CN104784699B

CN104784699B - Folate receptor binding ligand-drug conjugates

Info

Publication number: CN104784699B
Application number: CN201410024349.9A
Authority: CN
Inventors: 袁建栋; 宋云松; 黄仰青; 朱锐; 胡晓伟; 方程
Original assignee: Borui Pharmaceutical (suzhou) Ltd By Share Ltd
Current assignee: Borui Pharmaceutical (suzhou) Ltd By Share Ltd
Priority date: 2014-01-20
Filing date: 2014-01-20
Publication date: 2019-05-03
Anticipated expiration: 2034-01-20
Also published as: WO2015106599A1; CN104784699A; CN109316605B; CN109316605A

Abstract

The present invention provides a kind of folate receptor binding ligand-drug conjugates, and the conjugate is by vitamin receptor bound fraction (F)_n, connector (L) and drug or its analog or derivative (D) composition, the integer that wherein n is 2 ~ 4.The preferred folic acid of vitamin receptor bound fraction or pteroic acid, connector (L) include polyvalent linkers L₁L at least containing a releasable connector₂, (F)_nIt is covalently attached to polyvalent linkers L₁On, drug or its analog or derivative (D) and L₂Releasable connector be covalently attached, L₁With L₂It is covalently attached.Conjugate provided by the invention greatly improves the tumour cell affinity of folate receptor-positive, and has significant anti-tumor activity, and side effect is substantially reduced.

Description

Folate receptor binding ligand-drug conjugates

Technical field

The present invention relates to the medical compounds and preparation method thereof for targeting drug delivery.It combines and matches more particularly to folacin receptor Body-drug conjugates, more specifically, the present invention relates to two or more folate receptor binding ligands (F) to pass through connector (L) It is coupled, is formed (F) with drug or its analog or derivative (D)_nLD conjugate, the present invention also relates to the conjugate is used for The disease condition as caused by Pathogenic cellular group is treated, and is related to the preparation method and its pharmaceutical composition of such conjugate Object.

Background technique

Although the treatment method of cancer and the research of anticancer drug have been achieved for remarkable progress, cancer is still serious prestige One of the principal disease for coercing human health, in the U.S., cancer is the second largest reason for leading to death after heart disease.It controls now The exemplary process for treating cancer includes surgical treatment, radiotherapy, chemotherapy, immunotherapy and above treatment method joint Using to anticancer.Most often, it using high potent drug, such as mitomycin, taxol and camptothecine, is treated by amic therapy method Cancer.While wherein the major defect of chemotherapeutics is to inhibit growth of tumour cell, normal cell is also seriously inhibited Proliferation, these inevitable side effects are particularly important the exploitation of new specific anti-cancer drugs.

At present carried out many effort by by anticancer drug be integrated to ligand such as hormone, antibody or vitamin come Develop tumor selective agents.For example, low molecular weight element-vitamine compound (such as folic acid) is used as cancer target agent.

Folic acid (FA) is also referred to as vitamin B₉, it is that all living cells maintain a carbon approach eubolism and nucleotide biosynthesis Required necessary nutrients.Folacin receptor (FR) is a kind of cross-film single chain glycoprotein, contains 3 kinds of hypotypes: α-FR, β-FR, γ- FR.Folic acid (FA) shows high affinity (KD ~ 100pM) to the glycoprotein of the cell surface orientation of folacin receptor (FR), The glycoprotein is the albumen that the glycosyl-phosphatidyl inositol connection of its ligand (folic acid) is captured from extracellular environment.Folacin receptor (FR) be with folic acid (FA) combine tumourassociated membrane proteins and can in the cell by endocytic mechanisms transhipment in conjunction with folic acid Molecule.In conjunction with later, the plasma membrane for surrounding FR- ligand complex will be invaginated immediately to form internal compartment, referred to as inner body.Vesica The pH of chamber is slightly lower by the effect of the proton pump in common location interior body film, and this acidification may mediate FR albumen In conformational change to discharge ligand that it is combined to allow access into cytoplasm.

Research shows that in 90% oophoroma, advanced breast cancer, cervical carcinoma, carcinoma of endometrium, colon cancer, lung cancer, choroid Occurs highly expressed α-FR in cancer, ependymocytoma；In leucocyte (leukaemia) surface of malignant proliferation and rheumatoid arthrosis The Macrophage Surface of autoimmune disease patients' vivo activation such as inflammation has highly expressed β-FR, and folacin receptor is at normal group It is hardly expressed in knitting.Therefore folic acid and folacin receptor have good Research Prospects in targeted therapy technology, especially exist In terms of the treatment of tumour and autoimmune disease, FR is greatly paid close attention to as the target spot of anti-tumor drug, becomes novel One of the hot spot of anti-tumor drug research (Hilgenbrink, A. and P. Low (2005) Folate receptor mediated drug targeting: From therapeutics to diagnostics. Journal of Pharmaceutical Sciences 94 (10): 2135-2146.).It is attempting to utilize folic acid-radioactive nucleus in the world Imaging agent conjugate, as folic acid with¹²⁵I、⁶⁷Ga、¹¹¹The detection human body height expression folacin receptor of the conjugate energy Noninvasive of In Tumor tissues.Meanwhile at present in the world extensively with regard to folic acid-proteotoxin, folic acid-small molecule chemotherapeutic drug, folate liposome (packet chemotherapeutics or genomic medicine in liposome) and folic acid-immunotherapeutic agent etc. expand further investigation (bibliography: Hilgenbrink, A. and P. Low (2005). Folate receptor mediated drug targeting: From therapeutics to diagnostics. Journal of Pharmaceutical Sciences 94(10): 2135-2146.WO2012065085, WO2012065079, US2012022245A1).

Summary of the invention

There is specificity it is an object of the present invention to provide a kind of pair of pathogenic cell and to the folacin receptor of normal cell small toxicity Binding partner-drug conjugates, preparation method and its application in preparation tumor.

In an illustrated embodiment of the invention, the compound with following below formula is described:

(F)_nLD

Folate receptor binding ligand-drug conjugates, wherein n be 2,3 or 4；F is folate receptor binding ligand, is selected from Folic acid or pteroic acid； (F)_nIn each F it is independent be selected from folic acid and pteroic acid；D is drug or its analog or derivative；L is to include With the connector of flowering structure:

L₁-L₂

Wherein, L₁It is polyvalent linkers, L₂Include at least one releasable connector, L₁With L₂It is covalently attached；

It should be understood that heretofore described (F)_nRefer to each F respectively with polyvalent linkers L₁It is covalently attached, D and L₂Altogether Valence connection.

In another embodiment, the folate receptor binding ligand-drug conjugates having following structure are described:

,

Wherein F₁、F₂It is independent to be selected from folic acid and pteroic acid；D is drug or its analog or derivative；

L is comprising with the connector of flowering structure:

L₁-L₂

The F₁、F₂Respectively with polyvalent linkers L₁It is covalently attached, D and L₂It is covalently attached.

Polyvalent linkers (the L₁) connect containing the multiple connectors being mutually covalently attached, including one or more intervals Head, releasable connector, heteroatom linker and its combination arranged in any order.In an alterable mode, releasable connector It is to form connector L with the optional mutual covalent bond of spacer nipple₁；Can change in mode at another, ligand F by one or Multiple spacer nipples are covalently attached to polyvalent linkers L₁；2 or 2 or more the direct phases of ligand F in mode be can change at another Mutually adhere to or pass through multiple spacer nipples and/or releasable connector covalent attachment；In another alterable mode, two or more A releasable connector is mutually covalently attached, and wherein one or more releasable connectors pass through heteroatom linker and/or interval Connector is spaced from each other.

It should be understood that the changeability of the arrangement of one or more releasable connectors and optional spacer nipple is very big. Hetero atom in the heteroatom linker is nitrogen, oxygen, sulphur, phosphorus, silicon etc..Further, the heteroatom linker (except oxygen) It can be different oxidation state, such as N (OH), S (O), S (O)₂、P(O) 、P(O)₂、P(O)₃Deng.It is described in another alterable mode Heteroatom linker is that azanol, hydrazine, hydrazone, sulphonic acid ester, Asia are seen acid esters, acid esters of seeing etc..

In one embodiment, polyvalent linkers L₁It is described comprising at least one by amino acids formed peptide spacer nipple Amino acid it is independent be selected from natural amino acid and non-natural alpha-amino acid.In another embodiment, the polyvalent linkers L₁Including containing 1 ~ 40 amino acids formed peptide spacer nipple, the preferred natural amino acid of amino acid is further, described Polyvalent linkers L₁Including containing 1 ~ 20 amino acids formed peptide spacer nipple；Further, the L₁It preferably includes containing 10 ~ 15 amino acids formed peptide spacer nipples.Further, L₁Including at least two amino acid selected from the group below: asparagus fern ammonia Acid, arginine, cysteine, lysine, asparagine, arginine, threonine, glutamic acid, serine, citrulling, valine And glutamine.

Further, the L₁It preferably includes one or more by selected from aspartic acid, arginine, cysteine, melon The amino acids formed dipeptides of propylhomoserin, valine and lysine and combinations thereof, tripeptides, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, The spacer nipple of decapeptide, 11 peptides and dodecapeptide.

In an illustrative embodiment, combined with heteroatom linker one by amino acids formed peptide spacer nipple Act the polyvalent linkers L formed such as following formula₁:

Or

,

Wherein, * indicates open valency.

The polyvalent linkers L₁Optional includes spacer nipple chosen from the followings: polyethers, sugar, thiocarbonyl, alkylidene, 1- alkylenesuccinimid -3- base, 1- (carbonylic alkyl) succinimide -3- base, carbonylic alkyl carbonyl, 1- (carbonyl tetrahydro - 2H- pyranose) succinimide -3- base and 1- (carbonyl tetrahydrofuran base) succinimide -3- base, wherein it is described it is each between It is optionally substituted by one or more substituents every connector；

Wherein the substituent group it is independent selected from be alkyl, alkoxy, alkoxyalkyl, hydroxyl, hydroxyalkyl, amino, Aminoalkyl, alkyl aminoalkyl, dialkyl aminoalkyl, mercaptoalkyl, alkyl-thio-alkyl, aryl, substituted aryl, aralkyl, Substituted aralkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, alkyl carboxylates, chain acid alkyl ester, guanidine radicals alkane Base or the carbonyl replaced by amino acid and its derivative and peptide or acylamino- or amidoalkyl.

In the present invention, L₂Comprising at least one releasable connector, " the releasable connector " refer to including at least one The connector for the key (such as pH labile bond, sour labile bond, the unstable or enzyme labile bond of oxidation) that can be broken under physiological condition. Self-evident, this physiological condition for causing key to be broken includes occurring at physiological ph, or dissolve into organelle, example as compartment As endosome its pH lower than cytoplasm pH result and occur standard chemical hydrolysis reaction.It is self-evident, as described herein, Scissionable bond can connect two adjacent atoms in releasable connector, and/or either end or both ends in the releasable connector Connect other connectors or ligand F and/or drug D.Two adjacent atoms in releasable connector are keyed in this cleavable In the case of, after key fracture, the releasable joint breaking is at two or more segments.Alternatively, existing in this cleavable key Releasable connector and another part such as heteroatom linker, spacer nipple, another releasable connector, drug or its analog spread out In the case where between biology or folate receptor binding ligand, after key fracture, releasable connector is separated from other parts. The unstability of cleavable key can be adjusted, such as be changed by the substitution in cleavable key position or near it, such as wrap The α branch for adjoining cleavable disulfide bond is included, homologization forms hydrolyzable part ketal or alkoxy of acetal etc..

The releasable connector is selected from: 1- alkoxyalkylene, 1- alkoxyalkylenecarbonyl, 1- alkoxy Asia ring Alkyl-carbonyl, double carbonyl aryl, double carbonyl carboxyl aryls, double carbonyls double carboxyl aryl, haloalkylenecarbonyl, Epoxide carbonyl oxygen Base, Epoxide carbonyl oxygroup alkyl, imino alkyl, carbonyl alkylen group imino group, imino group cycloalkylidene, carbonyl cycloalkylidene are sub- Amino, alkylenethio, alkylidene aryl sulfenyl and carbonylalkylthio, wherein each releasable connector is optionally by one Or multiple substituent groups replace；

Preferably, in compound of the present invention, the releasable connector includes disulphide, carbonic ester, hydrazides, acyl Amine, hydrazone, amino-acid ester, urea or combinations thereof.

It is further preferred that releasable connector of the present invention includes one or more formulas having the following structure:

、、、、、、、Or。

Wherein, 0,1,2,3 or 4 n；R is H, alkyl, the acyl group optionally replaced or nitrogen-protecting group；X is O, CH₂Or NH；Y For O or S, Z NH, O or S；R₁Replace alkyl for alkyl, carboxy substituted alkyl or acyl group；* open valency is indicated.

Further, connecing of having the following structure is formed with heteroatom linker or spacer nipple by the releasable connector Head L₂:

、、、、Or

Wherein, the integer that m is 0 ~ 4, W are selected from NH or O；* open valency is indicated.

Authorization Notice No. is that releasable connector and spacer nipple, the document is described in detail in the patent of CN100381177C Disclosure be incorporated by reference into the present invention.

The polyvalent linkers L₁With the L comprising releasable connector₂Optionally connect by one or more spacer nipples, hetero atom Head, releasable connector connect to form connector L.

Illustratively by L₁With L₂It is directly covalently attached or by one or more spacer nipples, heteroatom linker, releasable It includes following structure that connector, which connects the connector L to be formed:

、

Or

；

Wherein, W is selected from NH or O；* open valency is indicated.

In folate receptor binding ligand-drug conjugates provided by the present invention, include folate receptor binding ligand (F), Connector (L) and drug (D), any mode that can be mentioned according to the present invention forms connector L or usable this field recognize it is known between Connector L, folate receptor binding ligand F or medicine are formed by being covalently attached between connector, releasable connector and heteroatom linker Object D can be matched with the connection of heteroatom linker by being present in conjunction with the drug D or folacin receptor that have been converted to heteroatom linker Reactive functional groups on body F are made.Wherein, the reactive functional groups on the folate receptor binding ligand F are carboxyl or ammonia Base, the carboxyl or amino that polyvalent linkers can attach on the folate receptor binding ligand F form ester or amide；The wherein medicine When object includes double bond nitrogen-atoms, releasable connector attaches on the drug nitrogen and forms hydrazone；When the drug may include sulphur atom, The releasable connector is selected from alkylenethio or carbonylalkylthio, and releasable connector is connected on the sulphur of the drug Form disulfide bond；The drug may include oxygen atom, and the releasable connector is to be substituted with a substituent or unsubstituted alkylene Epoxide carbonyl or haloalkylenecarbonyl, the releasable connector, which is connected on the oxygen of drug, forms carbonic ester or ester；The medicine Object D may include nitrogen-atoms, and releasable connector is the haloalkylenecarbonyl of haloalkylenecarbonyl or acquirement, described releasably to connect Head, which is connected on the nitrogen of drug D, forms amide.

Spacer nipple and releasable connector and heteroatom linker can combine in different ways.Illustrative explanation, institute State connector and pass through heteroatom linker and be connected with each other, such as alkylene-amino-alkylenecarbonyl, wherein x, y be respectively 1 ~ 5 it is whole Number.

Or

It is illustrative illustrative, in another embodiment, describe the compound of following formula:

、

With

；

Wherein, W is NH or O；M is 0 or 1；F₁、F₂It is independent to be selected from following formula:

With。

Heretofore described drug D can be any molecule that can be adjusted or change cell function in another manner, including Pharmaceutical active compounds.The pharmaceutical active compounds can be known in the art drug or its derivatization form, the drug It is living for anti-apoptotic in cytotoxicity, raising tumor permeability, inhibition tumor cell proliferation, promotion Apoptosis, reduction packet cell The drug of property.It include but is not limited to hormone, antibiotic, antimicrobial, antiviral agent, anticarcinogen suitable for drug of the invention. Itself it is cytotoxicity or the chemotherapeutic that can be used to increase tumor permeability, also uses in the methods of the invention.Described is thin The drug of cellular toxicity is for example including CBI(cyclopropyl benzo [e] indolone) analog or derivatives thereof, open loop-cyclopropyl benzo [e] indolone analog, O-Ac- open loop-cyclopropyl benzo [e] indolone analog, dolastatin (Dolastatin) class (such as dolastatin -10), ori inhibin (auristatin) class (such as monomethyl ori inhibin E(MMAE), monomethyl Europe Auspicious inhibin F(MMAF)), pipe dissolves plain class (Tubulysins), combretastatin, maytansine, maytansine analog, DM1, Ai Bo Mycin, taxol and paclitaxel derivatives (such as docetaxel), vincaleukoblastinum and the like (such as vincristine, deacetylated Changchun One hydrazides of alkali (DAVLBH)), camptothecine and camptothecin derivative, colchicin, different colchicin, muscoril, autumn Narcissamine, daunomycin, rhizomycin, cyclophosphamide, methotrexate, bleomycin, tesirolimus (Temsirolimus), silk Rimocidin, microtubule inhibitors, Pyrrolobenzodiazepines tall and erect (PBD) dimer class, open ring-ring at cyclopropyl benzo [e] indolone Propyl benzo [e] indolone, Calicheamicin (Calicheamicin).It includes big ring that other, which are applicable to drug of the invention, Lactone antineoplastic, chemotherapeutic such as alkylating agent, mustargen, nitroso ureas, busulfan, carboplatin, Chlorambucil, cis-platinum and other Platinum compounds, antimetabolite such as cytarabine, purine analogue, pyrimidine analogue and penicillin, cephalosporin, vancomycin, It is erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics and acyclovir, trifluridine, Ganciclovir, neat The Antimicrobe compound that more husbands are fixed, amantadine, Ribavirin gemcitabine and any other this field are approved.

Further, the preferred tesirolimus of drug of the present invention (Temsirolimus), open loop-cyclopropyl benzo Tall and erect (PBD) the dimer class of [e] indolone analog, Pyrrolobenzodiazepines, Calicheamicin (Calicheamicin), 7- Ethyl-10-hydroxycamptothecin (SN-38), vinca, dolastatin (Dolastatin), ori inhibin (auristatin) class, didemnun B (Didemnin B), pipe dissolve element B(Tubulysin B), CHROMATOGRAPHIC FRACTIONATION AND MASS, taxol Class, daunorubicin, Doxorubicin, epirubicin, ai sibo mycin or D actinomycin D.

Further, the preferred open loop of drug of the present invention-cyclopropyl benzo [e] indole ketone compound, PBD dimerization Body class, Calicheamicin, SN-38, DAVLBH, Tubulysin B, Didemnin B, MMAE, MMAF, MMAF derivative, DM1, Taxanes, vincristine, daunorubicin, Doxorubicin or epirubicin.

The patent of Publication No. WO2009/064913A1 and US2010113476A1 are to CBI class compound and open loop-cyclopropyl Base benzo [e] indole ketone compound is described in detail, and disclosed part is incorporated by reference into the present invention, shows Open loop-cyclopropyl benzo [e] indole ketone compound of example property is shown below:

Wherein R₁For H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or；R₂For NH₂Or OMe。

PBD refers to the knot such as lower unit in tall and erect (PBD) dimer class compound of the Pyrrolobenzodiazepines Structure:

The difference of PBD dimer class compound is number, type and the position of the substituent group of both aromatic ring A and pyrrole ring C Set and the saturation degree of pyrrole ring C be different, illustrative PBD dimer class compound such as:

Or

Wherein, R₁For hydroxyl, amino, C₁~C₆Alkoxy, C₁~C₆Alkyl, C₁~C₆Amino replace alkyl, C₁~C₃ Aldehyde radical or carbonyl or C₁~C₆Naphthenic base or heterocycle；R₂It is that 2-, 3- or 4- replace, is-N=NH- or-NH-；N be 0 ~ 3 it is whole Number.

Such compound is described in detail in the patent of Publication No. CN201180029867, and discloses its preparation Method is incorporated by reference into the present invention in wherein disclosure.

The SN-38 is the structure with following formula:

；

The DAVLBH is the structure with following formula:

Tubulysin B and MMAE specific structure is as follows:

、

MMAF and its derivative refer to the compound having the following structure:

、

Wherein, R_yFor C₁~C₆Alkyl or halogenated alkyl or the carbonyl containing 1 ~ 4 carbon atom arbitrarily replaced；Work as R_yFor hydrogen When, as MMAF.

The Didemnin B is the structure with following formula:

Wherein, R_xFor p-methoxyphenyl.

The DM1 is the structure with following formula:

In the folate receptor binding ligand conjugate, drug D and releasable connector connector L₂Connection, presence can be passed through Drug D or the reactive functional groups of its derivatization form are made, such as the hydroxyl of Didemnin B is converted to corresponding carbonic ester, Another illustrative embodiments is that first derivative turns to hydrazides to the terminal carboxyl group of Tubulysin B, and then the nitrogen-atoms on hydrazides is made It is connect again with the releasable connector on L for heteroatom linker, folic acid is converted to corresponding amide etc., as illustrated by following formula:

、、

Following folate receptor binding ligand-drug conjugates are illustrative drug conjugates provided by the invention, it is believed that They are in range of the present invention, scheme or of the present invention that these folate-drug conjugates can be approved according to this field Method preparation.

In another embodiment, the present invention describes the compound such as flowering structure:

、

With

Wherein, R_xFor p-methoxyphenyl；X₁For Cl or Br；R be H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or；R_yFor hydrogen, C₁~C₆Alkyl or halogenated alkyl or the carbonyl containing 1 ~ 4 carbon atom arbitrarily replaced Base；R₁For C₁~C₆Alkoxy or alkyl or the amino alkyl or C that replace₁~C₃Aldehyde radical or carbonyl, hydroxyl, amino or C₁~C₆ Naphthenic base or heterocycle；R₂It is the position 2-, the 3- or 4- substitution of benzene, is N=NH or NH；The integer that n is 0 ~ 3.

Wherein ONPhth is indicated with flowering structure:

Further, the present invention provides the compounds of such as flowering structure:

Wherein, L₄Structure selected from the following:

、 Or；

F₁, F₂It is independent to be selected from following formula:

With；

* link position is indicated.

Further, the present invention provides the compounds of such as following formula:

、

Or

。

On the other hand, the present invention provides the compounds with following general formula: F₃LD and F₄LD,

Wherein the connection type between F, L, D is similar to compound F₂Connection type in LD between F, L, D.

Illustrative F₃LD and F₄LD compound is as follows:

With

Wherein, D is selected from previously described drug or derivatives thereof or the like；F₁、F₂、F₃、F₄It is independent to be selected from:

With；

L₂It is connector, includes at least one releasable connector, optional connects comprising one or more heteroatom linkers, interval Head.

Illustratively, L₂It has the following structure:

、、、、 Or

Further, in detail below compound to illustrate F₃LD and F₄LD:

、

With

The synthetic method system that folate receptor binding ligand-drug conjugates of the present invention can be approved by this field It is standby.The selection of the synthetic method depends on the selection of heteroatom linker, the design feature of drug and the spacer nipple and Functional group on the releasable connector.In general, relative keys, which form reaction, is described in Richard C. Larock. Comprehensive Organic Transformations, a guide to functional group Preparations. VCH Publishers, Inc. New York (1989) and Theodora E. Greene & The Peter G.M. Wuts. Protective Groups ion Organic Synthesis. second edition, John Wiley & Sons, Inc. New York (1991), the disclosure of the document are partially incorporated by reference into the present invention.

Wherein the heteroatom linker is sulphur atom, and the functional group for being present in the releasable connector is that alkylidene mercaptan spreads out Biology, the disulphide group can pass through corresponding heteroaryl disulfide group derivative such as pyridine -2- base disulfide group alkyl derivative Object etc. reacts to be formed with alkylidene thiol derivative.Tetrahydrofuran (THF), N,N-dimethylformamide may be selected in its reaction dissolvent (DMF), methylene chloride, dimethyl sulfoxide (DMSO) etc., reaction temperature changes between 0 DEG C ~ 80 DEG C.

The formation of conventional carbonic ester, sulfocarbonate and carbamate, usually can by make hydroxyl replace compound, The compound that the compound or amine that sulfenyl replaces replace is reacted with activation alkoxycarbonyl derivative respectively, forms carbonic ester, sulphur For carbonic ester and carbamate.Reaction can in Conventional solvents such as THF, DMF, DMSO, methylene chloride, ethyl acetate into Any basic catalyst such as inorganic base, amine base, polymer combination alkali etc. can be used in 0 ~ 80 DEG C of range changing in row, reaction temperature Promote the reaction.

The formation of common amide and ester is present in spacer nipple or can for example, wherein the heteroatom linker is nitrogen-atoms Functional end-group on releasing-joint is carbonyl, the amide groups can by corresponding carboxyl or derivatives thereof esterification or Acylation reaction obtains, and includes methylene chloride, THF, DMF, DMSO etc. at amide reaction dissolvent, the illustrative amide can- Reaction preparation is carried out in 15 ~ 80 DEG C of mobility scales.Coupling agent includes DCC, EDC, HBTU, TBTU, HOBT/DCC, HOBT/ EDC Deng or parent acid can be converted to carbonyl derivative, such as acyl chlorides, n-hydroxysuccinimide base ester etc. of activation, at acyl Amine reaction can also carry out in N'- diisopropylethylamine etc. under alkaline condition such as triethylamine, N.

Likewise, the heteroatom linker is oxygen atom, there are the end groups on spacer nipple or releasable connector to be Carbonyl, required ester group can be prepared by corresponding carboxylic acid or derivatives thereof and alcohol coupling reaction.The coupling agent includes DCC, EDC, CDI, BOP, EEDQ, DEAD, triphenylphosphine etc., solvent include methylene chloride, THF, DMF, DMSO, acetonitrile, acetic acid Ethyl ester etc., alkali include triethylamine, diisopropylethylamine etc..Or parent acid can be converted to the carbonyl derivative of activation, such as Acyl chlorides, n-hydroxysuccinimide base ester etc..

The drug includes nitrogen-atoms, and releasable connector or spacer nipple can attach on the nitrogen-atoms of the drug D Hydrazone is formed, required hydrazone group can react to be formed by corresponding aldehydes or ketones and hydrazine or hydrazide derivatives.It can include two with solvent Chloromethanes, THF, DMF, DMSO, chloroform, ethyl acetate etc., reaction temperature can use any acidic catalyst in 0 ~ 80 DEG C of range changing Agent such as mineral acid, glacial acetic acid, trifluoroacetic acid etc. is used as catalyst.Acylhydrazone can be acylated hydrazine by suitable carboxylic acid or derivatives thereof, Then hydrazides is made to react to form acylhydrazone with corresponding aldehydes or ketones.Alternatively, the hydrazone functional group can by make hydrazine and corresponding aldehyde or Reactive ketone is formed.Gained hydrazone is acylated followed in turn by suitable carboxylic acid or derivatives thereof.

The formation of conventional succinimide: the heteroatom linker is nitrogen-atoms, oxygen atom or sulphur atom, exist and It is succinimide derivatives every the functional group on connector or releasable connector, resulting carbon-hetero atom can pass through corresponding amine, alcohol Or the Michael's addition of mercaptan and maleic imide derivative is formed.The solvent for forming Michael's addition includes THF, acetic acid second Ester (EtOAc), CH₂Cl₂、DMF、DMSO、H₂O etc..This kind of being formed for Michael's addition compound can use addition equimolar amounts Alkali such as triethylamine is completed by the pH to 6.0 ~ 7.4 of adjusting aqueous solution.It will be apparent that when the heteroatom linker is oxygen Or when nitrogen-atoms, be adjusted reaction condition easyization Michael's addition, such as by using higher reaction temperatures, addition catalyst, Maleimide is activated using the stronger solvent of polarity such as DMF, DMSO etc., and with silylating agent.

Symmetrical acetal or ketal group can be formed by corresponding pure and mild aldehydes or ketones by acetal and ketal reaction.Generally follow R. Schmidt etc., Chem. Rev., 2000,100,4423-42 it is summarized the step of, the disclosure of the document It is incorporated by reference into the present invention.

Conventional folic acid-peptide preparation: it is tactful with Fmoc- by polymer-carrier sequential grammar, in acid labile 2Cl-Trt Resin resin on prepare the peptidyl fragments Pte- γ Glu- (AA) containing folic acid_n- Cys-OH, shown in following scheme:

scheme 1

(a) 20% piperidines/DMF；(b) Fmoc-AA-OH, HOBT, DIC, DMF；(c) Fmoc-Glu-OtBu, HOBT, DIC, DMF；(d) N¹⁰-TFA-Pteroic acid；PyBop, DIPEA, DMF/DMSO；(e) 2%NH₂NH₂/DMF;(f) TFA/H₂O/ benzene Phenol/thioanisole/EDT (82.5:5:5:5:2.5)；

In the illustrated embodiment of the method for the invention, R₁For Fmoc, R₂Trityl group, DIC are diisopropyl Carbodiimide, DIPEA N, N'- diisopropylethylamine make activator with PyBop to ensure effectively to be coupled.In standard conditions Under, after each coupling step, Fmoc protecting group is removed.According to described in scheme 1, the amino acid of suitable protecting is used Component such as Fmoc-Glu-OtBu, N¹⁰- TFA-Pteroic acid etc., and using Fmoc-AA-OH in step (b) as representative.Cause This, AA refers to the amino acid starting material of any suitable protecting.Appoint it should be understood that term used herein " amino acid " refers to The amine or carboxylic acid functional that there are one or more carbon to separate for what, and including naturally occurring α and beta amino acids and these ammonia The derivative and analogue of base acid.Specifically, with side chain by protected amino acid for example protected serine, threonine, Cysteine, aspartic acid etc. can also be used for the synthesis of folic acid-peptide of the present invention.In addition, γ, δ or longer homologous side chains Or the amino acid analogue of the branched structure of substitution, such as nor-leucine, isovaline, Beta-methyl threonine, half Guang of Beta-methyl Propylhomoserin, β, it includes in the synthesis of folic acid-peptide of the present invention that Beta-Dimethylcysteine etc., which also can be used as raw material,.

The coupling sequence (step (a) and (b)) for being related to Fmoc-AA-OH carries out n times, supports peptide (II) to prepare solid, Middle n is integer and can be 0 ~ 100.After last coupling step, remaining Fmoc base (step (a)) is removed, the peptide is subsequent It is coupled to glutamate derivatives (step (c)), is deprotected, and be coupled to the pteroic acid (step (d)) of TFA- protection.The TFA Protecting group with alkali process (step (e)) by being removed, then through step (f) obtains (III) containing peptidyl fragments.

Then, the peptide is by with trifluoroacetic acid (TFA), H₂O, phenol, thioanisole and EDT processing, from polymer supported (step (f)) is cut on body.These reaction conditions cause to remove t-Bu, t-Boc, Pbf and Trt protecting group simultaneously, the protection Base can form the part of the amino acid side chain of suitable protecting.

(III)

Segment containing three or four folic acid peptides can be prepared in a similar way, and polyvalent linkers therein can be by containing 3 A or 3 or more reactive functional groups (such as amino, hydroxyl, carboxyl) amino acid construct, the amino acid for example can be Lysine, glutamic acid, serine, asparagine, aspartic acid, tyrosine, arginine glutamate etc..

Illustratively the segment III-2 containing three folic acid peptides can be prepared according to following scheme:

scheme 2

First two lysines are connected, obtain the compound c of the reactive functional groups containing there are four, then compound c is added Reaction column, ninhydrin monitoring, is successively condensed Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N¹⁰- TFA-pteroic acid, (wherein, amino acid and The dosage of HOBT/DIC is respectively 15eq and 16.5eq/16.5eq) condensation after be added 2% hydrazine hydrate of 10ml/DMF solution it is anti- It answers 5 minutes, is repeated 3 times, DMF washing, DCM is washed, and MeOH washing is drained, and synthesis finishes, and lytic reagent, TBME precipitating is added Polypeptide obtains III-2.

Illustratively the segment III-3 containing four folic acid peptides can be prepared according to method shown in following scheme 3:

scheme 3

Similar, first four lysines are condensed, obtain compound f, reaction column, indenes three is being added in compound f Ketone monitoring, is successively condensed Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc- Asp(OtBu)-OH, Fmoc-Glu-OtBu, N¹⁰- TFA-pteroic acid, (wherein, the dosage of amino acid and HOBT/DIC Respectively 15eq and 16.5eq/16.5eq) it 10 2% hydrazine hydrates of ml/DMF solution is added after condensation reacts 5 minutes, repeat 3 Secondary, DMF washing, DCM is washed, and MeOH washing is drained, and synthesis finishes, and lytic reagent is added, and TBME precipitated polypeptide obtains target Compound III-3.

The amino acid or substituted amino acid that wherein any one of c and f lysine can also be similar with other structures replace Generation, to prepare it is similar containing there are four or five reactive functional groups segment, and the condensation sequence of different aminoacids, connection The spatial configuration of mode and amino acid is not intended to restrict the invention.

N¹⁰- TFA-pteroic acid refers to the compound having the following structure:

,

Preparation method is referring specifically to entitled " Efficient Syntheses of Pyrofolic Acid and Pteroyl Azide, Reagents for the Production of Carboxyl-Differentiated Derivatives of Folic Acid " (J. Am. Chem. Soc., Vol. 119, No. 42,1997,10004- 10013) document, the document discloses prepare N by folic acid¹⁰The specific method of-TFA-pteroic acid, disclosed part It is incorporated by reference into the present invention.

In another scheme, the present invention provides a kind of pharmaceutical compositions, and it includes folacin receptor knots described herein Close ligand-drug conjugate and pharmaceutically acceptable carrier, diluent or excipient, or combinations thereof.

In another scheme, the present invention provides a kind of described pharmaceutical compositions to treat and/or prevent by causing a disease carefully The purposes of disease caused by born of the same parents group.The population of pathogenic cells refers to cancer cell, infectious substance such as bacterium and virus, thin The cell of bacterium or virus infection, can cause illness activation macrophage and any unique, priority expression or excessively Express the other types pathogenic cell of folacin receptor.The population of pathogenic cells can be tumorigenesis (including benign tumour and pernicious swollen Tumor) cancer cell population, or can be not tumorigenesis.The cancer cell population includes but is not limited to, carcinoma of mouth, thyroid cancer, Endocrine gland cancer, cutaneum carcinoma, gastric cancer, cancer of the esophagus, laryngocarcinoma, cancer of pancreas, colon cancer, bladder cancer, osteocarcinoma, oophoroma, uterine cancer, cream Gland cancer, carcinoma of testis, prostate cancer, the carcinoma of the rectum, kidney, liver cancer and lung cancer.

According to the present invention, the folate receptor binding ligand-drug conjugates can be used for treating to exist in host and cause a disease The disease that cell colony is characterized, wherein the member of population of pathogenic cells has folic acid or pteroic acid binding site, wherein the knot Close position expressed by the pathogenic cell uniqueness, overexpression or priority expression.The selectivity of the pathogenic cell is eliminated, by institute It states folate receptor binding ligand-drug conjugates vitamin moieties and is connected to folacin receptor.High-affinity folacin receptor surface Express vitamin receptor overexpression on tumour cell.Ovary, mammary gland, colon, lung, nose, pharynx and brain epithelioma are all reported Express high-level folacin receptor.In fact, known is more than that 90% human ovarian tumor is expressed at high levels this receptor.Therefore, Drug delivery conjugates of the present invention can be used for treating various tumor cell types and other pathogenic cell type such as infectants, institute State cell type priority expression folacin receptor, and therefore with surface can and vitamin or vitamin D 3-analogies or derivative knot Close position.

Term used in the present invention " folate or folic acid, pteroic acid salt or pteroic acid " is that independent reference is used to form coupling The folic acid of object or pteroic acid part.

Abbreviation:

" Boc " indicates tertbutyloxycarbonyl；

" CDI " indicates N, N- carbonyl dimidazoles；

" DDQ " indicates the chloro- 5,6- dicyano -1,4- benzoquinones of 2,3- bis-；

" DCC " indicates N, N'- dicyclohexylcarbodiimide；

" DEAD " indicates diethyl azodiformate；

" DIPEA " and " DIEA " indicates N, N'- diisopropylethylamine；

" DIC " indicates N, N'- diisopropylcarbodiimide；

" EDCI " indicates 1- (3- dimethylaminopropyl) -3- ethyl carbodiimide；

" EDC " indicates (1- ethyl -3-(3- dimethylaminopropyl) carbodiimide hydrochloride)；

" EEDQ " indicates 2- ethyoxyl -1- ethoxy carbonic acyl radical -1,2- dihydroquinoline；

" Fmoc " indicates fluorenylmethyloxycarbonyl；

" HBTU " indicates (2-(1H- benzotriazole -1- base) -1,1,3,3- tetramethyl tetrafluoro boric acid urea；

" HATU " indicates N- [[(dimethylamino) -1H-1,2,3- triazols [4,5-b] pyridine -1- base] methylene]-N- Methyl hexafluorophosphoric acid first ammonium N- oxide；

" HOAt " indicates 7- azepine-I-hydroxybenzotriazole；

" HOBT " indicates I-hydroxybenzotriazole；

" HOSU " indicates n-hydroxysuccinimide；

" ivDde " indicates 1-(4,4- dimethyl -2,6- dioxo cyclohexylene) -3- methyl butyl；

" NMP " indicates N-Methyl pyrrolidone；

" OtBu " indicates tert-butoxy；

" PABA " indicates p-aminobenzoic acid；

" TBTU " indicates 2- (1H- benzothiazolyl) -1,1,3,3- tetramethylurea tetrafluoro boric acid；

" TBME " indicates t-butyl methyl ether.

Folate receptor binding ligand is 2 or more in folate receptor binding ligand-drug conjugates provided by the invention It is a, preferably 2 ~ 4, and each ligand is covalently attached with polyvalent linkers respectively, and drug D covalently connects with releasable connector It connects, compared with the ligand for containing only the combination of a receptor in the prior art, conjugate provided by the invention and folacin receptor cell Affinity greatly improves, and polyvalent linkers L of the present invention include releasable connector, when conjugate folic acid and/or Pteroic acid part is integrated on pathogenic cell, and conjugate is combined closely with pathogenic cell surface, and subsequent endocytosis is to intracellular, in cell Interior releasable connector cracks rapidly, and drug D is discharged, and plays the drug action of drug, it is surprising that provided by the invention Conjugate shows significant anti-tumor activity in the tumour cell that folacin receptor is overexpressed, and is changed based on the weight of animals Out as a result, it has been found that, conjugate provided by the present invention while significantly improving anti-tumor activity, to drug dose under, Animal can be resistant to well.Also, compared with existing folate receptor binding ligand-drug conjugates (such as EC145), on an equal basis Under molar dose, the combination probability of conjugate and folacin receptor provided by the invention is bigger；Even more it is surprising that Under excessive folic acid existence condition, when equivalent molar dosage, (such as with existing folate receptor binding ligand-drug conjugates EC145 it) compares, conjugate exhibits provided by the invention go out more stable anti-tumor activity, this shows even if there are a certain amount of Folic acid race condition under, conjugate provided by the invention can also play good anti-tumor activity, these of the invention are excellent Point can be further proven by subsequent specific embodiment.

Compared with folate receptor binding ligand-drug conjugates in the prior art, Conjugate Molecules provided by the invention Amount is significant to be increased, and is conducive to conjugate by passive target mechanism selectivity and is gathered in tumor cell surface, while this hair Conjugate provided by bright contains 2 or multiple folate receptor binding ligands, further enhances conjugate and folacin receptor sun Property expression tumour cell residence time in tumour cell of affinity and conjugate, it is further, covalent with drug The releasable connector such as other releasable connectors in hydrazides key and connector L such as disulfide bond of connection under the conditions of cellular acid, Hydrazides key just disconnects in a few minutes, while disulfide bond is reduced, and discharges drug, plays the anti-tumor activity of drug rapidly.This Outside, inventor also found, conjugate provided by the present invention is low with plasma albumin Percentage bound, this also implies that the present invention is mentioned Clearance rate is fast in vivo for the conjugate of confession, small toxicity.

Beneficial effects of the present invention are further illustrated below in conjunction with the drawings and specific embodiments, following embodiment is only to say Bright property, does not limit the invention.

Detailed description of the invention

Fig. 1 shows the mass spectrogram of the compound 15 prepared by embodiment 4, wherein 2969.351 be the molecule of compound 15 Quasi-molecular ions.

Fig. 2 shows 1 μM of BP111b(the compound of the present invention 15) and reference substance BP111a(compound EC145) exist With there is no, to the inhibiting effect of KB cell, wherein "-" expression is added without, and "+" indicates to be added under the conditions of excessive folic acid.

Fig. 3 shows BP111b(the compound of the present invention 15) and reference substance BP111a(compound EC145) to folacin receptor The inhibiting effect of negative A549 cell growth, abscissa indicate the concentration that BP111a and BP111b is added.

Fig. 4 is shown gives 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg BP111b(compounds 15 respectively) and reference substance Inhibiting effect of the BP111a to mouse KB tumour growth, Vehicle expression blank control, it may be assumed that do not give drug therapy, be given only Equivalent solvent, ordinate TV(mm3) indicate gross tumor volume (Tumor Volume), abscissa: day (Day).

Fig. 5 is shown gives 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg BP111b(compounds 15 respectively) and reference substance BP111a influences KB mice with tumor weight, and Vehicle indicates blank control, it may be assumed that does not give drug therapy, it is molten to be given only equivalent Matchmaker, ordinate BW(g) indicate weight (Body Weight), abscissa: day (Day).

Specific embodiment

Technical solution of the present invention and advantage are further illustrated below by way of specific embodiment, it is believed that they are in the present invention In the range, it is not intended as limitation of the present invention.The method is illustrative method, can be used for preparing of the present invention Drug conjugate.

The preparation of 1 compound III of embodiment

(III)

According to the scheme of scheme 1, into 50ml reaction flask, 1000mg is added in conventional steps according to the present invention 2Cl-Trt Resin(1.5eq), add 234mg Fmoc-Cys(Trt)-OH(1.0eq), 8ml methylene chloride (DCM), 640 μ L DIEA(2.0eq), it is completely dissolved, after 8ml DCM, 1ml MeOH, 1ml DIEA reaction 20min are added after reaction 50min, It is transferred in self-control solid phase synthesis column, the Piperidine/DMF solution of 10ml 20% is added after DMF washing, reacts 20min, DMF is clear It is positive to wash rear ninhydrin monitoring, takes 709mg Fmoc-Lys(Fmoc)-OH (3.0eqiv, 178mg HOBT (3.3eqiv), 6ml Reaction column is added after 230 μ L DIC (3.3eq) mixing is added after dissolving in DMF, and ninhydrin monitors after reacting 1H, adds after DMF washing Enter 20% Piperidine/DMF solution.Above step is repeated, Fmoc-Asp(OtBu is successively condensed)-OH, Fmoc-Asp(OtBu)-OH , Fmoc-Arg(Pbf) and-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N¹⁰- TFA-pteroic acid is (wherein, The dosage of amino acid and HOBT/DIC are respectively 6eq and 6.6eq/6.6eq), 2% hydrazine hydrate of 10ml/DMF is added after condensation It solution reaction 5 minutes, being repeated 3 times, DMF washing, DCM washing, MeOH washing is drained, and synthesis finishes, lytic reagent is added, TBME precipitated polypeptide obtains crude product III, after HPLC prepares chromatographic separation and purification, obtains sterling III, MS [M]⁺: 2097.74.

The preparation of 2 compound 33 of embodiment

10.0ml DCM, 1.0ml MeOC (O) SCl(1.0eq are added into 100ml three-necked flask) ice bath is cooled to 0 DEG C, It is added dropwise 0.76ml mercaptoethanol (1.0eq), keeps the temperature 0 DEG C of reaction 30min, be added 1.22g 2- mercaptopyridine (1.0eq), 16ml DCM is added dropwise into reaction flask, is warmed to room temperature TLC raw material end of reaction after 1h after keeping the temperature 0 DEG C of reaction 1h, post-processing: is concentrated into 16ml Left and right filters, and filtrate obtains faint yellow micro- stink solid 2.0g after being washed with DCM.

3.0ml DCM, 0.46g surpalite (0.55eq) are added into 100ml three-necked flask, ice bath is cooled to 0 DEG C, will 1.0g compound 33-2(1.1eq) it is dissolved in 13ml DCM, then 0.45g triethylamine (1.0eq) is added thereto, it is dripped after dissolution It is added in reaction flask, 0 DEG C of temperature control when dropwise addition is hereinafter, be warmed to room temperature TLC raw material end of reaction after 1h (up to 33) yield 96%, MS [M]⁺: 248.97。

The other details of this method are shown in United States Patent (USP) US2007276018 and I. R. Vlahov,et al. Bioorg. Med. described in Chem. Lett. 16 (2006): 5093-5096, this two documents are incorporated integrally into the present invention by reference In.

The preparation of 3 compound 15-3 of embodiment

1.5g vincaleukoblastinum, 5 ml anhydrous methanols and 5ml anhydrous hydrazine are added into 100ml three-necked flask, is warming up to about 60 DEG C, TLC detection reaction process pours into 50ml water after reaction after reaction for 24 hours, and DCM is extracted 3 times, is washed 3 times, saturated common salt Washing 3 times obtains white solid 1.1g, i.e. compound 15-2 after the dry concentration of anhydrous sodium sulfate.

Compound 15-2 0.98g(1.0eq is added into 100ml three-necked flask), 33 0.67g(1.5eq of compound), 0.44ml triethylamine (2.5eq) is added dropwise into reaction flask after stirring and dissolving in 10g DCM, end of reaction after room temperature 2h.

Post-processing: being added 50ml DCM, washes 3 times, and saturated common salt is washed 3 times, is concentrated to give after anhydrous sodium sulfate is dry thick Product 1.2g, column obtain the sterling 750mg of 15-3 after chromatographing.MS[M]⁺: 959.43.

The preparation of 4 compound 15 of embodiment

7.5ml water is added into 100ml there-necked flask, 200mg compound III(1.2eq is added after being bubbled 30min in nitrogen), With the NaHCO of 0.1N₃It is about 6.9 that aqueous solution, which adjusts pH,；78mg compound 15-3 is dissolved in 15ml MeOH, and is added into Into reaction flask, react about 10min after end of reaction, HPLC preparation after be lyophilized compound 15 sterling 39mg.HPLC: 98.1%, the mass spectrogram of compound 15 is measured using Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF-MS) See attached drawing 1, MS [M+H]⁺: 2969.35.

The preparation of 5 compound 16 of embodiment

The preparation method prepare compound 16 of similar compound 15.

MS [M+H]⁺: 3043.18.

The preparation of 6 compound 17 of embodiment

The preparation method prepare compound 17 of similar compound 15.

MS[M+H]⁺: 2819.98.

Preparation (the R of 7 compound 18 of embodiment_xFor p-methoxyphenyl)

By compound 33 and DMAP(4- dimethyl aminopyridine) it is added in the dichloromethane solution of Didemnin B 0 DEG C reaction 2h, compound 18-1 is obtained after isolating and purifying；Compound III is soluble in water, under condition of nitrogen gas, with sodium bicarbonate tune Saving pH value of solution is 7, compound 18-1 is dissolved in acetonitrile, and under a nitrogen atmosphere, is added in the aqueous solution of compound III, It is isolated and purified after reaction using inverse analysis HPLC detection reaction process, obtains target product 18, MS [M+H]⁺: 3312.36。

The preparation of 8 compound 19 of embodiment

Compound 19-1(7.7mg, 10 μm of ol under the conditions of 0 DEG C in 5% ethanol/methylene) mixture in The trifluoroacetate (6.4mg, 20 μm of ol) of compound 19-2 is added.Reaction mixture is warming up to environment temperature and stirs 5h, Then it is concentrated under reduced pressure, silica gel column chromatography (eluant, eluent: 3% ethanol/methylene) obtains 3.3mg target compound 19-3, MS [M+ H]⁺: 974.47.

The sulfydryl of compound 19-3 and compound III is subjected to Michael addition reaction, prepares compound 19, MS [M+H]⁺: 3058.16.

The preparation of 9 compound 20 of embodiment

The preparation of compound 20 can be prepared by compound 20-1 by the reaction of 8 steps according to above-mentioned route；Wherein, by changing It closes object 20-1 to be described in detail in EP0624377A2 by the concrete operation method of 6 steps reaction prepare compound 20-9, this article The disclosure offered partially is incorporated by reference into the present invention.

The preparation of compound 20-10: MMAE(100.5mg, 0.14mmol, 1eq), compound 20-9(110.6mg, 0.15mmol, 1.1eq) and HOBt(19mg, 0.14mmol, 1.0eq) use DMF(2ml) dissolution.After 2min, pyridine is added (0.5ml) monitors reaction process with reversed-phase HPLC.Reaction about for 24 hours after, reaction terminates, and reaction solution is concentrated, residue reverse phase HPLC purifying (Varian Dynamax chromatographic column 21.4mm × 25cm, 5 μ, 100 holes) is prepared, acetonitrile and Et are used₃N-CO₂(pH 7) gradient elution, flow velocity 20ml/min, elution time 40min are carried out from 10% to 100%.Fractional Collections, concentration, obtain class White solid is compound 20-10, MS [M]⁺:1315.78。

The sulfydryl of compound 20-10 and compound III is subjected to Michael addition reaction, obtains compound 20.MS [M+ H]⁺ :3414.52。

The preparation of 10 compound 21 of embodiment

Referring to the method prepare compound 21 of embodiment 9.MS [M+H]⁺:3428.50。

The preparation of 11 compound 22 of embodiment

The synthetic method of the amine-modified valine-citrulline dipeptides (compound 22-1) of maleimide used in the present invention Bibliography: Gene M.D., et al.Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconjμgates:Model Studies of Enzymatic Drμg Release and Antigen-Specific In Vitro Anticancer Activity. Bioconjugate Chem.13(4)855-869(2002)。

The reacting of compound 22-1 and CBI is reacted the method for preparing amide with carboxyl according to conventional amino and is operated, Compound 22-2 is obtained, compound 22-2 is reacted with the sulfydryl of compound III, obtained by the preparation method of similar compound 19 Compound 22.MS [M+H]⁺: 3065.16.

The preparation of 12 compound 23 of embodiment

(Boc) is used according to conventional method in that art under the conditions of pyridine₂SN-38 10 hydroxyls are protected to obtain by O Compound 23-2, by compound 23-2(0.358g, 0.073mmol), DMAP (0.266,0.218mmol) and triphosgene (0.0095g, 0.032mmol) is added in reaction flask, then be added the initiation reaction of 1.5ml methylene chloride, TLC detection react into Journey is quenched with anhydrous methanol after reaction, obtains compound 23-3, is directly used in without processing and is reacted in next step.

Compound 20-8(0.768g, 0.883mmol) is added in the reaction solution of above-mentioned 23-3, reaction about 5min reaction Terminate, with flash column purified (ethanol/methylene gradient elution), obtains 20 by boc-protected compound 23-4, so Protecting group is sloughed with TFA hydrolysis again afterwards, obtains the tfa salt of target compound 23-4 with Diethyl ether recrystallization.

According to the scheme of scheme 6, compound 23-4 is reacted with-the SH of the cysteine of compound III is prepared mesh Mark compound 23.MS [M+H]⁺: 3089.15.

The preparation of 13 compound 24 of embodiment

The preparation method prepare compound 24 of similar compound 15.MS [M+H]⁺: 2946.23.

The preparation of 14 compound 25 of embodiment

The preparation method prepare compound 25 of similar compound 15.

MS[M+H]⁺: 2932.20.

The preparation of 15 compound 26 of embodiment

It is prepared by the preparation method of similar compound 15.

MS[M+H]⁺: 2918.22.

The preparation of 16 compound 27 of embodiment

According to the scheme of scheme1, into 50ml reaction flask, 1000mg is added in conventional steps according to the present invention 2Cl-Trt Resin(1.5eq), add 234mg Fmoc-Cys(Trt)-OH(1.0eq), 8ml methylene chloride (DCM), 640 μ L DIEA(2.0eq), it is completely dissolved, 8ml DCM, 1ml MeOH, 1ml DIEA reaction 20min is added after reacting 50min Afterwards, it is transferred in self-control solid phase synthesis column, the Piperidine/DMF solution of 10ml20% is added after DMF washing, reacts 20min, Ninhydrin monitoring is positive after DMF cleaning, takes 709mg Fmoc-Lys(Fmoc)-OH (3.0eqiv, 178mg HOBT Reaction column is added after 230 μ L DIC (3.3eq) mixing is added after 6ml DMF dissolution in (3.3eqiv), and ninhydrin is supervised after reacting 1H It surveys, 20% Piperidine/DMF solution is added after DMF washing.Above step is repeated, Fmoc-Asp(OtBu is successively condensed)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, N¹⁰- TFA-pteroic acid, (its In, the dosage of amino acid and HOBT/DIC are respectively 6eq and 6.6eq/6.6eq), after condensation be added 2% hydrazine hydrate of 10ml/ DMF solution reacts 5 minutes, is repeated 3 times, DMF washing, DCM washing, and MeOH washing is drained, and synthesis finishes, and cracking examination is added Agent, TBME precipitated polypeptide obtain III-1, MS [M]⁺:1839.65。

The preparation method of compound 15 in similar embodiment 4, prepare compound 27 are a difference in that and are substituted in fact with III-1 Apply III in example 4.MS[M+H]⁺: 2711.05.

17 compound III-1 ' of embodiment

Full guard peptide Asp(OtBu)-Asp(OtBu)-Arg(Pbf)-Asp(OtBu)-pteroic acid synthesis: adopt Fmoc-Asp(OtBu is successively condensed with 2Cl-Trt Resin, SPPS)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Asp(OtBu)-OH, N¹⁰It is molten that 2% hydrazine hydrate/DMF is added in-TFA-pteroic acid after condensation Liquid reacts 5 minutes, is repeated 3 times, DMF washing, DCM washing, and MeOH washing is drained.1% TFA/DCM solution full guard is added 5min is cut, is repeated several times, TLC is until without product, and filtrate is washed after being neutralized with pyridine, after saturated common salt water washing Concentration, column chromatograph pure to full guard peptide Asp(OtBu)-Asp(OtBu)-Arg (Pbf)-Asp(OtBu)-pteroic acid Product, MS [M]⁺: 981.05.

The synthesis of III-1 ': into 50ml reaction flask, 1000mg2Cl-Trt Resin(2.0eq is added), it adds 117mg Fmoc-Cys(Trt)-OH(1.0eq), 8ml DCM, 320 μ L DIEA(2.0eq) and, it is completely dissolved, after reacting 50min 8ml DCM, 1ml MeOH, 1ml DIEA is added, is transferred in self-control solid phase synthesis column after reacting 20min, DMF washing The Piperidine/DMF solution of 10ml20% is added afterwards, reacts 20min, ninhydrin monitoring is positive after DMF cleaning, takes Fmoc-Lys (ivDde) after 230 μ L DIC (3.3eq) mixing is added after-OH (3.0eq), 178mg HOBT (3.3eq), 6ml DMF dissolution Reaction column is added, ninhydrin monitors after reacting 1h, and 20% Piperidine/DMF solution is added after DMF washing.Above step is repeated, according to Secondary condensation Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)- OH, Fmoc-Glu-OtBu, N¹⁰- TFA-pteroic acid, (wherein, amino acid and HOBT/DIC dosage be respectively 3eq and 3.3eq/3.3eq), 10ml2% hydrazine hydrate/DMF solution is added after condensation to react 5 minutes, is repeated 3 times, DMF washing, indenes Triketone monitoring is positive, addition compound Asp(OtBu) and-Asp(OtBu)-Arg(Pbf)-Asp(OtBu)-pteroic acid } (2.0eq), HOAT (2.2eq), DIC (2.2eq), DMF makees solvent.Ninhydrin monitors after reaction overnight, after completion of the reaction DMF Washing, DCM washing, MeOH washing are drained, and synthesis finishes, and lytic reagent is added, and TBME precipitated polypeptide obtains III-1 '.MS[M]⁺: 1968.69。

Embodiment 18

The preparation method of compound 15 in similar embodiment 4, prepare compound 28, MS [M+H]⁺: 2840.10.

The preparation of 19 compound III-2 of embodiment

III-2 is prepared according to method shown in scheme2, the specific steps are as follows:

The preparation of compound c: 10g a, the THF of the HOSU of 1.1eq, 100ml, ice after dissolution are added in 250ml reaction flask Bath is cooled to 0 DEG C, and the DCC that 1.1eq is added, which is warmed to room temperature, to be stirred overnight.TLC raw material end of reaction.Post-processing: it filters, filter cake It is washed with THF, obtains 12g compound b after organic phase concentration.

The d of 3.0g is added in 100ml reaction flask, is dissolved in 30ml water, the NaHCO of 1.1eq is added₃, it is cooled to 10 degree；It will The b of 1.1eq is dissolved in 30ml DME, is added dropwise into reaction flask, adds 10mlTHF ambient temperature overnight.TLC end of reaction is added after concentration EA, dilute hydrochloric acid washing, is then washed, and salt washing, dry, concentration, column chromatographs to obtain compound c.

The preparation of III-2: into 50ml reaction flask, 1000mg2Cl-Trt Resin(2.0eqiv is added), it adds 117mg Fmoc-Cys(Trt)-OH(1.0eq), 8ml DCM, 320 μ L DIEA(2.0eq) and, it is completely dissolved, after reacting 50min 8ml DCM, 1ml MeOH, 1ml DIEA is added, is transferred in self-control solid phase synthesis column after reacting 20min, DMF washing The Piperidine/DMF solution of 10ml20% is added afterwards, reacts 20min, ninhydrin monitoring is positive after DMF cleaning, takes compound c Reaction column is added after 230 μ L DIC (3.3eq) mixing is added after 178mg HOBT (3.3eq), 6ml DMF dissolution in (3.0eq), Ninhydrin monitors after reacting 1h, and 20% Piperidine/DMF solution is added after DMF washing.Above step is repeated, Fmoc- is successively condensed Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Glu- OtBu, N¹⁰- TFA-pteroic acid, (wherein, amino acid and HOBT/DIC dosage are respectively 15eq and 16.5eq/ 16.5eq), 10ml2% hydrazine hydrate/DMF solution is added after condensation to react 5 minutes, is repeated 3 times, DMF washing, DCM washing, MeOH washing, is drained, and synthesis finishes, and lytic reagent is added, and TBME precipitated polypeptide obtains III-2, MS [M+H]⁺: 3151.15.

The preparation of 20 compound III-3 of embodiment

III-2 is prepared according to method shown in scheme3, the specific steps are as follows:

The preparation of compound f: 10g a, the THF of the HOSU of 1.1eq, 100ml, ice after dissolution are added in 250ml reaction flask Bath is cooled to 0 degree, and the DCC that 1.1eq is added, which is warmed to room temperature, to be stirred overnight.TLC raw material end of reaction.Post-processing: it filters, filter cake It is washed with THF, obtains 12g compound b after organic phase concentration.

Into 100ml reaction flask, the d of 3.0g is added, is dissolved in 30ml water, the NaHCO of 1.1eq is added₃, it is cooled to 10 DEG C； The b of 1.1eq is dissolved in 30ml DME, is added dropwise into reaction flask, 10mlTHF ambient temperature overnight is added.TLC end of reaction adds after concentration Enter EA(ethyl acetate), then dilute hydrochloric acid washing is washed, salt washing, dry, concentration, column chromatographs to obtain compound c.

5g c is added into 10ml reaction flask, the THF of the HOSU of 1.1eq, 50ml, ice bath is cooled to 0 DEG C after dissolution, adds The DCC for entering 1.1eq, which is warmed to room temperature, to be stirred overnight.TLC raw material end of reaction.Post-processing: it filters, filter cake is washed with THF, organic 6g compound e is obtained after being mutually concentrated.

3.0g compound e is added into 100ml reaction flask, is dissolved in 30ml water, the NaHCO of 1.1eq is added₃, it is cooled to 10 ℃；The b of 1.1eq is dissolved in 30ml DME, is added dropwise into reaction flask, ambient temperature overnight.EA is added after concentration in TLC end of reaction, dilute salt Then acid elution is washed, salt washing, dry, concentration, column chromatographs to obtain compound f.

The preparation of III-3: into 50ml reaction flask, 1000mg2Cl-Trt Resin(2.0eq is added), add 117mg Fmoc-Cys(Trt)-OH(1.0eq), 8ml DCM, 320 μ L DIEA(2.0eq), it is completely dissolved, is added after reacting 50min It is transferred in self-control solid phase synthesis column after 8ml DCM, 1ml MeOH, 1ml DIEA reaction 20min, DMF washing, then The Piperidine/DMF solution of 10ml20% is added, reacts 20min, ninhydrin monitoring is positive after DMF cleaning, compound f (3.0eq) is taken, After 178mg HOBT (3.3eq), 6ml DMF dissolution, reaction column is added after 230 μ L DIC (3.3eq) mixing is added, reacts 1h Ninhydrin monitors afterwards, and 20% Piperidine/DMF solution is added after DMF washing.Above step is repeated, Fmoc-Asp is successively condensed (OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu- OtBu, N¹⁰- TFA-pteroic acid, (amino acid be respectively 15eq and 16.5eq/16.5eq with HOBT/DIC) condensation finish 10ml2% hydrazine hydrate/DMF solution is added afterwards to react 5 minutes, repeats 3 times, DMF washing, DCM is washed, and MeOH washing is drained, closed At finishing, lytic reagent is added, TBME precipitated polypeptide obtains target compound III-3, MS [M+H]⁺: 4203.55.

The preparation of 21 compound 29 of embodiment

The preparation method of compound 15 in similar embodiment 4, prepare compound 29 are a difference in that and are substituted in fact with III-2 Apply III in example 4.MS[M+H]⁺: 4021.54.

Embodiment 22

Compound 30:MS [M+2H]²⁺:4481.91。

Compound 31:MS [M+2H]²⁺:5074.95。

Compound 32:MS [M+2H]²⁺:5520.33。

The test of 1 relative affinity of test example

FR positive KB cell is intensively planted in 20 porocyte culture plates, and allows the cell adherence in plastic continuing 18h.The culture medium consumed is placed in specified hole, is used in the case where improving and not improving by test product or folic acid concentration 100nM3H- folic acid supplement is free of the RPMI(FFRPMI of folic acid).By cell in 37 DEG C of culture 60min, then with pH7.4's PBS is rinsed 3 times.The PBS solution of the pH7.4 containing 1% dodecyl sodium sulfate (SDS) of 500 μ L is added to every hole.Then, it receives Collection cell pyrolysis liquid is simultaneously added into the independent pipe containing 5ml fluorescent mixture, then counts its radioactivity.It is negative right Look after the folic acid for containing only and being dissolved in FFRPMI (without competitor).Positive control pipe contains the folic acid of final concentration of 1mM, and And the CPM(measured in these samples represents the non-specific binding of label) it is deducted from all samples.It should be evident that phase The anti-molar ratio of compound needed for being defined as the 3H- folic acid for being incorporated into FR on KB cell of displacement 50% to affinity (inverse molar ratio), folic acid is set as 1 to the relative affinity of FR.

Relative affinity test result of the compound 15 in 10% serum/FDRPMI show compared with folic acid, compound 15 show 157% folacin receptor relative affinity.

Similar method, measures relative affinity of the compound 16~32 in 10% serum/FDRPMI, and test result is aobvious Show, compared with folic acid, compound 16~32 show be more than 100% folacin receptor relative affinity, wherein compound 20, 21,31 show respectively 162%, 127% and 187% folacin receptor relative affinity.

2 cell activity assays of test example

Cell activity assays 1:

The compounds of this invention 15~32 is evaluated with vitro cytotoxicity test, which predicts Drug inhibition folacin receptor sun Property the growth of KB cell ability, be inoculated with 100 μ l cell containing KB 5*10 in every hole³A PBS solution after culture for 24 hours, is drawn Medium, is divided into blank control group, control group a, test group b, and control group a and test group b are respectively divided into 10 groups, number a-1, A-2, a-3, a-4, a-5, a-6, a-7, a-8, a-9, a-10 and b-1, b-2, b-3, b-4, b-5, b-6, b-7, b-8, b-9, b- 10, control group and test group are sequentially added into 0,0.1,0.3,1,3,10,30,100,300 micromole's (μM) of folic acid (FA), Continue to cultivate 2h, the preparation method of control compound BP111a(compound EC145, EC145 is then separately added into control group a And structure is shown in Chinese patent CN100381177C) 1 μM, the compound of the present invention is added to test group b (indicates) 1 μ with BP111b Folic acid is also not added in M, blank control group neither dosing object；Continue to cultivate 70h, cell survival rate is evaluated with mtt assay, exempted from enzyme-linked The light absorption value in each hole is measured at epidemic disease detector OD570nm.

Mtt assay is also known as MTT colorimetric method, and testing principle is that the succinate dehydrogenase in living cells mitochondria can make external source Property MTT be reduced to the bluish violet crystallization of water-insoluble and first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function Energy.Dimethyl sulfoxide (DMSO) can dissolve the first a ceremonial jade-ladle, used in libation in cell, its light is measured at 5700nm wavelength with enzyme-linked immunosorbent assay instrument Absorption value (i.e. OD570 value), can reflect living cells quantity indirectly.Within the scope of certain cell number, MTT crystallize the amount to be formed with Cell number is directly proportional.

For example, the drug (being indicated with BP111b) using compound 15 as test group b, operates according to the method described above, For test result as shown in Fig. 2, wherein BP111b indicates the compound of the present invention 15, BP111a indicates compound EC145 (EC145 Preparation method see Chinese patent CN100381177C).As can be seen from Figure 2, under the same conditions, BP111b (change of the invention Close object 15) compound BP111a(compound EC145 is better than to the inhibiting effect of KB cell), also, with the increase of FA, BP111b reduces the inhibiting effect of KB tumour cell, this shows that observed killing functions of immunocytes is by being incorporated into leaf What acid acceptor mediated.It is thin to KB tumour even BP111b is existing for the excessive folic acid also, compared with BP111a Born of the same parents also have preferable inhibitory activity, also as shown in Figure 2.

Cell activity assays 2:

The ability of the A549 cell growth of Drug inhibition folacin receptor feminine gender is predicted in the test, is inoculated with 100 μ l in every hole and is contained The PBS solution that A549 cell is 5*103, after culture for 24 hours, extracting medium is divided into blank control group, control group a, test group b, Control group a and test group b is respectively divided into 10 groups, number a-1, a-2, a-3, a-4, a-5, a-6, a-7, a-8, a-9, a-10 With b-1, b-2, b-3, b-4, b-5, b-6, b-7, b-8, b-9, b-10, control group a and test group b are separately added into control chemical combination The preparation method of object BP111a(EC145, EC145 are shown in Chinese patent CN100381177C) and test compound BP111b10000、3333.33、111.111、370.370、123.4567、41.152、13.717、4.572、13.717、 4.5724,1.5241 nanomoles (nM), blank control group be both not added BP111a or BP111b are not added；Continue to cultivate 70h, cell Survival rate is evaluated with mtt assay, and the light absorption value in each hole is measured at enzyme-linked immunosorbent assay instrument OD570nm.

For using compound 15 as test group drug BP111b, test result is as shown in figure 3, BP111b(of the present inventionization Close object 15) it is unobvious to the tumour cell A549 inhibitory activity of FR- feminine gender, these are the result shows that compound 15 is to pass through folic acid Selectivity or folic acid specificity mechanism plays a role.

Compound 16~32 of the present invention is all obtained in the test of this type similar as a result, in most cases Under, 16~32 pairs of KB cells of compound provided by the present invention have significant inhibitory activity, and present dose-dependent thin Cellular toxicity, IC₅₀(IC₅₀Rate i.e. half-suppressed means the concentration of drug when generating the effect of 50% Tumor growth inhibition) In low nanomolar range, as shown in table 1 below.

1 compound of table, 16~32 pairs of KB cell inhibitory activity data (IC50:nM)

Compound	IC₅₀(nM)	Compound	IC₅₀(nM)
				16	1.4	25	1.7
17	2.2	26	1.2

18	10.2	27	1.9
				19	1.8	28	1.7
20	2.1	29	1.2
				21	2.6	30	1.3
22	5.9	31	0.9
				23	3.8	32	0.8
24	1.1

Test example 3 tests the inhibition of mice tumors grew

Compound 15(of the present invention is indicated with BP111b) intravenous (i.v.) when giving tumor animal, with subcutaneous Its anti-tumor activity is had rated on the Balb/c mouse of the mouse of KB tumour.In right axillary subcutaneous tissue 1*10⁶KB cell tumour About 11 days (t after inoculation₀When mean tumour volume=60mm³), 9 groups is divided into mouse, every group of mouse is 6, wherein a-1, a-2, A-3 group is drug control group a；B-1, b-2, b-3 are test group b；Remaining three groups are blank control group.Control group a distinguishes vein 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg control drug BP111a are given in interior injection；Test group b is injected intravenously gives respectively Give 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg compound BP111b(the compounds of this invention 15)；Blank group gives suitable agent The PBS of volume is measured, is injected intravenously 3 times weekly, for 2 weeks, each treatment group measures tumour growth 2 times a week, with caliber gauge. With equation V=axb²/ 2 calculate gross tumor volume, wherein a is length of tumor, and b is width, and unit is millimeter.Simultaneously 2 times a week or 3 the weight of animals that weigh with scale.

When as shown in Figures 4 and 5, with BP111b0.5 μm of ol/kg treatment of compound of the present invention, KB can be effectively delayed The growth of tumour, drug dose have obviously anti-tumor activity when increasing to 1 μm of ol/kg.Also, as shown in Fig. 4, It is in dose dependent with inhibiting effect of the compound BP111b of the present invention to tumour, compared with control drug BP111a, together Under sample dosage, the compounds of this invention BP111b shows better anti-tumor activity.And compound provided by the invention BP111b does not have overt toxicity (based on the weight of animals), right when 1 μm of ol/kg is equally administered compared with control drug BP111a Also there is anti-tumor activity according to drug BP111a, but when being administered 18 days, BP111b is substantially better than the inhibiting effect of tumour BP111a, as shown in Figure 4；And based on the weight of animals variation discovery, BP111b is as BP111a, under institute's dosage, moves Object can be resistant to well, as shown in Figure 5.

The compounds of this invention 16~32 carries out similar test, test result discovery: using the compound of the present invention 16~ The mouse KB tumour growth of 32 pairs of FR positives has significant inhibiting effect, and not obviously with the toxicity of changes of weight.

It is illustrative, the PBS(control of compound 25 or free drug MMAF or suitable dose volume) with 500nmol/ The dosage of kg is given in right axillary subcutaneous tissue 1*10⁶About 11 days (t after the inoculation of KB cell tumour₀When mean tumour volume=60mm³) Mouse (6/group) intravenous injections, are injected intravenously 3 times weekly, after for 2 weeks, observe the inhibition knot to mice tumors grew Fruit and mouse weight variation.Each treatment group measures tumour growth every 3 days, with caliber gauge.With equation V=axb²/ 2 calculating are swollen Knurl product, wherein a is length of tumor, and b is width, and unit is millimeter.Simultaneously every 3 days the weight of animals that weigh with scale.

Experimental result discovery can effectively delay the growth of KB tumour with compound 25 of the present invention treatment, and not have Overt toxicity (is based on the weight of animals).Free drug MMAF also has anti-tumor activity, but has apparent toxicity, and dynamic Object poor resistance.

Similar approach measures anti-tumor activity of the compound 25 in FR- negative cells, in right axillary subcutaneous tissue 1* 10⁶(t after the inoculation of A549 cell tumour₀When mean tumour volume=50~80mm³), (6/group) intravenous injections of mouse are given Give the PBS(control of the compound 25 or quite dose volume of 500nmol/kg), it is injected intravenously 3 times weekly, it is for 2 weeks, often A treatment group measures tumour growth every 3 days, with caliber gauge.With equation V=axb²/ 2 calculate gross tumor volume, wherein a is tumour Length, b are width, and unit is millimeter.Test result show compound 25 to FR- negative cells almost without activity, these The result shows that compound 25 is played a role by the mechanism of folic acid selectivity or folic acid specificity.

Claims

1. a kind of compound with following general formula:

(F)_nLD

Wherein,

N is 2；

F is folic acid or pteroic acid；(F)_nIn each F it is independent be selected from folic acid and pteroic acid；

D is drug moiety, and the drug is cyclopropyl-phenyl diindyl ketone, Pyrrolobenzodiazepines Zhuo dimer class, 7- Ethyl-10-hydroxycamptothecin, dolastatin, didemnun B, CHROMATOGRAPHIC FRACTIONATION AND MASS, vincristine class, ori inhibin class, Guan Rong Solve plain class；L is comprising with the connector of flowering structure:

L₁-L₂

(F)_nRefer to each F respectively with L₁Polyvalent linkers be covalently attached, D and L₂It is covalently attached；

L is comprising with the connector of flowering structure:

Wherein W selects NH and O；

Wherein, * indicates open valency.

2. compound as described in claim 1, which is characterized in that the connector L includes following structure:

Wherein, W is selected from NH or O；* open valency is indicated.

3. compound as described in claim 1, which is characterized in that the compound is selected from:

4. compound as described in claim 1, which is characterized in that the drug is selected from open loop-cyclopropyl benzo [e] indole ketone Compound, Pyrrolobenzodiazepines Zhuo dimer class, 7-Ethyl-10-hydroxycamptothecin, one hydrazides of deacetylate vincaleukoblastinum, Monomethyl ori inhibin E, monomethyl ori inhibin F, pipe dissolution element B, film ascidian rope B, DM1 or vincristine.

5. compound as described in claim 1, which is characterized in that the compound is selected from:

Wherein, R_x: for p-methoxyphenyl；X₁For Cl or Br；R be H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth orR_yFor hydrogen, C₁~C₆Alkyl or halogenated alkyl or the carbonyl containing 1~4 carbon atom；R₁For C₁~ C₆Alkoxy or alkyl or the amino alkyl or C that replace₁~C₃Aldehyde radical or carbonyl, hydroxyl, amino or C₁~C₆Naphthenic base or Heterocycle；

R₂It is the position 2-, the 3- or 4- substitution of benzene, is NH；The integer that n is 0~3；

F₁, F₂It is independent to be selected from following formula:

6. compound as described in claim 1, which is characterized in that the compound is selected from:

Wherein, L₃Formula selected from the following:

Wherein, the integer that m is 0~4, * indicate link position.

7. compound, which is characterized in that the compound structure is as follows:

Wherein, R_xFor p-methoxyphenyl.

8. a kind of pharmaceutical composition, it includes the compounds as described in claim 1~7 is any of therapeutically effective amount, and pharmaceutically Acceptable carrier, diluent or excipient, or any combination thereof.