CN107029243A - A kind of double acylhydrazone connecting keys of polypeptide bridging are applied to the delivering of aldehyde drug derivative - Google Patents
A kind of double acylhydrazone connecting keys of polypeptide bridging are applied to the delivering of aldehyde drug derivative Download PDFInfo
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Abstract
A kind of double acylhydrazone connecting keys of polypeptide bridging are applied to the delivering of aldehyde drug derivative, are related to the double acylhydrazone connecting keys of pH response types.The invention be related to a class newly be connected chemically key, it is different from traditional single acylhydrazone key, the connecting key passes through the synergistic stability effect between two acylhydrazone keys, so that such PTA linkers (tumor tissues under neutral (blood circulation) or acid condition, inclusion body, lysosome) all there is the stability of superelevation.After the polypeptide chain of the double acylhydrazone keys of bridging is by specific protein enzyme hydrolysis, the synergistic stability effect between two acylhydrazone keys disappears, therefore can have good response again in acid condition.Using PTA linkers as connecting key, two kinds of prodrugs that extracellular and intracellular polypeptides chain pinpoints hydrolysis are designed, two kinds of prodrugs can promptly discharge aldehyde drug derivative molecule and kill tumour cell.
Description
Technical field
The present invention relates to the double acylhydrazone connecting keys of pH response types are related to, connected more particularly, to a kind of double acylhydrazones of polypeptide bridging
Key is applied to the delivering of aldehyde drug derivative.
Background technology
Malignant tumour be it is a kind of it is serious threaten human health common disease and frequently-occurring disease, be cause main causes of death it
One.Current chemotherapy is one of main path of clinical treatment tumour, is played an important role always in oncotherapy, but greatly
Most chemotherapeutics are because non-selectivity, toxic side effect are metabolized fast, the low shortcoming of drug effect greatly, in vivo and limit clinical practice.
Overcome drawbacks described above, the chemotherapeutics for finding new, effective treatment tumour is still the focus of researcher's concern.Anti-tumor target
It is exactly one of which to the design of prodrug.
Neoplasm targeted therapy accurately selects carcinogenic site based on prodrug, makes tumor cell specific dead, without ripple
And the normal tissue cell around tumour.This method depends on the connecting key of rational design stimuli responsive (as by enzyme
Overexpression, redox micro-environmental variation, pH value change etc.).At these for designing in the connecting key of prodrug, pH sensitivities
Acylhydrazone connecting key causes extensive concern always, on the one hand can be straight in a mild condition mainly due to aldehyde group modified medicine
Connect be coupled to the part containing hydrazides construct acylhydrazone key based on prodrug, on the other hand, aldehyde radical is the chemical official of the low polarity of a class
It can roll into a ball (compared to hydroxyl, carboxyl, amino), the hydrophobic upper aldehyde radical of cancer therapy drug modification does not change the diffusivity of medicine substantially
Matter.Moreover, recent studies have indicated that, aldehyde radical can be transformed into carboxyl in the presence of aldehyde dehydrogenase (ALDH), and carboxyl is in cell
Interior (pH=7.4) is negatively charged to be made it difficult to escape from cell.Therefore therapeutic reagent and developing agents include aldehyde radical
Label can show the cell retention time of superelevation to reach more preferable curative effect.Although many research report acylhydrazone keys exist
Hydrolysis dynamics are than relatively inert under neutrallty condition, but acylhydrazone key is thermodynamically unstable in weak solution, therefore very
It is difficult to preserve for a long time.In addition, in physiological conditions, acylhydrazone key, which can be hydrolyzed slowly, to be caused to discharge medicine too early to produce malicious secondary work
With so that it is difficult to be applied in life system.Nearest research report antibody drug coupling is related to and made using acylhydrazone connecting key
Obtain myotarg and inotuzumab ozogamicin and withdraw (Chari, R.V. with the clinical phase III from the market respectively；
Miller,M.L.；Widdison,W.C.Angew.Chem.Int.Ed.2014,53,3796).
In order to overcome this limitation of acylhydrazone connecting key.At present, two methods mainly can be applied to acylhydrazone key
Stability：1) the neighbouring chemical group of regulation and control aldehyde radical (Hamann, P.R.et al.Bioconjugate Chem.2002,13,
47)；2) other nucleopilic reagent (Casi, G. are utilized；Huguenin-Dezot,N.；Zuberbuhler,K.；
Scheuermann,J.；Neri,D.J.Am.Chem.Soc.2012,134,5887).Although however, these methods can be used for
Regulate and control the stability of acylhydrazone key, but this method for improving acylhydrazone key stability must be to sacrifice its response in target spot
For cost.And in actual applications, while acquisition stability and response always can not be in traditional single acylhydrazone key (SA-
Linkers realized in).
The content of the invention
The first object of the present invention is the double acylhydrazone connecting keys (PTA-linkers) for providing a class polypeptide bridging.
The second object of the present invention is the molecule for providing double acylhydrazone connecting keys (PTA-linkers) of a class polypeptide bridging
Skeleton formula.
The third object of the present invention is that providing prodrug design of the class based on double acylhydrazone connecting keys (PTA-linkers) leads to
Formula.
The fourth object of the present invention is to provide based on double acylhydrazone key (PTA-linkers) prodrugs (D1And D2) conjunction
Into method.
Double acylhydrazone connecting keys of the one class polypeptide bridging, are polypeptide chain bridging and double acylhydrazone keys, with protease hydrolytic
With sour water solution dual responsiveness, stability and response are had concurrently in biotic environment, the biotic environment include blood circulation,
Extracellular environment, intracellular environment etc..
The molecular skeleton formula of double acylhydrazone connecting keys (PTA-linkers) of the one class polypeptide bridging is：
Wherein：(x) any active peptides sequence is represented, can be degraded by specific proteases；(M) representing N-terminal can be further
The arbitrary amino acid being chemically modified, the amino acid includes lysine, cysteine, glutamic acid, aspartic acid etc.；R generations
Table cancer therapy drug, E* represents the amino acid that side chain is hydrazides.
Based on above-mentioned logical formula (I), the present invention uses hydroxy functionalized dialdehyde base small molecule (bis-CHO) and MMP-2 enzymes
Peptide molecule (the active fragment of Peptide 1, Peptide 1 of the hydrazides modification of high specific identification：PLGLA) design is synthesized
A kind of PTA-linker, matrix metalloproteinase (matrix metalloprotinase, MMP) be a class rely on metallic zinc from
The proteolytic enzyme of son, the regulation and control of the degraded of extracellular matrix (ECM), tissue reconstruction and intracellular a variety of soluble factors
Play an important role, be that a class occurs with tumour, attacks and shift closely related proteolytic enzyme, the hair with many malignant tumours
Life is closely related, therefore selection peptide 1 is used as enzymolysis lock unit.Two construction unit peptide 1 and bis-CHO are mixed
Close in DMSO solution and (contain 0.05%TFA), chromatogram shows that two construction units tend to form intermolecular pair of acylhydrazone key.This
Outside, the hydroxyl in dialdehyde base small molecule can be used for flexible modified medicaments molecule to build medicine derived from dialdehyde.
As with reference to experiment, relatively double acylhydrazone keys and single acylhydrazone key under condition of different pH (pH 7.0 simulates blood circulation,
PH 6.0 simulates tumor tissues microenvironment and inclusion body, the simulations of pH 4.8 lysosome) hydrolytic cleavage dynamics.PTA-linker
There is the stability of superelevation under the conditions of pH 4.8.On the contrary, SA-linker is in (the half-life period τ of pH 4.81/22.9h) with pH 6.0
(half-life period τ1/2Hydrolytic cleavage is all very fast under the conditions of 19.9h).In addition, under the conditions of pH 7.4, SA-linker can also delay
It is slow to hydrolyze.This slow hydrolysis limits such connecting key and is applied to albumin or antibody as the prodrug delivery of carrier
System, such part generally has extremely long blood circulatory half-life (19~23 days), so medicine can be caused to be released in non-target spot
Put.This ultrastability of PTA-linker derives from the synergy of two acylhydrazone keys, when double acylhydrazones of polypeptide bridging are by MMP
After enzymolysis lock, two rapid hydrolytic cleavages of acylhydrazone key under the conditions of pH 4.8.
It is logical there is provided prodrug design of the class based on PTA-linkers that the present invention investigates result for PTA-liners properties
Formula (II).The formula introduces part and medicine on the basis of logical formula (I).
Wherein：Any polypeptide chain is represented,Representing introns (is used to isolate dialdehyde base small molecule and medicine
Space length, minimize influence of the dialdehyde base small molecule to pharmaceutical activity).
The present invention is based on PTA-linkers prodrugs (D based on above-mentioned logical formula (II), present invention design two1With
D2), two kinds of prodrugs are made up of three parts：
I) RGD is as targeting ligand, and RGD is that a class contains arginine-glycine-aspartic acid (Arg-Gly-Asp) sequence
Small peptide, be the specific binding site of fibronectin and its acceptor.RGD peptide has highly affine with integrin receptor
Power, and acceptor has the expression of height on the endothelial cell during neonate tumour blood vessel.
II) MMAE derived from dialdehyde base is used as cancer therapy drug, monomethyl Austria profit statin E (monomethyl auristain
E, MMAE) be a kind of universal cancer therapy drug, it by with tubulin binding and suppress its polymerize it is follow-up thin so as to disturb
Born of the same parents' cycle events, and cause apoptosis of tumor cells.
III connecting key) is used as using PTA-linkers.
It is described to be based on double acylhydrazone key (PTA-linkers) prodrugs (D1And D2) synthetic method comprise the following steps：
Step 1) hydrazides modified polypeptide design：
If structural formula (a) and the designs of structural formula (b) Peptide 2 are for being overexpressed MMP-2 enzyme spcificitys outside tumour cell
Hydrolysis, the designs of peptide 3 are directed to intracellular cathepsin B's specific for hydrolysis.Cathepsin is that a major class is primarily present
In the cysteine proteinase enzyme of lysosome, exist more with inactive zymogen forms, protease zymogens pass through lysosome
Sour environment is activated.Because tumour cell can be acidified its surrounding environment, its cathepsin secreted also can be in this environment
In be activated, therefore, in tumor tissues and cell the activity of cathepsin be far above away from tumour other normal structures and
The cathepsin active of normal cell.Therefore, the feature based on above-mentioned tumor microenvironment, is devised extracellular or intracellular
Pinpoint the PTA-linkers of hydrolysis.In addition, the introducing design of lysine can be used for further modifying targeting ligand interested.
Step 2) dialdehyde drug derivative design：
MMAE is a kind of common cancer therapy drug, and the monomethylated amino of its N-terminal can be modified flexibly, in order to minimize
Influence to MMAE activity, it is flexible and polyethylene glycol of difunctionalization is used to be coupled MMAE and bis-CHO using a segment structure, obtain
(it is named as to MMAE derived from a kind of dialdehyde：bis-CHO-PEG3- MMAE, shown in its structural formula such as structural formula (C), cell
Toxicity profile shows bis-CHO-PEG3-MMAE (IC50=14.7nM) show to kill very much the ability of tumour cell by force, although its
Activity is compared to parent drug MMAE (IC50=0.75nM) decrease.
Step 3) part containing RGD prodrug D1 and D2 synthesis：
Pass through peptides 2/3 two hydrazides groups and bis-CHO-PEG3- MMAE two aldehyde radicals react to form two
Individual acylhydrazone key, is subsequently added NHS-PEG4- Maleimide, further the lysine side-chain with peptides 2/3 reacted,
Finally add c (RGDyC) part；In addition, as with reference to experiment, not being coupled MMAE prodrug C1(correspondence D1) and C2(correspondence
D2) while being also designed synthesis.As a result prodrug D is shown1And D2Ability with very strong kill tumour cell, in control experiment
C1And C2Substantially without cytotoxicity.
The principle of the present invention：
1) traditional SA-linkers can quickly form the transition state of positive tetrahedron in the case of neutral or weakly acidic,
Then occurs hydrolytic cleavage.
2) as shown in figure 1, PTA-liners hydrolytic cleavage process is different from SA-linkers, PTA-linkers is utilized
Cooperative effect between the double acylhydrazone keys of polypeptide bridging locking.In acid condition, occur portion fractures acylhydrazone key (i.e. one of them
Acylhydrazone key is broken) intermediate is due to the synergisticing stable effect between double acylhydrazone keys, it may occur that intramolecular aldehyde radical and hydrazides it is anti-
Should, so as to re-form double acylhydrazone keys；Therefore with ultrastability.After enzymolysis lock, the synergy of acylhydrazone key disappears, so that
Two acylhydrazone keys are caused to be broken in succession.
Double acylhydrazone connecting keys of heretofore described enzymolysis lock response, wherein unlocking manner has multiple choices, such as can be with
It is designed to that photodissociation is locked, H2O2The modes such as unblock.
The advantage of the invention is that：
1) cooperative effect between double acylhydrazone keys is locked using polypeptide so that acylhydrazone key has the stability of superelevation.
2) present invention breaches conventional connecting key stability and response is difficult to the defect that has concurrently, and it has the water of superelevation
Numerical solution, and can occur rapid hydrolytic cleavage after enzymolysis lock.This unique response not with sacrifice its
The response of target spot is cost.
3) need to only place two construction units just can directly form intermolecular dimer, therefore PTA- together
Linkers is very easy to be designed to be synthesized.
Brief description of the drawings
Fig. 1 is PTA-linkes reduction fracture mechanism.
Fig. 2 is PTA-linker formation chromatograms.
Fig. 3 is SA-linker and PTA-linker hydrolysis dynamics.
Fig. 4 is to hydrolyze chromatogram after PTA-linker enzymolysis is locked.
Fig. 5 is bis-CHO-PEG3-MMAE and MMAE cytotoxicity curves.
Fig. 6 is prodrug D1、D2、C1、C2Cytotoxicity curve.
Fig. 7 is bis-CHO-PEG3-MMAE mass spectral characteristi figures.
Fig. 8 is prodrug D1Mass spectral characteristi figure.
Fig. 9 is prodrug D2Mass spectral characteristi figure.
Figure 10 is prodrug C1Mass spectral characteristi figure.
Figure 11 is prodrug C2Mass spectral characteristi figure.
Embodiment
Following examples the present invention will be further described with reference to accompanying drawing.
Embodiment 1
Chromatogram monitoring PTA-linker forming process：
Take the μ L of 500 μ L (2mM is dissolved in containing 0.5%DMSO) peptide 1 and 500 (2mM is dissolved in containing 0.5%DMSO)
bis-CHO.To predetermined time point, each sample is carried out efficient liquid phase by 50 μ L of sampling to the interpolation pipe in chromatography column feed materials bottle
Chromatogram (HPLC) analyzes (the μ L of sampling volume 20), and monitors reaction product peak (such as Fig. 2) at 280nm.
Embodiment 2
Analyze SA-linker and PTA-linker hydrolysis dynamics：
1mL phosphate buffers (100mM, pH 7.4), 1mL phosphate buffers (100mM, pH 6), 1mL acetate are taken respectively
Buffer solution (100mM, pH 4.8) is separately added into 50 μ L SA-linker's and PTA-linker in 2.0mL chromatography column feed materials bottle
Solution (ultimate density is 10 μM).At regular intervals, chromatogram automatic sampling is analyzed (the μ L of sampling volume 80).And
Reaction product peak is monitored at 280nm.Record peak areas and drafting of the SA-linker and PTA-linker at differential responses time point
Connecting key breaking kinetics curve (such as Fig. 3).
Embodiment 3
Hydrolytic process after chromatogram monitoring PTA-linker enzymolysis locks：
Take 180 μ L acetate buffers (100mM, pH 4.8) in the low protein adsorption centrifuge tubes of 0.5mL, be separately added into 20
μ L PTA-linker enzymolysis locks product (ultimate density is 10 μM), to predetermined time point, 100 μ L of sampling to chromatography column feed materials bottle
In interpolation pipe, to sample carry out high performance liquid chromatography (HPLC) analysis (the μ L of sampling volume 70), and 280nm at monitoring react
Product peak (such as Fig. 4).
Embodiment 4
Cytotoxicity experiment：
U87 cells are inoculated in 96 orifice plates with every 85000/mL in hole density, 100 μ L DMEM cultures are added per hole
Base (contains 5% cow's serum, 1 ‰ is dual anti-), is placed in 37 DEG C, 5%CO2Incubator in be incubated 24h.Nutrient solution is abandoned in suction, is added to every hole
The solution for entering the various concentrations that 100 μ L are prepared using serum free medium and medicine is then placed in 37 DEG C, 5%CO2Incubator
In, each concentration sets 3 multiple holes.After cell and medicine are incubated 48h jointly, 10 μ L CCK-8 solution are added in every hole,
And put it into incubator be incubated 1h after by ELIASA detect 450nm under absorbance.Experiment is in triplicate.CCK-8
It is a kind of there is high sensitivity, favorable reproducibility, cell toxicity test method easy to operate.The activity that CCK-8 contains
Thing can be in the presence of electron carrier by the dehydrogenase reduction in cell mitochondrial, and generates the yellow first with high water soluble
Za product.Can reflecting the quantity of living cells according to the proportional relation of generation formazans thing and living cells quantity, (toxicity data is such as
Fig. 5 and Fig. 6).
Embodiment 5
Compound bis-CHO synthesis
The sodium hydroxide for weighing 1.31g (32.7mmol) is dissolved in 60mL pure water.4.0g is added into solution
(32.7mmol) parahydroxyben-zaldehyde, is slowly added dropwise 1.1mL epoxychloropropane at 60 DEG C after fully mixing, dropwise addition process is held
Continuous 2h.Reaction solution is then stirred into 3h at a temperature of 60 DEG C.After reaction terminates, with the mixing that first alcohol and water mixing ratio is 1 ︰ 1
Solvent is carried out being recrystallized to give the end-product that yield is 85% to product, and products therefrom is characterized using nuclear-magnetism.
The structural characterization of target product：
1H NMR(400MHz,CDCl3,δ,ppm):9.91 (d, J=1.7Hz, 2H), 7.85~7.87 (t, J=1.8Hz,
4H), 7.05~7.07 (s, 4H), 4.26~4.28 (s, 3H), 2.61 (s, 1H).
Embodiment 6
Peptide 1 synthesis
Weigh 5.0mg (5.29 μm of ol) polypeptide (sequence：Ac-C-G-G-P-L-G-L-A-G-G-C-NH2 400) are dissolved in
In μ L (100mM, pH 7.4) phosphate buffer solution.It is added dropwise into above-mentioned solution and is dissolved in containing in 400 μ L DMSO
Trifluoroacetate (BMPH) solution of 7.86mg (32.0 μm of ol) 3- maleimidopropionic acid hydrazides.Treat above-mentioned mixed liquor reaction
After 0.5h, purified with chromatogram (HPLC) is prepared.ESI-MS m/z(M)+calcd.1383.56,found(M)+1383.7。
Embodiment 7
PTA-linker synthesis:
By 500 μ L (2.0mM) of the above-mentioned synthesis μ L (2.0mM) of peptide 1 and 500 dialdehyde molecular mixing in 1mL
In DMSO containing 0.05% trifluoroacetic acid.Mixed solution is placed in stirring reaction 3h at room temperature, then, product entered by chromatogram
Row purifying.ESI-MS m/z(M)+calcd.1647.58(M)+,Found 1647.3(M)+,824.6(M+2H)2+。
Embodiment 8
SA-linker synthesis：
By 10.0mg (0.060mmol) 4- (2- hydroxyl-oxethyls) benzaldehydes and 70.9mg (0.60mmol) 4- hydroxyls
Butyric acid hydrazine is mixed in 5mL DMSO solution.Mixed solution is placed in stirring reaction 24h at room temperature, then, will be produced by chromatogram
Thing is purified.ESI-MS m/z(M)+calcd.266.29(M)+,Found 266.7(M)+。
Embodiment 9
Synthesis bis-CHO-PEG3-MMAE is performed the following steps：
1) synthesis of compound 1：
The bis-CHO for weighing 1.0g (3.3mmol) is dissolved in 20mL dichloromethane solution, is added then in solution
937 μ L (6.6mmol) triethylamine and 0.66g (1.5mmol) p-nitrophenyl chloroformate.Mixed solution is placed at room temperature
It is slowly added 20mL pure water into solution to be quenched reaction after reaction 4h.With 10mL saturated aqueous common salt washing reaction liquids, dichloromethane
After alkane extraction point liquid, organic phase is dried with anhydrous sodium sulfate.Solvent is spin-dried for, then product is divided using silicagel column
From purifying, 1.10g white powder compound 1 is finally given, yield is 75%.1H NMR(400MHz,Chloroform-
D) δ 9.93 (s, 2H), 8.35~8.29 (m, 2H), 7.92~7.87 (m, 4H), 7.47~7.41 (m, 2H), 7.12~7.07
(m, 4H), 5.56 (p, J=4.9Hz, 1H), 4.51 (d, J=4.9Hz, 4H).ESI-MS m/z(M)+calcd.465.41(M)+,
Found 465.88(M)+。
2) Fmoc-NH-PEG is synthesized3-MMAE:
By 10mg (14.0 μm of ol) MMAE, 12.4mg (28 μm of ol) Fmoc-NH-PEG3-CH2CH2COOH、10.6mg
HATU, 9.8 μ L (the 56 μm of ol) DIEA of (28 μm of ol) are added in 5ml round-bottomed flasks, are solvent with DMSO (1mL), at room temperature
React 1h.Then, prepare chromatogram (HPLC) with half to purify to product, 12.8mg white solid, yield are obtained after freezing
For 80%.ESI-MS m/z(M)+calcd.1143.45(M)+,Found 1144.6(M+H)+。
3) bis-CHO-PEG3-MMAE is synthesized：
It is 1 ︰'s 1 that diethylamine is added into 10mg (8.7 μm of ol) Fmoc-NH-PEG3-MMAE with dichloromethane mixing ratio
Mixed solvent.After reaction stirring 30min, solvent is removed in rotation.Then with 1mL DMSO dissolving crude products, 6.1 μ L are added into solution
The DIEA and 4.0mg (8.7 μm of ol) of (34.8 μm of ol) compound 1.Mixed solution was stirred at room temperature after 1 hour and made with half
Standby chromatogram (HPLC) is purified to product.ESI-MS m/z(M)+calcd.1247.51(M)+,Found1248.5(M+H)+。
Embodiment 10
Synthesize D1：
The μ L concentration of peptide 2 and 100 for being 10mM by 100 μ L concentration is the bis-CHO-PEG of the above-mentioned synthesis of 10mM3-
MMAE is mixed in the DMSO that 1mL contains 0.05% trifluoroacetic acid, and reaction 5h is stirred at room temperature.Then, 4mM NHS- is added
PEG4- Maleimide and 100 μ L concentration are mixed in 1mL DMSO for 10mM DIEA, and reaction 1h is stirred at room temperature.Most
Afterwards, the c (RGDyC) that 100 μ L concentration are 4mM, reaction stirring 1h are added into solution.Chromatogram (HPLC) is prepared with half to enter product
Row purifying.ESI-MS m/z(M+)calcd.3738.31(M)+,Found 1246.5(M+3H)3+,935.2(M+4H)4+,748.7
(M+5H)5+。
Embodiment 11
Synthesize D2：
D2Synthesis step and D1Synthesis step is identical.
Embodiment 12
Synthesize C1：
C1Synthesis step and D1Synthesis step is identical.
Embodiment 13
Synthesize C2：
C2Synthesis step and D1Synthesis step is identical.
Claims (7)
1. double acylhydrazone connecting keys of a class polypeptide bridging, it is characterised in that polypeptide chain bridging and double acylhydrazone keys, with protease water
Solution and sour water solution dual responsiveness, have stability and response concurrently, the biotic environment is followed including blood in biotic environment
Ring, extracellular environment, intracellular environment.
2. double acylhydrazone connecting keys of class polypeptide bridging as claimed in claim 1, it is characterised in that its molecular skeleton formula is：
Wherein：(x) any active peptides sequence is represented, is degraded by specific proteases；(M) represent N-terminal and further carry out chemistry and repair
The arbitrary amino acid of decorations, the amino acid includes lysine, cysteine, glutamic acid, aspartic acid；R represents cancer therapy drug, E*
Represent the amino acid that side chain is hydrazides.
3. double acylhydrazone connecting keys of class polypeptide bridging as claimed in claim 2, it is characterised in that the logical formula (I) uses hydroxyl
The dialdehyde base small molecule bis-CHO of functionalization and the base containing bishydrazide that can be hydrolyzed by matrix metalloproteinase MMP-2 high specifics
Peptide molecule design a kind of pair of acylhydrazone connecting key of synthesis；Matrix metalloproteinase be a class in tumor tissues overexpression, and with
Generation, transfer and the invasin protein hydrolase of tumour, will contain bishydrazide Quito peptide molecule and be mixed with bis-CHO in DMSO solution
Close, add 0.05%TFA, two construction units of chromatogram tend to form intermolecular pair of acylhydrazone key.
4. double acylhydrazone connecting keys of class polypeptide bridging as claimed in claim 2, it is characterised in that in the dialdehyde base small molecule
Hydroxyl be used for modified medicaments molecule and build medicine derived from dialdehyde, structural formula is：
5. prodrug design formula of the class based on double acylhydrazone connecting keys, it is characterised in that the formula draws on the basis of logical formula (I)
Enter part and medicine：
Wherein：Any peptide sequence is represented,Represent connection unit between medicine and bis-CHO.
6. prodrug design formula of the class as claimed in claim 5 based on double acylhydrazone connecting keys, it is characterised in that the class base
Lead to formula (II) in the prodrug design of double acylhydrazone connecting keys and design two based on double acylhydrazone connecting key prodrugs D1And D2, before two kinds
Medicine is made up of three parts：
I) there is the targeting ligand of tumour or cancer cell identification function：RGD is as targeting ligand, and RGD is a class containing arginine-sweet
The small peptide of propylhomoserin-aspartic acid sequence, it is the specific binding site of fibronectin and its acceptor；RGD peptide is with integrating
Plain acceptor has high-affinity, and acceptor altimeter on the endothelial cell during neonate tumour blood vessel reaches；
II) the anti-tumor drug molecule of strength：MMAE derived from dialdehyde base is as cancer therapy drug, and monomethyl Austria profit statin E is a kind of
General type cancer therapy drug, it is by with tubulin binding and suppressing its polymerization methodses interference cell cycle events, causing tumour
Apoptosis；
III) it is used as connecting key using double acylhydrazone connecting keys.
7. based on double acylhydrazone key prodrugs D1And D2Synthetic method, it is characterised in that comprise the following steps：
Step 1) bishydrazide base group modification peptide molecule design：
Structural formula is (a) and (b), and Peptide 2 design is directed to the MMP-2 enzymes being overexpressed outside tumour cell, peptide's 3
Design is directed to intracellular cathepsin B；Cathepsin is the cysteine proteinase that a class is primarily present in lysosome
Enzyme, exists with inactive zymogen forms more, and protease zymogens can be activated under lysosomal acid environment；Due to tumour cell
Its surrounding environment can be acidified, its cathepsin secreted can also be activated in extracellular environment, tumor tissues microenvironment
In and tumour cell in cathepsin active be far above away from tumor tissues other normal structures in and normal cell in
Cathepsin active；Based on the feature of above-mentioned tumor microenvironment, the double acylhydrazones for designing extracellular and intracellular fixed point hydrolysis connect
Connect key；The introducing of lysine is designed for further modifying targeting ligand interested；
Step 2) dialdehyde drug derivative design：
MMAE is a kind of common cancer therapy drug, and the amino that its N-terminal methylates flexibly is modified, in order to minimize chemical modification pair
The influence of MMAE activity, using a segment structure is flexible and polyethylene glycol conjugation MMAE and bis-CHO of difunctionalization, has obtained one
Plant MMAE, its structural formula (C) derived from dialdehyde；
Step 3) part containing RGD prodrug D1 and D2 synthesis：
Pass through peptides 2/3 two hydrazides groups and bis-CHO-PEG3- MMAE two aldehyde radicals react to form two acyls
Hydrazone key, is subsequently added NHS-PEG4- Maleimide, further the lysine side-chain with peptides 2/3 reacted, finally
Add c (RGDyC) part；In addition, being used as reference, prodrug C of the design synthesis without coupling MMAE1With prodrug C2, wherein prodrug
C1Correspondence D1, prodrug C2Correspondence D2。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109467517A (en) * | 2018-10-19 | 2019-03-15 | 华中科技大学 | A kind of acylhydrazone molecular switch, preparation method and application |
| CN114748640A (en) * | 2022-05-06 | 2022-07-15 | 南方医科大学珠江医院 | pH-responsive siRNA delivery system |
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| US8563698B2 (en) * | 2007-08-23 | 2013-10-22 | Amgen Inc. | Antigen binding proteins to proprotein convertase subtilisin kexin type 9 (PCSK9) |
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| US8563698B2 (en) * | 2007-08-23 | 2013-10-22 | Amgen Inc. | Antigen binding proteins to proprotein convertase subtilisin kexin type 9 (PCSK9) |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109467517A (en) * | 2018-10-19 | 2019-03-15 | 华中科技大学 | A kind of acylhydrazone molecular switch, preparation method and application |
| CN114748640A (en) * | 2022-05-06 | 2022-07-15 | 南方医科大学珠江医院 | pH-responsive siRNA delivery system |
| CN114748640B (en) * | 2022-05-06 | 2024-02-09 | 南方医科大学珠江医院 | PH-responsive siRNA delivery system |
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