CN109316605B - Folate receptor binding ligand-drug conjugates - Google Patents

Abstract

The present invention provides a folate receptor binding ligand-drug conjugate consisting of a vitamin receptor binding moiety (F) _n The linker (L) and the drug or the analogue or the derivative (D) thereof, wherein n is an integer of 2 to 4. The vitamin receptor binding moiety is preferably folic acid or pteroic acid and the linker (L) comprises a multivalent linker L ₁ And L containing at least one releasable linker ₂ ，(F) _n Covalent attachment to multivalent linker L ₁ Above, the drug or the analogue or derivative (D) and L ₂ Is covalently linked to a releasable linker, L ₁ And L is equal to ₂ Covalent attachment. The conjugate provided by the invention has greatly improved affinity to the folic acid receptor positive tumor cells, has obvious anti-tumor activity and obviously reduced side effect.

Description

Folate receptor binding ligand-drug conjugates

The application is a divisional application of the name of "folate receptor binding ligand-drug conjugate" of the applicant, the Borui biomedical (Suzhou) stock, with application number 201410024349.9 and application date 2014.01.20.

Technical Field

The present invention relates to pharmaceutical compounds for targeted drug delivery and methods of making the same. In particular to a folic acid receptor binding ligand-drug conjugate, more particularly, the invention relates to the coupling of two or more folic acid receptor binding ligands (F) with a drug or an analogue or derivative thereof (D) through a linker (L) to form (F) _n The invention also relates to LD conjugates for use in the treatment of disease conditions caused by pathogenic cell populations, as well as to methods of making such conjugates and pharmaceutical compositions thereof.

Background

Despite great progress in the treatment of cancer and in the research of anticancer drugs, cancer is one of the major diseases severely threatening human health, and in the united states, cancer is the second leading cause of death following heart disease. Representative methods of treating cancer include surgical therapy, radiation therapy, chemotherapy, immunotherapy, and combinations of the above methods of treatment are now used to combat cancer. Most often, cancers are treated by chemotherapy with highly potent drugs, such as mitomycin, paclitaxel, and camptothecin. The main disadvantage of the chemotherapeutic drugs is that the proliferation of normal cells is severely inhibited while the growth of tumor cells is inhibited, and the unavoidable side effects make the development of new specific anticancer drugs particularly important.

There have been many efforts to develop tumor selective drugs by binding anticancer drugs to ligands such as hormones, antibodies, or vitamins. For example, low molecular weight vitamin compounds (such as folic acid) are used as tumor targeting agents.

Folic Acid (FA) is also known as vitamin B ₉ Is an essential nutrient required for all living cells to maintain normal metabolism of the one-carbon pathway and nucleotide biosynthesis. Folate Receptor (FR) is a transmembrane single-chain glycoprotein containing 3 subtypes: alpha-FR, beta-FR, gamma-FR. Folate (FA) exhibits a very high affinity (KD-100 pM) for cell surface-directed glycoproteins of the Folate Receptor (FR), which are glycosyl phosphatidylinositol-linked proteins that capture their ligands (folic acid) from the extracellular environment. Folate Receptors (FR) are molecules that bind to Folate (FA) and are capable of transporting folate-binding molecules inside the cell via an endocytic mechanism. Upon binding, the plasma membrane surrounding the FR-ligand complex will immediately invaginate to form internal vesicles, called endosomes. The pH of the vesicle lumen is somewhat lower by the action of proton pumps in the co-localized endosomal membrane, and this acidification may mediate conformational changes in the FR protein to release its bound ligand allowing entry into the cytoplasm.

Studies have shown that highly expressed α -FR occurs in 90% of ovarian, advanced breast, cervical, endometrial, colon, lung, choroidal, ependymal cell tumors; beta-FR is highly expressed on the surface of macrophages activated in patients suffering from autoimmune diseases such as malignant and proliferative leukocytes (leukemia) and rheumatoid arthritis, while folate receptor is hardly expressed in normal tissues. Therefore, folic acid and folic acid receptors have good research prospect in the targeted therapy technology, especially in tumors And autoimmune diseases, FR has received great attention as a target for anti-tumor drugs, and has become one of the hot spots for research into novel anti-tumor drugs (Hilgenbrink, A.and P.Low (2005). Folate receptor mediated drug targeting: from therapeutics to diagnostics. Journal of Pharmaceutical Sciences (10): 2135-2146.). Internationally attempts have been made to use folic acid-radionuclide imaging agent conjugates, such as folic acid and ¹²⁵ I、 ⁶⁷ Ga、 ¹¹¹ the conjugate of In can non-invasively detect tumor tissues In which the folate receptor is highly expressed In humans. Meanwhile, intensive studies on folic acid-protein toxins, folic acid-small molecule chemotherapeutics, folic acid-liposomes (lipid body-encapsulated chemotherapeutics or gene drugs), folic acid-immunotherapeutics, etc. have been widely conducted internationally (references: hilgenbrink, A.and P.Low (2005): folate receptor mediated drug targeting: from therapeutics to diagnostics. Journal of Pharmaceutical Sciences (10): 2135-2146.WO2012065085, WO2012065079, U.S. Pat. No. 5, 2012022245A 1).

Disclosure of Invention

The invention aims to provide a folic acid receptor binding ligand-drug conjugate which has specificity to pathogenic cells and low toxicity to normal cells, a preparation method thereof and application thereof in preparing medicines for treating tumors.

In one illustrative embodiment of the invention, compounds having the following formula are described:

(F) _n LD

a folate receptor binding ligand-drug conjugate of (1), wherein n is 2, 3, or 4; f is a folate receptor binding ligand selected from folic acid or pteroic acid; (F) _n Each F of (a) is independently selected from folic acid and pteroic acid; d is a drug or analog or derivative thereof; l is a linker comprising the structure:

L ₁ -L ₂

wherein L is ₁ Is a multivalent linker, L ₂ Comprising at least one releasable linker, L ₁ And L is equal to ₂ Covalent attachment;

it should be understood that the invention described (F) _n Meaning that each F is separately linked to a multivalent linkerL ₁ Covalent linkage of D and L ₂ Covalent attachment.

In another embodiment, a folate receptor binding ligand-drug conjugate is described having the structure:

wherein F is ₁ 、F ₂ Independently selected from folic acid and pteroic acid; d is a drug or analog or derivative thereof;

l is a linker comprising the structure:

L ₁ -L ₂

wherein L is ₁ Is a multivalent linker, L ₂ Comprising at least one releasable linker, L ₁ And L is equal to ₂ Covalent attachment;

said F ₁ 、F ₂ Respectively with polyvalent linkers L ₁ Covalent linkage of D and L ₂ Covalent attachment.

Said multivalent linker (L ₁ ) May contain multiple linkers covalently attached to each other, including one or more spacer linkers, releasable linkers, heteroatom linkers, and combinations thereof in any order. In a variant, the releasable linker and optionally the spacer linker are covalently bound to each other to form linker L ₁ The method comprises the steps of carrying out a first treatment on the surface of the In another variant, ligand F is covalently linked to multivalent linker L via one or more spacer linkers ₁ The method comprises the steps of carrying out a first treatment on the surface of the In another variant 2 or more ligands F are attached directly to each other or covalently through a plurality of spacer linkers and/or releasable linkers; in another variation, two or more releasable linkers are covalently linked to each other, and wherein the one or more releasable linkers are separated from each other by a heteroatom linker and/or a spacer linker.

It will be appreciated that the arrangement of the one or more releasable linkers and optional spacer linkers is very variable. The hetero atom in the hetero atom joint is nitrogen, oxygen, sulfur, phosphorus, silicon and the like. Further, the heteroatom linkers (excluding oxygen) may be of different oxidation states, e.gN(OH)、S(O)、S(O) ₂ 、P(O)、P(O) ₂ 、P(O) ₃ Etc. In another variation, the heteroatom linker is hydroxylamine, hydrazine, hydrazone, sulfonate, phosphonite, phosphonate, or the like.

In one embodiment, the multivalent linker L ₁ Comprising at least one peptide spacer formed from an amino acid independently selected from the group consisting of a natural amino acid and a non-natural alpha-amino acid. In another embodiment, the multivalent linker L ₁ Comprising a peptide spacer comprising 1 to 40 amino acids, preferably natural amino acids, and further wherein the multivalent linker L ₁ Comprising a peptide spacer comprising 1 to 20 amino acids; further, the L ₁ Preferably comprises a peptide spacer comprising 10 to 15 amino acids. Further, L ₁ Comprising at least 2 amino acids selected from the group consisting of: aspartic acid, arginine, cysteine, lysine, asparagine, arginine, threonine, glutamic acid, serine, citrulline, valine, and glutamine.

Further, the L ₁ Preferably comprising one or more spacer linkers formed from amino acids selected from the group consisting of aspartic acid, arginine, cysteine, citrulline, valine, and lysine, and combinations thereof.

In one exemplary embodiment, peptide spacer linkers formed from amino acids are combined with heteroatom linkers to form multivalent linkers L of the formula ₁ ：

Wherein, represents open valency.

The multivalent linker L ₁ Optionally including a spacer linker selected from the group consisting of: polyethers, sugars, thiocarbonyl groups, alkylene groups, 1-alkylene-succinimid-3-yl groups, 1- (carbonylalkyl) succinimid-3-yl groups, carbonylalkylcarbonyl groups, 1- (carbonyltetray-l) s hydrogen-2H-pyranyl) succinimid-3-yl and 1- (carbonyl tetrahydrofuranyl) succinimid-3-yl, wherein each of the spacer linkers is optionally substituted with one or more substituents;

wherein the substituents are independently selected from alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, mercaptoalkyl, alkylthio alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, carboxylic acid alkyl ester, alkanoic acid alkyl ester, guanidinoalkyl, or carbonyl or amido or amidoalkyl substituted with amino acids and derivatives and peptides thereof.

In the present invention, L ₂ Comprising at least one releasable linker, by "releasable linker" is meant a linker comprising at least one bond cleavable under physiological conditions (e.g. a pH-labile bond, an acid-labile bond, an oxidative-labile or an enzymatic-labile bond). It goes without saying that such physiological conditions leading to bond cleavage include standard chemical hydrolysis reactions occurring at physiological pH or as a result of compartmentalization into organelles, e.g. endosomes whose pH is lower than cytoplasmic pH. It goes without saying that, as described in the present invention, the cleavable bond may connect two adjacent atoms within the releasable linker and/or connect the other linker or ligand F and/or drug D at either or both ends of the releasable linker. In the case where such cleavable bonds connect two adjacent atoms within a releasable linker, the releasable linker is cleaved into two or more fragments upon cleavage of the bond. Alternatively, in the case of such cleavable linkages between the releasable linker and another moiety, such as a heteroatom linker, a spacer linker, another releasable linker, a drug or analogue or derivative thereof or a folate receptor binding ligand, the releasable linker is separated from the other moiety upon cleavage of the linkage. The instability of the cleavable bond can be modulated, for example, by substitution changes at or near the cleavable tether position, e.g., including alpha branching adjacent to the cleavable disulfide bond, alkoxy groups homologating to form hydrolyzable partial ketals or acetals, and the like.

The releasable linker is selected from the group consisting of: 1-alkoxyalkylene, 1-alkoxyalkylenecarbonyl, 1-alkoxycycloalkylenecarbonyl, dicarbonylaryl, dicarbonylalkoxyaryl, dicarbonyladicarboxylaryl, haloalkylcarbonyl, oxycarbonyloxy, oxycarbonyloxyalkyl, iminoalkyl, carbonylalkyleneimino, iminocycloalkylene, carbonylcycloalkyleneimino, alkylenethio, alkylenearylthio, and carbonylalkylthio, wherein each of said releasable linkers is optionally substituted with one or more substituents;

wherein the substituents are independently selected from alkyl, alkoxy, alkoxyalkyl, hydroxy, hydroxyalkyl, amino, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl, mercaptoalkyl, alkylthio alkyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroaryl, substituted heteroaryl, carboxyl, carboxyalkyl, carboxylic acid alkyl ester, alkanoic acid alkyl ester, guanidinoalkyl, or carbonyl or amido or amidoalkyl substituted with amino acids and derivatives and peptides thereof.

Preferably, in the compounds of the present invention, the releasable linker comprises a disulfide, a carbonate, a hydrazide, an amide, a hydrazone, an amino acid ester, urea, or a combination thereof.

Further preferred, the releasable joint of the present invention comprises one or more of the formulas having the structure:

wherein n is 0, 1, 2, 3 or 4; r is H, alkyl, optionally substituted acyl or nitrogen protecting group; x is O, CH ₂ Or NH; y is O or S, Z is NH, O or S; r is R ₁ Alkyl, carboxy substituted alkyl or acyl substituted alkyl; * Indicating an open price.

Further, the releasable linker and the heteroatom linker or spacer form a compound havingJoint L of the following structure ₂ ：

Wherein m is an integer of 0 to 4, and W is selected from NH or O; * Indicating an open price.

The releasable and spacer connectors are described in detail in the patent publication CN100381177C, the disclosure of which is incorporated herein by reference.

The multivalent linker L ₁ And L comprising a releasable linker ₂ Optionally, the linker L is formed by one or more spacer linkers, heteroatom linkers, releasable linkers.

Exemplary by L ₁ And L is equal to ₂ The linker L, either directly covalently linked or formed by one or more spacer linkers, heteroatom linkers, releasable linkers, comprises the structure:

wherein W is selected from NH or O; * Indicating an open price.

The folate receptor binding ligand-drug conjugate provided by the present invention comprises a folate receptor binding ligand (F), a linker (L) and a drug (D), the linker L may be formed according to any of the means mentioned in the present invention or may be formed by covalent attachment between a spacer linker, a releasable linker and a heteroatom linker known in the art, the linkage of the folate receptor binding ligand F or drug D to the heteroatom linker being made by reactive functional groups present on the drug D or folate receptor binding ligand F which have been converted to a heteroatom linker. Wherein the reactive functional group on the folate receptor binding ligand F is a carboxyl or amino group, and a multivalent linker can be attached to the carboxyl or amino group on the folate receptor binding ligand F to form an ester or amide; wherein when the drug includes a double bond nitrogen atom, the releasable linker is attached to the drug nitrogen to form a hydrazone; when the drug may include a sulfur atom, the releasable linker is selected from the group consisting of an alkylene sulfide or a carbonylalkylsulfide, and the releasable linker is attached to the sulfur of the drug to form a disulfide bond; the drug may include an oxygen atom, the releasable linker being an alkyleneoxycarbonyl or haloalkylcarbonyl group substituted or unsubstituted with a substituent, the releasable linker being attached to the oxygen of the drug to form a carbonate or ester; the drug D may comprise a nitrogen atom and the releasable linker is a haloalkylcarbonyl or a haloalkylcarbonyl group obtained, the releasable linker being attached to the nitrogen of drug D to form an amide.

Spacer linkers and releasable linkers, as well as heteroatom linkers, may be combined in different ways. Illustratively, the linkers are attached to each other by a heteroatom linker, such as alkylene-amino-alkylene carbonyl, wherein x, y are each integers from 1 to 5.

In an exemplary illustrative embodiment, compounds of the formula:

wherein W is NH or O; m is 0 or 1; f (F) ₁ 、F ₂ Independently selected from the following formulae:

the drug D in the present invention may be any molecule capable of modulating or otherwise altering cellular function, including pharmaceutically active compounds. The pharmaceutically active compounds may be agents known in the art, or derived forms thereof, which are cytotoxic, enhance tumor permeability, inhibit tumor cell proliferation, promote apoptosis, reduce anti-apoptotic activity in the packaging cells. Drugs suitable for the present invention include, but are not limited to, hormones, antibiotics, antimicrobials, antivirals, anticancer drugs. Chemotherapeutic agents, which are themselves cytotoxic or may be used to increase tumor permeability, are also used in the methods of the invention. The cytotoxic drugs include, for example, CBI (cyclopropylbenzo [ E ] indolone) analogues or derivatives thereof, ring-opening-cyclopropylbenzo [ E ] indolone analogues, O-Ac-ring-opening-cyclopropylbenzo [ E ] indolone analogues, dolastatin (Dolastatin) analogues such as Dolastatin-10, auristatin (auristatin) analogues such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), tubular lysines such as Tubulysins, cobustatins, maytansine analogues, DM1, epothilones, paclitaxel and paclitaxel derivatives such as docetaxel, vinblastine and analogues thereof such as vincristine, desacetylvinblastine monoazide (DAVLBH), camptothecin and camptothecin derivatives, colchicine, isocolchicine, thiocolchicine, colchicine, dacarbazine, cyclophosphamide, cyclomycin, methotrexate, benzogliptin, mitomycin, and mitomycin (telbixaprop) and the like. Other drugs that may be suitable for use in the present invention include macrolide antineoplastic agents, chemotherapeutic agents such as alkylating agents, nitrogen mustard, nitrosoureas, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, antimetabolites such as cytarabine, purine analogues, pyrimidine analogues and penicillins, cephalosporins, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside antibiotics and acyclovir, trifluoretoside, ganciclovir, zidovudine, amantadine, ribavirin Lin Jixi tabine and any other art-recognized antimicrobial compound.

Further, the drug of the present invention is preferably Temsirolimus (Temsirolimus), a ring-opened-cyclopropylbenzo [ e ] indolone analog, a Pyrrolobenzodiazepine (PBD) dimer, calicheamicin (Calicheamicin), 7-ethyl-10-hydroxycamptothecin (SN-38), vinblastine, dolastatin (Dolastatin), auristatin (auristatin), ascin B (Didemnin B), a tube dissolvent B (Tubulysin B), a maytansinoid, a taxol, daunorubicin, doxorubicin, epirubicin, epothilone, or an actinomycin.

Still further, the agents of the present invention are preferably ring-opened-cyclopropyl-benzo [ e ] indolones, PBD dimers, calicheamicins, SN-38, DAVLBH, tubulysin B, didemnin B, MMAE, MMAF, MMAF derivatives, DM1, taxol, vincristine, daunorubicin, doxorubicin or epirubicin.

The CBI class of compounds and the ring-opened-cyclopropylbenzo [ e ] indolones are described in detail in publication nos. WO2009/064913A1 and US2010113476A1, the disclosures of which are incorporated herein by reference, exemplary ring-opened-cyclopropylbenzo [ e ] indolones are shown in the following formulas:

Wherein R is ₁ For H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or +.>

R ₂ Is NH ₂ Or OMe.

The PBD in the Pyrrolobenzodiazepine (PBD) dimer compound refers to a structure with the following units:

PBD dimers differ in the number, type and position of substituents on both aromatic ring a and pyrrole ring C and in the saturation of pyrrole ring C, exemplary PBD dimers such as:

wherein R is ₁ Is hydroxy, amino, C ₁ ～C ₆ Alkoxy, C ₁ ～C ₆ Alkyl, C of (2) ₁ ～C ₆ Amino-substituted alkyl, C ₁ ～C ₃ Aldehyde or carbonyl group, or C ₁ ～C ₆ Cycloalkyl or heterocyclyl; r is R ₂ Is 2-, 3-or 4-substituted, is-n=nh-or-NH-; n is an integer of 0 to 3.

Such compounds are described in detail in the publication CN201180029867, the disclosure of which is incorporated herein by reference, and methods of making the same are disclosed.

The SN-38 is a structure having the formula:

the DAVLBH is of the formula:

the specific structures of Tubulysin B and MMAE are shown below:

MMAF and its derivatives refer to compounds having the following structure:

wherein R is _y Is C ₁ ～C ₆ An alkyl or haloalkyl group or an optionally substituted carbonyl group having 1 to 4 carbon atoms; when R is _y When hydrogen, MMAF is the case.

The Didemnin B is of the formula:

wherein R is _x Is p-methoxyphenyl.

The DM1 is a structure having the formula:

in the folic acid receptor binding ligand conjugate, the medicine D is connected with a releasable joint L ₂ Can be made by the presence of reactive functionalities of drug D or a derivatized form thereof, for example, the hydroxyl group of Didemnin B is converted to the corresponding carbonate, another illustrative example is the derivatization of the terminal carboxyl group of Tubulysin B to a hydrazide followed by the attachment of the nitrogen atom on the hydrazide as a heteroatom linker to a releasable linker on L, the conversion of folic acid to the corresponding amide, etc., as illustrated by the following formula:

the following folate receptor binding ligand-drug conjugates are illustrative of the drug conjugates provided herein, and are considered to be within the scope of the present invention, these folate-drug conjugates being prepared according to art-recognized protocols or methods described herein.

In another embodiment, the invention describes a compound of the structure:

wherein R is _x Is p-methoxyphenyl; x is X ₁ Is Cl or Br; r is H, OMe, OH, ONHBoc, ONHAc, ONH (Ac) Boc, ONPhth or

R _y Is hydrogen, C ₁ ～C ₆ An alkyl or haloalkyl group or an optionally substituted carbonyl group having 1 to 4 carbon atoms; r is R ₁ Is C ₁ ～C ₆ Alkoxy or alkyl or amino substituted alkyl, or C ₁ ～C ₃ Aldehyde or carbonyl, hydroxy, amino or C ₁ ～C ₆ Cycloalkyl or heterocyclyl; r is R ₂ Is benzene 2-, 3-or 4-substituted, is n=nh or NH; n is an integer of 0 to 3. />

Wherein ONPhth represents the following structure:

further, the present invention provides a compound of the structure:

wherein L is ₄ A structure selected from the group consisting of:

F ₁ ，F ₂ independently selected from the following formulae:

* Indicating the connection location.

Further, the present invention provides a compound of the formula:

in another aspect, the present invention provides a compound having the formula:

F ₃ LD and F ₄ LD，

Wherein F, L, D is linked in a manner analogous to Compound F ₂ The connection mode between F, L, D in LD.

Exemplary F ₃ LD and F ₄ The LD compounds are shown below:

wherein D is selected from the drugs described hereinbefore or derivatives or analogues thereof; f (F) ₁ 、F ₂ 、F ₃ 、F ₄ Independently selected from:

L ₂ is a linker comprising at least one releasable linker, optionally comprising one or more heteroatom linkers, spacer linkers.

Exemplary, L ₂ The structure is as follows:

wherein m is an integer of 0 to 4, and W is selected from NH or O; * Indicating an open price.

Further, the following specific compounds are used to illustrate F ₃ LD and F ₄ LD：

The folate receptor binding ligand-drug conjugates of the present invention can be prepared by art-recognized synthetic methods. The choice of the synthetic method depends on the choice of the heteroatom linker, the structural characteristics of the drug, and the functional groups on the spacer linker and the releasable linker. In general, related bond formation reactions are described in Richard c.larock.completive Organic Transformations, a guide to functional group preparations.vch Publishers, inc.new York (1989), and Theodora E.Greene & Peter G.M.wuts.protective Groups ion Organic Synthesis, second edition, john Wiley & Sons, inc.new York (1991), the disclosures of which are incorporated herein by reference.

Wherein the heteroatom linker is a sulfur atom and the functional group present in the releasable linker is an alkylene thiol derivative, the disulfide group may be formed by reacting the corresponding heteroaryl disulfide derivative, such as pyridin-2-yl dithioalkyl derivative, and the like, with an alkylene thiol derivative. The reaction solvent can be Tetrahydrofuran (THF), N-Dimethylformamide (DMF), dichloromethane, dimethyl sulfoxide (DMSO) and the like, and the reaction temperature can be changed between 0 ℃ and 80 ℃.

The formation of conventional carbonates, thiocarbonates and carbamates can generally be accomplished by reacting a hydroxy-substituted compound, a thio-substituted compound or an amine-substituted compound, respectively, with an activated alkoxycarbonyl derivative to form carbonates, thiocarbonates and carbamates. The reaction may be carried out in a conventional solvent such as THF, DMF, DMSO, methylene chloride, ethyl acetate, etc., and the reaction temperature varies in the range of 0 to 80 c, and any basic catalyst such as an inorganic base, an amine base, a polymer-bound base, etc. may be used to promote the reaction.

Commonly used amides and esters are formed, for example, wherein the heteroatom linker is a nitrogen atom and the terminal functional group present on the spacer linker or releasable linker is a carbonyl group, the amide group may be obtained by esterification or acylation of the corresponding carboxyl group or derivative thereof, the amide forming reaction solvent comprises methylene chloride, THF, DMF, DMSO, and the like, and the amide may illustratively be prepared by reaction at a temperature ranging from-15 to 80 ℃. Coupling agents include DCC, EDC, HBTU, TBTU, HOBT/DCC, HOBT/EDC, etc., or the parent acid may be converted to an activated carbonyl derivative, such as an acid chloride, N-hydroxysuccinimidyl ester, etc., and the amide formation reaction may be performed under basic conditions such as triethylamine, N' -diisopropylethylamine, etc.

Similarly, where the heteroatom linker is an oxygen atom and the terminal group on the spacer linker or releasable linker is a carbonyl group, the desired ester group may be prepared by coupling the corresponding carboxylic acid or derivative thereof with an alcohol. The coupling agent comprises DCC, EDC, CDI, BOP, EEDQ, DEAD, triphenylphosphine and the like, the solvent comprises dichloromethane, THF, DMF, DMSO, acetonitrile, ethyl acetate and the like, and the base comprises triethylamine, diisopropylethylamine and the like. Or the parent acid may be converted to an activated carbonyl derivative such as an acyl chloride, an N-hydroxysuccinimide ester, or the like.

The drug includes a nitrogen atom to which a releasable or spacer linker may be attached to form a hydrazone, and the desired hydrazone group may be formed by reaction of the corresponding aldehyde or ketone with a hydrazine or hydrazide derivative. The solvent may include methylene chloride, THF, DMF, DMSO, chloroform, ethyl acetate, etc., and the reaction temperature may vary from 0 to 80 c, and any acidic catalyst such as mineral acid, glacial acetic acid, trifluoroacetic acid, etc. may be used as the catalyst. The acylhydrazone may be formed by acylating the hydrazine with a suitable carboxylic acid or derivative thereof, and then reacting the hydrazide with the corresponding aldehyde or ketone to form the acylhydrazone. Alternatively, the hydrazone functionality may be formed by reacting hydrazine with the corresponding aldehyde or ketone. The hydrazone obtained is then acylated with the appropriate carboxylic acid or derivative thereof.

Formation of conventional succinimides: the heteroatom linker is a nitrogen atom, an oxygen atom or a sulfur atom, the functional group present on the spacer linker or releasable linker is a succinimide derivative, and the resulting carbon-heteroatom may be formed by Michael addition of the corresponding amine, alcohol or thiol and maleimide derivative. Solvents for forming the Michael addition include THF, ethyl acetate (EtOAc), CH ₂ Cl ₂ 、DMF、DMSO、H ₂ O, etc. The formation of this Michael addition compound can be accomplished by adding an equimolar amount of a base such as triethylamine or by adjusting the pH of the aqueous solution to 6.0 to 7.4. It is apparent that when the heteroatom linker is an oxygen or nitrogen atom, the reaction conditions can be adjusted to facilitate Michael addition, such as by employing higher reaction temperatures, adding catalysts, using more polar solvents such as DMF, DMSO, etc., and activating maleimide with silylating agents.

Symmetrical acetals or ketals can be formed from the corresponding alcohols and aldehydes or ketones by the reaction of acetals and ketals. The procedures reviewed in r.r.schmidt et al, chem.rev.,2000,100,4423-42, the disclosure of which is incorporated by reference into the present invention, are generally followed.

Preparation of conventional folate-peptides: preparation of the folate-containing peptidyl fragment Pte-gamma on acid-sensitive 2Cl-Trt Resin by the Polymer-Carrier sequential method using Fmoc-strategy Glu-(AA) _n -Cys-OH, as shown in the scheme below:

(a) 20% piperidine/DMF; (b) Fmoc-AA-OH, HOBT, DIC, DMF; (c) Fmoc-Glu-OtBu, HOBT, DIC, DMF; (d) N (N) ¹⁰ -TFA-Pteroic acid；PyBop，DIPEA，DMF/DMSO；(e)2％NH ₂ NH ₂ /DMF；(f)TFA/H ₂ O/phenol/anisole/EDT (82.5:5:5:5:2.5);

in an illustrative embodiment of the method of the invention, R ₁ Fmoc, R ₂ Triphenylmethyl, DIC is diisopropylcarbodiimide, DIPEA is N, N' -diisopropylethylamine, and PyBop is used as an activator to ensure efficient coupling. Under standard conditions, after each coupling step, the Fmoc protecting group was removed. The use of appropriately protected amino acid building blocks, e.g. Fmoc-Glu-OtBu, N, as described in scheme 1 ¹⁰ -TFA-Pteroic acid, etc., and is represented by Fmoc-AA-OH in step (b). Thus, AA refers to any suitably protected amino acid starting material. It should be understood that the term "amino acid" as used herein refers to any amine or carboxylic acid functional group having one or more carbon separation and includes naturally occurring alpha and beta amino acids, as well as derivatives and analogs of such amino acids. Specifically, protected amino acids having side chains such as protected serine, threonine, cysteine, aspartic acid, and the like may also be used in the synthesis of the folate-peptides of the present invention. In addition, amino acid analogs of homologous side chains of γ, δ or longer or alternative branching structures, such as norleucine, isovaline, β -methyl threonine, β -methyl cysteine, β -dimethyl cysteine, and the like, may also be included as starting materials in the synthesis of the folate-peptides of the present invention.

The coupling sequences (steps (a) and (b)) involving Fmoc-AA-OH are performed n times to prepare a solid support peptide (II), wherein n is an integer and may be 0 to 100. After the final coupling step, the remaining Fmoc group is removed (step (a)), the peptide is then coupled to a glutamic acid derivative (step (c)), deprotected, and coupled to TFA-protected pteroic acid (step (d)). The TFA protecting group is removed by treatment with a base (step (e)) and then subjected to step (f) to provide peptide-containing substrate fragment (III).

The peptide is then prepared by reacting with trifluoroacetic acid (TFA), H ₂ O, phenol, anisole and EDT treatment, cut from the polymer support (step (f)). These reaction conditions result in the simultaneous removal of t-Bu, t-Boc, pbf and Trt protecting groups which may form part of the appropriately protected amino acid side chains.

Fragments containing three or four folic acid peptides can be prepared in a similar manner, wherein the multivalent linker can be constructed from amino acids containing 3 or more reactive functional groups (e.g., amino, hydroxyl, carboxyl) such as lysine, glutamic acid, serine, asparagine, aspartic acid, tyrosine, glutamic acid arginine, and the like.

Exemplary fragment III-2 containing three folic acid peptides can be prepared according to the following protocol:

Connecting two lysines to obtain a compound c containing four reactive functional groups, adding the compound c into a reaction column, monitoring by ninhydrin, and sequentially condensing Fmoc-Asp (OtBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Glu-OtBu, N ¹⁰ After condensation of TFA-pteroic acid (wherein the amounts of amino acid and HOBT/DIC are 15eq and 16.5eq, respectively), 10ml of 2% hydrazine hydrate/DMF solution is added to react for 5 minutes, and the reaction is repeated 3 times, DMF washing, DCM washing, meOH washing, draining, and after synthesis, a cleavage reagent is added, TBME precipitates the polypeptide to obtain III-2.

Exemplary fragment III-3 containing four folic acid peptides can be prepared as shown in scheme 3 below:

similarly, four lysines were first condensed to give compound f, which was then added to the reaction column and monitored by ninhydrin, followed by sequential condensation of Fmoc-Asp (OtBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Glu-OtBu, N ¹⁰ After the condensation of TFA-pteroic acid (wherein the amounts of amino acid and HOBT/DIC are 15eq and 16.5eq/16.5eq, respectively), 10ml of 2% hydrazine hydrate/DMF solution is added to react for 5 minutes, and the reaction is repeated 3 times, DMF washing, DCM washing, meOH washing, pumping-out, and after the synthesis, a cleavage reagent is added and TBME precipitates the polypeptide to obtain the target compound III-3.

Wherein either of c and f lysine can also be replaced with other structurally similar amino acids or substituted amino acids to prepare fragments similar to those containing four or five reactive functional groups, and the order of condensation, manner of attachment, and steric configuration of the amino acids are not intended to limit the present invention.

N ¹⁰ TFA-pteroic acid refers to a compound having the structure:

the preparation is described in particular in the literature under the title "Efficient Syntheses of Pyrofolic Acid and Pteroyl Azide, reagents for the Production of Carboxyl-Differentiated Derivatives of Folic Acid" (J.Am.chem.Soc., vol.119, no.42,1997, 10004-10013), which discloses the preparation of N from folic acid ¹⁰ Specific methods of TFA-pteroic acid, the disclosure of which is incorporated by reference in its part into the present invention.

In another aspect, the present invention provides a pharmaceutical composition comprising a folate receptor binding ligand-drug conjugate described herein and a pharmaceutically acceptable carrier, diluent, or excipient, or a combination thereof.

In another aspect, the invention provides the use of the pharmaceutical composition for the treatment and/or prophylaxis of a disease caused by a population of pathogenic cells. The pathogenic cell population refers to cancer cells, infectious agents such as bacteria and viruses, cells infected with bacteria or viruses, activated macrophages capable of causing a condition, and any other type of pathogenic cell that is unique, preferentially expresses or overexpresses a folate receptor. The population of pathogenic cells may be a population of cancerous cells that are tumorigenic (including benign tumors and malignant tumors), or may be non-tumorigenic. The cancer cell population includes, but is not limited to, oral cancer, thyroid cancer, endocrine gland cancer, skin cancer, stomach cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, colon cancer, bladder cancer, bone cancer, ovarian cancer, uterine cancer, breast cancer, testicular cancer, prostate cancer, rectal cancer, kidney cancer, liver cancer, and lung cancer.

According to the present invention, the folate receptor binding ligand-drug conjugate is useful for treating a disease characterized by the presence of a population of pathogenic cells in a host, wherein a member of the population of pathogenic cells has a folate or pteroic acid binding site, wherein the binding site is uniquely expressed, overexpressed, or preferentially expressed by the pathogenic cells. The selective elimination of the pathogenic cells is linked to the folate receptor by the vitamin moiety of the folate receptor binding ligand-drug conjugate. High affinity folate receptor surface expression vitamin receptors are overexpressed on tumor cells. Ovarian, breast, colon, lung, nasal, pharyngeal and brain epithelial cancers have all been reported to express high levels of folate receptors. Indeed, over 90% of human ovarian tumors are known to express this receptor at high levels. Thus, the drug delivery conjugates of the invention are useful in the treatment of a variety of tumor cell types, as well as other pathogenic cell types such as infectious agents, that preferentially express folate receptors and thus have surface accessible vitamin or vitamin analog or derivative binding sites.

The term "folate or folic acid, pteroate or pteroic acid" as used in the present invention is used independently to refer to the folic acid, or pteroic acid moiety, used to form the conjugate.

Abbreviations:

"Boc" means t-butoxycarbonyl; "CDI" means N, N-carbonyldiimidazole; "DDQ" means 2, 3-dichloro-5, 6-dicyano-1, 4-benzoquinone; "DCC" means N, N' -dicyclohexylcarbodiimide; "DEAD" means diethyl azodicarboxylate; "DIPEA" and "DIEA" both represent N, N' -diisopropylethylamine; "DIC" means N, N' -diisopropylcarbodiimide; "EDCI" means 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide; "EDC" means (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride); "EEDQ" means 2-ethoxy-1-ethoxycarbonyl-1, 2-dihydroquinoline; "Fmoc" means fluorenylmethoxycarbonyl; "HBTU" means (2- (1H-benzotriazol-1-yl) -1, 3-tetramethyltetrafluoroborate, "HATU" means N- [ [ (dimethylamino) -1H-1,2, 3-triazolo [4,5-b ] pyridin-1-yl ] methylene ] -N-methylparaben N-oxide, "HOAt" means 7-aza-1-hydroxybenzotriazole, "HOBT" means 1-hydroxybenzotriazole, "HOSU" means N-hydroxysuccinimide, "ivDde" means 1- (4, 4-dimethyl-2, 6-dioxocyclohexylidene) -3-methylbutyl; "NMP" means N-methylpyrrolidone, "OtBu" means tert-butoxy, "PABA" means p-aminobenzoic acid, "TBTU" means 2- (1H-benzothiazolyl) -1, 3-tetramethyluronium tetrafluoroborate "means tert-butyl methyl ether.

The folic acid receptor binding ligands in the folic acid receptor binding ligand-drug conjugate provided by the invention are 2 or more, preferably 2-4, and each ligand is respectively and covalently connected with a multivalent joint, and the drug D is covalently connected with a releasable joint. Moreover, compared with the existing folic acid receptor binding ligand-drug conjugate (such as EC 145), the conjugate provided by the invention has a larger binding probability with the folic acid receptor at the same molar dosage; even more unexpectedly, the conjugates provided herein exhibit more stable antitumor activity than existing folate receptor binding ligand-drug conjugates (e.g., EC 145) even at equivalent molar dosages in the presence of excess folate, indicating that the conjugates provided herein exert good antitumor activity even in the presence of amounts of folate in competition conditions, as demonstrated by the following examples.

Compared with the folic acid receptor binding ligand-drug conjugate in the prior art, the conjugate provided by the invention has the advantages that the molecular weight is obviously increased, the conjugate is favorable for selectively gathering on the surface of tumor cells through a passive targeting mechanism, meanwhile, the conjugate provided by the invention contains 2 or more folic acid receptor binding ligands, the affinity of the conjugate to the tumor cells positively expressed by the folic acid receptor and the residence time of the conjugate in the tumor cells are further enhanced, and further, releasable joints such as hydrazide bonds which are covalently connected with a drug and other releasable joints such as disulfide bonds in the joint L are disconnected within a few minutes under the condition of cell acidity, and simultaneously, the disulfide bonds are reduced, so that the drug is released, and the antitumor activity of the drug is rapidly exerted. In addition, the inventors also found that the conjugates provided by the invention have low binding rate to plasma albumin, which also indicates that the conjugates provided by the invention have fast in vivo clearance rate and low toxicity.

The beneficial effects of the present invention are further illustrated in the following description, taken in conjunction with the accompanying drawings and specific embodiments, which are given by way of illustration only and not by way of limitation.

Drawings

Fig. 1 shows a mass spectrum of compound 15 prepared from example 4, wherein 2969.35 is a molecular ion peak of compound 15.

FIG. 2 shows the inhibition of KB cells by 1. Mu.M BP111b (compound 15 of the invention) and by the control BP111a (compound EC 145) in the presence and absence of excess folic acid, where "-" indicates no addition and "+" indicates addition.

FIG. 3 shows the inhibition of folate receptor negative A549 cell growth by BP111b (compound 15 of the invention) and by control BP111a (compound EC 145), the abscissa indicates the concentrations of BP111a and BP111b added.

FIG. 4 shows the inhibition of KB tumor growth in mice by administration of BP111b (Compound 15) and control BP111a at 0.5. Mu. Mol/kg, 1. Mu. Mol/kg, 2. Mu. Mol/kg, respectively, vehicle representing a blank, namely: no drug treatment was given, only an equal amount of vehicle, the ordinate TV (mm 3) represents Tumor Volume (Tumor Volume), the abscissa: day (Day).

FIG. 5 shows the weight effects on KB tumor mice given 0.5. Mu. Mol/kg, 1. Mu. Mol/kg, 2. Mu. Mol/kg BP111b (Compound 15) and control BP111a, respectively, vehicle representing the blank, namely: no drug treatment was given, only an equal amount of vehicle, and the ordinate BW (g) represents Body Weight (Body Weight), and the abscissa: day (Day).

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The technical solutions and advantages of the present invention will be further illustrated by the following specific examples, which are considered to be within the scope of the present invention and are not to be construed as limiting the invention. The methods are illustrative and can be used to prepare the drug conjugates of the present invention.

EXAMPLE 1 preparation of Compound III

According to the general procedure of the invention, 1000mg of 2Cl-Trt Resin (1.5 eq) and then 234mg of Fmoc-Cys (Trt) -OH (1.0 eq), 8ml of Dichloromethane (DCM), 640. Mu.L of DIEA (2.0 eq) were added to a 50ml reaction flask according to the scheme of scheme 1, completely dissolved and invertedAfter 50min, 8ml DCM, 1ml MeOH and 1ml DIEA were added for 20min, the mixture was transferred to a self-made solid phase synthesis reaction column, after washing with DMF, 10ml 20% piperidine DMF solution was added, after 20min of reaction, after washing with DMF, ninhydrin was monitored positive, 709mg Fmoc-Lys (Fmoc) -OH (3.0eqiv,178mg HOBT) (3.3 eq) was taken, after 6ml DMF was dissolved, 230. Mu.L DIC (3.3 eq) was added and mixed, after 1H of reaction, ninhydrin was monitored, after washing with DMF, 20% piperidine DMF solution was added ¹⁰ TFA-pteroic acid (wherein the amount of amino acid and HOBT/DIC is 6eq and 6.6eq/6.6eq respectively), adding 10ml 2% hydrazine hydrate/DMF solution after condensation for reaction for 5 min, repeating for 3 times, DMF washing, DCM washing, meOH washing, pumping drying, adding cracking reagent after synthesis, TBME precipitating polypeptide to obtain crude product III, separating and purifying by HPLC preparation chromatography to obtain pure product III, MS [ M ] ] ⁺ ：2097.74。

Example 2 preparation of Compound 33

To a 100ml three-neck flask was added 10.0ml DCM,1.0ml MeOC (O) SCl (1.0 eq) cooled to 0℃in an ice bath, 0.76ml mercaptoethanol (1.0 eq) was added dropwise, the reaction was continued at 0℃for 30min, 1.22g 2-mercaptopyridine (1.0 eq) was added, 16ml DCM was added dropwise to the flask, the reaction was continued at 0℃for 1h and then allowed to stand at room temperature for 1h, after completion of TLC starting material reaction, post-treatment: concentrated to about 16ml, suction filtered and the filtrate washed with DCM to give 2.0g of pale yellow slightly odorous solid.

3.0ml DCM and 0.46g diphosgene (0.55 eq) are added into a 100ml three-neck flask, the temperature is reduced to 0 ℃ by ice bath, 1.0g compound 33-2 (1.1 eq) is dissolved in 13ml DCM, 0.45g triethylamine (1.0 eq) is added into the mixture, the mixture is added into a reaction bottle after dissolution, the temperature is lower than 0 ℃ when the solution is added dropwise, the reaction of TLC raw materials is completed (33) after the solution is heated to room temperature for 1h, the yield is 96%, MS [ M ] is obtained] ⁺ :248.97。

Additional details of this process are described in U.S. Pat. No. 3,182,62 and I.R. Vlahov, et al Bioorg.Med. Chem. Lett.16 (2006): 5093-5096, both of which are incorporated herein by reference in their entirety.

EXAMPLE 3 preparation of Compound 15-3

1.5g of vinblastine, 5ml of anhydrous methanol and 5ml of anhydrous hydrazine are added into a 100ml three-neck flask, the temperature is raised to about 60 ℃, TLC (thin layer chromatography) detects the reaction progress after the reaction is carried out for 24 hours, after the reaction is finished, the mixture is poured into 50ml of water, DCM (digital control program) is used for extraction for 3 times, water washing is carried out for 3 times, saturated salt water is used for 3 times, and anhydrous sodium sulfate is dried and concentrated to obtain 1.1g of white solid, namely the compound 15-2.

To a 100ml three-necked flask, 15-2.98 g (1.0 eq) of the compound, 33.67 g (1.5 eq) of the compound, and 10g of DCM were charged, and after stirring and dissolution, 0.44ml of triethylamine (2.5 eq) was added dropwise to the reaction flask, and after 2 hours at room temperature, the reaction was completed.

Post-treatment: 50ml of DCM was added, washed 3 times with saturated brine for 3 times, dried over anhydrous sodium sulfate and concentrated to give 1.2g of crude product, which was column chromatographed to give 750mg of pure 15-3. MS [ M ]] ⁺ ：959.43。

EXAMPLE 4 preparation of Compound 15

Into a 100ml three-necked flask, 7.5ml of water was added, and after 30 minutes of nitrogen bubbling, 200mg of Compound III (1.2 eq) was added, followed by 0.1N NaHCO ₃ The aqueous solution was adjusted to a pH of about 6.9; 78mg of compound 15-3 was dissolved in 15ml of MeOH and added to the reaction flask, and after about 10 minutes of reaction, 39mg of pure compound 15 was obtained by HPLC preparation and lyophilization. HPLC:98.1% of the mass spectrum of the compound 15 is shown in figure 1, MS [ M+H ] as measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS)] ⁺ ：2969.35。

EXAMPLE 5 preparation of Compound 16

Similar to the preparation of compound 15, compound 16 was prepared. MS [ M+H ]] ⁺ ：3043.18。

EXAMPLE 6 preparation of Compound 17

Similar to the preparation of compound 15, compound 17 was prepared. MS [ M+H ]] ⁺ ：2819.98。

Example 7 preparation of Compound 18 (R _x Is p-methoxyphenyl)

Adding the compound 33 and DMAP (4-dimethylaminopyridine) into a dichloromethane solution of Didemnin B to react for 2 hours at 0 ℃, and separating and purifying to obtain a compound 18-1; dissolving compound III in water, regulating pH to 7 with sodium bicarbonate under nitrogen condition, dissolving compound 18-1 in acetonitrile, adding into water solution of compound III under nitrogen condition, detecting reaction progress by reverse phase analytical HPLC, separating and purifying after reaction to obtain target product 18, MS [ M+H ]] ⁺ ：3312.36。

EXAMPLE 8 preparation of Compound 19

A mixture of compound 19-1 (7.7 mg, 10. Mu. Mol) in 5% methanol/dichloromethane at 0deg.C was added to the trifluoroacetate salt of compound 19-2 (6.4 mg, 20. Mu. Mol). The reaction mixture was warmed to ambient temperature and stirred for 5h, then concentrated under reduced pressure, and chromatographed on silica gel (eluent: 3% methanol/dichloromethane) to give 3.3mg of the title compound 19-3, MS [ M+H ]] ⁺ ：974.47。

The compound 19-3 and the sulfhydryl group of the compound III are subjected to Michael addition reaction to prepare the compound 19, MS [ M+H ]] ⁺ ：3058.16。

EXAMPLE 9 preparation of Compound 20

The preparation of the compound 20 can be obtained by 8 steps of reaction of the compound 20-1 according to the above route; the specific procedure for the preparation of compound 20-9 from compound 20-1 by a 6-step reaction is described in detail in EP0624377A2, the disclosure of which is incorporated herein by reference.

Preparation of Compounds 20-10: MMAE (100.5 mg,0.14mmol,1 eq) compound 20-9 (110.6 mg,0.15mmol,1.1 eq) and HOBt (19 mg,0.14mmol,1.0 eq) were dissolved in DMF (2 ml). After 2min pyridine (0.5 ml) was added and the progress of the reaction was monitored by reverse phase HPLC. After about 24h of reaction, the reaction was quenched, the reaction concentrated and the residue purified by reverse phase prep HPLC (Varian Dynamax column 21.4 mm. Times.25 cm, 5. Mu.100 well) using acetonitrile and Et ₃ N-CO ₂ (pH 7) gradient elution was performed from 10% to 100% at a flow rate of 20ml/min for 40min. Collecting and concentrating in segments to obtain off-white solid, which is compound 20-10, MS [ M ]] ⁺ :1315.78。

And carrying out Michael addition reaction on the compound 20-10 and the sulfhydryl group of the compound III to obtain the compound 20.MS [ M+H ]] ⁺ :3414.52。

EXAMPLE 10 preparation of Compound 21

Compound 21 was prepared according to the procedure of example 9. MS [ M+H ]] ⁺ :3428.50。

EXAMPLE 11 preparation of Compound 22

Synthetic method reference of maleimide-modified valine-citrulline dipeptide (compound 22-1) for use in the present invention: gene M.D., et al cathepsin B-Labile Dipeptide Linkers for Lysosomal Release of Doxorubicin from Internalizing Immunoconj. Mu. Gates: model Studies of Enzymatic Dr. Mu. g Release and Antigen-Specific In Vitro Anticancer Activity. Bioconjugate chem.13 (4) 855-869 (2002).

The reaction of compound 22-1 with CBI is carried out according to the conventional method for preparing amide by reacting amino with carboxyl to obtain compound 22-2, and similarly to the preparation method of compound 19, compound 22-2 is reacted with mercapto of compound III to obtain compound 22.MS [ M+H ]] ⁺ ：3065.16。

EXAMPLE 12 preparation of Compound 23

Under pyridine conditions, according to methods conventional in the art (Boc) ₂ O the hydroxyl group at the SN-38 position was protected to give compound 23-2, and compound 23-2 (0.358 g,0.073 mmol), DMAP (0.266, 0.218 mmol) and triphosgene (0.0095 g,0.032 mmol) were added to the reaction flask, then 1.5ml of dichloromethane was added to initiate the reaction, TLC examined the progress of the reaction, and after the completion of the reaction, quenched with anhydrous methanol to give compound 23-3, which was used directly in the next reaction without workup.

Compound 20-8 (0.768 g,0.883 mmol) was added to the reaction solution of 23-3, the reaction was completed for about 5min, and the reaction solution was purified by flash chromatography (methanol/dichloromethane gradient elution) to obtain compound 23-4 with Boc protected at 20-position, which was then hydrolyzed with TFA to remove the protecting group, and recrystallized from diethyl ether to obtain TFA salt of the target compound 23-4.

The title compound 23 was prepared according to the scheme of scheme 6 by reacting compound 23-4 with cysteine-SH of compound III. MS [ M+H ] ] ⁺ ：3089.15。

EXAMPLE 13 preparation of Compound 24

Similar to the preparation of compound 15, compound 24 was prepared. MS [ M+H ]] ⁺ ：2946.23。

EXAMPLE 14 preparation of Compound 25

Similar to the preparation of compound 15, compound 25 was prepared. MS [ M+H ]] ⁺ ：2932.20。

EXAMPLE 15 preparation of Compound 26

A process for the preparation of analogous compound 15. MS [ M+H ]] ⁺ ：2918.22。

EXAMPLE 16 preparation of Compound 27

According to the general procedure described in the present invention, 1000mg of 2Cl-Trt Resin (1.5 eq) was added to a 50ml reaction flask, followed by 234mg of Fmoc-Cys (Trt) -OH (1.0 eq), 8ml of Dichloromethane (DCM), 640. Mu.L of DIEA (2.0 eq), dissolved completely, after 50min of reaction, 8ml of DCM, 1ml of MeOH, 1ml of DIEA were added to react for 20min, transferred to a self-made solid phase synthesis reaction column, after washing with DMF, 10ml of 20% piperidine DMF solution was added, 20min of reaction, after washing with DMF, the ninhydrin was monitored positive, 709mg of Fmoc-Lys (Fmoc) -OH (3.0eqiv,178mg HOBT (3.3 eq), 6ml of DMF was dissolved, 230. Mu.L of DIC (3.3 eq) was added to the reaction column, after 1H of reaction was monitored, 20% piperidine DMF solution was added after washing with DMF, the above steps were repeated, fmoc-Asp (OtBu) -OH, fmoc-Asp (OH, fmoc-FbF) were taken, fmoc-Asp (FbFbF) was obtained by sequential condensation ¹⁰ TFA-pteroic acid (wherein the amounts of amino acid and HOBT/DIC are 6eq and 6.6eq/6.6eq, respectively), adding 10ml of 2% hydrazine hydrate/DMF solution after condensation for reaction for 5 minutes, repeating 3 times, DMF washing, DCM washing, meOH washing, draining, adding a cleavage reagent after synthesis, and precipitating the polypeptide by TBME to obtain III-1, MS [ M ]] ⁺ :1839.65。

Compound 27 was prepared similarly to the preparation of compound 15 in example 4, except that III-1 was used instead of III in example 4. MS [ M+H ]] ⁺ ：2711.05。

EXAMPLE 17 Compound III-1'

Synthesis of the full protection peptide Asp (OtBu) -Asp (OtBu) -Arg (Pbf) -Asp (OtBu) -pteroic acid: the SPPS is sequentially condensed with Fmoc-Asp (OtBu) -OH, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, N by using 2Cl-Trt Resin ¹⁰ TFA-pteroic acid, after condensation, was reacted with 2% hydrazine hydrate in DMF for 5min, repeated 3 times, DMF wash, DCM wash, meOH wash, and suction. Adding 1% TFA/DCM solution for full protection cleavage for 5min, repeating for several times, TLC until no product is present, neutralizing the filtrate with pyridine, washing with water, washing with saturated saline, concentrating, and column chromatography to pure product of full protection peptide Asp (OtBu) -Asp (OtBu) -Arg (Pbf) -Asp (OtBu) -pteroic acid, MS [ M ]] ⁺ ：981.05。

III-1' Synthesis: to a 50ml reaction flask, 1000mg of 2Cl-Trt Resin (2.0 eq) was added, followed by 117mg of Fmoc-Cys (Trt) -OH (1.0 eq), 8ml of DCM, 320. Mu.L of DIEA (2.0 eq) were completely dissolved, 8ml DCM,1ml MeOH,1ml DIEA was added after 50min of reaction, transferred to a self-made solid phase synthesis reaction column after 20min of reaction, 10ml of 20% piperidine DMF solution was added after DMF washing, 20min of reaction, ninhydrin monitoring was positive after DMF washing, fmoc-Lys (ivDde) -OH (3.0 eq), 178mg of HOBT (3.3 eq) was taken, 6ml of DMF was dissolved, 230. Mu.L of DIC (3.3 eq) was added to the reaction column after mixing, and 20% piperidine DMF solution was added after 1h of reaction. The steps are repeated, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Glu-OtBu, N ¹⁰ TFA-pteroic acid, (wherein the amino acid and HOBT/DIC amounts are 3eq and 3.3eq/3.3eq, respectively), 10ml of 2% hydrazine hydrate/DMF solution is added to react for 5 minutes after condensation, DMF washing is repeated 3 times, ninhydrin monitoring is positive, and the compound { Asp (OtBu) -Asp (OtBu) -Arg (Pbf) -Asp (OtBu) -pteroic acid } (2.0 eq), HOAT (2.2 eq), DIC (2.2 eq) and DMF are added as solvents. After the reaction is carried out overnight, ninhydrin is monitored, DMF is washed after the reaction is finished, DCM is used for washing, meOH is used for washing, pumping is carried out, and after the synthesis is finished, a cleavage test is addedAnd (3) precipitating the polypeptide by TBME to obtain III-1'. MS [ M ]] ⁺ ：1968.69。

EXAMPLE 18 preparation of Compound 28

A similar procedure as for the preparation of compound 15 in example 4, compound 28, MS [ M+H ]] ⁺ ：2840.10。

EXAMPLE 19 preparation of Compound III-2

III-2 was prepared according to the method shown in scheme 2, and the specific steps are as follows:

preparation of compound c: into a 250ml reaction flask, 10g a,1.1eq of HOSU and 100ml of THF were added, and after dissolution, the ice bath was cooled to 0℃and 1.1eq of DCC was added and stirred overnight at room temperature. TLC starting material was reacted. Post-treatment: suction filtration, washing the filter cake with THF, and concentrating the organic phase to obtain 12g of compound b.

Into a 100ml reaction flask was added 3.0g of d, dissolved in 30ml of water, and 1.1eq of NaHCO was added ₃ Cooling to 10 ℃; 1.1eq of b was dissolved in 30ml of DME and added dropwise to the reaction flask, with 10ml of THF being added overnight at room temperature. After the TLC reaction is finished, EA is added after concentration, diluted hydrochloric acid is used for washing, then water washing, common salt water washing, drying and concentration are carried out, and the compound c is obtained through column chromatography.

III-2 preparation: to a 50ml reaction flask, 1000mg of 2Cl-Trt Resin (2.0 eq) was added, followed by 117mg of Fmoc-Cys (Trt) -OH (1.0 eq), 8ml of DCM, 320. Mu.L of DIEA (2.0 eq) were completely dissolved, 8ml DCM,1ml MeOH,1ml DIEA was added after 50min of reaction, transferred to a self-made solid phase synthesis reaction column after 20min of reaction, 10ml of 20% piperidine DMF solution was added after DMF washing, 20min of reaction, ninhydrin monitoring was positive after DMF washing, compound c (3.0 eq), 178mg of HOBT (3.3 eq), 6ml of DMF was dissolved, 230. Mu.L of DIC (3.3 eq) was added, mixed and then added to the reaction column, and after 1h of reaction ninhydrin monitoring, 20% piperidine DMF solution was added after DMF washing. The steps are repeated, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Glu-OtBu, N ¹⁰ TFA-pteroic acid (wherein, the dosage of amino acid and HOBT/DIC is 15eq and 16.5eq/16.5eq respectively), adding 10ml 2% hydrazine hydrate/DMF solution to react for 5 minutes after condensation, repeating 3 times, DMF washing, DCM washing, meOH washing, pumping, adding after synthesis Cleavage reagent, TBME precipitate polypeptide to obtain III-2, MS [ M+H ]] ⁺ ：3151.15。

EXAMPLE 20 preparation of Compound III-3

III-2 was prepared according to the method shown in scheme 3, comprising the following steps:

preparation of compound f: into a 250ml reaction flask, 10g a,1.1eq of HOSU and 100ml of THF were added, and after dissolution, the ice bath was cooled to 0℃and 1.1eq of DCC was added and stirred overnight at room temperature. TLC starting material was reacted. Post-treatment: suction filtration, washing the filter cake with THF, and concentrating the organic phase to obtain 12g of compound b.

Into a 100ml reaction flask, 3.0g of d was added, dissolved in 30ml of water, and 1.1eq of NaHCO was added ₃ Cooling to 10 ℃; 1.1eq of b was dissolved in 30ml of DME and added dropwise to the reaction flask, with 10ml of THF being added overnight at room temperature. After the TLC reaction is finished, EA (ethyl acetate) is added after concentration, and then the mixture is washed by dilute hydrochloric acid, and then by common salt water, dried and concentrated, and the compound c is obtained by column chromatography.

To a 10ml reaction flask was added 5g c,1.1eq of HOSU,50ml of THF, the ice bath was cooled to 0℃after dissolution, 1.1eq of DCC was added and stirred at room temperature overnight. TLC starting material was reacted. Post-treatment: suction filtration, washing of the filter cake with THF and concentration of the organic phase gave 6g of compound e.

Into a 100ml reaction flask was charged 3.0g of Compound e, dissolved in 30ml of water, 1.1eq of NaHCO was added ₃ Cooling to 10 ℃;1.1eq of b was dissolved in 30ml of DME and added dropwise to the reaction flask at room temperature overnight. After the TLC reaction is finished, EA is added after concentration, diluted hydrochloric acid is used for washing, then salt water is used for washing, drying and concentration are carried out, and the compound f is obtained through column chromatography.

III-3 preparation: into a 50ml reaction flask, 1000mg of 2Cl-Trt Resin (2.0 eq) was added, 117mg of Fmoc-Cys (Trt) -OH (1.0 eq) was added, 8ml of DCM, 320. Mu.L of DIEA (2.0 eq) was completely dissolved, after 50min of reaction, 8ml DCM,1ml MeOH,1ml DIEA was added, after 20min of reaction, transferred to a self-made solid phase synthesis reaction column, DMF was washed, then 10ml of 20% piperidine DMF solution was added, after 20min of reaction, after DMF washing, ninhydrin was monitored positive, compound f (3.0 eq), 178mg of HOBT (3.3 eq) was taken, after 6ml of DMF was dissolved, 230. Mu.L of DIC (3.3 eq) was added, and after mixing, the mixture was added to the reaction column, and reactedAfter 1h ninhydrin monitoring, a 20% piperidine DMF solution was added after DMF washing. The steps are repeated, fmoc-Asp (OtBu) -OH, fmoc-Arg (Pbf) -OH, fmoc-Asp (OtBu) -OH, fmoc-Glu-OtBu, N ¹⁰ After condensation of TFA-pteroic acid (15 eq and HOBT/DIC, respectively) and 16.5eq/16.5eq, 10ml of 2% hydrazine hydrate/DMF solution is added to react for 5 min, and the reaction is repeated for 3 times, DMF washing, DCM washing, meOH washing, pumping, adding a cleavage reagent after synthesis, and TBME precipitating the polypeptide to obtain the target compound III-3, MS [ M+H ] ] ⁺ ：4203.55。

Example 21 preparation of Compound 29

Compound 29 was prepared similarly to the preparation of compound 15 in example 4, except that III-2 was used instead of III in example 4.

MS[M+H] ⁺ ：4021.54。

Example 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 1 relative affinity test

FR positive KB cells were densely seeded on 20-well cell culture plates and the cells were allowed to adhere to plastic for 18h. Spent medium was placed in designated wells with and without increasing test article or folic acid concentration by 100nM ³ H-folate supplements folate-free RPMI (FFRPMI). The cells were incubated at 37℃for 60min and then rinsed 3 times with PBS pH 7.4. To each well was added 500. Mu.L of a PBS solution pH7.4 containing 1% Sodium Dodecyl Sulfate (SDS). Then, cell lysates were collected and added to a separate tube containing 5ml of fluorescent mixture, and then their radioactivity was counted. Negative control tubes contained only folic acid dissolved in FFRPMI (no competitor). Positive control tubes contained folic acid at a final concentration of 1mM, and CPM (representing labeled non-specific binding) measured in these samples was subtracted from all samples. Obviously, relative affinity is defined as the displacement of 50% of the FRs bound to KB cells ³ The inverse molar ratio of the desired compound of H-folic acid (inverse molar ratio), the relative affinity of folic acid for FR was set to 1.

The results of the relative affinity assay of compound 15 in 10% serum/FDRPMI indicate that compound 15 exhibits 157% relative affinity for the folate receptor compared to folic acid.

In a similar manner, the relative affinities of compounds 16-32 in 10% serum/FDRPMI were determined, and the test results showed that compounds 16-32 each exhibited more than 100% folate receptor relative affinity compared to folic acid, with compounds 20, 21, 31 exhibiting 162%, 127% and 187% folate receptor relative affinities, respectively.

Test example 2 cell Activity test

Cell activity assay 1:

compounds 15 to 32 of the invention were evaluated using an in vitro cytotoxicity assay that predicts the ability of the drug to inhibit growth of KB cells positive for the folate receptor, with 100 μl KB-containing cells 5X 10 per well inoculated ³ After 24h of culture, the medium is sucked and divided into a blank control group, a control group a and a test group b, the control group a and the test group b are respectively divided into 10 groups, the serial numbers of the control group a and the test group b are 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 and b-10, folic Acid (FA) 0, 0.1, 0.3, 1, 3, 10, 30, 100 and 300 micromoles (mu M) are respectively added into the control group a, the culture is continued for 2h, then a control compound BP111a (the preparation method and the structure of the compound EC145 see Chinese patent CN 100381177C) 1 mu M is respectively added into the test group b, 1 mu M is added into the control group a, and the blank group neither folic acid nor the folic acid is added into the control group a; the culture was continued for 70 hours, the cell viability was evaluated by MTT method, and the absorbance of each well was measured at OD570nm of ELISA.

MTT method is also called MTT colorimetric method, and its detection principle is that succinic dehydrogenase in mitochondria of living cells can reduce exogenous MTT into water-insoluble blue-violet crystal Formazan (Formazan) and deposit it in cells, while dead cells have no such function. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value (namely OD570 value) of the formazan can be measured at 5700nm wavelength by an enzyme-linked immunosorbent assay, so that the number of living cells can be indirectly reflected. The amount of MTT crystals formed is proportional to the number of cells over a range of cell numbers.

For example, the drug of test group b (shown by BP111 b) was compound 15, and the test results are shown in FIG. 2, wherein BP111b represents compound 15 of the present invention, and BP111a represents compound EC145 (see Chinese patent CN100381177C for the preparation of EC 145). As can be seen from fig. 2, under the same conditions, the inhibition of KB cells by BP111b (compound 15 of the present invention) was superior to that of compound BP111a (compound EC 145), and as FA increased, the inhibition of KB tumor cells by BP111b was decreased, indicating that the observed cell killing effect was mediated by binding to folate receptors. Further, BP111b has a better inhibitory activity against KB tumor cells than BP111a even in the presence of excess folic acid, as shown in FIG. 2.

Cell activity assay 2:

the assay predicts the ability of the drug to inhibit growth of folate receptor negative a549 cells, with 100 μl of a 549-containing cells seeded per well 5 x 10 ³ After 24h of culture, the medium is sucked and divided into a blank control group, a control group a and a test group b, wherein the control group a and the test group b are respectively divided into 10 groups, the numbers of the control group a and the test group b are 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 and b-10, the control group a and the test group b are respectively added with a control compound BP111a (EC 145, the preparation method of EC145 is shown in China patent CN 100381177C) and test compounds BP111b, 3333.33, 111.111, 370.370, 123.4567, 41.152, 13.717, 4.572, 13.717, 4.5724 and 1.5241 nanomole (nM), and the blank control group is added with neither BP111a nor BP111b; the culture was continued for 70 hours, the cell viability was evaluated by MTT method, and the absorbance of each well was measured at OD570nm of ELISA.

Taking compound 15 as an example of test group drug BP111b, the test results are shown in FIG. 3, in which BP111b (compound 15 of the present invention) has insignificant inhibitory activity on FR-negative tumor cell A549, and these results indicate that compound 15 acts through a folate-selective or folate-specific mechanism.

Similar results were obtained for all of the compounds 16 to 32 of the present invention in this type of assay, and in most cases, the compounds 16 to 32 provided by the present invention have significant inhibitory activity against KB cells and exhibit dose-dependent cytotoxicity, IC ₅₀ (IC ₅₀ I.e., half-inhibition, meaning the concentration of drug at which 50% tumor growth inhibition occurs) is in the low nanomolar range, as shown in table 1 below.

TABLE 1 data on KB cytostatic Activity of Compounds 16 to 32 (IC) ₅₀ ：nM)

Compounds of formula (I)	IC 50 (nM)	Compounds of formula (I)	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 3 test for inhibition of tumor growth in mice

When the compound 15 of the present invention (represented by BP111 b) was administered intravenously (i.v.) to tumor-bearing animals, the antitumor activity was evaluated on Balb/c mice of mice bearing subcutaneous KB tumors. Use 1 x 10 in right underarm subcutaneous tissue ⁶ KB cell tumorsAbout 11 days (t) ₀ Time average tumor volume = 60mm ³ ) The mice are divided into 9 groups, and 6 mice are arranged in each group, wherein the groups a-1, a-2 and a-3 are drug control groups a; b-1, b-2, b-3 are test groups b; the remaining three groups are blank control groups. Control group a was given 0.5. Mu. Mol/kg, 1. Mu. Mol/kg, 2. Mu. Mol/kg of control drug BP111a by intravenous injection, respectively; test group b was given 0.5. Mu. Mol/kg, 1. Mu. Mol/kg, 2. Mu. Mol/kg of compound BP111b (compound 15 of the invention) by intravenous injection, respectively; the blank group was given a comparable dose volume of PBS, intravenously injected 3 times per week for 2 weeks, and tumor growth was measured with calipers 2 times per week for each treatment group. With equation v=axb ² Tumor volume was calculated, where a is tumor length and b is width in millimeters. While animal weights were weighed with a balance 2 or 3 times per week.

As shown in figures 4 and 5, the compound BP111b of the invention can effectively delay KB tumor growth when being used for treating 0.5 mu mol/kg, and has very obvious anti-tumor activity when the dosage of the drug is increased to 1 mu mol/kg. Furthermore, as shown in FIG. 4, the inhibition of tumor by the compound BP111b of the present invention was dose-dependent, and the compound BP111b of the present invention showed better antitumor activity at the same dose as that of the control drug BP111 a. In addition, the compound BP111b provided by the invention has no obvious toxicity (based on animal weight), compared with the control drug BP111a, when the compound BP111a is also administered with 1 mu mol/kg, the control drug BP111a also has anti-tumor activity, but when the compound BP111b is administered for 18 days, the inhibition effect of BP111b on tumors is obviously better than that of BP111a, as shown in figure 4; the animals were found to be well tolerated at the doses administered, as was found to be BP111b and BP111a based on the weight change of the animals, as shown in figure 5.

Similar experiments were performed with compounds 16 to 32 of the present invention, and the test results found that: the compounds 16-32 have remarkable inhibition effect on the growth of the KB tumor of the mice positive to FR, and have no obvious toxicity accompanied with weight change.

Illustratively, compound 25 or free drug MMAF or a comparable dose volume of PBS (control) is administered at a dose of 500nmol/kg to the right underarm subcutaneous tissue at 1X 10 ⁶ About 11 days after KB cell tumor inoculation(t ₀ Time average tumor volume = 60mm ³ ) Mice (6/group) were given i.v. injections 3 times a week for 2 weeks, and after 2 weeks, the results of inhibition of tumor growth and changes in body weight were observed. Tumor growth was measured every 3 days with calipers for each treatment group. With equation v=axb ² Tumor volume was calculated, where a is tumor length and b is width in millimeters. While weighing the animals with a balance every 3 days.

The experimental results show that the treatment with the compound 25 of the invention is effective in delaying KB tumor growth without significant toxicity (based on animal body weight). Free drug MMAF also has antitumor activity, but has significant toxicity and poor animal tolerance.

In a similar manner, compound 25 was assayed for anti-tumor activity in FR-negative cells using 1 x 10 in right underarm subcutaneous tissue ⁶ A549 cell tumor post-inoculation (t) ₀ Time average tumor volume=50-80 mm ³ ) Mice (6/group) were given 500nmol/kg of compound 25 or a comparable dose volume of PBS (control) by intravenous injection 3 times per week for 2 weeks, and tumor growth was measured with callipers every 3 days for each treatment group. With equation v=axb ² Tumor volume was calculated, where a is tumor length and b is width in millimeters. The results of the assay showed that compound 25 had little activity against FR-negative cells, and these results indicated that compound 25 acted through a folate-selective or folate-specific mechanism.

Claims (4)

1. A compound having the general formula:

(F) _n LD, wherein n is 3 or 4,

f is folic acid or pteroic acid; (F) _n Each F of (a) is independently selected from folic acid and pteroic acid;

d is a drug moiety, wherein the drug is cyclopropyl benzoindolone, pyrrolobenzodiazepine dimer, 7-ethyl-10-hydroxycamptothecin, dolastatin, ecteinascidin B, auristatin, tube dissolvins and vinblastine;

said (F) _n Refers to eachF is covalently linked to the polyvalent linker of L, respectively, and D is covalently linked to L;

wherein (F) ₃ LD (F) ₄ LD includes the following compounds:

wherein D is selected from the drugs described hereinbefore; f (F) ₁ 、F ₂ 、F ₃ 、F ₄ Independently selected from:

L ₂ is a joint, having the following structure:

wherein W is selected from NH or O; * Indicating an open price.

2. A compound characterized by having the structure:

3. a pharmaceutical composition comprising a therapeutically effective amount of a compound of any one of claims 1-2, and a pharmaceutically acceptable carrier, diluent, or any combination thereof.

4. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1-2, in any combination with a pharmaceutically acceptable excipient.

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