CN104693137A - Active metabolite of mosapride - Google Patents
Active metabolite of mosapride Download PDFInfo
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- CN104693137A CN104693137A CN201310666229.4A CN201310666229A CN104693137A CN 104693137 A CN104693137 A CN 104693137A CN 201310666229 A CN201310666229 A CN 201310666229A CN 104693137 A CN104693137 A CN 104693137A
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- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
- C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
- C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
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Abstract
The invention provides a low-toxicity and high-efficiency active metabolite of mosapride, which is about to clinically become a substitute of mosapride later. The metabolite provided by the invention is as shown in the specification.
Description
Technical field
The invention belongs to medical art, be specifically related to preparation method and the application thereof of the active metabolite of mosapride.
Background technology
Mosapride (mosapride), (±)-4-amino-5-chloro-2-ethoxy-N-[[4-(4-luorobenzyl)-2-morpholinyl] methyl] benzamide, it is potent, a selectivity benzamides 5-HT4 receptor stimulant, as novel medicine for stomach dynamic, it is more and more for Japan and some other Asian countries.Mosapride has following chemical structure:
D2 dopamine receptor on mosapride and brain nervous cell synaptic membrane, suprarenin α 1 acceptor, 5-HT1 and 5-HT2 acceptor without avidity, therefore can not cause Extra Pyramidal Syndrome and cardiovascular adverse effects.The incidence of untoward reaction is about 4%.Main manifestations is diarrhoea, stomachache, dry, fash, burnout, dizziness, discomfort, palpitaition etc.Separately have an appointment 3.8% patient there is test rating ANOMALOUS VARIATIONS, show as eosinophilia, triglyceride level raise, ALT raise etc.Based on above untoward reaction, therefore finding the research may with the compound of the dependency structure of equal or higher stomach motility enhancing effect just has important value.
Summary of the invention
This patent aims to provide the active metabolite of a kind of low toxicity, efficiently mosapride, thus for becoming the surrogate of mosapride clinically later.Active metabolite provided by the invention is as described below:
The preparation method of this metabolite comprises:
(1) early-stage preparations prepared of metabolite: after cultured cunninghamella elegans AS3.156 (buying from the market) preculture 24h, add mosapride DMF (N, dinethylformamide) solution (80mg/ml), concentration of substrate is made to reach 200mg/L, transform 120h, transformation culture, in the centrifugal 20min of 4000rpm, is collected supernatant conversion fluid and is about 3L, put-20 DEG C freezing, for the preparation of mosapride metabolite.Meanwhile, the mycelium precipitation after centrifugal is collected together, with the ultrasonic 20min of the ethyl acetate of 2 times of volumes, in the centrifugal 20min of 4000rpm, collect supernatant liquor, repeat above step twice, merge wash mycelial ethyl acetate, put-20 DEG C freezing, for the preparation of mosapride metabolite.
(2) separation and purification of metabolite: conversion fluid is used saturated Na
2cO
3alkalization is to pH=9, with 2 times of volume extraction into ethyl acetate 2 times, merge organic layer, and wash mycelial ethyl acetate layer and merge, anhydrous sodium sulfate drying, evaporated under reduced pressure, residue about 8.0mL methanol-water (7:3, v/v) redissolves, and loading is about the gel column of 5cm to the internal diameter being filled with Sephadex G10 filler, take methyl alcohol as eluting solvent, every 8mL collects one bottle.Wash-out stream part is detected through UPLC-MS, and the stream part containing metabolite merged, evaporated under reduced pressure, with dissolve with methanol, use the open column chromatography of ODS, carry out gradient elution (10:90,30:70,50:50 with Methanol+Water system, 70:30,90:10), every 50mL collects one bottle.Wash-out stream part is detected through UPLC-MS, merges the stream part containing metabolite, evaporated under reduced pressure; Redissolve, for further preparative liquid chromatography separation and purification with methyl alcohol.
Embodiment of the present invention will be set forth in the following description, and the following embodiment provided is not meaned and limited invention in any method, and be only illustrative objects.
Embodiment
The preparation method of embodiment 1 metabolite
(1) early-stage preparations prepared of metabolite: buy on cultured cunninghamella elegans AS3.156(market) after preculture 24h, add mosapride DMF (N, dinethylformamide) solution (80mg/ml), concentration of substrate is made to reach 200mg/L, transform 120h, transformation culture, in the centrifugal 20min of 4000rpm, is collected supernatant conversion fluid and is about 3L, put-20 DEG C freezing, for the preparation of mosapride metabolite.Meanwhile, the mycelium precipitation after centrifugal is collected together, with the ultrasonic 20min of the ethyl acetate of 2 times amount, in the centrifugal 20min of 4000rpm, collect supernatant liquor, repeat above step twice, merge wash mycelial ethyl acetate, put-20 DEG C freezing, for the preparation of mosapride metabolite.
(2) separation and purification of metabolite: conversion fluid is used saturated Na
2cO
3alkalization is to pH=9, with 2 times of volume maximum dose extraction into ethyl acetate 2 times, merge organic layer, and wash mycelial ethyl acetate layer and merge, anhydrous sodium sulfate drying, evaporated under reduced pressure, residue about 8.0mL methanol-water (7:3, v/v) redissolves, and loading is about the gel column of 5cm to the internal diameter being filled with Sephadex G10 filler, take methyl alcohol as eluting solvent, every 8mL collects one bottle.Wash-out stream part is detected through UPLC-MS, and the stream part containing metabolite merged, evaporated under reduced pressure, with dissolve with methanol, use the open column chromatography of ODS, carry out gradient elution (10:90,30:70,50:50 with Methanol+Water system, 70:30,90:10), every 50mL collects one bottle.Wash-out stream part is detected through UPLC-MS, merges the stream part containing metabolite, evaporated under reduced pressure; Redissolve, for further preparative liquid chromatography separation and purification with methyl alcohol.Methanol solution containing metabolite is repeated to enter preparative liquid chromatography repeatedly, collect the chromatographic effluent of different retention time respectively, retention time phase homogeneous turbulence part is merged, evaporated under reduced pressure organic solvent, remaining partial freeze is dry, obtains the sterling of this metabolite.The preparative liquid chromatography condition adopted is chromatographic column: ZorbaxC18 post (10.7 × 250mm I.D., 7 μm, Agilent company of the U.S.); Moving phase: methyl alcohol-10mmol/L ammonium acetate aqueous solution (45:55, v:v), isocratic elution; Flow velocity: 20.0mL/min; Determined wavelength 274nm; Column temperature: room temperature.
The structural confirmation of embodiment 2 metabolite
Comprehensive utilization NMR, TOF-MS, UPLC-MS/MS and UV carry out structural identification to the mosapride active metabolite that embodiment 2 obtains.
Fusing point: 141-143 DEG C.To be 2.81min, UV maximum absorption wavelength be retention time 217,273 and 308nm.TOF-MS analyzes and shows, quasi-molecular ion peak [M+H]
+m/z438.1592 (calculated value is 438.1590), show that molecular formula is C
21h
25clF N
3o
4, compared with original shape medicine, many O atoms, explanation is monohydroxy compound or the N-oxide metabolites of original shape medicine.MS/MS result shows, its fragmention m/z is 420,329,280,215,198,170,159 and 109.The existence of m/z198,170 and 109 fragmentions shows benzoyl and is not oxidized luorobenzyl part.The fragmention of m/z420 is sloughed a part water by original shape medicine and is generated, and the relative abundance in second order ms figure is only 2%, prompting MSP437 is N-oxide compound, but not hydroxylated metabolite, because in ESI-MS/MS analyzes, hydroxylated metabolite is easier than N-oxidative metabolites to dewater, and the relative abundance of the fragmention of generation is larger.The quasi-molecular ion [M+H] of MSP437
+slough fragmention luorobenzyl part being generated to m/z329, there is C1-C7 bond rupture in it, loses 4-amino-5-chloro-2-ethoxy part (m/z170) then, generates the fragmention of m/z159.The fragmention of m/z215 is then the benzamide Part after C7 '-N bond rupture.
With original shape medicine mosapride
1h with 13C-NMR data are compared, 3 ', 5 ', 7 ' ', all there is in various degree displacement, 6 ' and 1 ' in the C of 2 ' ' and 6 ' ' position and H signal to low field ' signal then move to High-Field.H-2 ' moves to 4.2ppm from 3.5 to low field, and corresponding C signal then moves to 69.6ppm by 73.9 to High-Field.There is oxidation Electron Affinities and strengthen in the atom N due to morpholine ring, and causes M4 that these changes occur.Other signal does not substantially change compared with original shape medicine.In addition, in hydrocarbon long-range Correlated Spectroscopy (HMBC), the H-6 ' at 3.4ppm place is long-range relevant to the C-2 ' at 69.6ppm place, and the H-5 ' at 4.10ppm place is relevant to the C-3 ' at 64.7ppm place; In hydrogen-hydrogen chemical shifts Correlated Spectroscopy (1H-1H COSY), H-5 ' and H-6 ' is correlated with; Undistorted polarization transfer strengthens spectrum (DEPT135) display, and only have C-2 ' (CH) to be positive signal, and other C-3 ', 5 ' and 6 ' (CH2) are negative signal, showing to be oxidized is not occur on four C atoms of morpholine ring.Hydrocarbon Correlated Spectroscopy (HSQC) further demonstrate that said structure.
Comprehensive ESI-MS, one dimension (1D) and two dimension (2D)-NMR data, determine that it is mosapride-4 '-N-oxide compound.
The activity research of embodiment 3 metabolite
Cavy 12, is divided into 2 groups at random, during experiment, cavy is caused dusk, cuts open the belly rapidly and gets the ileal segment at nearly caecum end 10cm place, put into the culture dish of tyrode.Except intestinal mucosa and the fat of attachment removal, wash down intestinal contents, be cut into 2-3cm intestinal segment for subsequent use, one end is fixed on ventilation hook, and the other end is connected and fixed on muscle tone transverter and causes RM6240 conductance physiograph interface.Stablize after intestinal tube normal creepage of gastrointestinal functions until sample and trace normal contraction curve, the spontaneous contractile activity of intestinal tube inputs physiograph by tonotransducer.Use accumulation dose regimen, the dosing interval of the mosapride metabolite I that mosapride and embodiment 1 obtain is 15s, 20s, 20s and 15s respectively.After each experiment, rinse sample with the tyrode of 37 ± 0.5 DEG C of preheatings, rinse 3-4 time, all need when giving same medicine and repeating experiment first to allow intestinal tube recover normal creepage of gastrointestinal functions balance, medication interval 15min.The result of each determinand is from 6 cavys.There is the contraction intensity action effect value in for some time of maximum effect (contraction) after adding each concentration.
Table 1 mosapride and metabolite strengthen per-cent to the contraction of isolated guinea pig ileum
Result shows, metabolite III all has activity under each cumulative concentration, but is all less than original shape medicine mosapride to the contraction enhancing per-cent of isolated guinea pig ileum.
Claims (3)
1. an active metabolite for mosapride, its structure is as follows:
。
2. the preparation method of compound as claimed in claim 1, step is as follows:
1) early-stage preparations prepared of metabolite: after cultured cunninghamella elegans AS3.156 (buying from the market) preculture 24h, add mosapride DMF (N, dinethylformamide) solution (80mg/ml), concentration of substrate is made to reach 200mg/L, transform 120h, transformation culture, in the centrifugal 20min of 4000rpm, is collected supernatant conversion fluid and is about 3L, put-20 DEG C freezing, for the preparation of mosapride metabolite.Meanwhile, the mycelium precipitation after centrifugal is collected together, with the ultrasonic 20min of the ethyl acetate of 2 times of volumes, in the centrifugal 20min of 4000rpm, collect supernatant liquor, repeat above step twice, merge wash mycelial ethyl acetate, put-20 DEG C freezing, for the preparation of mosapride metabolite.
(2) separation and purification of metabolite: conversion fluid is used saturated Na
2cO
3alkalization is to pH=9, with 2 times of volume extraction into ethyl acetate 2 times, merge organic layer, and wash mycelial ethyl acetate layer and merge, anhydrous sodium sulfate drying, evaporated under reduced pressure, residue about 8.0mL methanol-water (7:3, v/v) redissolves, and loading is about the gel column of 5cm to the internal diameter being filled with Sephadex G10 filler, take methyl alcohol as eluting solvent, every 8mL collects one bottle.Wash-out stream part is detected through UPLC-MS, and the stream part containing metabolite merged, evaporated under reduced pressure, with dissolve with methanol, use the open column chromatography of ODS, carry out gradient elution (10:90,30:70,50:50 with Methanol+Water system, 70:30,90:10), every 50mL collects one bottle.Wash-out stream part is detected through UPLC-MS, merges the stream part containing metabolite, evaporated under reduced pressure; Redissolve with methyl alcohol, for the preparation of liquid chromatography separation and purification, to obtain final product.
3. compound as claimed in claim 1 is preparing the application of medicine for stomach dynamic.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310666229.4A CN104693137A (en) | 2013-12-10 | 2013-12-10 | Active metabolite of mosapride |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310666229.4A CN104693137A (en) | 2013-12-10 | 2013-12-10 | Active metabolite of mosapride |
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| CN104693137A true CN104693137A (en) | 2015-06-10 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111285820A (en) * | 2018-12-07 | 2020-06-16 | 成都康弘药业集团股份有限公司 | Mosapride citrate related substance and preparation method thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002076462A1 (en) * | 2001-03-26 | 2002-10-03 | Dainippon Pharmaceutical Co., Ltd. | Drugs for improving insulin resistance |
| CN1526700A (en) * | 2003-03-03 | 2004-09-08 | 鲁南制药股份有限公司 | Synthesis of Important intermediate for mosapride citrate |
| US20090176857A1 (en) * | 2005-12-02 | 2009-07-09 | Stephane David Levy | Use of Organic Compounds |
| WO2011107903A1 (en) * | 2010-03-04 | 2011-09-09 | Ranbaxy Laboratories Limited | Highly pure mosapride citrate dihydrate and processes for its preparation |
-
2013
- 2013-12-10 CN CN201310666229.4A patent/CN104693137A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002076462A1 (en) * | 2001-03-26 | 2002-10-03 | Dainippon Pharmaceutical Co., Ltd. | Drugs for improving insulin resistance |
| CN1526700A (en) * | 2003-03-03 | 2004-09-08 | 鲁南制药股份有限公司 | Synthesis of Important intermediate for mosapride citrate |
| US20090176857A1 (en) * | 2005-12-02 | 2009-07-09 | Stephane David Levy | Use of Organic Compounds |
| WO2011107903A1 (en) * | 2010-03-04 | 2011-09-09 | Ranbaxy Laboratories Limited | Highly pure mosapride citrate dihydrate and processes for its preparation |
Non-Patent Citations (3)
| Title |
|---|
| SUN XIAOHONG ET AL: "Fungal biotransformation of mosapride by Cunninghamella elegans", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》, vol. 59, 6 February 2009 (2009-02-06), pages 82 - 89, XP026044085, DOI: doi:10.1016/j.molcatb.2009.01.009 * |
| SUN, XIAOHONG ET AL: "Characterization of metabolic profile of mosapride citrate in rat and identification of two new metabolites: Mosapride N-oxide and morpholine ring-opened mosapride by UPLC-ESI-MS/MS", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》, vol. 50, no. 1, 24 March 2009 (2009-03-24), pages 27 - 34, XP026133450, DOI: doi:10.1016/j.jpba.2009.03.011 * |
| 孙晓红: "莫沙必利的代谢和药物动力学研究", 《中国博士学位论文全文数据库 医药卫生科技辑》, no. 9, 15 September 2013 (2013-09-15) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111285820A (en) * | 2018-12-07 | 2020-06-16 | 成都康弘药业集团股份有限公司 | Mosapride citrate related substance and preparation method thereof |
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