CN109694404A - A kind of incretin peptide and its application - Google Patents
A kind of incretin peptide and its application Download PDFInfo
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- CN109694404A CN109694404A CN201910170129.XA CN201910170129A CN109694404A CN 109694404 A CN109694404 A CN 109694404A CN 201910170129 A CN201910170129 A CN 201910170129A CN 109694404 A CN109694404 A CN 109694404A
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- glu
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- methylene chloride
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
- C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
- C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
- C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Abstract
The present invention relates to a kind of incretin peptide and its applications, obtain the derivative of its long-actingization by carrying out structure of modification to insulin.A kind of new modification compound and preparation method thereof is also disclosed, incretin peptide of the invention is by orthogonal Preservation tactics synthesis in solid state, and synthetic method is simple, and aggregate velocity is fast, and the hypoglycemic effect of incretin peptide is significant, long action time.
Description
Technical field
The present invention relates to medical biochemistry fields, are specifically related to a kind of New-type long-actingization drop in treating diabetes field
The design and its application of glycopeptide.
Background technique
Diabetes are the third-largest chronic noncommunicable diseases for seriously threatening human health after tumour, cardiovascular disease
Disease.Currently, there are about 300,000,000 diabetics in the whole world, it is contemplated that will be increased to 500,000,000 by 2025.Clinically controlled using insulin strengthening
The method for the treatment of delays diabetes progression, but insulin injection has the risk of hypoglycemia.Therapeutic effect be even more by dosage,
The influence of the factors such as injection site, injecting pathway, and individual difference is larger, keeps insulin careless slightly, just will appear serious
Hypoglycemia side effect.
Glucagon-like-peptide-1 (GLP-1) is a kind of glucose dependency intestines rush blood sugar lowing polypeptide hormone, GLP-1 stimulation
Insulin secretion avoids treating diabetes without there is hypoglycemia, the insulin secretion accelerating characteristic of this glucose dependency
In the hypoglycemia that is commonly present it is dangerous.Therefore, GLP-1 has wide development prospect as a kind of diabetes B therapeutic agent.
Natural GLP-1 has many advantages, such as in treatment diabetes, promotes the effect of insulin secretion dependent on higher
Glucose level promotes insulin secretion and inhibits the effect of glucagons secretion under normal and lower glucose level
It is very weak, and it can protect β cell, start the regeneration of β cell.But it is in vivo easily by DPP IV (DPP-
IV) fast degradation, Half-life in vivo only 5min or so.The modification plan for the extension GLP-1 Half-life in vivo being commonly used
Slightly mainly 8 are modified, GLP-1 is enabled to resist the degradation of DPP-IV enzyme, in addition, by GLP-1 peptide chain N-terminal 8 and 9
The amino acid exchange of position also can achieve this purpose.
Exenatide ((Exendin-4)) is the short-acting GLP-1 receptor stimulating agent of typical case for reducing the metabolism of DPP-IV enzyme.Its by
39 amino acid residue compositions, there is 53% homology with GLP-1.Compared with natural GLP-1, Exenatide has stronger two
Peptidyl enzyme resistance (plasma half-life is 60~90min), can be more lasting in vivo, stable performance GLP-1 sample effect.But
It still needs to medication in one day 2 times, similar with natural GLP-1 class formation, equally exists the problem for complying with difference.
Insulin derivates are disclosed in EP 894095, wherein the N- end group of B- chain and/or B28, B29 or B30
The epsilon-amino of upper Lys has the substituent group of general formula-CO-W-COOH, and wherein W can be long chain hydrocarbon groups.These insulin derivates
It is with extended action characteristic and solvable under physiological ph.
Moral paddy insulin is the actrapid monotard that small molecule is fitted into, and has longer action characteristic and simultaneously in physiological pH
Solvable and effect is suitable with actrapid monotard under value.But its function and effect is unstable, there are certain use limitations.
Summary of the invention
The present invention relates to a kind of incretin peptides, which is characterized in that polypeptid acid sequence are as follows:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa1-Gln-Met-Glu-Glu-Glu-
Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-
Pro-Pro-Ser-(Xaa2)q-NH2
Wherein Xaa1 is Lys or Cys-AA1- (AA2)m-AA3；
Xaa2 is Cys-AA1- (AA2)m-AA3；
AA1 is
AA2 is methylene, and wherein AA2 is connected with the N-terminal of AA1；
AA3 is
Wherein, q is 0 or 1；Arbitrary integer of the m between 1-11；
Wherein, when Xaa1 is Lys, q is not 0.
In some embodiments, when Xaa1 is Cys-AA1- (AA2)m- AA3, q 0.
In some embodiments, m is preferably 5-11, and more preferable m is 11.
In some embodiments, Cys-AA1- (AA2)m- AA3 has structure as follows:
In some embodiments, the incretin peptide has structure as follows:
On the other hand, the present invention provides the compound of Formula X, and specific structure is as follows
Wherein arbitrary integer between n=1-11.
In some embodiments it is preferred that arbitrary integer between n=5-11.
In some embodiments, more preferable n is 11.
Further, the present invention also provides a kind of preparation methods of Formula X, with preparation route as follows:
Wherein, P is vector resin.
Wherein arbitrary integer between n=1-11.
In some embodiments it is preferred that arbitrary integer between n=5-11.
In some embodiments, more preferable n is 11.
Wherein, specific the preparation method is as follows:
A) Fmoc-Glu (otBu)-OH, non-nucleophilic organic base are dissolved in organic solvent, the vector resin with swelling
In conjunction with；
B) deprotection agent is added；
C) add after the carbochain alkanoic acid that 2,5- dioxo -2,5- dihydro -1H- pyrroles's -1- base replaces being dissolved in organic solvent
In vector resin after entering step b) reaction, react in the presence of a catalyst；
D) resin trap filtrate is eluted；
E) in organic solvent by the product dissolution in step d), acid adding, reaction.
Wherein, resin described in step a) is 2-CTC resin, HMBA-AM resin, Novasyn-KB resin, bromination Wang
One of resin or bromination PPOA resin；Wherein preferred 2-CTC resin.
The swelling method for the vector resin being swollen in step a) are as follows: be swollen with organic solvent, wherein described organic molten
Agent is selected from methylene chloride, chloroform, tetrahydrofuran, acetonitrile, dimethylformamide or dimethyl sulfoxide；Wherein preferred methylene chloride.
Non-nucleophilic organic base described in step a) is N, N- diisopropyl ethyl amine, 1,8- diazabicylo [5.4.0]
One of 11 carbon -7- alkene or 2,6 di tert butyl pyridine；Wherein preferred N, N- diisopropyl ethyl amine.
Organic solvent described in step a) is selected from methylene chloride, chloroform, tetrahydrofuran, acetonitrile, dimethylformamide or two
Any mixture of one or both of first sulfoxide；Wherein preferred methylene chloride.
Deprotecting regent described in step b) is one of piperidines, ethanol amine, ring amine or pyrrolidones；It is wherein excellent
Select piperidines.
Organic solvent described in step c) is selected from methylene chloride, chloroform, tetrahydrofuran, acetonitrile, Dimethylformamide dimethyl
Sulfoxide；Wherein preferred methylene chloride.Wherein the catalyst is one of EDC/NHS, EDC/HOBt or EDC/DMAP；Wherein
It is preferred that EDC/NHS.
Described in step d) elute resin method be with acid organic solvent eluted, wherein acid be selected from hydrochloric acid,
One of sulfuric acid, trifluoroacetic acid or acetic acid, organic solvent are selected from methylene chloride, chloroform, tetrahydrofuran, acetonitrile, dimethyl methyl
One of amide or dimethyl sulfoxide；The wherein dichloromethane solution of preferred trifluoroacetic acid.
Acid described in step e) is selected from one of hydrochloric acid, sulfuric acid, trifluoroacetic acid or acetic acid；Wherein preferred trifluoroacetic acid；
Organic solvent is selected from one of methylene chloride, chloroform, tetrahydrofuran, acetonitrile, dimethylformamide or dimethyl sulfoxide；It is wherein excellent
Select methylene chloride.
Wherein, further, specific the preparation method is as follows:
A-1) Fmoc-Glu (otBu)-OH and DIPEA is dissolved in methylene chloride, pours into the load being swollen with methylene chloride
In body resin, normal-temperature reaction；
B-1 above-mentioned vector resin successively) is cleaned with DMF and methylene chloride respectively, the dichloromethane solution of piperidines is added, often
Temperature reaction；
C-1) respectively with the vector resin after DMF and methylene chloride successively cleaning step b-1) reaction, by 2,5- dioxo-
Carbochain alkanoic acid, EDC, NHS of 2,5- dihydro -1H- pyrroles's -1- bases substitution dissolve in methylene chloride, after being then added to cleaning
Vector resin in, normal-temperature reaction；
D-1 trifluoroacetic acid) is added with the vector resin after methanol and methylene chloride successively cleaning step c-1) reaction respectively
Dichloromethane solution, room temperature concussion, elute resin trap filtrate；
E-1) in methylene chloride by the product dissolution in step d-1), add trifluoroacetic acid, react, vacuum distillation removal is molten
Agent is added the aqueous solution of sodium hydroxide, obtains white solid.
Wherein, further specific the preparation method is as follows:
A-2) Fmoc-Glu (otBu)-OH and DIPEA is dissolved in methylene chloride, pours into the load being swollen with methylene chloride
In body resin, normal-temperature reaction, nitrogen is bubbled；
B-2 above-mentioned vector resin successively) is cleaned with DMF and methylene chloride respectively, the dichloromethane solution of piperidines is added, often
Temperature reaction, nitrogen are bubbled；
C-2) respectively with the vector resin after DMF and methylene chloride successively cleaning step b-2) reaction, by 2,5- dioxo-
Carbochain alkanoic acid, EDC, NHS of 2,5- dihydro -1H- pyrroles's -1- bases substitution dissolve in methylene chloride, after being then added to cleaning
Vector resin in, normal-temperature reaction, nitrogen be bubbled；
D-2) successively clean c-2 with methanol and methylene chloride respectively) step reaction after vector resin, the dichloro of TFA is added
Dichloromethane, room temperature concussion, elutes resin, collects filtrate, repeats this step 2 times, merging filtrate, vacuum distillation removal solvent；
E-2) in methylene chloride by the product dissolution of step d-2), trifluoroacetic acid is slowly added dropwise, reacts, vacuum distillation is gone
Except solvent, sodium hydrate aqueous solution is added, there is white solid precipitation, be centrifuged, collects precipitating.
Wherein, the specific structure for the carbochain alkanoic acid that 2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base replaces is as follows:
Wherein arbitrary integer between n=1-11.
In some embodiments it is preferred that arbitrary integer between n=5-11.
In some embodiments, more preferable n is 11.
Further, the present invention provides the purposes that Formula X compound is used to prepare incretin peptide.
Further, the present invention also provides the preparation methods of above-mentioned incretin peptide, which is characterized in that by polypeptide chain and Formula X
Small molecule react conjugation in organic solvent.
Further, in some embodiments, conjugation is reacted in organic solvent with the small molecule of Formula X by polypeptide chain
Afterwards, through preparation liquid phase purifying.
Aforementioned polypeptides chain can be prepared by the method for synthesis in solid state；Wherein, the solid phase synthesis process specifically includes
Synthesis, cutting and the purifying of polypeptide chain.
The present invention also provides a kind of pharmaceutical compositions, the above-mentioned incretin peptide of at least one including therapeutically effective amount.
In some embodiments, described pharmaceutical composition also further includes pharmaceutically acceptable carrier.
In the present invention, unless stated otherwise, the therapeutically effective amount, be refer to effectively to realize its therapeutic purposes activity at
The content divided.Treated situation, etc. can be depended on to the effective actual amount of concrete application.For example, when in treatment glycosuria
In the method for disease when applying, this kind of composition (such as can reduce empty stomach in subject containing can effectively realize expected result
Blood-glucose) active principle.
The dosage and frequency (single dose or multi-agent) of the compound of application can change with many factors, the factor packet
Include but be not limited to administration method, the situation of recipient (such as physique, the age, gender, health, weight, body mass index and drink
Food), the property and degree of treat disease symptoms, there are Other diseases or other health related problems while the types treated
With the complication from any disease or therapeutic scheme.Other treatment sides can be used in combination with method and compound of the invention
Case or medicament.
The therapeutically effective amount of people can be determined from animal model.For example, can be to reach by people's dosage formulation
It is found that the effective concentration in animal.It can be by monitoring one or more physiologic parameters, including but not limited to blood Portugal
Grape sugar and weight, and the dosage is adjusted upward or downward to adjust people's dosage, in as described above and this field
Know.
As used in this article, term " pharmaceutical acceptable carrier " refers to pharmaceutical excipient, is for example adapted for intestines or parenteral
The acceptable organic or inorganic carrier substance of the pharmacy of application, physiology, does not react with activating agent.Suitable pharmacy can connect
By carrier include water, salting liquid (such as woods lattice (Ringer) family name's solution etc.), alcohol, oil, gelatin and carbohydrate such as lactose,
Amylose or starch, aliphatic ester, hydroxymethyl cellulose and polyvinylpyrrolidine.This kind of preparation can be sterilized, and
It is expected that when with the adjuvant that does not react deleteriously the compound of the present invention or not for example lubricant, preservative, stabilizer, wetting agent,
The mixing such as emulsifier, the salt for influencing osmotic pressure, buffer, dyestuff, and/or aromatic substance.
Further, there may also be other tension regulators such as sodium chloride and other known excipient.One
In a little situations, this kind of excipient can be used for maintaining the overall tension of compound.Excipient can be included in mesh with various concentration
In the preparaton of preceding description.For example, can include excipient with following concentration range: about 0.02% to about 20%w/w, preferably
Between about 0.02% and 0.5%w/w, about 0.02% to about 10%w/v, or about 1% to about 20%w/w.In addition, with current preparation
Itself is similar, can include excipient with solid-state (including powder), liquid, semisolid or gel form.
Described pharmaceutical composition can be presented in a variety of manners, such as solid, liquid, semisolid or liquid are constituted.Such as this
Used herein, term " solid " means all normal uses for covering this term, for example, powder or freeze-dried formulation.
Term buffer is made of the solution system that the salt or weak base of weak acid and the weak acid or the salt of the weak base form,
Ability with confrontation pH variation after being diluted after addition acid or alkali or with solvent.It such as can be Acetic acid-sodium acetate, lactic acid-
Sodium lactate, acetic acid-ammonium acetate etc.
Described pharmaceutical composition is tablet, capsule, injection, powder, granule, inhalant in any pharmacy
Or film.
Invention further provides above-mentioned incretin peptide or its pharmaceutical composition in preparation for preventing and treating diabetes
Drug in application.
Following abbreviation is used in this specification:
His: histidine；Gly: glycine；Glu: glutamy；Thr: threonine；Phe: phenylalanine；Ser: serine；
Asp: asparatate；Leu: leucine；Cys: cysteine；Gln: glutamine；Met: methionine；Ala: alanine；
Val: valine；Arg: arginine；Leu: leucine；Ile: isoleucine；Trp: tryptophan；Lys: lysine；Asn: asparagus fern
Amide；Pro: proline.
N- fluorenylmethyloxycarbonyl-Pidolidone GAMMA- the tert-butyl ester: Fmoc-Glu (otBu)-OH；2- chlorine trityl chloride tree
Rouge: 2-CTC resin；Methylene chloride: DCM；Chloroform: TCM；Tetrahydrofuran: THF；Acetonitrile: ACN；Dimethylformamide: DMF；Two
First sulfoxide: DMSO；N, N- diisopropyl ethyl amine: DIPEA；11 carbon -7- alkene of 1,8- diazabicylo [5.4.0]: DBU；
EDC:1- ethyl-(3- dimethylaminopropyl) carbodiimide；NHS:N- HOSu NHS；HOBt:1- hydroxyl-benzo three
Nitrogen azoles；DMAP:4- dimethylamino naphthyridine；Fmoc:N-9- fluorenylmethyloxycarbonyl；Trifluoroacetic acid: TFA.
Above-mentioned incretin peptide provided by the invention has significant hypoglycemic effect, and chemical property is stablized, and long half time reaches
39 hours, compared with endogenous GLP-1 (half-life period 2-3 minute) or marketed drug Exenatide (2.4 hours half-life period) have it is aobvious
The raising of work.
Detailed description of the invention
Fig. 1: 6 plasma stability experimental data figure of embodiment；
Fig. 2: sugar tolerance experimental data figure is injected intraperitoneally in 7 normal mouse of embodiment；
Fig. 3: long-actingization sugar tolerance experimental data figure is injected intraperitoneally in 8 normal mouse of embodiment.
Specific embodiment
The present invention is to be illustrated by the following example, but these embodiments do not do any restrictions solution of the invention
It releases.
The synthesis for the carbochain alkanoic acid that embodiment 1:2,5- dioxo -2,5- dihydro -1H- pyrroles's -1- base replaces,
The synthesis of mode 1.1:12- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) dodecanoic acid
Weighing 12-aminolauric acid (2151.9mg) uses acetic acid in three-necked bottle, weighs maleic anhydride
(980.6mg) is slowly added dropwise using constant pressure funnel into above-mentioned solution with after acetic acid, and 120 DEG C are heated to reflux 6h, is subtracted
Pressure distillation removal solvent, alkali alumina column chromatograph (ethyl acetate/petroleum ether gradient elution) after purification, obtain white powder
Solid, yield 81.9%.
1H-NMR(DMSO-d6,300MHz):δppm:12.42(s,1H,-COOH),7.50(s,2H,-COCH=CHCO-),
3.88 (t, 2H, J=7.0Hz ,-NCH2 ), 2.68 (t, J=7.3Hz, 2H ,-CH2 COOH),2.00-1.96(m,4H,-
NCH2CH2 (CH2)7CH2 ),1.73(s,14H,-NCH2CH2(CH2)7 CH2).MS(ESI,m/z):294.1[M-H]-,m.p.91-92
℃.
The synthesis of mode 1.2:10- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) ten alkanoic acids
Synthetic method reference pattern 1.1 is synthesized by starting material of amino-nonanoic acid.
The synthesis of mode 1.3:4- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) butane acid
Synthetic method reference pattern 1.1 is synthesized by starting material of Beta-alanine.
Embodiment 2: the preparation method of compound X
Reaction route is as follows:
Mode 2.1:12- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) dodecane acyl group)-Pidolidone conjunction
At
A) swelling of 2-CTC resin
It weighs 2-CTC resin (363.6mg, degree of substitution 0.55mmol/g) to be placed in polypeptides reactive pipe, methylene chloride swelling
30min, then resin is cleaned 3-5 times with methylene chloride, it is filtered dry spare.
B) synthesis of X-1
Take Fmoc-Glu (otBu)-OH (255.3mg) and DIPEA (1.6mmol, 264 μ L) molten with about 7mL methylene chloride
Solution, solution is poured into above-mentioned resin, normal-temperature reaction, and nitrogen is bubbled 4h, methanol/DIPEA=9:1 end socket 30min.
C) synthesis of X-2
Above-mentioned resin is respectively washed 3 times with DMF and methylene chloride, and into reactor plus about 10mL deprotection agent, room temperature are anti-
It answers, nitrogen is bubbled 30min, is filtered dry solvent.10mL deprotection agent, normal-temperature reaction are rejoined, nitrogen is bubbled 60min.
D) synthesis of X-3
Above-mentioned resin is respectively washed 3 times with DMF and methylene chloride, weighs 12- (2,5- dioxo -2,5- dihydro -1H- pyrroles
Cough up -1- base) dodecanoic acid (882mg), EDC (690mg), NHS (414mg) are dissolved with about 7mL methylene chloride, solution are poured into
In above-mentioned resin, normal-temperature reaction, nitrogen bubbled overnight.
E) synthesis of X-4
Above-mentioned resin is respectively washed 3 times with methanol and methylene chloride, is cleaned for the last time with methylene chloride.In the reactor
The dichloromethane solution of 0.5%TFA is added, room temperature shakes 30min, collects filtrate, repeats this step 2 times, merging filtrate, decompression
Distillation removal solvent.
F) synthesis of X
X-4 is redissolved with 2mL methylene chloride, 1ml TFA is slowly added dropwise, reacts 3h, vacuum distillation removal solvent.5mL is added
1M NaOH aqueous solution has white solid precipitation, and centrifugation collects precipitating, obtains white powder sterling, yield 94.2%.
Mode 2.2:12- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) dodecane acyl group)-Pidolidone conjunction
At
A) swelling of 2-CTC resin
It weighs 2-CTC resin (363.6mg, degree of substitution 0.55mmol/g) to be placed in polypeptides reactive pipe, methylene chloride swelling
30min, then resin is cleaned 3-5 times with methylene chloride, it is filtered dry spare.
B) synthesis of X-1
Fmoc-Glu (otBu)-OH (255.3mg) and DBU (1.6mmol, 264 μ L) about 7mL methylene chloride is taken to dissolve,
Solution is poured into above-mentioned resin, normal-temperature reaction, nitrogen is bubbled 4h, methanol/DIPEA=9:1 end socket 30min.
C) synthesis of X-2
Above-mentioned resin is respectively washed 3 times with DMF and methylene chloride, and into reactor plus about 10mL deprotection agent, room temperature are anti-
It answers, nitrogen is bubbled 30min, is filtered dry solvent.10mL deprotection agent, normal-temperature reaction are rejoined, nitrogen is bubbled 60min.
D) synthesis of X-3
Above-mentioned resin is respectively washed 3 times with DMF and methylene chloride, weighs 12- (2,5- dioxo -2,5- dihydro -1H- pyrroles
Cough up -1- base) dodecanoic acid (882mg), EDC (690mg), HOBt (121.5mg) are dissolved with about 7mL methylene chloride, solution are fallen
Enter in above-mentioned resin, normal-temperature reaction, nitrogen bubbled overnight.
E) synthesis of X-4
Above-mentioned resin is respectively washed 3 times with methanol and methylene chloride, is cleaned for the last time with methylene chloride.In the reactor
The dichloromethane solution of 0.5%TFA is added, room temperature shakes 30min, collects filtrate, repeats this step 2 times, merging filtrate, decompression
Distillation removal solvent.
F) synthesis of X
X-4 is redissolved with 2mL methylene chloride, 1ml TFA is slowly added dropwise, reacts 3h, vacuum distillation removal solvent.5mL is added
1M NaOH aqueous solution has white solid precipitation, and centrifugation collects precipitating, obtains white powder sterling.
Mode 2.3:10- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) ten alkanoyls)-Pidolidone synthesis
Reference pattern 2.1, by 12- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) dodecane in the d of step)
Acid replaces with 10- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) ten alkanoic acids.
Mode 2.4:4- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) butane acyl group)-Pidolidone synthesis
Reference pattern 2.1 replaces with 12- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) dodecanoic acid in the d of step)
4- (2,5- dioxo -2,5- dihydro -1H- pyrroles -1- base) butane acid.
The synthesis of 3 polypeptide chain of embodiment
3.1
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Cys-Gln-Met-Glu-Glu-Glu-
Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-
Pro-Pro-Ser-NH2
A) swelling of resin
Fmoc-Rink amide-MBHA resin (18.2g, degree of substitution 0.55mmol/g) is weighed to be placed in polypeptides reactive pipe,
It after methylene chloride is swollen 30min, is respectively washed 3 times, is placed spare with methanol and methylene chloride.
B) removing of 9- tablet held before the breast by officials methoxycarbonyl group (Fmoc)
The preparation of deprotection agent: weighing HOBt (6.75g), and after DMF 400mL dissolution, piperidinyl-1 00mL is added, mixes.
Deprotection carries out in two times.For the first time, deprotection about 50mL is added, nitrogen is bubbled, and comes into full contact with resin with deprotection agent, is taken off
After protecting 30min, deprotection agent is filtered off, new deprotection agent about 50mL is rejoined, second of deprotection 60min.Deprotection
After the completion, with alternately cleaning resin 3 times of DMF and methylene chloride, a small amount of resin is taken to be detected with 1% bromophenol blue/ethanol solution, if tree
Rouge is blue and solution is yellow clarified solution body, then enters in next step, otherwise re-starts deprotection operation.
C) coupling of the amino acid of Fmoc protecting group
It is sufficiently molten with the mixed solvent of DMF:DCM=1:1 to weigh fmoc-protected amino acid 25mmol and HOBt (4.05g)
It solves, after ice bath stirring 10min, 35% DIC/ ethanol solution is slowly added dropwise, continue to stir 10min, wait there is white insoluble matter to go out
It is existing, above-mentioned amino acid solution is poured into the polypeptides reactive device for filling resin.Nitrogen is bubbled 4h, after reaction, with DMF and two
Chloromethanes rotation is cleaned 2 times, and a small amount of resins is taken to be detected with 1% bromophenol blue/alcoholic solution, if resin is white clear, is coupled
Success, into next circulation, otherwise re-starts amino acid coupling operation.
D) synthesis of polypeptide
According to amino acid sequence, above-mentioned coupling and deprotection steps are repeated, corresponding amino acid, Zhi Daoduo are successively coupled
Peptide link coupling finishes, and obtains the resin for being embroidered with polypeptide chain.
E) cutting of polypeptide
The resin for being embroidered with polypeptide chain is placed in polypeptides reactive device, replaces cleaning resin 5 times with methylene chloride and methanol, most
Resin once is cleaned with methylene chloride afterwards.If dry resin, after being swollen 30min with methylene chloride, using methylene chloride and
Methanol rotation is washed resin 5 times.Cutting agent Reagent K (TFA/thioanisole/water/ is added into reactor
Phenol/EDT 82.5:5:5:2.5), after 4 DEG C of concussion 1h, room temperature shakes 3h, filters, and collects filtrate.Filtrate is poured into 5 times of bodies
In long-pending ice ether, -20 DEG C of standing 1h, 4 DEG C of centrifugations (5000rpm*5min) throw aside supernatant, clean precipitating 5-6 with ice ether
It is secondary, vacuum drying.
F) purifying of polypeptide
Using the method for preparation liquid phase.Carry out the purifying of polypeptide.Use Shimadzu 6A preparative efficient liquid phase, chromatography
Condition: chromatographic column: C18 reverse phase preparative column (5 μm, 340mm*28mm, Shimadzu), mobile phase A: 1 ‰ trifluoracetic acids/water (V/
V), Mobile phase B: 1 ‰ trifluoracetic acids/acetonitrile (V/V), wavelength: 214nm, flow velocity: 5mL/min, gradient elution, the preparation of sample:
Polypeptide to be detected is dissolved with 50% acetonitrile/water (V/V), is configured to the solution of 10mg/mL, 0.45 μm of filtering with microporous membrane is laggard
Sample.
3.2
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-
Ala-Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-
Pro-Pro-Ser-Cys-NH2The synthesis of peptide chain
It is synthesized referring to 3.1 mode.
The synthesis of 4 incretin peptide of embodiment
4.1 incretin peptide B-3
The polypeptide for weighing the synthesis of 100mg embodiment 3.1 is sufficiently dissolved with DMSO 3mL, weighs 4mg small molecule X (embodiment
2, the small molecule that mode 2.1 synthesizes) it is sufficiently dissolved with 200 μ L of DMSO, the two mixes, stirring, and room temperature reaction is overnight.Use UPLC-
MS is detected after the reaction was completed, reaction solution is poured into 30% acetonitrile/water solution (containing 1 ‰ trifluoracetic acids) 15mL, concussion is mixed immediately
It is even.Sample uses 0.45 μm of membrane filtration, is prepared liquid phase and is purified.It is high using Shimadzu, Japan's preparative reverse phase
Effect liquid phase chromatogram, chromatographic condition: chromatographic column: C18 reverse phase preparative column (5 μm, 340mm*28mm, Shimadzu), mobile phase A: 1 ‰
Trifluoracetic acid/water (V/V), Mobile phase B: 1 ‰ trifluoracetic acids/acetonitrile (V/V), wavelength: 214nm, flow velocity: 5mL/min, gradient are washed
It is de-, B 30%~90%30min, 90%~90%10min.
Polypeptide molecular weight, chromatographic condition: chromatographic column: C18 reversed-phase column (CSH, 1.7 μ are detected using LC-MS LC-MS instrument
M, 2.1 × 50mm, Waters), sample volume: 3.000 μ L, column temperature: 40 DEG C, mobile phase A: 0.1% formic acid/water (V/V), mobile phase
B: acetonitrile, wavelength: 214nm, flow velocity: 0.3mL/min, gradient elution: B 10%~90%3min, 90%~90%1min.Matter
Original figure spectrum is composed referring to annex.
The preparation of sample: polypeptide to be detected being dissolved with 50% acetonitrile/water (V/V), is configured to the solution of 0.5mg/mL,
Sample introduction is analyzed after 0.22 μm of filtering with microporous membrane.
Specific mass spectrometric data is as follows
4.2 incretin peptide B-4
The polypeptide for weighing the synthesis of 100mg embodiment 3.2 is sufficiently dissolved with DMSO 3mL, weighs 4mg small molecule X (embodiment
2, the small molecule that mode 2.1 synthesizes) it is sufficiently dissolved with 200 μ L of DMSO, the two mixes, stirring, and room temperature reaction is overnight.Use UPLC-
MS is detected after the reaction was completed, reaction solution is poured into 30% acetonitrile/water solution (containing 1 ‰ trifluoracetic acids) 15mL, concussion is mixed immediately
It is even.Sample uses 0.45 μm of membrane filtration, is prepared liquid phase and is purified.It is high using Shimadzu, Japan's preparative reverse phase
Effect liquid phase chromatogram, chromatographic condition: chromatographic column: C18 reverse phase preparative column (5 μm, 340mm*28mm, Shimadzu), mobile phase A: 1 ‰
Trifluoracetic acid/water (V/V), Mobile phase B: 1 ‰ trifluoracetic acids/acetonitrile (V/V), wavelength: 214nm, flow velocity: 5mL/min, gradient are washed
It is de-, B 30%~90%30min, 90%~90%10min.
Polypeptide molecular weight, chromatographic condition: chromatographic column: C18 reversed-phase column (CSH, 1.7 μ are detected using LC-MS LC-MS instrument
M, 2.1 × 50mm, Waters), sample volume: 3.000 μ L, column temperature: 40 DEG C, mobile phase A: 0.1% formic acid/water (V/V), mobile phase
B: acetonitrile, wavelength: 214nm, flow velocity: 0.3mL/min, gradient elution: B 10%~90%3min, 90%~90%1min.Matter
Original figure spectrum is composed referring to annex.
The preparation of sample: polypeptide to be detected being dissolved with 50% acetonitrile/water (V/V), is configured to the solution of 0.5mg/mL,
Sample introduction is analyzed after 0.22 μm of filtering with microporous membrane.
Specific mass spectrometric data is as follows
Other incretin peptides are referred to above method synthesis, need to only change the structure of specific binding compounds X.
The experiment of 5 receptor agonist activity of embodiment
GLP-1 receptor stimulating agent mainly passes through exciting GLP-1R and plays various physiological activity.Early stage passes through rotaring dyeing technology, at
The HEK293 cell of high expression GLP-1R is constructed to function, after receptor is activated, intracellular second messenger cAMP content is increased,
Close KATPWith valtage-gated type potassium-channel, cause membrane depolarization, activates the L-type Ca of voltage-dependent2+Channel increases
Add extracellular Ca2+Interior stream increases insulin releasing.Using cAMP detection kit, swashed by competitive ELISA measuring compound
The amount for the cAMP that receptor living generates assesses the receptor agonist activity of compound.
Concrete mode is as follows: HEK293 cell cotransfection encodes the cDNA of GLP-1R, and cell line is expressed and utilizes Western
The protein level of GLP-1R in the HEK293 cell that Blot detection has constructed, stablizes highly expressed GLP- to investigate whether to establish
R-HEK293 cell strain.In receptor agonist activity experiment, firstly, after 2h, compound is molten with DMSO by cell kind in 96 orifice plates
Solution, is diluted to different multiples using the culture medium containing 0.1% bovine serum albumin, the GLP-1R-HEK293 that cotransfection is added is thin
In born of the same parents.After being incubated for 20min, corresponding cAMP value is detected using the ELISA kit of Cisbo company, is calculated after nonlinear regression
The EC of compound50Numerical value.
1 receptor agonist activity of table
It as a result is average value ± SD, * * P < 0.01vs Exenatide, ##P < 0.01vs Liraglutide
As shown in table 1, all long-actingization incretin peptides all retain the agonist activity of GLP-1R, draw with marketed drug benefit
Shandong peptide is compared, and compound is all significantly improved to the agonist activity of GLP-1R, and is significantly improved, activity improve 6 times with
On.
The experiment of 6 plasma stability of embodiment
Incretin peptide B-3, B-4 and Exenatide, Liraglutide as a control group are weighed, it is molten with Tris-HCl buffer
Solution, is formulated as 1000ng/mL mother liquor, takes 400 μ L derivative solutions and 400 μ L rat plasmas, mixed with blending instrument, be placed in 37 DEG C
In water-bath, it is incubated for 0,0.5,1,2,4,6,8,12,24,36,48 and 72h, each time point takes out 50 μ L mixed liquors, adds 100 μ L
Acetonitrile (contain 0.1%TFA) protein precipitation is centrifuged 14000rpm*15min, is taken supernatant using blending instrument vortex 30min, again from
Heart 14000rpm*10min, is analyzed using UPLC-MS/MS, and confrontation spectral peak area is integrated, and is drawn degradation curve, is obtained mesh
Mark the plasma half-life of compound.Concrete condition is detailed in Fig. 1.
According to the data of Fig. 1 it is found that the half-life period of incretin peptide B-3 and B-4 are obviously prolonged, it is better than Exenatide, Li La
Shandong peptide.
Sugar tolerance experiment is injected intraperitoneally in 7 normal mouse of embodiment
Male ICR mouse (18-22g) is grouped at random by weight, and every group 6, adaptable fed is tested after 1 week.It opens
Begin before experiment, fasting 12h can't help water, and incretin peptide B-3, B-4 (25nmol/kg) to be measured, positive control Ai Saina is injected intraperitoneally
Peptide (25nmol/kg), negative control are physiological saline (saline).Blood glucose is measured after 30min, injects 18mmol/kg glucose,
It is time point 0min by injectable dextrose monohydrate time rule, measures the blood of 15min, 30min, 60min, 120min and 180min respectively
Sugar level, and blood glucose-time graph is drawn, it is detailed in Fig. 2.
It is hypoglycemic the experimental results showed that, long-actingization incretin peptide involved in the present invention, blood sugar decreasing effect and Exenatide phase
When.
The experiment of long-actingization sugar tolerance is injected intraperitoneally in 8 normal mouse of embodiment
In order to which the long-actingization hypoglycemic for detecting target incretin peptide B-3, B-4 is horizontal, carries out normal mouse abdominal cavity single injection and give
Medicine, multiple sugar tolerance test experience.First day intraperitoneal injection of saline, Exenatide (25nmol/kg), incretin peptide B-3
Glucose Tolerance is detected after (25nmol/kg) or incretin peptide B-4 (25nmol/kg), 30min, then free diet 12 hours, with
After be deprived of food but not water 12 hours；(i.e. next day) intraperitoneal injection glucose (18mmol/kg) later, detects Glucose Tolerance again；
Above-mentioned " free diet-fasting-sugar tolerance " circulation is repeated, so that the long-actingization hypoglycemic for probing into compound is horizontal.
Male ICR mouse (18-22g) is grouped at random by weight, and every group 6,12h replaces round the clock, and adaptable fed 1 week
After tested.Untested compound B-3 (25nmol/kg), compound B-4 (25nmol/kg) is injected intraperitoneally, positive control is Chinese mugwort
That peptide (25nmol/kg) is filled in, negative control is physiological saline (saline), injects 30min after compound, intraperitoneal injection
18mmol/kg glucose measures the blood glucose level of 15min, 30min, 60min, 120min and 180min respectively, then freely drinks
Food 12 hours, then be deprived of food but not water 12 hours, sugar tolerance is detected again, and monitoring repeats above-mentioned to the change of blood sugar situation after sugar
" free diet-fasting-sugar tolerance " circulation, draw blood glucose-time graph, to probe into the long-actingization hypoglycemic water of compound
It is flat.Specific effect data is detailed in Fig. 3.From the figure 3, it may be seen that the time of incretin peptide B-3 and B-4 stabilizing blood sugar, to can reach 25 small
When, much higher than 2 hours of Exenatide, hypoglycemic effect was continual and steady.
Incretin peptide i.e. provided by the invention, while guaranteeing effect, when effectively extending its half-life period and effect
Between.
Sequence table
<110>China Medicine University；Nanjing Zhengda Tianqing Pharmaceutical Co., Ltd
<120>a kind of incretin peptide and its application
<130> 01
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Xaa Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Tyr Ile Gln Trp Leu Lys Glu Gly Gly Pro Ser
20 25 30
Ser Gly Arg Pro Pro Pro Ser
35
<210> 2
<211> 40
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
His Gly Glu Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu
1 5 10 15
Glu Ala Val Arg Leu Tyr Ile Gln Trp Leu Lys Glu Gly Gly Pro Ser
20 25 30
Ser Gly Arg Pro Pro Pro Ser Xaa
35 40
Claims (10)
1. a kind of incretin peptide, which is characterized in that amino acid sequence are as follows:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Xaa1-Gln-Met-Glu-Glu-Glu-Ala-
Val-Arg-Leu-Tyr-Ile-Gln-Trp-Leu-Lys-Glu-Gly-Gly-Pro-Ser-Ser-Gly-Arg-Pro-Pro-
Pro-Ser-(Xaa2)q-NH2
Wherein Xaa1 is Lys or Cys-AA1- (AA2)m-AA3；
Xaa2 is Cys-AA1- (AA2)m-AA3；
AA1 is
AA2 is methylene, and wherein AA2 is connected with the N-terminal of AA1；
AA3 is
Wherein, q is 0 or 1；Arbitrary integer of the m between 1-11；
Wherein, when Xaa1 is Lys, q is not 0.
2. a kind of incretin peptide according to claim 1, which is characterized in that when Xaa1 is Cys-AA1- (AA2)m- AA3, q are
0。
3. a kind of incretin peptide according to claim 1, which is characterized in that any integer of the m between 5-11, preferably m are
11。
4. a kind of incretin peptide according to claim 1, which is characterized in that Cys-AA1- (AA2)mThe specific structure of-AA3 is such as
Under:
5. a kind of incretin peptide according to claim 1, which is characterized in that have the following structure:
6. a kind of compound of Formula X, feature, specific structure are as follows:
Wherein arbitrary integer between n=1-11, the integer between preferably n=5-11, more preferable n are 11.
7. a kind of preparation method of Formula X compound, which is characterized in that reaction route is as follows:
Wherein, P is vector resin；
Wherein arbitrary integer between n=1-11.
8. a kind of Formula X compound according to claim 6, is used to prepare the use of incretin peptide described in claim 1-5
On the way.
9. -5 any a kind of incretin peptides or its pharmaceutical composition are in preparation for preventing and treating sugar according to claim 1
Urinate the application in the drug of disease.
10. pharmaceutical composition prepared by -5 any incretin peptides according to claim 1, described pharmaceutical composition is any
Described tablet, capsule, injection, powder, granule, inhalant or film in a kind of pharmacy.
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|---|---|---|---|---|
| CN103626845A (en) * | 2013-09-30 | 2014-03-12 | 承德医学院 | Arylpyrrole-2-formamide dipeptide derivatives serving as glycogen phosphorylase inhibitor and preparation method and medical application thereof |
| CN105936647A (en) * | 2016-04-27 | 2016-09-14 | 中国药科大学 | Long-acting Exendin-4 analogue and application thereof |
| CN107056926A (en) * | 2017-06-23 | 2017-08-18 | 中国药科大学 | One class carries the Exenatide of ehter bond（Exendin-4）Analog and its application |
| CN107141348A (en) * | 2017-06-23 | 2017-09-08 | 中国药科大学 | One class long-actingization Exenatide（Exendin-4）Analog and its application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103626845A (en) * | 2013-09-30 | 2014-03-12 | 承德医学院 | Arylpyrrole-2-formamide dipeptide derivatives serving as glycogen phosphorylase inhibitor and preparation method and medical application thereof |
| CN105936647A (en) * | 2016-04-27 | 2016-09-14 | 中国药科大学 | Long-acting Exendin-4 analogue and application thereof |
| CN107056926A (en) * | 2017-06-23 | 2017-08-18 | 中国药科大学 | One class carries the Exenatide of ehter bond（Exendin-4）Analog and its application |
| CN107141348A (en) * | 2017-06-23 | 2017-09-08 | 中国药科大学 | One class long-actingization Exenatide（Exendin-4）Analog and its application |
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| CN113461669A (en) * | 2021-03-22 | 2021-10-01 | 中国药科大学 | Novel androgen receptor degradation agent, preparation method and medical application |
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