WO2022262825A1 - 含内酰胺桥的多肽化合物 - Google Patents
含内酰胺桥的多肽化合物 Download PDFInfo
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- 238000006798 ring closing metathesis reaction Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 239000011975 tartaric acid Substances 0.000 description 1
- 235000002906 tartaric acid Nutrition 0.000 description 1
- ISIJQEHRDSCQIU-UHFFFAOYSA-N tert-butyl 2,7-diazaspiro[4.5]decane-7-carboxylate Chemical class C1N(C(=O)OC(C)(C)C)CCCC11CNCC1 ISIJQEHRDSCQIU-UHFFFAOYSA-N 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
- UGNWTBMOAKPKBL-UHFFFAOYSA-N tetrachloro-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(Cl)=C(Cl)C1=O UGNWTBMOAKPKBL-UHFFFAOYSA-N 0.000 description 1
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- 239000013559 triple agonist Substances 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/26—Glucagons
-
- 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
-
- 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
Definitions
- the present invention relates to a class of polypeptide compounds containing lactam bridges and their application in the preparation of medicines for treating related diseases, in particular to the polypeptides of the sequence shown in formula (I).
- CVD cardiovascular disease
- GLP-1 protects islet ⁇ cells in islets, stimulates islet ⁇ cells to release insulin in a glucose-dependent manner, and effectively controls postprandial blood sugar. Because of its unique mechanism of action, the risk of hypoglycemia is greatly reduced. Although GLP-1R agonists have demonstrated excellent hypoglycemic effects in clinical practice, there are still many type 2 diabetics who have not achieved their hypoglycemic and weight loss goals. Therefore, combining GLP-1R agonists with other hypoglycemic targets such as GIP and GCG is an urgent and promising need.
- GIP is a polypeptide secreted by neuroendocrine K cells of the small intestine. Its physiological effects are mediated by GIPR, mainly non-glucose-dependent stimulation of insulin secretion, enhancement of glucagon secretion, and enhancement of lipid metabolism. Although the beneficial effects of GIPR agonists appear to be attenuated in hyperglycemic symptoms in patients with type 2 diabetes, studies have shown that the diminished insulinotropic effect of GIP can be fully restored after a period of normalization of plasma glucose levels.
- Glucagon is secreted by the pancreas and binds to the glucagon receptor (GCGR) to produce a hormone with physiological functions.
- Glucagon promotes the rise of blood sugar by increasing gluconeogenesis and glycogenolysis.
- GCG can also reduce fatty acid synthesis in liver adipose tissue and promote fat decomposition.
- the introduction of GCG agonistic activity into the drug can be more beneficial for patients to further control their weight on the basis of hypoglycemia. Therefore, GLP-1/GIP/GCG triple agonists will have a synergistic effect on the treatment of diabetes, obesity and related diseases.
- the present invention provides the polypeptide (SEQ ID NO:7) of the sequence shown in formula (I),
- a lactam bridge is formed between amino acids at positions 17 and 20 or between amino acids at positions 20 and 24; wherein,
- K 0 represents lysine, and the amino group on the lysine side chain is linked to -X 0 ;
- p 1, 2 or 3;
- s 1 or 2;
- t 8, 9 or 10.
- the above-mentioned polypeptide has the following sequence SEQ ID NO: 1-6,
- SEQ ID NO: 1 YAibEGT FTSDY SIAibLD KK 0 AQK AFVEW LIAGG PSSG-NH 2 ;
- SEQ ID NO: 2 YAibEGT FTSDY SIAibLD KK 0 AQK EFVEW LIAGG PSSG-NH 2 ;
- SEQ ID NO: 3 YAibEGT FTSDY SIAibLD KK 0 AQK AVIEW LIAGG PSSG-NH 2 ;
- SEQ ID NO: 4 YAibEGT FTSDY SIAibLD KEAQK AFVK 0 W LIAGG PSSG-NH 2 ;
- SEQ ID NO: 5 YAibEGT FTSDY SIAibLD KEAQK EFVK 0 W LIAGG PSSG-NH 2 ;
- SEQ ID NO: 6 YAibEGT FTSDY SIAibLD KEAQK AFIK 0 W LIAGG PSSG-NH 2 ;
- a lactam bridge is formed between the amino acids at positions 17 and 20 or between the amino acids at positions 20 and 24;
- Aib and K 0 are as defined in the present invention, and other variables are as defined in the present invention.
- the above-X is selected from
- the above-X is selected from
- the present invention also provides a polypeptide represented by the following formula,
- the present invention also provides a polypeptide represented by the following formula,
- the present invention also provides the above-mentioned pharmaceutical composition, which comprises, as an active ingredient, a therapeutically effective amount of the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.
- the above-mentioned polypeptide compound or a pharmaceutically acceptable salt thereof or the above-mentioned composition is used in the preparation of a drug for treating diabetes.
- the compound of the present invention has strong agonistic activity on GLP-1R/GIPR/GCGR.
- pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms, which are suitable for use in contact with human and animal tissues within the scope of sound medical judgment , without undue toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit/risk ratio.
- pharmaceutically acceptable salt refers to a salt of a compound of the present invention, which is prepared from a compound having a specific substituent found in the present invention and a relatively non-toxic acid or base.
- base addition salts can be obtained by contacting such compounds with a sufficient amount of base, either neat solution or in a suitable inert solvent.
- Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine or magnesium salts or similar salts.
- acid addition salts can be obtained by contacting such compounds with a sufficient amount of the acid, either neat or in a suitable inert solvent.
- Examples of pharmaceutically acceptable acid addition salts include salts of inorganic acids including, for example, hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, Hydrogen sulfate, hydriodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, Fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid and methanesulfonic acid and similar acids; also salts of amino acids such as arginine and the like , and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain basic and acidic functional groups and can thus be converted into
- the pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound containing acid groups or bases by conventional chemical methods.
- such salts are prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent or a mixture of both.
- amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids.
- Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine.
- Amino acid analogs are compounds that have the same basic chemical structure (such as the alpha carbon bound to a hydrogen, carboxyl group, amino group, and R group) as a naturally occurring amino acid, such as homoserine, norleucine, formazine Thionine sulfoxide, methionine methylsulfonium.
- Such analogs can have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
- Amino acid mimetics are chemical compounds whose structure differs from the general chemical structure of amino acids, but which function similarly to naturally occurring amino acids.
- a or Ala described herein represents alanine, and the structure is R or Arg means arginine, the structure is N or Asn means asparagine, the structure is D or Asp means aspartic acid, the structure is C or Cys means cysteine, the structure is Q or Gln means glutamine, the structure is E or Glu means glutamic acid, the structure is G or Gly means glycine, the structure is H or His means histidine, the structure is I or Ile means isoleucine, the structure is L or Leu means leucine, the structure is K or Lys means lysine, the structure is M or Met means methionine, the structure is F or Phe means phenylalanine, the structure is P or Pro means proline, the structure is S or Ser represents serine, the structure is T or Thr means threonine, the structure is W or Trp means tryptophan, the structure is Y or Tyr means tyrosine, the structure is V or Val represents valine, the structure is
- treating includes inhibiting, slowing, stopping or reversing the progression or severity of an existing symptom or condition.
- the compounds of the invention may exist in particular geometric or stereoisomeric forms.
- the present invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers isomers, (D)-isomers, (L)-isomers, and their racemic and other mixtures, such as enantiomerically or diastereomerically enriched mixtures, all of which are subject to the present within the scope of the invention.
- Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers, as well as mixtures thereof, are included within the scope of the present invention.
- enantiomer or “optical isomer” refer to stereoisomers that are mirror images of each other.
- cis-trans isomers or “geometric isomers” arise from the inability to rotate freely due to the double bond or the single bond of the carbon atoms forming the ring.
- diastereoisomer refers to stereoisomers whose molecules have two or more chiral centers and which are not mirror images of each other.
- keys with wedge-shaped solid lines and dotted wedge keys Indicates the absolute configuration of a stereocenter, with a straight solid-line bond and straight dashed keys Indicates the relative configuration of the stereocenter, with a wavy line Indicates wedge-shaped solid-line bond or dotted wedge key or with tilde Indicates a straight solid line key or straight dotted key
- the terms “enriched in an isomer”, “enriched in an isomer”, “enriched in an enantiomer” or “enantiomerically enriched” refer to one of the isomers or enantiomers
- the content of the enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or Greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.
- the terms “isomer excess” or “enantiomeric excess” refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if the content of one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomeric excess (ee value) is 80% .
- Optically active (R)- and (S)-isomers as well as D and L-isomers can be prepared by chiral synthesis or chiral reagents or other conventional techniques. If one enantiomer of a compound of the invention is desired, it can be prepared by asymmetric synthesis or derivatization with chiral auxiliary agents, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide pure desired enantiomer.
- a diastereoisomeric salt is formed with an appropriate optically active acid or base, and then a diastereomeric salt is formed by a conventional method known in the art. Diastereomeric resolution is performed and the pure enantiomers are recovered. Furthermore, the separation of enantiomers and diastereomers is usually accomplished by the use of chromatography using chiral stationary phases, optionally in combination with chemical derivatization methods (e.g. amines to amino groups formate).
- the compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compounds.
- compounds may be labeled with radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
- radioactive isotopes such as tritium ( 3 H), iodine-125 ( 125 I) or C-14 ( 14 C).
- heavy hydrogen can be used to replace hydrogen to form deuterated drugs.
- the bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon.
- deuterated drugs can reduce toxic side effects and increase drug stability. , enhance the efficacy, prolong the biological half-life of drugs and other advantages. All changes in isotopic composition of the compounds of the invention, whether radioactive or not, are included within the scope of the invention.
- linking group listed does not indicate its linking direction
- its linking direction is arbitrary, for example,
- the connecting group L in the middle is -MW-, at this time -MW- can connect ring A and ring B in the same direction as the reading order from left to right to form It can also be formed by connecting loop A and loop B in the opposite direction to the reading order from left to right
- any one or more sites of the group can be linked to other groups through chemical bonds.
- connection method of the chemical bond is not positioned, and there is an H atom at the connectable site, when the chemical bond is connected, the number of H atoms at the site will decrease correspondingly with the number of chemical bonds connected to become the corresponding valence group.
- the chemical bonds that the site connects with other groups can use straight solid line bonds Straight dotted key or tilde express.
- the straight-shaped solid-line bond in -OCH3 indicates that it is connected to other groups through the oxygen atom in the group;
- the straight dotted line bond in indicates that the two ends of the nitrogen atom in the group are connected to other groups;
- the wavy lines in indicate that the 1 and 2 carbon atoms in the phenyl group are connected to other groups.
- the structure of the compounds of the present invention can be confirmed by conventional methods known to those skilled in the art. If the present invention involves the absolute configuration of the compound, the absolute configuration can be confirmed by conventional technical means in the art. For example, in single crystal X-ray diffraction (SXRD), the cultured single crystal is collected with a Bruker D8 venture diffractometer to collect diffraction intensity data, the light source is CuK ⁇ radiation, and the scanning method is: After scanning and collecting relevant data, the absolute configuration can be confirmed by further analyzing the crystal structure by direct method (Shelxs97).
- SXRD single crystal X-ray diffraction
- the compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, the embodiments formed by combining them with other chemical synthesis methods, and the methods well known to those skilled in the art Equivalent alternatives, preferred embodiments include but are not limited to the examples of the present invention.
- the solvent used in the present invention is commercially available.
- the dried peptide resin was added to the prepared cutting solution, shaken on a shaker for 2.5 hours, filtered, and the filtrate was added to 10 times the volume of ice isopropyl ether, centrifuged, and washed 3 times with isopropyl ether.
- the crude peptide was obtained by drying in vacuo for 2 h and purified.
- the molecular weight of the polypeptide was confirmed by ESI-MS, the calculated value was 4361.9, and the detected value was 4362.0.
- Example 1 In vitro GLP-1R/GIPR/GCGR agonistic activity test
- the cell line was constructed by Shanghai WuXi PharmaTech. See the table below for details.
- the compound to be tested is diluted 4 times at 10 points, the initial concentration is 30 ⁇ M, and Bravo completes the dilution b) Transfer the compound:
- the compound of the present invention has strong agonistic activity on GLP-1R/GIPR/GCGR.
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Abstract
提供了一类含内酰胺桥的多肽化合物,及其在制备治疗相关病症的药物中的应用,具体提供了式(I)所示序列的多肽。 YAibEGT FTSDY SIAibLD KKAQK AFVEW LIAGG PSSG 0 (I)
Description
本申请主张如下优先权:
CN202110680696.7,申请日:2021年06月18日;
CN202210138364.0,申请日:2022年02月15日;
CN202210626502.X,申请日:2022年06月02日。
本发明涉及一类含内酰胺桥的多肽化合物,及其在制备治疗相关病症的药物中的应用,具体涉及式(I)所示序列的多肽。
中国预估有1.1亿糖尿病患者,占全世界糖尿病患者的24%。随着经济发展与生活方式的改变,我国糖尿病患病率增加至12.8%(部分省市高达19.9%),这些患者通常并伴有肥胖或心血管疾病(CVD)。
GLP-1在胰岛中发挥保护胰岛β细胞的作用,以葡萄糖依赖的方式刺激胰岛β细胞释放胰岛素,有效控制餐后血糖的作用。因其独特的作用机制,低血糖风险大降低。虽然GLP-1R激动剂在临床中展现了优秀的降糖效果,但还是有很多二型糖尿病人未达到降糖及减重目标。因此,将GLP-1R激动剂与其它降糖靶点结合如GIP及GCG是一个迫切且有前景的需求。
GIP是由小肠的神经内分泌K细胞分泌的多肽。由GIPR介导其生理作用,主要为非葡萄糖依赖的促胰岛素分泌、增强胰高血糖素分泌、增强脂质代谢等。虽然GIPR激动剂的有益作用似乎在2型糖尿病患者的高血糖症状中减弱,但研究表明,GIP减弱的促胰岛素分泌作用可以在血浆葡萄糖水平恢复正常一段时间后完全恢复。
胰高血糖素(GCG)是由胰腺分泌的,与胰高血糖素受体(GCGR)结合产生生理功能的激素。胰高血糖素通过增加糖异生及糖原分解的方式促进血糖的升高。另外,GCG还可以减少肝脏脂肪组织中的脂肪酸合成,促进脂肪的分解。药物中引入GCG激动活性能够更有利于患者在降糖基础上进一步控制体重。因此,GLP-1/GIP/GCG三重激动剂对于治疗糖尿病,肥胖及相关疾病将具有协同作用。
发明内容
本发明提供了式(I)所示序列的多肽(SEQ ID NO:7),
YAibEGT FTSDY SIAibLD KKAQK AFVEW LIAGG PSSG
0
(I)
其具有以下修饰:
1)上述第17位或第24位的氨基酸有且只有一个被替换为K
0;且
2)上述式(I)所示序列的0-2个天然氨基酸被替换;且
3)第17和第20位置的氨基酸之间或第20和第24位置的氨基酸之间形成内酰胺桥;其中,
K
0表示赖氨酸,且该赖氨酸侧链上的氨基与-X
0相连;
p为1、2或3;
s为1或2;
t为8、9或10。
在本发明的一些方案中,上述的多肽,其具有如下序列SEQ ID NO:1-6,
SEQ ID NO:1:YAibEGT FTSDY SIAibLD KK
0AQK AFVEW LIAGG PSSG-NH
2;
SEQ ID NO:2:YAibEGT FTSDY SIAibLD KK
0AQK EFVEW LIAGG PSSG-NH
2;
SEQ ID NO:3:YAibEGT FTSDY SIAibLD KK
0AQK AFIEW LIAGG PSSG-NH
2;
SEQ ID NO:4:YAibEGT FTSDY SIAibLD KEAQK AFVK
0W LIAGG PSSG-NH
2;
SEQ ID NO:5:YAibEGT FTSDY SIAibLD KEAQK EFVK
0W LIAGG PSSG-NH
2;
SEQ ID NO:6:YAibEGT FTSDY SIAibLD KEAQK AFIK
0W LIAGG PSSG-NH
2;
其中,
第17和20位置的氨基酸之间或第20和24位置的的氨基酸之间形成内酰胺桥;
Aib和K
0如本发明定义,其他变量如本发明所定义。
在本发明的一些方案中,上述p为2,其他变量如本发明所定义。
在本发明的一些方案中,上述s为1,其他变量如本发明所定义。
在本发明的一些方案中,上述t为9,其他变量如本发明所定义。
在本发明的一些方案中,上述-X
0选自
在本发明的一些方案中,上述-X
0选自
本发明还有一些方案由上述变量任意组合而来。
本发明还提供了下式所示多肽,
本发明还提供了下式所示多肽,
本发明还提供了上述药物组合物,包括作为活性成分的治疗有效量根据上述的多肽化合物或其药学上可接受的盐以及药学上可接受的载体。
在本发明的一些方案中,上述多肽化合物或其药学上可接受的盐或者上述的组合物在制备治疗糖尿病的药物上的应用。
技术效果
本发明化合物对GLP-1R/GIPR/GCGR具有很强的激动活性。
定义和说明
除非另有说明,本文所用的下列术语和短语旨在具有下列含义。一个特定的术语或短语在没有特别定义的情况下不应该被认为是不确定的或不清楚的,而应该按照普通的含义去理解。当本文中出现商品名时,意在指代其对应的商品或其活性成分。
这里所采用的术语“药学上可接受的”,是针对那些化合物、材料、组合物和/或剂型而言,它们在可靠的医学判断的范围之内,适用于与人类和动物的组织接触使用,而没有过多的毒性、刺激性、过敏性反应或其它问题或并发症,与合理的利益/风险比相称。
术语“药学上可接受的盐”是指本发明化合物的盐,由本发明发现的具有特定取代基的化合物与相对无毒的酸或碱制备。当本发明的化合物中含有相对酸性的功能团时,可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与这类化合物接触的方式获得碱加成盐。药学上可接受的碱加成盐包括钠、钾、钙、铵、有机胺或镁盐或类似的盐。当本发明的化合物中含有相对碱性的官能团时,可以通 过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐,所述无机酸包括例如盐酸、氢溴酸、硝酸、碳酸,碳酸氢根,磷酸、磷酸一氢根、磷酸二氢根、硫酸、硫酸氢根、氢碘酸、亚磷酸等;以及有机酸盐,所述有机酸包括如乙酸、丙酸、异丁酸、马来酸、丙二酸、苯甲酸、琥珀酸、辛二酸、反丁烯二酸、乳酸、扁桃酸、邻苯二甲酸、苯磺酸、对甲苯磺酸、柠檬酸、酒石酸和甲磺酸等类似的酸;还包括氨基酸(如精氨酸等)的盐,以及如葡糖醛酸等有机酸的盐。本发明的某些特定的化合物含有碱性和酸性的官能团,从而可以被转换成任一碱或酸加成盐。
本发明的药学上可接受的盐可由含有酸根或碱基的母体化合物通过常规化学方法合成。一般情况下,这样的盐的制备方法是:在水或有机溶剂或两者的混合物中,经由游离酸或碱形式的这些化合物与化学计量的适当的碱或酸反应来制备。
“氨基酸”是指天然存在的和合成的氨基酸,以及起到与天然存在的氨基酸类似的作用的氨基酸类似物和氨基酸模拟物。天然存在的氨基酸是由遗传密码编码的那些氨基酸,以及后来修饰的那些氨基酸,例如,羟脯氨酸、γ-羧基谷氨酸和O-磷酸丝氨酸。氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构(例如与氢、羧基基团、氨基基团和R基团结合的α碳)的化合物,例如高丝氨酸、正亮氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍。这样的类似物可以具有修饰的R基团(例如,正亮氨酸)或修饰的肽骨架,但保留与天然存在的氨基酸相同的基本化学结构。氨基酸模拟物是指其结构不同于一般的氨基酸化学结构,但起到与天然存在的氨基酸相似的作用的化学化合物。
本文所述的A或Ala表示丙氨酸,结构为
R或Arg表示精氨酸,结构为
N或Asn表示天冬酰胺,结构为
D或Asp表示天冬氨酸,结构为
C或Cys表示半胱氨酸,结构为
Q或Gln表示谷氨酰胺,结构为
E或Glu表示谷氨酸,结构为
G或Gly表示甘氨酸,结构为
H或His表示组氨酸,结构为
I或Ile 表示异亮氨酸,结构为
L或Leu表示亮氨酸,结构为
K或Lys表示赖氨酸,结构为
M或Met表示甲硫氨酸,结构为
F或Phe表示苯丙氨酸,结构为
P或Pro表示脯氨酸,结构为
S或Ser表示丝氨酸,结构为
T或Thr表示苏氨酸,结构为
W或Trp表示色氨酸,结构为
Y或Tyr表示酪氨酸,结构为
V或Val表示缬氨酸,结构为
Fmoc-AEEA-OH表示
术语“治疗”包括抑制、减缓、停止或逆转现有症状或病患的进展或严重程度。
除非另有说明,术语“异构体”意在包括几何异构体、顺反异构体、立体异构体、对映异构体、旋光异构体、非对映异构体和互变异构体。
本发明的化合物可以存在特定的几何或立体异构体形式。本发明设想所有的这类化合物,包括顺式和反式异构体、(-)-和(+)-对映体、(R)-和(S)-对映体、非对映异构体、(D)-异构体、(L)-异构体,及其外消旋混合物和其他混合物,例如对映异构体或非对映体富集的混合物,所有这些混合物都属于本发明的范围之内。烷基等取代基中可存在另外的不对称碳原子。所有这些异构体以及它们的混合物,均包括在本发明的范围之内。
除非另有说明,术语“对映异构体”或者“旋光异构体”是指互为镜像关系的立体异构体。
除非另有说明,术语“顺反异构体”或者“几何异构体”系由因双键或者成环碳原子单键不能自由旋转而引起。
除非另有说明,术语“非对映异构体”是指分子具有两个或多个手性中心,并且分子间为非镜像 的关系的立体异构体。
除非另有说明,“(+)”表示右旋,“(-)”表示左旋,“(±)”表示外消旋。
除非另有说明,用楔形实线键
和楔形虚线键
表示一个立体中心的绝对构型,用直形实线键
和直形虚线键
表示立体中心的相对构型,用波浪线
表示楔形实线键
或楔形虚线键
或用波浪线
表示直形实线键
或直形虚线键
除非另有说明,术语“富含一种异构体”、“异构体富集”、“富含一种对映体”或者“对映体富集”指其中一种异构体或对映体的含量小于100%,并且,该异构体或对映体的含量大于等于60%,或者大于等于70%,或者大于等于80%,或者大于等于90%,或者大于等于95%,或者大于等于96%,或者大于等于97%,或者大于等于98%,或者大于等于99%,或者大于等于99.5%,或者大于等于99.6%,或者大于等于99.7%,或者大于等于99.8%,或者大于等于99.9%。
除非另有说明,术语“异构体过量”或“对映体过量”指两种异构体或两种对映体相对百分数之间的差值。例如,其中一种异构体或对映体的含量为90%,另一种异构体或对映体的含量为10%,则异构体或对映体过量(ee值)为80%。
可以通过的手性合成或手性试剂或者其他常规技术制备光学活性的(R)-和(S)-异构体以及D和L异构体。如果想得到本发明某化合物的一种对映体,可以通过不对称合成或者具有手性助剂的衍生作用来制备,其中将所得非对映体混合物分离,并且辅助基团裂开以提供纯的所需对映异构体。或者,当分子中含有碱性官能团(如氨基)或酸性官能团(如羧基)时,与适当的光学活性的酸或碱形成非对映异构体的盐,然后通过本领域所公知的常规方法进行非对映异构体拆分,然后回收得到纯的对映体。此外,对映异构体和非对映异构体的分离通常是通过使用色谱法完成的,所述色谱法采用手性固定相,并任选地与化学衍生法相结合(例如由胺生成氨基甲酸盐)。
本发明的化合物可以在一个或多个构成该化合物的原子上包含非天然比例的原子同位素。例如,可用放射性同位素标记化合物,比如氚(
3H),碘-125(
125I)或C-14(
14C)。又例如,可用重氢取代氢形成氘代药物,氘与碳构成的键比普通氢与碳构成的键更坚固,相比于未氘化药物,氘代药物有降低毒副作用、增加药物稳定性、增强疗效、延长药物生物半衰期等优势。本发明的化合物的所有同位素组成的变换,无论放射性与否,都包括在本发明的范围之内。
当所列举的连接基团没有指明其连接方向,其连接方向是任意的,例如,
中连接基团L为-M-W-,此时-M-W-既可以按与从左往右的读取顺序相同的方向连接环A和环B构成
也可以按照与从左往右的读取顺序相反的方向连接环A和环B构成
所述连接基团、取代基和/或其变体的组合只有在这样的组合会产生稳定的化合物的情况下才是被允许的。
除非另有规定,当某一基团具有一个或多个可连接位点时,该基团的任意一个或多个位点可以通过化学键与其他基团相连。当该化学键的连接方式是不定位的,且可连接位点存在H原子时,则连接化学键时,该位点的H原子的个数会随所连接化学键的个数而对应减少变成相应价数的基团。所述位点与其他基团连接的化学键可以用直形实线键
直形虚线键
或波浪线
表示。例如-OCH
3中的直形实线键表示通过该基团中的氧原子与其他基团相连;
中的直形虚线键表示通过该基团中的氮原子的两端与其他基团相连;
中的波浪线表示通过该苯基基团中的1和2位碳原子与其他基团相连。
本发明的化合物可以通过本领域技术人员所熟知的常规方法来确认结构,如果本发明涉及化合物的绝对构型,则该绝对构型可以通过本领域常规技术手段予以确证。例如单晶X射线衍射法(SXRD),把培养出的单晶用Bruker D8 venture衍射仪收集衍射强度数据,光源为CuKα辐射,扫描方式:
扫描,收集相关数据后,进一步采用直接法(Shelxs97)解析晶体结构,便可以确证绝对构型。
本发明的化合物可以通过本领域技术人员所熟知的多种合成方法来制备,包括下面列举的具体实施方式、其与其他化学合成方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式,优选的实施方式包括但不限于本发明的实施例。
本发明所使用的溶剂可经市售获得。
本发明采用下述缩略词:aq代表水;eq代表当量、等量;DCM代表二氯甲烷;PE代表石油醚;DMSO代表二甲亚砜;MeOH代表甲醇;BOC代表叔丁氧羰基是一种胺保护基团;r.t.代表室温;O/N代表过夜;THF代表四氢呋喃;Boc
2O代表二叔丁基二碳酸酯;TFA代表三氟乙酸;DIEA代表二异丙基乙基胺;DMF代表N,N-二甲基甲酰胺;HBTU代表苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯;HOBT代表1-羟基苯并三唑;DIC代表N,N'-二异丙基碳二亚胺;PhSiH
3代表苯硅烷;Pd(PPh
3)
4代表四三苯基膦钯。
下面通过实施例对本发明进行详细描述,但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明,其中也公开了其具体实施例方式,对本领域的技术人员而言,在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1
1.称取1.10g Rink Amide MBHA Resin(替代度Sub=0.28mmol/g),加入到反应柱中,再加入DMF(20.0mL)至反应柱氮气鼓气体2h,排废,直至没有液体流出,加入DMF(50mL)洗涤5次每次1min,排废,直至没有液体流出。
2.加入20%的哌啶/DMF(20.0mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
3.氨基酸的偶联
3.1 Fmoc-Gly-OH的偶联
1.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
2.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
3.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.2 Fmoc-Ser(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.3 Fmoc-Ser(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。四氯苯醌检测,树脂蓝色。
2.称取Fmoc-Ser(tBu)-OH(3.0eq)入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次,每次1min,排废,直至没有液体流出。
3.4 Fmoc-Pro-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Pro-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.5 Fmoc-Gly-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。四氯苯醌检测,树脂蓝色。
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,四氯苯醌检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.6 Fmoc-Gly-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.7 Fmoc-Ala-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ala-OH(3.0eq)加入到上述树脂中,加入HOBT(3.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入DIC(3.00eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应过夜,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.8 Fmoc-Ile-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ile-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.9 Fmoc-Leu-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Leu-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.10 Fmoc-Trp(Boc)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。 加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。四氯苯醌检测,树脂蓝色。
2.称取Fmoc-Trp(Boc)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.11 Fmoc-Glu(OAll)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Glu(OAll)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.12 Fmoc-Val-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Val-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.13 Fmoc-Phe-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Phe-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.14 Fmoc-Ala-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ala-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.15 Fmoc-Lys(Alloc)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Lys(Alloc)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.16 Fmoc-Gln(Trt)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Gln(Trt)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.17 Fmoc-Ala-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ala-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.18 Fmoc-Lys(Dde)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Lys(Dde)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.19 Fmoc-Lys(Boc)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Lys(Boc)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.20 Fmoc-Asp(OtBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Asp(OtBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸和HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.21 Fmoc-Leu-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Leu-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.22 Fmoc-Aib-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Aib-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.23 Fmoc-Ile-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Lys(Dde)-OH(6.0eq)加入到上述树脂中,加入DIEA(12.0eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(5.70eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.24 Fmoc-Ser(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.25 Fmoc-Tyr(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。 加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Tyr(tBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.26 Fmoc-Asp(OtBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Asp(OtBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.27 Fmoc-Ser(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Ser(tBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HBTU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应2h,茚三酮检测,树脂蓝色。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.28 Fmoc-Thr(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Thr(tBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸后溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.29 Fmoc-Phe-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Phe-OH(3.0eq)加入到上述树脂中,加入DIEA(6.0eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.30 Fmoc-Thr(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Thr(tBu)-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.31 Fmoc-Gly-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Gly-OH(3.0eq)加入到上述树脂中,,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.32 Fmoc-Glu(OtBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Glu(OtBu)-OH(3.0eq)加入到上述树脂中,加入HOAT(3.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入DIC(3.00eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应过夜,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.33 Fmoc-Aib-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Aib-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.34 Boc-Tyr(tBu)-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Boc-Tyr(tBu)-OH(6.0eq)加入到上述树脂中,加入DIEA(12.0eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(5.70eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.35脱Alloc和OAll
1.加入PhSiH
3(20.0eq)和DCM(10mL)至反应柱中,氮气鼓起后加入Pd(PPh
3)
4(0.2eq),氮气鼓起20min,反应二次,排废,直至没有液体流出。
2.用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.36酰胺关环
1.加入DIEA(3.00eq)补加10mL DMF至反应柱中,鼓氮气,待溶解后加入HATU(1.5eq)。调节好氮气使树脂均匀鼓起。
2.在25℃的环境中反应0.5h,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.重复上述1和2操作,再关环一次。茚三酮检测,树脂无色透明。
4.用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.37脱Dde
1.加入3%的水合肼/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
3.38 Fmoc-AEEA-OH的偶联
1.称取Fmoc-AEEA-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
2.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
3.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.39 Fmoc-AEEA-OH的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-AEEA-OH(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.40 Fmoc-Glu-OtBu的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取Fmoc-Glu-OtBu(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
3.41 C20DA的偶联
1.加入20%的哌啶/DMF(50mL)至反应柱中,氮气鼓起20min,排废,直至没有液体流出。加入DMF(50mL)洗涤5次,每次1min,排废,直至没有液体流出。茚三酮检测,树脂蓝色。
2.称取C20DA(3.0eq)加入到上述树脂中,加入DIEA(6.00eq)补加10mL DMF至反应柱中,鼓氮气,待氨基酸溶解后加入HATU(2.85eq)。调节好氮气使树脂均匀鼓起。
3.在25℃的环境中反应0.5h,茚三酮检测,树脂无色透明。
4.抽掉反应液,用DMF洗涤5次(50mL每次),每次1min,排废,直至没有液体流出。
5.用MeOH(50mL)收缩树脂,每次3min,排废,直至没有液体流出,将树脂倒出干燥,备用。
4.切割及粗肽干燥
4.1.按如下体积配置切割液
试剂 | 比例% |
TFA | 91 |
Tis | 3 |
H 2O | 3 |
Mpr | 3 |
将干燥后的肽树脂加入到配好的切割液中,在摇床震荡2.5h,过滤,滤液加入到10倍体积冰异丙醚中,离心,再用异丙醚洗涤3次。在真空干燥2h得到粗肽,纯化。多肽分子量经ESI-MS进行确认,计算值为4361.9,检测值为4362.0。
实施例2
参考001的合成,在3.14步骤中将Fmoc-Ala-OH更换为Fmoc-Glu(OtBu)-OH得到002。多肽分子量经ESI-MS进行确认,计算值为4420.0,检测值为4419.3。
实施例3
参考001的合成,在3.12步骤中将Fmoc-Val-OH更换为Fmoc-Ile-OH得到003。多肽分子量经ESI-MS进行确认,计算值为4420.0,检测值为4419.3。
实施例4
参考001的合成,得到004。多肽分子量经ESI-MS进行确认,计算值为4361.9,检测值为4361.7。
实施例5
参考001的合成,得到005。多肽分子量经ESI-MS进行确认,计算值为4420.0,检测值为4419.3。
实施例6
参考001的合成,得到006。多肽分子量经ESI-MS进行确认,计算值为4376.0,检测值为4375.5。
生物测试数据
实施例1:体外GLP-1R/GIPR/GCGR激动活性测试
A:主要材料:
1)细胞株
该细胞株由上海药明康德构建。详情见下表。
靶点 | 宿主细胞 | 克隆 |
GLP-1R | HEK293 | N/A |
GCGR | HEK293 | N/A |
GIPR | CHO | N/A |
2)试剂与耗材
名称 | 批次. | 货号 | 厂家 |
cAMP检测盒 | 29F | 62AM4PEJ | Cisbio |
1M HEPES | 2120919 | 15630-106 | Invitrogen |
Hanks平衡盐溶液(HBSS) | 2185775 | 14025 | Invitrogen |
人血清白蛋白(HSA) | SLCF7301 | A1653-10G | 西格玛 |
酪蛋白 | SLCC9458 | C4765-10mL | 西格玛 |
3-异丁基-1-甲基黄嘌呤(IBMX) | STBF6061V | I5879-5G | 西格玛 |
ECHO qualified 384孔板 | 0006433672 | PP-0200 | Labcyte |
OptiPlate-384 | 8210-19481 | 6007299 | 珀金埃尔默 |
3)仪器
名称 | 型号 | 厂家 |
EnVision | envision2014 | 珀金埃尔默 |
Vi-cell counter | Vi-CELL TM XR Cell Viability Analyzer | 贝克曼 |
Bravo | Bravo V11 | 安捷伦 |
ECHO | ECHO 555 | Labcyte |
Centrifuge | Allegra TM 25R Centrifuge | 贝克曼 |
B.方法
1)实验材料
实验缓冲液
检测试剂制备
试剂 | 储存浓度 | 体积 | 终浓度 |
细胞裂解液 | 1× | 9.5mL | ≈1× |
D2-cAMP溶液 | 40× | 250μL | 1× |
cAMP-抗体溶液 | 40× | 250μL | 1× |
2)实验方法
a)制备化合物板:
待测化合物做10个点4倍稀释,起始浓度为30μM,Bravo完成稀释b)转移化合物:
1)使用Echo转移100nL化合物至OptiPlate-384 plate。
2)将OptiPlate-384 plate在1000rpm离心5s。
c)细胞悬液的制备
1)将一支GLP-1R/GIPR/GCGR细胞冻存管迅速置于37℃温水中解冻。
2)将细胞悬液转移至Transfer 15mL离心管中,用10mL HBSS轻柔冲洗。
3)将离心管在1000rpm室温离心1min。
4)弃去上清。
5)轻柔打散底部细胞并再用10mL HBSS轻柔冲洗,离心沉降细胞,最后用实验缓冲液重悬细胞。
6)利用Vi-cell测量细胞密度与活度。
7)用实验缓冲液将GLP-1R/GCGR细胞浓度稀释至2.0×10
5/mL。
8)在OptiPlate-384plate中转入100nL稀释好的细胞悬液。
9)室温孵育30min。
d)加入检测试剂:
1)在OptiPlate-384 plate空孔中加入10μL 800nM梯度稀释好的cAMP标准品。
2)加入10μL cAMP检测试剂。
3)用TopSeal-A film覆盖OptiPlate-384 plate,室温孵育60min。
揭去TopSeal-A,在EnVision读数。
C实验结果
结论:本发明化合物对GLP-1R/GIPR/GCGR具有很强的激动活性。
Claims (10)
- 根据权利要求1所述的多肽,其具有如下序列SEQ ID NO:1-6,SEQ ID NO:1:YAibEGT FTSDY SIAibLD KK 0AQK AFVEW LIAGG PSSG-NH 2;SEQ ID NO:2:YAibEGT FTSDY SIAibLD KK 0AQK EFVEW LIAGG PSSG-NH 2;SEQ ID NO:3:YAibEGT FTSDY SIAibLD KK 0AQK AFIEW LIAGG PSSG-NH 2;SEQ ID NO:4:YAibEGT FTSDY SIAibLD KEAQK AFVK 0W LIAGG PSSG-NH 2;SEQ ID NO:5:YAibEGT FTSDY SIAibLD KEAQK EFVK 0W LIAGG PSSG-NH 2;SEQ ID NO:6:YAibEGT FTSDY SIAibLD KEAQK AFIK 0W LIAGG PSSG-NH 2;其中,第17和20位置的氨基酸之间或第20和24位置的的氨基酸之间形成内酰胺桥;Aib和K 0如权利要求1所定义。
- 根据权利要求1或2所述的多肽,其中,p为2。
- 根据权利要求1或2所述的多肽,其中,s为1。
- 根据权利要求1或2所述的多肽,其中,t为9。
- 一种药物组合物,包括作为活性成分的治疗有效量的根据权利要求1~8任意一项所述的多肽化合物或其药学上可接受的盐以及药学上可接受的载体。
- 根据权利要求1~8任意一项所述的多肽化合物或其药学上可接受的盐或者根据权利要求9所述的组合物在制备治疗糖尿病药物上的应用。
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CN112608377A (zh) * | 2015-01-09 | 2021-04-06 | 伊莱利利公司 | Gip和glp-1共激动剂化合物 |
CN109310743A (zh) * | 2016-05-16 | 2019-02-05 | 因塔西亚制药公司 | 胰高血糖素受体选择性多肽及其使用方法 |
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