CN105884765A - N-[beta]-carboline-3-formyl-N'-amino acid acylhydrazine, nano structure, activity and application thereof - Google Patents
N-[beta]-carboline-3-formyl-N'-amino acid acylhydrazine, nano structure, activity and application thereof Download PDFInfo
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- CN105884765A CN105884765A CN201410562154.XA CN201410562154A CN105884765A CN 105884765 A CN105884765 A CN 105884765A CN 201410562154 A CN201410562154 A CN 201410562154A CN 105884765 A CN105884765 A CN 105884765A
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Abstract
The invention discloses two kinds of N-[beta]-carboline-3-formyl-N'-amino acid acyl hydrazines which are obtained through active pocket butt-connection of an average structure of P-selectin established by the inventor, wherein the amino acid acyl group is an L-serine acyl group when R is a hydroxymethyl group, and the amino acid acyl group is an L-histidine acyl group when R is 1H-imidazole-4-yl. The invention also discloses nano structures of the compounds, an inhibition effect of the compounds on blood platelet expressing the P-selectin, and also includes the application of the compounds in preparation of a P-selectin inhibitor.
Description
Technical field
The present invention relates to the active pocket analyzing the palatelet-selectin average structure that inventor the sets up structural requirement to smaller ligand, with DS software, the active pocket of the little molecule and the average structure of palatelet-selectin with antithrombotic acitivity is docked, search the little molecule of known antithrombotic of the active pocket space requirement meeting average structure, N-B-carboline-3-formoxyl-N '-(when R is methylol, amino acid acyl is Serine acyl group to amino acid acyl hydrazine to 2 kinds of locking following formula, when R is 1H-imidazol-4 yl, amino acid acyl is L-Histidine acyl group), relate to their nanostructured, relate to them and platelet is expressed the inhibitory action of palatelet-selectin, thus the present invention illustrates they application in preparing palatelet-selectin inhibitor.The invention belongs to biomedicine field.
Technical background
Palatelet-selectin exists with lysotype palatelet-selectin (also known as sP-selectin) and two kinds of forms of insoluble type palatelet-selectin (also known as P-selectin).Insoluble type palatelet-selectin under normal condition is stored in hematoblastic α-grain (α-granule).Under Platelet Activation, thrombosed stimulation makes cell exocytosis (exocytosis) order about the α-grain containing insoluble type palatelet-selectin and moves quickly into the hematoblastic skin covering of the surface of activation from hematoblastic theca interna.On platelet surface film, insoluble type palatelet-selectin is expressed, and cuts and is converted into lysotype palatelet-selectin and enter blood circulation.Knowing at present, in blood circulation, the morbidity of the multiple fatal disease such as the rising of lysotype palatelet-selectin level and inflammation, thrombosis, cancer, and cancerometastasis is relevant.Generally acknowledge at present, find the pure inhibitor (hereinafter referred to as palatelet-selectin inhibitor) of lysotype palatelet-selectin, be the key areas of new drug research.So far there is no successful story.
Inventor's long campaigns antithrombotic reagent is studied, and understands and has multiple pathogenic pathways to may result in thrombosis.Great majority are thought in the industry, and platelet membrane glycoproteins GPIIb/IIIa activates, and is thrombotic final common path.Inventor knows, this is incomplete understanding.It practice, thrombosis includes two kinds.A kind of be cross-linked by Fibrinogen and platelet by platelet the thrombosis constituted, i.e. platelet-fibrin former-platelet (platelet-fibrinogen-platelet, PFP) type thrombosis, GPIIb/IIIa activation cause.A kind of is to be cross-linked, with leukocyte, the thrombosis constituted by Fibrinogen by platelet, i.e. platelet-fibrin former-leukocyte (platelet-fibrinogen-leukocyte, PFL) type thrombosis, is caused by the lysotype palatelet-selectin in intra platelet free calcium to blood.As GPIIb/IIIa inhibitor, palatelet-selectin inhibitor is the member of antithrombotic reagent.Find palatelet-selectin inhibitor, need the theoretical model used.nullThen，The average structure of inventor's first palatelet-selectin that can be used for computational screening palatelet-selectin inhibitor disclosing Fig. 1 in the world is (see Haimei Zhu，Yuji Wang，Ming Zhao，Jianhui Wu，Xiaoyi Zhang，Guodong Yang，Shiqi Peng，Energy minimized crystal structures of P-selectins based on molecular dynamics simulation:Leading to two average structures capable of designing anti-thrombotic agents.MedChemComm 2013，4，1066-1072).Although inventor once utilized the crystal structure of palatelet-selectin to add glycoprotein receptor GPIIb/IIIa and screens the double inhibitor of following structure, but they are not the pure inhibitor of lysotype palatelet-selectin.Additionally, their structure can not be docked to globality can be used among the active pocket of the average structure of the palatelet-selectin of computational screening palatelet-selectin inhibitor.
AA in structural formula1During for L-Tyr residue, AA2For L-Phe residue, or L-Ser residue, or L-Val residue；AA in formula1During for L-Leu residue, AA2For L-Phe residue, or L-Ser residue, or L-Val residue；AA in formula1During for L-Pro residue, AA2For L-Phe residue, or L-Ser residue, or L-Val residue.
Hundreds of compounds with antithrombotic acitivity that oneself is invented by inventor and inventor in the world disclosed in can be used for computational screening palatelet-selectin inhibitor the active pocket of palatelet-selectin average structure dock; N-B-carboline-3-formoxyl-the N '-amino acid acyl hydrazine (when R is methylol, amino acid acyl is Serine acyl group, and when R is 1H-imidazol-4 yl, amino acid acyl is L-Histidine acyl group) locking following structure is the pure inhibitor of lysotype palatelet-selectin.Then, the present invention is inventors herein proposed.
Summary of the invention
First content of the present invention is the active pocket analyzing the palatelet-selectin average structure that inventor the sets up structural requirement to smaller ligand, with DS software, the active pocket of the little molecule and the average structure of palatelet-selectin with antithrombotic acitivity is docked, search the little molecule of known antithrombotic of the active pocket space requirement meeting average structure, N-B-carboline-3-formoxyl-N '-(when R is methylol, amino acid acyl is Serine acyl group to amino acid acyl hydrazine to the antithrombotic compound of locking following formula, when R is 1H-imidazol-4 yl, amino acid acyl is L-Histidine acyl group) it is outstanding part, thus be outstanding palatelet-selectin inhibitor.
Second content of the present invention is to evaluate N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine (when R is methylol, amino acid acyl is Serine acyl group, and when R is 1H-imidazol-4 yl, amino acid acyl is L-Histidine acyl group) platelet is expressed the inhibitory action of lysotype palatelet-selectin.
3rd content of the present invention is the nanostructured measuring N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine (when R is methylol, amino acid acyl is Serine acyl group, and when R is 1H-imidazol-4 yl, amino acid acyl is L-Histidine acyl group).
Accompanying drawing explanation
Fig. 1. the structure chart of palatelet-selectin inhibitor of the present invention.
Fig. 2. inventor in the world disclosed in can be used for the palatelet-selectin 1 (dirty-green) of computational screening palatelet-selectin inhibitor and the average structure of 2 (green) and the binding site .Ca of SLex2+Represent with orange chromosphere.
Fig. 3. inventor in the world disclosed in can be used for the Pharmacophore Model of smaller ligand of requirement of active pocket of average structure of palatelet-selectin of computational screening palatelet-selectin inhibitor.
Fig. 4 .N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine (when R is methylol, amino acid acyl is Serine acyl group, and when R is 1H-imidazol-4 yl, amino acid acyl is L-Histidine acyl group) mates best with Pharmacophore Model.
(R is 1H-imidazol-4 yl to-N '-group amino acid acyl hydrazine to Fig. 5 .N-(1-methyl-ss-carboline-3-formoxyl); left) and the details docked with the active pocket of the average structure of palatelet-selectin of N-(1-methyl-ss-carboline-3-formoxyl)-N '-Silk Amino Acids acid hydrazide (R is methylol, the right side).
Fig. 6 .N-B-carboline-3-formoxyl-N '-L-Histidine acid hydrazide amino acid acyl hydrazine (R is 1H-imidazol-4 yl, left) and N-B-carboline-3-formoxyl-N '-Serine acid hydrazide (right) are in pure water solution 1 × 10-6Transmission electron microscope photo under M concentration.
Detailed description of the invention
In order to the present invention is expanded on further, a series of embodiment is given below.These embodiments are entirely illustrative, and they are only used for being specifically described the present invention, are not construed as limitation of the present invention.
Embodiment 1 docking obtains N-(1-methyl-ss-carboline-3-formoxyl)-N '-amino acid acyl hydrazine
The active pocket of the average structure of palatelet-selectin based on inventor's foundation, inventor constructs Pharmacophore Model based on receptor structure, confirms that smaller ligand structure should meet 5 pharmacophore characteristic elements of Fig. 2., including 1 hydrophobic centers (No. 1 grid ball), 2 hydrogen bond receptors (No. 2 grid balls), and 2 carbonium centers (No. 3 grid balls).According to these structural requirements, the micromolecular compound storehouse with antithrombotic acitivity is screened, determines that N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine is satisfactory ligand classes.The amino acid acyl of N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine is Serine acyl group and L-Histidine acyl group mates best (see Fig. 3) with Pharmacophore Model.
N-(1-methyl-ss-carboline-3-formoxyl)-N '-Serine acid hydrazide with N-(1-methyl-ss-carboline-3-formoxyl)-N '-L-Histidine acid hydrazide respectively active pocket with the average structure of palatelet-selectin dock, docking result show that the interaction details of they and palatelet-selectin is shown in Fig. 4.The details of docking is, the Asn residue of their 1 '-O and palatelet-selectin 105 forms the Lys residue side chains NH of hydrogen bond, 4 '-O and palatelet-selectin 1113 +Form hydrogen bond, the side chain carboxyl group center of negative charge of 107 Glu residues of the center of positive charge of 2 '-N and palatelet-selectin is mated, the side chain carboxyl group center of negative charge of 88 Glu residues of the center of positive charge of 5 '-N and palatelet-selectin is mated, phenyl ring mates with hydrophobic centers, and 2-N forms hydrogen bond with the side chain carbonyl of 82 Asn residues of palatelet-selectin.It addition, the L-Histidine side chain N of the L-Histidine side chain N of N-(1-methyl-ss-carboline-3-formoxyl)-N '-L-Histidine acid hydrazide also with 84 Lys residue side chains NH of palatelet-selectin3 +Forming hydrogen bond, the Serine side chain OH of N-(1-methyl-ss-carboline-3-formoxyl)-N '-Serine acid hydrazide also forms hydrogen bond with the side chain carboxyl group of 88 Glu of palatelet-selectin.
Experimental example 1 measures the transmission electron microscope photo of N-(1-methyl-ss-carboline-3-formoxyl)-N '-amino acid acyl hydrazine
By N-(1-methyl-ss-carboline-3-formoxyl)-N '-amino acid acyl hydrazine (amino acid acyl is Serine acyl group, and amino acid acyl is L-Histidine acyl group) according to 1 × 10-6The concentration configuration pure water solution of M, is layered on uniformly on copper mesh, observes the self-assembly property of compound under transmission electron microscope (TEM, JEM-1230, JEOL).The photo obtained such as Fig. 5.Result shows, they can form the nano-particle of a diameter of 20-100nm in water.
Experimental example 2 measures N-(1-methyl-ss-carboline-3-formoxyl)-N '-amino acid acyl hydrazine inhibitory action to palatelet-selectin
1. draw palatelet-selectin standard curve
Description by palatelet-selectin ELISA kit (U.S. CUSABIO CO), use progressively dilution method to be coated plate at 96 hole enzyme marks and prepare palatelet-selectin standard solution, every hole solution amount is 50 μ L, the final concentration of 600ng/mL of palatelet-selectin, 300ng/mL, 150ng/mL, 75ng/mL, 37.5ng/mL, 18.75ng/mL and 9.37ng/mL.Microplate reader measures OD value.Each concentration is repeated 2 times, and averages.Draw palatelet-selectin standard curve.
2. prepare platelet sample
Blood is taken through carotid artery after the anesthesia of healthy SD male rat (body weight 180-220g) urethane.Blood 3.8% sodium citrate aqueous solution anticoagulant, every 4500 μ L whole bloods add 500 μ L sodium citrate aqueous solutions.Centrifugal blood 20 minutes (2000-3000 rev/min), obtains platelet rich plasma.Take the diluent mixing in 600 μ L platelet rich plasmas and 5400 μ L test kits, obtain platelet sample use to be determined.
3. prepare palatelet-selectin and measure sample
By 960 μ L platelet sample and 20 μ L NS (blank) or 20 μ LN-[(1S in PC pipe; 3S)-1-methyl isophthalic acid; 2; 3; 4-tetrahydro-beta-carboline-3-formoxyl] (amino acid acyl is Serine acyl group to-N '-amino acid acyl hydrazine; and amino acid acyl is L-Trp acyl group) NS solution mixing, hatch 5min in 37 DEG C of constant incubators.Add the NS solution (0.15g/L) of 20 μ L AA the most inward, then hatch 3min.
4. measure palatelet-selectin content
It is coated toward 96 hole enzyme marks at the bottom of the hole of plate and adds 100 μ L blanks or 100 μ L palatelet-selectins mensuration sample.120min is hatched in shrouding 37 DEG C of constant incubators of film shrouding juxtaposition.Taking shrouding film off, suck culture fluid, the culture fluid filter paper of residual blots.Residue 200+100 μ L weak wash (from the cleaning mixture distilled water diluting in test kit 20 times) is washed, and discards after standing 30 seconds.This washing operation is repeated 5 times, and pats dry.Add 100 μ L NS toward blank control wells, measure hole toward palatelet-selectin and add the biotin labelled antibodies in 100 μ L test kits.60min is hatched in shrouding 37 DEG C of constant incubators of film shrouding juxtaposition.Taking shrouding film off, suck culture fluid, the culture fluid filter paper of residual blots.Residue 200+100 μ L weak wash (from the cleaning mixture distilled water diluting in test kit 20 times) is washed, and discards after standing 30 seconds.This washing operation is repeated 3 times, and pats dry.In residue, add the Horseradish peroxidase-conjugated avidin in 100 μ L test kits, in shrouding 37 DEG C of constant incubators of film shrouding juxtaposition, hatch 60min.Taking shrouding film off, suck culture fluid, the culture fluid filter paper of residual blots.Residue 200+100 μ L weak wash (from the cleaning mixture distilled water diluting in test kit 20 times) is washed, and discards after standing 30 seconds.This washing operation is repeated 5 times, and pats dry.In residue, add the substrate solution in 90 μ L test kits, shake mixing, 37 DEG C of lucifuge colour developing 20min gently.Then every hole adds the stop buffer in 50 μ L test kits, color development stopping reaction (blueness turns yellow).Under 450nn wavelength, measure the absorbance (OD value) in each hole after 15min, obtained the palatelet-selectin content in each hole by palatelet-selectin standard curve.Result shows that (amino acid acyl is Serine acyl group to N-(1-methyl-ss-carboline-3-formoxyl)-N '-amino acid acyl hydrazine, is called for short compound A；And amino acid acyl is L-Histidine acyl group, it is called for short compound B) can substantially suppress palatelet-selectin to express (table 1).
Table 1 compound A and B to palatelet-selectin expression (Ng/ml) impact
N=4；A) with normal saline than p ＜ 0.01.
Claims (4)
1. the palatelet-selectin inhibitor of structure below, N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine,
When R is methylol, amino acid acyl is Serine acyl group, and when R is 1H-imidazol-4 yl, amino acid acyl is L-group ammonia
Acid acyl group.
2. obtain the palatelet-selectin inhibitor of claim 1, the docking side of N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine
Method, is characterized in the active pocket docking of the average structure of the palatelet-selectin utilizing inventor to set up, and the method is by following steps
Constitute:
(1) active pocket of the palatelet-selectin average structure that analysis inventor the sets up structural requirement to smaller ligand；
(2) with DS software, the active pocket of the little molecule and the average structure of palatelet-selectin with antithrombotic acitivity is docked,
Search the little molecule of known antithrombotic of the active pocket space requirement meeting average structure；
(3) the palatelet-selectin inhibitor of claim 1 is locked.
3. the palatelet-selectin inhibitor of claim 1, the nanostructured of N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine.
4. the palatelet-selectin inhibitor of claim 1, N-B-carboline-3-formoxyl-N '-amino acid acyl hydrazine selects at preparation P-
Application in element inhibitor.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106588928A (en) * | 2015-10-16 | 2017-04-26 | 首都医科大学 | Novel heptacyclic compound and synthesis, activity evaluation and application thereof |
| CN108969770A (en) * | 2017-05-31 | 2018-12-11 | 首都医科大学 | 1- methyl -3- methylol-tetrahydro-beta-carboline of dipeptides modification, synthesis and application |
| CN110577582A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | LDV modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof |
| CN112010936A (en) * | 2019-05-28 | 2020-12-01 | 首都医科大学 | Ethyl GRPAK modified bis-carbolino piperazine diketone and preparation, activity and application thereof |
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| CN102477034A (en) * | 2010-11-30 | 2012-05-30 | 首都医科大学 | 1-methyl-beta-carboline-3-carboxylic acid modified by amino acid as well as synthesizing method and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106588928A (en) * | 2015-10-16 | 2017-04-26 | 首都医科大学 | Novel heptacyclic compound and synthesis, activity evaluation and application thereof |
| CN106588928B (en) * | 2015-10-16 | 2018-07-27 | 首都医科大学 | Heptacyclic compound, synthesis, activity rating and application |
| CN108969770A (en) * | 2017-05-31 | 2018-12-11 | 首都医科大学 | 1- methyl -3- methylol-tetrahydro-beta-carboline of dipeptides modification, synthesis and application |
| CN108969770B (en) * | 2017-05-31 | 2021-07-27 | 首都医科大学 | Dipeptide modified 1-methyl-3-hydroxymethyl-tetrahydro-beta-carboline, synthesis and application thereof |
| CN110577582A (en) * | 2018-06-08 | 2019-12-17 | 首都医科大学 | LDV modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof |
| CN110577582B (en) * | 2018-06-08 | 2022-09-02 | 首都医科大学 | LDV modified heptacyclic aldehyde, synthesis, antithrombotic activity and application thereof |
| CN112010936A (en) * | 2019-05-28 | 2020-12-01 | 首都医科大学 | Ethyl GRPAK modified bis-carbolino piperazine diketone and preparation, activity and application thereof |
| CN112010936B (en) * | 2019-05-28 | 2022-04-22 | 首都医科大学 | Ethyl GRPAK modified bis-carbolino piperazine diketone and preparation, activity and application thereof |
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