CN103172726A - Secretin analogue, as well as preparation method and application thereof - Google Patents
Secretin analogue, as well as preparation method and application thereof Download PDFInfo
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- CN103172726A CN103172726A CN2011104510118A CN201110451011A CN103172726A CN 103172726 A CN103172726 A CN 103172726A CN 2011104510118 A CN2011104510118 A CN 2011104510118A CN 201110451011 A CN201110451011 A CN 201110451011A CN 103172726 A CN103172726 A CN 103172726A
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Abstract
The invention relates to a novel secretin analogue and a preparation method of the novel secretin analogue. Compared with natural secretin, the compound has improved chemical stability and maintains the pharmacological activity of the natural secretin.
Description
Technical field
The present invention relates to secretin analogue and preparation method thereof and therepic use.
Background technology
Secretin (Secretin) is a kind of enteron aisle hormone, and by forming in the mucous membrane of small intestine upper end, stimulating pancreas divides bleeding and supercarbonate.Secretin is a kind of polypeptide of 27 amino acid link.
People's secretin is the polypeptide compound with following sequence:
H
2N-His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-CONH
2
Secretin can stimulate, and promotes the pancreatic secretion weakly alkaline to contain the pancreatic juice of enzyme, is the reagent of the Diagnosis of Pancreatic function commonly used, studies show that, secretin also has the effect for the treatment of digestive tract ulcer.US2004241154 discloses the purposes of secretin treatment asthma.WO2004081195 discloses the purposes of secretin treatment infectious diseases.US2007154534 discloses secretin treatment psychosis, as the purposes of schizophrenia.
Yet secretin studies show that in a large number in the external utmost point unstable that shows, and secretin is external, especially in the aqueous solution, and can rapid deactivation.Even if at low-temperature condition, in the time of for example-20 ℃, secretin also can be degraded gradually, and loss of activity.
Natural secretin in the preparation preparation process, store in transportation, all can because its unstable causes bioactive reduction, can not satisfy the safety and effectiveness in diagnoses and treatment.And due to the unstable of natural secretin, must make transportation and store cost increases, and has indirectly caused the increase of cost of drugs.
Be all to improve the stability problem of natural secretin by preparation way at present, for example US3940480 provides a kind of particle that contains the secretin of L-Ala, improves the stability of secretin.
Therefore, be badly in need of developing a kind of have natural secretin activity, the polypeptide compound that stability is improved.
Summary of the invention
The analogue that the purpose of this invention is to provide a kind of secretin, its chemical structural formula is as follows:
Each R wherein
1, R
2Independently be selected from H, alkyl, thiazolinyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocyclic radical alkyl; R
1, R
2Preferred alkyl, more preferably C
1-C
4Alkyl, most preferable;
R
3Be selected from alkyl, thiazolinyl, alkynyl, [R
1'-K-R
1']
n
Described R
1' be selected from alkyl, alkynyl (thiazolinyl) or alkynyl;
Described K is O, S, SO, SO2, CO, CO2, CONR
2',
Described R
2' be H or alkyl;
N is the integer of 2-7.
R
3Preferred thiazolinyl.
The analogue of secretin of the present invention preferably has the compound of lower array structure:
Each R wherein
1, R
2Independently be selected from H, alkyl, thiazolinyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocyclic radical alkyl; Preferred alkyl, more preferably C
1-C
4Alkyl, most preferable;
N is selected from the integer of 2-7, and preferred n is 3.
Secretin analogue of the present invention more preferably has the compound of lower array structure:
Secretin analogue of the present invention has natural polypeptide secondary structure, and the α helix degree increases, and makes it closer to the quaternary structure characteristics of secretin, has reduced the degraded of secretin.Compare with natural secretin sequence, the chemical stability of polypeptide compound of the present invention is improved.And polypeptide compound of the present invention has kept the pharmacologically active of natural secretin.
Secretin analogue of the present invention especially preferably (S)-2-amino-2-methyl optically active to have-6-heptenoic acid (is the S in Fig. 1
5) replace the natural amino acid in sequence, the polypeptide compound that the mode of described substituting group by chemical catalysis obtains after cyclization each other.
Described substituting group is protected with Fmoc, participates in the synthetic of polypeptide, and described Fmoc-(S)-2-amino-2-methyl-6-heptenoic acid has following structure:
Another aspect of the present invention provides the method for preparing above-mentioned secretin analogue.Can adopt method well known to those skilled in the art, synthesis steps selecting sequence or that order is different produce the material with following chemical structure:
Described synthetic method comprises: for example liquid phase is synthetic or solid phase synthesis (SPPS) is prepared, preferred SPPS.Described solid phase synthesis can be selected conventional polystyrene-benzene divinyl cross-linked resin, polyacrylamide, polyethylene-glycols resin etc., for example: Wang resin (Wang Resin), Rink amide linker mbha resin etc., preferred Rink amide linker mbha resin.The ring texture of the compounds of this invention is that the mode by catalyzed cyclization prepares, and described catalyzer can be: ruthenium catalyst, preferred catalyzer are that (Grubbs 1 for two (thricyclohexyl phosphorus) ruthenous chlorides of phenylmethylene
st), or Grubbs 2
st
The present invention provides the pharmaceutical composition that comprises above-mentioned secretin analogue on the other hand.Described pharmaceutical composition comprises the polypeptide compound with said structure and the pharmaceutically acceptable carrier of significant quantity, described composition can be prepared according to the known technology of this area, for example mix with the pharmaceutical excipient of routine, be prepared into injection, injection liquid, or the formulation of preparation type example emulsion, paste, creme or the nose administration of topical etc., or have a preparation of slow release effect, such as microballoon, liposome etc.
" pharmaceutically acceptable carrier " of the present invention comprises any pharmaceutical excipient, such as weighting agent, thinner, vehicle etc.Such as including but not limited to: lactose, sucrose, glucose, starch, cellulose family (as carboxymethyl cellulose, HPMC etc.), ethylene glycol, soya-bean oil, sesame oil, ethanol, stroke-physiological saline solution, sterilized water, ethanol etc.Usually, pharmaceutically acceptable carrier is water miscible pH damping fluid, such as Citrate trianion, phosphoric acid salt etc.; Can also contain antioxidant, stablizer (as amino acid), fungistat etc.
Contriver's discovery, secretin analogue of the present invention has the biological activity similar to natural secretin, can promote that the experimenter secretes pancreatic juice, disease and illness that prevention is relevant to secretin with treatment, for example Diagnosis of Pancreatic function and/or treatment digestive tract ulcer.And the secretin analogue of invention has better more stable than natural secretin.
Description of drawings
Fig. 1 is the preparation flow of the preferred secretin analogue of the present invention, the S in Fig. 1 molecular formula
5It is (S)-2-amino-2-methyl-6-heptenyl
Fig. 2 is the preferred secretin analogue of the present invention stability experiment result
Fig. 3 is that the preferred secretin analogue of the present invention is on the impact of isolated rat exocrine pancreas
Specific embodiment
Following embodiment is used for further illustrating technical scheme of the present invention.In specification sheets of the present invention and embodiment with the amino acid monomer of protecting group and other chemical reagent etc.; all can buy from associated companies and obtain; the test method of unreceipted actual conditions, condition is carried out routinely, or is undertaken by the condition that goods supplier is advised.
The preparation of embodiment 1 secretin analogue
According to step shown in Figure 1, amino acid is connected on resin one by one, then use the catalyst cyclization, then slough protecting group and polypeptide is downcut from resin.
1, solid phase synthesis
Select 0.402g Rink amide linker mbha resin (substitution value: 0.373mmol/g) preparation; soak resin 60min with DMF at ambient temperature; to activate connection according to sequence arranged sequentially in synthesizer with the amino acid of Fmoc protection, obtain compound as shown in the figure.
2, catalyzed cyclization
Use successively DCM (20ml) and DCE (20ml) respectively to rinse twice the compound 2 of above-mentioned steps gained, then use DCE (8ml) to soak 30min, then with Grubbs 1
st(25mgx2,20mg/0.1mmol) adds in reaction mixture, room temperature jolting 3 hours.Detect the cyclisation situation with MS, if cyclisation is incomplete, repeat this step, usually repeat 2 times, obtain compound (0.817g) as shown in the figure.
3, excision resin
The reaction mixture that will contain above-claimed cpd 3 is placed in lysate, and under nitrogen protection, stirring at room is 2.5 hours.Filter, remove filtrate, filter cake rinses with TFA (2ml), then is placed on precipitation in cold ether (50ml), and is centrifugal; The filter cake that obtains rinses twice with cold ether (50ml), and vacuum-drying obtains SECRETIN polypeptide analog 389mg of the present invention, productive rate 83%, MW=3104.7, theoretical value MW=3104.7
4, purifying
Above-mentioned part crude product 191mg is carried out purifying with HPLC, obtain the sterling 26mg of purity more than 95%.
The vitro stability test of embodiment 2 secretin analogues
Respectively natural secretin secretin analogue of the present invention (embodiment 1 preparation) is placed in the phosphoric acid buffer (PH7.0) of 0.1M, placed 36 hours under 60 ℃, detected the residual quantity of secretin wherein or analogue in every three hours with RP-HPLC.
Get the sample of secretin and analogue, use CH
3CN and 1% HClO
4(2: 3, v/v) dilution, C
18Post is stationary phase, acetonitrile/0.005M phosphoric acid buffer/0.2MNaClO
4Be moving phase, 210nm detects at the place.
Result as shown in Figure 2, under experiment condition, natural secretin is degraded rapidly, degraded approximately 30% in 24 hours; And analogue of the present invention is not almost degraded.
The extracorporeal biology experiment of embodiment 3 secretin analogues
Select the Wistar rat, body weight 200 ± 20g, is divided into two groups (natural secretin group, analogue groups) at random by 10.Urethane (45mg/kg) anesthetized rat cuts the abdominal cavity, peels off pancreas, and the pancreas that will exsomatize is put into rapidly 37 ℃ of constant-temperature incubation casees that contain Krebs-Ringer damping fluid (KR liquid).With constant flow pump, PH7.2 is filled with 95%O
2And 5%CO
2KR liquid constant speed (1.0ml/min) pour into aorta abdominalis, hatch damping fluid in ware with the speed sustainable supply of 0.35ml/min.Natural secretin and the analogue of the present invention (embodiment 1) of two groups of independent perfusion 0.25CU/kg.h of difference.Micro-kapillary is inserted ductus pancreaticus, and pancreatic juice flows out along kapillary, and every 30min collects a pancreatic juice, and perfusion KR liquid 1.5h before administration collects and exsomatizes pancreatic juice in contrast.
Result as shown in Figure 3, natural secretin can the effective stimulus pancreatic secretion, the pancreatic juice amount is increased to 24.66 ± 4.02ul/30min by blank 10.52 ± 1.05; Analogue also can the effective stimulus pancreatic secretion, and the pancreatic juice amount is increased to 22.74 ± 1.02ul/30min by blank 10.06 ± 2.25.Analogue of the present invention has still kept the biological activity of natural secretin.
Claims (11)
1. secretin analogue, its chemical structural formula is as follows:
Each R wherein
1, R
2Independently be selected from H, alkyl, thiazolinyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocyclic radical alkyl;
R
3Be selected from alkyl, thiazolinyl, alkynyl, [R
1'-K-R
1']
n
Described R
1' be selected from alkyl, alkynyl (thiazolinyl) or alkynyl;
Described K is O, S, SO, SO2, CO, CO2, CONR
2',
Described R
2' be H or alkyl;
N is the integer of 2-7.
2. secretin analogue as claimed in claim 1, its chemical structural formula is as follows:
Each R wherein
1, R
2Independently be selected from H, alkyl, thiazolinyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl or heterocyclic radical alkyl;
N is selected from the integer of 2-7.
3. secretin analogue as claimed in claim 2, its chemical structural formula is as follows:
4. method for preparing the described secretin analogue of claim 3 is characterized in that:
(1) prepare the material with following structure
(2) by the compound of catalyst treatment step (1), make its cyclization.
5. method as claimed in claim 4, is characterized in that, step (1) prepares by solid phase synthesis technique.
6. method as claimed in claim 5, is characterized in that, step (1) adopts the solid phase synthesis technique of Fmoc protection.
7. method as claimed in claim 4, is characterized in that, the catalyzer of step (2) is ruthenium catalyst.
8. method as claimed in claim 7, is characterized in that, described ruthenium catalyst is Grubbs 1
st
9. as the compound of claim 1-3 any one, for the preparation of the application in the medicine of Diagnosis of Pancreatic function.
10. as the compound of claim 1-3 any one, for the preparation of the application in the medicine for the treatment of digestive tract ulcer.
11. a pharmaceutical composition comprises compound and pharmaceutical carrier as claim 1-3 any one.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110451011.8A CN103172726B (en) | 2011-12-22 | 2011-12-22 | Secretin analog and its production and use |
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|---|---|---|---|
| CN201110451011.8A CN103172726B (en) | 2011-12-22 | 2011-12-22 | Secretin analog and its production and use |
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|---|---|
| CN103172726A true CN103172726A (en) | 2013-06-26 |
| CN103172726B CN103172726B (en) | 2017-06-13 |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010080605A1 (en) * | 2008-12-19 | 2010-07-15 | Indiana University Research And Technology Corporation | Dipeptide linked medicinal agents |
-
2011
- 2011-12-22 CN CN201110451011.8A patent/CN103172726B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010080605A1 (en) * | 2008-12-19 | 2010-07-15 | Indiana University Research And Technology Corporation | Dipeptide linked medicinal agents |
| CN102300580A (en) * | 2008-12-19 | 2011-12-28 | 印第安纳大学研究及科技有限公司 | Dipeptide linked medicinal agents |
Non-Patent Citations (1)
| Title |
|---|
| 黄怀德: "消化道激素", 《浙江医学》 * |
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Effective date of registration: 20210507 Address after: Room 1-1115, Heda Medicine Valley Center, 291 Fucheng Road, Xiasha street, Qiantang New District, Hangzhou, Zhejiang 310000 Patentee after: Taide Pharmaceutical (Zhejiang) Co.,Ltd. Address before: 310018 room 5b03, building 1, 452, 6 Baiyang street, Hangzhou Economic and Technological Development Zone, Zhejiang Province Patentee before: Hangzhou Chuntai Technology Co.,Ltd. |
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