CN103172726B - Secretin analog and its production and use - Google Patents
Secretin analog and its production and use Download PDFInfo
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- CN103172726B CN103172726B CN201110451011.8A CN201110451011A CN103172726B CN 103172726 B CN103172726 B CN 103172726B CN 201110451011 A CN201110451011 A CN 201110451011A CN 103172726 B CN103172726 B CN 103172726B
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- secretin
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- 0 C*N[C@@](C)(CCCC=C)C(*)=O Chemical compound C*N[C@@](C)(CCCC=C)C(*)=O 0.000 description 2
- PJSNQCAWMYREAY-UHFFFAOYSA-N CC(OCC1c(cccc2)c2-c2ccccc12)=O Chemical compound CC(OCC1c(cccc2)c2-c2ccccc12)=O PJSNQCAWMYREAY-UHFFFAOYSA-N 0.000 description 1
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Abstract
The present invention relates to a kind of new analog of secretin and preparation method thereof, compared with natural secretin, the chemical stability of compound of the invention is improved, while remaining the pharmacological activity of natural secretin.
Description
Technical field
The present invention relates to secretin analog and preparation method thereof and therapeutical uses.
Background technology
Secretin (Secretin) is a kind of enteron aisle hormone, by being formed in upper small intestine mucous membrane, stimulating pancreas secretion water and
Bicarbonate.Secretin is a kind of polypeptide of 27 amino acid link.
People's secretin is the polypeptide compound with following sequence：
H2N-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-CONH2
Secretin can stimulate, and promote pancreatic juice of the pancreatic secretion alkalescent containing enzyme, be the examination of conventional Diagnosis of Pancreatic function
Agent, research shows that secretin also has the effect for the treatment of digestive tract ulcer.US2004241154 discloses secretin treatment asthma
Purposes.WO2004081195 discloses the purposes that secretin treats infectious diseases.US2007154534 discloses secretin
Treatment psychosis, such as purposes of schizophrenia.
However, secretin shows extremely unstable property in vitro, numerous studies show that secretin in vitro, especially exists
In the aqueous solution, can rapid deactivation.Even in low-temperature condition, such as at -20 DEG C, secretin also can gradually degrade, and lose and live
Property.
Naturally secretin all can cause bioactivity in formulation process, in storage transport because of its unstability
Reduction, it is impossible to meet the safety and effectiveness in diagnoses and treatment.The unstability of natural secretin is additionally, since, necessarily makes fortune
Defeated and storage cost increases, and the increase of cost of drugs is result in indirectly.
All it is at present the stability problem for improving natural secretin by preparation way, such as US3940480 provides one
The particle of the secretin containing alanine is planted, the stability of secretin is improved.
Therefore, it is badly in need of exploitation a kind of with natural secretin activity, the polypeptide compound that stability is improved.
The content of the invention
It is an object of the invention to provide a kind of analog of secretin, its chemical structural formula is as follows：
Wherein each R1、R2It is independent selected from H, alkyl, alkenyl, alkynyl, aryl alkyl, cycloalkyl-alkyl, heteroaryl alkyl
Or cycloheteroalkylalkyl；R1、R2It is preferred that alkyl, more preferably C1-C4Alkyl, most preferable；
R3Selected from alkyl, alkenyl, alkynyl, [R1’-K-R1’]n；
Described R1' it is selected from alkyl, alkynyl (alkenyl) or alkynyl；
Described K is O, S, SO, SO2, CO, CO2, CONR2’、
Described R2' it is H or alkyl；
N is the integer of 2-7.
R3It is preferred that alkenyl.
The analog of the secretin of the present invention preferably compound with having structure：
Wherein each R1、R2It is independent selected from H, alkyl, alkenyl, alkynyl, aryl alkyl, cycloalkyl-alkyl, heteroaryl alkyl
Or cycloheteroalkylalkyl；It is preferred that alkyl, more preferably C1-C4Alkyl, most preferable；
N is selected from the integer of 2-7, and preferably n is 3.
The secretin analog of the present invention more preferably compound with having structure：
Secretin analog of the invention has a natural polypeptide secondary structure, and α helix degrees increase, make its closer to
The quaternary structure feature of secretin, reduces the degraded of secretin.Compared with natural secretin sequence, peptide of the invention
The chemical stability of thing is improved.And polypeptide compound of the invention remains the pharmacological activity of natural secretin.
Secretin analog of the present invention is particularly preferably with optically active (S) -2- amino-2-methyl -6- heptenoic acids
(i.e. S in Fig. 15) natural amino acid in substitution sequence, described substitution base is by way of chemical catalysis each other after cyclization
The polypeptide compound of acquisition.
Described substitution base is protected with Fmoc, participates in the synthesis of polypeptide, described Fmoc- (S) -2- amino -2- first
Base -6- heptenoic acids have following structure：
Method another aspect provides above-mentioned secretin analog is prepared.People in the art can be used
Method known to member, synthesis step selecting sequence or that order is different produces the material with following chemical constitution：
Described synthetic method includes：For example liquid phase synthesis or synthesis in solid state (SPPS) are prepared, preferably SPPS.It is described
Synthesis in solid state can select normal polystyrene-benzene divinyl crosslinked resin, polyacrylamide, polyethylene-glycols tree
Fat etc., for example：Wang resin (Wang Resin), Rink amide linker mbha resins etc., preferably Rink amide
Linker mbha resins.The cyclic structure of the compounds of this invention is prepared by way of catalyzed cyclization, described catalyst
Can be：Ruthenium catalyst, preferred catalyst is double (thricyclohexyl phosphorus) ruthenous chloride (Grubbs 1 of phenylmethylenest), or
Grubbs 2st。
Another aspect of the present invention is provided includes the pharmaceutical composition of above-mentioned secretin analog.Described pharmaceutical composition
Polypeptide compound and pharmaceutically acceptable carrier with said structure comprising effective dose, described composition can be according to this
The known technology in field is prepared, for example, mixed with conventional pharmaceutic adjuvant, is prepared into injection, parenteral solution, or office
Preparation type such as formulation of emulsion, paste, creme or nose administration etc. of portion's administration, or the preparation with slow release effect, example
Such as microballoon, liposome.
" pharmaceutically acceptable carrier " of the present invention includes any pharmaceutic adjuvant, for example filler, diluent,
Excipient etc..For example include but is not limited to：Lactose, sucrose, glucose, starch, cellulose family (such as carboxymethylcellulose calcium, hydroxypropyl
Methylcellulose etc.), ethylene glycol, soya-bean oil, sesame oil, ethanol, SPSS, sterilized water, ethanol etc..Generally, pharmaceutically
Acceptable carrier is water miscible pH buffer solutions, such as citrate, phosphate etc.；Antioxidant can also be contained, stabilization
Agent (such as amino acid), bacteriostatic agent.
Inventor has found that secretin analog of the invention has the bioactivity similar to natural secretin, can promote
Enter subject's secretion pancreatic juice, prevent and treat the disease and illness related to secretin, such as Diagnosis of Pancreatic function and/or treatment
Digestive tract ulcer.And the secretin analog of invention has stability more more preferable than natural secretin.
Brief description of the drawings
Fig. 1 is the preparation flow of currently preferred secretin analog, the S in Fig. 1 molecular formula5Be (S) -2- amino -
2- methyl -6- heptenyls
Fig. 2 is preferably secretin analog stability experiment result of the invention
Fig. 3 is influence of the currently preferred secretin analog to isolated rat exocrine pancreas
Specific embodiment
Following embodiment is used to further illustrate technical scheme.Institute in description of the invention and embodiment
With the amino acid monomer of protection group and other chemical reagent etc., can be obtained from associated companies purchase, unreceipted actual conditions
Test method, routinely condition can carry out, or condition as proposed by goods supplier is carried out.
The preparation of the secretin analog of embodiment 1
According to the step shown in Fig. 1, amino acid is connected on resin one by one, then uses catalyst cyclization, then taken off
Deprotection base simultaneously cuts polypeptide from resin.
1st, synthesis in solid state
Selection 0.402g Rink amide linker mbha resin (substitution values：0.373mmol/g) prepare, in room temperature
Under the conditions of with DMF soak resin 60min, will carry Fmoc protection amino acid be arranged sequentially in synthesizer according to sequence
Row activation connection, obtains compound as depicted.
2nd, catalyzed cyclization
Compound 2 obtained by above-mentioned steps is respectively rinsed twice with DCM (20ml) and DCE (20ml) successively, DCE is then used
(8ml) soaks 30min, then by Grubbs 1st(25mgx2,20mg/0.1mmol) is added in reactant mixture, rocked at room temperature 3
Hour.Cyclisation situation is detected with MS, if cyclisation is incomplete, the step is repeated, is generally repeated 2 times, obtain as depictedization
Compound (0.817g).
3rd, resin is cut off
Reactant mixture containing above-claimed cpd 3 is placed in lysate, is stirred at room temperature 2.5 hours under nitrogen protection.
Filtering, removes filtrate, and filter cake is rinsed with TFA (2ml), is then placed over precipitation in cold ether (50ml), centrifugation；The filter for obtaining
The cold ether (50ml) of cake is rinsed twice, and vacuum drying obtains SECRETIN polypeptide analogs 389mg of the present invention, yield
83%, MW=3104.7, theoretical value MW=3104.7
4th, purify
Above-mentioned part crude product 191mg is purified with HPLC, the sterling 26mg of purity more than 95% is obtained.
The vitro stability experiment of the secretin analog of embodiment 2
Natural secretin secretin analog of the present invention (prepared by embodiment 1) is placed in the phosphate buffer of 0.1M respectively
(PH7.0), in placement 36 hours at 60 DEG C, the residual quantity for wherein secretin or the like being detected with RP-HPLC in every three hours.
The sample of secretin and analog is taken, CH is used3The HClO of CN and 1%4(2: 3, v/v) dilutes, C18Post is fixing phase,
Acetonitrile/0.005M phosphate buffers/0.2MNaClO4It is mobile phase, is detected at 210nm.
Result as shown in Fig. 2 under experimental conditions, natural secretin is degraded rapidly, degraded about 30% in 24 hours；And this hair
Bright analog is almost degraded.
The extracorporeal biology experiment of the secretin analog of embodiment 3
Wistar rats are selected, 200 ± 20g of body weight, is randomly divided into two groups (natural secretin group, analog groups) by 10.Crow
La Tan (45mg/kg) anesthetized rat, cuts abdominal cavity, peels off pancreas, and in vitro pancreas is put into rapidly into 37 DEG C containing Krebs-Ringer
In the constant-temperature incubation case of buffer solution (KR liquid).With constant flow pump by PH7.2 filled with 95%O2And 5%CO2KR liquid constant speed (1.0ml/
Min abdominal aorta) is poured into, the buffer solution in ware is incubated then with the speed sustainable supply of 0.35ml/min.Two groups individually fill
Flow the natural secretin and analog of the present invention (embodiment 1) of 0.25CU/kg.h.Micro-capillary tubes are inserted into ductus pancreaticus, pancreatic juice edge
Capillary is flowed out, and a pancreatic juice is collected per 30min, and perfusion KR liquid 1.5h before administration collect in vitro pancreatic juice as control.
Result as shown in figure 3, natural secretin can with effective stimulus pancreatic secretion, pancreatic juice amount by blank 10.52 ±
1.05 increase to 24.66 ± 4.02ul/30min；Analog can also effective stimulus pancreatic secretion, pancreatic juice amount is by blank
10.06 ± 2.25 increase to 22.74 ± 1.02ul/30min.The biology that analog of the present invention still remains natural secretin is living
Property.
Claims (9)
1. a kind of secretin analog, its chemical structural formula is as follows：
2. a kind of method for preparing secretin analog described in claim 1, it is characterised in that：
(1) material with following structures is prepared
(2) by the material of catalyst processing steps (1), its cyclization is made.
3. method as claimed in claim 2, it is characterised in that step (1) is prepared by solid phase synthesis technique.
4. method as claimed in claim 3, it is characterised in that step (1) using Fmoc protections solid phase synthesis technique.
5. method as claimed in claim 2, it is characterised in that the catalyst of step (2) is ruthenium catalyst.
6. method as claimed in claim 5, it is characterised in that described ruthenium catalyst is Grubbs 1st。
7. secretin analog as claimed in claim 1, the application in the medicine for Diagnosis of Pancreatic function is prepared.
8. secretin analog as claimed in claim 1, prepare for treat digestive tract ulcer medicine in application.
9. a kind of pharmaceutical composition, including secretin analog as claimed in claim 1 and pharmaceutical carrier.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201110451011.8A CN103172726B (en) | 2011-12-22 | 2011-12-22 | Secretin analog and its production and use |
Applications Claiming Priority (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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| CN103172726A CN103172726A (en) | 2013-06-26 |
| CN103172726B true 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 |
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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 |
|---|
| 消化道激素;黄怀德;《浙江医学》;19831201;第05卷(第06期);47 * |
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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. |