first naphthyl, benzyl, halogen substitution phenyl,Benzyl containing substituted base, contain
Replace the anilino- of base, the furyl containing substituted base, the thiadiazolyl group containing substituted base, the thiazolyl containing substituted base, contain
The pyridine radicals of substituted base, the alkyl of the C1-C6 containing substituted base,Quinary heterocyclic radical and hexa-member heterocycle base；
Wherein, the benzyl containing substituted base, the anilino- containing substituted base, the furyl containing substituted base, containing substituted
In the thiadiazolyl group of base, the thiazolyl containing substituted base, the pyridine radicals containing substituted base, the alkyl of the C1-C6 containing substituted base,
Substitution base is selected from phenyl, methyl, ethyl, amino, halogen, group-4 ethyl formate, dioxanes base, methoxyphenyl, piperidyl, methyl
Amido, methoxyl group, isopropyl, hydroxyl, carbonyl, acetyl group, acetamido, methyl formate base, group-4 ethyl formate and ethyl acetate
At least one of base.
2. application according to claim 1, it is characterised in that：In the Formulas I, R1Selected from least one of following radicals：
R2Any one selected from following radicals：
3. application according to claim 1, it is characterised in that：Compound shown in the Formulas I is compound shown in Formulas I -1：
4. according to any described applications of claim 1-3, it is characterised in that：In the urea hydrolytic velocity inhibitor, urea
Channel protein is UT-B and/or UT-A.
5. application according to claim 4, it is characterised in that：The UT-A is UT-A1.
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