CN103374057A - New compound with inhibiting activity to glycine transporters - Google Patents
New compound with inhibiting activity to glycine transporters Download PDFInfo
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- CN103374057A CN103374057A CN2012101099049A CN201210109904A CN103374057A CN 103374057 A CN103374057 A CN 103374057A CN 2012101099049 A CN2012101099049 A CN 2012101099049A CN 201210109904 A CN201210109904 A CN 201210109904A CN 103374057 A CN103374057 A CN 103374057A
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Abstract
The invention discloses a new compound with inhibiting the activity to glycine transporters, which is shown in a formula (I). The compound can be used for treating mental disorders associated with NMDA glycine receptors. The invention also discloses a preparation method of the compound shown in the formula (I), a pharmaceutical composition taking the compound as an active ingredient, and the application of the compound in preparation of drugs for treating mental disorders reacting with NMDA glycine receptors.
Description
Technical field
What the present invention relates to microorganisms has the new compound that suppresses active to the mammalian nervous spongiocyte glycine transport factor.
Technical background
The schizophrenia physiology cause of disease that academic circles at present is generally acknowledged is due to the L-glutamic acid Neurotransmission function that depends on N-methyl-D-aspartate (NMDA) in the brain reduces.Wherein nmda receptor contains glycine and polyamines combining site, can activate nmda receptor by being combined with glycine, plays treatment schizophrenia and other mental diseases relevant with the nmda receptor dysfunction.
In recent years, find that through a large amount of molecular biology researches there are two types of GlyT-1 and GlyT-2 in the NMDA Glycine Receptors, wherein GlyT-1 is divided into again three kinds of GlyT-1a, GlyT-1b and GlyT-1c.These nmda receptors can be blocked by some inhibitor or antagonist, are similar to schizoid negative symptoms thereby induce to produce.These studies show that the nmda receptor dysfunction is relevant with schizoid physiopathology reason.
In addition, further clinical studies show, the relative normal population of nmda receptor quantity in schizophreniac's brain significantly increases.At present existing clinical report is pointed out, can effectively improve the symptoms of schizophrenia by using high dosage glycine therapeutics.(such as D.C Javitt, I Zylberman etc., Amelioration of negative symptoms in schizophrenia by glycine.Am.J.Psychiatry, 1994; The glycine quantity that 151:1234-1236) is present in the myelencephalon gap is regulated by the glycine transporter that is present in the neurogliocyte, therefore, the glycine transporter inhibitor can be by increasing the quantity of glycine in the myelencephalon gap, reach treatment and alleviate negative schizophrenia, and the symptoms such as other mental anomaly, Alzheimer, many infraction dementias, AIDS with the nmda receptor functional correlation are dull-witted, Huntington's disease and Parkinson's disease.
The compounds of this invention is based upon on the Chinese patent application CN1535980A Research foundation, adopting exogenous amino acid to mix fermentation technique obtains, not yet discovery is identical with the compounds of this invention structure at present, and the compound that glycine transporter is had remarkable inhibiting activity.
Summary of the invention
Purpose of the present invention is for providing a class neurogliocyte glycine transporter to be had the novel physiologically active substance that suppresses active.
The contriver finds that Cucumber has excellent inhibition activity to the glycine transporter of neurogliocyte, thereby has finished the present invention by various researchs.
That is, the present invention is the compound with represented by formula I, and compound number is C6035G, and chemistry is by name: 3-Benzyl-9,12-di-sec-butyl-6-(3-oxo-but-1-enyl)-Isosorbide-5-Nitrae, 7,10-tetraaza-cyclododecane-2,5,8,11-tetraone.
The contriver is in order to finish above-mentioned purpose, patent microorganism strains CGMCCNo.0792 to report among the Chinese patent application CN1535980A (glycine transporter being had the new compound that suppresses active) has adopted the external source ILE to mix fermentation technique, namely promote the biosynthesizing of target compound C6035G by adding exogenous ILE on the basis of former fermention medium, and through separation and purification, structure elucidation and active determination in vitro, find that new compound C6035G has remarkable inhibiting activity to the glycine uptake of rat neurogliocyte, thereby finished the present invention.
According to conventional aerobic fermentation condition, bacterial strain CGMCCNo.0792 has been carried out fermentation culture in the substratum that contains the multiple nutrients material.
The main liquid nutrient medium that uses, substratum is comprised of carbon source, nitrogenous source, amino acid and inorganic salt, can add VITAMIN, precursor substance and defoamer as required.Carbon source is used alone or as a mixture glucose, glycerine and starch etc.; Nitrogenous source is used alone or as a mixture oat, yeast water, soyflour, meat soup, peptone, urea, ammonium salt and is rich in compound nitrogen source of ILE etc.; Amino acid adds separately ILE; Inorganic salt are used alone or as a mixture sodium phosphate, sal epsom, sodium-chlor and salt of wormwood etc.; Defoamer can use alcohols and silicide; Fermention medium adopts hydrochloric acid or sodium hydroxide to regulate pH to 7.0.
Fermentation culture method adopts the aerobic modes such as shaking culture and aeration-agitation cultivation, and pH is controlled at 4-10, and temperature 25-35 ℃, time 2-8 days, optimal pH 6-7, cultivated under the condition of 6 days time by optimum temperuture 25-28 ℃.
Its tunning of compound of the present invention can be made with extra care with ordinary method and obtain.After namely cultivating end, adopt centrifugation or filtration to obtain culturing filtrate, make it to be adsorbed in HP-20 (trade(brand)name, Mitsubishi Chemical Ind makes) polyvinyl resin, with lower alcohol and acetone and other organic solvent wash-out.Thalline extracts with lower alcohol and acetone and other organic solvent.Subsequently, the extracting solution of thalline and the elutriant of polymeric adsorbent are merged, concentrating under reduced pressure is removed organic solvent, residue is changed in the water-insoluble organic solvents such as ethyl acetate, chloroform and propyl carbinol to dissolve, and is condensed into pasty state.After this mashed prod being dissolved in again the organic solvents such as benzene, ethyl acetate, acetone, methyl alcohol and chloroform, adopt silica gel column chromatography, reversed-phase silica gel chromatography and preparation HPLC separation and purification the compounds of this invention.
Resulting Compound C 6035G warp as stated above
1The H-NMR nuclear magnetic resonance spectrum,
13After the spectrum such as C-NMR nuclear magnetic resonance spectrum is measured, resolve and prove conclusively its chemical structure.
Compound C 6035G physico-chemical property is as follows:
1, outward appearance: white powder
2, molecular weight: 498
3, molecular formula: C
27H
38N
4O
5
4, HR-TOF mass spectrum:
Measured value: 498.2822; Calculated value: 498.2842 (according to C
27H
38N
4O
5H+ calculates)
5, fusing point:＞300 ℃
6, ultra-violet absorption spectrum λ
MAX(nm):
Diox: 206.5,223.5 (sh)
7,
1The H-NMR nuclear magnetic resonance spectrum: sample is dissolved in the dimethyl sulfoxide (DMSO) (DMSO), measures under 500MHz, the results are shown in Fig. 1.
8,
13The C-NMR nuclear magnetic resonance spectrum: sample is dissolved in the dimethyl sulfoxide (DMSO) (DMSO), measures under 125MHz, the results are shown in Fig. 2.
9, solubleness: be dissolved in dioxane, methyl-sulfate, dimethyl formamide; Indissoluble and chloroform; Water insoluble, normal hexane, methyl alcohol, ether, acetoneand ethyl acetate.
10, acid-basicity: neutrality
The compounds of this invention has inhibition to the glycine transporter of finding in neurogliocyte.So useful to schizoid treatment.
Description of drawings
Fig. 1 represents what Compound C 6035G measured with 500MHz in dimethyl sulfoxide (DMSO) (DMSO)
1The H-NMR spectrum
Fig. 2 represents what Compound C 6035G measured with 125MHz in dimethyl sulfoxide (DMSO) (DMSO)
13The C-NMR spectrum
Embodiment
Below the present invention is further described in detail explanation.
The preparation of embodiment 1 Compound C 6035G
The liquid nutrient medium that 1, will contain 1% glucose, 2.5% Zulkovsky starch, 0.3% cottonseed meal, 0.5% fish meal, 0.3%NZCASE, 0.2% yeast extract and 0.2% calcium carbonate was regulated pH to 7.0, in 120 ℃ of lower moist heat sterilizations 20 minutes.Then an amount of bacterial strain CGMCCNo.0792 of inoculation under aseptic technique, 28 ℃, 180rpm shaking culture 3 days are as seed culture fluid.
Then will contain 1% glycerine, 0.5% glucose, 1% Zulkovsky starch, 0.1% (NH
4)
2SO
4, 0.05%MgSO
47H
2The liquid nutrient medium 100ml that O, 0.2% calcium carbonate and ILE 0.2% form regulated in the 500ml triangle shaking flask of packing into behind the pH to 7.0, altogether prepares 100 bottles, in 120 ℃ of lower moist heat sterilizations 20 minutes.Then under aseptic technique, aforementioned seed culture fluid is added wherein 28 ℃, 180rpm shaking culture 6 days by 10% inoculum size.
After cultivating end, gained 10L nutrient solution is added propyl carbinol by 2: 1 (V/V), get the n-butanol layer concentrating under reduced pressure after stirring, the centrifugation and get brown oily crude product 5.71g.
2, aforementioned brown oily crude product 5.71g is dissolved in the 10ml chloroform, be splined on 400ml silica gel G el 60 (manufacturing of Merck company) chromatography column bed behind the ultrasonic dissolution, adopt chloroform-methanol binary elution system by certain ratio of mixture (98: 2-50: 50) carry out gradient elution.To wherein merge take the elution fraction of mixed solvent ratio as 90: 10, concentrating under reduced pressure is dry, gets 88.4mg cut Fr-1.
3, above-mentioned cut Fr-1 is dissolved in a small amount of methyl alcohol, adopt acetonitrile-0.01% trifluoroacetic acid aqueous solution (45: 65) as the isocratic elution mode of moving phase, LC-8A prepares liquid phase systems in Shimadzu, separation and purification on YMC ODS-AM (150 * 20mm, the 5 μ) preparative column.Flow velocity 5ml/min, ultraviolet detection wavelength 210nm.Collecting retention time is the component of 12.8-13.4min, after concentrating under reduced pressure is removed acetonitrile, adds ethyl acetate extraction 2 times by partly measuring volume.The combined ethyl acetate extraction liquid, dried with being evaporated to behind the anhydrous sodium sulfate dehydration, obtain 18.4mg Compound C 6035G.
The inhibition experiment of 2 pairs of rat neurogliocyte of embodiment
1, a corpse or other object for laboratory examination and chemical testing
Compound C 6035G is dissolved in dimethyl sulfoxide (DMSO) and is mixed with the concentration of 10mg/ml, be diluted to purpose concentration with aqua sterilisa and use.
2, test cell
Rat neurogliocyte C6.
3, test method
In the flat 96 hole microwell plates (Cytostar-T:Sigma Co.) that contain liquid scintillator, add rat neuroglia C6 cell 5 * 10
4Cell/ware.In containing 10% calf serum (Gibco Co.) substratum, in 37 ℃ and 5%CO
2Cultivated 24 hours in the incubator.After this use BBS damping fluid (10mMHepes-tris (pH7.4), 1mM calcium chloride, 2.5mM Repone K, 2.5mM sal epsom) and 10mM glucose replacement medium.At the test compound C6035G 10 μ l that add different concns and 375KBq/ml
[14C]Behind the glycine 10 μ l, cultivated 50 minutes in 30 ℃.After the cultivation, discard damping fluid, drying is 5 hours under the room temperature.Then use TopCount Isotope analyzer (Perkin Elmer Co.) to measure its radioactivity.To not add the blank group radioactivity of medicine as 0%, excessive glycine (10mM) control group radioactivity is as 100%, compare with medicine group radioactivity, be calculated according to the following formula inhibiting rate, show that the compound concentration of 50% inhibiting rate is decided to be IC
50Value.
Average through three mensuration, Compound C 6035G is at 0.5nM, 1nM, 2.5nM, 5nM, 10nM, the inhibiting rate under the 20nM concentration is respectively 9.4%, 11.9%, and 31.8%, 58.2%, 78.4% and 77.9%, adopt logarithm concentration and inhibiting rate mapping the Fitting Calculation, its IC
50Value is 4.2nM.
Claims (4)
1. one kind has the new compound that suppresses active to glycine transporter, and structural formula I is as follows:
2. new compound according to claim 1 is characterized in that pharmaceutical composition adds pharmaceutically acceptable medicinal adjuvant by this new compound by activeconstituents and formed.
3. new compound according to claim 1 is for the preparation of the purposes in the mental disorder medicine that reacts with the NMDA Glycine Receptors for the treatment of.
4. new compound according to claim 1 is characterized in that the preparation method is as follows:
The liquid nutrient medium that 1) will contain 1% glucose, 2.5% Zulkovsky starch, 0.3% cottonseed meal, 0.5% fish meal, 0.3%NZ CASE, 0.2% yeast extract and 0.2% calcium carbonate was regulated pH to 7.0, in 120 ℃ of lower moist heat sterilizations 20 minutes; Then an amount of bacterial strain CGMCCNo.0792 of inoculation under aseptic technique, 28 ℃, 180rpm shaking culture 3 days are as seed culture fluid;
2) then will contain 1% glycerine, 0.5% glucose, 1% Zulkovsky starch, 0.1% (NH
4)
2SO
4, 0.05%MgSO
4The liquid nutrient medium 100ml that 7H2O, 0.2% calcium carbonate and ILE 0.2% form regulated in the 500ml triangle shaking flask of packing into behind the pH to 7.0, altogether prepares 100 bottles, in 120 ℃ of lower moist heat sterilizations 20 minutes; Then under aseptic technique, aforementioned seed culture fluid is added wherein 28 ℃, 180rpm shaking culture 6 days by 10% inoculum size;
3) after cultivation finishes, gained 10L nutrient solution is added propyl carbinol by 2: 1 (V/V), get the n-butanol layer concentrating under reduced pressure after stirring, the centrifugation and get brown oily crude product 5.71g;
4) aforementioned brown oily crude product 5.71g is dissolved in the 10ml chloroform, is splined on 400ml silica gel G el 60 chromatography column beds behind the ultrasonic dissolution, adopt chloroform-methanol binary elution system by certain ratio of mixture (98: 2-50: 50) carry out gradient elution; To wherein merge take the elution fraction of mixed solvent ratio as 90: 10, concentrating under reduced pressure is dry, gets 88.4mg cut Fr-1;
5) above-mentioned cut Fr-1 is dissolved in a small amount of methyl alcohol, adopt acetonitrile-0.01% trifluoroacetic acid aqueous solution (45: 65) as the isocratic elution mode of moving phase, LC-8A prepares liquid phase systems in Shimadzu, separation and purification on YMC ODS-AM (150 * 20mm, the 5 μ) preparative column; Flow velocity 5ml/min, ultraviolet detection wavelength 210nm; Collecting retention time is the component of 12.8-13.4min, after concentrating under reduced pressure is removed acetonitrile, adds ethyl acetate extraction 2 times by partly measuring volume; The combined ethyl acetate extraction liquid, dried with being evaporated to behind the anhydrous sodium sulfate dehydration, obtain the 18.4mg target compound.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210109904.9A CN103374057B (en) | 2012-04-16 | 2012-04-16 | A kind of compound glycine transporter to inhibit activities |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1535980A (en) * | 2003-04-08 | 2004-10-13 | 国家药品监督管理局四川抗菌素工业研 | New compound with cativity for inhibiting glycine transport factor |
| CN101479243A (en) * | 2006-06-28 | 2009-07-08 | 安姆根有限公司 | Glycine transporter-1 inhibitors |
| JP2010059182A (en) * | 2001-02-16 | 2010-03-18 | Nps Pharmaceuticals Inc | Glyt-1 inhibitor |
-
2012
- 2012-04-16 CN CN201210109904.9A patent/CN103374057B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2010059182A (en) * | 2001-02-16 | 2010-03-18 | Nps Pharmaceuticals Inc | Glyt-1 inhibitor |
| CN1535980A (en) * | 2003-04-08 | 2004-10-13 | 国家药品监督管理局四川抗菌素工业研 | New compound with cativity for inhibiting glycine transport factor |
| CN101479243A (en) * | 2006-06-28 | 2009-07-08 | 安姆根有限公司 | Glycine transporter-1 inhibitors |
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Effective date of registration: 20210421 Address after: Outside the East Shiling Town of Longquanyi District of Chengdu city of Sichuan Province in 610000 Patentee after: CHENGDU University Address before: 610052 No. 168 Hua Guan Road, Longtan economic zone, Chenghua District, Sichuan, Chengdu Patentee before: SICHUAN INDUSTRIAL INSTITUTE OF ANTIBIOTICS, CHINA NATIONAL PHARMACEUTICAL Group Corp. |
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