CN106317067B - A kind of antitumor medicine conjugate, preparation method, preparation and application - Google Patents
A kind of antitumor medicine conjugate, preparation method, preparation and application Download PDFInfo
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- CN106317067B CN106317067B CN201610700195.XA CN201610700195A CN106317067B CN 106317067 B CN106317067 B CN 106317067B CN 201610700195 A CN201610700195 A CN 201610700195A CN 106317067 B CN106317067 B CN 106317067B
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- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 claims abstract description 5
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- PSHKMPUSSFXUIA-UHFFFAOYSA-N n,n-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
- C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Prepare and apply the invention discloses a kind of antitumor medicine conjugate, preparation method and its nano-micelle.Shown in the structural formula of conjugate provided by the invention such as formula (I), it is formed by connecting by 7 ethyl, 10 hydroxycamptothecin and taxanes antitumor drug by connecting key, wherein, L is connecting key；R1For phenyl or tert-butoxy, R2For acetyl group, H or methyl, R3For H or methyl, step is simple, and manufacturing cost is low, and stability is high, and security is good, meets the requirement of clinical application, suitable for large-scale industrial production.Invention additionally discloses nano-micelle and its antitumor application of the antitumor drug coupling with amphipathic nature polyalcohol composition, in vivo studies as shown in formula (I) to show the good antitumous effect of nano-micelle, have preferable market prospects and value.
Description
Technical field
The invention belongs to antitumor drug research and development technology field, a kind of antitumor medicine conjugate, preparation are specifically related to
Method, preparation and application.
Background technology
Cancer is the common disease of serious threat human life safety.In current oncotherapy, the chemistry based on medicine
Therapy plays irreplaceable effect, but long-term single drug easily makes human body produce drug resistance to certain drug.And combine
Therapeutic regimen can then improve the antitumor curative effect of medicine, delay the generation of body drug resistance, reduce adverse reaction and toxic side effect
(Ma L,Kohli M,Smith A.Nanoparticles for Combination Drug Therapy.ACS
nano.2013,7:9518-25.) at present, the treatment of tumour changes from initial single drug to drug combination direction.
7-Ethyl-10-hydroxycamptothecin (SN38) is the active ingredient of clinical antineoplastic medication Irinotecan (CPT-11),
Its anti tumor activity in vitro is 100-1000 times of CPT-11.But water is dissolved in since its is extremely difficult, a small number of organic solvents can only be dissolved in,
So its use in biological medicine is extremely limited.The SN38 delivery systems reported both at home and abroad at present mainly include thing
Two kinds of forms of reason embedding and chemical bonding.However, how unstable the drugloading rate and carrier medicine carrying efficiency of the SN38 medicine-carried systems of physically trapping be
Fixed, insoluble drug release is uncontrollable, and there are the phenomenon of burst release.Then there are drugloading rate is low, synthesis is tired for the SN38 medicine-carried systems of chemical bonding
The problems such as difficult and molecular structure is complicated.
Taxol is initially a kind of natural antitumor medicine extracted from the trunk, bark of Taxus various plants,
There is the effect of obvious to many cancers.So far, taxol and its semi-synthetic analog Docetaxel have become in history
The most salable cancer therapy drug, is widely used in antitumor treatment.But taxol is water-soluble extremely low, its application is also subject to one
Fixed limitation.
Polymer micelle is a kind of novel nano carrier that fast development is got up in recent years, by amphipathic block copolymer
It is self-assembly of in water, there is the nucleocapsid structure of hydrophobic cores and hydrophily shell, hydrophobic inner core can loads slightly solubility
Medicine, improves the stability of insoluble drug, and hydrophilic outer shell can be effectively prevented from the phagocytosis of reticuloendothelial system, extend medicine
Circulation time in vivo, improves medicine pharmacokinetics behavior in vivo, improves target-oriented drug.Antineoplastic combination is used
The Research Challenges of medicine body system are how two or more medicines are effectively encapsulated in same liposome, make it in blood circulation
Middle stabilization, and ensure proportional action that antitumor active ingredient acts synergistically according to it in tumour cell.
The research of our early periods finds, the unique chemical moieties of camptothecine and taxol make its be difficult to directly encapsulating with it is same
In nano-carrier, in the case of can ensureing the antitumor activity of medicine by the way of the chemical modification, allow medicament to seal jointly
Loaded in nano-carrier.
Up to the present, be not related to the combination of camptothecin and taxanes antitumor drug and its nano-micelle prepare and
The report of application.
The content of the invention
The present invention provides a kind of antitumor medicine conjugate, it is resisted by 7-Ethyl-10-hydroxycamptothecin and taxanes
Tumour medicine is formed by connecting by connecting key, and step is simple, and manufacturing cost is low.
Present invention also offers a kind of antitumor medicine conjugate nano-micelle preparations, preparation method is simple, and stability is high,
Security is good, meets the requirement of clinical application, suitable for large-scale industrial production.
Present invention also offers a kind of application of antitumor medicine conjugate nano-micelle preparations in antitumor, have compared with
It is safe for the rate of release of mitigation, it is without side-effects.
A kind of antitumor medicine conjugate, the antitumor medicine conjugate have such as lower structure：
A—L—B
Wherein：The structure of A is：
The structure of B is：
Setback line position is the connection position with L；
That is, the general structure such as (I) of described antitumor medicine conjugate：
Wherein, L is linkage section, R1For phenyl or tert-butoxy, R2For acetyl group, H or methyl, R3For H or methyl.
Preferably, in structure formula (I), work as R1For phenyl when, R2For acetyl group, R3For H；R1For tert-butoxy when, R2For
H, R3For H；Alternatively, R1For tert-butoxy when, R2For methyl, R3For methyl；
Preferably, the linkage section has such as lower structure：
N=2-6；
As further preferred, the n=2.
The structure difference of i.e. preferable antitumor medicine conjugate is as follows：
Present invention also offers a kind of preparation method of above-mentioned antitumor medicine conjugate, including：The precursor raw material of A structures
Diacid anhydride reactant corresponding with L, is prepared midbody compound (II), midbody compound (II) precursor with B structure again
Raw material reacts, and obtains final antitumor medicine conjugate；
Midbody compound (II) structure is as follows：
The precursor raw material structure of the A structures is as follows：
The precursor raw material structure of the B structure is as follows：
Preferably, as n=2, midbody compound (II) structure is as follows：
Corresponding, the corresponding dicarboxylic anhydride of the L is succinic anhydride；When n takes other numerical value, corresponding diacid can be used
Acid anhydride, prepares according to the method described above.
Preferably, the precursor raw material of the A structures is selected from taxol (PTX), Docetaxel (DTX), Cabazitaxel
(DTX) etc..The precursor raw material of the B structure is 7-Ethyl-10-hydroxycamptothecin.
Preferably, in the reaction of the precursor raw material of A structures dicarboxylic anhydride corresponding with succinic anhydride or other L, one
As add DMAP as catalyst；Reaction can be completed at room temperature.The precursor raw material of the A structures and the molar ratio of succinic anhydride
For 1：(1~4).
Preferably, when the precursor raw material of the midbody compound (II) and B structure reacts, it is general add condensing agent and
Catalyst etc., condensing agent are typically chosen EDCHCl, and catalyst is typically chosen, DMAP, while by DIEA and EDCHCl
In hydrochloric acid.Reaction can select to carry out at room temperature, and reaction condition is gentle.Preferably, the midbody compound (II)
Precursor raw material molar ratio with B structure is 1:(1~1.5), more preferably 1:1.
A kind of antitumor medicine conjugate nano-micelle preparations, the nano-micelle is by antitumor medicine conjugate and amphiphilic
Property macromolecule composition, the mass ratio of the amphipathy macromolecule and medicine is 5:1~1:10.
Preferably, the amphipathy macromolecule be selected from DSPE-PEG, PEG-PLA, PEG-PLGA, mPEG-PLA,
Any one in mPEG-PLGA.It is DSPE-PEG as further preferred, described amphipathy macromolecule2000。
Preferably, the preparation is pulvis or tablet, injection, pill etc..
A kind of preparation method of above-mentioned antitumor medicine conjugate nano-micelle preparations, including, antitumor drug is coupled
Thing and the dissolving of amphipathy macromolecule material in organic solvent, are then added in water, you can obtain Combretastatin nanometer formulation.
A kind of application of above-mentioned antitumor medicine conjugate nano-micelle in antitumor.
The present invention is formed by connecting by 7-Ethyl-10-hydroxycamptothecin and taxanes antitumor drug by connecting key, is walked
Rapid simple, manufacturing cost is low, and stability is high, and security is good, meets the requirement of clinical application, suitable for large-scale industry metaplasia
Production.Nano-micelle is made in antitumor drug coupling and amphipathic nature polyalcohol by the present invention, and in vivo studies shows that nano-micelle is good
Antitumous effect, have preferable market prospects with value.
Brief description of the drawings
Fig. 1 is the synthetic route of SN38-PTX conjugates 1；
Fig. 2 is the synthetic route of SN38-DTX conjugates 2；
Fig. 3 is the synthetic route of SN38-CTX conjugates 3；
Fig. 4 is the Electronic Speculum grain-size graph of nano-micelle 1-NM in embodiment 4；
Fig. 5 is the Electronic Speculum grain-size graph of nano-micelle 2-NM in embodiment 5；
Fig. 6 is the Electronic Speculum grain-size graph of nano-micelle 3-NM in embodiment 6；
Fig. 7 is the vitro stability test result of nano-micelle in embodiment 5,7,8,9；
Fig. 8 is the release in vitro of nano-micelle in embodiment 5；
Fig. 9 is the release in vitro of nano-micelle in embodiment 7；
Figure 10 is the extracorporeal suppression tumor cell cultivation effect of conjugate in embodiment 2；
Figure 11 is the tumor-targeting test result of nano-micelle in embodiment 5；
Figure 12 is the internal antitumous effect of nano-micelle in embodiment 4,5,6；
In figure, SN38 represents 7-Ethyl-10-hydroxycamptothecin, and PTX represents taxol, and DTX represents Docetaxel, CTX
Representing Cabazitaxel, CPT-11 represents Irinotecan, and DMF represents dimethylformamide, and DMAP represents dimethyl aminopyridine,
EDCHCl represents the hydrochloride of 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides, and HOBT represents I-hydroxybenzotriazole,
HBTU represents benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphate, and DSPE-PEG represents distearyl acyl group phosphatidyl
Monoethanolamine-polyethylene glycol, DIEA represent n,N-diisopropylethylamine.
Embodiment
The present invention is described in further detail with reference to the accompanying drawings and detailed description, but the present invention and from its limit
System.
Embodiment 1
The synthetic route of SN38-PTX conjugates is as shown in Figure 1, comprise the following steps that：
PTX (500mg, 0.59mmol) and succinic anhydride (176mg, 1.77mmol) are added in 100mL round-bottomed flasks, it is molten
Solution adds DMAP (7.2mg, 0.06mmol), 25 DEG C of stirring 3h in 8mL anhydrous pyridines, and oil pump removes pyridine, 0.1N
HCl, saturated salt solution respectively clean 1 time；Organic phase is dried with anhydrous sodium sulfate, and filtering, solvent is removed under reduced pressure after collecting filtrate；Gu
Body isolates and purifies (DCM with column chromatography chromatogram:MeOH=80:1) product 5 (550mg, yield 98%) is obtained after.
Product 51H NMR nuclear magnetic datas and mass spectrometric data are as follows：
1H NMR(400MHz,CDCl3)：δ1.14(s,3H),1.23(s,3H),1.69(s,3H),1.89-1.92(d,4H,
), J=11.2 2.20-2.22 (d, 4H, J=9.6), 2.44 (s, 3H), 2.54-2.64 (m, 4H), 2.67-2.70 (t, 2H),
3.68 (s, 2H), 3.80-3.82 (d, 2H, J=6.8), 4.20-4.23 (d, 1H, J=8.8), 4.30-4.32 (d, 1H, J=
8.4), 4.42-4.46 (m, 1H), 4.97-5.00 (d, 1H, J=8.8), 5.53-5.54 (d, 1H, J=3.2), 5.69-5.71
(d, 1H, J=7.6), 5.98-6.01 (m, 1H), 6.30 (s, 1H), 7.13-7.15 (d, 1H, J=9.2), 7.40-7.45 (m,
6H), 7.50-7.54 (t, 3H), 7.60-7.62 (t, 1H), 7.76-7.77 (d, 1H, J=7.2), 8.10-8.12 (d, 2H, J=
7.2).
HRMS：Calculated value C51H55NO17]+[M+H]+=954.3548；Observation：954.3534.
Product 5 (550mg, 0.58mmol) and SN38 (226mg, 0.58mmol), dissolving are added in 100mL round-bottomed flasks
In 18mL anhydrous DMFs, EDCHCl (122mg, 0.64mmol), DMAP (7.2mg, 0.06mmol) and DIEA (144 are added
μ L, 0.87mmol), 25 DEG C are stirred overnight, and oil pump removes solvent, add DCM, and successively with 5% citric acid, saturation NaHCO3、
Saturated salt solution respectively cleaning 1 time；Organic phase is dried with anhydrous sodium sulfate, and filtering, solvent is removed under reduced pressure after collecting filtrate；Solid is used
Column chromatography chromatogram isolates and purifies (DCM:MeOH=100:1) product SN38-PTX conjugates 1 (320mg, yield 42%) are obtained after.
Product SN38-PTX conjugates 11H NMR nuclear magnetic datas and mass spectrometric data are as follows：
1H NMR(400MHz,CDCl3):δ0.84-0.90(m,3H),1.04-1.07(t,3H),1.17(s,3H),1.70
(s,3H),1.89-1.93(m,5H),1.99(s,4H),2.24(s,4H),2.50-2.53(m,4H),2.89-2.95(m,4H),
2.99-3.08 (m, 3H), 3.77 (s, 1H), 3.84-3.85 (d, 1H, J=6.8), 4.21-4.23 (d, 1H, J=8.4),
4.32-4.35 (d, 1H, J=8.8), 4.46-4.49 (m, 1H), 4.98-5.01 (d, 1H, J=9.2), 5.17-5.21 (m,
2H),5.30(s,1H),5.34(s,1H),5.53-5.54(m,1H),5.71-5.77(m,2H),6.05-6.07(m,1H),
6.32 (s, 2H), 7.00-7.04 (m, 3H), 7.11-7.14 (t, 3H), 7.42-7.43 (d, 4H, J=4), 7.49-7.52 (m,
3H),7.59-7.61(d,3H),7.64(s,1H),7.86(s,1H),8.16-8.19(m,3H)。
HRMS：Calculated value [C73H73N3O21]+[M+H]+=1328.4815；Observation：1328.4798.
Embodiment 2
The synthetic route of SN38-DTX conjugates is as shown in Fig. 2, comprise the following steps that：
DTX (500mg, 0.62mmol) and succinic anhydride (186mg, 1.86mmol) are added in 100mL round-bottomed flasks, it is molten
Solution adds DMAP (7.2mg, 0.06mmol), 25 DEG C of stirring 3h in 8mL anhydrous pyridines, and oil pump removes pyridine, 0.1N
HCl, saturated salt solution respectively clean 1 time；Organic phase is dried with anhydrous sodium sulfate, and filtering, solvent is removed under reduced pressure after collecting filtrate；Gu
Body isolates and purifies (DCM with column chromatography chromatogram:MeOH=80:1) product 6 (500mg, yield 91%) is obtained after.
Product 51H NMR nuclear magnetic datas and mass spectrometric data are as follows：
1H NMR(400MHz,CDCl3):δ 1.08-1.13 (t, 3H), 1.20 (s, 3H), 1.32-1.34 (d, 9H, J=
7.2), 1.74-1.76 (d, 3H, J=6.4), 1.83-1.86 (d, 2H, J=10.0), 1.89-1.91 (d, 3H, J=7.2),
2.27-2.28 (t, 2H), 2.37-2.39 (d, 2H, J=8.4) 2.53-2.64 (m, 5H), 2.77-2.83 (m, 2H), 3.89-
3.92 (t, 1H), 4.17-4.20 (t, 1H), 4.23-4.32 (m, 2H), 4.95-4.97 (d, 1H, J=8.4), 5.26 (s, 1H),
5.39-5.48 (m, 2H), 5.66-5.67 (d, 1H, J=7.2), 6.18-6.21 (t, 1H), 7.29-7.31 (d, 1H, J=
7.6), 7.37-7.40 (m, 2H), 7.49-7.54 (q, 2H), 7.61-7.63 (t, 1H), 8.09-8.10 (d, 2H, J=6.8).
HRMS：Calculated value [C47H57NO17]+[M+H]+=908.3704；Observation：908.3680.
Product 6 (420mg, 0.46mmol) and SN38 (180mg, 0.46mmol), dissolving are added in 100mL round-bottomed flasks
In 18mL anhydrous DMFs, add HOBT (68mg, 0.51mmol), HBTU (193mg, 0.51mmol) and DIEA (69 μ L,
0.42mmol), it is stirred overnight for 25 DEG C, oil pump removes solvent, adds DCM, and successively with 5% citric acid, saturation NaHCO3, saturation
Saline solution respectively cleaning 1 time；Organic phase is dried with anhydrous sodium sulfate, and filtering, solvent is removed under reduced pressure after collecting filtrate；Solid column layer
Analyse chromatographic separation and purification (DCM:MeOH=100:1) product SN38-DTX conjugates 2 (220mg, yield 37%) are obtained after.
Product SN38-DTX conjugates 21H NMR nuclear magnetic datas and mass spectrometric data are as follows：
1H NMR(400MHz,CDCl3):δ1.02-1.06(t,3H),1.11(s,3H),1.21(s,3H),1.26(s,
2H),1.29(s,2H),1.31(s,9H),1.37-1.41(t,3H),1.76(s,3H),1.82-1.97(m,7H),2.43(s,
3H),2.58-2.65(m,1H),2.81-2.88(m,1H),2.93-2.99(m,3H),3.11-3.18(m,2H),3.82-3.84
(m, 1H), 3.91-3.93 (d, 1H, J=6.8), 4.18-4.20 (d, 1H, J=7.6), 4.26-4.34 (m, 2H), 4.97-
4.99 (d, 1H, J=8.0), 5.15 (s, 1H), 5.25 (s, 2H), 5.33 (s, 1H), 5.39-5.48 (m, 3H), 5.67-5.71
(m,1H),5.73-5.77(t,1H),6.22(m,1H),7.28-7.32(t,3H),7.36-7.40(t,2H),7.48-7.52
(t, 2H), 7.53-7.56 (d, 1H, J=8.8), 7.59-7.63 (m, 1H) 7.64 (s, 1H), 7.82-7.83 (d, 1H, J=
2.4), 8.10-8.12 (d, 2H, J=7.2), 8.21-8.23 (d, 1H, J=9.2).
HRMS：Calculated value [C69H75N3O21]+[M+H]+=1282.4971；Observation：1282.4947.
Embodiment 3
The synthetic route of SN38-CTX conjugates is as shown in figure 3, comprise the following steps that：
CTX (500mg, 0.60mmol) and succinic anhydride (180mg, 1.80mmol) are added in 100mL round-bottomed flasks, it is molten
Solution adds DMAP (7.2mg, 0.06mmol), 25 DEG C of stirring 3h in 8mL anhydrous pyridines, and oil pump removes pyridine, adds DCM
Dissolving, is respectively cleaned 1 time with 0.1N HCl, saturated salt solution successively；Organic phase is dried with anhydrous sodium sulfate, and filtering, collects filtrate
After solvent is removed under reduced pressure；Solid isolates and purifies (DCM with column chromatography chromatogram:MeOH=80:1) product 7 (549mg, yield are obtained after
98%).
Product 71H NMR nuclear magnetic datas and mass spectrometric data are as follows：
1H NMR(400MHz,CDCl3):δ1.21-1.22(s,6H),1.36(s,9H),1.57(s,7H),1.72(s,
3H), 1.87-1.88 (d, 3H, J=1.2), 2.25-2.27 (d, 1H, J=9.2), 2.66-2.73 (m, 5H), 3.30 (s, 3H),
3.45 (s, 4H), 3.80-3.88 (m, 2H), 4.16-4.18 (d, 1H, J=8.4), 4.29-4.31 (d, 1H, J=8.4), 4.80
(s, 1H), 4.96-4.98 (d, 1H, J=8.0), 5.62-5.64 (d, 1H, J=7.2), 6.19-6.23 (t, 1H), 7.31-
7.33 (m, 1H), 7.38-7.42 (m, 4H), 7.47-7.50 (t, 2H), 7.59-7.63 (t, 1H), 8.08-8.10 (d, 2H, J=
7.2)。
HRMS：Calculated value [C49H61NO17]+[M+H]+=936.4017；Observation：936.4026.
Product 7 (548mg, 0.59mmol) and SN38 (229mg, 0.59mmol), dissolving are added in 100mL round-bottomed flasks
In 18mL anhydrous DMFs, EDCHCl (125mg, 0.65mmol), DMAP (79mg, 0.65mmol) and DIEA (146 μ are added
L, 0.89mmol), 25 DEG C are stirred overnight, and oil pump removes solvent, add DCM, and successively with 5% citric acid, saturation NaHCO3, it is full
Respectively cleaned with saline solution 1 time；Organic phase is dried with anhydrous sodium sulfate, and filtering, solvent is removed under reduced pressure after collecting filtrate；Solid column
Thin layer chromatography isolates and purifies (DCM:MeOH=100:1) product SN38-CTX conjugates 3 (321mg, yield 42%) are obtained after.
Product SN38-CTX conjugates 11H NMR nuclear magnetic datas and mass spectrometric data are as follows：1H NMR(400MHz,
CDCl3):δ1.03-1.06(t,3H),1.20(s,3H),1.22(s,3H),1.25(s,2H),1.33(s,9H),1.38-1.41
(t,3H),1.72(s,3H),1.86-1.94(m,2H),2.00(s,3H),2.44(s,3H),2.67-2.75(m,1H),2.85-
2.94(m,2H),2.96(s,2H),3.13-3.18(q,2H),3.30(s,3H),3.42(s,3H),3.74(s,1H),3.84-
3.86 (d, 1H, J=7.2), 3.88-3.93 (m, 1H), 4.16-4.18 (d, 1H, J=8.4), 4.30-4.32 (d, 1H, J=
8.4), 4.81 (s, 1H), 4.99-5.01 (d, 1H, J=9.2), 5.26 (s, 2H), 5.30 (s, 2H), 5.42 (s, 1H), 5.64-
5.66 (d, 1H, J=7.2), 5.74-5.78 (d, 1H, J=16.0), 6.25-6.29 (t, 1H), 7.29-7.33 (t, 3H),
7.37-7.40 (t, 2H), 7.47-7.53 (m, 3H), 7.58-7.62 (m, 1H), 7.65 (s, 1H), 7.84-7.85 (d, 1H, J=
2.4), 8.10-8.12 (d, 2H, J=7.2), 8.21-8.23 (d, 1H, J=9.2).
HRMS：Calculated value [C71H79N3O21]+[M+H]+=1310.5284；Observation：1310.5251.
Embodiment 4
SN38-PTX conjugates 1 and DSPE-PEG prepared by Example 12000(conjugate and DSPE-PEG2000Mass ratio
For 1:5) DMSO (40mg/mL) in right amount, is dissolved in, (conjugate final concentration 0.5mg/mL) is diluted in water, obtains containing SN38-
The nano-micelle (being denoted as 1-NM) of PTX conjugates 1.Its Electronic Speculum particle diameter is as shown in figure 4, the results show that obtained nano-micelle is big
Small uniform, shape is in regular circle.
Embodiment 5
SN38-DTX conjugates 2 and DSPE-PEG prepared by Example 22000(conjugate and DSPE-PEG2000Mass ratio
For 1:5) DMSO (40mg/mL) in right amount, is dissolved in, (medicine final concentration 0.5mg/mL) is diluted in water, obtains containing SN38-DTX
The nano-micelle (being denoted as 2-NM) of conjugate 2, envelop rate 99.0%, drugloading rate 19.3%.Its Electronic Speculum particle diameter is as shown in figure 5, knot
Fruit shows that obtained nano-micelle is uniform in size, and shape is in regular circle.
Embodiment 6
SN38-CTX conjugates 3 and DSPE-PEG prepared by Example 32000(conjugate and DSPE-PEG2000Mass ratio
For 1:5) DMSO (40mg/mL) in right amount, is dissolved in, (medicine final concentration 0.5mg/mL) is diluted in water, obtains containing SN38-CTX
The nano-micelle (being denoted as 3-NM) of conjugate 3.Its Electronic Speculum particle diameter is as shown in fig. 6, the results show that obtained nano-micelle size is equal
Even, shape is in regular circle.
Embodiment 7
SN38-DTX conjugates 2 and DSPE-PEG prepared by Example 22000(conjugate and DSPE-PEG2000Mass ratio
For 10:1) DMSO (40mg/mL) in right amount, is dissolved in, (medicine final concentration 0.1mg/mL) is diluted in water, obtains containing SN38-DTX
The nano-micelle of conjugate 2, envelop rate 99.4%, drugloading rate 92.3%.Measure particle size 79 ± 18nm, PDI (0.237) table
Bright particle diameter distribution is uniform.
Embodiment 8
SN38-DTX conjugates 2 and DSPE-PEG prepared by Example 22000(conjugate and DSPE-PEG2000Mass ratio
For 5:1) DMSO (40mg/mL) in right amount, is dissolved in, (medicine final concentration 0.1mg/mL) is diluted in water, obtains containing SN38-DTX
The nano-micelle of conjugate 2, envelop rate 99.6%, drugloading rate 85.6%.Measure particle size 86 ± 18nm, PDI (0.208) table
Bright particle diameter distribution is uniform.
Embodiment 9
DTX-SN38 conjugates 2 and DSPE-PEG prepared by Example 22000(conjugate and DSPE-PEG2000Mass ratio
For 1:1) DMSO (40mg/mL) in right amount, is dissolved in, (medicine final concentration 0.1mg/mL) is diluted in water, obtains containing DTX-SN38
The nano-micelle of conjugate 2, envelop rate 99.3%, drugloading rate 54.6%.Measure particle size 74 ± 15nm, PDI (0.138) table
Bright particle diameter distribution is uniform.
Feature below by way of test case the present invention is furture elucidated the nano-micelle and its therapeutic effect to tumour：
Test case 1
Nano-micelle room temperature prepared by embodiment 5,7,8,9 is placed, and at regular intervals by particle instrument, detects nanometer
The vitro stability of micella, testing result as shown in fig. 7, the results show that each group nanometer under the long-time room temperature environment of 14 days,
Particle diameter is relatively stablized, it is shown that nanoparticle prepared by embodiment 5,7,8,9 has preferable stability.
Test case 2
Nano-micelle (0.1mg/mL) prepared by embodiment 5 is respectively placed in the bag filter that molecular weight is 7000kDa, outside
Boundary 25mL pH are 37 DEG C in the phosphate buffer of 7.4 (containing 0.2% tween), and rotating speed is interval one in the environment of 150r/min
The extraneous phosphate buffer of taking-up of fixing time, measures SN38 contents, so as to obtain the external of Nano medication with ultraviolet specrophotometer
Release conditions.It can be seen that from experimental result Fig. 8, nano-micelle prepared by embodiment 5 can be released slowly for a long time in an in vitro environment
Put, and without obvious phenomenon of burst release, be conducive to the long-time circulation of medicine in vivo.
Test case 3
Nano-micelle (0.1mg/mL) prepared by embodiment 5 is respectively placed in the bag filter that molecular weight is 7000kDa, outside
Boundary 25mL pH are 37 DEG C in the phosphate buffer of 7.4 or 4.6 (containing 0.2% tween), and rotating speed is in the environment of 150r/min, is divided
Extraneous phosphate buffer is not taken out in 2h, 4h, 8h, 12h, 24h, 48h, 72h and 120h, SN38 is measured with ultraviolet specrophotometer
Content, so as to obtain release in vitro situation of the Nano medication of the preparation of embodiment 5 under two kinds of pH environment.From experimental result Fig. 9
Can be seen that, nano-micelle prepared by embodiment 5 can slowly discharge for a long time in an in vitro environment, and without obvious phenomenon of burst release,
Be conducive to the long-time circulation of medicine in vivo.
Test case 4
Take the logarithm growth period cell (HCT-116, LoVo, A549), after pancreatin digestion, kind plate (96 orifice plates, 5 × 103It is a thin
Born of the same parents/hole), 37 DEG C are incubated 24h and make cell attachment, using SN38, DTX as control group, add in the embodiment 2 of various concentrations and prepare
DTX-SN38 conjugates 2 (100 μ L/ holes), after cultivating 48h, 30 μ L tetrazolium bromides (MTT, 5mg/mL) are added per hole, continue to cultivate 4h
After remove supernatant, per hole add 100 μ L dimethyl sulfoxide (DMSO)s (DMSO), vibration, crystallized product is fully dissolved, 492nm is in enzyme
Each hole absorbance is detected on mark instrument, draws out cell survival curve, and calculate 503nhibiting concentration of each group nanoparticle to cell
(IC50) value.In vitro toxicity the result is shown in Figure 10 and table 1 of the antitumor medicinal liposome to various tumour cells.
Table 1：IC of the DTX-SN38 conjugates 2 to tumour cell in embodiment 250(μM)
It can be seen that from Figure 10 and table 1, by preparing DTX-SN38 conjugates 2 in embodiment 2 to free SN38 with similar
Cytotoxicity, there is stronger anti-tumour cell proliferative.
Test case 5
Balb/c nude mices (5 week old) subcutaneous vaccination HCT-116 colon-cancer cells, are divided into three groups：(fluorescence contaminates free Cy 5.5
Material), the nano-micelle (being denoted as Cy 5.5-NM) of load C y 5.5 and while load C y 5.5-DTX conjugates 4 and embodiment 5 in
Nano-micelle (being denoted as 2/4-NM).Tail vein single medication (5.5 dosage of Cy, 20 μ g/ are only), certain intervals after medication, use
Living imaging instrument analyzes distribution situation of the medicine in nude mouse, and 24h takes each group nude mice conscience spleen and lung kidney and tumor tissues, analyzes
Medicine fluorescence intensity.As a result it is as shown in figure 11.2/4-NM has stronger distribution in tumor tissues after display 24h, it was demonstrated that real
The nano-micelle for applying the preparation of example 5 has stronger tumor-targeting and anelasticity.
Test case 6
Tumor suppression is carried out to intestinal cancer HCT-116 models under Animal Skin to nano-micelle 1-NM, 2-NM, 3-NM in embodiment 4,5
Evaluation.Balb/c nude mice models tumour is after 2 weeks, a tail vein administration every three days, altogether three times：Physiological saline, CPT-11
(11.5mg/kg), 1-NM, 2-NM and 3-NM (SN38 equivalent dose 10mg/kg), 6 groups altogether.To be administered for the first time as 0 day, survey
Measure tumor volume change and carry out result statistics.In the evaluating drug effect the result is shown in Figure 12 of subcutaneous tumor (a) and (b).By in Figure 12
(a) understand, nano-micelle 1-NM, 2-NM and 3-NM are aobvious to suppressing tumour growth tool relative to clinical CPT-11 in embodiment 4,5
The effect of work, and the effect of 2-NM is better than the Nano medication in embodiment 4 in 1-NM in embodiment 5.From Figure 12 (b),
A middle of the month is treated, nano-micelle 1-NM, 2-NM and 3-NM have not significant impact nude mice weight in embodiment 4,5, it changes
Trend is similar with physiological saline group, and display medicine has preferable internal compatibility, to medication nude mice without obvious toxic-side effects.
Claims (10)
1. a kind of antitumor medicine conjugate, it is characterised in that the antitumor medicine conjugate has such as lower structure：
A—L—B
Wherein：The structure of A is：
The structure of B is：
Wherein, L is linkage section, R1For phenyl or tert-butoxy, R2For acetyl group, H or methyl, R3For H or methyl；For A
With the connection position of B and L.
2. antitumor medicine conjugate according to claim 1, it is characterised in that in A structures：Work as R1For phenyl when, R2For
Acetyl group, R3For H；R1For tert-butoxy when, R2For H, R3For H；Or R1For tert-butoxy when, R2For methyl, R3For methyl.
3. antitumor medicine conjugate according to claim 1, it is characterised in that the linkage section has such as lower structure：
N=2-6.
A kind of 4. preparation method of any one of claims 1 to 3 antitumor medicine conjugate, it is characterised in that including：A
Precursor raw material diacid anhydride reactant corresponding with L, is prepared midbody compound (II), midbody compound (II) is again with B's
Precursor raw material reacts, and obtains final antitumor medicine conjugate, above-mentioned reaction carries out at room temperature；
Midbody compound (II) structure is as follows：
The precursor raw material structure of the A structures is as follows：
The precursor raw material structure of the B structure is as follows：
5. any one of a kind of claims 1 to 3 antitumor medicine conjugate nano-micelle preparations, it is characterised in that described
Nano-micelle is made of antitumor medicine conjugate and amphipathy macromolecule, and the mass ratio of the amphipathy macromolecule and medicine is
5:1~1:10.
6. antitumor medicine conjugate nano-micelle preparations according to claim 5, it is characterised in that described is amphipathic
Any one of macromolecule in DSPE-PEG, PEG-PLA, PEG-PLGA, mPEG-PLA, mPEG-PLGA.
7. antitumor medicine conjugate nano-micelle preparations according to claim 6, it is characterised in that described is amphipathic
Macromolecule is DSPE-PEG2000。
8. according to claim 5~7 any one of them antitumor medicine conjugate nano-micelle preparations, it is characterised in that institute
The preparation stated is pulvis or tablet, injection, pill.
9. a kind of preparation method of any one of claim 5~8 antitumor medicine conjugate nano-micelle preparations, its feature
It is, including, antitumor medicine conjugate and amphipathy macromolecule material are dissolved in organic solvent, are then added to water
In, you can obtain medicament-carried nano preparation.
10. a kind of any one of claim 5~8 antitumor medicine conjugate nano-micelle preparations are preparing antitumor drug
In application.
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