CN108727582A

CN108727582A - Target anticancer conjugate

Info

Publication number: CN108727582A
Application number: CN201710263113.4A
Authority: CN
Inventors: 袁建栋; 黄仰青; 宋云松; 丁海峰
Original assignee: Borui Pharmaceutical (suzhou) Ltd By Share Ltd
Current assignee: Borui Pharmaceutical (suzhou) Ltd By Share Ltd; Brightgene Bio Medical Technology Co Ltd
Priority date: 2017-04-21
Filing date: 2017-04-21
Publication date: 2018-11-02
Anticipated expiration: 2037-04-21
Also published as: CN108727582B

Abstract

The invention discloses the highly branched chain drug conjugates of the formula that has the following structure (I) or its pharmaceutically acceptable salts：R is organic core, and POLY is polymer, and L is polyvalent linkers, and T is targeted molecular, and D is activating agent, any integers of the q between 3-8, and wherein L is：

Description

Target anticancer conjugate

Technical field

The present invention relates to the target anticancer conjugates of multiarm polymers modification, it is more particularly related to will targeting Molecule connects into conjugate by multiarm polymers and anticancer drug.

Background technology

For many years, it has been proposed that a variety of methods of stability and delivering for improving bioactivator.With medicinal examination It the preparation of agent and delivers associated challenge and may include：The water solubility of the difference of the medicinal reagent, toxicity, low biological utilisation Rate, unstability and rapidly degradation in vivo.Although having devised thousand and one way to improve the delivering of medicinal reagent, Be a kind of no individual method it is without its disadvantage.For example, the delivery method of generally use aims at solution Or at least improve one or more problems with, include the medicine such as in a kind of liposome, polymer substrate or unimolecular micelle Composite capsule, be covalently attached on a kind of water-soluble polymer such as polyethylene glycol, the use of gene target agent, salt structure, Etc..

WO2005028539, WO2010019233, WO2011063156, WO2011063158 disclose a kind of in clinic The drug nktr 102 of three phases, the drug are mainly used for metastatic breast cancer, are researched and developed by NektarTherapeutics.The drug It is a kind of water-soluble multibranched polymer prodrug, to improve the load of drug, structure is as follows：

The compound is connect with Irinotecan using multi-arm PEG, to improve water solubility, increases drugloading rate, is made in anticancer In the case of constant, side effect is reduced.But the drug still has the disadvantage, for example, targeting is poor, cannot act on specific Cancer cell can also influence the performance of normal cell while killing cancer cell, and adverse reaction rate is made still to compare It is high.

Invention content

The invention discloses a kind of completely new highly branched chain drug conjugates with targeting, there are three conjugate tools or More branches, the conjugate are represented by following formula：

R is organic core, and POLY is polymer, and L is polyvalent linkers, and T is targeted molecular, and D is activating agent, and q is between 3-8 Any integer, wherein L is：

Symbol " * " represents the tie point of polyvalent linkers L and targeted molecular T, and " # " represents the company of polyvalent linkers L and activating agent D Contact, " % " represent the tie point of polyvalent linkers L and POLY, wherein any integers of the l between 2-20, m, n are respectively 0-10 Between any integer；

D is the camptothecine as shown in formula (II)：

R₁-R₅It is independently selected from following group：Hydrogen, halogen, acyl group, alkyl, substitution alkyl, alkoxy, substitution alkane Oxygroup, alkenyl, alkynyl, naphthenic base, hydroxyl, cyano, nitro, azido, amide groups, hydrazine, amido, substituted amido, hydroxycarbonyl group, Alkoxy carbonyl group, alkyl oxy carbonyl oxygen, carbamoyloxy, aryl-sulfonyl oxygen, alkylsulfonyloxy；R₆For H or OR₈, R₈For alkane Base, alkenyl, naphthenic base, halogenated alkyl or hydroxyalkyl；R₇For hydroxyl, amino or mercaptan.

POLY is polymer, and L is polyvalent linkers, and T is targeted molecular, and D is activating agent, and four collectively form the highly branched chain medicine " branch " of object conjugate.Each branch of the highly branched chain drug conjugates and other branches are independent from each other.It is each A branch is sent out from organic core " R ".However, in general, each branch of the conjugate is identical.

Describe each variable part in structural formula (I) in detail now.

Organic core, " R "

In structural formula (I), " R " is organic core group of a 1-100 atom.It is that R contains 3-50 better A atom is preferably that R contains about 3-30 atom.R can be the core of all carbon atoms, also optionally contain There are one or more hetero atoms, for example, O, S, N, P etc., depend on used special central element.R can be straight chain, branch Or it is cricoid, send out the independent polymer branch of at least three.In structural formula (I), " q " and the polymer branch that is sent out from " R " Quantity is corresponding.

Organic core " R " is derived from a molecule, which provides the position of many polymer connections, closely The approximately equal to quantity of polymer branch.Preferably it is that the central core molecule formula of multichain polymer structure is at least provided with being suitable as For 3 of polymer branch and 3 or more hydroxyls, the polyol of sulfenyl or amino, polysulfide or polyaminated Close the residue of object.One " polyol " is a molecule being made of multiple (being more than 2) available hydroxyl groups. One " polysulfide " is a molecule being made of multiple (being more than 2) available thio groups.One " polyamines chemical combination Object " is a molecule being made of multiple (being more than 2) available amine groups.According to the quantity of polymer branch, polyhydroxy The parent of based compound, polyamine compounds or polysulfide typically comprises 3-25 hydroxyl (before POLY Covalent bonding togethers) Base, sulfenyl or amino, preferably 3-10 hydroxyl, sulfenyl or amino preferably include from 3 to about 8 (such as 3,4,5,6, 7 or 8) it is suitble to and hydroxyl, sulfenyl or the amino of POLY Covalent bonding togethers.

Typically there are one structures before being acted on polymer for polyol or the parent at polyamine compounds center Formula R- (OH) p or R- (NH₂)p.In structural formula (I), p value and q values are corresponding, because in parent organic molecule Each functional groups, typically there is-OH and-NH₂If position susceptible easily reacts, they just and polymerization The POLY Covalent bonding togethers of object branch.In structural formula (I), after being connect with POLY, the hydroxyl of the polyol of R parents is all It is converted at a polymer branch, described R be connection after residue.For example, if organic core molecules are from season Penta tetrol derivative is got, and the parent of polyol possesses structural formula C (CH₂OH)₄, organic core base R is expressed as：

Illustrative polyol preferably as polymer core includes containing 1 to 10 carbon atoms and 1 to 10 The aliphatic polyhydroxy compound of a hydroxyl group, for example, ethylene glycol, alkane glycol, alkyl ethylene glycol, alkyliden two Alcohol, alkyl cycloalkane diols, 1,5- naphthalanes glycol, 4,8- bis- (methylol) tristane, ring aklylene glycol, dihydroxy alkane Hydrocarbon, trihydroxy alkane, tetrahydroxy alkane etc..Cycloaliphatic polyols include straight chain or closed-ring sugars and sugar alcohol, Such as mannitol, D-sorbite, inositol, xylitol, quebrachite, threitol, arabite, erythritol, hexitol, core Sugar, pectinose, xylose, lyxose, rhamnose, galactolipin, glucose, fructose, sorbose, mannose, pyranose, A Zhuo Sugar, talose, Tagatose, pyranoside, sucrose, lactose, maltose etc..Aromatic polyhydroxy compounds, such as benzene can also be used Phosphorus diphenol, alkyl benzene phosphorus diphenol, pyrogaelol, fluoro glycine phenol, oxyhydroquinone, resorcinol, alkyl resorcinol, two hydrocarbon Base resorcinol, orcin monohydrate, olive phenol, hydroquinone, alkyl hydroquinone, phenyl hydroquinone etc..Other can The polyol center that can be used may include crown ether, cyclodextrin, dextrin or other carbohydrate.

In structural formula (I), q is the number of the polymer branch connected on corresponding " R ", specific number can be 3~ 20.Typically, the specific number of " q " is 3,4,5,6,7,8.Specifically, send out three centered on " R ", four, five, six, seven, Eight polymer branches.

In some specific schemes, " R " has there are three polymer branch, and " R " is preferably：

In some specific schemes, " R " has there are four polymer branch, and " R " is preferably：

In some specific schemes, " R " has there are six polymer branch, and " R " is preferably：

In some specific schemes, " R " is preferably with eight polymer branches, " R "：

Polymer, " POLY "

In structural formula (I), " POLY " is polymer, and the POLY in each polymer branch is to be selected independently out , preferably, each polymer is identical polymer, and more appropriately, the polymer branch in each structural formula (I) is It is identical.Preferred polymer is water-soluble, and arbitrary water-soluble polymer can be used for being formed the conjugate of the present invention, this The signified polymer of invention can be arbitrary geometric shape or form.Representative polymer includes but is not limited to：Poly- second two Alcohol, polyvinylpyrrolidone), poly- (hydroxyalkylmethacrylamide), poly- (hydroxyalkylmethacrylate), gathers polypropylene glycol (carbohydrate), poly- (±-carboxylic acid), poly- (acrylic acid), poly- (vinyl acetate), poly-phosphine piperazine, polyoxazoline, poly- (N- acryloyl morpholines) Deng.

In typical compound, " POLY " is polyethylene glycol (PEG), can be arbitrary geometric shape or form, including straight Chain, branch, fork-shaped chain etc., it is " polyethylene glycol " as used herein, it means and covers any water-soluble poly (ethylene oxide).It is typical Ground will include two one kind with lower structure for the PEG in the present invention："(CH₂CH₂O)_k" or " (CH₂CH₂O)_k- CH₂CH₂", whether for example it has been replaced during synthesis conversion at one depending on one or more end oxygen.Variable k ranges It is from 5 to about 500, and the structure of these end groups and totality PEG can change.The polyethylene glycol structures are usual Also contain portion distal end moiety, is similar to the terminal group of POLY, can be H, NH₂、OH、CO₂H、C_1-6Alkyl (for example, Methyl, ethyl, propyl), C_1-6Alkoxy (for example, methoxyl group, ethyoxyl), acyl group or aryl terminate.

Currently preferred " POLY " is linear polyethylene glycol, and typical structure is：

The junction for representing atom, the oxygen atom for marking ampersand are and have The atom of machine center " R " connection.Wherein the value range of k is about 5~500, preferably 50~200, r appointing between 1-10 One integer, it is furthermore preferred that " POLY " of the invention is：

POLY of the present invention can also be：

Deng.

Signified activating agent " D " of the invention is camptothecin anticancer agent, and camptothecine is different for clinical topology I inhibitor of structure enzyme has the shortcomings that poorly water-soluble, big to normal body tissue toxic side effect, greatly while high activity Limit the clinical application of camptothecin anticancer agent.

R in D structures₇For the group being covalently attached with polyvalent linkers L, such as hydroxyl, amino or mercaptan, preferably hydroxyl. When activating agent D is connected to polyvalent linkers L, bioactivity that should be ungreat is lost.

The preferred Irinotecan of activating agent of the present invention, SN-38,10-hydroxycamptothecine, Rubitecan.

Wherein, Irinotecan structure is as follows：

The structure of SN-38 is as follows：

The structure of 10-hydroxycamptothecine is as follows：

The structure of Rubitecan is as follows：

In the present invention, " T " is targeted molecular, and with or without pharmacologic action, the effect of the targeted molecular is to increase target Tropism so that the conjugate improves physiological activity or pharmacologic action in the concentration higher of destination organization." T " can be unifunctional Targeted molecular or multi-functional targeted molecular, in some selectable schemes, " T " can also be two or more The targeting moiety of targeted molecular composition.For example, T can be Angiopep2.

The polypeptide sequence of Angiopep2 is：TFFYGGSRGKRNNFKTEEY, structure are as follows：

Signified activating agent " D " of the invention refers to a part for non-modified parent active agent or by drug with the present invention's Polyvalent linkers be covalently attached caused by covalently chain (or its activation or chemical modification form) before it is unmodified The residue of parent active agent.When linker between active agent moiety and polyvalent linkers is hydrolyzed or digested, activating agent inherently obtains To release.

Purpose according to the present invention, term " residue " are construed as a part for compound, it refer to experienced with separately Residue after one compound substitution reaction.

Conjugate of the present invention enters in organism, and when reaching target cell or target tissue, activating agent D is broken with polyvalent linkers L, Activating agent D is not improved with it, i.e., the form for not forming covalent bond is released, and is detached with parent, plays physiological activity.

In preferred embodiments of the present invention, " POLY " is linear polyethylene glycol linking arm, i.e., conjugate of the present invention include with The compound of lower several types：

Four arms：

Three arms：

Eight arms：

Wherein the value range of k is about 5~500, any integers of preferably 50~200, the r between 1-10.

Preferred formula (III) compound of the present invention, on the basis of formula (III), k preferably 113.Those skilled in the art should know Dawn, in the field of polymers, k represents the degree of polymerization of the polymer, and depending on the molecular weight of the polymer, is not Absolute numerical value, when k values 113, refer to average value be 113.

In preferred scheme, the targeting moiety " T " of conjugate of the present invention is Angiopep2, and activating agent " D " is selected from her Vertical one kind in health, SN-38,10-hydroxycamptothecine, Rubitecan.

In preferred scheme, L is selected from：

In one kind.

Based on formula (III), in some specific schemes, the compounds of this invention is as follows：

Compound e：D is Irinotecan, T Angiopep-2

Compound E is the pharmaceutical salts hydrochlorate of compound e

More specifically, compound e can be written as following form：

Herein it should be noted that when at salt, be conjugate of the present invention branch and HCl respectively at salt, such as chemical combination Object E, carries 6 molecule HCl on each branch, and entire molecule is by the HCl with 24 molecules.

Spirit according to the present invention, in addition to particular compound disclosed above, those skilled in the art can also basis Technical solutions according to the invention and preparation method prepare more conjugates, such as：

1. D is SN-38, T is the conjugate of Angiopep2；

2. D is 10-hydroxycamptothecine, T is the conjugate of Angiopep2；

3. D is Rubitecan, T is the conjugate of Angiopep2.

Conjugate of the present invention is typical prodrug, and by hydrolysis or enzymolysis, activating agent D is released Come, detached with parent, plays physiological activity.

Conjugate of the present invention shows high loadability, can thus reduce accumulated dose to treat a kind of special disease Disease, such as cancer etc..That is, conjugate active agent carrier of the present invention can be effectively with various active agent molecule with covalent Bond is closed, and allows that per certain coupling object amount further amounts of therapeutic dosage forms (namely active agent moiety) can be taken.The present invention Conjugate be substantially conjugate is also hydrophilic by the modification of water-soluble polymer, and especially activating agent is water difficult-soluble medicine When object, the bioavilability of conjugate is improved.

Compared to the drug not being coupled, conjugate of the present invention can show stronger effect, in human body or other animal bodies Inner tissue is more enriched with.

Conjugate prodrug unique property containing there are many in the present invention is especially an anti-cancer compounds in activating agent In the case of object.This prodrug can inhibit the growth of tumour with greater efficiency.This small molecule that we use is one Small molecule with anticancer property known to kind.However, by being combined as described above with multibranched polymer, curative effect and drug generation It thanks to dynamics compared with the small molecule (for example, anticancer compound itself), there is prodigious improvement.Suitable variety of solid tumor types Including colon cancer, breast cancer, oophoroma, cancer of pancreas, gastric cancer, glioma and breast, ovary, colon, kidney, bile duct, lung and brain Malignant sarcomas, cancer and lymthoma.

To sum up, the present invention is the target anticancer conjugate of multiarm polymers modification, wherein water-soluble polymer is repaiied Decorations can enhance the water solubility of the conjugate, to improve drugloading rate；Targeted molecular increases targeting so that the conjugate is in target The concentration higher of tissue；L be arbitrary jointing, effect be targeted molecular and anticancer drug are connected first, then By " targeted molecular, anticancer drug and polymeric arms connect so that entire conjugate forms an organic whole.

Conjugate of the present invention, pharmaceutically acceptable salt are preferably hydrochloride, can use the conventional means of medicinal chemistry art Can also be trifluoroacetate, sulfate, phosphate, acetate etc. at salt.

On the other hand, the present invention provides the preparation methods of the conjugate.In conjugate preparation process of the present invention, POLY and organic core R actually constitute multiarm polymers, and in the preferred embodiment of the invention, which is more Arm polyethylene glycol can be obtained from commercially available raw material, for example, can be bought from Jiankai Science and Technology Co., Ltd., Beijing more Four arms, three arms, the eight arm polyethyleneglycol derivatives of type.Commercially available these multi-arms PEG can directly participate in reacting.

When formula (III) conjugate, it is preferable to use four arm polyethylene glycol it is as follows：

The preferred four arms polyethylene glycol is referred to as 4ARM-PEG20K-SCM, and molecular weight is about 20kDa or so.Similarly, When formula (IV) and formula (V) conjugate, the molecular weight of three arms and eight arm polyethylene glycol that use is preferably also 20kDa or so.

Specific embodiment

It present invention will be described in detail below.However, the present invention may be embodied as many different forms, And it is not necessarily limited in embodiment described herein, and it is to make disclosure to provide the purpose in these embodiments More completely with comprehensively.Agents useful for same and raw material, except providing preparation method, remaining is commercially available, wherein 4ARM- PEG20K-SCM is purchased from Jiankai Science and Technology Co., Ltd., Beijing

Explanation of nouns

DMF：N,N-dimethylformamide

DCM：Dichloromethane

Boc-Gly-OH：

DMAP：4-dimethylaminopyridine

DCC：Dicyclohexylcarbodiimide

IPA：Isopropanol

TFA：Trifluoroacetic acid

TBME：T-butyl methyl ether

EA：Ethyl acetate

DME：Glycol dimethyl ether

Fmoc-OSU：9- fluorene methyl-N- succinimdyl carbonates THF：Tetrahydrofuran

H-Lys(Boc)-OBzl·HCl：

DIEA：N, N- diisopropylethylamine

DEPC：Diethyl phosphorocyanidate

DEA：Triethylamine

Pbf：

HOBT：I-hydroxybenzotriazole

DIC：N, N- diisopropylcarbodiimide

TFE：Trifluoroethanol

DPPA：Diphenyl phosphate azide

SPPS：Solid-phase organic synthesis

NMM：N-methylmorpholine

TIS：Tri isopropyl silane

MTBE：T-butyl methyl ether

Embodiment 1

The preparation of compound 2

3.50g compounds 1 (1.0eq) are added into 250mL round-bottomed flasks, 52.5mlDMF is heated to 60 DEG C of dissolvings, 5- Decompression boils off DMF after 10min, and the vacuum distillation of 300ml normal heptanes is added, in triplicate, 105mlDCM, 1.08g are added after being spin-dried for Boc-Gly-OH (1.2eq), 63mg DMAP (0.1eq) are added dropwise 1.59gDCC (1.5eq) and are dissolved in the solution of 10mlDCM, 20 DEG C Reaction 4 hours, TLC are monitored after completion of the reaction, and filtering is added 120ml IPA when being concentrated into remaining 25% volume, boils off 75% Solvent is added 150ml normal heptanes, is stirred at room temperature 1 hour, filters, and normal heptane washs 2 times, dry faint yellow solid 4.02gization Close object 2.

The preparation of compound 3

4.02g compounds 2,50mlDCM are added into 100mL there-necked flasks, 11.6mlTFA, room temperature is added dropwise after stirring and dissolving 2h is reacted, TLC monitoring is added 150ml acetonitriles, is poured into 320mlTBME solution after being evaporated under reduced pressure 120ml solvents after completion of the reaction, 30min, filtering are stirred, filter cake washs to obtain 4.00g faint yellow solids compound 3 with TBME.

Embodiment 2

The preparation of compound 5

It is added 6.9g compounds 4 into 250mL there-necked flasks, 30ml EA are cooled to 0 DEG C after stirring and dissolving, add 40ml The HCl/EA of 0.3M, insulation reaction 2h, TLC monitoring reaction, which finishes, to be concentrated to dryness, and is obtained compound 5, is directly carried out anti-in next step It answers.

The preparation of compound 6

Compound 5 (1.0eq) 50ml is purified into water dissolution, 3.96g sodium bicarbonates (2.0eq) are added, it will with 50mlDME 5.30g Fmoc-OSU (1.0eq) dissolve, and are added in the reaction bulb of compound 5, add 25mlTHF, are stirred at room temperature 2 hours, TLC is monitored after completion of the reaction, boils off organic solvent, EA extracting impurities, and water phase dilute hydrochloric acid adjusts pH to 3-4, and EA is extracted 2 times, Merge organic phase, washing is primary, and anhydrous sodium sulfate is dried after saturated common salt water washing, is concentrated to give 8.4g pale yellow oil chemical combination Object 6.

The preparation of compound 7

In 100ml reaction bulbs be added 4.00g compounds 6 (1.0eq), 2.92g H-Lys (Boc)-OBzlHCl, 40mlDCM dissolves, and 2.76gDIEA (3.0eq) is added, and 1.74g DEPC (1.5eq) are stirred at room temperature 2 hours, TLC monitoring reactions After, aqueous acetic acid washing, sodium bicarbonate solution washing, washing is primary, and saturated salt solution washed once rear anhydrous slufuric acid Sodium is dried, and 7.0g pale yellow oils compound 7 is concentrated to give, and is not purified and is directly carried out next step reaction.(utilize same method Prepare compound 16)

The preparation of compound 8

7.0g compounds 7 are dissolved with the DEA/DCM of 140ml 25%, are stirred at room temperature 6 hours, TLC monitoring reactions finish Afterwards, it is concentrated to dryness, 100ml, 50mlEA is added, pH is adjusted to 3-4, liquid separation with dilute hydrochloric acid, water phase concentrates after being extracted 2 times with EA To doing, 3.5g faint yellow solids compound 8 is obtained.(utilizing same method prepare compound 17)

Embodiment 3

It is connected with the preparation of the targeted molecular Angiopep-2 (compound 50) of protecting group

The sequence of Angiopep-2 is TFFYGGSRGKRNNFKTEEY

Using 2Cl-Trt Resin, it is reaction dissolvent that coupling reagent, which uses HOBT/DIC, DMF, and reaction monitoring uses indenes three Following protected amino acid is connected on resin by ketone detection method successively：Fmoc-Tyr(tBu)-OH,Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Phe-OH、Fmoc-Asn (Trt)-OH、Fmoc-Asn(Trt)-OH、Fmoc-Arg(Pbf)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Gly-OH、Fmoc- Arg(Pbf)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Gly-OH、Fmoc-Gly-OH、Fmoc-Tyr(tBu)-OH、Fmoc- Lytic reagent is added in Phe-OH, Fmoc-Phe-OH, Boc-Thr (tBu)-OH：Acetic acid/TFE/DCM=1/2/7 reacts 2 hours, The MTBE precipitations of ice, washing, dry off-white powder compound 50.

The preparation of embodiment 4 compound e and compound E

The preparation of compound 51

Addition 6.20g compounds 50 (1.0eq) in 100ml reaction bulbs, 1.39g compounds 17 (1.1eq), 62mlDMF, 0.60gDIEA (3.0eq), 0.38g DEPC (1.5eq) react at room temperature 2h, and TLC is poured into after completion of the reaction in 10mL water, EA extractions It takes 2 times, aqueous acetic acid washing, sodium bicarbonate solution washing, anhydrous sodium sulfate is dried after saturated sodium-chloride washing, is concentrated to give fruit Freeze 51 6.5g of shape solid chemical compound, directly carries out next step reaction.

The preparation of compound 52

6.53g compounds 51,150ml methanol are added in the hydriding reactor of 200ml, 0.33gPd/C adds hydrogen to stay overnight, TLC reactions After filter, be concentrated to give 52 6.50g of gray solid compound

The preparation of compound 53

Addition 6.50g compounds 52 (1.0eq) in 100ml reaction bulbs, 1.08g compounds 3 (1.05eq), 65mlDMF, 0.54gDIEA (3.0eq), 0.34g DEPC (1.5eq) react at room temperature 2h, and TLC is poured into after completion of the reaction in 650ml TBME, It is filtered after mashing, solid pours into after being dissolved with 100ml DCM in 1.0L TBME, and mashing filters, dry grey powder 53 6.71g directly carries out next step reaction.

The preparation of compound 54

50ml lytic reagents are added in 2.5g compounds 53：Acetic acid/TFE/DCM=1/2/7 reacts 2 hours, and the MTBE of ice is heavy It forms sediment, washing, dry and HPLC purifies to obtain 0.97g off-white powders compound 54.

The preparation of compound 55

Addition 2.91g compounds 54 (4.5eq) in 50ml reaction bulbs, 3.00g 4ARM-PEG20K-SCM (1.0eq), 30ml DMF, 0.13gTEA (9.0eq), room temperature reaction are poured into after HPLC monitoring reaction is without apparent progress in 300ml TBME, Mashing filters, dry 55 5.83g, directly progress next step reaction.

The preparation of compound e

In 50ml reaction bulbs, 2.04g compounds 55,30ml lytic reagents is added：92.5%TFA/2.5% water/2.5% 2h is stirred at room temperature in TIS, and with the MTBE precipitations of ice, centrifugation, washing, the inverted HPLC of crude product desalinations after purification, concentration removes organic Off-white powder E 0.42g are lyophilized to obtain in solvent.

The preparation of compound E

The inverted HPLC of compound e crude products desalinations after purification, concentration remove organic solvent, with dilute hydrochloric acid adjust pH=5~ 6, yellow greenish powder E 0.42g are lyophilized to obtain.

MALDI-TOF detection molecules amounts are 33812.65.

5 compound of embodiment evaluating drug effect in HT-29 Nude Mouse Model bodies

1. experiment purpose

It evaluates after compound e and compound E is administered in human colon carcinoma HT-29 cell strain heterograft BALB/cnude mouse Evaluating drug effect in animal model.

2. experiment material

2.1 test sample

Irinotecan (bulk pharmaceutical chemicals) system purchase gained, nktr-102 and 2 test-compound is by rich auspicious biological medicine (Soviet Union State) limited liability company's offer.

Wherein, method of the preparation method of nktr-102 with reference to disclosed in CN102711837A is as follows：

Compound 3 (829mg, 4.5eq) in embodiment 1 is added in the reaction bulb of 250mL, DCM (50mL) is added, Triethylamine (221mg, 9.0eq) is added 4ARM-PEG20K-SCM (5.00g, 1.0eq) and is added in the reaction bulb after dissolving. After HPLC monitoring reactions are without being obviously in progress, about 20mL DCM are evaporated under reduced pressure away, solution is poured into 300mL TBME and is stirred Precipitation, filtering obtain 5.4g crude products, and crude product is prepared through HPLC and purified, desalination, adjust pH to 5-6 with dilute hydrochloric acid, freeze-drying obtains 2.71g pale green powder nktr-102.

2.2 reagent

McCoy ' s 5A culture solutions, fetal calf serum (FBS), trypsase, blueness-chain is dual anti-, water for injection, physiological saline, Lactic acid, D-sorbite.

2.3 experimental animal

Female BAl BIc/c nude mouse (numbers of elements：150；Week old：6~7 weeks) have from Beijing dimension tonneau China experimental animal technology Limit company buys, and raising illuminates each 12 hours in SPF animal houses, 20~25 DEG C of temperature, relative humidity 40%~70%, light and shade； Animal free water and feeding.After normal nursing about 1 week, through veterinary inspector, mouse can enter anthology experiment to sign in order.Point It is identified in animal root of the tail portion using marking pen before group, every animal cuts scarce mode with ear and identifies after grouping.

2.4 transplanted tumor tumor strains

Human colon cancer cell HT-29 derives from the American Type Culture Collection committee of the Chinese Academy of Sciences cell bank (CAS, this reality Test room liquid nitrogen cryopreservation).

3. experimental method

3.1HT-29 cell culture

In 5%CO₂, under 37 DEG C of condition of culture, HT-29 cells in containing 10% fetal calf serum McCoy ' s 5A culture solutions into Row regular growth culture；With 0.25% pancreatin had digestive transfer culture；It according to cell growth status, passes on 2 to 3 times weekly, passes on ratio It is 1:4 to 1:6.

It is prepared by 3.2 animal models

Exponential phase HT-29 cells are collected, are resuspended in after cell count in serum-free McCoy ' s 5A culture mediums, are adjusted Cell concentration is to 4 × 10⁷Cell/mL；It is fitted into 50mL centrifuge tubes, will centrifuge after so that it is uniformly dispersed with pipettor piping and druming cell Pipe is placed in ice chest；Cell suspension is drawn with 1mL syringes, it is subcutaneous to be injected into nude mice front right limb armpit, every animal inoculation pvaccination 100 μL(4×10⁶Cell/only), establish HT-29 Nude Mouse Models.Routine observation animal state and tumour growth feelings after inoculation Condition measures knurl footpath using electronic vernier caliper, and data directly input Excel electrical forms, calculate gross tumor volume.Wait for gross tumor volume Reach 100~300mm³, select be in a good state of health, animal 30 similar in gross tumor volume, be divided into 5 groups using randomized blocks (n=6).Experiment measures weekly 2 knurl footpaths after starting, calculate gross tumor volume, while weighing the weight of animals and recording.

Gross tumor volume (TV) calculation formula is as follows：

TV(mm³)=l × w²/2

Wherein, l indicates tumour major diameter (mm)；W indicates tumour minor axis (mm).

3.3 solvents are prepared

It weighs 0.5g D-sorbites to be fitted into 50mL centrifuge tubes, 50mL waters for injection, vortex oscillation is added in centrifuge tube So that solid matter is completely dissolved, is configured to the aqueous sorbitol solution (w/v) of concentration 1%, it is spare to be stored in 4 DEG C of refrigerators.

3.4 drug-delivery preparations are prepared

3..4.1 Irinotecan drug-delivery preparation is prepared

1% lactic acid of 0.15mL is added in the Irinotecan for weighing 12.0mg, and vortex oscillation makes complete drug dissolution, then divides Not Jia Ru 2.85mL 1% aqueous sorbitol solution, vortex oscillation be uniformly mixed, 1% lactic acid, 1% D-sorbite water in solution The ratio of solution is about 5:95(v/v).Irinotecan effective concentration is 4.0mgmL in solution^-1。

3.4.2 nktr-102 drug-delivery preparations are prepared

Every time before administration, the physiological saline of 2.5mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine Object is completely dissolved, and Irinotecan effective concentration is 4.0mgmL in solution^-1。

3.4.3 compound a and compound A drug-delivery preparations are prepared

Every time before administration, the compound a and compound A of precise 120.3mg, is added the physiological saline of 2.5mL respectively, Vortex oscillation, ultrasound (as need) make complete drug dissolution, and Irinotecan effective concentration is 4.0mgmL in solution^-1。

3.4.4 compound e and compound E drug-delivery preparations are prepared

Every time before administration, the compound E of precise 159.6mg respectively is added the physiological saline of 2.5mL, vortex oscillation, Ultrasound (as need) makes complete drug dissolution, and Irinotecan effective concentration is 4.0mgmL in solution^-1。

3.5 animal packets and administration

Animal packet and dosage regimen are shown in Table 1.Started to be administered for the first time in the grouping same day, rear end experiment in 21 days or so, Administered volume is 10mLkg^-1.The effective dose of equivalent Irinotecan is 40mgkg^-1.First group is solvent control Group, tail vein injection give physiological saline, every 4 days 1 time, 3 times (Q4D × 3) are administered altogether.2-5 groups respectively give by tail vein injection Given the test agent Irinotecan, nktr-102, compound e, compound E are given, is to be administered once for every 4 days, Q4D × 3.

1. Nude Mouse Model effect experiment dosage regimen of table

3.6 experiments terminate

Last day is tested, weighs in, measure animal euthanasia (CO after knurl footpath₂).It strips tumor tissues and weighs, count Calculate knurl weight tumour inhibiting rate.

4. data record, calculation formula

The calculation formula of relative tumour volume (RTV) is：

RTV=TV_t/TV_initial

Wherein, TV_initialThe gross tumor volume measured when being administered for grouping；TV_tIt is swollen when to be measured each time during administration Knurl is accumulated.

The calculation formula of Relative tumor proliferation rate (%T/C) is：

%T/C=100% × (RTV_T/RTV_C)

Wherein, RTV_TIndicate treatment group RTV；RTV_CIndicate solvent control group RTV.

The calculation formula of inhibition rate of tumor growth TGI (%) is：

TGI=100% × [1- (TV_t(T)-TV_initial(T))/(TV_t(C)-TV_initial(C))]

Wherein, TV_t(T)Indicate the gross tumor volume that treatment group measures every time；TV_initial(T)Indicate treatment group when grouping administration Gross tumor volume；TV_t(C)Indicate the gross tumor volume that solvent control group measures every time；TV_initial(C)Indicate solvent control when grouping administration The gross tumor volume of group.

The calculation formula of the weight of animals rate of descent is：

The weight of animals rate of descent=100% × (BW_initial-BW_t)/BW_initial

Wherein, BW_tIndicate the weight of animals measured every time during administration；BW_initialIndicate animal body when grouping administration Weight.

The calculation formula of knurl weight tumour inhibiting rate IR (%) is：

IR (%)=100% × (W_C-W_T)/W_C

Wherein, W_CIndicate control group knurl weight；W_TIndicate treatment group's knurl weight.

5. statistical analysis technique

Test data with 2007 softwares of Microsoft Office Excel calculate and ASSOCIATE STATISTICS is handled.Number It according to unless otherwise indicated, is indicated with mean ± standard error (Mean ± SE), two comparison among groups are examined using t.6. Germicidal efficacy

During the experiment, experimenter and animal doctor need to carry out continuous observation to the sign and health status of experimental animal. Any Novel presentation of animal, such as pain, depression, activity reduction etc., need to be recorded in experiment original record.If experiment is dynamic The Novel presentation of object is more than the file regulation of IACUC relevant animal welfares, can judge whether to stop experiment via animal doctor, and be notified to Experimental project responsible person.

7. result

For people's cancer heteroplastic transplantation tumor model, recommend to be used as test evaluation index using Relative tumor proliferation rate T/C (%), Proliferation rate is lower, illustrates to inhibit tumor effect the better, is shown in Table 2.

Table 2 is to tumor proliferation rate T/C (%)

* compared with the RTV of blank solvent, Irinotecan and nktr-102 groups, P < 0.05

# is compared with the %T/C of blank solvent, Irinotecan and nktr-102 groups, P < 0.05

Experimental result shows that the compounds of this invention has human colon carcinoma HT-29 Nude Mouse Model tumor growth in vivo Good inhibiting effect, and it is better than Irinotecan and nktr-102.

The inhibiting effect of 6 human breast carcinoma MDA-MB-231 nude mouse xenograft models of embodiment

1. experiment purpose

This research is with human breast carcinoma MDA-MB-231 Nude Mouse Models to the internal anti-swollen of compound e and compound E Tumor activity is evaluated.

2. experimental animal

2.1 animal species

Mouse.

2.2 kind

BALB/c nude mices.

2.3 gender

Female.

2.4 quantity

Inoculation 150 is tested with 30.

2.5 the age

6-8 weeks.

2.6 weight

± 20% weight mean values of 20-22g.

2.7 animal origins (supplier)

Shanghai western Poole-Bi Kai experimental animals Co., Ltd (BK), credit number SCXK (Shanghai) 2008-0016.2.8 experiment The care of animal

All experimental animals are raised in SPF ranks laboratory.Experimenter is responsible for daily nursing and experimental study.

2.8.1 animal identification identification method

Each equal wearing of mouse cage has the information such as experiment numbers, experiment group, experimenter's name, mouse strain and gender Identity card, mouse are marked with ear nail.

2.8.2 random grouping

When gross tumor volume reaches 150-200mm³It is divided into 5 groups with randomized blocks afterwards, every group of 6 mouse ensure between each group Gross tumor volume and mouse weight are uniform.The mean value of each group gross tumor volume and the mean value difference of all experimental animal tumor volumes do not surpass Cross ± 10%.

2.8.3 operational administrative specification

The operation and management of all experimental animals strictly observe animal and use and management guidance principle.

2.8.4 rearing conditions

Living conditions：IVC systems, per 6, cage

Temperature：20℃-26℃

Humidity：40% ± 70%

Illumination：Day alternates with night within 12 hours

2.8.5 feed

Size mouse feed is irradiated, is pulled together feed corporation,Ltd purchased from Beijing Australia of section.Ad lib.

2.8.6 drinking-water

City tap-water is drunk after filtering high pressure sterilization.

2.8.7 bedding and padding

Corncob, Shanghai Mao Sheng derivatives Science and Technology Ltd. use after high pressure sterilization.Bedding and padding twice are changed weekly.

2.8.8 the laundering period

Give mouse most short one week environment laundering period before experiment.

3. experiment material

3.1 test drugs

3.2 other chemical reagent and material

3.2.1 physiological saline

Physiological saline is purchased from Shanghai Hua Yuanchang richnesses medicine company (group) Co., Ltd.

3.2.2 asepsis injector

1ml asepsis injectors are purchased from Shanghai Kindly Enterprise Development Group (Shanghai, China).

3.2.3 cell strain

Human breast carcinoma MDA-MB-231 is purchased from cell biological research institute of the Shanghai Chinese Academy of Sciences.

MDA-MB-231 is incubated at DMEM culture mediums (GIBCO, the U.S.), (GIBCO, the U.S.) containing 10% fetal calf serum FBS, It is incubated at and contains 5%CO₂37 DEG C of incubators.

3.2.4 matrigel (BD Matrigel)

Matrigel Matrigel is purchased from U.S. company BD

3.3 instrument

Biohazard Safety Equipment (model：AC2-6E1), it is purchased from ESCO；

CO₂Water proof cell incubator (model：3111) Thermo Scientific Forma, are purchased from；

Inverted microscope (model：CKX41SF), it is purchased from Olympus；

Electric suction apparatus (model YX930D) is purchased from Shanghai Medical appliance industry (group) Co., Ltd；

Balance (plum Teller-support benefit AB135-S) is purchased from plum Teller-support benefit；

Low speed centrifuge (model LD5-2A) is purchased from Beijing Lei Boer centrifuges Co., Ltd；

Digimatic calipers (model：SF2000), it is purchased from Guanglu Digital Measure-Control Co., Ltd., Guilin.

4. experimental design

Establish human breast carcinoma MDA-MB-231 Xenografts in nude mice models, every inoculation 1 × 10⁶A cell.

Following (table 3) dosage of this experimental design and dosage regimen.

Table 3：Antitumor action in human breast carcinoma MDA-MB-231 Nude Mouse Models

5. compound is prepared

Preparation method is provided by Borui Biomedical Technology (Suzhou) Co., Ltd.

The volume 3mL that single-dose needs.

5.1 Irinotecan drug-delivery preparations are prepared

1% lactic acid of 0.15mL is added in the Irinotecan for weighing 12.0mg, and vortex oscillation makes complete drug dissolution, then divides Not Jia Ru 2.85mL 1% aqueous sorbitol solution, vortex oscillation be uniformly mixed, 1% lactic acid, 1% D-sorbite water in solution The ratio of solution is about 5:95(v/v).A concentration of 4.0mgmL of Irinotecan free form in solution^-1。

5.2 nktr-102 drug-delivery preparations are prepared

Every time before administration, the physiological saline of 2.5mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine Object is completely dissolved, a concentration of 4.0mgmL of Irinotecan free form in solution^-1。

5.3 compound e, compound E drug-delivery preparations are prepared：Precise respectively, is added the physiological saline of 2.5mL, is vortexed Vibrating makes complete drug dissolution, a concentration of 4.0mgmL of Irinotecan free form in solution^-1。

6. experimental method

MDA-MB-231 cell culture is in DMEM, (GIBCO, the U.S.) containing 10% fetal calf serum FBS.Cell is positioned over 5% CO₂37 DEG C of cultures of incubator.

Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model：The tumour cell for collecting exponential phase, weight after counting It is suspended from 1 × PBS, adjustment concentration of cell suspension to 1 × 10⁷/ml.With 1ml syringes (No. 4 syringe needles) dorsal sc on the right side of nude mice Inoculated tumour cell, 1 × 10⁶/ 0.1ml/ mouse.

Reach 100-200mm in gross tumor volume³When, animal is grouped at random by randomized blocks, is divided into 5 groups, makes Each group tumour difference is less than the 10% of mean value, and every group 6, the grouping same day is as Day1, grouping same day administration.

Experimental period carries out 3 weeks, measures the weight of animals twice and tumor size during experiment weekly.Daily observe and record is faced Bed symptom.It tests last day and puts to death animal, weigh in, remove tumour, weigh and photograph to record.

All zoopery operations strictly observe animal use and management regulation.The calculating of tumour relevant parameter is with reference to China CFDA《Cell toxicant series antineoplastic medicament non-clinical study technological guidance's principle》.

The calculation formula of gross tumor volume (Tumor volume, TV) is：TV=a × b²/2.Wherein a, b respectively represent tumour It measures long and wide.Relative tumour volume (relative tumor volume, RTV) calculation formula is：RTV=Vt/V₀.Wherein V₀ Gross tumor volume when being administered for grouping, Vt are gross tumor volume when measuring.The evaluation index of antitumor activity increases for Relative tumor Value rate T/C (%) and tumour inhibiting rate (%), calculation formula are respectively：T/C (%)=(T_RTV/C_RTV) × 100%.T_RTVFor treatment group RTV, C_RTVFor negative control group RTV；Tumour inhibiting rate (%)=(negative control group average knurl weight-administration group average knurl weight)/feminine gender is right According to group average knurl weight × 100%.

The changes of weight (%) of tumor animal calculates as follows：(weight when weight-grouping when measurement)/grouping when weight × 100。

7. data analysis

Test data with 2007 softwares of Microsoft Office Excel calculate and ASSOCIATE STATISTICS is handled.Number It according to unless otherwise indicated, is indicated with mean ± standard error (Mean ± SE), two comparison among groups are examined using t.8. result and report

According to Chinese CFDA《Cell toxicant series antineoplastic medicament non-clinical study technological guidance's principle》(in November, 2006), T/ C (%)≤40% and through statistical analysis p<0.05 is effective, is shown in Table 4.

Table 4 is to tumor proliferation rate T/C (%)

Experimental result shows that the compounds of this invention has good inhibition to make human breast carcinoma MDA-MB-231 transplanted tumor in nude mice With, and it is better than Irinotecan and nktr-102.

Inhibiting effect of the embodiment 7 to human pancreas cancer MIA Paca-2 nude mouse xenograft models

1. experiment purpose

This research is with human pancreas cancer MIA Paca-2 Nude Mouse Models to the internal anti-swollen of compound e and compound E Tumor activity is evaluated.

2. experimental animal

2.1 animal species

Mouse.

2.2 kind

BALB/c-nu/nu nude mices.

2.3 gender

Female.

2.4 quantity

150。

2.5. the age

6-8 weeks.

2.6. weight

± 20% weight mean values of 20-22g.

2.7. animal origin (supplier)

Shanghai western Poole-Bi Kai experimental animals Co., Ltd (BK), credit number SCXK (Shanghai) 2008-0016.2.8. real Test the care of animal

All experimental animals are raised in SPF ranks laboratory.

2.8.1 animal identification identification method

2.8.2 random grouping

2.8.3 operational administrative specification

The operation and management of all experimental animals strictly observe experimental animal and use and management guidance principle.

2.8.4 rearing conditions

Living conditions：IVC systems, per 6, cage

Temperature：25℃±1℃

Humidity：65% ± 10%

Illumination：Day alternates with night within 12 hours

2.8.5 feed

2.8.6 drinking-water

City tap-water is drunk after filtering high pressure sterilization.

2.8.7 bedding and padding

2.8.8 the laundering period

Give mouse most short one week environment laundering period before experiment.

3. experiment material

3.1 test drugs

3.2 other chemical reagent and material

3.2.1 physiological saline

Physiological saline is purchased from Shanghai Hua Yuanchang richnesses medicine company (group) Co., Ltd (Shanghai, China).

3.2.2 asepsis injector

3.2.3 cell strain

Human pancreas cancer MIA Paca-2 are purchased from cell biological research institute of the Shanghai Chinese Academy of Sciences.

MIA Paca-2 are incubated at DMEM culture mediums (GIBCO, the U.S.), (GIBCO, the U.S.) containing 10% fetal calf serum FBS And 2.5%HS, it is incubated at containing 5%CO₂37 DEG C of incubators.

3.2.4 matrigel (BD Matrigel)

Matrigel Matrigel is purchased from U.S. company BD

3.3 instrument

Biohazard Safety Equipment (model：AC2-6E1), it is purchased from ESCO；

Inverted microscope (model：CKX41SF), it is purchased from Olympus；

4. experimental design

Establish human pancreas cancer MIA Paca-2 Xenografts in nude mice models, every inoculation 3 × 10⁶A cell.

Following (table 5) dosage of this experimental design and dosage regimen.

Table 5：Antitumor action in human pancreas cancer MIA Paca-2 Nude Mouse Models

Tumor strain：MIAPaca-2；It is inoculated with 30 altogether

5. compound is prepared

The volume that single-dose needs is 3mL.

5.1 Irinotecan drug-delivery preparations are prepared

5.2 nktr-102 drug-delivery preparations are prepared

Every time before administration, the physiological saline of 2.5mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine Object is completely dissolved, a concentration of 4.0mgmL-1 of Irinotecan free form in solution.

5.3 compound e and compound E drug-delivery preparations are prepared：Precise respectively, is added the physiological saline of 2.5mL, is vortexed Vibrating makes complete drug dissolution, a concentration of 4.0mgmL of Irinotecan free form in solution^-1。

6. experimental method

MIA Paca-2 cell culture contains 10% fetal calf serum FBS (GIBCO, the U.S.) and 2.5%HS in DMEM.Cell It is positioned over 5%CO₂37 DEG C of cultures of incubator.

Cell inoculation method establishes tumour nude mice by subcutaneous transplantation model：The tumour cell for collecting exponential phase, weight after counting It is suspended from 1 × PBS, adjustment concentration of cell suspension to 3 × 10⁷/ml.With 1ml syringes (No. 4 syringe needles) dorsal sc on the right side of nude mice Inoculated tumour cell, 3 × 10⁶/ 0.1ml/ mouse.

All zoopery operations strictly observe animal use and management regulation.The calculating of tumour relevant parameter is with reference to China SFDA《Cell toxicant series antineoplastic medicament non-clinical study technological guidance's principle》.

According to Chinese SFDA《Cell toxicant series antineoplastic medicament non-clinical study technological guidance's principle》(in November, 2006), T/ C (%)≤40% and through statistical analysis P<0.05 is effective.

7. data analysis

Test data with 2007 softwares of Microsoft Office Excel calculate and ASSOCIATE STATISTICS is handled.Number It according to unless otherwise indicated, is indicated with mean ± standard error (Mean ± SE), comparison among groups are examined using t-, P<0.05 is conspicuousness Difference.

8. result and report

According to Chinese CFDA《Cell toxicant series antineoplastic medicament non-clinical study technological guidance's principle》(in November, 2006), T/ C (%)≤40% and through statistical analysis P<0.05 is effective, is shown in Table 6.

Table 6 is to tumor proliferation rate T/C (%)

Experimental result shows that the compounds of this invention has good inhibition to make human pancreas cancer MIA Paca-2 transplanted tumor in nude mice With, and it is better than Irinotecan and nktr-102.

Inhibiting effect of the embodiment 8 to human gastric cancer NCI-N87 cell strain Nude Mouse Model tumor growth in vivo.1. real Test purpose

Tested drug compound e and compound E are evaluated in human gastric cancer NCI-N87 cell strain Nude Mouse Model tumour bodies The inhibiting effect of growth.

2. experiment material

2.1 test sample

Irinotecan (bulk pharmaceutical chemicals) system purchase gained, nktr-102 and 2 test-compound be by rich auspicious biological medicine (Suzhou) limited liability company provides.

2.2 reagent

RPMI-1640 culture solutions, fetal calf serum (FBS), trypsase, blueness-chain is dual anti-, physiological saline.

2.3 experimental animal

Female BAl BIc/c nude mouse (numbers of elements：150；Week old：6~8 weeks) have from Beijing dimension tonneau China experimental animal technology Limit company buys, and raises in Suzhou Sheng Su new drug developments Co., Ltd SPF animal houses, 20~25 DEG C of temperature, relative humidity 40% ~70%, light and shade illuminates each 12 hours；Animal free water and feeding.After normal nursing about 1 week, through veterinary inspector, sign shape The good mouse of condition can enter anthology experiment.It is identified in animal root of the tail portion using marking pen before grouping, every animal is equal after grouping Scarce mode is cut with ear to identify.

2.4 transplanted tumor tumor strains

Gastric carcinoma cells NCI-N87 derives from the American Type Culture Collection committee of the Chinese Academy of Sciences cell bank (CAS, this reality Test room liquid nitrogen cryopreservation).

3 experimental methods

3.1 NCI-N87 cell culture

In 5%CO₂, under 37 DEG C of condition of culture, NCI-N87 cells are in containing 10% fetal calf serum RPMI-1640 culture solutions Carry out regular growth culture；With 0.25% pancreatin had digestive transfer culture；It according to cell growth status, passes on 1 to 2 times weekly, passes on ratio Example is 1:2 to 1:6.

It is prepared by 3.2 animal models

Exponential phase NCI-N87 cells are collected, are resuspended in after cell count in the RPMI-1640 culture mediums of serum-free, Cell concentration is adjusted to 5 × 10⁷Cell/mL；It is fitted into 50mL centrifuge tubes after so that it is uniformly dispersed with pipettor piping and druming cell, it will Centrifuge tube is placed in ice chest；Cell suspension is drawn with 1mL syringes, is injected into that nude mice front right limb armpit is subcutaneous, and every animal connects 100 μ L (5 × 10 of kind⁶Cell/only), establish NCI-N87 Nude Mouse Models.Routine observation animal state and tumour after inoculation Growing state measures knurl footpath using electronic vernier caliper, and data directly input Excel electrical forms, calculate gross tumor volume.It waits swelling Knurl product reaches 100~300mm³, select be in a good state of health, animal 30 similar in gross tumor volume, using randomized blocks It is divided into 5 groups (n=6).Experiment measures weekly 2 knurl footpaths after starting, calculate gross tumor volume, while weighing the weight of animals and recording.

Gross tumor volume (TV) calculation formula is as follows：

TV(mm³)=l × w²/2

3.3 solvents are prepared

3.4 drug-delivery preparations are prepared

3..4.1 Irinotecan drug-delivery preparation is prepared

3.4.2 nktr-102 drug-delivery preparations are prepared

Every time before administration, the physiological saline of 2.3mL is added in the nktr-102 of precise 101.5mg, and vortex oscillation makes medicine Object is completely dissolved, a concentration of 4.0mgmL of Irinotecan free form in solution^-1。

3.4.3 compound e and compound E drug-delivery preparations are prepared：Precise respectively, the physiological saline of 2.5mL is added, Vortex oscillation makes complete drug dissolution, a concentration of 4.0mgmL of Irinotecan free form in solution^-1。

3.5 animal packets and administration

Animal packet and dosage regimen are shown in Table 7.Started to be administered for the first time in the grouping same day, terminates experiment, administration after 21 days Volume is 10mLkg^-1.1st group is solvent control group, and tail vein injection is given blank solvent, every 4 days 1 time, is administered 3 times altogether (Q4D×3).Tail vein injection gives given the test agent Irinotecan, nktr-102, test-compound to 2-5 groups respectively, gives medicament Amount is 40mgkg^-1(with Irinotecan content calculation), Q4D × 3.

7. Nude Mouse Model effect experiment dosage regimen of table

3.6 experiments terminate

It after experiment, weighs in, measure animal euthanasia (CO after knurl footpath₂).It strips tumor tissues and weighs, calculate Knurl weight tumour inhibiting rate.Tumor tissues are transferred to after weighing less than being preserved in -70 DEG C of refrigerators, for subsequent analysis.

4. data record, calculation formula

The calculation formula of relative tumour volume (RTV) is：

RTV=TV_t/TV_initial

The calculation formula of Relative tumor proliferation rate (%T/C) is：

%T/C=100% × (RTV_T/RTV_C)

The calculation formula of inhibition rate of tumor growth TGI (%) is：

TGI=100% × [1- (TV_t(T)-TV_initial(T))/(TV_t(C)-TV_initial(C))]

The calculation formula of the weight of animals rate of descent is：

The weight of animals rate of descent=100% × (BW_initial-BW_t)/BW_initial

The calculation formula of knurl weight tumour inhibiting rate IR (%) is：

IR (%)=100% × (W_C-W_T)/W_C

5. statistical analysis technique

Test data with 2007 softwares of Microsoft Office Excel calculate and ASSOCIATE STATISTICS is handled.

Data unless otherwise indicated, indicate that two comparison among groups use t- with mean ± standard error (Mean ± SE)

It examines.

6. Germicidal efficacy

7. result

For people's cancer heteroplastic transplantation tumor model, recommend to be used as test evaluation index using Relative tumor proliferation rate T/C (%), Proliferation rate is lower, illustrates to inhibit tumor effect the better, is shown in Table 8.

Table 8 is to tumor proliferation rate T/C (%)

Experimental result shows that the compounds of this invention is to human gastric cancer NCI-N87 cell strain Nude Mouse Model tumour growths There is good inhibiting effect, and is better than Irinotecan and nktr-102.

Influence of the embodiment 9 to U87MG nude mice brain model in situ survival rates.

1. experiment purpose

Evaluate the influence of tested drug compound e and compound E to U87MG nude mice brain model in situ survival rates.2. testing material Material

2.1 test sample

2.2 reagent

RPMI-1640 culture solutions, trypsase, blueness-chain is dual anti-, physiological saline.

2.3 experimental animal

Female BAl BIc/c nude mouse (numbers of elements：150；Week old：6~8 weeks) have from Beijing dimension tonneau China experimental animal technology Limit company buys, and raising illuminates each 12 hours in SPF animal houses, 20~25 DEG C of temperature, relative humidity 40%~70%, light and shade； Animal free water and feeding.After normal nursing about 1 week, through veterinary inspector, mouse can enter anthology experiment to sign in order.Point It is identified in animal root of the tail portion using marking pen before group, every animal cuts scarce mode with ear and identifies after grouping.

2.4 transplanted tumor tumor strains

Brain glioblastoma cell U87MG derives from the American Type Culture Collection committee of Chinese Academy of Sciences cell bank

(CAS, this laboratory liquid nitrogen cryopreservation).

3. experimental method

NCI-N87 cell culture

In 5%CO₂, under 37 DEG C of condition of culture, NCI-N87 cells carry out regular growth training in RPMI-1640 culture solutions It supports；With 0.25% pancreatin had digestive transfer culture；It according to cell growth status, passes on 1 to 2 times weekly, passage ratio is 1:2 to 1:6.

It is prepared by 3.1 animal models

Exponential phase NCI-N87 cells are collected, are resuspended in after cell count in the RPMI-1640 culture mediums of serum-free, Cell concentration is adjusted to 1 × 10⁸Cell/mL；It is fitted into 50mL centrifuge tubes after so that it is uniformly dispersed with pipettor piping and druming cell, it will Centrifuge tube is placed in ice chest；Cell suspension is drawn with 1mL syringes, by the guiding of animal stereotactic apparatus, using microinjection Method is by 1 μ L (1 × 10 of in vitro culture human glioma cell U87MG cells⁵Cell/only), it is in situ to establish U87MG gliomas Model, routine observation animal state after inoculation.The 12nd day after inoculation, animal 30 is selected, 5 groups of (n are divided into using randomized blocks =6).

3.2 drug-delivery preparations are prepared

3.2.1 Irinotecan drug-delivery preparation is prepared

3.2.2nktr-102 drug-delivery preparation is prepared

3.2.3 compound e and compound E drug-delivery preparations are prepared：Precise respectively, is added the physiological saline of 2.5mL, whirlpool Rotation, which is vibrated, makes complete drug dissolution, a concentration of 4.0mgmL of Irinotecan free form in solution^-1。

3.3 animal packet and administration

Animal packet and dosage regimen are shown in Table 9.Started to be administered for the first time in the grouping same day, terminates experiment, administration after 21 days Volume is 10mLkg^-1.1st group is solvent control group, and tail vein injection is given blank solvent, every 4 days 1 time, is administered 3 times altogether (Q4D×3).Tail vein injection gives given the test agent Irinotecan, nktr-102, test-compound to 2-5 groups respectively, gives medicament Amount is 40mgkg^-1(with Irinotecan content calculation), Q4D × 3.

9. Nude Mouse Model effect experiment dosage regimen of table

4. data record, calculation formula

Record survival time of animals.

5. statistical analysis technique

Test data with 2007 softwares of Microsoft Office Excel calculate and ASSOCIATE STATISTICS is handled.Two Comparison among groups are examined using t.

6. result

It is shown in Table 10

10 survival time of animals of table (day)

* compared with the median survival time of blank solvent, Irinotecan and nktr-102 groups, P < 0.05

Experimental result shows, the compounds of this invention has a good inhibiting effect to glioma, and better than Irinotecan and nktr-102。

Claims

1. having the following structure the highly branched chain drug conjugates or its pharmaceutically acceptable salt of formula (I)：

R is organic core, and POLY is polymer, and L is polyvalent linkers, and T is targeted molecular, and D is activating agent, q appointing between 3-8 One integer, wherein L are：

Symbol " * " represents the tie point of polyvalent linkers L and targeted molecular T, and " # " represents the connection of polyvalent linkers L and activating agent D Point, " % " represent the tie point of polyvalent linkers L and POLY, wherein any integers of the l between 2-20, m, n be respectively 0-10 it Between any integer；

T is Angiopep2；

D is the camptothecine as shown in formula (II)：

R₁-R₅It is independently selected from following group：Hydrogen, halogen, acyl group, alkyl, substitution alkyl, alkoxy, substituted alcoxyl Base, alkenyl, alkynyl, naphthenic base, hydroxyl, cyano, nitro, azido, amide groups, hydrazine, amido, substituted amido, hydroxycarbonyl group, alkane Oxygen carbonyl, alkyl oxy carbonyl oxygen, carbamoyloxy, aryl-sulfonyl oxygen, alkylsulfonyloxy；R₆For H or OR₈, R₈For alkyl, Alkenyl, naphthenic base, halogenated alkyl or hydroxyalkyl；R₇For hydroxyl.

2. highly branched chain drug conjugates as described in claim 1 or its pharmaceutically acceptable salt, POLY are polyethylene glycol.

3. highly branched chain drug conjugates as claimed in claim 2 or its pharmaceutically acceptable salt, POLY are

Wherein,The junction for representing atom, the oxygen atom for marking ampersand are the atom being connect with organic core " R ", k 50 An integer in~200 ranges, any integers of the r between 1-10.

4. highly branched chain drug conjugates as claimed in claim 3 or its pharmaceutically acceptable salt, be structural formula (III), (IV) or shown in (V)：

5. highly branched chain drug conjugates as claimed in claim 4 or its pharmaceutically acceptable salt, POLY are

6. highly branched chain drug conjugates as claimed in claim 5 or its pharmaceutically acceptable salt, are：

7. highly branched chain drug conjugates as claimed in claim 6 or its pharmaceutically acceptable salt, the average value that k has are 113。

8. highly branched chain drug conjugates as claimed in claim 7 or its pharmaceutically acceptable salt, L are：

9. highly branched chain drug conjugates as claimed in claim 8 or its pharmaceutically acceptable salt, D is Irinotecan, SN- 38,10-hydroxycamptothecine or Rubitecan.

10. highly branched chain drug conjugates as claimed in claim 9 or its pharmaceutically acceptable salt, are：

And its salt.

11. highly branched chain drug conjugates as claimed in claim 10 or its pharmaceutically acceptable salt, are：

12. a kind of pharmaceutically acceptable composition, it includes any highly branched chain drug conjugates of claim 1-11, And pharmaceutically acceptable excipient.

13. highly branched chain drug conjugates as described in claim 1-11 is any answering in the preparation of medicament for cancer treatment With.

14. the highly branched chain drug conjugates as described in claim 1-11 is any are being prepared for treating colon cancer, lung cancer, mammary gland Cancer, oophoroma, cancer of pancreas, gastric cancer, glioma and breast, ovary, colon, kidney, bile duct, the malignant sarcomas of lung and brain, cancer and Application in the drug of lymthoma.

15. the application of composition as claimed in claim 12 in the preparation of medicament for cancer treatment.

16. composition as claimed in claim 12 is being prepared for treating colon cancer, lung cancer, breast cancer, oophoroma, pancreas Cancer, gastric cancer, glioma and breast, ovary, colon, kidney, bile duct, the malignant sarcomas of lung and brain, cancer and lymthoma drug in Application.