CN114642718B - Drug target for treating cancer and application thereof - Google Patents

Drug target for treating cancer and application thereof Download PDF

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CN114642718B
CN114642718B CN202210377514.3A CN202210377514A CN114642718B CN 114642718 B CN114642718 B CN 114642718B CN 202210377514 A CN202210377514 A CN 202210377514A CN 114642718 B CN114642718 B CN 114642718B
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methyl
mmol
microcolin
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polypeptide
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CN114642718A (en
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王锐
张海龙
杨瀚舸
王聪
侯彦喆
黄珊
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Lanzhou University
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Abstract

The invention relates to a drug target for treating cancer and application thereof. The polypeptide Microcolin H and the application of the analogues thereof in the aspect of preparing medicaments, wherein the medicaments comprise: medicaments for treating neurodegenerative diseases; a medicament for treating duchenne muscular dystrophy; medicaments for the treatment of inflammatory diseases; medicaments for treating cardiovascular diseases; a medicament for treating cancers. The invention provides the application of an inhibitor of phosphatidyl alcohol transport protein as an anti-tumor drug, and the inhibitor is used as a drug target to develop a therapeutic drug for diseases such as cancers, cardiovascular diseases and the like; the invention also screens out natural polypeptide Microcolin H and analogues and derivatives thereof, can precisely target phosphatidyl alcohol transport protein, further realizes high-efficiency inhibition on tumor cells, has strong pharmacological action, can be used as the first high-activity high-selectivity inhibitor of a novel drug target phosphatidyl alcohol transport protein, and has good medicinal prospect.

Description

Drug target for treating cancer and application thereof
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a drug target for treating cancer and application thereof.
Background
Lipid signaling regulation is widespread and involved in cellular processes, including regulation of G protein-coupled receptors and receptor tyrosine kinases on the plasma membrane, actin dynamics, transcription and membrane trafficking. Eukaryotic lipid signaling relies primarily on the synthesis of inositol phosphates (PtdIns) and soluble inositol phosphates derived from them.
Phosphatidylinositol is an essential phospholipid which acts as a metabolic precursor for phosphoinositides and Ins-phosphates. Although inositol-phosphate forms are chemically diverse, the phosphoinositide molecular chain is relatively simple. The mammal produces seven types of phosphoinositides: including phosphatidyl alcohol-3-phosphate, phosphatidyl alcohol-4-phosphate, phosphatidyl alcohol-5-phosphate, phosphatidyl alcohol-4, 5-diphosphate, phosphatidyl alcohol-3, 5-diphosphate phosphatidyl alcohol-3, 4-diphosphate, and PtdIns-3,4, 5-triphosphate. These limited phosphoinositide sequences support lipid signaling that regulates the action of hundreds of proteins.
Phosphatidylinositol transfer proteins (Phosphatidylinositol transfer proteins, PITPs) integrate different regions of intracellular lipid metabolism with stimulation of phosphatidylinositol 4-phosphate production, a key regulator of phosphoinositide signaling. The phosphatidyl alcohol transporter is capable of binding and exchanging a molecule of phosphatidyl alcohol (PI) or Phosphatidyl Choline (PC) and facilitating the transfer of these two lipid molecules between intracellular membrane components.
Several members of the PITP family have now been identified and classified into two different types based on homology. Members of both classes have a lipid binding site that can transfer PI and PC between membranes in vitro, but both classes have no sequence homology. Class I includes, among other things, two mammalian subtypes, the PITP-alpha (PITPNA) and PITP-beta (PITPNB) membrane-specific integral membrane protein RdgB. It has been found that PITP has an important effect on physiological and biochemical processes such as lipid transport and metabolism between intracellular membrane components, formation and transport of secretory vesicles, phospholipase C (PLC) regulated signal transduction, and neurodegeneration. Mutations in PITP or PITP-like proteins are also the root cause of neurodegenerative and lipid homeostatic diseases in mammals, and pleiotropic effects of PITPs on phosphoinositide levels and phosphoinositide signals suggest that PITPs play an important role in critical cellular processes such as cell growth, migration and invasion, which are abnormally regulated in tumor cells.
Various evidences suggest that PITP as a key factor in phosphoinositide signaling may be a target for a variety of diseases, and targeted inhibition of PITP may have potentially beneficial effects on the treatment of cancer, neurodegenerative diseases, du's muscular dystrophy, inflammatory diseases, and cardiovascular diseases (e.g., atherosclerosis).
No inhibitors of PITPs have been found so far, so it is very important to develop corresponding high-selectivity inhibitors with PITP as a target, regulate specific phosphoinositide signaling pathways in cells, and further realize treatment of related diseases.
Disclosure of Invention
Microcollins (A-M) is a polypeptide compound with good biological activity and peculiar structure extracted and separated from marine microorganism Sphingomonas megatherium. Structurally, microcollins contain unsaturated butyrolactams, hydroxyproline, N-methylated amino acids and long chain carboxylic acid compositions, as well as unsaturated butyrolactams.
In nineties of the last century, moore and Koehn et al separated into Majusculamide D and Microcolin A/B, and the study on biological activity showed that they had activity against P388 leukemia cells and mixed lymphocyte reactionActivity (MLR, EC) 50 = 1.5,42.7nM,Microcolin A,B). The recent Gerwick panel, in turn, isolated 9 new analogs (Microcolin E-M), and activity tests showed that these compounds had half-Inhibitory Concentrations (IC) on non-small cell lung cancer cells H460 50 ) In the nanomolar to micromolar range.
According to the invention, the ocean polypeptide Microcolin H and the derivatives thereof are synthesized by a chemical synthesis method, and the probe molecules of the ocean polypeptide Microcolin H are utilized to explore the anticancer action targets. Experiments prove that the direct targets of the ocean polypeptide microcollin H playing an anticancer role are phosphatidyl alcohol transporter alpha (PITPNA) and phosphatidyl alcohol transporter beta (PITPNA), so that the ocean polypeptide microcollins and derivatives thereof can be used as first high-activity and high-selectivity covalent binding inhibitors of the target phosphatidyl alcohol transporter alpha (PITPNA) and the phosphatidyl alcohol transporter beta (PITPNA). The invention further utilizes computer simulation, and the covalent binding site of microcollins and derivatives thereof and target proteins phosphatidyl alcohol transporter alpha (PITPNA) and phosphatidyl alcohol transporter beta (PITPNA) is Cys187, which is an important drug design site and region.
Based on the research results, the invention aims to provide an application of polypeptide Microcolin H and analogues thereof in preparation of medicines.
It is a further object of the present invention to provide the use of a phosphatidyl alcohol transporter as a drug target for screening anticancer drugs.
It is a further object of the present invention to provide the use of inhibitors of phosphatidyl alcohol transport proteins for the preparation of a medicament.
The application of the polypeptide Microcolin H and analogues thereof in the preparation of medicaments according to the specific embodiment of the invention comprises any one or more of the following steps:
(1) Medicaments for treating neurodegenerative diseases;
(2) A medicament for treating duchenne muscular dystrophy;
(3) Medicaments for the treatment of inflammatory diseases;
(4) Medicaments for treating cardiovascular diseases;
(5) A medicament for treating cancers.
Among them, neurodegenerative diseases are those in which the nerve cells and/or their myelin sheath are lost, and the nerve cells deteriorate over time, and thus, dysfunction occurs. It can be classified into acute neurodegenerative diseases and chronic neurodegenerative diseases, the former mainly including Cerebral Ischemia (CI), brain Injury (BI), epilepsy; the latter include Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), amyotrophic Lateral Sclerosis (ALS), different types of spinocerebellar ataxia (SCA), pick's disease, and the like.
Du's muscular dystrophy (Duchenne Muscular Dystrophy, DMD) is a hereditary muscular dystrophy. Its gene (Dystrophin gene) is present in the X sex chromosome (Xp 21), and Du's muscular dystrophy can lead to muscular fiber inoxism, atrophy. Mainly progressive muscle weakness and atrophy.
Cardiovascular and cerebrovascular diseases are the general terms of cardiovascular and cerebrovascular diseases, and refer broadly to ischemic or hemorrhagic diseases of heart, brain and systemic tissues caused by hyperlipidemia, blood viscosity, atherosclerosis, hypertension, etc. Cardiovascular and cerebrovascular diseases are manifestations of systemic vascular lesions or systemic vascular lesions in the heart and brain. The etiology of the disease has 4 main aspects: (1) vascular factors such as atherosclerosis, hypertensive arteriole arteriosclerosis, arteritis, etc.; (2) hemodynamic factors such as hypertension; (3) blood rheology abnormality such as hyperlipidemia and diabetes; (4) leukemia, anemia, thrombocytosis, etc.
Inflammatory diseases, also known as inflammatory diseases, are a class of inflammation that occurs in multiple joints, e.g., rheumatism, hot air damp-heat refers to inflammation of joints (arthritis) and heart (cardioinflammation) that is usually caused by streptococcal infection of the pharynx.
The cancer is selected from: ovarian cancer; lung cancer; stomach cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer; malignant melanoma; cancer of the head and neck; sarcoma; bile duct cancer; bladder cancer; renal cancer; colon cancer; small intestine cancer; testicular cancer; placental choriocarcinoma; cervical cancer; testicular cancer; uterine cancer; prostate cancer; leukemia; multiple myeloma; malignant lymphoma; and their transferred forms.
The application of the polypeptide Microcolin H and the analogues thereof in the aspect of preparing antitumor drugs is provided in the specific embodiment of the invention, wherein the structure of the polypeptide Microcolin H and the analogues thereof is shown in the formula I:
wherein R is saturated alkyl or unsaturated alkyl.
The derivatives refer to various changes of microcollins fatty long-chain acid, including saturated and unsaturated fatty acids such as acetic acid, n-butyric acid, pentynoic acid, hexynoic acid, n-caproic acid, n-caprylic acid, heptynoic acid, tetradecynoic acid and the like, substitution or modification of each amino acid in the tripeptide part of the middle fragment, modification and change of the cyclic amino acid of the tail fragment and the like.
The polypeptide Microcolin H and the analogues thereof according to the specific embodiments of the present invention are used for preparing antitumor drugs, wherein the saturated alkyl group is an alkyl group, preferably a saturated straight-chain alkyl group having 1 to 14 carbon atoms or a saturated branched-chain alkyl group having 1 to 14 carbon atoms, for example, methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl.
Preferably, saturated branched alkyl groups containing 1 to 14 carbon atoms include ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl and alkyl groups modified with a fatty chain side chain.
The polypeptide Microcolin H and analogues thereof according to the specific embodiment of the invention are applied to the preparation of antitumor drugs, wherein R is 1-methylheptyl ((R) -1-methylheptyl or (L) -1-methylheptyl), methyl, n-propyl, n-pentyl, n-heptyl, n-nonyl and undecyl.
The application of the polypeptide Microcolin H and analogues thereof in preparing antitumor drugs is characterized in that unsaturated alkane is C1-C14 alkynyl, such as propynyl, n-butynyl, n-pentynyl, n-hexynyl, n-heptynyl, n-octynyl, n-nonynyl, n-decynyl, n-undecynyl, n-dodecenyl, n-tridecylynyl, n-tetradecynyl and alkynyl modified by fatty chain side chains.
The polypeptide Microcolin H and the analogues thereof in the specific embodiment of the invention are applied to the preparation of antitumor drugs, wherein R is butynyl, hexynyl, n-13-alkynyl, n-16-alkyne-12-bisazepine.
The application of the polypeptide Microcolin H and the analogues thereof in the aspect of preparing antitumor drugs is provided, wherein the targets of the polypeptide Microcolin H and the analogues thereof are phosphatidyl alcohol transport proteins.
Phosphatidylinositol transfer proteins (Phosphatidylinositol transfer proteins, PITPs) are specifically referred to as phosphatidylinositol transporter α (PITPNA) and phosphatidylinositol transporter β (PITPNB).
The invention also provides application of the phosphatidyl alcohol transport protein as a drug target in screening anticancer drugs.
Use of a phosphatidyl alcohol transporter according to an embodiment of the present invention as a drug target for screening for an anti-cancer drug having a binding site for Cys187 with the phosphatidyl alcohol transporter. Cys187 is not a pocket site for binding PITPNA and PITPNB to Phosphatidylinositol (PI), and therefore the anticancer drug binds to the allosteric site of PITPNA and PITPNB, and is a covalent bond to the anticancer drug, which is a covalently bound drug.
The invention also provides the use of an inhibitor of a phosphatidyl alcohol transporter in the manufacture of a medicament which is any one or more of:
(1) Medicaments for treating neurodegenerative diseases;
(2) A medicament for treating duchenne muscular dystrophy;
(3) Medicaments for the treatment of inflammatory diseases;
(4) Medicaments for treating cardiovascular diseases;
(5) A medicament for treating cancers.
The invention has the beneficial effects that:
the invention provides application of inhibitors of phosphatidyl alcohol transport proteins (PITPNA and PITPNB) as antitumor drugs, and the inhibitors of the PITPNA and the PITPNB are used as drug targets to develop drugs for treating diseases such as cancers, cardiovascular diseases and the like. According to the invention, natural polypeptide Microcolin H and analogues and derivatives thereof can be screened out, and phosphatidyl alcohol transport proteins (PITPNA and PITPNB) can be precisely targeted, so that high-efficiency inhibition on tumor cells is realized. Microcollins and derivatives thereof have strong pharmacological actions as polypeptide compounds, are the first high-activity and high-selectivity inhibitors of novel drug target phosphatidyl alcohol transport proteins (PITPNA and PITPNB), and have good medicinal prospects.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of a protein labelling experiment of probe 5 (probe 5) with cell lysate;
FIG. 2 shows the results of Western-blot validation of target proteins;
fig. 3 shows the binding patterns of PITPNA and PITPNB and CYS187.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
In an embodiment of the present invention, the cancer cells used include:
human liver cancer cells: huH-7, hepaRG;
human gastric cancer cells: HGC-27, AGS, MKN-28;
human lung cancer cells: a549, H460;
human pancreatic cancer cells: asPC-1, PANC-1;
human cervical cancer cells: hela;
all purchased from cell banks of the national academy of sciences.
EXAMPLE 1 Synthesis of Microcolin H and derivatives
According to the invention, the natural product Microcolin H is decomposed into three fragments M1, M2 and M3, the synthesis of the three fragments M1, M2 and M3 is respectively completed, and then the three fragments are condensed step by step, so that the natural product Microcolin H is finally obtained.
1.1 preparation of Microcolin H
1) Preparation of M1:
(R) -2-methyl octanoic acid (M1)
3.5g (11.6 mmol) of (R) -4-benzyl-3-octanoyloxazolidine-2-one (3) are dissolved in 15mL THF, 12.7mL (12.7 mmol) of NaHMDS are added dropwise at-78℃and stirred for 30min, 1.45mL (23.2 mmol) of MeI are then added and reacted at-78℃for 5h. After completion of the reaction, quenched with saturated ammonium chloride, extracted 3 times with EtOAc (100 mL), the organic phases combined, using 5% na 2 S 2 O 3 Extracting with saturated saline once, and purifying by column chromatography. The product obtained after purification was dissolved in 90mL THF and 30mL H 2 To the mixed solvent of O, 0.62g (15.8 mmol) of lithium hydroxide, 7mL (63mmol)H 2 O 2 The reaction was carried out for 4 hours. After completion of the reaction, 5% Na was added 2 S 2 O 3 The solution was adjusted to pH 1.0 using 6N HCl, extracted 3 times (100 mL) with DCM, and the organic phases combined, extracted with saturated brine and purified by column chromatography to give 550mg (3.5 mmol 30%) of (R) -2-methyl octanoic acid (M1).
1 H NMR(300MHz,Chloroform-d)δ2.39(q,J＝6.9Hz,1H),1.61(dd,J＝14.0,7.2Hz,1H),1.44–1.32(m,1H),1.22(dt,J＝10.6,4.5Hz,9H),1.11(d,J＝7.0Hz,3H),0.85–0.74(m,3H).
13 C NMR(75MHz,Chloroform-d)δ183.21,39.94,32.54,31.83,29.35,26.56,22.77,16.63,14.11.
2) Preparation of M2
Benzyl N- (tert-Butoxycarbonyl) -N-methyl-L-valine ester (5)
4.64g (20 mmol) of N- (t-butoxycarbonyl) -N-methyl-L-valine, 6.07g (44 mmol) of potassium carbonate were added to 100mL of acetonitrile, 4.27mL (32 mmol) of benzyl bromide was added dropwise thereto, the reaction was stirred at room temperature for 4 hours, acetonitrile was removed under reduced pressure, and the product was purified by column chromatography to give 5.64g (17.6 mmol, 88%) of benzyl N- (t-butoxycarbonyl) -N-methyl-L-valine ester.
1 H NMR(300MHz,Chloroform-d)δ7.34(s,5H),5.16(s,2H),4.33(dd,J＝107.1,10.5Hz,1H),2.80(d,J＝21.9Hz,3H),2.19(dq,J＝11.3,6.5,5.7Hz,1H),1.44(d,J＝11.4Hz,9H),0.96(d,J＝6.5Hz,3H),0.89(d,J＝5.3Hz,3H).
13 C NMR(75MHz,Chloroform-d)δ128.54,128.44,128.25,128.06,127.97,80.25,66.34,66.19,65.11,63.24,30.57,30.44,28.35,28.33,27.82,27.64,20.00,19.75,19.07,18.83.
Benzyl N- (tert-Butoxycarbonyl) threonyl) -N-methyl-L-valine ester (7)
5.64g (17.6 mmol) of benzyl N- (t-butoxycarbonyl) threonyl) -N-methyl-L-valine ester (5) was dissolved in 40mL of DCM, 10mL of TFA was slowly added dropwise thereto at 0℃and reacted at room temperature for 1 hour, the solvent was removed under reduced pressure, and the remaining solid (3X 30 mL) was dissolved again with DCM and removed under reduced pressure to give methyl-L-valine benzyl ester. 4.7g (21.3 mmol) of 2- (tert-butoxycarbonyl) amino) -3-hydroxybutyric acid (6) was dissolved in 50mL of DCM and 5.5g (21.3 mmol) of Bop-Cl was added at 0deg.C and activated for 10min at 0deg.C. Subsequently methyl-L-valine benzyl ester was dissolved in 50mL DCM, 9.3mL (53.3 mmol) DIPEA was added, and after mixing well, the activated 2- (t-butoxycarbonyl) amino) -3-hydroxybutyric acid solution was added and reacted overnight at room temperature. The solvent was extracted under reduced pressure, a small amount of water was added, the mixture was extracted 3 times with EtOAc (100 mL), and the organic phases were combined, extracted once with saturated brine, concentrated under reduced pressure, and the product was purified by column chromatography to give 2.8g (6.7 mmol, 38%) of benzyl N- (tert-butoxycarbonyl) threonyl) -N-methyl-L-valine ester (7).
1 H NMR(300MHz,Chloroform-d)δ7.34(d,J＝1.0Hz,5H),5.41(d,J＝9.5Hz,1H),5.25–5.06(m,2H),4.90(d,J＝10.5Hz,1H),4.42(dd,J＝9.5,2.1Hz,1H),3.97(dd,J＝6.4,2.0Hz,1H),3.72(s,1H),3.03(s,3H),2.32–2.16(m,1H),1.40(d,J＝13.3Hz,9H),1.14(d,J＝6.4Hz,3H),1.00(d,J＝6.6Hz,3H),0.84(d,J＝6.7Hz,3H).
13 C NMR(75MHz,Chloroform-d)δ173.61,170.31,156.15,135.43,128.59,128.48,128.41,128.04,80.07,67.26,66.72,61.74,53.53,31.55,28.21,28.16,27.29,19.80,18.63.
Benzyl N- (O-N- (t-Butoxycarbonyl) -L-threonyl) -N-methyl-L-valine benzyl ester (8)
1.4g (3.31 mmol) of benzyl N- (tert-butoxycarbonyl) threonyl) -N-methyl-L-valine ester, 0.485g (3.97 mmol) of DMAP were dissolved in 20mL of EtOAc and 0.38mL (3.97 mmol) of Ac 2 O is added dropwise, and the reaction is carried out for 5h at room temperature. Concentrating under reduced pressure, and purifying the product by column chromatography to obtain benzyl1.26g (79%) of N- (O-N- (t-butoxycarbonyl) -L-threonyl) -N-methyl-L-valine benzyl ester.
1 H NMR(300MHz,Chloroform-d)δ7.34(s,5H),5.40(d,J＝9.2Hz,1H),5.15(s,2H),4.92(d,J＝10.6Hz,1H),4.67(dd,J＝9.3,5.4Hz,1H),3.05(s,3H),2.30–2.15(m,1H),1.96(s,3H),1.42(s,9H),1.26(d,J＝4.6Hz,1H),1.16(d,J＝6.4Hz,3H),0.99(d,J＝6.5Hz,3H),0.83(d,J＝6.7Hz,3H).
13 C NMR(75MHz,Chloroform-d)δ170.59,170.26,170.17,155.74,135.51,128.51,128.46,128.35,79.98,69.46,66.71,61.77,54.04,31.35,28.23,27.17,20.95,19.70,18.60,16.71.
Benzyl N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -L-threonyl) -N-methyl-L-valine ester (9)
1g (2.08 mmol) of benzyl N- (O-N- (t-butoxycarbonyl) -L-threonyl) -N-methyl-L-valine benzyl ester was dissolved in 20mL of DCM, 10mL of TFA was slowly added dropwise thereto at 0℃and reacted at room temperature for 1 hour, the solvent was removed under reduced pressure, and the remaining solid (3X 20 mL) was dissolved again by adding DCM and removed under reduced pressure to give benzyl N- (O-acetyl-L-threonyl) -N-methyl-L-valine. 0.61g (2.5 mmol) of N- (tert-butoxycarbonyl) -N-methylleucine was dissolved in 15mL of DMF, 0.95g (2.5 mmol) of HATU,0.83mL (5.0 mmol) of DIPEA were added at 0deg.C and activated for 10min. Benzyl N- (O-acetyl-L-threonyl) -N-methyl-L-valine was then dissolved in 15mL DMF, added to 0.83mL (5.0 mmol) DIPEA, mixed well and added to the activated N- (tert-butoxycarbonyl) -N-methylleucine solution and reacted overnight at room temperature. A small amount of water was added, extracted 3 times with EtOAc (50 mL), and the organic phases were combined, extracted once with saturated brine, concentrated under reduced pressure, and the product was purified by column chromatography to give 0.8g (1.35 mmol, 65%) of benzyl N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -N-methyl-L-valine ester.
1 H NMR(300MHz,Chloroform-d)δ7.34(s,5H),5.25(s,1H),5.15(d,J＝1.3Hz,2H),4.99(dd,J＝8.7,5.0Hz,1H),4.91(d,J＝10.6Hz,1H),4.77–4.49(m,1H),3.06(s,3H),2.76(s,3H),1.93(s,3H),1.75(s,2H),1.58–1.41(m,11H),1.12(d,J＝6.4Hz,3H),1.02–0.85(m,10H),0.81(d,J＝6.7Hz,3H).
13 C NMR(75MHz,Chloroform-d)δ170.02,169.72,135.50,128.51,128.44,128.35,80.39,66.72,61.75,52.46,36.52,31.25,29.93,29.21,28.33,27.15,24.48,23.24,21.31,20.87,19.63,18.63,16.74.
N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -L-threonyl) -N-methyl-L-valine (M2)
0.72g (1.22 mmol) of benzyl N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -L-threonyl) -N-methyl-L-valine ester was dissolved in 30mL of methanol, 160mg of Pd/C was added thereto, and H was introduced 2 The reaction was carried out at room temperature for 2 hours. After the reaction, the mixture was filtered through celite, and the methanol was removed under reduced pressure to give 0.55g (1.1 mmol, 90%) of N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -L-threonyl) -N-methyl-L-valine.
3) Preparation of M3
(S) -5-methylpyrrolidin-2-one (13)
4g (22.6 mmol) of (R) -5- (bromomethyl) pyrrolidin-2-one, 40mg (0.24 mmol) of AIBN were dissolved in 40mL of toluene and protected with argon, 7.25mL (26.9 mmol) of tri-n-butylstannum hydrogen were added and reacted at 80℃for 7 hours. After completion of the reaction, toluene was removed under reduced pressure, and purified by column chromatography to give (S) -5-methylpyrrolidin-2-one 1.78g (18.0 mmol, 80%).
1 H NMR(300MHz,Chloroform-d)δ6.39(s,1H),3.77(h,J＝6.5Hz,1H),2.40–2.31(m,1H),2.30–2.22(m,1H),1.76–1.59(m,1H),1.22(d,J＝6.3Hz,2H)；
13 C NMR(75MHz,Chloroform-d)δ178.63,50.25,30.77,29.21,22.24.
Tert-butyl (2S, 4S) -4- ((tert-butyldimethylsilyl) oxy) -2- ((S) -2-methyl-5-oxopyrrolidine-1-carbonyl) pyrrolidine-1-carboxylic acid ester (14)
0.55g (5.5 mmol) of (R) -5-methylpyrrolidin-2-one (13) are dissolved in 10ml of anhydrous THF, 2.3ml (5.7 mmol) of 2.5M n-butyllithium are slowly added dropwise at-78℃and stirred for 15min, then (2.36 g,4.59 mmol) of 1- (tert-butyl) 2- (perfluorophenyl) (2S, 4S) -4- ((tert-butyldimethylsilyloxy) pyrrolidine-1, 2-dicarboxylic acid ester are dissolved in 10ml of anhydrous THF and added to the previous reaction, stirred for 4h at-78℃and then the temperature is raised to 0℃and stirred (12) for 30min.40ml of saturated NH 4 Cl solution quenching, etOAc extraction (3X 50 ml), combined organic phases, 15% K 2 CO 3 Washing 3 times, saturated NaCl washing 1 time, anhydrous Na 2 SO 4 Drying and removing the solvent under reduced pressure. Purification by column chromatography, dry loading, and passing P: e=10:1→5:1 through the column gave product (14) as a nearly transparent oil (1.38 g (3.235 mmol, 70.5%).
1 H NMR(300MHz,Chloroform-d,mixture of two rotamers in a ratio of ca.2:1)δ5.20(dd,J＝8.5,6.0Hz,1H,minor rotamer),5.17(dd,J＝8.9,5.2Hz,1H,major rotamer),4.55–4.41(m,2H),4.33(dq,J＝11.0,5.9Hz,2H),3.78(dd,J＝11.0,6.0Hz,1H,major rotamer),3.70(dd,J＝10.8,6.2Hz,1H,minor rotamer),3.34(dd,J＝11.0,5.0Hz,1H,major rotamer),3.28(dd,J＝10.7,5.7Hz,1H,minor rotamer),2.74(dddd,J＝17.7,14.9,11.9,9.1Hz,2H),2.64–2.39(m,3H),2.24–2.11(m,2H),1.83–1.70(m,3H),1.46(s,9H,minor rotamer),1.39(s,9H,major rotamer),1.35(d,J＝6.4Hz,3H),0.86(s,9H,minor rotamer),0.84(s,9H,major rotamer),0.05(s,3H,minor rotamer),0.04(s,3H,major rotamer),0.04(s,3H,minor rotamer),0.02(s,3H,major rotamer).
13 C NMR(75MHz,Chloroform-d)δ175.26,175.21,172.71,172.23,154.24,153.90,79.80,79.69,70.35,69.61,59.78,59.40,54.63,54.37,53.62,39.78,38.84,32.04,28.55,28.43,25.76,25.73,25.61,25.50,19.48,19.32,17.97,-4.72,-4.94.
Tert-butyl (2S, 4S) -4- ((tert-butyldimethylsilyl) oxy) -2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidine-1-carboxylate (15)
610mg (1.43 mmol) of tert-butyl (2S, 4S) -4- (tert-butyldimethylsilyloxy) -2- ((R) -2-methyl-5-oxopyrrolidine-1-carbonyl) pyrrolidine-1-carboxylate (14) are dissolved in 7ml THF, 2.4ml (2.4 mmol) of 1M LiHMDS are added dropwise at-78℃and stirred for 15min, 453mg (1.9 mmol) of phenylselenium bromide in 4.5ml THF are then added and the temperature is raised to 0℃and stirred for 1h at 0 ℃. 10ml of ultrapure water, 2.95ml of acetic acid, 13.23ml of 30% hydrogen peroxide solution were added in this order, and stirred at room temperature for 30 minutes. Then saturated NaHCO 3 :H 2 O=1:1 the above was added, extracted with ethyl acetate (3 x 80 ml), washed with saturated NaCl, anhydrous Na 2 SO 4 Drying and spin drying. P: e=5:1, spin-dry to give 225mg (0.53 mmol, 37.06%) of product (15).
1 H NMR(300MHz,Chloroform-d,mixture of two rotamers in a ratio of ca.2:1)δ7.26(dd,J＝6.0,2Hz,1H,major rotamer),7.22(dd,J＝6.0,2.0Hz,1H,minor rotamer),6.07(dd,J＝6.0,1.5Hz,1H,major rotamer),6.04(dd,J＝6.0,1.5Hz,1H,minor rotamer),5.24(m,1H),4.79–4.72(m,1H),4.41–4.28(m,1H),3.83(dd,J＝10.8,6.2Hz,1H,major rotamer),3.73(dd,J＝10.5,6.5Hz,1H,minor rotamer),3.32(dd,J＝10.8,6.0Hz,1H,major rotamer),3.28(dd,J＝10.6,6.6Hz,1H,minor rotamer),2.70–2.62(m,1H),2.70–2.62(m,1H),1.82–1.71(m,1H),1.64–1.61(m,1H),1.51–1.46(m,3H),1.43(s,9H,major rotamer),1.37(s,9H,minor rotamer),0.85(s,9H,major rotamer),0.83(s,9H,minor rotamer),0.05(s,3H,minor rotamer),0.04(s,3H,major rotamer),0.03(s,3H,minor rotamer),0.01(s,3H,major rotamer)；
13 C NMR(125MHz,CDCl 3 ,mixture of two rotamers)δ172.25,171.85,169.94,154.22,154.04,153.80,125.66,79.88,79.81,70.30,69.58,58.99,58.60,58.36,54.43,54.19,49.65,39.26,38.30,30.46,29.85,28.55,28.47,25.81,25.80,22.84,18.27,18.04,17.49,-4.71,-4.85.
The compound 15 was added with 4M dioxane solution of hydrochloric acid in ice bath, reacted for one hour in ice bath, and the solvent was removed under reduced pressure to give the product M3, which was used directly in the subsequent reaction without further purification.
4) Synthesis of fragment M4:
(2R, 3S) -3- ((S) -2- (tert-Butoxycarbonyl) (methyl) amino) -4-methylpentanamido) -4- (((2S) -1- ((4S) -4-hydroxy-2- (((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -4-oxobutan-2-oic acid methyl ester (M4)
250mg (0.62 mmol) of N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -L-threonyl) -N-methyl-L-valine (M2) are dissolved in 5mL of DCM, 185mg (0.73 mmol) of Bop-Cl are added at 0deg.C and activated at 0deg.C for 10min. 220mg (0.52 mmol) of (S) -1- ((2S, 4S) -4-hydroxypyrrolidine-2-carbonyl) -5-methyl-1, 5-dihydro-2H-pyrrol-2-one (M3) are then dissolved in 5mL DCM, 0.26mL DIPEA is added, after mixing well activated N- (O-acetyl-N- (N-t-butoxycarbonyl) -N-methyl-L-leucyl) -L-threonyl) -N-methyl-L-valine solution and reacted overnight at room temperature. The product was purified using column chromatography to give (2 r, 3S) -4- (((S) -1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- ((S) -4-methyl-2- (methylamino) pentanamido) -4-oxobutane-2-acetate (M4) 90mg (0.13 mmol, 25%).
1 H NMR(300MHz,Chloroform-d)δ6.78(d,J＝48.8Hz,1H),6.09(dd,J＝6.1,1.6Hz,1H),5.67(d,J＝9.7Hz,1H),5.28(s,1H),5.06–4.91(m,1H),4.32(q,J＝8.8,6.7Hz,1H),3.87(s,1H),3.11(s,2H),2.94–2.82(m,1H),2.78(s,2H),2.60–2.37(m,1H),2.26(td,J＝12.2,5.9Hz,1H),2.05(s,3H),2.00(s,2H),1.81–1.60(m,4H),1.48(dd,J＝10.1,6.4Hz,10H),1.38–1.10(m,10H),1.06–0.75(m,12H).
13 C NMR(75MHz,Chloroform-d)δ174.59,174.47,173.47,168.97,168.89,168.82,167.83,154.38,153.22,124.28,79.45,70.79,70.73,67.56,58.26,58.06,57.61,57.13,55.85,55.06,52.48,50.98,35.72,35.54,30.85,29.45,27.32,26.12,24.21,19.96,18.09,17.80,17.34,15.87.
90mg (0.13 mmol) of (2R, 3S) -4- (((S) -1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- ((S) -4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate was dissolved in 3mL of DCM, 1mL of TFA was slowly added dropwise thereto at 0℃and reacted at room temperature for 1 hour, the solvent was removed under reduced pressure, the DCM was again added to dissolve the remaining solid (3X 10 mL) and removed under reduced pressure to give (2R, 3S) -4- (((2S) -1- ((4S) -4-hydroxy-2- (((S) -2-methyl-5-oxo-2), 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (((S) -4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-oic acid ester. 25mg (0.16 mmol) of (R) -2-methyloctanoic acid was dissolved in 3mL of DCM, 51mg (0.2 mmol) of Bop-Cl was added at 0deg.C and activated for 10min at 0deg.C. (2R, 3S) -4- (((2S) -1- ((4S) -4-hydroxy-2- (((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (((S) -4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-oic acid ester was then dissolved in 3mLDCM, 0.065mL DIPEA was added, mixed well and then added to the activated (R) -2-methyloctanoic acid solution, reacted overnight at room temperature, concentrated under reduced pressure, and the product was purified using column chromatography to give Microcolin H10 mg (0.014 mmol, 11%).
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.1Hz,1H),6.98(d,J＝8.9Hz,1H),6.09(dd,J＝6.1,1.6Hz,1H),5.67(dd,J＝10.0,2.1Hz,1H),5.32–5.20(m,2H),5.02(d,J＝11.1Hz,1H),4.95(dd,J＝8.9,2.9Hz,1H),4.87–4.73(m,1H),4.46–4.29(m,1H),3.92–3.77(m,2H),3.54(d,J＝11.0Hz,1H),3.10(s,3H),2.94(s,3H),2.72(q,J＝6.8Hz,1H),2.48(ddd,J＝14.6,10.0,4.7Hz,1H),2.26(tt,J＝12.9,6.6Hz,1H),2.12–2.01(m,1H),2.00(s,3H),1.79(d,J＝8.8Hz,1H),1.71(dd,J＝10.1,4.2Hz,1H),1.65–1.52(m,1H),1.47(d,J＝6.8Hz,3H),1.43–1.37(m,1H),1.35–1.19(m,9H),1.14(t,J＝6.5Hz,6H),0.97(dd,J＝12.5,6.5Hz,6H),0.92–0.78(m,9H).
13 C NMR(75MHz,Chloroform-d)δ177.95,174.62,171.27,170.26,169.75,168.64,154.23,125.42,71.97,68.45,59.12,58.68,58.12,57.75,53.83,51.95,36.64,36.21,35.82,34.23,31.84,30.55,30.47,29.34,27.41,27.24,24.88,23.48,22.63,21.72,21.16,18.87,18.42,17.64,16.95,16.21,14.16.HRMS(ESI)calcd for C 38 H 63 N 5 NaO 9 [M + Na + ]756.4518；found,756.4502.
1.2 preparation of derivatives
The intermediate M4 in example 1.1 was used as starting material and condensed with different carboxylic acids R-COOH to give derivatives of Microcolin H (compounds probe 1-5, MIC 1-5).
4- ((1- ((2S, 4S) -4-hydroxy-2- ((R) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (N-methylacetamido) penta-2-acetate 13mg (0.0225 mmol) 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) penta-2-acetate and 2mg (0.03375 mmol) acetic acid were dissolved in 1ml DCM at 0℃and 6.5. Mu.l of TEA was added without stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography. Yield 5.5mg (0.0086 mmol, 38.5%).
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝1.9Hz,1H),6.91(d,J＝8.6Hz,1H),6.09(dd,J＝6.0,1.6Hz,1H),5.78–5.51(m,1H),5.30–5.13(m,2H),5.09–4.88(m,2H),4.88–4.74(m,1H),4.57–4.24(m,1H),3.92–3.71(m,2H),3.52(d,J＝11.0Hz,1H),3.11(s,3H),2.93(s,3H),2.48(ddd,J＝14.5,10.0,4.8Hz,2H),2.34–2.20(m,1H),2.02(s,3H),1.88–1.75(m,1H),1.71–1.60(m,3H),1.47(d,J＝6.8Hz,3H),1.35(d,J＝6.4Hz,2H),1.22–1.12(m,3H),0.99(dd,J＝6.4,1.7Hz,3H),0.92(dd,J＝13.0,6.6Hz,6H),0.81(dd,J＝6.6,1.7Hz,3H).
HRMS(ESI)calcd for C 31 H 49 N 5 NaO 9 [M + Na + ]658.3422；found,658.3407.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 3mg (0.03375 mmol) of n-butyric acid, 8.0mg (0.03375 mmol) of Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA are added without ice-bath stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography. Yield 5.4mg (0.0081 mmol, 36%).
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.0Hz,1H),6.94(d,J＝8.9Hz,1H),6.09(dd,J＝6.1,1.6Hz,1H),5.66(dd,J＝10.0,2.2Hz,1H),5.24(q,J＝7.9,6.6Hz,2H),5.06–4.99(m,1H),4.95(dd,J＝8.9,3.5Hz,1H),4.86–4.75(m,1H),4.38(s,1H),3.83(dd,J＝11.0,6.9Hz,2H),3.11(s,3H),2.92(s,3H),2.53–2.44(m,1H),2.28–2.20(m,1H),2.05(s,1H),2.01(s,3H),1.84–1.75(m,2H),1.47(d,J＝6.7Hz,3H),1.42(d,J＝6.4Hz,1H),1.34(d,J＝6.4Hz,2H),1.25(s,3H),1.16(dd,J＝6.6,1.5Hz,3H),1.00–0.86(m,12H),0.83–0.80(m,3H).HRMS(ESI)calcd for C 33 H 53 N 5 NaO 9 [M + Na + ]686.3735；found,686.3725.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 5.8mg (0.03375 mmol) of n-hexanoic acid, 8.6mg (0.03375 mmol) of Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA is added without removing ice bath and stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography. Yield 13mg (0.0172 mmol, 76.5%).
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.2Hz,1H),6.91(d,J＝8.8Hz,1H),6.09(dd,J＝6.1,1.7Hz,1H),5.63(dd,J＝23.1,9.7Hz,1H),5.23(q,J＝7.7Hz,2H),5.01(dd,J＝11.1,5.6Hz,1H),4.95(dd,J＝8.7,3.4Hz,1H),4.81(d,J＝7.1Hz,1H),4.46(dd,J＝8.7,7.1Hz,1H),3.83(dd,J＝10.8,6.5Hz,2H),3.68–3.61(m,1H),3.11(s,3H),2.92(s,3H),2.50–2.44(m,1H),2.28–2.22(m,2H),2.05(s,1H),2.01(s,3H),1.71(dd,J＝10.1,4.2Hz,1H),1.65–1.52(m,1H),1.47(d,J＝6.8Hz,3H),1.43–1.37(m,1H),1.35–1.19(m,3H),1.14(t,J＝6.5Hz,6H),0.97(dd,J＝12.5,6.5Hz,6H),0.92–0.78(m,9H).
HRMS(ESI)calcd for C 35 H 57 N 5 NaO 9 [M + Na + ]714.4048；found,714.4035.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanoylamino) -4-oxobutan-2-acetate and 6.8mg of n-octanoic acid, 8.6mg (0.03375 mmol) Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA is added without removing the ice bath and stirred overnight. Concentrating under reduced pressure, and purifying by column chromatography. 14mg of product was obtained.
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.0Hz,1H),6.90(d,J＝8.9Hz,1H),6.09(dd,J＝6.0,1.6Hz,1H),5.67(dd,J＝10.1,2.2Hz,1H),5.32–5.18(m,2H),5.02(d,J＝11.1Hz,1H),4.95(dd,J＝8.8,3.5Hz,1H),4.82(dt,J＝6.7,1.8Hz,1H),4.45–4.34(m,1H),3.93–3.77(m,2H),3.54(d,J＝11.0Hz,1H),3.11(s,3H),2.92(s,3H),2.48(ddd,J＝14.5,10.0,4.7Hz,1H),2.36(dd,J＝8.6,6.6Hz,2H),2.31–2.19(m,1H),2.05(s,1H),2.00(d,J＝3.4Hz,3H),1.67(q,J＝8.2,7.5Hz,5H),1.47(d,J＝6.8Hz,3H),1.42(d,J＝6.2Hz,1H),1.34–1.25(m,10H),1.17(d,J＝6.5Hz,3H),0.99(d,J＝6.5Hz,3H),0.94(d,J＝6.6Hz,3H),0.90–0.87(m,3H),0.81(d,J＝6.6Hz,3H).
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 5.8mg (0.03375 mmol) of n-decanoic acid, 8.6mg (0.03375 mmol) of Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA are added without removing the ice bath and stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography. Yield 13mg (0.0172 mmol, 76.5%).
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.1Hz,1H),6.91(d,J＝8.7Hz,1H),6.09(dd,J＝6.1,1.6Hz,1H),5.72–5.63(m,1H),5.23(q,J＝7.9Hz,2H),5.06–5.00(m,1H),4.95(dd,J＝8.8,3.4Hz,1H),4.81(q,J＝6.7Hz,1H),3.85(dd,J＝12.0,4.3Hz,2H),3.11(s,3H),2.92(s,3H),2.49(t,J＝7.1Hz,1H),2.27–2.22(m,1H),2.05(s,1H),2.01(s,3H),1.72–1.51(m,3H),1.49–1.40(m,11H),1.36–0.92(m,21H),0.88(d,J＝1.1Hz,3H),0.81(dd,J＝6.7,1.7Hz,3H).
HRMS(ESI)calcd for C 39 H 65 N 5 NaO 9 [M + Na + ]770.4647；found,770.4655.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 6.8mg (0.03375 mmol) of dodecanoic acid, 8.6mg (0.03375 mmol) of Bop-Cl are dissolved in 1ml DCM at 0℃and 6.5. Mu.l TEA are added without removing the ice bath and stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography. 14mg (0.018 mmol, 80.3%) of product are obtained.
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.1Hz,1H),6.92(d,J＝8.8Hz,1H),6.09(dd,J＝6.0,1.6Hz,1H),5.66(dd,J＝10.1,2.2Hz,1H),5.23(q,J＝7.8,6.4Hz,2H),5.06–4.99(m,1H),4.95(dd,J＝8.9,3.4Hz,1H),4.81(d,J＝6.9Hz,1H),4.46(dd,J＝8.8,7.1Hz,1H),3.83(dd,J＝10.6,6.5Hz,2H),3.66(d,J＝7.9Hz,1H),3.10(d,J＝1.4Hz,3H),2.92(s,3H),2.48–2.42(m,1H),2.27–2.22(m,1H),2.05(s,1H),2.01(s,3H),1.71(dd,J＝10.1,4.2Hz,1H),1.65–1.52(m,1H),1.47(d,J＝6.8Hz,3H),1.43–1.37(m,1H),1.35–1.19(m,16H),1.14(t,J＝6.5Hz,6H),0.97(dd,J＝12.5,6.5Hz,6H),0.92–0.78(m,9H).
HRMS(ESI)calcd for C 41 H 69 N 5 NaO 9 [M + Na + ]798.4987；found,798.4970.
1.3 preparation of probes
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanoylamino) -4-oxobutan-2-acetate and 3.8mg of valerenic acid, 8.6mg (0.03375 mmol) Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA is added without removing the ice bath and stirred overnight. Concentrating under reduced pressure, and purifying by column chromatography to obtain product Probe 1.
1 H NMR(300MHz,Chloroform-d)δ7.31–7.27(m,1H),6.84(d,J＝8.8Hz,1H),6.09(dd,J＝6.1,1.6Hz,1H),5.66(dd,J＝10.0,2.1Hz,1H),5.30–5.16(m,2H),5.02(d,J＝11.1Hz,1H),4.95(dd,J＝8.8,3.7Hz,1H),4.87–4.77(m,1H),4.39(s,1H),3.89(d,J＝11.6Hz,1H),3.80(dd,J＝11.5,4.3Hz,1H),3.54(d,J＝11.0Hz,1H),3.49(s,1H),3.11(s,3H),2.94(s,3H),2.66–2.55(m,J＝4.0Hz,4H),2.48(ddd,J＝14.5,10.0,4.8Hz,1H),2.34–2.21(m,1H),2.06(d,J＝2.3Hz,1H),2.01(d,J＝4.8Hz,3H),1.99(d,J＝2.5Hz,1H),1.66(ddd,J＝9.3,6.1,3.1Hz,2H),1.47(d,J＝6.8Hz,3H),1.17(d,J＝6.5Hz,3H),0.99(d,J＝6.5Hz,3H),0.94(d,J＝6.6Hz,3H),0.89(d,J＝6.5Hz,3H),0.81(d,J＝6.7Hz,3H).
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 4.6mg heptynoic acid, 8.6mg (0.03375 mmol) Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA is added without removing the ice bath and stirred overnight. Concentrating under reduced pressure, and purifying by column chromatography to obtain product Probe2.
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝2.2Hz,1H),6.92(d,J＝8.8Hz,1H),6.09(dd,J＝6.0,1.6Hz,1H),5.66(dd,J＝10.1,2.2Hz,1H),5.24(dt,J＝14.0,5.7Hz,2H),5.02(d,J＝11.1Hz,1H),4.95(dd,J＝8.9,3.5Hz,1H),4.86–4.76(m,1H),4.39(s,1H),3.93–3.78(m,2H),3.58(s,1H),3.11(s,3H),2.93(s,3H),2.49(td,J＝9.9,5.0Hz,1H),2.40(t,J＝7.4Hz,2H),2.24(td,J＝6.9,2.9Hz,4H),2.05(s,1H),2.02(s,3H),1.96(t,J＝2.7Hz,1H),1.78(q,J＝7.6,7.2Hz,2H),1.64(q,J＝7.6Hz,4H),1.47(d,J＝6.8Hz,3H),1.17(d,J＝6.5Hz,3H),0.99(d,J＝6.5Hz,3H),0.94(d,J＝6.6Hz,3H),0.89(d,J＝6.4Hz,3H),0.81(d,J＝6.6Hz,3H).
13 C NMR(75MHz,Chloroform-d)δ174.59,173.68,171.16,169.87,168.88,154.19,125.33,84.04,71.85,68.61,68.55,59.20,58.59,58.12,56.93,54.16,51.99,36.57,36.32,33.21,30.76,30.45,28.02,27.15,24.93,24.10,23.13,21.92,21.08,18.84,18.36,18.24,17.42,16.92.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-yl acetate and 7.5mg of n-14-alkynoic acid, 8.6mg (0.03375 mmol) Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA is added without ice-bath stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography to obtain product Probe 3.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 7.8mg of photocrosslinked alkynoic acid, 8.6mg (0.03375 mmol) of Bop-Cl are dissolved in 1ml DCM and 6.5. Mu.l TEA are added without removing the ice bath and stirring overnight. Concentrating under reduced pressure, and purifying by column chromatography to obtain product Probe 4.
1 H NMR(300MHz,Chloroform-d)δ7.29(d,J＝5.5Hz,1H),6.89(d,J＝8.8Hz,1H),6.09(dd,J＝6.0,1.6Hz,1H),5.66(dd,J＝10.0,2.2Hz,1H),5.29–5.18(m,2H),5.06–4.99(m,1H),4.95(dd,J＝8.9,3.3Hz,1H),4.82(dddd,J＝8.9,6.9,4.4,1.9Hz,1H),4.45–4.31(m,1H),3.93–3.73(m,2H),3.55(d,J＝11.0Hz,1H),3.11(s,3H),2.92(s,3H),2.48(ddd,J＝14.5,10.1,4.6Hz,1H),2.35(dd,J＝8.7,6.7Hz,2H),2.27(d,J＝6.0Hz,1H),2.17(dt,J＝6.8,3.4Hz,1H),2.01(s,3H),2.00–1.94(m,2H),1.65(t,J＝7.4Hz,4H),1.47(dd,J＝7.7,3.9Hz,5H),1.39–1.20(m,19H),1.17(d,J＝6.5Hz,3H),1.08(d,J＝7.6Hz,1H),0.99(d,J＝6.5Hz,3H),0.94(d,J＝6.6Hz,3H),0.89(d,J＝6.4Hz,3H),0.81(d,J＝6.6Hz,3H). 13 C NMR(75MHz,Chloroform-d)δ174.62,174.18,171.21,169.85,168.89,154.16,125.33,83.46,71.88,68.89,68.56,60.40,59.19,58.60,58.12,56.96,54.09,51.96,36.59,36.29,33.85,32.82,31.81,30.76,30.44,29.69,29.43,29.36,29.17,28.46,27.15,25.11,24.92,23.82,23.15,22.74,21.93,21.06,18.84,18.37,17.95,17.41,16.93,14.20.
HRMS(ESI)calcd for C 46 H 73 N 7 NaO 9 [M + Na + ]890.5362；found,890.5345.
13mg (0.0225 mmol) of 4- ((1- ((2S, 4S) -4-hydroxy-2- ((S) -2-methyl-5-oxo-2, 5-dihydro-1H-pyrrole-1-carbonyl) pyrrolidin-1-yl) -3-methyl-1-oxobutan-2-yl) (methyl) amino) -3- (4-methyl-2- (methylamino) pentanamido) -4-oxobutan-2-acetate and 15.3mg of biotin-derived acid, 8.6mg (0.03375 mmol) Bop-Cl are dissolved in 1ml DCM at 0℃and 6.5. Mu.l TEA is added without removing the ice bath and stirring overnight. Concentrating under reduced pressure, and preparing by HPLC to obtain product Probe 5.
EXAMPLE 2 measurement of Microcolin H and derivative molecules for Activity on tumor cells
Preparing 50000 cancer cells/ml cell suspension, adding into 96-well plate cell culture dish, respectively adding the compound synthesized in example 1, each test concentration being 6 holes, placing at 37deg.C, 5% CO 2 Culturing for 48 hours under saturated humidity, measuring absorbance A value at 450nm wavelength of an enzyme-linked detector by using a CCK8 method, and calculating the inhibition effect of the compound on the tested cancer cells.
TABLE 1 inhibitory Activity of Microcolin H against various tumor cells
Cells HuH-7 HepaRG HGC-27 AGS MKN-28 A549 H460 AsPC-1 PANC-1 Hela
IC 50 (nM) 1.3 5.7 3.5 8.6 50.2 0.7 10.1 3.5 2.5 132.6
The activity test shows that the marine polypeptide Microcolin H has nanomolar growth inhibition activity on human liver cancer cells (HuH-7 and HepaRG), human gastric cancer cells (HGC-27, AGS and MKN-28), human lung cancer cells (A549 and H460), human pancreatic cancer cells (AsPC-1 and PANC-1) and human cervical cancer cells (Hela).
TABLE 2 inhibitory Activity of Microcolin H derivatives on tumor cells HGC-27
Compounds of formula (I) Probe1 Probe2 Probe3 Probe4 Probe5 MIC-1 MIC-2 MIC-3 MIC-4 MIC-5 MIC-6
IC 50 (nM) 1300 270 36 8.6 >10000 9000 120 75 2.5 1.3 0.9
Note that: the data in tables 1 and 2 are the average of three results.
The growth inhibition activity evaluation of the derivative MIC (1-6) of the synthesized marine polypeptide Microcolin H and the Probe molecule Probe (1-5) on human gastric cancer cells (HGC-27) shows that the longer the carbon chain, the stronger the influence of the fatty acid chain carbon number on the derivative and the Probe molecule on the growth inhibition of the derivative and the Probe on tumor cells.
Example 3 investigation of Microcolin H direct action target in tumor cells
According to the invention, probe molecule Probe 5 synthesized in example 1 is utilized to incubate with cell lysate or living cells, after incubation, an alkynyl part of the Probe and a reagent rhodamine-azide are subjected to click chemistry reaction, a target protein combined with Microcolin H is provided with a rhodamine fluorescent tag, and after SDS-PAGE separation, a target protein of Microcolin H can be found through fluorescent imaging, and the specific experimental process is as follows:
different concentrations of probe molecules (probe 5 concentration: 0.01. Mu.M, 0.1. Mu.M, 1.0. Mu.M) were incubated with gastric cancer cell HGC lysate, while the control group (25. Mu.M concentration) was competed with 25. Mu.M of the parent molecule Microcolin H as cold probe, after 1 hour, the fluorescent reagents rhodamine-azide (10. Mu.M), 100. Mu.M TBTA, 1mM TCEP and 1mM CuSO were added 4 Click reactions were pooled for one hour and then electrophoresed by SDS-PAGE and exposed to light using a chemiluminescent gel imaging system to reveal fluorescent signal bands.
As a result, as shown in FIG. 1, the probe molecules can be labeled with a clear protein band (about 30 kDa) with a high specificity, even if the probe molecule concentration is 10nM, and the parent molecule Microcolin H can compete very effectively for the probe-labeled band in the competition group experiment, which indicates that the drug Microcolin H has very high target selectivity and effectiveness.
Example 4 target enrichment Pull-Down experiments and Western-blot validation
The frozen cell pellet was resuspended in PBS containing 0.1% Triton X-100 (Sigma-Aldrich), sonicated, and separated into soluble and insoluble fractions by ultracentrifugation at 15000g for 45 minutes. Using BCA protein assay (Pierce TM BCA protein assay kit, thermo Fisher Scientific) soluble protein concentration was measured on a microplate reader (Bio-Rad).
Lysates were adjusted to 2mg/mL and treated with 25. Mu.M Microcolin H or DMSO, then labeled with 200nM probe 5 or DMSO, then 300. Mu.M biotin-azide, 100. Mu.M TBTA, 1mM TCEP and 1mM CuSO were added 4 The combination was performed for 1 hour. After this reaction, the proteome was extracted with chloroform-methanol to remove excess reagents.
The protein intermediate phase was washed with cold methanol, dissolved in 1.2% SDS/PBS, and diluted 5-fold with PBS. The solubilized protein was incubated with streptavidin beads for 3 hours at room temperature with rotation. The streptavidin beads were then washed three times with 5mL PBS and three times with 5mL water before being transferred to a spiral Eppendorf tube.
The enriched proteins are added into Loading buffer, and then subjected to SDS-PAGE, and then subjected to in-gel digestion, and protein mass spectrum LC-MS/MS is used for identifying target proteins as phosphatidyl alcohol transporter alpha (PITPNA) and phosphatidyl alcohol transporter beta (PITPNB).
And simultaneously, carrying out Western-blot verification on the target proteins enriched on the gel.
The result is shown in figure 2, the target proteins enriched by the probe molecules are PITPNB and PITPNA through Western-blot verification, and the competitive group and the blank group show that the PITPNB and the PITPNA are not enriched, and the result proves that the probe molecules can be effectively and covalently combined with the PITPNB and the PITPNA and can be competed by Microcolin H, and the direct acting target of the drug Microcolin H is PITPNB and PITPNA.
The target proteins were identified as phosphatidyl alcohol transporter α (PITPNA) and phosphatidyl alcohol transporter β (PITPNB).
EXAMPLE 5 investigation of the binding site of Microcolin H to target protein
The sequence of human PITPNB (number: P48739) was downloaded from the Uniport database, and the sequence of murine PITPNB (PDB ID:2A 1L) was found to be the highest in sequence identity by sequence alignment, thus murine PITPNB was selected as the template. Homologous modeling of the human PITPNB is performed automatically through the SWISS-Model website.
Covalent docking was performed using the CovDock version of Schrodinger 2020 software. The cysteines 94, 191, 187, and 230 were used to create a lattice with a radius of 15A for covalent docking of the PITPNB to MHC. The reaction type is Michael addition. All other parameters of the CovDock docking procedure are set to default values.
It was found by binding pattern analysis that Microcolin H formed a covalent bond with CYS187 in PITPNA, PITPNB. The space matching degree of the binding pocket of Microcolin H and PITPNB is higher; interaction level PITPNA residue LYS164 forms a hydrogen bond with Microcolin H, while PITPNB residue GLU180 forms two hydrogen bonds with Microcolin H. The binding docking score and binding pattern initially indicate that compared to the PITPNA protein, PITPNB and Microcolin H are covalently bound better.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims (5)

1. The polypeptide Microcolin H derivative is characterized in that the structure of the polypeptide Microcolin H derivative is shown as a formula I:
（Ⅰ）
wherein R is C1-C14 saturated alkane or C1-C14 alkynyl.
2. The polypeptide Microcolin H derivative according to claim 1, characterized in that it is selected from any one of the following structures:
。
3. use of a polypeptide Microcolin H derivative according to any one of claims 1-2 for the preparation of a medicament, characterized in that the medicament is a medicament for the treatment of gastric cancer.
4. Use of a phosphatidyl alcohol transporter as a drug target for screening an anti-cancer drug, characterized in that the drug is a polypeptide Microcolin H derivative according to any one of claims 1-2.
5. The use of a phosphatidylol transporter according to claim 4 as a drug target for screening for an anti-cancer drug, wherein the binding site of the anti-cancer drug to the phosphatidylol transporter is Cys187.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091368A (en) * 1990-08-08 1992-02-25 Harbor Branch Oceanographic Institution, Inc. Biologically active compounds from blue-green algae
CN115721700A (en) * 2022-11-23 2023-03-03 兰州大学 Application of polypeptide Microcolin H and analogue thereof in preparation of autophagy inducer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5091368A (en) * 1990-08-08 1992-02-25 Harbor Branch Oceanographic Institution, Inc. Biologically active compounds from blue-green algae
CN115721700A (en) * 2022-11-23 2023-03-03 兰州大学 Application of polypeptide Microcolin H and analogue thereof in preparation of autophagy inducer

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Cytotoxic Microcolin Lipopeptides from the Marine Cyanobacterium Moorea producens;Yu HB, et al.;《J Nat Prod》;第82卷(第9期);摘要、图表1、表6、第2615页右栏末段 *
Hippo pathway regulation by phosphatidylinositol transfer protein and phosphoinositides;Fulong Li et al.;《Nat Chem Biol》;第18卷(第10期);全文 *
Microcolins A and B, New Immunosuppressive Peptides from the Blue-Green Alga Lyngbya majuscula;Frank E. Koehn et al.;《J. Nat. Prod》;第55卷(第5期);全文 *

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