CN104784699A - Folate receptor binding ligand-drug conjugate - Google Patents

Folate receptor binding ligand-drug conjugate Download PDF

Info

Publication number
CN104784699A
CN104784699A CN201410024349.9A CN201410024349A CN104784699A CN 104784699 A CN104784699 A CN 104784699A CN 201410024349 A CN201410024349 A CN 201410024349A CN 104784699 A CN104784699 A CN 104784699A
Authority
CN
China
Prior art keywords
compound
acid
joint
arbitrary
reaction
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201410024349.9A
Other languages
Chinese (zh)
Other versions
CN104784699B (en
Inventor
袁建栋
宋云松
黄仰青
朱锐
胡晓伟
方程
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Borui Bio-Medical Technology (jiangsu) Co Ltd
Brightgene Bio Medical Technology Co Ltd
Original Assignee
Borui Bio-Medical Technology (jiangsu) Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Borui Bio-Medical Technology (jiangsu) Co Ltd filed Critical Borui Bio-Medical Technology (jiangsu) Co Ltd
Priority to CN201410024349.9A priority Critical patent/CN104784699B/en
Priority to CN201811352537.9A priority patent/CN109316605B/en
Priority to PCT/CN2014/091690 priority patent/WO2015106599A1/en
Publication of CN104784699A publication Critical patent/CN104784699A/en
Application granted granted Critical
Publication of CN104784699B publication Critical patent/CN104784699B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

The invention provides a folate receptor binding ligand-drug conjugate. The conjugate is composed of a vitamin receptor binding part (F)n, a linker (L), and a drug or its analog or derivative (D), wherein n is an integer in a range of 2-4. The vitamin receptor binding part preferably selects folic acid or technical pteroic acid, the linker (L) comprises a multivalent linker L1 and at least one releasable linker L2, the (F)n is covalently connected to the multivalent linker L1, the drug or its analog or derivative (D) is covalently connected with the releasable linker L2, and the L1 is covalently connected with the L2. The conjugate provided by the invention has great improved affinity to folate receptor positive tumor cells, has substantial antitumor activity and has obviously reduced side effects.

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.Be specifically related to folate receptor binding ligand-drug conjugates, more specifically, the present invention relates to two or more folate receptor binding ligand (F) by joint (L) and medicine or its analog or derivant (D) coupling, formed (F) nlD conjugate, the present invention also relates to, and described conjugate is used for the treatment of the disease condition caused by Pathogenic cellular colony, and relates to preparation method and its pharmaceutical composition of such conjugate.
Background technology
Although the research of the Therapeutic Method of cancer and cancer therapy drug has made great progress, cancer is still one of principal disease of serious threat human health, and in the U.S., cancer is the second largest reason causing death after heart disease.The exemplary process of present Therapeutic cancer comprises surgical treatment, actinotherapy, chemotherapy, immunotherapy and above Therapeutic Method use in conjunction to anticancer.The most frequently, utilize high potent drug, such as mitomycin, paclitaxel and camptothecine, by amic therapy method Therapeutic cancer.While wherein the major defect of chemotherapeutics is inhibition tumor cell growth, also seriously inhibit Normocellular propagation, these inevitable side effect make the exploitation of new specific anti-cancer drugs seem particularly important.
Many effort are carried out at present by cancer therapy drug being attached to ligand as hormone, antibody or support one's family and usually develop tumor selective agents.Such as, by low-molecular-weight element-vitamine compound (as folic acid) as cancer target agent.
Folic acid (FA) also claim vitamin B 9, be the necessary nutrient that all living cells maintain needed for a carbon approach homergy and nucleotide biosynthesis.Folacin receptor (FR) is a kind of cross-film single chain glycoprotein, containing 3 kinds of hypotypes: α-FR, β-FR, γ-FR.The glycoprotein of folic acid (FA) to the cell surface orientation of folacin receptor (FR) shows high affinity (KD ~ 100pM), and this glycoprotein is the albumen that the glycosyl-phosphatidyl inositol catching its part (folic acid) from extracellular environment connects.Folacin receptor (FR) is the tumourassociated membrane proteins that combines with folic acid (FA) and in cell, can transports the molecule be combined with folic acid by endocytic mechanisms.In conjunction with after, the plasma membrane surrounding FR-ligand complex will cave in form internal compartment immediately, be called endosome.The pH in vesicle chamber is by locating the effect step-down a little of the proton pump in endosome film altogether, and this acidization may mediate conformational change in FR albumen with the part discharging it and combine allows to enter kytoplasm.
Research show 90% ovarian cancer, advanced breast cancer, cervical cancer, carcinoma of endometrium, colon cancer, pulmonary carcinoma, choroidcarcinoma, there is the α-FR of high expressed in ependymoma; Have the β-FR of high expressed at the Macrophage Surface of autoimmune disease patient's vivo activations such as leukocyte (leukemia) surface of malignant proliferation and rheumatoid arthritis, and folacin receptor is expressed in the normal tissue hardly.Therefore folic acid and folacin receptor have good Research Prospects in targeted therapy technology, especially in the treatment of tumor and autoimmune disease, FR receives great concern as the target spot of antitumor drug, become new type antineoplastic medicine research one of focus (Hilgenbrink, A. and P. Low (2005). Folate receptor mediated drug targeting:From therapeutics to diagnostics. Journal of Pharmaceutical Sciences 94 (10): 2135-2146.).In the world trial utilize folic acid-radionuclide imaging agent conjugate, as folic acid with 125i, 67ga, 111the tumor tissues of the detection human body high expressed folacin receptor of the conjugate energy Noninvasive of In.Simultaneously, at present in the world extensive with regard to folic acid-proteotoxin, folic acid-small molecule chemotherapeutic medicine, folate liposome (in liposome bag chemotherapeutics or genomic medicine) and folic acid-immunotherapeutic agent etc. expand further investigation (list of references: 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
The object of the invention is to provide a kind ofly has specificity to pathogenic cell and the folate receptor binding ligand-drug conjugates little to normal cytotoxicity, its preparation method and its application in preparation tumor.
In an illustrated embodiment of the present invention, describe the compound with following chemical formula:
(F) nLD
Folate receptor binding ligand-drug conjugates, wherein n is 2,3 or 4; F is folate receptor binding ligand, is selected from folic acid or pteroic acid; (F) nin each F be independently selected from folic acid and pteroic acid; D is medicine or its analog or derivant; L is the joint comprising following structure:
L 1-L 2
Wherein, L 1polyvalent linkers, L 2comprising at least one can releasing-joint, L 1with L 2covalently bound;
Should be understood that, (F) described in the present invention nrefer to each F respectively with polyvalent linkers L 1covalently bound, D and L 2covalently bound.
In another embodiment, the folate receptor binding ligand-drug conjugates with following structure is described:
，
Wherein F 1, F 2independently be selected from folic acid and pteroic acid; D is medicine or its analog or derivant;
L is the joint comprising following structure:
L 1-L 2
Wherein, L 1polyvalent linkers, L 2comprising at least one can releasing-joint, L 1with L 2covalently bound;
Described F 1, F 2respectively with polyvalent linkers L 1covalently bound, D and L 2covalently bound.
Described polyvalent linkers (L 1) can containing mutually covalently bound multiple joints, comprise one or more spacer nipples, can releasing-joint, heteroatom linker and the combination with any sequential arrangement thereof.In an alterable mode, can releasing-joint and the mutual covalent bond of optional spacer nipple for forming joint L 1; In another alterable mode, part F is covalently attached to polyvalent linkers L by one or more spacer nipple 1; More than 2 or 2 part F directly attachment or by multiple spacer nipple and/or can releasing-joint covalent attachment mutually in another alterable mode; In another alterable mode, two or more can releasing-joint mutually covalently bound, and wherein one or more can be spaced from each other by heteroatom linker and/or spacer nipple by releasing-joint.
Should be understood that, one or more can the transmutability of arrangement of releasing-joint and optional spacer nipple very large.Hetero atom in described heteroatom linker is nitrogen, oxygen, sulfur, phosphorus, silicon etc.Further, described heteroatom linker (except oxygen) can be different oxidation state, as N (OH), S (O), S (O) 2, P (O), P (O) 2, P (O) 3deng.In another alterable mode, described heteroatom linker is azanol, acid esters, acid esters etc. of seeing are seen in hydrazine, hydrazone, sulphonic acid ester, Asia.
In one embodiment, polyvalent linkers L 1comprise at least one by amino acids formed peptide spacer nipple, described aminoacid is independently selected from natural amino acid and non-natural alpha-amino acid.In another embodiment, described polyvalent linkers L 1comprise containing 1 ~ 40 amino acids formed peptide spacer nipple, the preferred natural amino acid of described aminoacid, further, described polyvalent linkers L 1comprise containing 1 ~ 20 amino acids formed peptide spacer nipple; Further, described L 1preferably include containing 10 ~ 15 amino acids formed peptide spacer nipples.Further, L 1comprise the aminoacid that at least 2 are selected from lower group: aspartic acid, arginine, cysteine, lysine, agedoite, arginine, threonine, glutamic acid, serine, citrulline, valine and glutamine.
Further, described L 1preferably include one or more spacer nipple by being selected from the amino acids formed dipeptides of aspartic acid, arginine, cysteine, citrulline, valine and lysine and combination thereof, tripeptides, tetrapeptide, pentapeptide, six peptides, seven peptides, octapeptide, decapeptide, 11 peptides and dodecapeptide.
In an exemplary embodiment, by amino acids formed peptide spacer nipple and heteroatom linker combine formed as shown in the formula polyvalent linkers L 1:
or
，
Wherein, * represents open valency.
Described polyvalent linkers L 1optional comprising is selected from following spacer nipple: polyethers, sugar, thiocarbonyl, alkylidene, 1-alkylenesuccinimid-3-base, 1-(carbonylic alkyl) butanimide-3-base, carbonylic alkyl carbonyl, 1-(carbonyl tetrahydrochysene-2H-pyranose) butanimide-3-base and 1-(carbonyl tetrahydrofuran base) butanimide-3-base, and wherein said each spacer nipple is optionally replaced by one or more substituent group;
It is alkyl that wherein said substituent group is independently selected from, alkoxyl, alkoxyalkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, alkyl aminoalkyl, dialkyl aminoalkyl, mercaptoalkyl, alkyl-thio-alkyl, aryl, the aryl replaced, aralkyl, the aralkyl replaced, heteroaryl, the heteroaryl replaced, carboxyl, carboxyalkyl, alkyl carboxylates, chain acid alkyl ester, guanidine alkylation, or the carbonyl to be replaced by aminoacid and derivant thereof and peptide or acylamino-or amidoalkyl.
In the present invention, L 2comprising at least one can releasing-joint, described " can releasing-joint " refers to the joint of the key (such as pH labile bond, sour labile bond, oxidation unstable or enzyme labile bond) comprising at least one and can rupture in physiological conditions.Self-evident, this physiological condition of bond fission that causes comprises generation at physiological ph, or dissolves organelle as compartment, the result of endosome that such as its pH is lower than kytoplasm pH and the standard chemical hydrolysis reaction that occurs.Self-evident, as described herein, scissionable bond can connect can two adjacent atoms in releasing-joint, and/or the either end of releasing-joint or two ends can connect other joint or part F and/or medicine D described.When this cleavable key connect can two adjacent atoms in releasing-joint, after this bond fission, describedly can fragment into two or more fragments by releasing-joint.Or, when this cleavable key can releasing-joint and another part such as heteroatom linker, spacer nipple, another can between releasing-joint, medicine or its analog or derivant or folate receptor binding ligand, after this bond fission, can separate from other parts by releasing-joint.The unstability of cleavable key can adjust, and such as, by changing in Ke Lie Xie Key position or the replacement near it, such as comprise the alpha-branching adjoining cleavable disulfide bond, homologization forms the alkoxyl etc. of hydrolyzable part ketal or acetal.
Described can be selected from by releasing-joint: 1-alkoxyalkylene, 1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, two carbonyl aryl, two carbonyl carboxyl aryl, two carbonyl two carboxyl aryl, haloalkylenecarbonyl, oxygen base ketonic oxygen base, oxygen base ketonic oxygen base alkyl, imino alkyl, carbonyl alkylen group imino group, imino group cycloalkylidene, carbonylcycloalkylideniminyl, alkylenethio, alkylidene aryl sulfenyl and carbonylalkylthio, wherein eachly describedly can optionally to be replaced by one or more substituent group by releasing-joint;
It is alkyl that wherein said substituent group is independently selected from, alkoxyl, alkoxyalkyl, hydroxyl, hydroxyalkyl, amino, aminoalkyl, alkyl aminoalkyl, dialkyl aminoalkyl, mercaptoalkyl, alkyl-thio-alkyl, aryl, the aryl replaced, aralkyl, the aralkyl replaced, heteroaryl, the heteroaryl replaced, carboxyl, carboxyalkyl, alkyl carboxylates, chain acid alkyl ester, guanidine alkylation, or the carbonyl to be replaced by aminoacid and derivant thereof and peptide or acylamino-or amidoalkyl.
Preferably, in compound of the present invention, described can releasing-joint comprise disulphide, carbonic ester, hydrazides, amide, hydrazone, amino-acid ester, urea or its combination.
Further preferred, of the present inventionly can comprise one or more formula with following structure by releasing-joint:
, , , , , , , or .
Wherein, n is 0,1,2,3 or 4; R is H, the acyl group of alkyl, optional replacement or nitrogen-protecting group; X is O, CH 2or NH; Y is O or S, Z is NH, O or S; R 1for alkyl, carboxy substituted alkyl or acyl substituted alkyl; * open valency is represented.
Further, releasing-joint and heteroatom linker or spacer nipple the joint L with following structure can be formed by described 2:
, , , , or
Wherein, m is the integer of 0 ~ 4, and W is selected from NH or O; * open valency is represented.
Authorization Notice No. is that the patent of CN100381177C describes in detail can releasing-joint and spacer nipple, and the disclosure of described document is attached in the present invention by reference.
Described polyvalent linkers L 1with comprise can the L of releasing-joint 2optionally through one or more spacer nipple, heteroatom linker, joint L can be connected to form by releasing-joint.
Exemplary by L 1with L 2directly covalently bound or by one or more spacer nipple, heteroatom linker, following structure can be comprised by the joint L that is connected to form of releasing-joint:
, , or or
；
Wherein, W is selected from NH or O; * open valency is represented.
In folate receptor binding ligand-drug conjugates provided by the present invention, comprise folate receptor binding ligand (F), joint (L) and medicine (D), the any mode can mentioned according to the present invention forms joint L and this area maybe can be used to recognize known spacer nipple, can form joint L by covalently bound between releasing-joint and heteroatom linker, the connection of folate receptor binding ligand F or medicine D and heteroatom linker by be present in change into heteroatom linker medicine D or folate receptor binding ligand F on reactive functional groups make.Wherein, the reactive functional groups on described folate receptor binding ligand F is carboxyl or amino, and polyvalent linkers can attach to carboxyl on described folate receptor binding ligand F or aminoly form ester or amide; When wherein said medicine comprises double bond nitrogen-atoms, can attach on described medicine nitrogen and form hydrazone by releasing-joint; When described medicine can comprise sulphur atom, describedly can be selected from alkylenethio or carbonylalkylthio by releasing-joint, and can releasing-joint be connected to described medicine sulfur on form disulfide bond; Described medicine can comprise oxygen atom, described can releasing-joint for being substituted with a substituent or unsubstituted alkylene oxide group carbonyl or haloalkylenecarbonyl, described can releasing-joint be connected to medicine oxygen on form carbonic ester or ester; Described medicine D can comprise nitrogen-atoms, can the releasing-joint haloalkylenecarbonyl that is haloalkylenecarbonyl or obtains, described can releasing-joint be connected to medicine D nitrogen on form amide.
Spacer nipple and can releasing-joint, and heteroatom linker can combine in a different manner.Exemplary explanation, described joint is interconnected by heteroatom linker, such as alkylene-amino-alkylenecarbonyl, and wherein, x, y are respectively the integer of 1 ~ 5.
or
Exemplary is illustrative, in another embodiment, describes the compound of following formula:
、
、
With
；
Wherein, W is NH or O; M is 0 or 1; F 1, F 2independently be selected from following formula:
with .
The D of medicine described in the present invention can be any molecule that can regulate or change cell function in another manner, comprises pharmaceutical active compounds.Described pharmaceutical active compounds can be known in the art medicine or its derivatization form, and described medicine is cytotoxicity, improves the medicine of anti-apoptotic activities in tumor permeability, inhibition tumor cell propagation, promotion apoptosis, reduction bag cell.Be suitable for medicine of the present invention and include but not limited to hormone, antibiotic, antimicrobial drug, antiviral agents, anticarcinogen.Itself be Cytotoxic or can be used to increase the chemotherapeutic of tumor permeability, also use in the methods of the invention.Described Cytotoxic medicine such as comprises 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 (as dolastatin-10), ALLRED inhibin (auristatin) class (as monomethyl ALLRED inhibin E(MMAE), monomethyl ALLRED inhibin F(MMAF)), pipe dissolves plain class (Tubulysins), combretastatin, maytansine, maytansine analog, DM1, Epothilones, paclitaxel and paclitaxel derivant (as docetaxel), vinblastine and analog thereof are (as vincristine, de-acetyl vinblastine one hydrazides (DAVLBH)), camptothecine and camptothecin derivative, colchicine, different colchicine, muscoril, Demecolcine, daunomycin, rhizomycin, cyclophosphamide, methotrexate, bleomycin, CCI-779 (Temsirolimus), mitomycin, microtubule inhibitors, Pyrrolobenzodiazepines Zhuo (PBD) dimer class, cyclopropyl benzo [e] indolone, open loop-cyclopropyl benzo [e] indolone, Calicheamicin (Calicheamicin).Other are applicable to medicine of the present invention and comprise Macrolide antineoplastic agent, chemotherapeutic is as alkylating agent, chlormethine, nitroso ureas, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, antimetabolite is as cytosine arabinoside, purine analogue, pyrimidine analogue and penicillin, cephalosporin, vancomycin, erythromycin, clindamycin, rifampicin, chloromycetin, aminoglycoside antibiotics and acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, the Antimicrobe compound of ribavirin gemcitabine and other this area any accreditation.
Further, the preferred CCI-779 of medicine of the present invention (Temsirolimus), open loop-cyclopropyl benzo [e] indolone analog, Pyrrolobenzodiazepines Zhuo (PBD) dimer class, Calicheamicin (Calicheamicin), SN38 (SN-38), vinca, dolastatin (Dolastatin), ALLRED inhibin (auristatin) class, didemnun B (Didemnin B), pipe dissolves plain B(Tubulysin B), CHROMATOGRAPHIC FRACTIONATION AND MASS, taxanes, daunorubicin, doxorubicin, epirubicin, Ai Sibo mycin or D actinomycin D.
Further, the preferred open loop of medicine of the present invention-cyclopropyl benzo [e] indole ketone compound, PBD dimer class, Calicheamicin, SN-38, DAVLBH, Tubulysin B, Didemnin B, MMAE, MMAF, MMAF derivant, DM1, taxanes, vincristine, daunorubicin, doxorubicin or epirubicin.
Publication number is that the patent of WO2009/064913A1 and US2010113476A1 is described in detail CBI compounds and open loop-cyclopropyl benzo [e] indole ketone compound, part disclosed in it, be attached to by reference in the present invention, exemplary open loop-cyclopropyl benzo [e] indole ketone compound is shown below:
Wherein R 1for H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or ; R 2for NH 2or OMe.
In described Pyrrolobenzodiazepines Zhuo (PBD) dimer compounds, PBD refers to the structure had as lower unit:
The difference of PBD dimer compounds be the substituent number of aromatic ring A and pyrrole ring C, type and position and pyrrole ring C saturation different, exemplary PBD dimer compounds as:
or
Wherein, R 1for hydroxyl, amino, C 1~ C 6alkoxyl, C 1~ C 6alkyl, C 1~ C 6amino replace alkyl, C 1~ C 3aldehyde radical or carbonyl or C 1~ C 6cycloalkyl or heterocyclic radical; R 2being that 2-, 3-or 4-replace, is-N=NH-or-NH-; N is the integer of 0 ~ 3.
Publication number is that the patent of CN201180029867 has been described in detail this compounds, and discloses its preparation method, and disclosed content is attached in the present invention by reference wherein.
Described SN-38 is the structure with following formula:
；
Described DAVLBH is the structure with following formula:
Tubulysin B and MMAE concrete structure as follows:
、
MMAF and derivant thereof refer to the compound with following structure:
、
Wherein, R yfor C 1~ C 6alkyl or haloalkyl or the carbonyl containing 1 ~ 4 carbon atom that replaces arbitrarily; Work as R yduring for hydrogen, be MMAF.
Described Didemnin B is the structure with following formula:
Wherein, R xfor p-methoxyphenyl.
Described DM1 is the structure with following formula:
In described folate receptor binding ligand conjugate, medicine D with can releasing-joint joint L 2connection, reactive functional groups by there is medicine D or its derivatization form is made, such as the conversion of hydroxyl of Didemnin B becomes corresponding carbonic ester, another illustrative embodiment is, the terminal carboxyl group of Tubulysin B is first derivative turns to hydrazides, then the nitrogen-atoms on hydrazides as heteroatom linker again with can be connected by releasing-joint on L, folic acid changes into corresponding amide etc., as shown in the formula illustrated:
、 、
Following folate receptor binding ligand-drug conjugates is illustrative drug conjugates provided by the invention, thinks that they are in scope of the present invention, and these folate-drug conjugates can according to the scheme of this area accreditation or the method for the invention preparation.
In another embodiment, the invention describes the compound of following structure:
、
、 、
、
、
、
、
、
with
Wherein, R xfor p-methoxyphenyl; X 1for Cl or Br; R be H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or ; R yfor hydrogen, C 1~ C 6alkyl or haloalkyl or the carbonyl containing 1 ~ 4 carbon atom that replaces arbitrarily; R 1for C 1~ C 6alkoxyl or alkyl or the amino alkyl that replaces or C 1~ C 3aldehyde radical or carbonyl, hydroxyl, amino or C 1~ C 6cycloalkyl or heterocyclic radical; R 2being 2-, 3-or 4-position replacement of benzene, is N=NH or NH; N is the integer of 0 ~ 3.
Wherein ONPhth represents following structure:
Further, the invention provides the compound of following structure:
Wherein, L 4be selected from following structure:
, or ;
F 1, F 2independently be selected from following formula:
with ;
* link position is represented.
Further, the invention provides as shown in the formula compound:
、
、
、
、
、
、
、
、
、
, or
。
On the other hand, the invention provides the compound with following general formula: F 3lD and F 4lD,
Connected mode wherein between F, L, D is similar to compound F 17-hydroxy-corticosterone 2connected mode in LD between F, L, D.
Exemplary F 3lD and F 4lD compound is as follows:
With
Wherein, D is selected from previously described medicine or derivatives thereof or analog; F 1, F 2, F 3, F 4independently be selected from:
with ;
L 2be joint, comprising at least one can releasing-joint, and optional comprises one or more heteroatom linker, spacer nipple.
Exemplary, L 2there is following structure:
, , , , or
Wherein, m is the integer of 0 ~ 4, and W is selected from NH or O; * open valency is represented.
Further, following particular compound is in order to illustrate F 3lD and F 4lD:
、
、
With
The synthetic method preparation that folate receptor binding ligand-drug conjugates of the present invention is approved by this area.The selection of heteroatom linker is depended in the selection of described synthetic method, the construction features of medicine, and described spacer nipple and described can functional group on releasing-joint.Generally speaking, relative keys forming reactions is described in Richard C. Larock. Comprehensive Organic Transformations, a guide to functional group preparations. VCH Publishers, Inc. New York (1989), with the Theodora E. Greene & Peter G.M. Wuts. Protective Groups ion Organic Synthesis. second edition, John Wiley & Sons, Inc. New York (1991), the open part of described document is attached in the present invention by reference.
Wherein said heteroatom linker is sulphur atom, being present in described can the functional group of releasing-joint be alkylidene thiol derivative, and described disulphide group is formed as pyridine-2-base disulfide group alkyl derivative etc. reacts with alkylidene thiol derivative by corresponding heteroaryl disulfide group derivant.Its reaction dissolvent can select oxolane (THF), DMF (DMF), dichloromethane, dimethyl sulfoxide (DMSO) etc., and reaction temperature changes between 0 DEG C ~ 80 DEG C.
The formation of conventional carbonic ester, sulfocarbonate and carbamate, usually compound, the compound of sulfenyl replacement or the compound of amine replacement by making hydroxyl replace react with activation alkoxycarbonyl derivative respectively, form carbonic ester, sulfocarbonate and carbamate.Reaction can be carried out in Conventional solvents is as THF, DMF, DMSO, dichloromethane, ethyl acetate etc., and reaction temperature, at 0 ~ 80 DEG C of range changing, can use any base catalyst such as inorganic base, amine alkali, polymer combined alkali etc. to promote this reaction.
The formation of conventional amide and ester; such as; wherein said heteroatom linker is nitrogen-atoms; be present in spacer nipple or can the functional end-group on releasing-joint be carbonyl; described amide groups obtains by corresponding carboxyl or derivatives thereof esterification or acylation reaction; become amide reaction dissolvent to comprise dichloromethane, THF, DMF, DMSO etc., illustrative described amide can carry out reaction preparation in-15 ~ 80 DEG C of mobility scales.Coupling agent comprises DCC, EDC, HBTU, TBTU, HOBT/DCC, HOBT/ EDC etc., or parent acid can be converted to the carbonyl derivative of activation, such as acyl chlorides, N-hydroxy-succinamide base ester etc., becoming amide to react also can in the basic conditions as carried out in triethylamine, N, N'-diisopropylethylamine etc.
Same, described heteroatom linker is oxygen atom, there is spacer nipple or can the end group on releasing-joint be carbonyl, and required ester group is prepared by corresponding carboxylic acid or derivatives thereof and alcohol coupling reaction.Described coupling agent comprises DCC, EDC, CDI, BOP, EEDQ, DEAD, triphenylphosphine etc., and solvent comprises dichloromethane, THF, DMF, DMSO, acetonitrile, ethyl acetate etc., and alkali comprises triethylamine, diisopropylethylamine etc.Or parent acid can be converted to the carbonyl derivative of activation, such as acyl chlorides, N-hydroxy-succinamide base ester etc.
Described medicine comprises nitrogen-atoms, can releasing-joint or spacer nipple can attach to described medicine D nitrogen-atoms on form hydrazone, required hydrazone group is formed by corresponding aldehydes or ketones and hydrazine or hydrazide derivatives reaction.Useable solvents comprises dichloromethane, THF, DMF, DMSO, chloroform, ethyl acetate etc., and reaction temperature is at 0 ~ 80 DEG C of range changing, and available any acidic catalyst is if mineral acid, glacial acetic acid, trifluoroacetic acid etc. are as catalyst.Acylhydrazone, by suitable carboxylic acid or derivatives thereof acidylate hydrazine, then makes hydrazides and corresponding aldehydes or ketones react and forms acylhydrazone.Or described hydrazone functional group is formed by making hydrazine and corresponding aldehydes or ketones react.Gained hydrazone is subsequently again by suitable carboxylic acid or derivatives thereof acidylate.
The formation of conventional butanimide: described heteroatom linker is nitrogen-atoms, oxygen atom or sulphur atom, exist with spacer nipple or can the functional group on releasing-joint be succinimide derivatives, the carbon-hetero atom of gained be formed by the Michael addition of corresponding amine, alcohol or mercaptan and maleic imide derivative.The solvent forming Michael addition comprises THF, ethyl acetate (EtOAc), CH 2cl 2, DMF, DMSO, H 2o etc.The available alkali adding equimolar amounts of the formation of this kind of Michael addition compound has come as triethylamine or by pH to 6.0 ~ 7.4 of adjustment aqueous solution.Apparent, when described heteroatom linker be oxygen or nitrogen-atoms time, scalable reaction condition easily changes Michael addition, as by adopting higher reaction temperatures, adding catalyst, using the stronger solvent of polarity as DMF, DMSO etc., and activates maleimide with silylating agent.
Symmetrical acetal or ketal group can be formed by acetal and ketal reaction by corresponding alcohols and aldehydes or ketone.Generally follow R. R. Schmidt etc., Chem. Rev., 2000,100, the 4423-42 steps summarized, the disclosure of described document is attached in the present invention by reference.
The preparation of conventional folic acid-peptide: by the sequential grammar of polymer-carrier, with Fmoc-strategy, on the 2Cl-Trt Resin resin of acid labile, preparation is containing peptidyl fragments Pte-γ Glu-(AA) of 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) N 10-TFA-Pteroic acid; PyBop, DIPEA, DMF/DMSO; (e) 2%NH 2nH 2/ DMF; (f) TFA/H 2o/ phenol/THIOANISOLE/EDT (82.5:5:5:5:2.5);
In the illustrated embodiment of the method for the invention, R 1for Fmoc, R 2trityl group, DIC is DIC, and DIPEA is N, N'-diisopropylethylamine, for guaranteeing effective coupling, makes activator with PyBop.At the standard conditions, after each coupling step, Fmoc protecting group is removed.Described in scheme 1, use aminoacid component such as Fmoc-Glu-OtBu, N of suitably protection 10-TFA-Pteroic acid etc., and with Fmoc-AA-OH in step (b) for representative.Therefore, AA refers to the amino acid starting material of any suitable protection.Should be understood that, term used herein " aminoacid " refers to any amine or the carboxylic acid functional with the separation of one or more carbon, and comprises naturally occurring α and beta amino acids, and these amino acid whose derivant and analog.Particularly, protected aminoacid such as protected serine, threonine, cysteine, the aspartic acid etc. with side chain also can be used for the synthesis of folic acid-peptide of the present invention.In addition, the homologous side chains of γ, δ or longer or the amino acid analogue of alternative branched structure, such as nor-leucine, 2-amino-3-methylpentanoic acid, Beta-methyl threonine, Beta-methyl cysteine, β, Beta-Dimethylcysteine etc. also can be used as raw material and are included in the synthesis of folic acid-peptide of the present invention.
The coupling sequence (step (a) and (b)) relating to Fmoc-AA-OH carries out n time, and support peptide (II) to prepare solid, wherein n is integer and can be 0 ~ 100.After last coupling step, remove remaining Fmoc base (step (a)), described peptide is coupled to glutamate derivatives (step (c)) subsequently, deprotection, and is coupled to the pteroic acid (step (d)) of TFA-protection.Described TFA protecting group by removing with alkali treatment (step (e)), then obtains containing peptidyl fragments (III) through step (f).
Then, described peptide is by using trifluoroacetic acid (TFA), H 2o, phenol, THIOANISOLE and EDT process, cut (step (f)) from polymer support.These reaction conditions cause removing t-Bu, t-Boc, Pbf and Trt protecting group simultaneously, and described protecting group can form the part of the amino acid side chain of suitably protection.
（III）
Fragment containing three or four folic acid peptides can be prepared according to similar method, polyvalent linkers wherein builds by the aminoacid containing more than 3 or 3 reactive functional groups (as amino, hydroxyl, carboxyl), and described aminoacid can be such as lysine, glutamic acid, serine, agedoite, aspartic acid, tyrosine, arginine glutamate etc.
The exemplary fragment 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 containing four reactive functional groups, again compound c is added reaction column, 1,2,3-indantrione monohydrate is monitored, condensation Fmoc-Asp(OtBu successively)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N 10-TFA-pteroic acid, add 10ml 2% hydrazine hydrate/DMF solution reaction 5 minutes after (wherein, the consumption of aminoacid and HOBT/DIC is respectively 15eq and 16.5eq/16.5eq) condensation, repeat 3 times, DMF washs, DCM washs, and MeOH washs, and drains, synthesize complete, add lytic reagent, TBME precipitated polypeptide, obtains III-2.
The exemplary fragment 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 carried out condensation, obtain compound f, compound f is being added reaction column, 1,2,3-indantrione monohydrate is monitored, condensation Fmoc-Asp(OtBu successively)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N 10-TFA-pteroic acid, add 10 ml 2% hydrazine hydrates/DMF solution reaction 5 minutes after (wherein, the consumption of aminoacid and HOBT/DIC is respectively 15eq and 16.5eq/16.5eq) condensation, repeat 3 times, DMF washs, DCM washs, and MeOH washs, and drains, synthesize complete, add lytic reagent, TBME precipitated polypeptide, obtain target compound III-3.
Any one lysine wherein in c and f also can with the amino acid replacement of the aminoacid of other similar or replacement, in order to prepare the similar fragment containing four or five reactive functional groups, and the condensation of different aminoacids order, connected mode and amino acid whose spatial configuration are not limited to the present invention.
N 10-TFA-pteroic acid refers to the compound with following structure:
，
Its preparation method is specifically " Efficient Syntheses of Pyrofolic Acid and Pteroyl Azide; Reagents for the Production of Carboxyl-Differentiated Derivatives of Folic Acid " (J. Am. Chem. Soc. see exercise question, Vol. 119, No. 42,1997, document 10004-10013), the document discloses and prepare N by folic acid 10the concrete grammar of-TFA-pteroic acid, its disclosed part is attached in the present invention by reference.
In another scheme, the invention provides a kind of pharmaceutical composition, it comprises folate receptor binding ligand-drug conjugates described here and pharmaceutically acceptable carrier, diluent or excipient or its and combines.
In another scheme, the invention provides a kind of described pharmaceutical composition in the purposes treating and/or preventing the disease caused by population of pathogenic cells.Described population of pathogenic cells refer to cancerous cell, infectious substance as antibacterial and virus, the cell be infected by bacteria or virus, the macrophage of the activation of disease can be caused, and any uniqueness, preferentially to express or the other types pathogenic cell of overexpression folacin receptor.Described population of pathogenic cells can be the cancer cell population of tumorigenesis (comprising benign tumor and malignant tumor), or can be not tumorigenesis.Described cancer cell population includes but not limited to, oral cancer, thyroid carcinoma, endocrine adenocarcinoma, skin carcinoma, gastric cancer, esophageal carcinoma, laryngeal carcinoma, cancer of pancreas, colon cancer, bladder cancer, osteocarcinoma, ovarian cancer, uterus carcinoma, breast carcinoma, carcinoma of testis, carcinoma of prostate, rectal cancer, renal carcinoma, hepatocarcinoma and pulmonary carcinoma.
According to the present invention, described folate receptor binding ligand-drug conjugates can be used for treatment there is the disease that population of pathogenic cells is feature in host, wherein the member of population of pathogenic cells has folic acid or pteroic acid binding site, and wherein said binding site is by the expression of described pathogenic cell uniqueness, overexpression or preferentially express.The selectivity of described pathogenic cell is eliminated, and is connected to folacin receptor by the vitamin moieties of described folate receptor binding ligand-drug conjugates.High-affinity folacin receptor surface-expressed vitamin receptor overexpression on tumor cell.Ovary, mammary gland, colon, lung, nose, pharynx and brain epithelial cancer are all in the news and express high-level folacin receptor.In fact, the known human ovarian tumor more than 90% is with this receptor of high level expression.Therefore, drug delivery conjugates of the present invention can be used for treating various tumor cell type, and other pathogenic cell type is as infectant, described cell type preferentially expresses folacin receptor, and therefore have surface can and vitamin or vitamin D 3-analogies or derivant binding site.
The term " folate or folic acid, pteroic acid salt or pteroic acid " used in the present invention independently refers to the folic acid for the formation of conjugate, or pteroic acid part.
Abbreviation:
" Boc " represents tertbutyloxycarbonyl;
" CDI " represents N, N-carbonyl dimidazoles;
" DDQ " represents 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone;
" DCC " represents N, N'-dicyclohexylcarbodiimide;
" DEAD " represents diethyl azodiformate;
" DIPEA " and " DIEA " all represents N, N'-diisopropylethylamine;
" DIC " represents N, N'-DIC;
" EDCI " represents 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide;
" EDC " represents (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride);
" EEDQ " represents 2-ethyoxyl-1-ethoxy carbonic acyl radical-1,2-dihydroquinoline;
" Fmoc " represents fluorenylmethyloxycarbonyl;
" HBTU " represents (2-(1H-benzotriazole-1-base)-1,1,3,3-tetramethyl Tetrafluoroboric acid urea;
" HATU " represents N-[[(dimethylamino)-1H-1,2,3-triazol [4,5-b] pyridine-1-base] methylene]-N-methyl hexafluorophosphoric acid first ammonium N-oxide;
" HOAt " represents 7-azepine-I-hydroxybenzotriazole;
" HOBT " represents I-hydroxybenzotriazole;
" HOSU " represents N-hydroxy-succinamide;
" ivDde " represents 1-(4,4-dimethyl-2,6-dioxo cyclohexylene)-3-methyl butyl;
" NMP " represents N-Methyl pyrrolidone;
" OtBu " represents tert-butoxy;
" PABA " represents para-amino benzoic acid;
" TBTU " represents 2-(1H-benzothiazolyl)-1,1,3,3-tetramethylurea Tetrafluoroboric acid;
" TBME " represents t-butyl methyl ether.
Folate receptor binding ligand provided by the invention-drug conjugates Folic Acid receptor-binding ligands is 2 or more, preferably 2 ~ 4, and described each part is covalently bound with polyvalent linkers respectively, medicine D with can releasing-joint covalently bound, compared with the part only containing a receptors bind in prior art, conjugate provided by the invention and folacin receptor cellular affinity improve greatly, and polyvalent linkers L of the present invention comprises can releasing-joint, when the folic acid of conjugate and/or pteroic acid part are attached on pathogenic cell, conjugate is combined with pathogenic cell intimate surface, endocytosis is in cell subsequently, can the rapid cracking of releasing-joint in cell, medicine D is discharged, play the drug action of medicine, unexpectedly, conjugate provided by the invention shows significant anti-tumor activity in the tumor cell of folacin receptor process LAN, and based on the weight of animals change draw found that, conjugate provided by the present invention is while significantly improving anti-tumor activity, under institute's drug dosage, animal all can well tolerate.Further, compared with existing folate receptor binding ligand-drug conjugates (as EC145), under equivalent molar dosage, conjugate provided by the invention and folacin receptor larger in conjunction with probability; More unexpectedly, even under excessive folic acid existence condition, during equivalent molar dosage, compared with existing folate receptor binding ligand-drug conjugates (as EC145), conjugate exhibits provided by the invention goes out more stable anti-tumor activity, and this shows, even if under the race condition that there is a certain amount of folic acid, conjugate provided by the invention also can play good anti-tumor activity, and these advantages of the present invention can be proven further by specific embodiment below.
Compared with folate receptor binding ligand-drug conjugates of the prior art, Conjugate Molecules amount provided by the invention increases significantly, be conducive to conjugate and be optionally gathered in tumor cell surface by passive target mechanism, conjugate provided by the present invention contains 2 or multiple folate receptor binding ligand simultaneously, further enhancing affinity and the holdup time of conjugate in tumor cell of the tumor cell that conjugate and folate receptor-positive are expressed, further, with medicine covalently bound can releasing-joint as other in hydrazides key and joint L can releasing-joint if disulfide bond is under cellular acid condition, in a few minutes, hydrazides key just disconnects, disulfide bond is reduced simultaneously, release medicine, the anti-tumor activity of rapid performance medicine.In addition, inventor also finds, conjugate provided by the present invention and plasma albumin combination rate low, this also imply that conjugate provided by the present invention, and clearance rate is fast in vivo, and toxicity is little.
Further illustrate beneficial effect of the present invention below in conjunction with the drawings and specific embodiments, following examples are only illustrative, do not limit the invention.
Accompanying drawing explanation
Fig. 1 shows the mass spectrum of the compound 15 prepared by embodiment 4, and wherein 2969.351 is the molecular ion peak of compound 15.
Fig. 2 shows 1 μM of BP111b(compound of the present invention 15) and reference substance BP111a(compd E C145) there is and not exist the inhibitory action to KB cell under excessive folic acid condition, wherein "-" expression does not add, and "+" represents and add.
Fig. 3 shows BP111b(compound 15 of the present invention) and reference substance BP111a(compd E C145) inhibitory action to the A549 Growth of Cells of folacin receptor feminine gender, abscissa represents the concentration adding BP111a and BP111b.
Fig. 4 display give 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg BP111b(compounds 15 respectively) and reference substance BP111a to the inhibitory action of mice KB tumor growth, Vehicle represents blank, that is: Drug therapy is not given, only give equivalent solvent, vertical coordinate TV(mm3) represent gross tumor volume (Tumor Volume), abscissa: sky (Day).
Fig. 5 display give 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg BP111b(compounds 15 respectively) and reference substance BP111a KB mice with tumor body weight is affected, Vehicle represents blank, that is: Drug therapy is not given, only give equivalent solvent, vertical coordinate BW(g) represent body weight (Body Weight), abscissa: sky (Day).
Specific embodiment
Below by way of specific embodiment, technical scheme of the present invention and advantage are further illustrated, think that they are in scope of the present invention, not as limitation of the present invention.Described method is illustrative method, can be used for preparing drug conjugate of the present invention.
embodiment 1the preparation of compound III
（III）
According to conventional steps of the present invention, according to the scheme of scheme 1, to in 50ml reaction bulb, add 1000mg 2Cl-Trt Resin(1.5eq), add 234mg Fmoc-Cys(Trt again)-OH(1.0eq), 8ml dichloromethane (DCM), 640 μ L DIEA(2.0eq), dissolve completely, 8ml DCM is added after reaction 50min, 1ml MeOH, after 1ml DIEA reacts 20min, be transferred in self-control solid phase synthesis post, the Piperidine/DMF solution of 10ml 20% is added after DMF washing, reaction 20min, after DMF cleaning, 1,2,3-indantrione monohydrate monitoring is positive, get 709mg Fmoc-Lys(Fmoc)-OH (3.0eqiv, 178mg HOBT (3.3eqiv), 6ml DMF adds reaction column after adding 230 μ L DIC (3.3eq) mixing after dissolving, 1,2,3-indantrione monohydrate monitoring after reaction 1H, the Piperidine/DMF solution of 20% is added after DMF washing.Repeat above step, successively condensation Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N 10-TFA-pteroic acid (wherein, the consumption of aminoacid and HOBT/DIC is respectively 6eq and 6.6eq/6.6eq), adds 10ml 2% hydrazine hydrate/DMF solution reaction 5 minutes after condensation, repeat 3 times, DMF washs, and DCM washs, MeOH washs, and drains, synthesizes complete, add lytic reagent, TBME precipitated polypeptide, obtains crude product III, after the separation and purification of HPLC preparative hplc, obtain sterling III, MS [M] +: 2097.74.
embodiment 2the preparation of compound 33
10.0ml DCM is added in 100ml there-necked flask, 1.0ml MeOC (O) SCl(1.0eq) ice bath is cooled to 0 DEG C, drip 0.76ml mercaptoethanol (1.0eq), be incubated 0 DEG C of reaction 30min, add 1.22g 2-mercaptopyridine (1.0eq), 16ml DCM drips into reaction bulb, after rising to room temperature 1h after being incubated 0 DEG C of reaction 1h, TLC raw material reaction is complete, post processing: be concentrated into about 16ml, sucking filtration, filtrate obtains faint yellow micro-stink solid 2.0g with after DCM washing.
3.0ml DCM, 0.46g surpalite (0.55eq) is added in 100ml there-necked flask, ice bath is cooled to 0 DEG C, by 1.0g compound 33-2(1.1eq) be dissolved in 13ml DCM, add 0.45g triethylamine (1.0eq) more wherein, be added drop-wise to after dissolving in reaction bulb, temperature control less than 0 DEG C during dropping, TLC raw material reaction complete (obtaining 33) yield 96%, MS [M] after rising to room temperature 1h +: 248.97.
Other details of the method are shown in US Patent No. 2007276018 and I. R. Vlahov, et al. described in Bioorg. Med. Chem. Lett. 16 (2006): 5093 – 5096, these two sections of documents by reference entirety are attached in the present invention.
embodiment 3the preparation of compound 15-3
1.5g vinblastine, 5 ml absolute methanols and 5ml anhydrous hydrazine is added in 100ml there-necked flask, be warming up to about 60 DEG C, TLC detection reaction process after reaction 24h, after reaction terminates, pour in 50ml water, DCM extracts 3 times, wash 3 times, saturated common salt washes 3 times, obtains white solid 1.1g, i.e. compound 15-2 after anhydrous sodium sulfate drying is concentrated.
In 100ml there-necked flask, add compound 15-2 0.98g(1.0eq), compound 33 0.67g(1.5eq), 10g DCM, in reaction bulb, drip 0.44ml triethylamine (2.5eq) after stirring and dissolving, react complete after room temperature 2h.
Post processing: add 50ml DCM, wash 3 times, saturated common salt washes 3 times, concentrates and to obtain crude product 1.2g, obtain the sterling 750mg of 15-3 after column chromatography after anhydrous sodium sulfate drying.MS[M] +：959.43。
embodiment 4the preparation of compound 15
In 100ml there-necked flask, add 7.5ml water, after nitrogen bubble 30min, add 200mg compound III (1.2eq), with the NaHCO of 0.1N 3aqueous solution regulates pH to be about 6.9; Be dissolved in 15ml MeOH by 78mg compound 15-3, and joined in reaction bulb, react complete after reacting about 10min, after HPLC preparation, lyophilizing obtains the sterling 39mg of compound 15.HPLC:98.1%, the mass spectrum adopting Matrix Assisted Laser Desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) to record compound 15 is shown in accompanying drawing 1, MS [M+H] +: 2969.35.
embodiment 5the preparation of compound 16
The preparation method of similar compound 15 prepares compound 16.
MS [M+H] +：3043.18。
embodiment 6the preparation of compound 17
The preparation method of similar compound 15 prepares compound 17.
MS[M+H] +：2819.98。
embodiment 7preparation (the R of compound 18 xfor p-methoxyphenyl)
By compound 33 and DMAP(4-dimethyl aminopyridine) join in the dichloromethane solution of Didemnin B and react 2h at 0 DEG C, obtain compound 18-1 after separation and purification; Compound III is soluble in water, under condition of nitrogen gas, be 7 with manganese hydrogen sodium regulating solution pH, compound 18-1 is dissolved in acetonitrile, and under a nitrogen atmosphere, join in the aqueous solution of compound III, utilize inverse analysis HPLC detection reaction process, after reaction terminates, separation and purification, obtain target product 18, MS [M+H] +: 3312.36.
embodiment 8the preparation of compound 19
Under 0 DEG C of condition, be in the compound 19-1(7.7mg in 5% ethanol/methylene, 10 μm of ol) mixture in add the trifluoroacetate (6.4mg, 20 μm of ol) of compound 19-2.Reactant mixture is warming up to ambient temperature and stirs 5h, then concentrating under reduced pressure, silica gel column chromatography (eluant: 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 carried out Michael addition reaction, prepares compound 19, MS [M+H] +: 3058.16.
embodiment 9the preparation of compound 20
The preparation of compound 20 can be prepared through 8 steps reactions by compound 20-1 according to above-mentioned route; Wherein, react through 6 steps the concrete operation method preparing compound 20-9 by compound 20-1 and be described in detail in EP0624377A2, the open part of the document is attached in the present invention by reference.
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) dissolve.After 2min, add pyridine (0.5ml), monitor reaction process with reversed-phase HPLC.After reacting about 24h, reaction terminates, and is concentrated by reactant liquor, and residue, with reverse phase preparative HPLC (Varian Dynamax chromatographic column 21.4mm × 25cm, 5 μ, 100 holes), uses acetonitrile and Et 3n-CO 2(pH 7) carries out gradient elution from 10% to 100%, and flow velocity is 20ml/min, and elution time is 40min.Fractional Collections, concentrated, obtain off-white color solid, be compound 20-10, MS [M] +: 1315.78.
The sulfydryl of compound 20-10 and compound III is carried out Michael addition reaction, obtains compound 20.MS [M+H] +:3414.52。
embodiment 10the preparation of compound 21
Method with reference to embodiment 9 prepares compound 21.MS [M+H] +:3428.50。
embodiment 11the preparation of compound 22
The synthetic method list of references of the valine-citrulline dipeptides (compound 22-1) that maleimide used in the present invention is amine-modified: Gene M.D., et al.Cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconj μ gates:Model Studies of Enzymatic Dr μ g Release and Antigen-Specific In Vitro Anticancer Activity. Bioconjugate Chem.13 (4) 855-869 (2002).
The method that the reaction of compound 22-1 and CBI amino conveniently and carboxyl reaction prepare amide operates, and obtains compound 22-2, the preparation method of similar compound 19, is reacted by the sulfydryl of compound 22-2 and compound III, obtain compound 22.MS [M+H] +：3065.16。
embodiment 12the preparation of compound 23
(Boc) is used according to this area conventional method under pyridine condition 2the hydroxyl of SN-38 10 is carried out protection and obtains compound 23-2 by O; by compound 23-2(0.358g; 0.073mmol), DMAP (0.266,0.218mmol) and triphosgene (0.0095g; 0.032mmol) join in reaction bulb; then add the initiation reaction of 1.5ml dichloromethane, TLC detection reaction process, reaction terminates rear absolute methanol cancellation; obtain compound 23-3, not treated be directly used in next step reaction.
By compound 20-8(0.768g; 0.883mmol) join in the reactant liquor of above-mentioned 23-3; react about 5min and react end; with flash column purified (ethanol/methylene gradient elution); obtain 20 compound 23-4 protected by Boc; and then slough protecting group with TFA hydrolysis, the tfa salt of target compound 23-4 is obtained with Diethyl ether recrystallization.
According to the scheme of scheme 6 ,-the SH of the cysteine of compound 23-4 and compound III reacts and prepares target compound 23.MS [M+H] +：3089.15。
embodiment 13the preparation of compound 24
The preparation method of similar compound 15 prepares compound 24.MS [M+H] +：2946.23。
embodiment 14the preparation of compound 25
The preparation method of similar compound 15 prepares compound 25.
MS[M+H] +：2932.20。
embodiment 15the preparation of compound 26
The preparation method preparation of similar compound 15.
MS[M+H] +：2918.22。
embodiment 16the preparation of compound 27
According to conventional steps of the present invention, according to the scheme of scheme 1, to in 50ml reaction bulb, add 1000mg 2Cl-Trt Resin(1.5eq), add 234mg Fmoc-Cys(Trt again)-OH(1.0eq), 8ml dichloromethane (DCM), 640 μ L DIEA(2.0eq), dissolve completely, 8ml DCM is added after reaction 50min, 1ml MeOH, after 1ml DIEA reacts 20min, be transferred in self-control solid phase synthesis post, the Piperidine/DMF solution of 10ml 20% is added after DMF washing, reaction 20min, after DMF cleaning, 1,2,3-indantrione monohydrate monitoring is positive, get 709mg Fmoc-Lys(Fmoc)-OH (3.0eqiv, 178mg HOBT (3.3eqiv), 6ml DMF adds reaction column after adding 230 μ L DIC (3.3eq) mixing after dissolving, 1,2,3-indantrione monohydrate monitoring after reaction 1H, the Piperidine/DMF solution of 20% is added after DMF washing.Repeat above step, successively condensation Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, N 10-TFA-pteroic acid, (wherein, the consumption of aminoacid and HOBT/DIC is respectively 6eq and 6.6eq/6.6eq), add 10ml 2% hydrazine hydrate/DMF solution reaction 5 minutes after condensation, repeat 3 times, DMF washs, DCM washs, MeOH washs, and drains, synthesizes complete, add lytic reagent, TBME precipitated polypeptide, obtains III-1, MS [M] +: 1839.65.
The preparation method of compound 15 in similar embodiment 4, prepares compound 27, and difference is with III in III-1 alternate embodiment 4.MS [M+H] +：2711.05。
embodiment 17compound III-1 '
III-1’
Full guard peptide Asp(OtBu)-Asp(OtBu)-Arg(Pbf)-Asp(OtBu) synthesis of-pteroic acid: adopt 2Cl-Trt Resin; SPPS is condensation Fmoc-Asp(OtBu successively)-OH; Fmoc-Asp(OtBu)-OH; Fmoc-Arg (Pbf)-OH; Fmoc-Asp(OtBu)-OH, N 10-TFA-pteroic acid, adds 2% hydrazine hydrate/DMF solution reaction 5 minutes after condensation, repeat 3 times, and DMF washs, and DCM washs, and MeOH washs, and drains.Add the TFA/DCM solution full guard cutting 5min of 1%; repeat for several times; TLC is not till having product; in filtrate pyridine and after washing; concentrated after saturated common salt water washing; column chromatography to full guard peptide Asp(OtBu)-Asp(OtBu)-Arg(Pbf)-Asp(OtBu)-pteroic acid sterling, MS [M] +: 981.05.
The synthesis of III-1 ': in 50ml reaction bulb, add 1000mg 2Cl-Trt Resin(2.0eq), add 117mg Fmoc-Cys(Trt again)-OH(1.0eq), 8ml DCM, 320 μ L DIEA(2.0eq), dissolve completely, 8ml DCM is added after reaction 50min, 1ml MeOH, 1ml DIEA, be transferred in self-control solid phase synthesis post after reaction 20min, the Piperidine/DMF solution of 10ml 20% is added after DMF washing, reaction 20min, after DMF cleaning, 1,2,3-indantrione monohydrate monitoring is positive, get Fmoc-Lys(ivDde)-OH (3.0eq), 178mg HOBT (3.3eq), 6ml DMF adds reaction column after adding 230 μ L DIC (3.3eq) mixing after dissolving, 1,2,3-indantrione monohydrate monitoring after reaction 1h, the Piperidine/DMF solution of 20% is added after DMF washing.Repeat above step, successively condensation Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N 10-TFA-pteroic acid, (wherein, aminoacid and HOBT/DIC consumption are respectively 3eq and 3.3eq/3.3eq), add 10ml 2% hydrazine hydrate/DMF solution reaction 5 minutes after condensation, repeat 3 times, DMF washs, 1,2,3-indantrione monohydrate monitoring is positive, add compound Asp(OtBu)-Asp(OtBu)-Arg(Pbf)-Asp(OtBu)-pteroic acid}(2.0eq), HOAT (2.2eq), DIC (2.2eq), DMF makees solvent.React 1,2,3-indantrione monohydrate monitoring after spending the night, DMF washing after completion of the reaction, DCM washs, and MeOH washs, and drain, synthesize complete, add lytic reagent, TBME precipitated polypeptide, obtains III-1 '.MS [M] +：1968.69。
embodiment 18
The preparation method of compound 15 in similar embodiment 4, prepares compound 28, MS [M+H] +: 2840.10.
embodiment 19the preparation of compound III-2
Prepare III-2 according to the method shown in scheme 2, concrete steps are as follows:
The preparation of compound c: add 10g a in 250ml reaction bulb, the THF of the HOSU of 1.1eq, 100ml, after dissolving, ice bath is cooled to 0 DEG C, and the DCC adding 1.1eq rises to stirred overnight at room temperature.TLC raw material reaction is complete.Post processing: sucking filtration, filter cake THF washs, and obtains 12g compound b after organic facies is concentrated.
Add the d of 3.0g in 100ml reaction bulb, be dissolved in 30ml water, add the NaHCO of 1.1eq 3, be cooled to 10 degree; The b of 1.1eq is dissolved in 30ml DME, drips into reaction bulb, add 10mlTHF ambient temperature overnight.TLC reaction is complete, adds EA after concentrated, and dilute hydrochloric acid washs, and then washes, and Sal is washed, dry, and concentrated, column chromatography obtains compound c.
The preparation of III-2: in 50ml reaction bulb, add 1000mg 2Cl-Trt Resin(2.0eqiv), add 117mg Fmoc-Cys(Trt again)-OH(1.0eq), 8ml DCM, 320 μ L DIEA(2.0eq), dissolve completely, 8ml DCM is added after reaction 50min, 1ml MeOH, 1ml DIEA, be transferred in self-control solid phase synthesis post after reaction 20min, the Piperidine/DMF solution of 10ml 20% is added after DMF washing, reaction 20min, after DMF cleaning, 1,2,3-indantrione monohydrate monitoring is positive, get compound c (3.0eq), 178mg HOBT (3.3eq), 6ml DMF adds reaction column after adding 230 μ L DIC (3.3eq) mixing after dissolving, 1,2,3-indantrione monohydrate monitoring after reaction 1h, the Piperidine/DMF solution of 20% is added after DMF washing.Repeat above step, successively condensation Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N 10-TFA-pteroic acid, (wherein, aminoacid and HOBT/DIC consumption are respectively 15eq and 16.5eq/16.5eq), add 10ml 2% hydrazine hydrate/DMF solution reaction 5 minutes after condensation, repeat 3 times, DMF washs, DCM washs, MeOH washs, and drains, synthesizes complete, add lytic reagent, TBME precipitated polypeptide, obtains III-2, MS [M+H] +: 3151.15.
embodiment 20the preparation of compound III-3
Prepare III-2 according to the method shown in scheme 3, concrete steps are as follows:
The preparation of compound f: add 10g a in 250ml reaction bulb, the THF of the HOSU of 1.1eq, 100ml, after dissolving, ice bath is cooled to 0 degree, and the DCC adding 1.1eq rises to stirred overnight at room temperature.TLC raw material reaction is complete.Post processing: sucking filtration, filter cake THF washs, and obtains 12g compound b after organic facies is concentrated.
To in 100ml reaction bulb, add the d of 3.0g, be dissolved in 30ml water, add the NaHCO of 1.1eq 3, be cooled to 10 DEG C; The b of 1.1eq is dissolved in 30ml DME, drips into reaction bulb, add 10mlTHF ambient temperature overnight.TLC reaction is complete, adds EA(ethyl acetate after concentrated), dilute hydrochloric acid washs, and then washes, and Sal is washed, dry, and concentrated, column chromatography obtains compound c.
In 10ml reaction bulb, add 5g c, the THF of the HOSU of 1.1eq, 50ml, after dissolving, ice bath is cooled to 0 DEG C, and the DCC adding 1.1eq rises to stirred overnight at room temperature.TLC raw material reaction is complete.Post processing: sucking filtration, filter cake THF washs, and obtains 6g Verbindung after organic facies is concentrated.
In 100ml reaction bulb, add 3.0g Verbindung, be dissolved in 30ml water, add the NaHCO of 1.1eq 3, be cooled to 10 DEG C; The b of 1.1eq is dissolved in 30ml DME, drips into reaction bulb, ambient temperature overnight.TLC reaction is complete, adds EA after concentrated, dilute hydrochloric acid washs, and then washes, and Sal is washed, dry, and concentrated, column chromatography obtains compound f.
The preparation of III-3: in 50ml reaction bulb, add 1000mg 2Cl-Trt Resin(2.0eq), add 117mg Fmoc-Cys(Trt again)-OH(1.0eq), 8ml DCM, 320 μ L DIEA(2.0eq), dissolve completely, 8ml DCM is added after reaction 50min, 1ml MeOH, 1ml DIEA is transferred in self-control solid phase synthesis post after reacting 20min, DMF washs, then the Piperidine/DMF solution of 10ml 20% is added, reaction 20min, after DMF cleaning, 1,2,3-indantrione monohydrate monitoring is positive, get compound f (3.0eq), 178mg HOBT (3.3eq), after 6ml DMF dissolves, reaction column is added after adding 230 μ L DIC (3.3eq) mixing, 1,2,3-indantrione monohydrate monitoring after reaction 1h, the Piperidine/DMF solution of 20% is added after DMF washing.Repeat above step, successively condensation Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Glu-OtBu, N 10-TFA-pteroic acid, add 10 ml 2% hydrazine hydrates/DMF solution reaction 5 minutes after (aminoacid and HOBT/DIC are respectively 15eq and 16.5eq/16.5eq) condensation, repeat 3 times, DMF washs, DCM washs, MeOH washs, and drains, synthesizes complete, add lytic reagent, TBME precipitated polypeptide, obtains target compound III-3, MS [M+H] +: 4203.55.
embodiment 21the preparation of compound 29
The preparation method of compound 15 in similar embodiment 4, prepares compound 29, and difference is with III in III-2 alternate embodiment 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.
test example 1relative affinity is tested
Be planted in intensive for positive for FR KB cell on 20 porocyte culture plates, and allow described cell adhesion in plastic continuing 18h.The culture medium consumed is placed in the hole of specifying, when raising and do not improve by 100nM when test product or folic acid concentration 3h-folic acid supplements not containing the RPMI(FFRPMI of folic acid).Cell is cultivated 60min at 37 DEG C, then uses the PBS rinsing 3 times of pH7.4.The PBS solution of the pH7.4 containing 1% dodecyl sodium sulfate (SDS) of 500 μ L is added to every hole.Then, collecting cell lysate is also added in the independent pipe containing 5ml fluorescent mixture, then counts its radioactivity.Negative control pipe is not only containing the folic acid be dissolved in FFRPMI (containing competitor).Positive control pipe contains the folic acid that final concentration is 1mM, and the CPM(recorded in these samples represents the non-specific binding of labelling) deduct from all samples.Apparently, what relative affinity was defined as displacement 50% is incorporated into FR's on KB cell 3the anti-mol ratio (inverse molar ratio) of H-folic acid required compound, the relative affinity of folic acid to FR is set as 1.
The relative affinity result of the test of compound 15 in 10% serum/FDRPMI shows, compared with folic acid, compound 15 shows the folacin receptor relative affinity of 157%.
Similar method, measure the relative affinity of compound 16 ~ 32 in 10% serum/FDRPMI, result of the test shows, compared with folic acid, compound 16 ~ 32 all exhibits greater than the folacin receptor relative affinity of 100%, and wherein compound 20,21,31 shows the folacin receptor relative affinity of 162%, 127% and 187% respectively.
test example 2 cell activity assays
cell activity assays 1:
The compounds of this invention 15 ~ 32 vitro cytotoxicity test evaluation, the ability of the KB Growth of Cells of this test prediction Drug inhibition folate receptor-positive, inoculates 100 μ l containing KB cell 5*10 in every hole 3individual PBS solution, after cultivating 24h, extracting medium, be divided into blank group, matched group a, test group b, matched group a and test group b is divided into 10 groups respectively, be numbered 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, matched group and test group add folic acid (FA) 0 respectively successively, 0.1, 0.3, 1, 3, 10, 30, 100, 300 micromoles' (μM), continue to cultivate 2h, then control compound BP111a(compd E C145 is added respectively to matched group a, the preparation method of EC145 and structure are shown in Chinese patent CN100381177C) 1 μM, compound of the present invention (representing with BP111b) 1 μM is added to test group b, blank group neither adds medicine and does not also add folic acid, continue to cultivate 70h, cell survival rate mtt assay evaluation, measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD570nm place.
Mtt assay is also known as MTT colorimetry, and its Cleaning Principle is that the succinate dehydrogenase in living cells mitochondrion can make exogenous MTT be reduced to water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation (Formazan) and be deposited in cell, and dead cell is without this function.First a ceremonial jade-ladle, used in libation in dimethyl sulfoxide (DMSO) energy dissolved cell, measures its absorbance value (i.e. OD570 value) with enzyme-linked immunosorbent assay instrument at 5700nm wavelength place, can indirectly reflect living cells quantity.Within the scope of certain cell number, the amount that MTT crystallization is formed is directly proportional to cell number.
Illustrate, medicine (representing with BP111b) using compound 15 as test group b, operate according to the method described above, result of the test as shown in Figure 2, wherein BP111b represents that compound 15, BP111a of the present invention represents that the preparation method of compd E C145(EC145 is shown in Chinese patent CN100381177C).As can be seen from Figure 2, under the same conditions, BP111b(compound 15 of the present invention) compd B P111a(compd E C145 is better than to the inhibitory action of KB cell), and, along with the increase of FA, BP111b reduces the inhibitory action of KB tumor cell, and this shows, viewed killing functions of immunocytes is folacin receptor mediated by being incorporated into.Further, compared with BP111a, even BP111b is under excessive folic acid existent condition, to KB tumor cell, also there is good inhibit activities, also as shown in Figure 2.
cell activity assays 2:
The ability of the A549 Growth of Cells of this test prediction Drug inhibition folacin receptor feminine gender, inoculates 100 μ l containing A549 cell 5*10 in every hole 3individual PBS solution, after cultivating 24h, extracting medium, be divided into blank group, matched group a, test group b, matched group a and test group b is divided into 10 groups respectively, be numbered 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, matched group a and test group b adds control compound BP111a(EC145 respectively, the preparation method of EC145 is shown in Chinese patent CN100381177C) and test compound BP111b 10000, 3333.33, 111.111, 370.370, 123.4567, 41.152, 13.717, 4.572, 13.717, 4.5724, 1.5241 nanomole (nM), blank group neither adds BP111a and does not also add BP111b, continue to cultivate 70h, cell survival rate mtt assay evaluation, measure the light absorption value in each hole at enzyme-linked immunosorbent assay instrument OD570nm place.
For compound 15 as test group medicine BP111b, result of the test as shown in Figure 3, BP111b(the compounds of this invention 15) not obvious to the tumor cell A549 inhibit activities of FR-feminine gender, these results show compound 15 be by folic acid optionally or the specific mechanism of folic acid play a role.
In the test of this type, all obtain similar result to compound 16 ~ 32 of the present invention, as a rule, compound provided by the present invention 16 ~ 32 pairs of KB cells have significant inhibit activities, and present dose-dependent cytotoxicity, its IC 50(IC 50i.e. half suppression ratio, is meant to the concentration of medicine when generation 50% Tumor growth inhibition effect) all in low nanomolar range, as shown in table 1 below.
Table 1 compound 16 ~ 32 couples of KB cell inhibitory activity data (IC 50: nM)
Compound IC 50 （nM） Compound IC 50 （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 3to the inhibition test of mice tumors grew
Compound 15(BP111b of the present invention represents) intravenous (i.v.) is when giving tumor animal, and the Balb/c mice of the mice with subcutaneous KB tumor have rated its anti-tumor activity.At right subcutaneous tissue of axilla 1*10 6about 11 days (t after the inoculation of KB cell tumour 0time mean tumour volume=60mm 3), be divided into 9 groups to mice, often organizing mice is 6, and 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 is blank group.Matched group a respectively intravenous injection gives 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg control drug BP111a; Test group b respectively intravenous injection gives 0.5 μm of ol/kg, 1 μm of ol/kg, 2 μm of ol/kg compd B P111b(the compounds of this invention 15); Blank group gives the PBS of suitable dose volume, and intravenous injection 3 times weekly, continues about 2 weeks, and each treatment group measures tumor growth with caliber gauge 2 times weekly.Use equation V=axb 2/ 2 calculate gross tumor volume, and wherein, a is length of tumor, and b is width, and unit is millimeter.Simultaneously 2 times or 3 the weight of animals that weigh with scale weekly.
As shown in Figures 4 and 5, during with compd B P111b of the present invention 0.5 μm of ol/kg treatment, effectively can postpone the growth of KB tumor, there is when drug dose is increased to 1 μm of ol/kg obviously anti-tumor activity.Further, as shown in Figure 4, be dose dependent by the inhibitory action of compd B P111b of the present invention to tumor, compared with control drug BP111a, under same dosage, the compounds of this invention BP111b shows better anti-tumor activity.And compd B P111b provided by the invention does not have overt toxicity (based on the weight of animals), compared with control drug BP111a, during same administration 1 μm of ol/kg, control drug BP111a also has anti-tumor activity, but when administration 18 days, the inhibitory action of BP111b to tumor is obviously better than BP111a, as shown in Figure 4; And find based on the weight of animals change, BP111b with BP111a is the same, and under institute's dosage, animal all can well tolerate, as shown in Figure 5.
The compounds of this invention 16 ~ 32 carries out similar test, and result of the test finds: use the mice KB tumor growth of compound of the present invention 16 ~ 32 pairs of FR positives to have significant inhibitory action, and do not have the obvious toxicity with body weight change.
Illustrative, the PBS(contrast of compound 25 or free medicine MMAF or suitable dose volume) give at right subcutaneous tissue of axilla 1*10 with the dosage of 500nmol/kg 6about 11 days (t after the inoculation of KB cell tumour 0time mean tumour volume=60mm 3) mice (6/group) intravenous injection, weekly intravenous injection 3 times, continue after 2 weeks, observe the suppression result to mice tumors grew and Mouse Weight change.Each treatment group, every 3 days, measures tumor growth with caliber gauge.Use equation V=axb 2/ 2 calculate gross tumor volume, and 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 finds, effectively can postpone the growth of KB tumor, and do not have overt toxicity (based on the weight of animals) with compound 25 treatment of the present invention.Free drug MMAF also has anti-tumor activity, but has obvious toxicity, and animal toleration is poor.
Similar approach, measures the anti-tumor activity of compound 25 in FR-negative cells, at right subcutaneous tissue of axilla 1*10 6(t after the inoculation of A549 cell tumour 0time mean tumour volume=50 ~ 80mm 3), give the compound 25 of 500nmol/kg or the PBS(contrast of suitable dose volume to mice (6/group) intravenous injection), intravenous injection 3 times weekly, continue 2 weeks, each treatment group, every 3 days, measures tumor growth with caliber gauge.Use equation V=axb 2/ 2 calculate gross tumor volume, and wherein, a is length of tumor, and b is width, and unit is millimeter.Result of the test shows, compound 25 pairs of FR-negative cells almost do not have activity, these results show compound 25 be by folic acid optionally or the specific mechanism of folic acid play a role.

Claims (19)

1. one kind has the compound of following general formula:
(F) nLD
Wherein,
N is 2,3 or 4;
F is folic acid or pteroic acid; (F) nin each F be independently selected from folic acid and pteroic acid;
D is medicine or its analog or derivant;
L is the joint comprising following structure:
L 1-L 2
Wherein, L 1polyvalent linkers, L 2comprising at least one can releasing-joint, L 1with L 2covalently bound;
Described (F) nrefer to each F respectively with L 1polyvalent linkers covalently bound, D and L 2covalently bound.
2. compound as claimed in claim 1, is characterized in that, comprise the compound of following structure:
Wherein F 1, F 2independently be selected from folic acid and pteroic acid;
D is medicine or its analog or derivant;
L is the joint comprising following structure:
L 1-L 2
Wherein, L 1polyvalent linkers, L 2comprising at least one can releasing-joint;
Described F 1, F 2respectively with L 1polyvalent linkers covalently bound, D and L 2covalently bound.
3. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise at least one by amino acids formed peptide spacer nipple, described aminoacid is independently selected from natural amino acid and non-natural alpha-amino acid.
4. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise at least one by 1 ~ 40 amino acids formed peptide spacer nipple, described aminoacid is independently selected from natural amino acid and non-natural alpha-amino acid.
5. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise at least one by 1 ~ 20 amino acids formed peptide spacer nipple.
6. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise at least one by 10 ~ 15 amino acids formed peptide spacer nipples.
7. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise the aminoacid that at least 2 are selected from lower group: aspartic acid, arginine, cysteine, lysine, agedoite, arginine, threonine, glutamic acid, serine, citrulline, valine and glutamine.
8. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise one or more spacer nipple by being selected from the amino acids formed dipeptides of aspartic acid, arginine, cysteine, citrulline, valine and lysine and combination thereof, tripeptides, tetrapeptide, pentapeptide, six peptides, seven peptides, octapeptide, decapeptide, 11 peptides and dodecapeptide.
9. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, described L 1comprise the structure of following formula:
or
Wherein, * represents open valency.
10. compound as described in as arbitrary in claim 1 ~ 2, is characterized in that, describedly can comprise disulphide, carbonic ester, hydrazides, amide, hydrazone, amino-acid ester, urea or its combination by releasing-joint.
11. as arbitrary in claim 1 ~ 2 as described in compound, it is characterized in that, describedly can to comprise and one or more there is following structure by releasing-joint:
, , , , , , , or
Wherein, n is 0,1,2,3 or 4; R is H, the acyl group of alkyl, optional replacement or nitrogen-protecting group; X is O, CH 2or NH; Y is O or S; Z is NH, O or S; R 1for alkyl, carboxy substituted alkyl or acyl substituted alkyl; * open valency is represented.
12. compounds as claimed in claim 1 or 2, it is characterized in that, described joint L comprises following structure:
、
、
、
or
Wherein, W is selected from NH or O; * open valency is represented.
13. compounds as claimed in claim 1 or 2, it is characterized in that, described compound is selected from:
、
、
With
；
Wherein, W is NH or O; M is 0 or 1; F 1, F 2independently be selected from following formula:
with .
14. as arbitrary in claim 1 ~ 2 as described in compound, it is characterized in that, described medicine is selected from cyclopropyl-phenyl diindyl ketone and derivant thereof, the tall and erect dimer class of Pyrrolobenzodiazepines, Calicheamicin, SN38, vinca, dolastatin, didemnun B, maytansine and derivant thereof, taxanes, daunorubicin, doxorubicin, epirubicin, Ai Sibo mycin, D actinomycin D, ALLRED inhibin class, pipe dissolve plain class, azaserine class, bleomycin, tamoxifen or idarubicin.
15. as arbitrary in claim 1 ~ 2 as described in compound; it is characterized in that, described medicine is selected from open loop-cyclopropyl benzo [e] indole ketone compound, the tall and erect dimer class of Pyrrolobenzodiazepines, Calicheamicin, SN38, deacetylate vinblastine one hydrazides, monomethyl ALLRED inhibin E, monomethyl ALLRED inhibin F, monomethyl ALLRED inhibin F derivative, pipe dissolve plain B, film ascidean rope B, DM1, taxanes, vincristine, daunorubicin, doxorubicin or epirubicin.
16. compounds as claimed in claim 1 or 2, it is characterized in that, described compound is selected from:
、
、
、
、
、
、
、
、
、
with
Wherein, R x: be p-methoxyphenyl; X 1for Cl or Br; R be H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or ; R yfor hydrogen, C 1~ C 6alkyl or haloalkyl or the carbonyl containing 1 ~ 4 carbon atom that replaces arbitrarily; R 1for C 1~ C 6alkoxyl or alkyl or the amino alkyl that replaces or C 1~ C 3aldehyde radical or carbonyl, hydroxyl, amino or C 1~ C 6cycloalkyl or heterocyclic radical; R 2being 2-, 3-or 4-position replacement of benzene, is N=NH or NH; N is the integer of 0 ~ 3;
F 1, F 2independently be selected from following formula:
with .
17. compounds as claimed in claim 1 or 2, it is characterized in that, described compound is selected from:
Wherein, L 3be selected from following formula:
, or ;
Wherein, m is the integer of 0 ~ 4, and * represents link position.
Its structure of 18. compounds is:
、
、
、
、
、
、
、
、
、
, or
Wherein, R xfor p-methoxyphenyl.
19. 1 kinds of pharmaceutical compositions, its comprise treatment effective dose as arbitrary in claim 1 ~ 2 as described in compound, and pharmaceutically acceptable carrier, diluent or excipient or its combination in any.
CN201410024349.9A 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates Active CN104784699B (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CN201410024349.9A CN104784699B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates
CN201811352537.9A CN109316605B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates
PCT/CN2014/091690 WO2015106599A1 (en) 2014-01-20 2014-11-19 Conjugates and compositions for drug delivery

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201410024349.9A CN104784699B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN201811352537.9A Division CN109316605B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates

Publications (2)

Publication Number Publication Date
CN104784699A true CN104784699A (en) 2015-07-22
CN104784699B CN104784699B (en) 2019-05-03

Family

ID=53542374

Family Applications (2)

Application Number Title Priority Date Filing Date
CN201811352537.9A Active CN109316605B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates
CN201410024349.9A Active CN104784699B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN201811352537.9A Active CN109316605B (en) 2014-01-20 2014-01-20 Folate receptor binding ligand-drug conjugates

Country Status (2)

Country Link
CN (2) CN109316605B (en)
WO (1) WO2015106599A1 (en)

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017025057A1 (en) * 2015-08-11 2017-02-16 Coherent Biopharma Multi-ligand drug conjugates and uses thereof
CN106466484A (en) * 2015-08-11 2017-03-01 同宜医药开曼有限公司 A kind of many targeting ligand-drug coupling body with cell endocytic mediation function
CN106466485A (en) * 2015-08-11 2017-03-01 同宜医药开曼有限公司 A kind of targeting ligand-drug coupling body with cell endocytic mediation function
WO2017198124A1 (en) * 2016-05-16 2017-11-23 博瑞生物医药（苏州）股份有限公司 Multi-arm polymeric targeting anti-cancer conjugate
CN108117566A (en) * 2017-12-28 2018-06-05 广州白云山汉方现代药业有限公司 A kind of preparation method of desacetyl vinblastine hydrazides
WO2018192550A1 (en) * 2017-04-21 2018-10-25 博瑞生物医药（苏州）股份有限公司 Multi-arm targeted anti-cancer conjugate
CN108727583A (en) * 2017-04-21 2018-11-02 博瑞生物医药（苏州）股份有限公司 Multi-arm target anticancer conjugate
CN108727584A (en) * 2017-04-21 2018-11-02 博瑞生物医药（苏州）股份有限公司 Anticancer conjugate
CN109776787A (en) * 2017-11-14 2019-05-21 博瑞生物医药（苏州）股份有限公司 Multi-arm targeting conjugate
CN109771658A (en) * 2017-11-14 2019-05-21 博瑞生物医药（苏州）股份有限公司 Target multi-arm conjugate
WO2019096096A1 (en) * 2017-11-14 2019-05-23 博瑞生物医药（苏州）股份有限公司 Multi-arm targeting conjugate
WO2021212638A1 (en) * 2020-06-19 2021-10-28 Hangzhou Dac Biotech Co., Ltd. Conjugates of a cell-binding molecule with camptothecin analogs
WO2022268202A1 (en) * 2021-06-25 2022-12-29 同宜医药（苏州）有限公司 Ligand-drug conjugate and use thereof

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006012527A1 (en) 2004-07-23 2006-02-02 Endocyte, Inc. Bivalent linkers and conjugates thereof
CA2680535C (en) 2007-03-14 2016-09-20 Endocyte, Inc. Binding ligand linked drug delivery conjugates of tubulysins
WO2009002993A1 (en) 2007-06-25 2008-12-31 Endocyte, Inc. Conjugates containing hydrophilic spacer linkers
US9877965B2 (en) 2007-06-25 2018-01-30 Endocyte, Inc. Vitamin receptor drug delivery conjugates for treating inflammation
WO2013126797A1 (en) 2012-02-24 2013-08-29 Purdue Research Foundation Cholecystokinin b receptor targeting for imaging and therapy
US20140080175A1 (en) 2012-03-29 2014-03-20 Endocyte, Inc. Processes for preparing tubulysin derivatives and conjugates thereof
MX2018006218A (en) 2015-12-04 2018-09-05 Seattle Genetics Inc Conjugates of quaternized tubulysin compounds.
US11793880B2 (en) 2015-12-04 2023-10-24 Seagen Inc. Conjugates of quaternized tubulysin compounds
SG10202007520WA (en) 2016-03-02 2020-09-29 Eisai R&D Man Co Ltd Eribulin-based antibody-drug conjugates and methods of use
CN112569368A (en) * 2019-08-07 2021-03-30 烟台迈百瑞国际生物医药股份有限公司 Antibody drug conjugate and application thereof
WO2024105197A1 (en) * 2022-11-17 2024-05-23 Vincerx Pharma Gmbh Small molecule-drug-conjugates cleavable in a tumor microenvironment

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1761488A (en) * 2003-01-27 2006-04-19 恩多塞特公司 Vitamin receptor binding drug delivery conjugates
WO2007022493A2 (en) * 2005-08-19 2007-02-22 Endocyte, Inc. Ligand conjugates of vinca alkaloids, analogs, and derivatives
CN101495154A (en) * 2006-05-25 2009-07-29 百时美施贵宝公司 Conjugates of aziridinyl-epothilone analogs and pharmaceutical compositions comprising same

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2680535C (en) * 2007-03-14 2016-09-20 Endocyte, Inc. Binding ligand linked drug delivery conjugates of tubulysins

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1761488A (en) * 2003-01-27 2006-04-19 恩多塞特公司 Vitamin receptor binding drug delivery conjugates
CN101239190A (en) * 2003-01-27 2008-08-13 恩多塞特公司 Vitamin receptor binding drug delivery conjugates
WO2007022493A2 (en) * 2005-08-19 2007-02-22 Endocyte, Inc. Ligand conjugates of vinca alkaloids, analogs, and derivatives
CN101495154A (en) * 2006-05-25 2009-07-29 百时美施贵宝公司 Conjugates of aziridinyl-epothilone analogs and pharmaceutical compositions comprising same

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017025057A1 (en) * 2015-08-11 2017-02-16 Coherent Biopharma Multi-ligand drug conjugates and uses thereof
CN106466484A (en) * 2015-08-11 2017-03-01 同宜医药开曼有限公司 A kind of many targeting ligand-drug coupling body with cell endocytic mediation function
CN106466485A (en) * 2015-08-11 2017-03-01 同宜医药开曼有限公司 A kind of targeting ligand-drug coupling body with cell endocytic mediation function
CN106466485B (en) * 2015-08-11 2021-05-04 同宜医药（苏州）有限公司 Targeting ligand-drug conjugate with function of mediating cell endocytosis
CN106466484B (en) * 2015-08-11 2021-05-04 同宜医药（苏州）有限公司 Multi-target ligand-drug conjugate with function of cell endocytosis mediation
CN108135881B (en) * 2015-08-11 2020-11-13 同宜医药（苏州）有限公司 Multi-ligand drug couplet and application thereof
CN108135881A (en) * 2015-08-11 2018-06-08 同宜医药开曼有限公司 Multiple ligand drug coupling body and application thereof
CN111617250A (en) * 2015-08-11 2020-09-04 同宜医药（苏州）有限公司 Multi-ligand drug couplet and application thereof
RU2722449C2 (en) * 2015-08-11 2020-06-01 Кохерент Байофарма Poly-ligand medicinal conjugates and use thereof
CN107375288B (en) * 2016-05-16 2019-08-23 博瑞生物医药（苏州）股份有限公司 The polymerization target anticancer conjugate of multi-arm
CN107375288A (en) * 2016-05-16 2017-11-24 博瑞生物医药（苏州）股份有限公司 The polymerization target anticancer conjugate of multi-arm
AU2017265310B2 (en) * 2016-05-16 2019-05-16 Brightgene Bio-Medical Technology Co., Ltd. Multi-arm polymeric targeting anti-cancer conjugate
WO2017198124A1 (en) * 2016-05-16 2017-11-23 博瑞生物医药（苏州）股份有限公司 Multi-arm polymeric targeting anti-cancer conjugate
US10869863B2 (en) 2016-05-16 2020-12-22 Brightgene Bio-Medical Technology Co., Ltd. Multi-arm polymeric targeting anti-cancer conjugate
CN108727583B (en) * 2017-04-21 2022-03-22 高瑞耀业(北京)科技有限公司 Multi-arm targeted anticancer conjugate
US11191843B2 (en) 2017-04-21 2021-12-07 Brightgene Bio-Medical Technology Co., Ltd. Multi-arm targeting anti-cancer conjugate
CN108727584A (en) * 2017-04-21 2018-11-02 博瑞生物医药（苏州）股份有限公司 Anticancer conjugate
CN108727583A (en) * 2017-04-21 2018-11-02 博瑞生物医药（苏州）股份有限公司 Multi-arm target anticancer conjugate
JP2020516683A (en) * 2017-04-21 2020-06-11 博瑞生物医薬（蘇州）股▲分▼有限公司Ｂｒｉｇｈｔｇｅｎｅ Ｂｉｏ−Ｍｅｄｉｃａｌ Ｔｅｃｈｎｏｌｏｇｙ Ｃｏ．， Ｌｔｄ． Multi-arm targeted anti-cancer conjugate
WO2018192550A1 (en) * 2017-04-21 2018-10-25 博瑞生物医药（苏州）股份有限公司 Multi-arm targeted anti-cancer conjugate
CN109776787B (en) * 2017-11-14 2021-08-03 博瑞生物医药（苏州）股份有限公司 Multi-arm targeting conjugates
WO2019096096A1 (en) * 2017-11-14 2019-05-23 博瑞生物医药（苏州）股份有限公司 Multi-arm targeting conjugate
CN109771658A (en) * 2017-11-14 2019-05-21 博瑞生物医药（苏州）股份有限公司 Target multi-arm conjugate
CN109771658B (en) * 2017-11-14 2021-12-10 博瑞生物医药（苏州）股份有限公司 Targeted multi-arm conjugates
CN109776787A (en) * 2017-11-14 2019-05-21 博瑞生物医药（苏州）股份有限公司 Multi-arm targeting conjugate
CN108117566A (en) * 2017-12-28 2018-06-05 广州白云山汉方现代药业有限公司 A kind of preparation method of desacetyl vinblastine hydrazides
WO2021212638A1 (en) * 2020-06-19 2021-10-28 Hangzhou Dac Biotech Co., Ltd. Conjugates of a cell-binding molecule with camptothecin analogs
WO2022268202A1 (en) * 2021-06-25 2022-12-29 同宜医药（苏州）有限公司 Ligand-drug conjugate and use thereof

Also Published As

Publication number Publication date
CN109316605A (en) 2019-02-12
CN104784699B (en) 2019-05-03
WO2015106599A1 (en) 2015-07-23
CN109316605B (en) 2023-07-14

Similar Documents

Publication Publication Date Title
CN104784699B (en) Folate receptor binding ligand-drug conjugates
AU2018387418B2 (en) Bicyclic peptide ligands specific for EphA2
Vlahov et al. Design and regioselective synthesis of a new generation of targeted chemotherapeutics. Part 1: EC145, a folic acid conjugate of desacetylvinblastine monohydrazide
KR101413955B1 (en) Aziridinyl-epothilone compounds
WO2019034177A1 (en) Antibody drug conjugate having two different drugs
CN109106951A (en) A kind of camptothecine-antibody coupling matter
EP3152223B1 (en) Mitomycin conjugate
CN103948937A (en) Binding ligand linked drug delivery conjugates of tubulysins
CN103893778A (en) Multi-drug ligand conjugates
ES2821878T3 (en) Non-linear self-immolative connectors and their conjugates
KR20090025267A (en) Conjugates of aziridinyl-epothilone analogs and pharmaceutical compositions comprising same
CN111001012A (en) Hydrophilic carbonate type antibody coupling drug
JP2021501201A (en) Polypeptide conjugate for intracellular delivery of staple peptides
CN110974975B (en) Quick-release antibody drug conjugate
WO2016089879A1 (en) Conjugates of garftase inhibitors
CN101239190B (en) Vitamin receptor binding drug delivery conjugates
Zhou et al. A designed cyclic peptide based on Trastuzumab used to construct peptide-drug conjugates for its HER2-targeting ability
CN113663067A (en) Immune activation type antibody and application thereof
US20160303251A1 (en) Conjugates of garftase inhibitors
CA3127903A1 (en) Bi-ligand drug conjugate and use thereof
US20120094892A1 (en) Prodrugs
EP4201429A1 (en) Antibody-drug conjugate intermediate comprising sn38 and preparation method therefor
AU2022365991A1 (en) Epha2 bicyclic peptide ligand and conjugate thereof
CN104961800A (en) Polypeptide-antharcycline derivative

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
CB02 Change of applicant information

Address after: Suzhou City, Jiangsu Province, Suzhou Industrial Park 215123 Xinghu Street No. 218 Nano Technology Park building C25

Applicant after: Borui Pharmaceutical (Suzhou) Limited by Share Ltd

Address before: Xinghu Street Industrial Park of Suzhou city in Jiangsu province 215123 No. 218 BioBAY building C27

Applicant before: Borui Bio-medical Technology (Jiangsu) Co., Ltd.

COR Change of bibliographic data
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant