CN105906692A - cRGD-erlotinib conjugate and preparation method thereof - Google Patents
cRGD-erlotinib conjugate and preparation method thereof Download PDFInfo
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Abstract
The invention provides an anti-non-small cell lung cancer (NSCLC) tumor-targeting prodrug which is a cRGD-erlotinib/icotinib conjugate. cRGD is a cyclic polypeptide. Compared with the linear RGD polypeptide, a conjugate of the cRGD and an Integrin acceptor has better affinity and selectivity. Erlotinib and icotinib are small-molecule inhibitors of an epidermal growth factor receptor (EGFR) and are bonded to an EGFR catalytic structural domain through competitiveness so that ATP bonding is prevented, tyrosine residue autophosphorylation and proliferation signal transduction are further inhibited and cancer cell growth is interfered and thus the cRGD-erlotinib conjugate can be used for treating NSCLC. The cRGD-erlotinib/icotinib conjugate selectively transfers erlotinib/icotinib to NSCLC cells, improves locus transport efficiency and reduces cancer paint side effects.
Description
Technical field
The present invention relates to a kind of targeting type compound, be specifically a kind of cRGD-Erlotinib/erlotinib conjugate
And the preparation method of conjugate, belong to cancer target and deliver administration systems technology field.
Background technology
The delivery of conventional medicament is typically Passive intake through cell membrane.Generally, medicine can be along with systemic blood
Or body fluid circulatory and spread all over whole body most tissues or organ.For most drug, specific region produces minimum effective
Concentration is to need necessary to pharmacological effect.Therefore, cells in vivo function is inevitably produced by extra substantial amounts of medication amount
Toxicity.Targeted delivery of drugs is site-specific delivery, and this advantage is that therapeutic agent arrives disease location, is not affecting medicine
While curative effect, and decrease incoherent distribution in normal structure, thus to increase local drug concentration and to reduce side effect.
Erlotinib is the micromolecular inhibitor of EGF-R ELISA (EGFR), by competitive binding in EGFR's
Catalyst structure domain, prevents ATP from combining, and autophosphorylation and the proliferation signal of suppression tyrosine residue conducts further, and interference cancer is thin
The growth of born of the same parents, thus be used for treating Metastatic Nsclc (NSCLC).But, Erlotinib has other albumen much
Matter action site, can promote cancer cell multiplication signal connects or demonstrates the apoptosis-promoting effect to healthy cell each other, cause
Serious side effect.
Summary of the invention
The present invention is directed to Erlotinib tool and have other protein interaction sites many when treating nonsmall-cell lung cancer, aobvious
Apoptosis-promoting effect to healthy cell is shown, thus results in the weak point of serious side effect, it is provided that one has cancerous cell
The cRGD coupling Erlotinib/erlotinib conjugate of targeting.This conjugate passes through Erlotinib/erlotinib with ring-type
The direct coupling of non-natural lysine of the nitrine labelling introduced in rgd peptide forms.As a kind of targeted integration element receptor
Medicine targeted delivery system, the introducing of cRGD makes Erlotinib/erlotinib optionally for cancerous cell, and non-health is thin
Born of the same parents, thus improve therapeutic effect.
For realizing above technical purpose, the present invention will take following technical scheme:
A kind of compound selected from formula I-II and pharmaceutically acceptable structure thereof,
A kind of prepare such as the method for structural formula 1 or 2 compound, comprise the following steps:
(1) the ring type polypeptide cyclic RGDfK-azide[cyclo of nitrine labelling is synthesized according to Solid-phase Polypeptide technology order
(-Arg-Gly-Asp-D-Phe-Lys(N3)-)】；
(2) the cyclo (-Arg-Gly-Asp-D-Phe-Lys (N that will obtain in step 13)-) replaces with Erlotinib/angstrom sieve
Buddhist nun obtains end product by click chemistry conjugation.
The technical scheme that the present invention limits further as:
Further, step 1 includes the most step by step:
(1) Fmoc solid-phase synthesis synthesizes into linear polypeptide resin Fmoc-Asp (the OtBu)-D-Phe-Lys of nitrine labelling
(N3)-Arg(Pbf)-Gly-CTC Resin
2-chlorine trityl chloride resin, Fmoc-Glycine-OH and anhydrous DIPEA are dissolved in dichloro
Methane stirs appropriate time；Add the methanol prepared and anhydrous DIPEA mixed solution continues stirring；Then,
By resin respectively with dimethylformamide (DMF, 2 times), DCM (2 times) and methanol (2 times) washing, vacuum drying.Finally, by suitable
Sequence is introduced into the aminoacid of blocking group and (is followed successively by Fmoc-arginine (Pbf)-OH, Fmoc-lysine (N in order3)-OH,
Fmoc-aspartic acid (OtBu)-OH connects Fmoc-Gly-CTC resin.
(2) linear polypeptide [H-Asp (OtBu)-D-Phe-Lys (N3)-Arg (Pbf)-Gly-OH] of nitrine labelling is synthesized
Under the solution condition that DMF/DCM/ piperidines volume ratio is 1:1:2, solidify resin from the linear polypeptide DfKRG of nitrine labelling and remove
Fmoc group.Then under room temperature condition, in acetic acid/2, the mixture ratio of 2,2-trifluoroethanols (TFE) and DCM is that 3:1:6 is molten
Under the conditions of liquid, incubation is after 2 hours, and linear peptides splits in acetic acid solution together with the blocking group of the band side chain obtained from resin 3
Solve.After cracking reaction, resin uses the mixed solution of acetic acid/TFE/DCM to wash twice.The eluate of merging is mixed in 15 times
In the hexane of volume, Rotary Evaporators is used to be evaporated removing solvent to obtain the linear peptides 4 (H-with side chain protected effect
Asp(OtBu)-D-Phe-Lys(N3)-Arg(Pbf)-Gly-OH)。
(3) the ring type polypeptide cyclo (-Arg-Gly-Asp-D-Phe-Lys (N of liquid phase Cyclization nitrine labelling3)-)
Under nitrogen protective condition, by the linear polypeptide H-Asp (OtBu) of the nitrine labelling that finally gives in step by step 2-
D-Phe-Lys(N3)-Arg (Pbf)-Gly-OH carries out liquid phase cyclization using HOBt/HBTU/DIPEA as condensing agent.Then
After being concentrated under vacuum by reaction solution, concentrate solution further at tetrafluoroethene, water and dimercaptoethane mixed solvent
Middle deprotection, finally obtains the linear polypeptide cyclo (-Arg-Gly-Asp-D-Phe-Lys (N of nitrine labelling3)-)；
Further, step 2 includes the most step by step: a certain amount of Erlotinib or Conmana, cyclo (-Arg-
Gly-Asp-D-Phe-Lys(N3)-), second triethylenetetraminehexaacetic acid ammonium buffer and ascorbic acid are dissolved in hybrid reaction in dimethylformamide,
It is subsequently added copper (II)-three [(1-benzyl-1H-1,2,3-triazole-4-yl) methyl] amine complex and continues reaction to appropriate time；
Reversed-phase high-performance liquid chromatography RP-HPLC is used to concentrate and purify reactant mixture, it is thus achieved that cyclo (-Arg-Gly-Asp-D-Phe-
Lys(N3)-)-Erlotinib conjugate.
Further, the reaction of 1 is carried out under the protection of noble gas step by step.
Further, the reaction of step 2 is carried out under the protection of noble gas.
The application application protection above two compound purposes in preparing anti-lung-cancer medicament.
Ring type polypeptide RGD demonstrates higher affinity and selectivity to combining integrin Integrin receptor.Aobvious and easy
Seeing, compared to Erlotinib monomer, the Erlotinib conjugate of ring type polypeptide RGD coupling has specific binding cancerous cell Gao Feng
The integrin Integrin receptor that degree is expressed, promotes the picked-up of cancerous cell, thus shows more preferable curative effect.Meanwhile, strategic point Lip river is replaced
Buddhist nun or Conmana all comprise alkynyl group, it is allowed to alkynes azide cyclization addition coupling another kind based on click chemistry
Compound or biomolecule.
Accompanying drawing explanation
Fig. 1 is the structure chart of cRGD-Erlotinib conjugate in embodiment 1；
Fig. 2 is the synthetic route chart of cRGD-Erlotinib conjugate in embodiment 1；
Fig. 3 is the structure chart of cRGD-Conmana conjugate in embodiment 2；
Fig. 4 is the synthetic route chart of cRGD-Conmana conjugate in embodiment 2；
Fig. 5 is the ESI high resolution mass spec report of peptide 6 in embodiment 1；
Fig. 6 is the HRMS examining report of peptide 6 in embodiment 1；
Fig. 7 is the ESI high resolution mass spec report of final finished in embodiment 1；
Fig. 8 is the HRMS examining report of final finished in embodiment 1；
Fig. 9 is the ESI high resolution mass spec report of final finished in embodiment 2；
Figure 10 is the HRMS examining report of final finished in embodiment 2.
Figure 11 a is that the cRGD-Erlotinib that embodiment 1 manufactures is strengthening the sensitivity contrast of PC9 cell with Erlotinib
Lab diagram；(Erlotinib and the cRGD-Erlotinib inhibited proliferation to PC9 cell)
Figure 11 b is that the cRGD-Erlotinib that embodiment 1 manufactures is strengthening the sensitivity contrast of PC9 cell with Erlotinib
Lab diagram；(clone's survival measures)
Figure 12 a is that the cRGD-Erlotinib that embodiment 1 manufactures induces the apoptotic contrast experiment of PC9 with Erlotinib；
(flow cytometry detection DMSO, Erlotinib, and cRGD-Erlotinib process impact apoptotic on PC9)
Figure 12 b is that the cRGD-Erlotinib that embodiment 1 manufactures induces the apoptotic contrast experiment of PC9 with Erlotinib；
(PI staining analysis detection DMSO, Erlotinib, cRGD-Erlotinib processes impact apoptotic on PC9)
Figure 12 c is that the cRGD-Erlotinib that embodiment 1 manufactures induces the apoptotic contrast experiment of PC9 with Erlotinib.
(immunoblotting assay cell death related protein, Erlotinib and cRGD-Erlotinib process)
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further elaborated, but the present invention is not limited to following embodiment party
Formula.
Embodiment 1
First aspect, the invention provides the cancer target precursor medicine of a kind of anti-nonsmall-cell lung cancer, and it is cRGD-strategic point Lip river
For Buddhist nun's conjugate, concrete structure is shown in accompanying drawing 1.Wherein, cRGD is ring-type Arg-Gly-Asp tripeptide, it is possible to the most main
Moving-target is to the polypeptide of tumor tissues；Erlotinib is treatment non-small cell lung cancer drug.
Second aspect, the invention provides the preparation method of the cancer target precursor medicine of a kind of anti-nonsmall-cell lung cancer, its
Synthetic route is shown in accompanying drawing 2, specifically includes following steps:
(1) initial action resin (Fmoc-Gly-CTC Resin) is prepared
In round-bottomed flask, 1.0g resin is dissolved in 7 milliliters of anhydrous methylene chlorides (DCM).Add the most under nitrogen protection
Fmoc-glycine-OH (891.9 milligrams, 3.0 mMs) and anhydrous DIPEA (DIPEA) (1.00 milliliters,
5.74 mM).This reaction solution is stirred at room temperature 4 hours under nitrogen protection.Finally, the MeOH/DIPEA of 10 milliliters is added
(9:1) termination mix reaction, the single step reaction 20 minutes of going forward side by side.After being filtered to remove solution, respectively with 5 milliliters of DCM and 5 milliliter two
Methylformamide (DMF) respectively washes twice, and washed once by 5 ml methanol subsequently.After washing, resin is dried under vacuum 24
Hour, and be stored in 4 DEG C standby
(2) solid-phase synthesis synthesizes into linear polypeptide resin Fmoc-Asp (the OtBu)-D-Phe-Lys of nitrine labelling
(N3)-Arg(Pbf)-Gly-CTC Resin
Under room temperature condition, by the initial action resin (Fmoc-Gly-CTC Resin) that obtains in step by step 1 at DMF/
DCM/ piperidines volume ratio be 2:1:1 solution condition under carry out Fmoc group deprotection reaction from aminoacid.Reaction 30min
After, use DMF that resin is carried out twice washing.Then, hard resin exists with aminoacid or the amino acid derivativges of Fmoc protection
Coupling reaction 3 hours in DCM solution, the most amino acid whose equivalent is 1, and the equivalent of N-hydroxybenzotriazole (HOBt) is 2, O-
(benzotriazole-1-base)-N, the equivalent of N, N', N-tetramethylurea hexafluorophosphate (HBTU) and DIPEA is respectively 2 and 5.At this
In coupling reaction, successively the aminoacid with N end Fmoc protection and side chain protected (is followed successively by Fmoc-arginine in order
(Pbf)-OH, Fmoc-lysine (N3)-OH, Fmoc-aspartic acid (OtBu)-OH) connect Fmoc-Gly-CTC resin.This is even
Connection reaction is analyzed by ninhydrin test.It is of particular importance that lysine introduces nitrine to facilitate in step 2 by side chain
Erlotinib conjugate is prepared in click chemistry coupling in by.After coupling reaction terminates, resin use respectively 5 milliliters of DMF and
DCM washes twice, and is dried 12 hours under vacuum condition, to obtain linear RGDfK solidification resin 3.
(3) linear polypeptide 6[H-Asp (OtBu)-D-Phe-Lys (N of nitrine labelling is synthesized3)-Arg(Pbf)-Gly-
OH)】
Under the solution condition that DMF/DCM/ piperidines volume ratio is 1:1:2, remove Fmoc base from linear DfKRG solidification resin
Group.Then under room temperature condition, the DfKRG solidification resin of de-Fmoc in acetic acid/2,2,2-trifluoroethanols (TFE) and the mixing of DCM
In solution, (volume ratio is 3:1:6) carries out cracking reaction.After 2 hours, there is the linear peptides RGDfK of side chain protecting group from resin
It is cleaved in 3.After cracking reaction, resin uses the mixed solution of acetic acid/TFE/DCM to wash twice.The eluate that will merge
It is mixed in the hexane of 15 times of volumes, uses Rotary Evaporators to be evaporated removing solvent, to obtain, there is nitrine labelling and side chain
The linear peptides RGDfK 4 of protective effect.
(4) ring type polypeptide [cyclo (-Arg-Gly-Asp-D-Phe-Lys (N3)-)] of liquid phase Cyclization nitrine labelling
Under nitrogen protective condition, by the nitrine labelling obtained in step by step 3 and the linear polypeptide 4[H-Asp of side chain protected
(OtBu)-D-Phe-Lys(N3)-Arg (Pbf)-Gly-OH] to be dissolved in DMF solution to concentration be 1.25 × 10-3M.Then, with
HOBt/HBTU/DIPEA carries out liquid phase cyclization as condensing agent.This cyclization adds 1 equivalent in DMF solution
HOBt, the HBTU and the DIPEA of 5 equivalents of 2 equivalents, under nitrogen protection reaction 24 hours, to obtain nitrine labelling and side chain guarantor
The ring type polypeptide (-arginine (Pbf)-Gly-Asp (OtBu)-D-phenylalanine-lysine (N3)-) 5 protected.Will be anti-
After answering solution to be concentrated under vacuum, at ambient temperature, the residue of cyclic peptide 5 be used in 15mL deprotection solution (95%TFA,
2.5% water and the dimercaptoethylsulfide of 2.5%) complete deprotection 24 hours, finally obtain the linear of nitrine labelling and side chain protected
Polypeptide 6[cyclo (-Arg-Gly-Asp-D-Phe-Lys (N3)-)], i.e. ring (-arginine-glycine-aspartic acid-D-phenylpropyl alcohol
Propylhomoserin-lysine (N3)-).Rough deprotection cyclic peptide 6, by reversed-phase high-performance liquid chromatography RP-HPLC purification, utilizes linear ladder
Degree buffer (90% acetonitrile of 20-45% is dissolved in 0.05%TFA) obtains the cyclic peptide of 98% purity at Agela C18 post.Peptide 6 leads to
Cross ESI high resolution mass spec to analyze.Its retention time is 25 minutes.Its chemical molecular formula is C27H40N11O7, mass spectrum [M+H]+'s
HRMS value of calculation is 630.31, and detected value is 629.98.
Table 1 is the sample message of peptide 6:
Table 2 is the testing result of peptide 6:
| Detection | Result |
| Outward appearance | Freeze-dried material |
| Molecular weight (MS, M+H+) | 629.98 |
| Molecular weight (MS, M+Na+) | |
| Molecular weight (MS, M+K+) | |
| Purity (byHPLC) | 97.02% (HPLC, 220nm, C18, linear gradient elution) |
；
Table 3 is the storage condition of peptide 6:
| Storage condition | -20℃ |
；
(4) preparation of target product
A certain amount of Erlotinib is dissolved in 8 milliliters of DMF (20 milligrams, 0.051 mM), add a certain amount of cyclo (-
Arg-Gly-Asp-D-Phe-Lys (N3)-) (26.7 milligrams, 0.043 mM), it is subsequently added 5.6 milliliters of second triethylenetetraminehexaacetic acid ammoniums and delays
Rush liquid (pH7.0,2M) and 1.6 milliliters of ascorbic acid (5.0 mMs).Solution mixes 30 minutes at ambient temperature, subsequently, adds
Enter 0.8 milliliter of 10mM copper (II)-three [(1-benzyl-1H-1,2,3-triazole-4-yls) methyl] amine complex (10mM, 55%
DMSO).The mixture of reaction leads to argon and continues stirring 24 hours after saturated 20 minutes the most under nitrogen protection.The product that reaction obtains
Thing is evaporated removing solvent through Rotary Evaporators.Finally utilize reversed-phase high-performance liquid chromatography, linear by AgelaC18 post
Gradient buffering liquid (90% acetonitrile of 25-47%, be dissolved in 0.05%TFA) carries out isolated and purified product, finally obtains cyclo
(-Arg-Gly-Asp-D-Phe-Lys (N3)-)-Erlotinib conjugate 1 37.5mg (yield is 85%) altogether.End product leads to
Crossing ESI high resolution mass spec to be analyzed, its retention time is 22 minutes, and purity is 100%, and chemical molecular formula is
C49H63N14O11, mass spectrum [M+H]+HRMS value of calculation be 1024.48, detected value is 1024.75, C49H62N14NaO11[M+Na]+
HRMS value of calculation be 1046.09, detected value is 1046.50.
Table 4 is the sample message of embodiment 1 final finished:
Table 5 is the testing result of embodiment 1 final finished:
| Detection | Result |
| Outward appearance | Freeze-dried material |
| Molecular weight (MS, M+H+) | 1024.75 |
| Molecular weight (MS, M+Na+) | 1046.50 |
| Molecular weight (MS, M+K+) | |
| Purity (HPLC) | 100% (HPLC, 220nm, C18, linear gradient elution) |
；
Table 6 is the storage condition of embodiment 1 final finished:
| Storage condition | -20℃ |
Cell is cultivated and treatment
Non-small cell lung carcinoma PC9 cell is grown on containing 10% hyclone, 100U/ milliliter penicillin and 100U/ milliliter
In the DMEM culture medium of streptomycin.Cell is in 37 bases, 5%CO2Incubator is cultivated and within 48 hours, reaches 80% cell confluency.So
After, replace with replacing containing DMSO solvent control, Erlotinib and cRGD-strategic point Lip river of variable concentrations for cultivating the culture medium of cell
Buddhist nun, processes different time respectively.96 well culture plates measure for cell death.Six well culture plates are used for AnnexinV-
FITC-PI dyes, and 35 millimeters of culture dishs are for confocal laser scanning microscope, CLSM (CLSM) imaging.
Cytotoxicity detects
Cell in vitro viability is checked, to assess the sensitivity of medicine with mtt assay.PC9 cell is seeded in 24 orifice plates (2 plates
(planting in cells/well), and (0 arrives to hatch 72 hours in the Erlotinib containing progressive concentration or cRDG-Erlotinib culture medium
2uM).After hatching, every hole add 600 educate after MTT solution and cultivate 3 hours.Then, every hole adds the DMSO of 150 microlitres
(SIGMA, D2650) is also stirred at room temperature 10 minutes.Exist in the upper measurement of Bio-Rad company microplate reader (Hercules, the U.S.)
The absorbance A (OD) of 570nm.According to A value calculating cell proliferation inhibition rate: cell proliferation inhibition rate (%)=(control wells A value-
Experimental port A value)/(control wells A value-blank well A value) white hole=(system.Carry out three independent cell toxicity test (every kind of medicines
Concentration is each repeated four times), to calculate Erlotinib and cRDG-Erlotinib IC 50, the latter is defined as in inducing cell colony
Drug level in the cell death of 50%.
Clone's survival analysis
The PC9 cell sensitivity to process medicine, uses colony formation analysis, and the latter is used for determining cytotoxicity
Agent, as chemotherapy effectiveness is measured.Collecting cell and be seeded in 60 to millimeter culture dish, after 24 hours, cell is exposed to 2uM
In Erlotinib and/or cRDG-Erlotinib 72 hours.Then remove medicine, and make cell at fresh growth medium
Breed 10 days.Population of cells is fixing with the methanol (25%V/V) containing crystal violet (0.05% weight/volume) and dyes 30 points
Clock.The microscope meter counting colony containing at least 30 cells.
Flow cytometry analysis
PC9 cell is exposed to DMSO (solvent control), the Erlotinib of 20nM or cRDG-Erlotinib 72 hours.Cell
The annexinV puted together with Fluorescein isothiocyanate (FITC) and propidium iodide (PI) apoptosis detection kit (BD
Biosciences, California, the U.S.) detection.Then apoptotic cell passes through flow cytometry analysis.
Cell death quantitative
For quantifying the cell death caused by Erlotinib and/or cRDG-Erlotinib, PC9 cell, at 37 DEG C, is exposed to
30nM Erlotinib and/or cRDG-Erlotinib 48 hours.Culture medium is changed to containing 50 mcg/ml propidium iodides (PI)
The Extracellular solution of (Invitrogen company, California), at 37 DEG C, incubation is after 48 minutes, the fluorescence intensity in each hole
(Victor3 is measured by microplate reader；PerkinElmer company, Massachusetts).Dead cell number presses total cellular score standardization.
Western blot analyzes
The impact of PC9 cancer cell-apoptosis is passed through apoptosis-related protein by cRGD-Erlotinib conjugate further
The expression of caspase-3 and PARP carries out immunoblotting assay.After cell processes, the PBS of cell pre-cooling is washed twice by apoptosis
After, it is dissolved in the RIPA buffer containing Phenylmethanesulfonyl fluoride, at 4 degree, 12000 acyl fluorides are centrifuged 20 minutes.Albumen BCA method
After quantitatively and separated by 6-15%SDS-PAGE, transfer on nitrocellulose filter.At 37 DEG C, hatch 2 with 5% skim milk
Hour to block non-specific binding.Subsequently, caspase-3 anti-with rabbit, rabbit anti-PARP or little mouse-anti GAPDH antibody is with 1:2000
Diluent, 4 degree of overnight incubation.Then film is washed three times in TBS T, and the two of labelling resist the dilution times with 1:5000
Number at room temperature hatches 1 hour.Protein is shown by chemoluminescence method (Millipore).
Statistical analysis
Result is expressed as meansigma methods ± standard error, and experimental result is from the independent experiment of at least three times.Use single factor test side
Difference analysis analysis result.For clonal analysis, use two-way analysis of variance.After detecting significant difference, use Newman
Keuls checks difference two-by-two.Data analysis and statistical use carry out GraphPad Prism program draw (Gradphad,
California).< 0.05 is that difference is statistically significant to P value.
Result:
CRGD-Erlotinib strengthens the sensitivity of PC9 cell
MTT and clone's survival experiment are with detection cRGD-Erlotinib and the Erlotinib transplanting effect to cell viability.No
PC9 cell is processed respectively with concentration c RGD-Erlotinib and Erlotinib (0,0.2nm, 2nm, 20nm, 200nm and 2um).
CRGD-Erlotinib and Erlotinib demonstrate the dose-response curve to cell viability.Such as Figure 11 a, survival in 72 hours measures
Finding, Erlotinib and cRGD-Erlotinib respectively may be about 30.35nM and 5.56nM to the IC50 of PC9 cell, show cRGD-
Erlotinib there is significant reinforced effects.
As shown in figures 11a and 11b: PC9 cell is to Erlotinib and the sensitivity of cRGD-Erlotinib.Figure 11 a shows,
Erlotinib and the cRGD-Erlotinib inhibited proliferation to PC9 cell.The Erlotinib of PC9 cell prescribed concentration and
CRGD Erlotinib processes 72 hours.Cell viability measures optical density value (490 nanometer) with mtt assay and determines.Figure 11 b shows, gram
Grand survival measures 2uM Erlotinib and cRGD-Erlotinib processes PC9 cell 7 days.In Figure 11 b, 1000,10000,100000
Cell is layered in triplicate soft agar culture dish, and the number of survivaling cell is by each group DMSO solvent control standardization.*P<
0.05, one factor analysis of variance, Newman-Keuls checks difference (n=3) two-by-two.
In order to study the sensitizing cells sex differernce observed further, We conducted clone's survival and measure.PC9 cell exists
After the Erlotinib of 2uM or cRGD-Erlotinib process, measure its Clone formation.Result shows, compares and individually uses Erlotinib
The cell processed, the cell clone existence after cRGD-Erlotinib processes substantially reduces.
CRGD-Erlotinib induction PC9 apoptosis
Then, we study cRGD-Erlotinib and compare whether Erlotinib increases PC9 apoptosis.CRGD-strategic point Lip river is replaced
Buddhist nun and Erlotinib effect apoptotic to PC9 are by the double dye of annexin V and PI, and use flow cytomery.As
Figure 12 a and 12b shows, the cRGD-Erlotinib of 20nM and the treatment of Erlotinib result in 36.56% He in 72 hours respectively
The apoptosis of 79.24%, and the apoptosis of solvent control cell is 2.08% (P < 0.05).These results indicate that cRGD-
Erlotinib can induce the apoptosis rate that PC9 cell is higher.Additionally, as shown in Figure 12b, cRGD-Erlotinib and Erlotinib energy
Activate caspase-3 and the PARP cracking of PC9 cell, show that cRGD-Erlotinib and Erlotinib trigger caspase protease
The apoptosis of cascade.
Figure 12: Erlotinib and cRGD-Erlotinib induction PC9 apoptosis.Figure 12 a, flow cytometry detects
DMSO, Erlotinib and cRGD-Erlotinib process impact apoptotic on PC9.DMSO, 20nM Erlotinib, 20nM
CRGD-Erlotinib process cell is after 72 hours, and harvesting also dyes with annexin-V/ propidium iodide (PI), leads to subsequently
Overflow-type Cytometric Analysis.Figure 12 b, DMSO, after Erlotinib treatment, cRGD-Erlotinib processes PC9 cell after 48 hours,
PI dyes.After Figure 12 c, immunoblotting assay cell death related protein, Erlotinib and cRGD-Erlotinib process 48 hours
The activation of caspase-3 and PARP.GAPDH is used as loading control.*Difference significant with Erlotinib group (P < 0.05, n=3).
Embodiment 2
First aspect, the invention provides the cancer target precursor medicine of a kind of anti-nonsmall-cell lung cancer, and it is cRGD-angstrom gram
For Buddhist nun's conjugate, concrete structure is shown in accompanying drawing 3.Wherein, cRGD is ring-type Arg-Gly-Asp tripeptide, it is possible to the most main
Moving-target is to the polypeptide of tumor tissues；Conmana is treatment non-small cell lung cancer drug.
Second aspect, the invention provides the preparation method of the cancer target precursor medicine of a kind of anti-nonsmall-cell lung cancer, its
Synthetic route is shown in accompanying drawing 4, specifically includes following steps:
(1) initial action resin (Fmoc-Gly-CTC Resin) is prepared
In round-bottomed flask, 1.0g resin is dissolved in 7 milliliters of anhydrous methylene chlorides (DCM).Add the most under nitrogen protection
Fmoc-glycine-OH (891.9 milligrams, 3.0 mMs) and anhydrous DIPEA (DIPEA) (1.00 milliliters,
5.74 mM).This reaction solution is stirred at room temperature 4 hours under nitrogen protection.Finally, the MeOH/DIPEA of 10 milliliters is added
(9:1) termination mix reaction, the single step reaction 20 minutes of going forward side by side.After being filtered to remove solution, respectively with 5 milliliters of DCM and 5 milliliter two
Methylformamide (DMF) respectively washes twice, and washed once by 5 ml methanol subsequently.After washing, resin is dried under vacuum 24
Hour, and be stored in 4 DEG C standby
(2) solid-phase synthesis synthesizes into linear polypeptide resin Fmoc-Asp (the OtBu)-D-Phe-Lys of nitrine labelling
(N3)-Arg(Pbf)-Gly-CTC Resin
Under room temperature condition, by the initial action resin (Fmoc-Gly-CTC Resin) that obtains in step by step 1 at DMF/
DCM/ piperidines volume ratio be 2:1:1 solution condition under carry out Fmoc group deprotection reaction from aminoacid.Reaction 30min
After, use DMF that resin is carried out twice washing.Then, hard resin exists with aminoacid or the amino acid derivativges of Fmoc protection
Coupling reaction 3 hours in DCM solution, the most amino acid whose equivalent is 1, and the equivalent of N-hydroxybenzotriazole (HOBt) is 2, O-
(benzotriazole-1-base)-N, the equivalent of N, N', N-tetramethylurea hexafluorophosphate (HBTU) and DIPEA is respectively 2 and 5.At this
In coupling reaction, successively the aminoacid with N end Fmoc protection and side chain protected (is followed successively by Fmoc-arginine in order
(Pbf)-OH, Fmoc-lysine (N3)-OH, Fmoc-aspartic acid (OtBu)-OH) connect Fmoc-Gly-CTC resin.This is even
Connection reaction is analyzed by ninhydrin test.It is of particular importance that lysine introduces nitrine to facilitate in step 2 by side chain
Erlotinib conjugate is prepared in click chemistry coupling in by.After coupling reaction terminates, resin use respectively 5 milliliters of DMF and
DCM washes twice, and is dried 12 hours under vacuum condition, to obtain linear RGDfK solidification resin 3.
(3) linear polypeptide 6[H-Asp (OtBu)-D-Phe-Lys (N of nitrine labelling is synthesized3)-Arg(Pbf)-Gly-
OH)】
Under the solution condition that DMF/DCM/ piperidines volume ratio is 1:1:2, remove Fmoc base from linear DfKRG solidification resin
Group.Then under room temperature condition, the DfKRG solidification resin of de-Fmoc in acetic acid/2,2,2-trifluoroethanols (TFE) and the mixing of DCM
In solution, (volume ratio is 3:1:6) carries out cracking reaction.After 2 hours, there is the linear peptides RGDfK of side chain protecting group from resin
It is cleaved in 3.After cracking reaction, resin uses the mixed solution of acetic acid/TFE/DCM to wash twice.The eluate that will merge
It is mixed in the hexane of 15 times of volumes, uses Rotary Evaporators to be evaporated removing solvent, to obtain, there is nitrine labelling and side chain
The linear peptides RGDfK 4 of protective effect.
(4) ring type polypeptide [cyclo (-Arg-Gly-Asp-D-Phe-Lys (N3)-)] of liquid phase Cyclization nitrine labelling
Under nitrogen protective condition, by the nitrine labelling obtained in step by step 3 and the linear polypeptide 4[H-Asp of side chain protected
(OtBu)-D-Phe-Lys(N3)-Arg (Pbf)-Gly-OH] to be dissolved in DMF solution to concentration be 1.25 × 10-3M.Then, with
HOBt/HBTU/DIPEA carries out liquid phase cyclization as condensing agent.This cyclization adds 1 equivalent in DMF solution
HOBt, the HBTU and the DIPEA of 5 equivalents of 2 equivalents, under nitrogen protection reaction 24 hours, to obtain nitrine labelling and side chain guarantor
The ring type polypeptide (-arginine (Pbf)-Gly-Asp (OtBu)-D-phenylalanine-lysine (N3)-) 5 protected.Will be anti-
After answering solution to be concentrated under vacuum, at ambient temperature, the residue of cyclic peptide 5 be used in 15mL deprotection solution (95%TFA,
2.5% water and the dimercaptoethylsulfide of 2.5%) complete deprotection 24 hours, finally obtain the linear of nitrine labelling and side chain protected
Polypeptide 6[cyclo (-Arg-Gly-Asp-D-Phe-Lys (N3)-)], i.e. ring (-arginine-glycine-aspartic acid-D-phenylpropyl alcohol
Propylhomoserin-lysine (N3)-).Rough deprotection cyclic peptide 6, by reversed-phase high-performance liquid chromatography RP-HPLC purification, utilizes linear ladder
Degree buffer (90% acetonitrile of 20-45% is dissolved in 0.05%TFA) obtains the cyclic peptide of 98% purity at Agela C18 post.Peptide 6 leads to
Cross ESI and high resolution mass spec analysis.Its retention time is 25 minutes.C27H40N11O7[M+H]+HRMS value of calculation be
630.31, detected value is 629.98.
(5) Conmana (40 milligrams, 0.102 mM) is dissolved in 15ml DMF, adds ring (-Arg-glycine-Radix Asparagi
Propylhomoserin-D-phenylalanine-lysine (N 3)-) in solution (42.8 milligrams, 0.068 mM) be subsequently adding, be subsequently added
10.5 milliliters of triethylammonium acetate buffer (pH7.0,0.57M) and 3.0 milliliters of ascorbic acid (5.0 mMs).By solution
Mix 30 minutes under room temperature.Then, 1.5 milliliter of 10 milli copper (II)-tris [(1-benzyl-1H-1,2,3-triazole-4-yls) is added
Methyl] amine complex (10 millimeters, 55%DMSO).By the logical argon 20 minutes of the mixture obtained and be stirred for 24 under argon gas
Hour.By reactant mixture C18 post linear gradient buffer (the 0.05%TFA solution in 90% acetonitrile by RP-HPLC
25-45%) concentrate and purify, the productivity with 61% obtain 42.3 milligrams of rings (-Arg-Gly-Asp-D-phenylalanine-
Lysine (N 3)-)-Conmana conjugate 1.Final conjugate 1 is analyzed by ESI high resolution mass spec.HPLC TR=20
Minute.Purity: 89.35% (HPLC).HRMS: calculate C49H61N14O11:[M+H] 1022.09, measured value 1022.96.
Table 7 is the sample message of embodiment 2 final finished:
Table 8 is the testing result of embodiment 2 final finished:
| Detection | Testing result |
| Outward appearance | Freeze-dried material |
| Molecular weight (byMS, M+H+) | 1022.96 |
| Molecular weight (byMS, M+Na+) | 1044.06 |
| Molecular weight (byMS, M+K+) | |
| Purity (byHPLC) | 89.35% (HPLC, 220nm, C18, linear gradient elution) |
；
Table 9 is the storage condition of embodiment 2 final finished:
| Storage condition | -20℃ |
。
Claims (7)
1. selected from compound and the pharmaceutically acceptable structure thereof of formula I-II,
Formula 1,
Formula 2.
2. prepare a compound method as described in structural formula 1 or 2 in claim 1, comprise the following steps:
Ring type polypeptide cyclic RGDfK-azide [the cyclo (-Arg-of nitrine labelling is synthesized according to Solid-phase Polypeptide technology order
Gly-Asp-D-Phe-Lys(N3)-)】；
Cyclo (-Arg-Gly-Asp-D-Phe-Lys (the N that will obtain in step 13)-) is with Erlotinib by click chemistry altogether
Yoke obtains end product.
Compound method the most according to claim 2, it is characterised in that: step 1 includes the most step by step:
(1) Fmoc solid-phase synthesis synthesizes into linear polypeptide resin [Fmoc-Asp (the OtBu)-D-Phe-Lys of nitrine labelling
(N3)-Arg(Pbf)-Gly-CTC Resin】
2-chlorine trityl chloride resin, Fmoc-Glycine-OH and anhydrous DIPEA are dissolved in dichloromethane
Middle stirring appropriate time；Add the methanol prepared and anhydrous DIPEA mixed solution continues stirring；Then, will tree
Fat respectively with dimethylformamide (DMF, 2 times), DCM(2 time) and methanol (2 times) wash, be vacuum dried；
The aminoacid being finally introduced sequentially into protection (is followed successively by Fmoc-arginine (Pbf)-OH, Fmoc-lysine in order
(N3)-OH, Fmoc-aspartic acid (OtBu)-OH connects Fmoc-Gly resin；
(2) linear polypeptide H-Asp (OtBu)-D-Phe-Lys (N of nitrine labelling is synthesized3)-Arg(Pbf)-Gly-OH
In DMF/DCM/ piperidines mixed system, the linear polypeptide DfKRG solidification resin of nitrine labelling removes Fmoc group；
Then under room temperature condition, in acetic acid/2, the mixed solution of 2,2-trifluoroethanols (TFE)/DCM carries out cracking reaction；
After cracking reaction, resin uses the mixed solution of acetic acid/TFE/DCM to wash twice；
Finally by merge eluate mix with the hexane of 15 times of volumes in, use Rotary Evaporators be evaporated removing solvent with
Obtain linear peptides 4 (H-Asp (OtBu)-D-Phe-Lys (N with side chain protected effect3)-Arg(Pbf)-Gly-OH)；
Liquid phase Cyclization cyclo (-Arg-Gly-Asp-D-Phe-Lys (N3)-)
Under nitrogen protective condition, linear polypeptide H-Asp (the OtBu)-D-of nitrine labelling that will finally give in step by step 2
Phe-Lys(N3)-Arg (Pbf)-Gly-OH carries out liquid phase cyclization using HOBt/HBTU/DIPEA as condensing agent；
Then, after being concentrated under vacuum by reaction solution, solution is concentrated further at tetrafluoroethene, water and dimercaptoethane
Deprotection in mixed solvent, finally obtains linear polypeptide the cyclo (-Arg-Gly-Asp-D-Phe-Lys of nitrine labelling
(N3)-)。
Compound method the most according to claim 2, it is characterised in that: step 2 includes the most step by step: a certain amount of strategic point
Buddhist nun/erlotinib, cyclo (-Arg-Gly-Asp-D-Phe-Lys (N are replaced in Lip river3)-), second triethylenetetraminehexaacetic acid ammonium buffer and Vitamin C
Acid is dissolved in hybrid reaction in dimethylformamide, is subsequently added copper (II)-three [(1-benzyl-1H-1,2,3-triazole-4-yls) first
Base] amine complex continue reaction to appropriate time；Reversed-phase high-performance liquid chromatography RP-HPLC is used to concentrate and purify reactant mixture,
Obtain cyclo (-Arg-Gly-Asp-D-Phe-Lys (N3)-)-Erlotinib conjugate.
Compound method the most according to claim 3, it is characterised in that: the reaction of 1 is in the protection of noble gas step by step
Under carry out.
Compound method the most according to claim 4, it is characterised in that: the reaction of step 2 is under the protection of noble gas
Carry out.
7. the purposes in preparing anti-lung-cancer medicament of the compound in claim 1.
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|---|---|---|---|---|
| WO2017152756A1 (en) * | 2016-03-11 | 2017-09-14 | 徐州瑞康生物科技有限公司 | Crgd-erlotinib conjugate and preparation method thereof |
| CN110143997A (en) * | 2019-04-18 | 2019-08-20 | 湖州泽溪源生物科技有限公司 | A kind of operating method of expansible synthetic oligonucleotide peptide conjugate |
| CN117224697A (en) * | 2023-09-20 | 2023-12-15 | 安徽省立医院(中国科学技术大学附属第一医院) | cRGD modified xanthine and its derivative nano medicine and preparation method |
| WO2024087332A1 (en) * | 2022-10-26 | 2024-05-02 | 深圳先进技术研究院 | Bifunctional compound used as target protein degradation agent and use thereof in target protein lysosomal degradation |
| CN117224697B (en) * | 2023-09-20 | 2024-06-07 | 安徽省立医院(中国科学技术大学附属第一医院) | CRGD modified xanthine and its derivative nano medicine and preparation method |
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| CN115894490B (en) * | 2022-10-27 | 2024-05-24 | 陕西中医药大学 | Hydroxy evodiamine and erlotinib split drug containing indoloquinone unit, and preparation method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017152756A1 (en) * | 2016-03-11 | 2017-09-14 | 徐州瑞康生物科技有限公司 | Crgd-erlotinib conjugate and preparation method thereof |
| CN110143997A (en) * | 2019-04-18 | 2019-08-20 | 湖州泽溪源生物科技有限公司 | A kind of operating method of expansible synthetic oligonucleotide peptide conjugate |
| WO2024087332A1 (en) * | 2022-10-26 | 2024-05-02 | 深圳先进技术研究院 | Bifunctional compound used as target protein degradation agent and use thereof in target protein lysosomal degradation |
| CN117224697A (en) * | 2023-09-20 | 2023-12-15 | 安徽省立医院(中国科学技术大学附属第一医院) | cRGD modified xanthine and its derivative nano medicine and preparation method |
| CN117224697B (en) * | 2023-09-20 | 2024-06-07 | 安徽省立医院(中国科学技术大学附属第一医院) | CRGD modified xanthine and its derivative nano medicine and preparation method |
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