CN104987383A - GLP-1 derivate - Google Patents
GLP-1 derivate Download PDFInfo
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- CN104987383A CN104987383A CN201510393214.4A CN201510393214A CN104987383A CN 104987383 A CN104987383 A CN 104987383A CN 201510393214 A CN201510393214 A CN 201510393214A CN 104987383 A CN104987383 A CN 104987383A
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- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 title claims abstract description 23
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- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 10
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- 229910052757 nitrogen Inorganic materials 0.000 claims description 8
- HXMVNCMPQGPRLN-UHFFFAOYSA-N 2-hydroxyputrescine Chemical compound NCCC(O)CN HXMVNCMPQGPRLN-UHFFFAOYSA-N 0.000 claims description 6
- 125000000539 amino acid group Chemical group 0.000 claims description 6
- YZGQDNOIGFBYKF-UHFFFAOYSA-N Ethoxyacetic acid Chemical compound CCOCC(O)=O YZGQDNOIGFBYKF-UHFFFAOYSA-N 0.000 claims description 4
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- MFFMDFFZMYYVKS-SECBINFHSA-N sitagliptin Chemical compound C([C@H](CC(=O)N1CC=2N(C(=NN=2)C(F)(F)F)CC1)N)C1=CC(F)=C(F)C=C1F MFFMDFFZMYYVKS-SECBINFHSA-N 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 229960001254 vildagliptin Drugs 0.000 description 1
- SYOKIDBDQMKNDQ-XWTIBIIYSA-N vildagliptin Chemical compound C1C(O)(C2)CC(C3)CC1CC32NCC(=O)N1CCC[C@H]1C#N SYOKIDBDQMKNDQ-XWTIBIIYSA-N 0.000 description 1
Classifications
-
- 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
- C07K14/605—Glucagons
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Toxicology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Endocrinology (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a GLP-1 derivate. At least one of an N-terminal amino group, a side chain amino group, a side chain carboxyl group, a C-terminal carboxyl group and a histidine side chain imidazolyl group in the derivate is rejoined with a conjugate K1-K2 (I) with a formula (I), wherein K1 is modifying group containing a DPP-4 enzyme inhibitor, K2 is a divider, and the tail end of K2 is rejoined with a main chain.
Description
technical field:
The present invention relates to glucagon-like-peptide-1 (GLP-1) (7-37) derivative and pharmacy acceptable salt thereof, and relate to its some therepic use.
background technology:
Glucagon-like-peptide-1 (GLP-1) is the gang of incretin, by enteron aisle L emiocytosis, being a kind of hormone secreting after the meal, contributing to controlling blood sugar, can as an important target spot of diabetes B treatment.But GLP-1 in vivo rapidly degrade by a kind of enzyme (DPP-4), (t1-2 minute plasma half-life in vivo.This metabolism instability limit the therapeutic efficacy of natural GLP-1.Therefore, need a kind of more natural GLP-1 more effectively or GLP-1 derivative more stable in metabolism.
DPP-4 enzyme inhibitors, has restraining effect to DPP-4, can improve and extend the hormonal activity of GLP-1, thus stimulates the release of Regular Insulin, reduces the secretion of hyperglycemic-glycogenolytic factor.These two kinds effects are all conducive to the high-caliber adjustment of diabetes B patient blood glucose.
The present invention is by GLP-1(7-37) structure transform, and on side chain, introduce DPP-4 enzyme inhibitors, can obtain than GLP-1 derivative more stable in natural GLP-1 metabolism.
compound characteristic:
A kind of GLP-1 (7-37) or have the derivative of the most nearly GLP-1 (7-37) analogue of 8 amino acid changes compared with GLP-1 (7-37), described GLP-1 (7-37) analogue has the function of people GLP-1, it is characterized in that in the N Amino End Group in described derivative, side-chain amino group, side chain carboxyl group, C end carboxyl, histidine side chains imidazolyl, at least one and a kind of conjugate with formula I are puted together;
K1-K2 (Ⅰ)
Wherein K1 is the modification group comprising DPP-4 enzyme inhibitors, and K2 is spacer, and K2 end and main chain are puted together;
K1 is preferably:
Z-NH
2for DPP-4 enzyme inhibitors [comprise Sitagliptin(and be abbreviated as SI), Vildagliptin(is abbreviated as VI), Saxagliptin(is abbreviated as SA) etc.];
N is the integer of 0-14;
X is C, N or O;
Y is O or S;
R is preferably:
K2 is preferably the compound described by following general formula:
①（AEEA）
a-Xaa7- Xaa8 -Xaa9 -（AEEA）
b
Above-mentioned involved AEEA{ [2-(2-is amino) oxyethyl group] ethoxyacetic acid } structure is
A is the integer of 0-8;
B is the integer of 0-6;
Xaa7 is selected from: Glu, Gln, Phe, Tyr or disappearance;
Xaa8 is selected from: Asn, Asp, Thr or disappearance;
Xaa9 is selected from: Lys, Asn, Ser, Cys or disappearance;
2.-(CH
2)
pcO-Xaa10-, for the acyl group of straight chain or branched fatty acid is connected with amino-acid residue
Above-mentioned involved p is the integer of 4-30;
Xaa10 is selected from: Glu, Asp, Gln or disappearance;
compound structure example:
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
2】-Glu-γ-(AEEA)
3} }-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
2】-Glu-γ-【(AEEA)
2-NH-（CH
2）
2-NH-】}}Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile- Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{N-α-【SI-CO-(CH
2)
2-CO-NH-(CH
2)
15-CO-】L-γ-glutamyl} }-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-(CH
2)
2-CO- NH-(CH
2)
15-CO-】Glu-γ-【NH-（CH
2）
2-NH-】}}-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Arg-Gly-Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
2】-Glu-γ-(AEEA)
5}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
5】-Glu-γ-(AEEA)
2}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
8】-Glu-γ-(AEEA)
6}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
2】-Glu-γ-(AEEA)
4}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
5】-Glu-γ-(AEEA)
5}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2）
2-CO-(AEEA)
2】-Glu-γ-(AEEA)
2}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Lys{N-ε-{ N-α-[SI-CO-(CH
2)
2-CO-pentadecanoyl]-L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{ N-α-[SI-CO-(CH
2)
2-CO-myristoyl]-L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{ N-α-[SI-CO-(CH
2)
2-CO-O-heptadecanoyl]-L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{ N-α-[SI-CO-(CH
2)
2-CO-stearyl]-L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{ N-α-[SI-CO-(CH
2)
2-O-stearyl]-L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Ile-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{ N-α-[SI-CO-(CH
2)
2-CO-stearyl]-L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-(CH
2)
2-CO- NH-(CH
2)
15-CO-】Glu-γ-【NH-（CH
2）
2-NH-】}}-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Arg-Gly-Arg-Gly
His-Leu-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-(CH
2)
2-CO- NH-(CH
2)
15-CO-】Glu-γ-【NH-（CH
2）
2-NH-】}}Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Arg-Gly-Arg-Gly
His-Ile-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-(CH
2)
2-CO- NH-(CH
2)
15-CO-】Glu-γ-【NH-（CH
2）
2-NH-】}}Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Arg-Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{N-α-【SI-CO-NH-(CH
2)
2-CO- NH-(CH
2)
15-CO-】L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg- Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{N-α-【SI-CO-O-(CH
2)
2-CO- NH-(CH
2)
15-CO-】L-γ-glutamyl} }-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg- Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{N-α-【SI-CH
2-CO-O-(CH
2)
2-CO- NH-(CH
2)
15-CO-】L-γ-glutamyl} }-Glu-Phe-Ile-Ala-Trp-Leu-Val- Arg-Gly-Arg-Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{N-α-【SI- (CH
2)
3-CO- NH-(CH
2)
15-CO-】L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg- Gly
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-{N-α-【SI -O-(CH
2)
2-CO- NH-(CH
2)
15-CO-】L-γ-glutamyl}}-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
The implication of the abbreviation used in claims and specification sheets is listed in the following table:
| Boc | Tertbutyloxycarbonyl |
| DIC | DIC |
| DCC | N, N '-dicyclohexylcarbodiimide |
| DIEA | Diisopropylethylamine |
| DMF | DMF |
| DCM | Methylene dichloride |
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| HOBt | I-hydroxybenzotriazole |
| EDT | Dithioglycol |
| TFA | Trifluoracetic acid |
| Tis | Tri isopropyl silane |
| Palmitoyl | Palmityl |
| glutamyl | Glutamy |
| Fmoc-Asp(OBzL) | |
| SI | |
| VI | |
| SA |
2, preparation method
(1) preparation method of conjugate
1. the preparation of K1:
A) SI(1eq is taken) and Succinic anhydried (1eq), dissolve with Glacial acetic acid (2mol/L), 50 DEG C of reactions are spent the night;
B) reaction solution adds in ethyl acetate, washes acetic acid with water, concentrated, dries, obtains product;
2. the preparation of K1-K2:
A) according to required K2, with the method synthesis K2 of solid phase synthesis;
B) solid phase coupling K1 is continued;
C) cut by peptide resin, process, obtains conjugate;
(2) preparation method of main chain
A) 2-CTC resin DCM washing 2-3 time, swelling with DCM;
B) take the amino acid (3eq) that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
C) Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
D) repeating step A, B, C, but condensing agent changes HOBt (3.3eq) and DIC(3.3eq) into, solvent changes DMF into, 0-5 DEG C activates 5 minutes, adds reaction column, in 25-30 DEG C of reaction 2.5h, according to described aminoacid sequence, the corresponding amino acid of coupling one by one, and will the side chain carboxyl group connecting conjugate be needed to make benzyl ester, continue coupling until last amino acid couplings completes in sequence;
E) resin cutting liquid (TFA and DCM volume ratio the is 1:99) cutting obtained, cuts down polypeptide from resin, washes the TFA in solution with water, concentrated, obtains the thick peptide of main chain cut; Or resin is used TFA cracking, polypeptide is cut down from resin, and takes off all Side chain protective groups, ether sedimentation, obtain the thick peptide of main chain of cracking;
(3) method of attachment of side chain carboxyl group, C end carboxyl and conjugate
A) the side chain carboxyl group protecting group benzyl of the thick peptide of main chain of cutting is removed by the method for hydrogenolysis;
B) coupling Boc-quadrol on carboxyl, takes Boc-quadrol (1.1eq), is cooled to-5-0 DEG C, and add DCC (1eq), feed intake, room temperature reaction spends the night;
C) use TFA lysate sample, ether sedimentation, obtain thick peptide;
D) take conjugate (1eq), synthesized active ester;
E) take thick peptide (1eq), be dissolved in triethylamine, adjustment pH is 9-11, adds conjugate active ester, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects;
(4), the method that is connected with conjugate of N Amino End Group, side-chain amino group, histidine side chains imidazolyl
A) take conjugate (1eq), synthesized active ester;
B) take the thick peptide of main chain (1eq) of cracking, be dissolved in triethylamine, adjustment pH is 9-11, adds conjugate active ester, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects;
(5), purification process
A) use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, and gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, and collect cut, freeze-drying obtains the finished product;
B) its purity and content is detected by HPLC method, by mass spectrograph and its sequence of Edman degradation analysis;
4, the plant and instrument used
Peptide synthesizer: hyperchannel Peptide synthesizer is used in U.S. CsBio CS336X research;
HPLC: Japanese Shimadzu LC-15C high performance liquid chromatography;
Chromatographic column: YMC-Pack ODS-A-HG 10um 200 4.6mm*250mm;
Mass spectrograph: AB SCIEX 5800 MALDI-TOF-TOF;
5, implication explanation
(1) glucagon-like peptide 1 (GLP-1) is by a kind of very important incretin of enteron aisle internal secretion L emiocytosis, its main physiological effect in vivo stimulates secretion and the release of Regular Insulin after comprising feed, promote the propagation of pancreatic beta cell and suppress its apoptosis, the secretion of glucagon suppression, suppress the emptying of stomach, promote satietion generation etc.
(2) GLP-1(7-37) peptide sequence is: HAEGT-FTSDV-SSYLE-EQAAK-EFIAW-LVKGR-G
(3) DPP-4 is a kind of serine protease of cell surface.The DPP-4 of solubility has enzymic activity, N-terminal the first two amino acid shear removal of its Main Function to be preferential by N-terminal the 2nd amino acid be oligopeptides of L-Ala or proline(Pro).
(4) analogue: the term " analogue " relating to polypeptide refers to that one or more amino-acid residues of parent peptide are replaced by another or multiple amino-acid residue, and/or one or more amino-acid residue disappearances of parent tire, and/or parent peptide with the addition of one or more amino-acid residue wherein.
(5) derivative: the term " derivative " relating to polypeptide represents that wherein at least one substituting group is not present in not modified peptide or its analogue, namely represents through the peptide of covalent modification through the peptide of chemically modified or its analogue.
(6) in the present invention, " conjugate " of indication refers to the modification group that can be connected with the form of covalency with polypeptide, and described conjugate includes but not limited to example mentioned above.
(7) peptide of the present invention can be the form of pharmacy acceptable salt.These salt such as include, but not limited to acetic acid, lactic acid, toxilic acid, citric acid, oxysuccinic acid, xitix, succsinic acid, phenylformic acid, methylsulfonic acid, toluenesulphonic acids.The typical method preparing the salt of peptide of the present invention is well known in the art, and it has come by the salt exchange process of standard.
Accompanying drawing explanation
Fig. 1 is preparation technology's schema of the present invention.
the synthesis of embodiment one: GLP-1 derivative
His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{ N-ε-{ N-α-【SI-CO-（CH
2)
2-CO-(AEEA)
2】-Glu-γ-(AEEA)
3} }-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
Conjugate intermediate one [(SI-CO-(CH
2)
2cOOH) preparation]:
Take SI 90mg and Succinic anhydried 22mg, dissolve with Glacial acetic acid, 50 DEG C of reactions are spent the night;
Then monitor response situation (chloroform: methyl alcohol: acetic acid=5:1:2d) with TLC, reaction conversion ratio reaches after more than 95%, in reaction solution, add ethyl acetate, wash acetic acid with water, after ethyl acetate layer adds anhydrous sodium sulfate drying, suction filtration, collect filtrate, reduced vacuum is condensed into oily, after adding sherwood oil crystallization, filter, collect solid, solid is dried 40 DEG C of air blast, obtains product 100mg, it is 99% that yield about 90%, HPLC detects purity.Obtain conjugate intermediate one;
Conjugate [N-α-[SI-CO-(CH
2)
2-CO-(AEEA)
2]-Glu-γ-(AEEA)
3] preparation:
2-CTC resin (substitution degree 0.25mmol/g) 265mg DCM washing 2-3 time, swelling with DCM;
Take Fmoc-AEEA 0.2mmol, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA 0.27mmol, activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add activation solution (the Fmoc-AEEA 0.2mmol of Fmoc-AEEA, HOBt 0.22mmol and DIC 0.22mmol, solvent is DMF, 0-5 DEG C activates 5 minutes) in reaction column, in 25-30 DEG C of reaction 2.5h, sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, coupling Fmoc-AEEA, Fmoc-Glu-otBu, two Fmoc-AEEA in order, the intermediate one that last coupling is above-mentioned; Cut down from resin by product, obtain 65mg after obtaining conjugate forced air drying, HPLC detects about 95%, yield about 70%;
The preparation of main chain:
2-CTC resin (substitution degree 0.25mmol/g) 120mg, with DCM washing 2-3 time, swelling with DCM;
Take the amino acid 0.09mmol that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add amino acid whose activation solution (the protected amino acid 0.09mmol that N holds Fmoc protection; HOBt 0.1mmol and DIC 0.1mmol; solvent is DMF; 0-5 DEG C activates 5 minutes) in reaction column; in 25-30 DEG C of reaction 2.5h; sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, hold coupling protected amino acid one by one to hold (involved protected amino acid has Fmoc-Gly, Fmoc-Arg(pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc to N according to the order of main chain from C), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(otBu), Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Asp(otBu), Fmoc-Thr(tBu), Fmoc-His(Trt));
After completing, resin TFA cracking, cuts down polypeptide from resin, and takes off all Side chain protective groups, ether sedimentation, obtains the thick peptide of main chain of cracking, dries up rear detection purity about 56%, yield about 50% with nitrogen;
The synthesis of conjugate and main chain:
Taking thick peptide is dissolved in N-Methyl pyrrolidone, adds triethylamine, and adjustment pH is 9-11, and conjugate is beforehand with Viability ester, joins in reaction system, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects.
The purifying of product:
Use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, collects cut, freeze-drying obtains the finished product 12mg, HPLC about 96%, total recovery about 8%, MALDI-TOF-TOF mass spectroscopy molecular amount is: 4714.74.
the synthesis of embodiment two: GLP-1 derivative
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-（CH
2)
2-CO-(AEEA)
2】-Glu-γ-【(AEEA)
2-NH-（CH
2)
2-NH-】}}-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile- Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly
Conjugate intermediate one [(SI-CO-(CH
2)
2cOOH) preparation]:
Take SI 90mg and Succinic anhydried 22mg, dissolve with Glacial acetic acid, 50 DEG C of reactions are spent the night;
Then monitor response situation (chloroform: methyl alcohol: acetic acid=5:1:2d) with TLC, reaction conversion ratio reaches after more than 95%, in reaction solution, add ethyl acetate, wash acetic acid with water, after ethyl acetate layer adds anhydrous sodium sulfate drying, suction filtration, collect filtrate, reduced vacuum is condensed into oily, after adding sherwood oil crystallization, filter, collect solid, solid is dried 40 DEG C of air blast, obtains product 100mg, it is 99% that yield about 90%, HPLC detects purity.Obtain conjugate intermediate one;
Conjugate [N-α-[SI-CO-(CH
2)
2-CO-(AEEA)
2]-Glu-γ-(AEEA)
2] preparation:
2-CTC resin (substitution degree 0.25mmol/g) 265mg DCM washing 2-3 time, swelling with DCM;
Take Fmoc-AEEA 0.2mmol, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA 0.27mmol, activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add activation solution (the Fmoc-AEEA 0.2mmol of Fmoc-AEEA, HOBt 0.22mmol and DIC 0.22mmol, solvent is DMF, 0-5 DEG C activates 5 minutes) in reaction column, in 25-30 DEG C of reaction 2.5h, sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, coupling Fmoc-Glu-otBu, two Fmoc-AEEA in order, the intermediate one that last coupling is above-mentioned; Cut down from resin by product, obtain 54mg after obtaining conjugate forced air drying, HPLC detects about 95%, yield about 69%;
The preparation of main chain:
2-CTC resin (substitution degree 0.25mmol/g) 120mg, with DCM washing 2-3 time, swelling with DCM;
Take the amino acid 0.09mmol that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add amino acid whose activation solution (the protected amino acid 0.09mmol that N holds Fmoc protection; HOBt 0.1mmol and DIC 0.1mmol; solvent is DMF; 0-5 DEG C activates 5 minutes) in reaction column; in 25-30 DEG C of reaction 2.5h; sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, hold coupling protected amino acid one by one to hold (involved protected amino acid has Fmoc-Gly, Fmoc-Arg(pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc to N according to the order of main chain from C), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(otBu), Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Asp(OBzl), Fmoc-Thr(tBu), Fmoc-His(Trt));
Obtained the thick peptide protected after coupling completes by cutting, through hydrogenolysis remove after benzyl with Boc-reacting ethylenediamine, carry out cracking after having reacted and obtain thick peptide product nitrogen and dry up rear detection purity about 55%, yield about 50%; .
The synthesis of conjugate and main chain:
Taking thick peptide is dissolved in N-Methyl pyrrolidone, adds triethylamine, and adjustment pH is 9-11, and conjugate is beforehand with Viability ester, joins in reaction system, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects.
The purifying of product:
Use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, collects cut, freeze-drying obtains the finished product 10.5mg, HPLC about 96%, total recovery about 7.6%, MALDI-TOF-TOF mass spectroscopy molecular amount is: 4625.73.
the synthesis of embodiment three: GLP-1 derivative
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-(N-α-【SI-CO-(CH
2)
2-CO-NH-(CH
2)
15-CO-】-L-γ-glutamyl) }-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg- Gly-Arg-Gly
Conjugate intermediate one [(SI-CO-(CH
2)
2cOOH) preparation]:
Take SI 90mg and Succinic anhydried 22mg, dissolve with Glacial acetic acid, 50 DEG C of reactions are spent the night;
Then monitor response situation (chloroform: methyl alcohol: acetic acid=5:1:2d) with TLC, reaction conversion ratio reaches after more than 95%, in reaction solution, add ethyl acetate, wash acetic acid with water, after ethyl acetate layer adds anhydrous sodium sulfate drying, suction filtration, collect filtrate, reduced vacuum is condensed into oily, after adding sherwood oil crystallization, filter, collect solid, solid is dried 40 DEG C of air blast, obtains product 100mg, it is 99% that yield about 90%, HPLC detects purity.Obtain conjugate intermediate one;
Conjugate [N-α-[SI-CO-(CH
2)
2-CO-NH-(CH
2)
15-CO-] L-γ-Glu] preparation:
2-CTC resin (substitution degree 0.25mmol/g) 265mg DCM washing 2-3 time, swelling with DCM;
Take Fmoc-Glu-otBu 0.2mmol, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA 0.27mmol, activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add activation solution (the Fmoc-16-aminohexadecanoic acid 0.2mmol of Fmoc-16-aminohexadecanoic acid, HOBt 0.22mmol and DIC 0.22mmol, solvent is DMF and NMP, 0-5 DEG C activates 5 minutes) in reaction column, in 25-30 DEG C of reaction 2.5h, sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, the intermediate one that coupling is above-mentioned; Cut down from resin by product, obtain 40mg after obtaining conjugate forced air drying, HPLC detects about 95%, yield about 65%;
The preparation of main chain:
2-CTC resin (substitution degree 0.25mmol/g) 120mg, with DCM washing 2-3 time, swelling with DCM;
Take the amino acid 0.09mmol that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add amino acid whose activation solution (the protected amino acid 0.09mmol that N holds Fmoc protection; HOBt 0.1mmol and DIC 0.1mmol; solvent is DMF; 0-5 DEG C activates 5 minutes) in reaction column; in 25-30 DEG C of reaction 2.5h; sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, hold coupling protected amino acid one by one to hold (involved protected amino acid has Fmoc-Gly, Fmoc-Arg(pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc to N according to the order of main chain from C), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(otBu), Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Asp(OtBu), Fmoc-Thr(tBu), Fmoc-His(Trt));
After completing, resin TFA cracking, cuts down polypeptide from resin, and takes off all Side chain protective groups, ether sedimentation, obtains the thick peptide of main chain of cracking, dries up rear detection purity about 53%, yield about 48% with nitrogen;
The synthesis of conjugate and main chain:
Taking thick peptide is dissolved in N-Methyl pyrrolidone, adds triethylamine, and adjustment pH is 9-11, and conjugate is beforehand with Viability ester, joins in reaction system, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects.
The purifying of product:
Use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, collects cut, freeze-drying obtains the finished product 11mg, HPLC about 96%, total recovery about 8.5%, MALDI-TOF-TOF mass spectroscopy molecular amount is: 4311.6.
the synthesis of embodiment four: GLP-1 derivative
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-CO-(CH
2)
2-CO- NH-(CH
2)
15-CO-】Glu-γ-【-NH-（CH
2)
2-NH-】}}-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val- Arg-Gly-Arg-Gly
Conjugate intermediate one [(SI-CO-(CH
2)
2cOOH) preparation]:
Take SI 90mg and Succinic anhydried 22mg, dissolve with Glacial acetic acid, 50 DEG C of reactions are spent the night;
Then monitor response situation (chloroform: methyl alcohol: acetic acid=5:1:2d) with TLC, reaction conversion ratio reaches after more than 95%, in reaction solution, add ethyl acetate, wash acetic acid with water, after ethyl acetate layer adds anhydrous sodium sulfate drying, suction filtration, collect filtrate, reduced vacuum is condensed into oily, after adding sherwood oil crystallization, filter, collect solid, solid is dried 40 DEG C of air blast, obtains product 100mg, it is 99% that yield about 90%, HPLC detects purity.Obtain conjugate intermediate one;
Conjugate [N-α-[SI-CO-(CH
2)
2-CO-NH-(CH
2)
15-CO-] L-γ-Glu] preparation:
2-CTC resin (substitution degree 0.25mmol/g) 265mg DCM washing 2-3 time, swelling with DCM;
Take Fmoc-Glu-otBu 0.2mmol, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA 0.27mmol, activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add activation solution (the Fmoc-16-aminohexadecanoic acid 0.2mmol of Fmoc-16-aminohexadecanoic acid, HOBt 0.22mmol and DIC 0.22mmol, solvent is DMF and NMP, 0-5 DEG C activates 5 minutes) in reaction column, in 25-30 DEG C of reaction 2.5h, sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, the intermediate one that coupling is above-mentioned; Cut down from resin by product, obtain 41mg after obtaining conjugate forced air drying, HPLC detects about 95%, yield about 66%;
The preparation of main chain:
2-CTC resin (substitution degree 0.25mmol/g) 120mg, with DCM washing 2-3 time, swelling with DCM;
Take the amino acid 0.09mmol that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add amino acid whose activation solution (the protected amino acid 0.09mmol that N holds Fmoc protection; HOBt 0.1mmol and DIC 0.1mmol; solvent is DMF; 0-5 DEG C activates 5 minutes) in reaction column; in 25-30 DEG C of reaction 2.5h; sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, hold coupling protected amino acid one by one to hold (involved protected amino acid has Fmoc-Gly, Fmoc-Arg(pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc to N according to the order of main chain from C), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(otBu), Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Asp(OBzl), Fmoc-Thr(tBu), Fmoc-His(Trt));
Obtained the thick peptide protected after coupling completes by cutting, through hydrogenolysis remove after benzyl with Boc-reacting ethylenediamine, carry out cracking after having reacted and obtain thick peptide product nitrogen and dry up rear detection purity about 55%, yield about 51%; .
The synthesis of conjugate and main chain:
Taking thick peptide is dissolved in N-Methyl pyrrolidone, adds triethylamine, and adjustment pH is 9-11, and conjugate is beforehand with Viability ester, joins in reaction system, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects.
The purifying of product:
Use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, collects cut, freeze-drying obtains the finished product 11.5mg, HPLC about 96%, total recovery about 8.9%, MALDI-TOF-TOF mass spectroscopy molecular amount is: 4296.15.
the synthesis of embodiment five: GLP-1 derivative
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys{N-ε-(N-α-【SI- (CH
2)
3-CO- NH-(CH
2)
15-CO-】L-γ-glutamyl) }-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly- Arg-Gly
Conjugate intermediate one [(SI-(CH
2)
3cOOH) preparation]:
Reacted by 4-bromo-butyric acid 66.8mg and cylite 68.4mg and generate benzyl ester, then with SI 138mg, substitution reaction occurs, hydrogenolysis removes benzyl, obtains intermediate one about 115mg, yield 49.5%;
Conjugate [N-α-[SI-(CH
2)
3-CO-NH-(CH
2)
15-CO-]-L-γ-Glu] preparation:
2-CTC resin (substitution degree 0.25mmol/g) 265mg DCM washing 2-3 time, swelling with DCM;
Take Fmoc-Glu-otBu 0.2mmol, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA 0.27mmol, activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add activation solution (the Fmoc-16-aminohexadecanoic acid 0.2mmol of Fmoc-16-aminohexadecanoic acid, HOBt 0.22mmol and DIC 0.22mmol, solvent is DMF and NMP, 0-5 DEG C activates 5 minutes) in reaction column, in 25-30 DEG C of reaction 2.5h, sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, the intermediate one that coupling is above-mentioned; Cut down from resin by product, obtain 42mg after obtaining conjugate forced air drying, HPLC detects about 95%, yield about 66%;
The preparation of main chain:
2-CTC resin (substitution degree 0.25mmol/g) 120mg, with DCM washing 2-3 time, swelling with DCM;
Take the amino acid 0.09mmol that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add amino acid whose activation solution (the protected amino acid 0.09mmol that N holds Fmoc protection; HOBt 0.1mmol and DIC 0.1mmol; solvent is DMF; 0-5 DEG C activates 5 minutes) in reaction column; in 25-30 DEG C of reaction 2.5h; sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, hold coupling protected amino acid one by one to hold (involved protected amino acid has Fmoc-Gly, Fmoc-Arg(pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc to N according to the order of main chain from C), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(otBu), Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Asp(OtBu), Fmoc-Thr(tBu), Fmoc-His(Trt));
After completing, resin TFA cracking, cuts down polypeptide from resin, and takes off all Side chain protective groups, ether sedimentation, obtains the thick peptide of main chain of cracking, dries up rear detection purity about 56%, yield about 50% with nitrogen;
The synthesis of conjugate and main chain:
Taking thick peptide is dissolved in N-Methyl pyrrolidone, adds triethylamine, and adjustment pH is 9-11, and conjugate is beforehand with Viability ester, joins in reaction system, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects.
The purifying of product:
Use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, collects cut, freeze-drying obtains the finished product 12.5mg, HPLC about 96%, total recovery about 9.8%, MALDI-TOF-TOF mass spectroscopy molecular amount is: 4241.15.
the synthesis of embodiment six: GLP-1 derivative
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp{C-β-{N-α-【SI-(CH
2)
3-CO- NH-(CH
2)
15-CO-】Glu-γ-【- NH-（CH
2)
2-NH-】}}-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu- Val-Arg-Gly-Arg-Gly
Conjugate intermediate one [(SI-(CH
2)
3cOOH) preparation]:
Reacted by 4-bromo-butyric acid 66.8mg and cylite 68.4mg and generate benzyl ester, then with SI 138mg, substitution reaction occurs, hydrogenolysis removes benzyl, obtains intermediate one about 114mg, yield about 49%;
Conjugate [N-α-[SI-(CH
2)
3-CO-NH-(CH
2)
15-CO-] L-γ-Glu] preparation:
2-CTC resin (substitution degree 0.25mmol/g) 265mg DCM washing 2-3 time, swelling with DCM;
Take Fmoc-Glu-otBu 0.2mmol, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA 0.27mmol, activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add activation solution (the Fmoc-16-aminohexadecanoic acid 0.2mmol of Fmoc-16-aminohexadecanoic acid, HOBt 0.22mmol and DIC 0.22mmol, solvent is DMF and NMP, 0-5 DEG C activates 5 minutes) in reaction column, in 25-30 DEG C of reaction 2.5h, sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, the intermediate one that coupling is above-mentioned; Cut down from resin by product, obtain 40mg after obtaining conjugate forced air drying, HPLC detects about 95%, yield about 64%;
The preparation of main chain:
2-CTC resin (substitution degree 0.25mmol/g) 120mg, with DCM washing 2-3 time, swelling with DCM;
Take the amino acid 0.09mmol that N holds Fmoc protection, dissolve (amino acid concentration is 0.3mol/L) with DCM, add DIEA (3eq), activate 5 minutes, add reaction column, in 25-30 DEG C of reaction 20-22h;
Fmoc protecting group is removed 20 minutes with the mixed solution (DBLK) that piperidines and DMF volume ratio are 1:4;
Add amino acid whose activation solution (the protected amino acid 0.09mmol that N holds Fmoc protection; HOBt 0.1mmol and DIC 0.1mmol; solvent is DMF; 0-5 DEG C activates 5 minutes) in reaction column; in 25-30 DEG C of reaction 2.5h; sampling ninhydrin method detects color of resin, determines whether to arrive reaction end, then washs 6 times with DMF and removes impurity.Repeat the step of deprotection and coupling, hold coupling protected amino acid one by one to hold (involved protected amino acid has Fmoc-Gly, Fmoc-Arg(pbf), Fmoc-Val, Fmoc-Leu, Fmoc-Trp(Boc to N according to the order of main chain from C), Fmoc-Ala, Fmoc-Ile, Fmoc-Phe, Fmoc-Glu(otBu), Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Tyr(tBu), Fmoc-Ser(tBu), Fmoc-Asp(OBzl), Fmoc-Thr(tBu), Fmoc-His(Trt));
Obtained the thick peptide protected after coupling completes by cutting, through hydrogenolysis remove after benzyl with Boc-reacting ethylenediamine, carry out cracking after having reacted and obtain thick peptide product nitrogen and dry up rear detection purity about 53%, yield about 50%; .
The synthesis of conjugate and main chain:
Taking thick peptide is dissolved in N-Methyl pyrrolidone, adds triethylamine, and adjustment pH is 9-11, and conjugate is beforehand with Viability ester, joins in reaction system, room temperature reaction 20min, and add water termination reaction, and tune pH is 7-8, obtains the thick peptide that conjugate connects.
The purifying of product:
Use C18 chromatographic column, with the 0.1%TFA aqueous solution for mobile phase A, acetonitrile is Mobile phase B, linear gradient elution, gradient is that the volume ratio of Mobile phase B in 30min rises to 70% from 30%, collects cut, freeze-drying obtains the finished product 10.5mg, HPLC about 96%, total recovery about 8.2%, MALDI-TOF-TOF mass spectroscopy molecular amount is: 4283.15.
Claims (5)
1. a GLP-1 derivative, is characterized in that: in the N Amino End Group in described derivative, side-chain amino group, side chain carboxyl group, C end carboxyl, histidine side chains imidazolyl, at least one and a kind of conjugate with formula I are puted together;
K1-K2 (Ⅰ)
Wherein K1 is the modification group comprising DPP-4 enzyme inhibitors, and K2 is spacer, and K2 end and main chain are puted together.
2. a kind of GLP-1 derivative as claimed in claim 1, is characterized in that: K1 is selected from:
In one;
Wherein, Z-NH
2for DPP-4 enzyme inhibitors;
N is the integer of 0-14;
X is C, N or O;
Y is O or S.
3. a kind of GLP-1 derivative as claimed in claim 2, is characterized in that: R is selected from:
In one.
4. a kind of GLP-1 derivative as claimed in claim 1, is characterized in that: the compound of K2 described by following general formula:
（AEEA）
a-Xaa7- Xaa8 -Xaa9 -（AEEA）
b
AEEA{ [2-(2-amino) oxyethyl group] ethoxyacetic acid } structure is
A is the integer of 0-8;
B is the integer of 0-6;
Xaa7 is selected from: Glu, Gln, Phe, Tyr or disappearance;
Xaa8 is selected from: Asn, Asp, Thr or disappearance;
Xaa9 is selected from: Lys, Asn, Ser, Cys or disappearance.
5. a kind of GLP-1 derivative as claimed in claim 1, is characterized in that: the compound-(CH of K2 described by following general formula
2)
pcO-Xaa10-, for the acyl group of straight chain or branched fatty acid is connected with amino-acid residue
Wherein, p is the integer of 4-30;
Xaa10 is selected from: Glu, Asp, Gln or disappearance.
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| CN201410322342 | 2014-07-08 | ||
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ID=54299298
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115806593A (en) * | 2022-09-23 | 2023-03-17 | 成都普康生物科技有限公司 | Fatty acid chain modified double-receptor agonist and application thereof |
| CN116848243A (en) * | 2022-03-30 | 2023-10-03 | 北京质肽生物医药科技有限公司 | Liquid pharmaceutical compositions of polypeptide conjugates and methods of use thereof |
| WO2023186003A1 (en) * | 2022-03-30 | 2023-10-05 | Beijing Ql Biopharmaceutical Co., Ltd. | Liquid pharmaceutical compositions of polypeptide conjugates and methods of uses thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232470A (en) * | 1996-08-30 | 1999-10-20 | 诺沃挪第克公司 | GLP-1 derivs. |
| WO2009109927A2 (en) * | 2008-03-05 | 2009-09-11 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Glp-1 receptor agonists and related active pharmaceutical ingredients for treatment of cancer |
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2015
- 2015-07-07 CN CN201510393214.4A patent/CN104987383A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1232470A (en) * | 1996-08-30 | 1999-10-20 | 诺沃挪第克公司 | GLP-1 derivs. |
| WO2009109927A2 (en) * | 2008-03-05 | 2009-09-11 | Tel Hashomer Medical Research Infrastructure And Services Ltd. | Glp-1 receptor agonists and related active pharmaceutical ingredients for treatment of cancer |
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| 王泽想等: "胰高血糖素样肽1及其相关制剂的临床应用", 《医学综述》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116848243A (en) * | 2022-03-30 | 2023-10-03 | 北京质肽生物医药科技有限公司 | Liquid pharmaceutical compositions of polypeptide conjugates and methods of use thereof |
| WO2023186003A1 (en) * | 2022-03-30 | 2023-10-05 | Beijing Ql Biopharmaceutical Co., Ltd. | Liquid pharmaceutical compositions of polypeptide conjugates and methods of uses thereof |
| CN116848243B (en) * | 2022-03-30 | 2024-03-19 | 北京质肽生物医药科技有限公司 | Liquid pharmaceutical compositions of polypeptide conjugates and methods of use thereof |
| CN115806593A (en) * | 2022-09-23 | 2023-03-17 | 成都普康生物科技有限公司 | Fatty acid chain modified double-receptor agonist and application thereof |
| CN115806593B (en) * | 2022-09-23 | 2023-09-05 | 成都普康生物科技有限公司 | Fatty acid chain modified dual-receptor agonist and application thereof |
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