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WO2022263627A1 - Polymer derivatives of mertansine and therapeutic uses thereof - Google Patents

Polymer derivatives of mertansine and therapeutic uses thereof Download PDF

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Publication number
WO2022263627A1
WO2022263627A1 PCT/EP2022/066552 EP2022066552W WO2022263627A1 WO 2022263627 A1 WO2022263627 A1 WO 2022263627A1 EP 2022066552 W EP2022066552 W EP 2022066552W WO 2022263627 A1 WO2022263627 A1 WO 2022263627A1
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Prior art keywords
polymer compound
mmol
formula
cancer
polymer
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PCT/EP2022/066552
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French (fr)
Inventor
Tanguy BOISSENOT
Alexandre BORDAT
Nada IBRAHIM
Amani MOUSSA
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Imescia
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Publication of WO2022263627A1 publication Critical patent/WO2022263627A1/en

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    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D498/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F20/00—Homopolymers and copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride, ester, amide, imide or nitrile thereof
    • C08F20/02—Monocarboxylic acids having less than ten carbon atoms, Derivatives thereof
    • C08F20/52—Amides or imides
    • C08F20/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
    • C08F20/56—Acrylamide; Methacrylamide

Definitions

  • the present invention relates to polymer derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4), particularly polyacrylamide derivatives of mertansine or of analogues thereof.
  • DM1 mertansine
  • analogues thereof e.g., DM4
  • the mertansine polymer derivatives of the invention are useful in the treatment of cancers.
  • the invention also relates to a manufacturing process of the mertansine polymer derivatives by RAFT polymerization, as well as to the corresponding mertansine RAFT agents.
  • HP APIs have extreme physicochemical properties that prevent their administration by classic routes. Particularly, they tend to be poorly soluble, which compromises intravenous administration. They may also present a poor intestinal permeability, limiting oral administration.
  • HP APIs have a very narrow therapeutic index. This means that the difference between the effective dose and the toxic dose is small. This, coupled with unfavorable pharmacokinetics ( e.g ., short half-life in blood), implies difficulties in providing controlled and prolonged exposure to effective but non-toxic doses.
  • HP APIs are under investigation in order to increase their bioavailability or to achieve controlled exposure with limited toxicity.
  • some anticancer HP APIs were formulated as nanoparticles, nanocrystals or as amorphous solids for oral or intravenous administration.
  • the use of prodrugs of HP APIs is also considered for enabling their development.
  • PEG polymer prodrugs did not enable to achieve satisfying results.
  • Antibody-drug conjugates are also considered for HPAPIs.
  • ADC use antibodies as vectors in order to target HP APIs directly towards tumors overexpressing particular receptors.
  • Mertansine also called DM1
  • DM1 is a thiol-containing maytansinoid having properties that make it fall into the category of HPAPIs.
  • mertansine is a tubulin inhibitor that induces mitotic arrest and kills tumor cells at sub-nanomolar concentrations. It was evidenced that mertansine has in vitro efficiencies 100 to 1000 times superior to other tubulin-binding cytotoxics such as vincristine or paclitaxel. Nevertheless, its systemic toxicity hinders its use under free form.
  • Mertansine ADCs are thus under investigations in order to overcome this toxicity issue, such as lorvotuzumab mertansine, bivatuzumab mertansine, or cantuzumab mertansine.
  • Mertansine was also linked to an antibody using the SMCC (4-(3-mercapto- 2,5-dioxo-l-pyrrolidinylmethyl)-cylohexanecarboxylic acid) linker, in which case the INN of the conjugate formed contains the word emtansine.
  • trastuzumab emtansine T-DM1
  • T-DM1 trastuzumab emtansine
  • MSC metastatic breast cancer
  • ADC technology it does not improve the toxicity profile of the HP API (no improvement in maximum tolerated dose relative to the free molecule) and it is limited to tumors that overexpress the targeted receptor. [0007] Therefore, there is a need for other forms of mertansine in order to expand its access to patients.
  • the Applicant herein provides polymer prodrugs of mertansine overcoming above drawbacks.
  • the polymer derivatives of mertansine according to the invention are based on the introduction of a hydrophilic polymeric moiety (e.g ., a polyacrylamide moiety) on mertansine.
  • W02019/097025 discloses polymer prodmgs for subcutaneous and/or intramuscular administration when the direct subcutaneous and/or intramuscular administration of the drug is problematic or impossible in particular because of the toxicity at the site of injection.
  • prodrugs comprising polymeric chains, such as polyacrylamide chains, were evidenced to be suitable for subcutaneous administration and to present reduced toxicity at the site of injection.
  • W02019/097025 is silent relative to a possible modification of mertansine by this method, particularly in order to favor its specific accumulation in tumors, whatever the type of administration.
  • the polymer derivatives of mertansine of the invention enable to achieve a prolonged and controlled exposition to mertansine with an important tumor accumulation, while limiting related toxicity, thereby increasing the therapeutic index compared to free mertansine.
  • This invention thus relates to a polymer compound of formula (A) or a pharmaceutically acceptable salt and/or solvate thereof, wherein m is 0 or 1 ;
  • L is a linker; preferably a linker which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties;
  • M is a hydrophilic polymeric moiety selected from polyacrylamide, polyacrylic acid, poly (N-(2-hydroxypropyl)methacrylamide) , poly (oligo(ethylene glycol)methyl ether methacrylate), poly(2-methacryloxyethyl phosphorylcholine), and copolymers thereof; and
  • R B is alkyl, aryl, or heteroaryl
  • R c is hydrogen, alkyl, aryl, or heteroaryl
  • R D is an optionally substituted aryl
  • R E and R F are independently selected from alkyl, arylalkyl, and dialky lpho sphory lalky 1. or a pharmaceutically acceptable salt and/or solvate thereof, wherein n is an integer ranging from 10 to 1400; preferably from 50 to 700.
  • the polymer compound of the invention is of formula (1-1) or a pharmaceutically acceptable salt and/or solvate thereof, wherein L 1 is selected from moieties (i), (ii), (iii) and (iv): wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L 2 ; and
  • L 2 is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties, or L 2 is absent.
  • the polymer compound of the invention is of formula (1-3) or a pharmaceutically acceptable salt and/or solvate thereof, wherein X is O, N or S; preferably X is O or N. or a pharmaceutically acceptable salt and/or solvate thereof, wherein R G is selected from -S-R B , -0-R B , -NR B R C and R°; preferably R G is -S-R B .
  • the polymer compound of the invention is selected from compounds Pl-Pll and pharmaceutically acceptable salts and/or solvates thereof.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polymer compound according to the invention and at least one pharmaceutically acceptable carrier.
  • the present invention further relates to the polymer compound or the pharmaceutical composition according to the invention, for use as a medicament.
  • the present invention further relates to the polymer compound or the pharmaceutical composition according to the invention, for use in the treatment of cancer; preferably breast cancer, pancreatic cancer, ovarian cancer or lung cancer.
  • the polymer compound or the pharmaceutical composition for use according to the invention is to be administered by parenteral route; preferably intravenously, subcutaneously, intramuscularly or intratumorally.
  • the invention also provides a method of manufacturing of a polymer compound of formula (I) or subformulae thereof according to the invention, comprising a step of controlled radical polymerization performed by contacting a source of radicals, acrylamide monomers and a compound of formula (B) or a salt and/or solvate thereof, under conditions suitable to obtain the polymer compound of formula (1-4); optionally followed by a step of removal or modification of the thiocarbonylthio terminal group, leading to the polymer compound of formula (I) according to the invention.
  • the invention also provides a functionalized RAFT agent of formula (B) or a salt and/or solvate thereof, wherein m is 0 or 1 ;
  • L is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; and R G is selected from -S-R B , -0-R B , -NR B R C , and R° wherein
  • R B is alkyl, aryl, or heteroaryl
  • R c is hydrogen, alkyl, aryl, or heteroaryl; and R D is an optionally substituted aryl.
  • the functionalized RAFT agent is selected from RAFT-1, RAFT-2, RAFT-3, RAFT-4, RAFT-5, RAFT-6, RAFT-7 and salts and/or solvates thereof.
  • administering refers to providing the compound, alone or as part of a pharmaceutically acceptable composition, to the subject in whom/which the condition, symptom, or disease is to be treated or prevented.
  • alkenyl refers to an unsaturated hydrocarbyl group, which may be linear or branched, wherein the unsaturation arises from the presence of one or more carbon- carbon double bonds. Suitable alkenyl groups comprise between 2 and 6 carbon atoms. Non-limiting examples of alkenyl groups are ethenyl, propenyl, butenyl, pentenyl and hexenyl.
  • Alkyl refers to a hydrocarbyl radical of formula CiFhi +i wherein “i” is a number greater than or equal to 1.
  • alkyl groups of this invention comprise from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 15 carbon atoms. Alkyl groups may be linear or branched.
  • the alkyl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like.
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl and its isomers (e.g., n-pentyl, iso-pentyl), hexyl and its isomers (e.g., n-hexyl, iso-hexyl), heptyl and its isomers (e.g., n-heptyl), octyl and its isomers (e.g., n-octyl), nonyl and its isomers (e.g., n-nonyl), decyl and its isomers (e.g., n-decyl), undecyl and its isomers (e.g., n-undecyl), dodecyl and its isomers (e.g
  • Alkylamino refers to a group -NH-alkyl wherein alkyl is as herein defined.
  • Alkoxy refers to a group -O-alkyl wherein alkyl is as herein defined.
  • Alkynyl refers to a monovalent unsaturated hydrocarbyl group, wherein the unsaturation arises from the presence of one or more carbon-carbon triple bonds. Alkynyl groups typically, and preferably, have the same number of carbon atoms as described above in relation to alkenyl groups. Non limiting examples of alkynyl groups are ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl and its isomers, 2-hexynyl and its isomers- and the like. [0031] “Amidine” refers to the group .
  • Amino refers to the group -Nth.
  • Aryl refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e., phenyl) or multiple aromatic rings fused together (e.g., naphthyl) or linked covalently, typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic.
  • the aryl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like.
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3
  • Arylalkyl refers to a group -alkyl-aryl wherein aryl and alkyl are as herein defined.
  • Carboxy refers to the group -COOH.
  • Controlled radical polymerization refers to a living polymerization where the active polymer chain end is a free radical.
  • Controlled radical polymerization includes the following techniques: atom transfer radical polymerization (ATRP), reversible addition/fragmentation chain transfer polymerization (RAFT), and nitroxide-mediated polymerization (NMP).
  • ATRP atom transfer radical polymerization
  • RAFT reversible addition/fragmentation chain transfer polymerization
  • NMP nitroxide-mediated polymerization
  • the controlled radical polymerization particularly refers to RAFT polymerization.
  • Cycloalkyl refers to a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 or 2 cyclic structures. Cycloalkyl includes monocyclic or bicyclic hydrocarbyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention, comprise from 3 to 10, more preferably from 3 to 8 carbon atoms still more preferably from 3 to 6 carbon atoms.
  • the cycloalkyl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like.
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)
  • substituent(s) for example 1 to 4 substituent(s), or for example
  • Halo or ‘halogen” refers to fluoro, chloro, bromo, or iodo.
  • Haloalkoxy refers to a group -O-haloalkyl wherein haloalkyl is as herein defined.
  • Haloalkyl refers to an alkyl group as herein defined wherein one or more hydrogen atoms are replaced with a halogen as defined above.
  • Non-limiting examples of such haloalkyl radicals include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoro methyl, 1,1,1-trifluoroethyl and the like.
  • Heteroaryl refers to a 5 to 12 carbon-atom aromatic ring or ring system containing 1 to 2 rings which are fused together or linked covalently, typically containing 5 to 6 atoms on each ring; at least one of which is aromatic and in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
  • Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring.
  • Non-limiting examples of such heteroaryl groups include: triazolyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2,l-b][l,3]thiazolyl, thieno[3,2-b]furanyl, thieno [3 ,2-b] thiophenyl, thieno[2,3-d] [ 1 ,3 ]thiazolyl, thieno[2,3-d]imid
  • Heteroatom refers to one or more of oxygen, nitrogen, sulfur, phosphorus, selenium, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, selenium, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl)).
  • Heterocyclyl refers to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 7 member monocyclic, 7 to 1 1 member bicyclic, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring.
  • Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized.
  • the heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows.
  • the rings of multi-ring heterocycles may be fused, bridged and/or joined through one or more spiro atoms.
  • the heterocyclyl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like.
  • Non limiting exemplary heterocyclic groups include aziridinyl, oxiranyl, thiiranyl, piperidinyl, azetidinyl, 2- imidazolinyl, pyrazolidinyl imidazolidinyl, isoxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, succinimidyl, 3H-indolyl, indolinyl, isoindolinyl, 2H-pyrrolyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 4H- quinolizinyl, 2-oxopiperazinyl, piperazinyl, homopiperazinyl, 2-pyrazolinyl, 3-pyrazolinyl, tetrahydro-2H-pyranyl, 2H-pyranyl, 4
  • “Human” refers to a subject of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult).
  • “Hydroxyalkylamino” refers to a group -NH-alkyl-OH wherein alkyl is as herein defined.
  • “Pharmaceutically acceptable” refers to ingredients that are compatible with each other and not deleterious to the subject to which they are administered thereof.
  • “Pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio.
  • Pharmaceutically acceptable salts are well known in the art.
  • Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and bases. Suitable acid addition salts are formed from acids which form non-toxic salts.
  • Examples include the acetate, adipate, ammonium, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, bitartrate/tartrate, borate, bromide, calcium edetate, camsylate, chloride, citrate, clavulanate, cyclamate, dihydrochloride, edetate, edisylate, estolate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hibenzate, hydrochloride/chloride, hydrabamine, hydrobromide/bromide, hydroiodide/iodide, hydroxynaphthoate, isethionate, lactate, lactobionate, laurate, malate, maleate
  • Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
  • salts of the compounds of the invention are preferred, it should be noted that the invention in its broadest sense also includes non-pharmaceutically acceptable salts, which may for example be used in the isolation and/or purification of the compounds of the invention.
  • non-pharmaceutically acceptable salts which may for example be used in the isolation and/or purification of the compounds of the invention.
  • salts formed with optically active acids or bases may be used to form diastereoisomeric salts that can facilitate the separation of optically active isomers of the compounds of the invention.
  • “Pharmaceutically acceptable carrier” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human.
  • inactive substances such as for example solvents, cosolvents, antioxidants, surfactants, stabilizing agents, emulsifying agents, buffering agents, pH modifying agents, preserving agents (or preservating agents), antibacterial and antifungal agents, isotonifiers, granulating agents or binders, lubricants, disintegrants, glidants, diluents or fillers, adsorbents, dispersing agents, suspending agents, coating agents, bulking agents, release agents, absorption delaying agents, sweetening agents, flavoring agents and the like.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, e.g., FDA Office or EMA.
  • Polyacrylamide or “PAAm” refers to a polymer or a polymeric moiety formed from acrylamide monomers, of formula:
  • Polymer compound refers to a compound comprising a polymeric moiety, i.e., a moiety made of several repeating subunits.
  • the polymer compound of the present invention preferably comprises a hydrophilic polymeric moiety, such as a polyacrylamide moiety, as polymeric moiety.
  • Prodrug refers to a pharmacologically acceptable derivative of a drug whose in vivo biotransformation generates the biologically active drug.
  • Solvate refers to a compound in the invention that contains stoichiometric or sub-stoichiometric amounts of one or more pharmaceutically acceptable solvent molecule such as ethanol or water.
  • solvent molecule such as ethanol or water.
  • hydrate refers to when the said solvent is water.
  • Subject refers to a mammal, preferably a human.
  • a subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease
  • “Therapeutically effective amount” or “therapeutically effective dose” refer to the amount or dose of active ingredient that is aimed at, without causing significant negative or adverse side effects to the subject, (1) delaying or preventing the onset of a cancer in the subject; (2) reducing the severity or incidence of a cancer; (3) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of a cancer affecting the subject; (4) bringing about ameliorations of the symptoms of a cancer affecting the subject; or (5) curing a cancer affecting the subject.
  • a therapeutically effective amount may be administered prior to the onset of a cancer for a prophylactic or preventive action. Alternatively, or additionally, a therapeutically effective amount may be administered after initiation of a cancer for a therapeutic action.
  • “Treatment” or “treating” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder (e.g ., cancer).
  • Those in need of treatment include those already with the disorder (e.g., cancer) as well as those prone to have the disorder (e.g., cancer) or those in whom the disorder (e.g., cancer) is to be prevented.
  • a subject is successfully "treated” for cancer if, after receiving a therapeutic amount of a polymeric compound according to the present invention, the subject shows observable and/or measurable occurrence of one or more of the following: (1) reduction in the number of cancer cells; (2) reduction of tumor size; (3) relief to some extent one or more of the symptoms associated with cancer; (4) reduced morbidity and mortality; and/or (5) improvement in quality of life issues.
  • the above parameters for assessing successful treatment and improvement in the targeted disease are readily measurable by routine procedures familiar to a physician.
  • the present disclosure also includes compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13 C- or 14 C-enriched carbon are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present disclosure.
  • This invention thus relates to polymer derivatives of mertansine (DM1) or of analogues thereof (e.g ., DM4).
  • the polymer compounds of the invention are derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4) comprising a polymeric moiety, preferably a hydrophilic polymeric moiety.
  • the polymer compound of the invention are polyacrylamide derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4).
  • DM1 mertansine
  • analogues thereof e.g., DM4
  • the introduction of a hydrophilic polymeric moiety (e.g., a polyacrylamide moiety) on mertansine leads to the formation of a prodrug thereof.
  • the introduction of the hydrophilic polymeric moiety (e.g., polyacrylamide moiety) on mertansine advantageously leads to an increased accumulation in the tumor rather than in other organs. This enables to limit deleterious side effects of mertansine.
  • the introduction of the hydrophilic polymeric moiety (e.g., polyacrylamide moiety) on mertansine advantageously increases its circulating time which is remedi to the pharmacokinetic properties.
  • hydrophilic polymeric moiety e.g., polyacrylamide moiety
  • DM1 hydrophilic polymeric moiety
  • DM4 differs from DM1 only on the alkyl arm bearing the free thiol group.
  • the free thiol group of DM 1 or DM4 can be modified to form derivatives thereof, including prodmgs such as the polymer compounds of the invention.
  • the polymeric moiety is introduced by covalent coupling on the free thiol group.
  • the polymeric moiety may be introduced through a linker on the thiol group.
  • a terminal group may also be present at the terminal end of the polymeric moiety.
  • the polymer compound of the invention is of formula (A): or a pharmaceutically acceptable salt and/or solvate thereof, wherein: m is 0 or 1 ;
  • L is a linker
  • M is a polymeric moiety; preferably M is a hydrophilic polymeric moiety; and R A is a terminal group.
  • the polymer derivatives of mertansine (DM1) or of analogues thereof (e.g ., DM4) according to the invention comprise a polymeric moiety.
  • the polymeric moiety is a hydrophilic polymeric moiety, such as for example a polyacrylamide moiety, a copolymer thereof or a derivative thereof.
  • polymeric moiety it is referred to a moiety made of several repeating subunits.
  • the polymeric moiety can be made from a single monomer or from two or more monomers, and in such case, it can be referred to as a copolymeric moiety.
  • references made to “polymeric moieties” include copolymeric moieties.
  • hydrophilic polymeric moiety it is referred to a polymeric moiety that is typically charge-polarized and capable of hydrogen bonding, enabling it to dissolve more readily in water than in oil or other hydrophobic solvents.
  • hydrophilic polymeric moieties include polyacrylamide (PAAm), polyacrylic acid (PAAc), poly(N- (2-hydroxypropyl)methacrylamide) (PHPMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(2-methacryloxyethyl phosphorylcholine) (PMPC), poly(ethylene glycol) (PEG) and copolymers thereof.
  • PAAm polyacrylamide
  • PAAc polyacrylic acid
  • PPMA poly(N- (2-hydroxypropyl)methacrylamide)
  • POEGMA poly(oligo(ethylene glycol)methyl ether methacrylate)
  • PMPC poly(2-methacryloxyethyl phosphorylcholine)
  • PEG poly(ethylene glycol)
  • the polymeric moiety M is selected from polyacrylamide (PAAm), polyacrylic acid (PAAc), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(2-methacryloxyethyl phosphorylcholine) (PMPC), poly(ethylene glycol) (PEG) and copolymers thereof.
  • PAAm polyacrylamide
  • PAAc polyacrylic acid
  • PPMA poly(N-(2-hydroxypropyl)methacrylamide)
  • POEGMA poly(oligo(ethylene glycol)methyl ether methacrylate)
  • PMPC poly(2-methacryloxyethyl phosphorylcholine)
  • PEG poly(ethylene glycol) and copolymers thereof.
  • the polymeric moiety M is selected from polyacrylamide (PAAm), polyacrylic acid (PAAc), poly(N-(2- hydroxypropyl)methacrylamide) (PHPMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(2-methacryloxyethyl phosphorylcholine) (PMPC), and copolymers thereof.
  • PAAm polyacrylamide
  • PAAc polyacrylic acid
  • PEGMA poly(N-(2- hydroxypropyl)methacrylamide)
  • POEGMA poly(oligo(ethylene glycol)methyl ether methacrylate)
  • PMPC poly(2-methacryloxyethyl phosphorylcholine)
  • the polymeric moiety M is polyacrylamide (PAAm) or a copolymer thereof.
  • the polymeric moiety M does not comprise or consists of an amphiphilic polymeric moiety.
  • amphiphilic polymeric moiety it is referred to a polymeric moiety with both hydrophilic and lipophilic properties.
  • the polymeric moiety M does not comprise or consists of a polylactic acid (PLA) moiety.
  • the polymeric moiety M does not comprise or consists of polylactic acid or a copolymer thereof.
  • the polymeric moiety M does not comprise or consists of hyaluronic acid.
  • the polymeric moiety M does not comprise or consists of polyethylene glycol (PEG).
  • the polymeric moiety M does not comprise or consists of a polyether.
  • the polymer derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4) according to the invention comprise a polyacrylamide moiety, i.e., a moiety formed by the polymerization of acrylamide monomers.
  • the polymer compound of the invention is of formula (I): or a pharmaceutically acceptable salt and/or solvate thereof, wherein: m is 0 or 1; n is an integer ranging from 10 to 1400; L is a linker; and
  • R A is a terminal group.
  • the choice of the size of the polymeric moiety e.g., the polyacrylamide moiety, enables to vary the overall solubility of the polymer compound. It also impacts the absorption rate of the polymer compound.
  • the size of the polymeric moiety e.g., the polyacrylamide moiety, is ranging from 1 000 kDa to 100000 kDa in order to increase the absorption rate of the polymer compound.
  • the polymeric moiety e.g., the polyacrylamide moiety
  • the polymeric moiety is preferably obtained by controlled radical polymerization, for example by reversible addition-fragmentation chain-transfer (RAFT) polymerization, by atom transfer radical polymerization (ATRP) or by nitroxide-mediated polymerization (NMP).
  • RAFT reversible addition-fragmentation chain-transfer
  • ATRP atom transfer radical polymerization
  • NMP nitroxide-mediated polymerization
  • the synthesis of the polymer compounds of the invention is advantageously performed by the method of drug-initiated polymerization, as detailed hereafter. This method enables to control the size of the polymer and to ensure that only one DM1 or DM4 is present per polymer compound.
  • the polymeric moiety, e.g., the polyacrylamide moiety, of the polymer compound of the invention is a simple linear chain.
  • the number of acrylamide subunits in the polymer compound of the invention is ranging from 10 to 1 400; preferably from 50 to 700; more preferably from 70 to 500; even more preferably from 280 to 600.
  • n is an integer ranging from 10 to 1 400; preferably from 50 to 700; more preferably from 70 to 500; even more preferably from 280 to 600.
  • the polymer compound of the invention has a molecular weight ranging from 0.7 to 100 kDa; preferably from 3.5 to 50 kDa; more preferably from 5 to 35.5 kDa; even more preferably from 20 to 43 kDa.
  • the polymer compound of the invention comprises a terminal group at the terminal end of the polymeric moiety (e.g ., the polyacrylamide moiety).
  • the terminal group may be of any type and is typically selected depending on the physicochemical and biological properties which are sought when modifying mertansine or its analogues. Particularly, the choice of the terminal group enables to control the capacity of the polymer compound to enter into the tumor.
  • the terminal group R A of the polymer compounds of the invention comprises a moiety introduced during the polymerization of the polymeric moiety and being characteristic of the polymerization method.
  • moieties characteristic of the polymerization method can be removed or modified by methods known by one skilled in the art, leading to other terminal groups of interest.
  • the polymer compound of the invention can be obtained by RAFT polymerization.
  • S thiocarbonylthio group
  • the presence of a thiocarbonylthio group in the polymer compound of the invention does not adversely affect its properties and there is thus no need to remove this residue of polymerization. Nevertheless, the thiocarbonylthio group can be removed or modified by various methods, leading to modified terminal groups, depending on the targeted properties of the polymer compound.
  • the terminal group R A is typically of formula (a), (b), (c) or (d): wherein: represents the point of attachment to the polymeric moiety (e.g., the polyacrylamide moiety);
  • R B is alkyl, aryl or heteroaryl
  • R c is hydrogen, alkyl, aryl or heteroaryl
  • R D is an optionally substituted aryl.
  • R B is alkyl, preferably a C2-C20 alkyl, more preferably a C2-C12 alkyl. In one embodiment, R B is ethyl. In one embodiment, R B is butyl. In one embodiment, R B is dodecyl.
  • R c is hydrogen. In another embodiment, R c is alkyl, preferably a C2-C20 alkyl, more preferably a C2-C12 alkyl. In one embodiment, R c is ethyl. In one embodiment, R c is butyl. In one embodiment, R c is dodecyl.
  • R D is phenyl or substituted phenyl, e.g., substituted by cyano.
  • the thiocarbonylthio terminal groups can be removed or modified by several methods known by one skilled in the art (for example, it can be referred to Moad, G. et ah, Polymer International, 2011, Vol. 60, pp. 9-25; and Willcock, H. et ah, Polymer Chemistry, 2010, Vol. 1, pp. 149-157).
  • thiocarbonylthio terminal groups transformations include, without being limited to: reaction with nucleophiles, leading to the replacement of the thiocarbonylthio terminal group by a thiol group (-SH), which can further react in coupling reactions such as disulfide formation and Michael addition; thermolysis, leading to the elimination of the thiocarbonylthio group and thereby to the formation of an unsaturated end-chain at the extremity of the polymeric moiety; radical-induced reduction, leading to the replacement of the thiocarbonylthio terminal group by a hydrogen; addition-fragmentation-coupling, leading to the replacement of the thiocarbonylthio terminal group by a group brought under the form of a radical, usually using a functionalized azo-initiator; and hetero Diels-Alder reaction, leading to transformation of the thiocarbonylthio terminal group with a dienophile.
  • thiocarbonylthio terminal groups transformations include, without being limited to: reaction with nucle
  • ACVA 4,4'-azobis(4-cyanovaleric acid)
  • V-50 2,2'-azobis(2-methylpropionamidine) dihydrochloride
  • VA-086 2,2'-azobis [2-methyl- N- (2-hydroxyethyl)propionamide]
  • VA-044 2,2'-Azobis[2-(2-imidazolin-2- yl)propane] dihydrochloride
  • V-65 2,2'-azobis(2,4-dimethylvaleronitrile)
  • V-70 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile)
  • the terminal group R A is selected from: wherein --- represents the point of attachment to the polymeric moiety (e.g., the polyacrylamide moiety.
  • the polymer compound of the invention can alternatively be obtained by ATRP polymerization.
  • the terminal group R A is typically a halogen atom, such as bromine or fluorine.
  • Such terminal groups can be modified, for example to form copolymers.
  • the polymer compound of the invention can also be obtained by NMP polymerization.
  • the terminal group R A is typically a nitroxide group, for example of formula -0-NR E R F wherein R E and R F are independently selected from alkyl, arylalkyl, and dialkylphosphorylalkyl.
  • Such terminal groups can be modified, for example to form copolymers.
  • the polymer compound of the invention comprises a linker which links DM1 or an analog thereof to the polymeric moiety, e.g., the polyacrylamide moiety.
  • the linker may be of any type and is typically selected depending on the physicochemical and biological properties which are sought when modifying mertansine or its analogues.
  • the linker may be any chemical chain of at least two covalently linked atoms, which can comprise heteroatoms (e.g., O, NH, S, Se or P) as well as cyclic moieties such as cycloalkyl or heterocyclyl groups.
  • the linker may comprise up to 100 carbon atoms and even more. The length and the chemical nature of the linker may be optimized depending on the biological effect which is sought. Particularly, the nature of the linker impacts the kinetics of release of the drug and thus the efficacy of the compound. The choice of the linker also enables to control the capacity of the polymer compound to enter into the tumor.
  • L comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties.
  • L is a chemical chain comprising from 2 to 100 carbon atoms, preferably from 2 to 50 carbon atoms, from 2 to 30 carbon atoms, e.g., from 5 to 25 carbon atoms, or from 5 to 20 carbon atoms.
  • L may be selected from alkyl (e.g., Ci-20 alkyl, Ci-12 alkyl or Ci- 6 alkyl), ether, polyether, alkyl amide, alkylene diamide or a combination thereof.
  • alkyls is contemplated, including, but not limited to, -(CH2) P- , wherein “p” is from about 2 to about 20 or more.
  • L comprises a C2-20 straight or branched alkyl chain.
  • L is a polyether (e.g., polyethylene or polypropylene glycol).
  • PEG polyethylene glycol
  • p is an integer from 1-10
  • PEG polyethylene glycol
  • PEG polypropylene glycol
  • L is an alkyl amide.
  • alkyl amides include, but not limited to, -(CH 2 ) m- C(0)NH-(CH 2 ) P- and -(0CH 2 CH 2 ) P- C(0)NH-(0CH 2 CH 2 )q-, wherein “p” and “q” can be the same or different and “p” and “q” are from about 1 to about 20 or more.
  • L may also comprise an alkylene diamine, e.g., -NH-(CH2) r -NH-, where “r” is an integer from 2 to 20, for instance from 2 to 10, or an integer selected from 2, 3, 4, or 5.
  • L may comprise one or more cyclic moieties such as cycloalkyl, heterocyclyl, aryl, or heteroaryl groups.
  • L comprises an optionally substituted cycloalkyl group, such as cyclohexyl.
  • L comprises an optionally substituted heterocyclyl group, such as succinimidyl.
  • the polymer compound of the invention can be obtained by RAFT polymerization.
  • the linker L typically comprises moieties usually found in chain-transfer agents (RAFT agents), such as for example: wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety (e.g., the polyacrylamide moiety) of the polymer compound of the invention.
  • RAFT agents chain-transfer agents
  • L is of formula -L 2 -L 1 - wherein: L 2 links to the thiol group of DM1 or DM4 and L 1 links to the polymeric moiety
  • L 1 is selected from moieties (i), (ii), (iii) and (iv): wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L 2 ; and
  • L 2 is a linker, which preferably comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; or L 2 is absent. [0106] In one embodiment, L is of formula: wherein
  • L 3 is a linker, which preferably comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; [0107] In one embodiment, L 3 is -L 3 ’-L 1 - wherein
  • L 3 ’ links to the succinimide group and is a linker, which preferably comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; and L 1 is as defined above, wherein the carbonyl group links to L 3 ’.
  • linkers and/or to linker L apply similarly to linkers L 2 , L 3 and L 3 ’.
  • L is selected from:
  • -- - represents the points of attachment; wherein the point of attachment on the left is to the thiol group of DM1 or DM4 and the point of attachment on the right is to the polymeric moiety (e.g., the polyacrylamide moiety).
  • the polymer compound of the invention can alternatively be obtained by ATRP polymerization.
  • the linker L typically comprises moieties usually found in ATRP initiators, such as for example: (iii); as already listed above for RAFT polymerization, wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety (e.g., the polyacrylamide moiety) of the polymer compound of the invention.
  • the polymer compound of the invention can alternatively be obtained by NMP polymerization.
  • the linker L typically comprises moieties usually found in NMP initiators, such as for example: (iii); as already listed above for RAFT polymerization, wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety (e.g ., the polyacrylamide moiety) of the polymer compound of the invention.
  • NMP initiators such as for example: (iii); as already listed above for RAFT polymerization, wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety (e.g ., the polyacrylamide moiety) of the polymer compound of the invention.
  • the polymer compound according to the invention is of formula (1-1): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, and R A are as defined and described in classes and subclasses above and herein;
  • L 1 is selected from moieties (i), (ii), (iii) and (iv): wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L 2 ; and
  • L 2 is a linker, defined and described in classes and subclasses above and herein; or L 2 is absent.
  • the polymer compound according to the invention is of formula (I-li): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, L 2 , and R A are as defined and described in classes and subclasses above and herein.
  • the polymer compound according to the invention is of formula (1-2): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, and R A are as defined and described in classes and subclasses above and herein;
  • L 3 is a linker, defined and described in classes and subclasses above and herein.
  • the polymer compound according to the invention is of formula (1-3): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, and R A are as defined and described in classes and subclasses above and herein; and
  • X is O, N or S; preferably X is O or N.
  • X is O. In one embodiment, X is N. In one embodiment, X is S.
  • the polymer compound according to the invention is of formula (1-4): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m and n are as defined and described in classes and subclasses above and herein; and
  • R G is selected from -S-R B , -0-R B , -NR B R C and R°, wherein R B , R c and R D are as defined and described in classes and subclasses above and herein.
  • the polymer compound of the invention is of formula (I- a): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, L and R B are as defined and described in classes and subclasses above and herein.
  • the polymer compound according to the invention is of formula (I-l-a): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n and R B are as defined and described in classes and subclasses above and herein;
  • L 1 is selected from moieties (i), (ii), (iii) and (iv): wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L 2 ; and
  • L 2 is a linker, defined and described in classes and subclasses above and herein; or L 2 is absent.
  • the polymer compound according to the invention is of formula (I-li-a): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, L 2 and R B are as defined and described in classes and subclasses above and herein.
  • the polymer compound according to the invention is of formula (I-2-a): or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n and R B are as defined and described in classes and subclasses above and herein;
  • L 3 is a linker, defined and described in classes and subclasses above and herein.
  • the polymer compound according to the invention is of formula (1-3 -a):
  • X is O, N or S; preferably X is O or N.
  • the polymer compound according to the invention is selected from:
  • the present invention also relates to methods of manufacturing the polymer compounds according to the invention.
  • the polymer compounds of the invention can be manufactured by radical polymerization, preferably by controlled radical polymerization, for example by reversible addition-fragmentation chain-transfer (RAFT) polymerization, by atom transfer radical polymerization (ATRP) or by nitroxide-mediated polymerization (NMP).
  • RAFT reversible addition-fragmentation chain-transfer
  • ATRP atom transfer radical polymerization
  • NMP nitroxide-mediated polymerization
  • drug-initiated polymerization it is referred to a controlled radical polymerization technique using a control agent (such as a RAFT agent in a RAFT polymerization, or ATRP or NMP initiator in ATRP and NMP polymerizations) modified by chemical coupling with an active ingredient.
  • a control agent such as a RAFT agent in a RAFT polymerization, or ATRP or NMP initiator in ATRP and NMP polymerizations
  • the modified control agent carries an active ingredient molecule, and the resulting polymer also carries the active ingredient.
  • the polymer compounds of the invention are manufactured by reversible addition-fragmentation chain-transfer (RAFT) polymerization.
  • RAFT reversible addition-fragmentation chain-transfer
  • a RAFT polymerization system usually comprises: a source of radicals
  • radical polymerization initiators e.g., radical polymerization initiators
  • monomers e.g., monomers
  • RAFT agent e.g., RAFT agent
  • solvent e.g., solvent
  • radical polymerization initiator it is referred to compounds used to produce radicals and thus initiate radical polymerization. These compounds have a chemical function capable of releasing radicals under the action of heat, irradiation of light, oxidation-reduction reaction, ionizing radiation, electrochemical reactions and/or sonication.
  • Non-limiting examples of initiators include compounds of the azo type, such as 2,2'-azobis(2-methylpropionitrile) (also known as azobisisobutyronitrile, abbreviated as AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), l,T-azobis(cyclohexanecarbonitrile); of the inorganic peroxide type; or of the organic peroxide type such as benzoyl peroxide, lauroyl peroxide, methyl ethyl ketone peroxide, and tert-butyl peroxybenzoate.
  • azo type such as 2,2'-azobis(2-methylpropionitrile) (also known as azobisisobutyronitrile, abbreviated as AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), l,T-azobis(cyclohexanecarbonitrile)
  • AIBN azobisisobutyronitrile
  • ACVA
  • RAFT polymerization involves the use of a chain-transfer agent, named “RAFT agent”, to mediate the polymerization via a reversible chain-transfer process and to afford control over the generated molecular weight and polydispersity during a free-radical polymerization.
  • the RAFT agent comprises the active ingredient, herein mertansine or an analogue thereof.
  • RAFT agents suitable for the manufacturing of the polymer compounds according to the invention are detailed hereafter. Particularly, functionalized RAFT agent of formula (II), and subformulae thereof, are suitable for the manufacturing of the polymer compounds of the invention.
  • the polymer compounds contain a polyacrylamide moiety. Therefore, the monomer used for their manufacturing is acrylamide monomer.
  • method of manufacturing of a polymer compound according to the invention comprises a step of controlled radical polymerization performed by contacting a source of radicals, acrylamide monomers and a compound of formula (B): or a salt and/or solvate thereof, wherein m and L are as defined and described in classes and subclasses above and herein; and
  • R G is selected from -S-R B , -0-R B , -NR B R C and R°, wherein R B , R c and R D are as defined and described in classes and subclasses above and herein; under conditions suitable to obtain the polymer compound of formula (1-4) as herein defined.
  • the polymer compound of formula (I) can be obtained from the compound of formula (1-4) by the removal or modification of the thiocarbonylthio terminal group. Methods of such removal or modifications are known by one skilled in the art (for example, it can be referred to Moad, G. et al, Polymer International, 2011, Vol. 60, pp. 9-25; and Willcock, H. et ah, Polymer Chemistry, 2010, Vol. 1, pp. 149-157).
  • the method of manufacturing of the invention advantageously makes it possible to provide a polymer compound having only one mertansine (or analogue thereof) residue at one end. This control of the drug loading facilitates the purification of the polymer compound and is essential for therapeutic use.
  • the source of radicals is a radical polymerization initiator.
  • the radical polymerization initiator is selected from compounds of the azo type, such as azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), 1 , 1 '-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50), 2,2'-azobis[2-methyl-/V-(2-hydroxyethyl)propionamide] (VA- 086), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), 2,2'- azobis(2,4-dimethylvaleronitrile) (V-65), 2,2'-azobis(4-methoxy-2,4- dimethylvaleronitrile) (V-70); of the inorganic peroxide type; or
  • the amount of acrylamide monomers used in the method of manufacturing of the invention depends on the expected length of the polymer and can be estimated by one skilled in the art accordingly. As an illustration, it is referred to the synthesis of polymer compounds (PI) to (P3) reported in the Example section.
  • the temperature of polymerization is selected such that (a) chain growth occurs at an appropriate rate, (b) the radical polymerization initiator delivers radicals at an appropriate rate and (c) the RAFT equilibrium favors the active rather than dormant state to an acceptable extent.
  • the temperature of the controlled radical polymerization of the method of the invention ranges from 10 °C to 100 °C, preferably from 50°C to 100°C, preferably about 70°C.
  • the polymer compound obtained by the method of manufacturing of the invention is purified by precipitation and/or by dialysis.
  • Precipitation may be conducted in alcohol solvents such as for example methanol.
  • Dialysis may be conducted after solubilization of the polymerization product in water using a semipermeable membrane of suitable pore size (e.g., from 1 kDa to 15 kDa, preferably from 1 kDa to 10 kDa).
  • suitable pore size e.g., from 1 kDa to 15 kDa, preferably from 1 kDa to 10 kDa.
  • the invention further relates to compounds which are useful for the manufacturing of the polymer compounds of the invention.
  • the invention provides RAFT agents functionalized with mertansine or an analogue thereof (e.g., DM4).
  • the present invention provides compounds of formula (B): or a salt and/or solvate thereof, wherein m, L and R G are as defined and described in classes and subclasses above and herein.
  • compounds are of formula (B-a): or a salt and/or solvate thereof, wherein m, L and R B are as defined and described in classes and subclasses above and herein.
  • compounds are of formula (B-l-a): or a salt and/or solvate thereof, wherein m, L 1 , L 2 and R B are as defined and described in classes and subclasses above and herein.
  • compounds are of formula (B-li-a): -li-a) or a salt and/or solvate thereof, wherein m, L 2 and R B are as defined and described in classes and subclasses above and herein.
  • compounds are of formula (lib): or a salt and/or solvate thereof, wherein m, L 3 and R B are as defined and described in classes and subclasses above and herein.
  • compounds are of formula (lie): or a salt and/or solvate thereof, wherein m, X and R B are as defined and described in classes and subclasses above and herein.
  • the compounds are selected from:
  • the compounds of formula (B) can be manufactured by methods known by one skilled in the art. As an illustration, one can refer to the synthesis of compounds (RAFT-1), (RAFT-2), (RAFT-3), (RAFT-4), (RAFT-5), (RAFT-6) and (RAFT-7) described in the Example section.
  • the invention also relates to the use of the compounds of formula (B) according to the invention to manufacture the polymer compounds according to the invention, preferably by controlled radical polymerization.
  • the invention also relates to a polymer compound according to the invention, as described hereinabove, for use as a medicament.
  • This invention also relates to a polymer compound according to the invention, as described hereinabove, for use in the treatment of cancer.
  • This invention also relates to the use of a polymer compound according to the invention, as described hereinabove, in the manufacture of a medicament for the treatment of cancer.
  • This invention also relates to a method for the treatment of cancer in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of a polymer compound according to the invention, as described hereinabove.
  • the polymer compounds of the invention are effective for use in humans.
  • Cancers that can be treated using the methods of the invention include solid cancers and non-solid cancers, especially benign and malignant solid tumors and benign and malignant non- solid tumors.
  • the cancer may be metastatic or non-metastatic.
  • the cancer may be may be familial or sporadic.
  • the cancer to be treated according to the present invention is a solid cancer.
  • solid cancer encompasses any cancer (also referred to as malignancy) that forms a discrete tumor mass, as opposed to cancers (or malignancies) that diffusely infiltrate a tissue without forming a mass.
  • solid tumors include, but are not limited to: biliary tract cancer, brain cancer (including glioblastomas and medulloblastomas), breast cancer, carcinoid, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, intraepithelial neoplasms (including Bowen’s disease and Paget’s disease), liver cancer, lung cancer, neuroblastomas, oral cancer (including squamous cell carcinoma), ovarian cancer (including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells), pancreatic cancer, prostate cancer, rectal cancer, renal cancer (including adenocarcinoma and Wilms tumor), sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma), skin
  • the cancer to be treated according to the present invention is a non- solid cancer.
  • non- solid tumors include but are not limited to hematological neoplasms.
  • a hematologic neoplasm is a term of art which includes lymphoid disorders, myeloid disorders, and AIDS associated leukemias.
  • Lymphoid disorders include but are not limited to acute lymphocytic leukemia and chronic lymphoproliferative disorders (e.g., lymphomas, myelomas, and chronic lymphoid leukemias).
  • Lymphomas include, for example, Hodgkin’s disease, non- Hodgkin’s lymphoma lymphomas, and lymphocytic lymphomas).
  • Chronic lymphoid leukemias include, for example, T cell chronic lymphoid leukemias and B cell chronic lymphoid leukemias.
  • the cancer to be treated according to the present invention is selected from breast cancer, pancreatic cancer, ovarian cancer and lung cancer.
  • the polymer compound according to the invention is administered to the subject as sole therapeutic agent.
  • the polymer compound according to the invention is administered to the subject in combination with at least another therapeutic agent.
  • suitable other therapeutic agents include anticancer agents, such as immunotherapeutic agents, chemotherapeutic agents, antiangiogenic agents, multidrug resistance-associated proteins inhibitors, radio therapeutic agents, or any combination thereof.
  • the polymer compound according to the invention is for use in a patient treated by immunotherapy, a chemotherapy, radiotherapy or a combination thereof.
  • polymer compounds of the invention may be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant), topical, oral, sublingual, inhalation spray, nasal, vaginal, or rectal routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant
  • topical e.g., oral, sublingual, inhalation spray, nasal, vaginal, or rectal routes of administration
  • suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
  • the polymer compound of the invention is administered by parenteral route, preferably by intravenous injection or infusion, subcutaneous injection, intramuscular injection, or a combination thereof. In one embodiment, the polymer compound of the invention is administered by intravenous injection or infusion. In one embodiment, the polymer compound of the invention is administered by subcutaneous injection. In one embodiment, the polymer compound of the invention is administered by intramuscular injection.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polymer compound according to the invention, as described hereinabove, and at least one pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises 1 to 99 % of polymer compound according to the invention in weight to the total weight of the composition; and 1 to 99 % of pharmaceutically acceptable carrier in weight to the total weight of the composition.
  • the pharmaceutical composition comprises the polymer compound according to the invention as sole therapeutic agent. In another embodiment, the pharmaceutical composition further comprises at least another therapeutic agent.
  • the pharmaceutical compositions for the administration of the polymer compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
  • composition is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.
  • the pharmaceutical compositions may be in the form of a sterile injectable preparation.
  • the sterile injectable preparation may be a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol.
  • a non-toxic parenterally- acceptable diluent or solvent for example as a solution in 1,3-butane diol.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the pharmaceutical compositions may be in a form of creams, ointments, jellies, solutions or suspensions, etc., containing the polymer compounds of the invention.
  • the pharmaceutical compositions may be in a form of tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • the polymer compound of the invention is included in an amount sufficient to produce the desired effect upon the process or condition of diseases.
  • the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific polymer compound employed, the metabolic stability and length of action of that polymer compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.
  • a person skilled in the art can adjust dosing and/or determine a dose range to treat a particular subject and/or a particular disease based on the aforementioned factors, as well as other factors that are well known in the art.
  • the use of the mertansine polymer compound according to the present invention leads to administering equivalent doses of mertansine, or analogues thereof, being from significantly higher compared to the maximum tolerated dose of mertansine, or analogues thereof, for example 5- to 20-fold higher.
  • Figure 2 is a graph showing the survival rate overtime of mice bearing tumor model MDA-MB-231 and treated with DM1, paclitaxel (PTX) or polymer compounds (PI), (P2) or (P3) of the invention.
  • PTX paclitaxel
  • PI polymer compounds
  • P2 polymer compounds
  • P3 polymer compounds
  • Figure 3 is a graph showing the tumor volume (mm 3 ) overtime of mice bearing tumor model Calu-6 and treated with a vehicle [black circles], comparative polymer compound DM1-AEMI-PEG (12) [squares], or polymer compounds (P3) or (P10) of the invention [triangles and hexagons respectively] .
  • Figure 4 is a graph showing the tumor volume (mm 3 ) overtime of mice bearing tumor model HER2+ and treated with a vehicle [circles], comparative compound trastuzumab emtansine (T-DM1) [white circles], or polymer compounds (P3) or (P10) of the invention administered in combination with trastuzumab [triangles and hexagons respectively] .
  • T-DM1 comparative compound trastuzumab emtansine
  • P3 or (P10) of the invention administered in combination with trastuzumab [triangles and hexagons respectively] .
  • AAm acrylamide monomer
  • ACN acetonitrile
  • CEOEC 2-cyano-5-(ethylamino)-5-oxopentan-2-yl ethyl carbonotrithioate
  • AEMI N- (2- aminoethy l)maleimide
  • AIBN azobisisobutyronitrile
  • CDP 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid
  • CEP 4-cyano-4-[(ethylsulfanylthiocarbonyl)sulfanyl]pentanoic acid
  • DCC dicyclohexylcarbodiimide
  • DCM dichloromethane
  • DM1 mertansine
  • EtOAc ethyl acetate
  • FBS fetal bovine serum
  • CDP (AB252723) was obtained from abcr GmbH, CEP (A718802) from
  • H NMR spectroscopy of polymers acquisition was performed in 5 mm diameter tubes in D2O at 70 °C (128 scans) on a Bruker Avance 3 HD 400 spectrometer operating at 400 MHz. High resolution mass spectra (HR-MS) were recorded on a MicroMass LCT Premier Spectrometer.
  • CDP-NHS (1) 2,5 -dioxopyrrolidin- 1 -yl 4-cyano-4-
  • HEMI-CDP (3) 2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethyl 4-cyano-4- ((dodecylthio)carbonothioyl)thio)pentanoate
  • CDP-NHS (1) 0.4 mmol, 206.8 mg
  • HEMI 0.59 mmol, 82.9 mg
  • Na2CO3 2.13 mmol, 224.5 mg
  • the yellow mixture was stirred at r.t. for 24 hours.
  • HEMI (0.57 mmol; 80 mg
  • Na2CO3 (0.95 mmol; 100 mg) were added and the mixture was stirred again for 48 hours.
  • HEMI-CEP (4) 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl 4-cyano-4- (((ethylthio)carbonothioyl)thio)pentanoate [0178]
  • CEP-NHS (2) (0.28 mmol, 100 mg) were dissolved in anhydrous DCM (4 mL) under argon atmosphere.
  • HEMI (0.28 mmol, 39.5 mg) was dissolved in 2 mL of anhydrous DCM and added in the round bottom flask. The mixture was stirred and Na 2 CO 3 (1.4 mmol, 147 mg) was added and stirred at room temperature.
  • AEMI- CDP (6) 2-cyano-5 - ((2- (2,5 -dioxo-2,5-dihydro- lH-pyrrol- 1 -yl)ethyl)amino) - 5-oxopentan-2-yl dodecyl carbonotrithioate
  • MEP-HEMI (14) 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl 2- (((ethylthio)carbonothioyl)thio)propanoate [0182] In a round bottom flask was added was added MEP-NHS (13) (1 mmol, 307 mg), HEMI (1.5 mmol, 210 mg) and Na2CO3 (2.95 mmol, 313 mg) in DCM (15 mL).
  • Disulfide bond RAFT agent 2-cyano-5-oxo-5-((2-(pyridin-2- yldisulfaneyl)ethyl)amino)pentan-2-yl ethyl carbonotrithioate
  • CEP-NHS (2) (0.53 mmol, 201 mg
  • S)-2-Pyridylthio cysteamine hydrochloride 0.53 mmol, 119 mg
  • Triethylamine (0.53 mmol, 70 ⁇ L) diluted in anhydrous DCM (2 mL) was then added drop by drop and the mixture let to stir overnight at room temperature. After one day, 0.3 equivalent of CEP-NHS (2) (0.16 mmol, 60 mg) and triethylamine (0.16 mmol, 22 ⁇ L) were added and the yellow mixture was let to stir at room temperature for an additional 24 hours. The mixture was dried under vacuum. It was purified on silica gel column using a gradient of DCM-MeOH (0% to 2 % MeOH) to give the expected product Disulfide bond RAFT agent (15) (0.33 mmol, 144.3 mg) as a yellow oil. Yield 63%.
  • MEP-NHS (13) (0.78 mmol, 240.6 mg) was dissolved in anhydrous DCM (20 mL) and cooled down to 0°C.
  • a solution of N-Boc-Ethylenediamine (0.87 mmol, 138.8 mg) dissolved in DCM anhydrous (5 mL) was added dropwise to the previous solution. The reaction was stirred overnight at room temperature.
  • MEP-Boc (16) (0.28 mmol, 100 mg) was dissolved in anhydrous DCM (10 mL) the mixture is cooled down to 0°C. A solution of HC1 Dioxane (16 eq, 1.05 mL) in DCM anhydrous (3 mL) was added. The mixture was stirred at room temperature for 4 hours. It was evaporated and precipitated in petroleum ether to yield MEP-NH2 (17) (0.24 mmol, 61 mg) as a yellow solid. Yield 86%.
  • MEP-MCC (18): l-((2-(4-((2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)methyl)cyclohexane-l-arboxamido)ethyl)amino)-l-oxopropan-2-yl ethyl carbonotrithioate [0186] In a round bottom flask, MEP-NH2 (17) (0.2 mmol, 51 mg) and MCC
  • RAFT-1 2-(3-((3-((l-(((l 4 S,3 2 R,3 3 S,10£,12£,14R)-8 6 -chloro-l 4 - hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-l 2 ,6-dioxo-7-aza-l(6,4)-oxazinana-
  • DM1-HEMI-CEP (RAFT-2): 2-(3-((3-((l-(((l 4 5,3 2 R,3 3 5,10E,12E,14R)-8 6 - c hloro-l 4 - hydroxy-8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-l 2 ,6-dioxo-7-aza-l(6,4)-oxazinana- 3(2,3)-oxirana-8(l,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-l- oxopropan-2-yl)(methyl)amino)-3-oxopropyl)thio)-2,5-dioxopyrrolidin-l-yl)ethyl 4- cyano-4-(((ethylthio)carbonothioyl)thio)pentanoate [0189] In a round bottom flask
  • DM1-AEMI-CDP (RAFT-3): (1 4 S,3 2 R,3 3 S,10E,12E,14R)-8 6 -chloro-1 4 -hydroxy- 8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2-(4-cyano-4- (((dodecylthio)carbonothioyl)thio)pentanamido)ethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoyl)-N-methylalaninate [0190] In a round bottom flask was dissolved AEMI-CDP (6) (0.072 mmol, 38 mg) in anhydr
  • DM1-AEMI-CEP (1 4 S,3 2 R,3 3 S,10E,12E,14R)-8 6 -chloro-1 4 -hydroxy- 8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2-(4-cyano-4- (((ethylthio)carbonothioyl)thio)pentanamido)ethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoyl)-N-methylalan
  • DM1-HEMI-MEP (RAFT-5): (1 4 S,3 2 R,3 3 S,10E,12E,14R)-8 6 -chloro-1 4 -hydroxy- 8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2-((2- (((ethylthio)carbonothioyl)thio)propanoyl)oxy)ethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoyl)-N-methylalaninate [0192] In a round bottom flask was dissolved MEP-HEMI (14) (0.21 mmol, 68.5 mg) in anhydrous DCM (
  • DM4-S-S-CEOEC (1 4 S,3 2 R,3 3 S,10E,12E,14R)-8 6 -chloro-1 4 -hydroxy- 8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl 6-cyano-6,15,15,19,20- pentamethyl-9,18-dioxo-4-thioxo-3,5,13,14-tetrathia-10,19-diazahenicosan-21-oate [0193] In a round bottom flask was dissolved Dis
  • DM1-MCC-MEP (RAFT-7): (1 4 S,3 2 R,3 3 S,10E,12E,14R)-8 6 -chloro-1 4 -hydroxy- 8 5 ,14-dimethoxy-3 3 ,2,7,10-tetramethyl-1 2 ,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-((4-((2-(2- (((ethylthio)carbonothioyl)thio)propanamido)ethyl)carbamoyl)cyclohexyl)methyl)- 2,5-dioxopyrrolidin-3-yl)thio)propanoyl)-N-methylalaninate
  • the polymer was further solubilized in milliQ water and placed in a 3.5 kDa Spectra/Por 3 dialysis bag for dialysis against de-ionized water for 3 days, with dialysis water changed twice/thrice per day. The dialysate was then freeze-dried.
  • 1 H NMR spectroscopy of polymers acquisition was performed in 5 mm diameter tubes in D2O at 70 °C (128 scans) on a Bruker Avance 3 HD 400 spectrometer operating at 400 MHz.
  • NMR determination of the number-average molar mass (M n,NMR ) of the DM1 polymer derivatives was achieved by integrating the singlet at 4.03 ppm corresponding to 3 protons of methoxy-phenyl from the DM1.
  • the ratio between this integral and the integral of the broad peak between 2.62 and 1 ppm allowed determination of the number average degree of polymerization (DPn) of AAm.
  • the MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay was used to evaluate the cytotoxicity of the polymer compounds of the invention. This colorimetric test measures the mitochondrial deshydrogenase cell activity, an indicator of cell viability. The assay is based on the reduction, by living cells, of the tetrazolium salt, MTT, which forms a blue formazan product.
  • the human breast adenocarcinoma cell line MCF7 was obtained from ATCC (catalog number HTB-22) and grown in Eagle's Minimum Essential Medium (EMEM, Sigma M-5660) supplemented with 1% non-essential amino acids (NEAA, Sigma M- 7145) and 10 % FBS.
  • the human breast adenocarcinoma cell line MDA-MB-231 was obtained from ATCC (catalog number HTB-26) and grown in Leibovitz’s LI 5 medium (Sigma L-5520) supplemented with 1% of 200 mM L-glutamine solution (Sigma G- 7513), 20 mM sodium bicarbonate and 15 % FBS.
  • PTX paclitaxel (reference compound);
  • DM1 mertansine (free drug);
  • Table 1 Inhibitory Concentration 50% (IC 50 in nM) of tested compounds on MCF-7 and MDA-MB-231 breast cancer cells.
  • DM1 and PTX have comparable IC50.
  • the addition of a small hydrophilic group to DM1 increases the IC50 as the molecule is more soluble and less prone to diffuse inside the cells.
  • the addition of the very hydrophilic polyacrylamide chain (compounds PI, P2 and P3) further increases the IC50 as the proper DM1 metabolite must be cleaved from the prodrug to be able to diffuse inside the cells.
  • the in vitro tests confirm that the polymer compounds of the invention allow for a decrease in toxicity compared to free DM1, while retaining its activity.
  • the in vivo tests presented hereafter confirm the anticancer activity of the polymer compounds.
  • the tumor accumulation of the polymer compounds of the invention was assessed using fluorescent polymer compounds as model compounds. Cyanine 5.5 was used as fluorescent dye to form Cyanine-PAAm-C12 (10) and Cyanine-PAAm-C2 (11). Fluorescent polymer compounds 10 and 11 were administered to mice bearing 4T1 tumors (murine breast cancer cells) and accumulation in tumors and in other organs was measured by fluorescence. Material
  • Cyanine-PAAm-C12 (10) [0215] In a 25 ml round bottom flask NHS-PAAm-C12 (8) (0.025 mmol, 503mg) was dissolved in DMSO (7.5 mL), the mixture as heated until it becomes completely soluble. Argon was bubbled inside for 5 minutes. In a separate vial, Cyanine 5.5 amine (0.029 mmol, 19.35 mg) was dissolved in DMSO (3 mL) and add triethylamine (7.64 ⁇ L), this solution was added dropwise to the solution of compound 8. The mixture was stirred 24 h at r.t. in the dark. The solution was poured dropwise into cold methanol (200 mL).
  • Cyanine-PAAm-C2 (11) [0216] Compound Cyanine-PAAm-C2 (1) was obtained with a procedure similar to the one used for the synthesis of compound 14, using NHS-PAAm-C2 (9) as starting material. The final product Cyanine-PAAm-C2 (11) was isolated as blue powder in 25 % yield. The cyanine loading is 1.44%. Method [0217] Nine weeks old BALB/cAnNRj female mice were orthotopically inoculated with 10 6 4T1 cells (ATCC, catalog number CRL-2539) in the fourth inguinal mammary fat pad (50 ⁇ L/mouse).
  • ROI region of interest
  • Organs spleen, pancreas, kidney, liver, uterus-ovary, intestine, thymus, lung, heart, bladder, brain and tumor
  • IVIS Lumina LT Series III system Perkin Elmer
  • the vehicle used in the study is PBS (phosphate-buffered saline). Each dose corresponds to the maximal tolerated dose in mice. Tested compounds are administered by intravenous route (IV), once weekly (qw) for 4 weeks.
  • IV intravenous route
  • qw once weekly
  • MDA-MB-231 human breast cancer cells were amplified in vitro in DMEM supplied with at least 1% Penicillin-Streptomycin, 10% of Heat Inactivation of Fetal Bovine Serum and 5mg/mL of Plasmocin prior implantation. On the day of injection, cells were harvested, counted including a trypan blue viability dye (cut-off 80%), and resuspended in serum-free medium at the appropriate concentration. The cells were orthotopically implanted in the mammary fat pad SCID mice at 5xl0 6 cells/mice in 200m1 PBS within 30 minutes after harvesting.
  • mice monitoring Mice were randomized when the tumors reach a mean volume of 100 mm 3 for the 7 groups (for a total of 70 mice). After implantation, all the mice were observed in order to detect any toxic effects of the product.
  • the endpoints are defined by animal ethics as a tumor diameter of > 18mm, significant weight loss or alteration of animal well-being.
  • the vehicle used in the study was PBS (phosphate-buffered saline) IX.
  • Tumor cells were grown as monolayer at 37°C in a humidified atmosphere (5% CO 2 , 95% air).
  • the culture medium was RPMI 1640 containing 2 mM L-glutamine supplemented with 10% fetal bovine serum. Tumor cells are adherent to plastic flasks.
  • tumor cells were detached from the culture flask by a 5-minute treatment with Trypsin-Versene®, in Hanks' medium without calcium or magnesium and neutralized by addition of complete culture medium. Cells were counted and viability assessed using a 0.25% trypan blue exclusion assay. [0238] Animals.
  • Humane endpoints The human endpoints requiring specific action or euthanasia were defined by animal ethics and included: a body weight loss > 15% (compared to a reference day, e.g., the first day of treatment, tumor exceeding 10% of normal body weight or exceeding 2000 mm 3 and > 8 mm ulcerated tumor, infection, bleeding, tissue erosion. Euthanasia of animals was performed by over dosage on gas anesthesia (isoflurane).
  • T-DM1 brand name: Kadcyla®
  • P3 phosphate-buffered saline
  • P10 phosphate-buffered saline
  • NCIN-87 (gastric carcinoma cancer) cells were amplified in vitro in RPMI supplied with at 1% Penicillin-Streptomycin, 10% of Heat Inactivated Fetal Bovine Serum and 5 ⁇ g/mL of Plasmocin prior implantation. On the day of injection, cells were harvested, counted including a trypan blue viability dye (cut-off 80%), and resuspended in serum-free medium at the appropriate concentration. The cells injected subcutaneously in the right flank at 5.10 6 cells/mouse in 200 ⁇ L PBS within 30 minutes after harvesting. A margin of 2 mice were injected with cells (total of 50 mice implanted). [0251] Mice monitoring.
  • mice After implantation, mice were monitored thrice weekly for tumor uptake, and general health and behavior. After treatment onset, they were observed daily in order to detect any toxic effects of the product or deleterious effect of the tumors. They were weighted twice weekly.
  • the endpoints are defined by animal ethics as a tumor diameter of >18mm, significant weight loss or alteration of animal well-being.
  • mice randomization Animals were randomized based on their individual tumor volume. Randomization was performed when values reach at a median tumor volume of 226 to 242 mm 3 . Animals were randomized into groups of 8 animals each.

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Abstract

The present invention relates to polymer derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4), preferably to polyacrylamide derivatives, of formula (A), or a pharmaceutically acceptable salt and/or solvate thereof, wherein m is 0 or 1, L is a linker, M is a hydrophilic polymeric moiety selected from polyacrylamide, polyacrylic acid, poly(N-(2-hydroxypropyl)methacrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), poly(2-methacryloxyethyl phosphorylcholine), and copolymers thereof, and RA is a terminal group, as defined in the application. The mertansine polymer derivatives of the invention are useful in the treatment of cancers. The invention also relates to a manufacturing process of the mertansine polymer derivatives.

Description

POLYMER DERIVATIVES OF MERTANSINE AND THERAPEUTIC USES
THEREOF
FIELD OF INVENTION [0001] The present invention relates to polymer derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4), particularly polyacrylamide derivatives of mertansine or of analogues thereof. The mertansine polymer derivatives of the invention are useful in the treatment of cancers. The invention also relates to a manufacturing process of the mertansine polymer derivatives by RAFT polymerization, as well as to the corresponding mertansine RAFT agents.
BACKGROUND OF INVENTION
[0002] Despite the importance of investments in oncology research programs over the years, some cancers are still resistant to available treatments. For such cancers, it is necessary to provide more active anticancer agents that limit resistance to treatments and improve the chances of patient survival. Treatments that exhibit greater anticancer activity compared to currently available treatments do exist: these are molecules with high activity referred to as “highly potent active pharmaceutical ingredients” (HP APIs). These molecules are characterized by in vitro efficiencies 100 to 1000 times superior to traditional chemotherapy.
[0003] Nevertheless, very few of these HP APIs obtained marketing authorizations. The development of HP APIs is limited at least for two reasons. First, HP APIs often have extreme physicochemical properties that prevent their administration by classic routes. Particularly, they tend to be poorly soluble, which compromises intravenous administration. They may also present a poor intestinal permeability, limiting oral administration. Secondly, HP APIs have a very narrow therapeutic index. This means that the difference between the effective dose and the toxic dose is small. This, coupled with unfavorable pharmacokinetics ( e.g ., short half-life in blood), implies difficulties in providing controlled and prolonged exposure to effective but non-toxic doses.
[0004] Various formulations of HP APIs are under investigation in order to increase their bioavailability or to achieve controlled exposure with limited toxicity. For examples, some anticancer HP APIs were formulated as nanoparticles, nanocrystals or as amorphous solids for oral or intravenous administration. The use of prodrugs of HP APIs is also considered for enabling their development. However, recent attempts with PEG polymer prodrugs did not enable to achieve satisfying results. Antibody-drug conjugates (ADC) are also considered for HPAPIs. ADC use antibodies as vectors in order to target HP APIs directly towards tumors overexpressing particular receptors.
[0005] Mertansine, also called DM1, is a thiol-containing maytansinoid having properties that make it fall into the category of HPAPIs. Particularly, mertansine is a tubulin inhibitor that induces mitotic arrest and kills tumor cells at sub-nanomolar concentrations. It was evidenced that mertansine has in vitro efficiencies 100 to 1000 times superior to other tubulin-binding cytotoxics such as vincristine or paclitaxel. Nevertheless, its systemic toxicity hinders its use under free form.
[0006] Mertansine ADCs are thus under investigations in order to overcome this toxicity issue, such as lorvotuzumab mertansine, bivatuzumab mertansine, or cantuzumab mertansine. Mertansine was also linked to an antibody using the SMCC (4-(3-mercapto- 2,5-dioxo-l-pyrrolidinylmethyl)-cylohexanecarboxylic acid) linker, in which case the INN of the conjugate formed contains the word emtansine. This leads to the development of trastuzumab emtansine (T-DM1) as an anti-HER2 antibody-drug conjugate which was approved by the FDA for the treatment of patients with (HER2)-positive metastatic breast cancer (MBC) who previously received trastuzumab and a taxane, separately or in combination. However, the main limitations of ADC technology are that it does not improve the toxicity profile of the HP API (no improvement in maximum tolerated dose relative to the free molecule) and it is limited to tumors that overexpress the targeted receptor. [0007] Therefore, there is a need for other forms of mertansine in order to expand its access to patients.
[0008] The Applicant herein provides polymer prodrugs of mertansine overcoming above drawbacks. The polymer derivatives of mertansine according to the invention are based on the introduction of a hydrophilic polymeric moiety ( e.g ., a polyacrylamide moiety) on mertansine.
[0009] W02019/097025 discloses polymer prodmgs for subcutaneous and/or intramuscular administration when the direct subcutaneous and/or intramuscular administration of the drug is problematic or impossible in particular because of the toxicity at the site of injection. Particularly, prodrugs comprising polymeric chains, such as polyacrylamide chains, were evidenced to be suitable for subcutaneous administration and to present reduced toxicity at the site of injection. Nevertheless, W02019/097025 is silent relative to a possible modification of mertansine by this method, particularly in order to favor its specific accumulation in tumors, whatever the type of administration. [0010] The polymer derivatives of mertansine of the invention enable to achieve a prolonged and controlled exposition to mertansine with an important tumor accumulation, while limiting related toxicity, thereby increasing the therapeutic index compared to free mertansine. SUMMARY
[0011] This invention thus relates to a polymer compound of formula (A)
Figure imgf000004_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m is 0 or 1 ;
L is a linker; preferably a linker which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; M is a hydrophilic polymeric moiety selected from polyacrylamide, polyacrylic acid, poly (N-(2-hydroxypropyl)methacrylamide) , poly (oligo(ethylene glycol)methyl ether methacrylate), poly(2-methacryloxyethyl phosphorylcholine), and copolymers thereof; and
RA is a terminal group, preferably a terminal group selected from halo, -S-(C=S)-S-Rb, -S-(C=S)-0-Rb, -S-(C=S)-NRBRc, -S-(C=S)-Rd, -0-NRERf, hydrogen, -OH, -SH, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups; each of which group being optionally substituted by one or more substituent(s) selected from halo, cyano, alkoxy, haloalkoxy heterocyclyl, carboxy, -OH, oxo, amino, alkylamino, hydroxyalkylamino, and amidine; wherein
RB is alkyl, aryl, or heteroaryl;
Rc is hydrogen, alkyl, aryl, or heteroaryl;
RD is an optionally substituted aryl; and
RE and RF are independently selected from alkyl, arylalkyl, and dialky lpho sphory lalky 1.
Figure imgf000005_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein n is an integer ranging from 10 to 1400; preferably from 50 to 700. [0013] In one embodiment, L comprises one or more group(s) selected from optionally substituted, saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains; optionally substituted cycloalkyl; and optionally substituted heterocyclyl; wherein said groups are linked through single bond, -0-, -S-, -NHC(O)-, -OC(O)-, -C(0)-0-C(0)-, -NH-, -NH-C(0)-NH-, -NH-(CS)-NH-, -C(O)-, =N-NH-, and combinations thereof.
[0014] In one embodiment, the polymer compound of the invention is of formula (1-1)
Figure imgf000006_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein L1 is selected from moieties (i), (ii), (iii) and (iv):
Figure imgf000006_0002
wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L2; and
L2 is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties, or L2 is absent.
[0015] In one embodiment, the polymer compound of the invention is of formula (1-3)
Figure imgf000007_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein X is O, N or S; preferably X is O or N.
Figure imgf000007_0002
or a pharmaceutically acceptable salt and/or solvate thereof, wherein RG is selected from -S-RB, -0-RB, -NRBRC and R°; preferably RG is -S-RB.
[0017] In one embodiment, the polymer compound of the invention is selected from compounds Pl-Pll and pharmaceutically acceptable salts and/or solvates thereof.
[0018] The present invention also relates to a pharmaceutical composition comprising a polymer compound according to the invention and at least one pharmaceutically acceptable carrier.
[0019] The present invention further relates to the polymer compound or the pharmaceutical composition according to the invention, for use as a medicament. The present invention further relates to the polymer compound or the pharmaceutical composition according to the invention, for use in the treatment of cancer; preferably breast cancer, pancreatic cancer, ovarian cancer or lung cancer. In one embodiment, the polymer compound or the pharmaceutical composition for use according to the invention is to be administered by parenteral route; preferably intravenously, subcutaneously, intramuscularly or intratumorally.
[0020] The invention also provides a method of manufacturing of a polymer compound of formula (I) or subformulae thereof according to the invention, comprising a step of controlled radical polymerization performed by contacting a source of radicals, acrylamide monomers and a compound of formula (B)
Figure imgf000008_0001
or a salt and/or solvate thereof, under conditions suitable to obtain the polymer compound of formula (1-4); optionally followed by a step of removal or modification of the thiocarbonylthio terminal group, leading to the polymer compound of formula (I) according to the invention.
[0021] The invention also provides a functionalized RAFT agent of formula (B)
Figure imgf000008_0002
or a salt and/or solvate thereof, wherein m is 0 or 1 ;
L is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; and RG is selected from -S-RB, -0-RB, -NRBRC, and R° wherein
RB is alkyl, aryl, or heteroaryl;
Rc is hydrogen, alkyl, aryl, or heteroaryl; and RD is an optionally substituted aryl.
[0022] In one embodiment, the functionalized RAFT agent is selected from RAFT-1, RAFT-2, RAFT-3, RAFT-4, RAFT-5, RAFT-6, RAFT-7 and salts and/or solvates thereof.
DEFINITIONS
[0023] In the present invention, the following terms have the following meanings: [0024] “Acrylamide monomer” or “AAm” refers to the compound of formula:
Figure imgf000009_0001
[0025] “Administration” or a variant thereof ( e.g ., “administering"), refers to providing the compound, alone or as part of a pharmaceutically acceptable composition, to the subject in whom/which the condition, symptom, or disease is to be treated or prevented.
[0026] “Alkenyl” refers to an unsaturated hydrocarbyl group, which may be linear or branched, wherein the unsaturation arises from the presence of one or more carbon- carbon double bonds. Suitable alkenyl groups comprise between 2 and 6 carbon atoms. Non-limiting examples of alkenyl groups are ethenyl, propenyl, butenyl, pentenyl and hexenyl.
[0027] “Alkyl” refers to a hydrocarbyl radical of formula CiFhi+i wherein “i” is a number greater than or equal to 1. Generally, alkyl groups of this invention comprise from 1 to 30 carbon atoms, preferably from 1 to 20 carbon atoms, more preferably from 1 to 15 carbon atoms. Alkyl groups may be linear or branched. The alkyl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like. Suitable alkyl groups include methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl, t-butyl, pentyl and its isomers (e.g., n-pentyl, iso-pentyl), hexyl and its isomers (e.g., n-hexyl, iso-hexyl), heptyl and its isomers (e.g., n-heptyl), octyl and its isomers (e.g., n-octyl), nonyl and its isomers (e.g., n-nonyl), decyl and its isomers (e.g., n-decyl), undecyl and its isomers (e.g., n-undecyl), dodecyl and its isomers (e.g., n-dodecyl).
[0028] “Alkylamino” refers to a group -NH-alkyl wherein alkyl is as herein defined.
[0029] “Alkoxy” refers to a group -O-alkyl wherein alkyl is as herein defined.
[0030] “Alkynyl” refers to a monovalent unsaturated hydrocarbyl group, wherein the unsaturation arises from the presence of one or more carbon-carbon triple bonds. Alkynyl groups typically, and preferably, have the same number of carbon atoms as described above in relation to alkenyl groups. Non limiting examples of alkynyl groups are ethynyl, 2-propynyl, 2-butynyl, 3-butynyl, 2-pentynyl and its isomers, 2-hexynyl and its isomers- and the like. [0031] “Amidine” refers to the group
Figure imgf000010_0001
.
[0032] “Amino” refers to the group -Nth.
[0033] “Aryl” refers to a polyunsaturated, aromatic hydrocarbyl group having a single ring (i.e., phenyl) or multiple aromatic rings fused together (e.g., naphthyl) or linked covalently, typically containing 5 to 12 atoms; preferably 6 to 10, wherein at least one ring is aromatic. The aryl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like. A non-limiting example of aryl group is phenyl.
[0034] “Arylalkyl” refers to a group -alkyl-aryl wherein aryl and alkyl are as herein defined. [0035] “Carboxy” refers to the group -COOH.
[0036] “Controlled radical polymerization” refers to a living polymerization where the active polymer chain end is a free radical. Controlled radical polymerization includes the following techniques: atom transfer radical polymerization (ATRP), reversible addition/fragmentation chain transfer polymerization (RAFT), and nitroxide-mediated polymerization (NMP). In the present invention, the controlled radical polymerization particularly refers to RAFT polymerization.
[0037] “Cycloalkyl” refers to a cyclic alkyl group, that is to say, a monovalent, saturated, or unsaturated hydrocarbyl group having 1 or 2 cyclic structures. Cycloalkyl includes monocyclic or bicyclic hydrocarbyl groups. Cycloalkyl groups may comprise 3 or more carbon atoms in the ring and generally, according to this invention, comprise from 3 to 10, more preferably from 3 to 8 carbon atoms still more preferably from 3 to 6 carbon atoms. The cycloalkyl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like. Examples of cycloalkyl groups include but are not limited to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
[0038] “Dialkylphosphorylalkyl” refers to a group -alkyl-P(=0)(0-alkyl)(0-alkyl), wherein alkyl is as herein defined. [0039] “Halo” or ‘halogen” refers to fluoro, chloro, bromo, or iodo.
[0040] “Haloalkoxy” refers to a group -O-haloalkyl wherein haloalkyl is as herein defined. [0041] “Haloalkyl” refers to an alkyl group as herein defined wherein one or more hydrogen atoms are replaced with a halogen as defined above. Non-limiting examples of such haloalkyl radicals include chloromethyl, 1-bromoethyl, fluoromethyl, difluoromethyl, trifluoro methyl, 1,1,1-trifluoroethyl and the like. [0042] “Heteroaryl” refers to a 5 to 12 carbon-atom aromatic ring or ring system containing 1 to 2 rings which are fused together or linked covalently, typically containing 5 to 6 atoms on each ring; at least one of which is aromatic and in which one or more carbon atoms in one or more of these rings is replaced by oxygen, nitrogen or sulfur atoms where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. Non-limiting examples of such heteroaryl groups include: triazolyl, pyrrolyl, furanyl, thiophenyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl, thiatriazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, oxazinyl, dioxinyl, thiazinyl, triazinyl, imidazo[2,l-b][l,3]thiazolyl, thieno[3,2-b]furanyl, thieno [3 ,2-b] thiophenyl, thieno[2,3-d] [ 1 ,3 ]thiazolyl, thieno[2,3-d]imidazolyl, tetrazolo [ 1 ,5-a]pyridinyl, indolyl, indolizinyl, isoindolyl, benzofuranyl, isobenzofuranyl, benzothiophenyl, isobenzothiophenyl, indazolyl, benzimidazolyl, 1,3-benzoxazolyl, 1,2-benzisoxazolyl, 2,1-benzisoxazolyl, 1 ,3 -benzothiazolyl, 1,2-benzoisothiazolyl, 2,1-benzoisothiazolyl, benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzothiadiazolyl, 2,1,3-benzothiadiazolyl, thienopyridinyl, purinyl, imidazo[ 1 ,2-a]pyridinyl, 6-oxo-pyridazin-l(6H)-yl, 2-oxopyridin-l (2H)-yl, 6-oxo-pyrudazin-l(6H)-yl, 2-oxopyridin-l(2H)-yl, 1,3-benzodioxolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl. [0043] “Heteroatom” refers to one or more of oxygen, nitrogen, sulfur, phosphorus, selenium, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, selenium, or silicon; the quaternized form of any basic nitrogen or a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as in N-substituted pyrrolidinyl)). [0044] “Heterocyclyl” refers to non-aromatic, fully saturated or partially unsaturated cyclic groups (for example, 3 to 7 member monocyclic, 7 to 1 1 member bicyclic, or containing a total of 3 to 10 ring atoms) which have at least one heteroatom in at least one carbon atom-containing ring. Each ring of the heterocyclic group containing a heteroatom may have 1, 2, 3 or 4 heteroatoms selected from nitrogen atoms, oxygen atoms and/or sulfur atoms, where the nitrogen and sulfur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. The heterocyclic group may be attached at any heteroatom or carbon atom of the ring or ring system, where valence allows. The rings of multi-ring heterocycles may be fused, bridged and/or joined through one or more spiro atoms. The heterocyclyl group may optionally be substituted by one or more substituent(s) (for example 1 to 4 substituent(s), or for example 1, 2, 3 or 4 substituent(s)), which are for example selected from oxo, halogen, hydroxyl, nitro, amino, cyano, alkyl, alkylamino, dialkylamino, alkoxy, haloalkyl, acyl, carbamoyl, alkylsulfoxide, sulfamoyl, alkylthio, carboxyl, and the like. Non limiting exemplary heterocyclic groups include aziridinyl, oxiranyl, thiiranyl, piperidinyl, azetidinyl, 2- imidazolinyl, pyrazolidinyl imidazolidinyl, isoxazolinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, piperidinyl, succinimidyl, 3H-indolyl, indolinyl, isoindolinyl, 2H-pyrrolyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl, 4H- quinolizinyl, 2-oxopiperazinyl, piperazinyl, homopiperazinyl, 2-pyrazolinyl, 3-pyrazolinyl, tetrahydro-2H-pyranyl, 2H-pyranyl, 4H-pyranyl, 3,4-dihydro-2H-pyranyl, oxetanyl, thietanyl, 3-dioxolanyl, 1,4-dioxanyl, 2,5-dioximidazolidinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, indolinyl, tetrahydropyranyl, tetrahydrofuranyl, tetrahydrothiophenyl, tetrahydroquinolinyl, tetrahydroisoquinolin- 1 -yl, tetrahydroisoquinolin-2-yl, tetrahydroisoquinolin-3-yl, tetrahydroisoquinolin-4-yl, thiomorpholin-4-yl, thiomorpholin-4-ylsulfoxide, thiomorpholin-4-ylsulfone,
1, 3-dioxolanyl, 1,4-oxathianyl, 1,4-dithianyl, 1,3,5-trioxanyl, 1 H-pyrrolizinyl, tetrahydro- 1 , 1 -dioxothiophenyl, N-formylpiperazinyl, and morpholin-4-yl.
[0045] “Human” refers to a subject of both genders and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult). [0046] “Hydroxyalkylamino” refers to a group -NH-alkyl-OH wherein alkyl is as herein defined.
[0047] “Pharmaceutically acceptable” refers to ingredients that are compatible with each other and not deleterious to the subject to which they are administered thereof. [0048] “Pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. Pharmaceutically acceptable salts include those derived from suitable inorganic and organic acids and bases. Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, ammonium, aspartate, benzenesulfonate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, bitartrate/tartrate, borate, bromide, calcium edetate, camsylate, chloride, citrate, clavulanate, cyclamate, dihydrochloride, edetate, edisylate, estolate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hibenzate, hydrochloride/chloride, hydrabamine, hydrobromide/bromide, hydroiodide/iodide, hydroxynaphthoate, isethionate, lactate, lactobionate, laurate, malate, maleate, malonate, mandelate, mesylate, methylbromide, N-methylglucamine, methylnitrate, methylsulphate, mucate, naphthylate, napsylate, nicotinate, nitrate, oleate, orotate, oxalate, palmitate, pamoate, pantothenate, phosphate/hydrogen phosphate/dihydrogen phosphate, polygalacturonate, pyroglutamate, saccharate, salicylate, stearate, succinate, sulfate, subacetate, tannate, teoclate, tosylate, triethiodide, trifluoroacetate, valerate, and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, ammonia, arginine, benzathine, N-benzylphenethylamine, calcium, choline, chloroprocaine,
N,N -dibenzylethylenediamine, diethanolamine, diethylamine, 2-(diethylamino)ethanol, diolamine, ethylenediamine, ethanolamine, glycine, 4-(2-hydroxyethyl)morpholine, lithium, lysine, magnesium, meglumine, N-methyl-glutamine, morpholine, olamine, ornithine, potassium, piperazine, procaine, sodium, tetramethylammonium hydroxide, tris(hydroxymethyl)aminomethane, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulphate and hemicalcium salts.
[0049] Although generally, with respect to the salts of the compounds of the invention, pharmaceutically acceptable salts are preferred, it should be noted that the invention in its broadest sense also includes non-pharmaceutically acceptable salts, which may for example be used in the isolation and/or purification of the compounds of the invention. For example, salts formed with optically active acids or bases may be used to form diastereoisomeric salts that can facilitate the separation of optically active isomers of the compounds of the invention. [0050] “Pharmaceutically acceptable carrier” refers to an excipient that does not produce an adverse, allergic or other untoward reaction when administered to an animal, preferably a human. It includes any and all inactive substances such as for example solvents, cosolvents, antioxidants, surfactants, stabilizing agents, emulsifying agents, buffering agents, pH modifying agents, preserving agents (or preservating agents), antibacterial and antifungal agents, isotonifiers, granulating agents or binders, lubricants, disintegrants, glidants, diluents or fillers, adsorbents, dispersing agents, suspending agents, coating agents, bulking agents, release agents, absorption delaying agents, sweetening agents, flavoring agents and the like. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by regulatory offices, such as, e.g., FDA Office or EMA.
[0051 ] “Polyacrylamide” or “PAAm” refers to a polymer or a polymeric moiety formed from acrylamide monomers, of formula:
Figure imgf000015_0001
[0052] “Polymer compound” refers to a compound comprising a polymeric moiety, i.e., a moiety made of several repeating subunits. The polymer compound of the present invention preferably comprises a hydrophilic polymeric moiety, such as a polyacrylamide moiety, as polymeric moiety. [0053] “Prodrug” refers to a pharmacologically acceptable derivative of a drug whose in vivo biotransformation generates the biologically active drug.
[0054] “Solvate” refers to a compound in the invention that contains stoichiometric or sub-stoichiometric amounts of one or more pharmaceutically acceptable solvent molecule such as ethanol or water. The term "hydrate" refers to when the said solvent is water.
[0055] “Subject” refers to a mammal, preferably a human. In one embodiment, a subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who/which is awaiting the receipt of, or is receiving medical care or was/is/will be the object of a medical procedure, or is monitored for the development of a disease [0056] “Therapeutically effective amount” or “therapeutically effective dose” refer to the amount or dose of active ingredient that is aimed at, without causing significant negative or adverse side effects to the subject, (1) delaying or preventing the onset of a cancer in the subject; (2) reducing the severity or incidence of a cancer; (3) slowing down or stopping the progression, aggravation, or deterioration of one or more symptoms of a cancer affecting the subject; (4) bringing about ameliorations of the symptoms of a cancer affecting the subject; or (5) curing a cancer affecting the subject. A therapeutically effective amount may be administered prior to the onset of a cancer for a prophylactic or preventive action. Alternatively, or additionally, a therapeutically effective amount may be administered after initiation of a cancer for a therapeutic action. [0057] “Treatment” or “treating” refers to both therapeutic treatment and prophylactic or preventative measures; wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder ( e.g ., cancer). Those in need of treatment include those already with the disorder (e.g., cancer) as well as those prone to have the disorder (e.g., cancer) or those in whom the disorder (e.g., cancer) is to be prevented. A subject is successfully "treated" for cancer if, after receiving a therapeutic amount of a polymeric compound according to the present invention, the subject shows observable and/or measurable occurrence of one or more of the following: (1) reduction in the number of cancer cells; (2) reduction of tumor size; (3) relief to some extent one or more of the symptoms associated with cancer; (4) reduced morbidity and mortality; and/or (5) improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in the targeted disease are readily measurable by routine procedures familiar to a physician.
[0058] Unless otherwise stated, chemical structures depicted herein are also meant to include all isomeric ( e.g ., enantiomeric, diastereomeric, and geometric
(or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the present disclosure. Unless otherwise stated, all tautomeric forms are within the scope of the disclosure. Additionally, unless otherwise stated, the present disclosure also includes compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this disclosure. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present disclosure.
DETAILED DESCRIPTION Mertansine polymer compounds
[0059] This invention thus relates to polymer derivatives of mertansine (DM1) or of analogues thereof ( e.g ., DM4). Particularly, the polymer compounds of the invention are derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4) comprising a polymeric moiety, preferably a hydrophilic polymeric moiety. In one embodiment, the polymer compound of the invention are polyacrylamide derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4).
[0060] Unless otherwise specified hereafter, all references made to mertansine (DM1) also relate to analogues thereof (e.g., DM4).
[0061] In some aspects of the invention, the introduction of a hydrophilic polymeric moiety (e.g., a polyacrylamide moiety) on mertansine leads to the formation of a prodrug thereof. Particularly, the introduction of the hydrophilic polymeric moiety (e.g., polyacrylamide moiety) on mertansine advantageously leads to an increased accumulation in the tumor rather than in other organs. This enables to limit deleterious side effects of mertansine. Moreover, the introduction of the hydrophilic polymeric moiety (e.g., polyacrylamide moiety) on mertansine advantageously increases its circulating time which is benefic to the pharmacokinetic properties. Further, the introduction of the hydrophilic polymeric moiety (e.g., polyacrylamide moiety) on mertansine advantageously increases its solubility. An increased solubility enables to provide compositions of high concentration. This is especially useful for subcutaneous administration wherein the maximum injection volume is limited (to about 2 ml). Moreover, the increased solubility enables to achieve a better bioavailability, which is of particular interest for subcutaneous administration since it thereby avoids toxicities at the site of injection. [0062] Mertansine, also named DM1, has the following formula:
Figure imgf000019_0001
DM1.
[0063] A known analogue of DM1 is DM4, having the following formula:
Figure imgf000019_0002
DM4. [0064] DM4 differs from DM1 only on the alkyl arm bearing the free thiol group.
[0065] The free thiol group of DM 1 or DM4 can be modified to form derivatives thereof, including prodmgs such as the polymer compounds of the invention. In the present invention, the polymeric moiety is introduced by covalent coupling on the free thiol group. The polymeric moiety may be introduced through a linker on the thiol group. A terminal group may also be present at the terminal end of the polymeric moiety. [0066] In one embodiment, the polymer compound of the invention is of formula (A):
Figure imgf000020_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein: m is 0 or 1 ;
L is a linker;
M is a polymeric moiety; preferably M is a hydrophilic polymeric moiety; and RA is a terminal group.
Polymeric moiety
[0067] The polymer derivatives of mertansine (DM1) or of analogues thereof ( e.g ., DM4) according to the invention comprise a polymeric moiety. Preferably, the polymeric moiety is a hydrophilic polymeric moiety, such as for example a polyacrylamide moiety, a copolymer thereof or a derivative thereof.
[0068] By “polymeric moiety”, it is referred to a moiety made of several repeating subunits. The polymeric moiety can be made from a single monomer or from two or more monomers, and in such case, it can be referred to as a copolymeric moiety. Unless otherwise stated, references made to “polymeric moieties” include copolymeric moieties.
[0069] By “hydrophilic polymeric moiety”, it is referred to a polymeric moiety that is typically charge-polarized and capable of hydrogen bonding, enabling it to dissolve more readily in water than in oil or other hydrophobic solvents. Examples of hydrophilic polymeric moieties include polyacrylamide (PAAm), polyacrylic acid (PAAc), poly(N- (2-hydroxypropyl)methacrylamide) (PHPMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(2-methacryloxyethyl phosphorylcholine) (PMPC), poly(ethylene glycol) (PEG) and copolymers thereof. [0070] In one embodiment, the polymeric moiety M is a hydrophilic polymeric moiety. In one embodiment, the polymeric moiety M is selected from polyacrylamide (PAAm), polyacrylic acid (PAAc), poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(2-methacryloxyethyl phosphorylcholine) (PMPC), poly(ethylene glycol) (PEG) and copolymers thereof. In one embodiment, the polymeric moiety M is selected from polyacrylamide (PAAm), polyacrylic acid (PAAc), poly(N-(2- hydroxypropyl)methacrylamide) (PHPMA), poly(oligo(ethylene glycol)methyl ether methacrylate) (POEGMA), poly(2-methacryloxyethyl phosphorylcholine) (PMPC), and copolymers thereof. In one embodiment, the polymeric moiety M is polyacrylamide (PAAm) or a copolymer thereof.
[0071] In one embodiment, the polymeric moiety M does not comprise or consists of an amphiphilic polymeric moiety. By “amphiphilic polymeric moiety”, it is referred to a polymeric moiety with both hydrophilic and lipophilic properties. Especially, the polymeric moiety M does not comprise or consists of a polylactic acid (PLA) moiety. In one embodiment, the polymeric moiety M does not comprise or consists of polylactic acid or a copolymer thereof. According to one embodiment, the polymeric moiety M does not comprise or consists of hyaluronic acid. According to one embodiment, the polymeric moiety M does not comprise or consists of polyethylene glycol (PEG). In one embodiment, the polymeric moiety M does not comprise or consists of a polyether.
[0072] In one embodiment, the polymer derivatives of mertansine (DM1) or of analogues thereof (e.g., DM4) according to the invention comprise a polyacrylamide moiety, i.e., a moiety formed by the polymerization of acrylamide monomers.
[0073] In one embodiment, the polymer compound of the invention is of formula (I):
Figure imgf000021_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein: m is 0 or 1; n is an integer ranging from 10 to 1400; L is a linker; and
RA is a terminal group.
[0074] The choice of the size of the polymeric moiety, e.g., the polyacrylamide moiety, enables to vary the overall solubility of the polymer compound. It also impacts the absorption rate of the polymer compound. Advantageously, the size of the polymeric moiety, e.g., the polyacrylamide moiety, is ranging from 1 000 kDa to 100000 kDa in order to increase the absorption rate of the polymer compound.
[0075] The polymeric moiety, e.g., the polyacrylamide moiety, is preferably obtained by controlled radical polymerization, for example by reversible addition-fragmentation chain-transfer (RAFT) polymerization, by atom transfer radical polymerization (ATRP) or by nitroxide-mediated polymerization (NMP). The synthesis of the polymer compounds of the invention is advantageously performed by the method of drug-initiated polymerization, as detailed hereafter. This method enables to control the size of the polymer and to ensure that only one DM1 or DM4 is present per polymer compound.
[0076] In one embodiment, the polymeric moiety, e.g., the polyacrylamide moiety, of the polymer compound of the invention is a simple linear chain.
[0077] In one embodiment, the number of acrylamide subunits in the polymer compound of the invention is ranging from 10 to 1 400; preferably from 50 to 700; more preferably from 70 to 500; even more preferably from 280 to 600.
[0078] In one embodiment, n is an integer ranging from 10 to 1 400; preferably from 50 to 700; more preferably from 70 to 500; even more preferably from 280 to 600.
[0079] In one embodiment, the polymer compound of the invention has a molecular weight ranging from 0.7 to 100 kDa; preferably from 3.5 to 50 kDa; more preferably from 5 to 35.5 kDa; even more preferably from 20 to 43 kDa. Terminal group
[0080] The polymer compound of the invention comprises a terminal group at the terminal end of the polymeric moiety ( e.g ., the polyacrylamide moiety).
[0081] The terminal group may be of any type and is typically selected depending on the physicochemical and biological properties which are sought when modifying mertansine or its analogues. Particularly, the choice of the terminal group enables to control the capacity of the polymer compound to enter into the tumor.
[0082] In some embodiments, the terminal group RA of the polymer compounds of the invention comprises a moiety introduced during the polymerization of the polymeric moiety and being characteristic of the polymerization method. Such moieties characteristic of the polymerization method can be removed or modified by methods known by one skilled in the art, leading to other terminal groups of interest.
[0083] For example, the polymer compound of the invention can be obtained by RAFT polymerization. In such case, the terminal group RA typically comprises a thiocarbonylthio group (S=C-S) at its extremity linking the polymeric moiety (e.g., the polyacrylamide moiety). The presence of a thiocarbonylthio group in the polymer compound of the invention does not adversely affect its properties and there is thus no need to remove this residue of polymerization. Nevertheless, the thiocarbonylthio group can be removed or modified by various methods, leading to modified terminal groups, depending on the targeted properties of the polymer compound.
[0084] In the case of RAFT polymerization, the terminal group RA is typically of formula (a), (b), (c) or (d):
Figure imgf000023_0001
wherein: represents the point of attachment to the polymeric moiety (e.g., the polyacrylamide moiety);
RB is alkyl, aryl or heteroaryl; Rc is hydrogen, alkyl, aryl or heteroaryl; and RD is an optionally substituted aryl.
[0085] In one embodiment, RB is alkyl, preferably a C2-C20 alkyl, more preferably a C2-C12 alkyl. In one embodiment, RB is ethyl. In one embodiment, RB is butyl. In one embodiment, RB is dodecyl.
[0086] In one embodiment, Rc is hydrogen. In another embodiment, Rc is alkyl, preferably a C2-C20 alkyl, more preferably a C2-C12 alkyl. In one embodiment, Rc is ethyl. In one embodiment, Rc is butyl. In one embodiment, Rc is dodecyl.
[0087] In one embodiment, RD is phenyl or substituted phenyl, e.g., substituted by cyano.
[0088] The thiocarbonylthio terminal groups can be removed or modified by several methods known by one skilled in the art (for example, it can be referred to Moad, G. et ah, Polymer International, 2011, Vol. 60, pp. 9-25; and Willcock, H. et ah, Polymer Chemistry, 2010, Vol. 1, pp. 149-157). Examples of thiocarbonylthio terminal groups transformations include, without being limited to: reaction with nucleophiles, leading to the replacement of the thiocarbonylthio terminal group by a thiol group (-SH), which can further react in coupling reactions such as disulfide formation and Michael addition; thermolysis, leading to the elimination of the thiocarbonylthio group and thereby to the formation of an unsaturated end-chain at the extremity of the polymeric moiety; radical-induced reduction, leading to the replacement of the thiocarbonylthio terminal group by a hydrogen; addition-fragmentation-coupling, leading to the replacement of the thiocarbonylthio terminal group by a group brought under the form of a radical, usually using a functionalized azo-initiator; and hetero Diels-Alder reaction, leading to transformation of the thiocarbonylthio terminal group with a dienophile. [0089] Examples of functionalized azo -initiators that may be used for addition- fragmentation-coupling include azobisisobutyronitrile (AIBN),
4,4'-azobis(4-cyanovaleric acid) (ACVA), l,l'-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50), 2, 2'-azobis [2-methyl- N- (2-hydroxyethyl)propionamide] (VA-086), 2,2'-Azobis[2-(2-imidazolin-2- yl)propane] dihydrochloride (VA-044), 2,2'-azobis(2,4-dimethylvaleronitrile) (V-65), 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70).
[0090] In some embodiments, particularly when the thiocarbonylthio terminal group is modified by addition-fragmentation-coupling using above listed functionalized azo-initiators, the terminal group RA is selected from:
Figure imgf000025_0001
wherein --- represents the point of attachment to the polymeric moiety (e.g., the polyacrylamide moiety. [0091] The polymer compound of the invention can alternatively be obtained by ATRP polymerization. In such case, the terminal group RA is typically a halogen atom, such as bromine or fluorine. Such terminal groups can be modified, for example to form copolymers.
[0092] The polymer compound of the invention can also be obtained by NMP polymerization. In such case, the terminal group RA is typically a nitroxide group, for example of formula -0-NRERF wherein RE and RF are independently selected from alkyl, arylalkyl, and dialkylphosphorylalkyl. Such terminal groups can be modified, for example to form copolymers.
[0093] In some embodiments, the terminal group RA may be selected from halo, -S-(C=S)-S-Rb, -S-(C=S)-0-Rb, -S-(C=S)-NRBRc, -S-(C=S)-RD wherein RB, Rc and RD are as defined above, -0-NRERF wherein RE and RF are as defined above, hydrogen, -OH, -SH, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups; each of which group being optionally substituted by one or more substituent(s) selected from halo, cyano, alkoxy, haloalkoxy heterocyclyl, carboxy, -OH, oxo, amino, alkylamino, hydroxyalkylamino, and amidine.
Linker
[0094] The polymer compound of the invention comprises a linker which links DM1 or an analog thereof to the polymeric moiety, e.g., the polyacrylamide moiety.
[0095] The linker may be of any type and is typically selected depending on the physicochemical and biological properties which are sought when modifying mertansine or its analogues.
[0096] In some embodiments, the linker may be any chemical chain of at least two covalently linked atoms, which can comprise heteroatoms (e.g., O, NH, S, Se or P) as well as cyclic moieties such as cycloalkyl or heterocyclyl groups. [0097] In some embodiments, the linker may comprise up to 100 carbon atoms and even more. The length and the chemical nature of the linker may be optimized depending on the biological effect which is sought. Particularly, the nature of the linker impacts the kinetics of release of the drug and thus the efficacy of the compound. The choice of the linker also enables to control the capacity of the polymer compound to enter into the tumor.
[0098] In one embodiment, L comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties.
[0099] In some embodiments, L is a chemical chain comprising from 2 to 100 carbon atoms, preferably from 2 to 50 carbon atoms, from 2 to 30 carbon atoms, e.g., from 5 to 25 carbon atoms, or from 5 to 20 carbon atoms.
[0100] In some embodiments, L may be selected from alkyl (e.g., Ci-20 alkyl, Ci-12 alkyl or Ci-6 alkyl), ether, polyether, alkyl amide, alkylene diamide or a combination thereof. As used herein, “combination” means that L may comprise several hydrocarbon chains, oligomer chains or polymeric chains ( e.g ., 2, 3, 4, 5 or 6) linked by any appropriate group, such as -0-, -S-, -NHC(O)-, -OC(O)-, -C(0)-0-C(0)-, -NH-, -NH-C(0)-NH-, -NH-(CS)-NH-, -C(O)-, =N-NH- groups. The use of a variety of alkyls is contemplated, including, but not limited to, -(CH2)P-, wherein “p” is from about 2 to about 20 or more. In some embodiments, L comprises a C2-20 straight or branched alkyl chain. In some embodiments, L is a polyether (e.g., polyethylene or polypropylene glycol). The use of a variety of ethers and polyethers is contemplated, including, but not limited to polyethylene glycol (“PEG”) of formula -(OCthCth)?-, wherein “p” is an integer from 1-10; and polypropylene glycol, of formula -(OCH(CH3)CH2)P-, wherein “p” is an integer from 1-10. In some embodiments, L is an alkyl amide. The use of a variety of alkyl amides is contemplated, including, but not limited to, -(CH2)m-C(0)NH-(CH2)P- and -(0CH2CH2)P-C(0)NH-(0CH2CH2)q-, wherein “p” and “q” can be the same or different and “p” and “q” are from about 1 to about 20 or more. In some embodiments, L may also comprise an alkylene diamine, e.g., -NH-(CH2)r-NH-, where “r” is an integer from 2 to 20, for instance from 2 to 10, or an integer selected from 2, 3, 4, or 5. The use of a variety of amides, ester or thioesters having the linking units of alkyl or ether bonds is contemplated, including, but not limited to, -R1-C(0)NH-R2-, -R1-C(0)0-R2- and -R1-C(0)S-R2- wherein “R1” and “R2” are each independently selected from optionally substituted alkyls, ethers, or polyethers.
[0101] In some embodiments, L is selected from an optionally substituted group comprising, or consisting of, one or more saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains, linked by any appropriate group, such as -0-, -S-, -NHC(O)-, -OC(O)-, -C(0)-0-C(0)-, -NH-, -NH-C(0)-NH-, -NH-(CS)-NH-, -C(O)-, =N-NH- and combinations thereof.
[0102] In some embodiments, L may comprise one or more cyclic moieties such as cycloalkyl, heterocyclyl, aryl, or heteroaryl groups. For example, in some embodiments, L comprises an optionally substituted cycloalkyl group, such as cyclohexyl. For example, in some embodiments, L comprises an optionally substituted heterocyclyl group, such as succinimidyl. [0103] In some embodiments, L comprises, or consists of, one or more group(s) selected from optionally substituted, saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains; optionally substituted cycloalkyl; and optionally substituted heterocyclyl; wherein said groups are linked through single bond, -0-, -S-, -NHC(O)-, -OC(O)-, -C(0)-0-C(0)-, -NH-, -NH-C(0)-NH-, -NH-(CS)-NH-, -C(O)-, =N-NH- and combinations thereof.
[0104] The polymer compound of the invention can be obtained by RAFT polymerization. In such case, the linker L typically comprises moieties usually found in chain-transfer agents (RAFT agents), such as for example:
Figure imgf000028_0001
wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety (e.g., the polyacrylamide moiety) of the polymer compound of the invention.
[0105] In one embodiment, L is of formula -L2-L1- wherein: L2 links to the thiol group of DM1 or DM4 and L1 links to the polymeric moiety
(e.g., the polyacrylamide moiety);
L1 is selected from moieties (i), (ii), (iii) and (iv):
Figure imgf000028_0002
wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L2; and
L2 is a linker, which preferably comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; or L2 is absent. [0106] In one embodiment, L is of formula:
Figure imgf000029_0001
wherein
--- represents the points of attachment; wherein the point of attachment on the succinimide moiety links to the thiol group of DM1 or DM4; and the point of attachment on L3 links to the polymeric moiety (e.g., the polyacrylamide moiety); and
L3 is a linker, which preferably comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; [0107] In one embodiment, L3 is -L3’-L1- wherein
L3’ links to the succinimide group and is a linker, which preferably comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; and L1 is as defined above, wherein the carbonyl group links to L3’. [0108] Above definitions and descriptions relative generally to “linkers” and/or to linker L apply similarly to linkers L2, L3 and L3’.
[0109] In one embodiment, L is selected from:
Figure imgf000029_0002
Figure imgf000030_0001
wherein -- - represents the points of attachment; wherein the point of attachment on the left is to the thiol group of DM1 or DM4 and the point of attachment on the right is to the polymeric moiety (e.g., the polyacrylamide moiety).
[0110] The polymer compound of the invention can alternatively be obtained by ATRP polymerization. In such case, the linker L typically comprises moieties usually found in ATRP initiators, such as for example:
Figure imgf000030_0002
(iii); as already listed above for RAFT polymerization, wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety (e.g., the polyacrylamide moiety) of the polymer compound of the invention. [0111] The polymer compound of the invention can alternatively be obtained by NMP polymerization. In such case, the linker L typically comprises moieties usually found in NMP initiators, such as for example:
Figure imgf000030_0003
(iii); as already listed above for RAFT polymerization, wherein --- represents the points of attachment; wherein the point of attachment opposite to the carbonyl group is linked to the polymeric moiety ( e.g ., the polyacrylamide moiety) of the polymer compound of the invention. Subformulae of polymer compounds
[0112] In one embodiment, the polymer compound according to the invention is of formula (1-1):
Figure imgf000031_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, and RA are as defined and described in classes and subclasses above and herein;
L1 is selected from moieties (i), (ii), (iii) and (iv):
Figure imgf000031_0002
wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L2; and
L2 is a linker, defined and described in classes and subclasses above and herein; or L2 is absent. [0113] In one embodiment, the polymer compound according to the invention is of formula (I-li):
Figure imgf000032_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, L2, and RA are as defined and described in classes and subclasses above and herein.
[0114] In one embodiment, the polymer compound according to the invention is of formula (1-2):
Figure imgf000032_0002
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, and RA are as defined and described in classes and subclasses above and herein;
L3 is a linker, defined and described in classes and subclasses above and herein. [0115] In one embodiment, the polymer compound according to the invention is of formula (1-3):
Figure imgf000033_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, and RA are as defined and described in classes and subclasses above and herein; and
X is O, N or S; preferably X is O or N.
[0116] In one embodiment, X is O. In one embodiment, X is N. In one embodiment, X is S.
[0117] In one embodiment, the polymer compound according to the invention is of formula (1-4):
Figure imgf000033_0002
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m and n are as defined and described in classes and subclasses above and herein; and
RG is selected from -S-RB, -0-RB, -NRBRC and R°, wherein RB, Rc and RD are as defined and described in classes and subclasses above and herein. [0118] In one embodiment, the polymer compound of the invention is of formula (I- a):
Figure imgf000034_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, L and RB are as defined and described in classes and subclasses above and herein.
[0119] In one embodiment, the polymer compound according to the invention is of formula (I-l-a):
Figure imgf000034_0002
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n and RB are as defined and described in classes and subclasses above and herein;
L1 is selected from moieties (i), (ii), (iii) and (iv):
Figure imgf000034_0003
wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L2; and
L2 is a linker, defined and described in classes and subclasses above and herein; or L2 is absent. [0120] In one embodiment, the polymer compound according to the invention is of formula (I-li-a):
Figure imgf000035_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n, L2 and RB are as defined and described in classes and subclasses above and herein.
[0121] In one embodiment, the polymer compound according to the invention is of formula (I-2-a):
Figure imgf000035_0002
or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n and RB are as defined and described in classes and subclasses above and herein;
L3 is a linker, defined and described in classes and subclasses above and herein. [0122] In one embodiment, the polymer compound according to the invention is of formula (1-3 -a):
Figure imgf000036_0001
3 -a) or a pharmaceutically acceptable salt and/or solvate thereof, wherein m, n and RB are as defined and described in classes and subclasses above and herein; and
X is O, N or S; preferably X is O or N.
[0123] In one embodiment, the polymer compound according to the invention is selected from:
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
and pharmaceutically acceptable salts and/or solvates thereof.
Manufacturing of the polymer compounds
[0124] The present invention also relates to methods of manufacturing the polymer compounds according to the invention.
[0125] The polymer compounds of the invention can be manufactured by radical polymerization, preferably by controlled radical polymerization, for example by reversible addition-fragmentation chain-transfer (RAFT) polymerization, by atom transfer radical polymerization (ATRP) or by nitroxide-mediated polymerization (NMP). Thereby, the dispersity of the polymer compound is low.
[0126] The synthesis of the polymer compounds of the invention is advantageously performed by drug-initiated polymerization. By “drug-initiated polymerization” it is referred to a controlled radical polymerization technique using a control agent (such as a RAFT agent in a RAFT polymerization, or ATRP or NMP initiator in ATRP and NMP polymerizations) modified by chemical coupling with an active ingredient. Thus, the modified control agent carries an active ingredient molecule, and the resulting polymer also carries the active ingredient.
[0127] In one embodiment, the polymer compounds of the invention are manufactured by reversible addition-fragmentation chain-transfer (RAFT) polymerization. [0128] A RAFT polymerization system usually comprises: a source of radicals
(e.g., radical polymerization initiators); monomers; a RAFT agent; and, optionally, a solvent. [0129] By “radical polymerization initiator” it is referred to compounds used to produce radicals and thus initiate radical polymerization. These compounds have a chemical function capable of releasing radicals under the action of heat, irradiation of light, oxidation-reduction reaction, ionizing radiation, electrochemical reactions and/or sonication. Non-limiting examples of initiators include compounds of the azo type, such as 2,2'-azobis(2-methylpropionitrile) (also known as azobisisobutyronitrile, abbreviated as AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), l,T-azobis(cyclohexanecarbonitrile); of the inorganic peroxide type; or of the organic peroxide type such as benzoyl peroxide, lauroyl peroxide, methyl ethyl ketone peroxide, and tert-butyl peroxybenzoate.
[0130] RAFT polymerization involves the use of a chain-transfer agent, named “RAFT agent”, to mediate the polymerization via a reversible chain-transfer process and to afford control over the generated molecular weight and polydispersity during a free-radical polymerization. Preferably the RAFT agent comprises a thiocarbonylthio group (S=C-S), such as trithiocarbonates (A), dithiocarbamates (B), xanthates (C) or dithiobenzoates (D):
Figure imgf000040_0001
[0131] When the RAFT polymerization is performed according to the method of the drug-initiated polymerization, the RAFT agent comprises the active ingredient, herein mertansine or an analogue thereof. [0132] RAFT agents suitable for the manufacturing of the polymer compounds according to the invention are detailed hereafter. Particularly, functionalized RAFT agent of formula (II), and subformulae thereof, are suitable for the manufacturing of the polymer compounds of the invention.
[0133] In the present invention, the polymer compounds contain a polyacrylamide moiety. Therefore, the monomer used for their manufacturing is acrylamide monomer. [0134] In one embodiment, method of manufacturing of a polymer compound according to the invention comprises a step of controlled radical polymerization performed by contacting a source of radicals, acrylamide monomers and a compound of formula (B):
Figure imgf000041_0001
or a salt and/or solvate thereof, wherein m and L are as defined and described in classes and subclasses above and herein; and
RG is selected from -S-RB, -0-RB, -NRBRC and R°, wherein RB, Rc and RD are as defined and described in classes and subclasses above and herein; under conditions suitable to obtain the polymer compound of formula (1-4) as herein defined.
[0135] The polymer compound of formula (I) can be obtained from the compound of formula (1-4) by the removal or modification of the thiocarbonylthio terminal group. Methods of such removal or modifications are known by one skilled in the art (for example, it can be referred to Moad, G. et al, Polymer International, 2011, Vol. 60, pp. 9-25; and Willcock, H. et ah, Polymer Chemistry, 2010, Vol. 1, pp. 149-157).
[0136] The method of manufacturing of the invention advantageously makes it possible to provide a polymer compound having only one mertansine (or analogue thereof) residue at one end. This control of the drug loading facilitates the purification of the polymer compound and is essential for therapeutic use.
[0137] In one embodiment, the source of radicals is a radical polymerization initiator. In one embodiment, the radical polymerization initiator is selected from compounds of the azo type, such as azobisisobutyronitrile (AIBN), 4,4'-azobis(4-cyanovaleric acid) (ACVA), 1 , 1 '-azobis(cyclohexanecarbonitrile), 2,2'-azobis(2-methylpropionamidine) dihydrochloride (V-50), 2,2'-azobis[2-methyl-/V-(2-hydroxyethyl)propionamide] (VA- 086), 2,2'-Azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), 2,2'- azobis(2,4-dimethylvaleronitrile) (V-65), 2,2'-azobis(4-methoxy-2,4- dimethylvaleronitrile) (V-70); of the inorganic peroxide type; or of the organic peroxide type such as benzoyl peroxide, lauroyl peroxide, methyl ethyl ketone peroxide, and tert- butyl peroxybenzoate. Preferably the radical polymerization initiator is AIBN.
[0138] The amount of acrylamide monomers used in the method of manufacturing of the invention depends on the expected length of the polymer and can be estimated by one skilled in the art accordingly. As an illustration, it is referred to the synthesis of polymer compounds (PI) to (P3) reported in the Example section.
[0139] The temperature of polymerization is selected such that (a) chain growth occurs at an appropriate rate, (b) the radical polymerization initiator delivers radicals at an appropriate rate and (c) the RAFT equilibrium favors the active rather than dormant state to an acceptable extent. In one embodiment, the temperature of the controlled radical polymerization of the method of the invention ranges from 10 °C to 100 °C, preferably from 50°C to 100°C, preferably about 70°C.
[0140] In one embodiment, the polymer compound obtained by the method of manufacturing of the invention is purified by precipitation and/or by dialysis. Precipitation may be conducted in alcohol solvents such as for example methanol. Dialysis may be conducted after solubilization of the polymerization product in water using a semipermeable membrane of suitable pore size (e.g., from 1 kDa to 15 kDa, preferably from 1 kDa to 10 kDa). Functionalized RAFT agents
[0141] The invention further relates to compounds which are useful for the manufacturing of the polymer compounds of the invention. Particularly, the invention provides RAFT agents functionalized with mertansine or an analogue thereof (e.g., DM4). [0142] In one embodiment, the present invention provides compounds of formula (B):
Figure imgf000043_0001
or a salt and/or solvate thereof, wherein m, L and RG are as defined and described in classes and subclasses above and herein.
[0143] In one embodiment, compounds are of formula (B-a):
Figure imgf000043_0002
or a salt and/or solvate thereof, wherein m, L and RB are as defined and described in classes and subclasses above and herein.
[0144] In one embodiment, compounds are of formula (B-l-a):
Figure imgf000043_0003
or a salt and/or solvate thereof, wherein m, L1, L2 and RB are as defined and described in classes and subclasses above and herein.
[0145] In one embodiment, compounds are of formula (B-li-a):
Figure imgf000044_0001
-li-a) or a salt and/or solvate thereof, wherein m, L2 and RB are as defined and described in classes and subclasses above and herein.
[0146] In one embodiment, compounds are of formula (lib):
Figure imgf000044_0002
or a salt and/or solvate thereof, wherein m, L3 and RB are as defined and described in classes and subclasses above and herein. [0147] In one embodiment, compounds are of formula (lie):
Figure imgf000045_0001
or a salt and/or solvate thereof, wherein m, X and RB are as defined and described in classes and subclasses above and herein.
[0148] In one embodiment, the compounds are selected from:
Figure imgf000045_0002
Figure imgf000046_0001
and salts and/or solvates thereof.
[0149] It will be appreciated that the compounds of formula (B) can be manufactured by methods known by one skilled in the art. As an illustration, one can refer to the synthesis of compounds (RAFT-1), (RAFT-2), (RAFT-3), (RAFT-4), (RAFT-5), (RAFT-6) and (RAFT-7) described in the Example section.
[0150] The invention also relates to the use of the compounds of formula (B) according to the invention to manufacture the polymer compounds according to the invention, preferably by controlled radical polymerization.
Uses of the polymer compounds
[0151] The invention also relates to a polymer compound according to the invention, as described hereinabove, for use as a medicament. This invention also relates to a polymer compound according to the invention, as described hereinabove, for use in the treatment of cancer. This invention also relates to the use of a polymer compound according to the invention, as described hereinabove, in the manufacture of a medicament for the treatment of cancer. This invention also relates to a method for the treatment of cancer in a subject in need thereof, comprising a step of administering to said subject a therapeutically effective amount of a polymer compound according to the invention, as described hereinabove.
[0152] In addition to the treatment of warm-blooded animals such as mice, rats, horses, cattle, sheep, dogs, cats, monkeys, etc., the polymer compounds of the invention are effective for use in humans.
[0153] Various cancers are known in the art. Cancers that can be treated using the methods of the invention include solid cancers and non-solid cancers, especially benign and malignant solid tumors and benign and malignant non- solid tumors. The cancer may be metastatic or non-metastatic. The cancer may be may be familial or sporadic.
[0154] In one embodiment, the cancer to be treated according to the present invention is a solid cancer. As used herein, the term “solid cancer” encompasses any cancer (also referred to as malignancy) that forms a discrete tumor mass, as opposed to cancers (or malignancies) that diffusely infiltrate a tissue without forming a mass. Examples of solid tumors include, but are not limited to: biliary tract cancer, brain cancer (including glioblastomas and medulloblastomas), breast cancer, carcinoid, cervical cancer, choriocarcinoma, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, gastric cancer, glioma, head and neck cancer, intraepithelial neoplasms (including Bowen’s disease and Paget’s disease), liver cancer, lung cancer, neuroblastomas, oral cancer (including squamous cell carcinoma), ovarian cancer (including those arising from epithelial cells, stromal cells, germ cells and mesenchymal cells), pancreatic cancer, prostate cancer, rectal cancer, renal cancer (including adenocarcinoma and Wilms tumor), sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma and osteosarcoma), skin cancer (including melanoma, Kaposi’s sarcoma, basocellular cancer and squamous cell cancer), testicular cancer including germinal tumors (seminomas, and non-seminomas such as teratomas and choriocarcinomas), stromal tumors, germ cell tumors, thyroid cancer (including thyroid adenocarcinoma and medullary carcinoma) and urothelial cancer.
[0155] In one embodiment, the cancer to be treated according to the present invention is a non- solid cancer. Examples of non- solid tumors include but are not limited to hematological neoplasms. As used herein, a hematologic neoplasm is a term of art which includes lymphoid disorders, myeloid disorders, and AIDS associated leukemias. Lymphoid disorders include but are not limited to acute lymphocytic leukemia and chronic lymphoproliferative disorders (e.g., lymphomas, myelomas, and chronic lymphoid leukemias). Lymphomas include, for example, Hodgkin’s disease, non- Hodgkin’s lymphoma lymphomas, and lymphocytic lymphomas). Chronic lymphoid leukemias include, for example, T cell chronic lymphoid leukemias and B cell chronic lymphoid leukemias.
[0156] In one embodiment, the cancer to be treated according to the present invention is selected from breast cancer, pancreatic cancer, ovarian cancer and lung cancer. [0157] According to one embodiment, the polymer compound according to the invention is administered to the subject as sole therapeutic agent. According to another embodiment, the polymer compound according to the invention is administered to the subject in combination with at least another therapeutic agent. Examples of suitable other therapeutic agents include anticancer agents, such as immunotherapeutic agents, chemotherapeutic agents, antiangiogenic agents, multidrug resistance-associated proteins inhibitors, radio therapeutic agents, or any combination thereof.
[0158] According to one embodiment, the polymer compound according to the invention is for use in a patient treated by immunotherapy, a chemotherapy, radiotherapy or a combination thereof.
[0159] The polymer compounds of the invention may be administered by parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracistemal injection or infusion, subcutaneous injection, or implant), topical, oral, sublingual, inhalation spray, nasal, vaginal, or rectal routes of administration and may be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration.
[0160] In one embodiment, the polymer compound of the invention is administered by parenteral route, preferably by intravenous injection or infusion, subcutaneous injection, intramuscular injection, or a combination thereof. In one embodiment, the polymer compound of the invention is administered by intravenous injection or infusion. In one embodiment, the polymer compound of the invention is administered by subcutaneous injection. In one embodiment, the polymer compound of the invention is administered by intramuscular injection.
[0161] The invention also relates to a pharmaceutical composition comprising a polymer compound according to the invention, as described hereinabove, and at least one pharmaceutically acceptable carrier.
[0162] In one embodiment, the pharmaceutical composition comprises 1 to 99 % of polymer compound according to the invention in weight to the total weight of the composition; and 1 to 99 % of pharmaceutically acceptable carrier in weight to the total weight of the composition.
[0163] In one embodiment, the pharmaceutical composition comprises the polymer compound according to the invention as sole therapeutic agent. In another embodiment, the pharmaceutical composition further comprises at least another therapeutic agent. [0164] The pharmaceutical compositions for the administration of the polymer compounds of this invention may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. In general, the pharmaceutical compositions are prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into the desired formulation.
[0165] As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts. For parenteral use, the pharmaceutical compositions may be in the form of a sterile injectable preparation. The sterile injectable preparation may be a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally- acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. For topical use, the pharmaceutical compositions may be in a form of creams, ointments, jellies, solutions or suspensions, etc., containing the polymer compounds of the invention. For oral use, the pharmaceutical compositions may be in a form of tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
[0166] In the pharmaceutical composition, the polymer compound of the invention is included in an amount sufficient to produce the desired effect upon the process or condition of diseases. It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific polymer compound employed, the metabolic stability and length of action of that polymer compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. A person skilled in the art can adjust dosing and/or determine a dose range to treat a particular subject and/or a particular disease based on the aforementioned factors, as well as other factors that are well known in the art.
[0167] Advantageously, the use of the mertansine polymer compound according to the present invention leads to administering equivalent doses of mertansine, or analogues thereof, being from significantly higher compared to the maximum tolerated dose of mertansine, or analogues thereof, for example 5- to 20-fold higher.
BRIEF DESCRIPTION OF THE DRAWINGS [0168] Figure 1 is a graph showing overtime the total radiant efficiency at the tumor region of interest (ROI) for mice bearing 4T1 breast tumors and injected with fluorescent model compounds (free Cyanine 5.5 or cyanine polymer compounds 10 or 11). Error bars are standard deviations (n=3).
[0169] Figure 2 is a graph showing the survival rate overtime of mice bearing tumor model MDA-MB-231 and treated with DM1, paclitaxel (PTX) or polymer compounds (PI), (P2) or (P3) of the invention.
[0170] Figure 3 is a graph showing the tumor volume (mm3) overtime of mice bearing tumor model Calu-6 and treated with a vehicle [black circles], comparative polymer compound DM1-AEMI-PEG (12) [squares], or polymer compounds (P3) or (P10) of the invention [triangles and hexagons respectively] .
[0171] Figure 4 is a graph showing the tumor volume (mm3) overtime of mice bearing tumor model HER2+ and treated with a vehicle [circles], comparative compound trastuzumab emtansine (T-DM1) [white circles], or polymer compounds (P3) or (P10) of the invention administered in combination with trastuzumab [triangles and hexagons respectively] .
EXAMPLES [0172] The present invention is further illustrated by the following examples.
[0173] The following abbreviations are used:
AAm: acrylamide monomer;
ACN: acetonitrile;
CEOEC: 2-cyano-5-(ethylamino)-5-oxopentan-2-yl ethyl carbonotrithioate; AEMI: N- (2- aminoethy l)maleimide ;
AIBN: azobisisobutyronitrile;
CDP: 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid; CEP: 4-cyano-4-[(ethylsulfanylthiocarbonyl)sulfanyl]pentanoic acid;
DCC: dicyclohexylcarbodiimide; DCM: dichloromethane;
DM1: mertansine;
DM4: ravtansine;
DMSO: dimethylsulfoxide;
EtOAc: ethyl acetate; FBS: fetal bovine serum;
HEMI: N- (2-h y dro x y ct h y 1 ) m a 1 ci m i dc ;
MCC: 4-(/V-maleimidomethyl)cyclohexane-l -carboxylic acid MeOH: methanol;
NHS: N- h y dro x y s ucc i n i m i dc ; PEG-NHS: polyethylene glycol NHS ester; r.t.: room temperature;
I. SYNTHESIS OF THE POLYMER COMPOUNDS OF FORMULA 111
Materials / Analytical methods [0174] CDP (AB252723) was obtained from abcr GmbH, CEP (A718802) from
Ambeed, Inc., HEMI (EN300-121975) and AEMI (EN300-7379578) from SIA Enamine, DM1 (DC8540) and DM4 (DC31423) from DC Chemicals Ltd., Cyanine 5.5 amine (670C0) from Lumiprobe GmbH. 1 H NMR and 13C NMR spectra of small molecules were recorded in CDCb, DMSO-c/r, or MeOD on a Bruker Avance spectrometer operating at 300 MHz (¾ and 75 MHz (13C) or 400 MHz (¾ and 100 MHz (13C) at room temperature. The chemical shifts of 1 H and 13C are reported in ppm relative to the solvent residual peaks. For ! H NMR spectroscopy of polymers, acquisition was performed in 5 mm diameter tubes in D2O at 70 °C (128 scans) on a Bruker Avance 3 HD 400 spectrometer operating at 400 MHz. High resolution mass spectra (HR-MS) were recorded on a MicroMass LCT Premier Spectrometer.
1.1. INTERMEDIATES OF SYNTHESIS
CDP-NHS (1): 2,5 -dioxopyrrolidin- 1 -yl 4-cyano-4-
(((dodecylthio)carbonothioyl)thio)pentanoate
Figure imgf000053_0001
[0175] To a solution of compounds CDP (5mmol, 2g) and NHS (6 mmol, 691.07 mg) in DCM (100 mL), a solution of DCC (5 mmol, 1.03g) was added dropwise at 0°C with an ice bath. The reaction mixture was stirred overnight at room temperature. A white precipitate was obtained, it was filtered off. The crude product was dissolved in DCM and washed with water (2 x 100 mL). The organic layer was evaporated and then the crude was triturated with diethyl ether. The crude mixture can be crystallized from cyclohexane to afford compound CDP-NHS (1) as yellow powder in 72 % yield. 1 H NMR (300 MHz, CDCb) d 3.36 (t, / = 7.4 Hz, 2H), 3.00 - 2.81 (m, 6H), 2.61 (m, 2H), 1.91 (s, 3H), 1.71 (dd, / = 14.7, 7.5 Hz, 2H), 1.51 - 1.13 (m, 18H), 0.90 (t, 7 = 6.5 Hz, 3H). 13C NMR (75 MHz, CDCb) d 216.65 (Cq), 168.86 (Cq), 167.17 (Cq), 118.79 (Cq), 46.19 (Cq), 37.32 (Cq), 34.05 (CH2), 33.40 (CH2), 32.06 (CH2), 29.76 (CH2), 29.68 (CH2), 29.56 (CH2), 29.47 (CH2), 29.21 (CH2), 29.08 (CH2), 27.80 (CH2), 26.94(CH2), 25.73
(CH3), 24.95 (CH2), 22.82 (CH2), 14.24 (CH3). HRMS calcd for C23H37N204S3 (M + H)+, 501.1915; found, 501.1903.
CEP-NHS (2): 2,5-dioxopyrrolidin-l-yl 4-cyano-4-
(((ethylthio)carbonothioyl)thio)pentanoate
Figure imgf000054_0001
[0176] In a round bottom flask, CEP (7.6 mol, 2.0 g), and NHS (9.12 mol, 1.05g) were dissolved in DCM (150 mL). The mixture was sonicated for 3.30 min. A solution of DCC (7.6 mol, 1571.5 mol) in DCM was added dropwise at 0°C. The reaction mixture was stirred overnight at room temperature. A white precipitate was observed. This precipitate was filtered off. The impurities were eliminated by extraction after adding water. The organic layer was evaporated and then the crude was triturated with diethyl ether. The product can be crystallized from DCM/cyclohexane to give CEP-NHS (2) as a yellow powder, yield 66%.
Figure imgf000054_0002
NMR (300 MHz, CDCb) d 3.38 (q, J=7.4 Hz, 2H), 3.05 - 2.78 (m, 6H), 2.77 - 2.45 (m, 2H), 1.93 (s, 3H), 1.40 (t, J=7.4 Hz, 3H).13C NMR (75 MHz, CDCb) d 216.30 (Cq), 168.72 (Cq), 167.00 (Cq), 118.60 (Cq), 46.00 (Cq), 33.70 (CH2), 33.20 (CH2), 31.42 (CH2), 26.84, 25.55 (CH2), 24.85, 24.75 (CH3), 12.67 (CH3). HRMS calcd for CI3HI7N204S3 (M + H)+, 361.0347; found, 361.0350.
HEMI-CDP (3): 2-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)ethyl 4-cyano-4- ((dodecylthio)carbonothioyl)thio)pentanoate
Figure imgf000054_0003
[0177] In a 25 mL round bottom flask, CDP-NHS (1) (0.4 mmol, 206.8 mg), HEMI (0.59 mmol, 82.9 mg), Na2CO3 (2.13 mmol, 224.5 mg) were dissolved in anhydrous DCM (10 mL). The yellow mixture was stirred at r.t. for 24 hours. Then HEMI (0.57 mmol; 80 mg) and Na2CO3 (0.95 mmol; 100 mg) were added and the mixture was stirred again for 48 hours. The crude mixture was purified on silica gel column chromatography using as eluant, cyclohexane:EtOAc (7:3) to give the expected product HEMI-CDP (3) as a yellow amorphous solid, yield 48%.1H NMR (300 MHz, CDCl3) ^ 6.73 (s, 2H), 4.25 (t, J = 5.2 Hz, 2H), 3.79 (t, J = 5.2 Hz, 2H), 3.32 (t, J = 7.4 Hz, 2H), 2.67 – 2.23 (m, 4H), 1.86 (s, 3H), 1.77 – 1.53 (m, 2H), 1.47 – 1.15 (m, 18H), 0.87 (t, J = 6.6 Hz, 3H). 13C NMR, JMOD (75 MHz, CDCl3) ^ 220.2 (Cq), 171.2 (Cq), 170.3 (Cq), 134.2 (CH), 119.0 (Cq), 62.09 (CH2), 46.4 (Cq) 37.2(CH2), 36.9 (CH2), 33.8 (CH2), 32.0 (CH2), 29.7 (CH2), 29.6 (CH2), 29.5 (CH2), 29.4 (CH2), 29.1 (CH2), 29.0 (CH2), 27.8(CH2), 24.9 (CH3), 22.8 (CH2),14.2 (CH3) HRMS calcd for C25H39N2O4S3 (M + H)+, 527.2072; found, 527.2067. HEMI-CEP (4): 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl 4-cyano-4- (((ethylthio)carbonothioyl)thio)pentanoate
Figure imgf000055_0001
[0178] In a round bottom flask, CEP-NHS (2) (0.28 mmol, 100 mg) were dissolved in anhydrous DCM (4 mL) under argon atmosphere. HEMI (0.28 mmol, 39.5 mg) was dissolved in 2 mL of anhydrous DCM and added in the round bottom flask. The mixture was stirred and Na2CO3 (1.4 mmol, 147 mg) was added and stirred at room temperature. After one day, 0.5 equivalent of HEMI (0.7 mmol, 20 mg) was added. The yellow mixture was stirred at 30°C for 48 hours. The crude mixture was purified by column chromatography using cyclohexane:EtOAc (7:3) to give the expected product HEMI- CEP (4) as a yellow solid, yield 61%.
Figure imgf000055_0002
NMR (300 MHz, DMSO-d6) ^ 7.04 (s, 2H), 4.17 (t, J = 5.3 Hz, 2H), 3.67 (t, J = 5.3 Hz, 2H), 3.39 (q, J = 7.4 Hz, 2H),2.49-2.32 (m, 2H), 2.57 – 2.29 (m, 3H), 1.84 (s, 3H), 1.30 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) ^ 216.7 (Cq) 171.2 (Cq), 170.3 (Cq), 134.2 (CH), 118.9 (Cq), 62.0 (CH2), 46.3 (Cq), 36.7, 33.6 (CH2), 31.3 (CH2), 29.5 (CH2), 24.8 (CH3), 12.7 (CH3). AEMI- CEP (5) : 2-cyano-5 - ((2- (2,5 -dioxo-2,5-dihydro- lH-pyrrol- 1 -yl)ethyl)amino) - 5-oxopentan-2-yl ethyl carbonotrithioate
Figure imgf000056_0001
[0179] In a round bottom flask was added was added CEP-NHS (2) (0.555 mmol, 200 mg), AEMI (0.83 mmol, 146 mg), in 5 mL DCM. NaiCCE (1.11 mmol, 116 mg) was added to the reaction mixture which was stirred at room temperature for 24 h. The mixture was dried under vacuum. It was purified on silica gel column using cyclohexane :EtO Ac (1:1). The expected product AEMI-CEP (5) was obtained as a yellow solid. Yield 46 %. JH NMR (300 MHz, Chloroform- J) d 6.74 (s, 2H), 6.04 (s, 1H), 3.71 (dd, 7= 6.7, 4.2 Hz, 2H), 3.46 (m, 2H), 3.35 (q, J = 7.5 Hz, 2H), 2.54 - 2.30 (m, 4H), 1.89 (s, 3H), 1.37 (t, J
= 7.4 Hz, 3H). LC/MS, XSELECT 01433023015 - 2.1x75mm 2.5pm - H2O/ACN/0.1 % AF, retention time 8.39 minutes, LRMS for C15H20N3O3S3, found 386.0673 (M+H)+.
AEMI- CDP (6): 2-cyano-5 - ((2- (2,5 -dioxo-2,5-dihydro- lH-pyrrol- 1 -yl)ethyl)amino) - 5-oxopentan-2-yl dodecyl carbonotrithioate
Figure imgf000056_0002
[0180] In a round bottom flask was added was added CDP-NHS (1) (0.4 mmol, 200 mg), AEMI (0.65 mmol, 100 mg), in 10 mL DCM. The mixture was not homogeneous the pyridine was added (1 equiv) as a co- solvent. NaiCCE (1.17 mmol, 123 mg) was added to the reaction mixture which was stirred at room temperature for 48 h. The mixture was dried under vacuum. It was purified on silica gel column using a gradient of DCM-MeOH (0% to 3 % MeOH). The expected product AEMI-CDP (6) was obtained as a yellow solid in 52 % yield. lH NMR (300 MHz, Methanol-d4) d 6.83 (s, 2H), 3.76 - 3.55 (m, 3H), 3.48 - 3.24 (m, 8H), 2.55 - 2.22 (m, 5H), 1.88 (s, 4H), 1.71 (q, J = 7.4 Hz, 4H), 1.33 (d, J = 8.6 Hz, 26H), 0.98 - 0.83 (m, 4H). HRMS calcd for CisHsgNsOsSsNa (M + Na)+,
548.2051; found, 548.2054. MEP-NHS (13): 2,5-dioxopyrrolidin-1-yl 2-(((ethylthio)carbonothioyl)thio)propanoate
Figure imgf000057_0001
[0181] In a round bottom flask NHS (5.73 mmol, 659.3 mg) was dissolved in DCM (50 mL) prior to adding MEP (4.77 mmol, 1002.8 mg). The solution was cooled down to 0°C before adding dropwise a solution of DCC (4.76 mmol, 982.6 mg) in DCM (25 mL). The mixture was stirred at r.t. for 12 h and then filtered through Büchner with a layer of silica. The organic layer was washed twice with brine and once with water. It was concentrated under vacuum and then crystallized from EtOH to obtain MEP-NHS (13) (2.93 mmol, 902 mg) as a yellow oil. Yield 65 %.
Figure imgf000057_0002
NMR (300 MHz, CDCl3), ^ 5.16 (q, J = 7.4 Hz, 1H), 3.40 (q, J = 7.4 Hz, 2H), 2.84 (s, 4H), 1.76 (d, J = 7.4 Hz, 3H), 1.38 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) ^ 208.59 – 205.44, 44.94, 31.79, 30.84, 25.56, 16.69, 12.82. MEP-HEMI (14): 2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl 2- (((ethylthio)carbonothioyl)thio)propanoate
Figure imgf000057_0003
[0182] In a round bottom flask was added was added MEP-NHS (13) (1 mmol, 307 mg), HEMI (1.5 mmol, 210 mg) and Na2CO3 (2.95 mmol, 313 mg) in DCM (15 mL). The reaction mixture was stirred at room temperature for 65 hours. The mixture was dried under vacuum. It was purified on silica gel column using a gradient of cyclohexane:EtOAc (100% ; 70% ; 50% ; 0% cyclohexane). The expected product MEP-HEMI (14) (0.21 mmol, 71 mg) was obtained as a yellow solid. Yield 21%. 1H NMR (300 MHz, CDCl3) ^ 6.72 (d, J = 4.0 Hz, 2H), 4.76 (q, J = 7.4 Hz, 1H), 4.41 – 4.16 (m, 2H), 3.84 – 3.78 (m, 2H), 3.34 (q, J = 7.5 Hz, 2H), 1.56 (d, J = 7.4 Hz, 3H), 1.34 (dd, J = 9.4, 5.5 Hz, 3H). Disulfide bond RAFT agent (15): 2-cyano-5-oxo-5-((2-(pyridin-2- yldisulfaneyl)ethyl)amino)pentan-2-yl ethyl carbonotrithioate
Figure imgf000058_0001
[0183] In a round bottom flask, CEP-NHS (2) (0.53 mmol, 201 mg) and (S)-2-Pyridylthio cysteamine hydrochloride (0.53 mmol, 119 mg) were dissolved in anhydrous DCM (8 mL) under argon atmosphere. The solution was cooled to 0°C. Triethylamine (0.53 mmol, 70 µL) diluted in anhydrous DCM (2 mL) was then added drop by drop and the mixture let to stir overnight at room temperature. After one day, 0.3 equivalent of CEP-NHS (2) (0.16 mmol, 60 mg) and triethylamine (0.16 mmol, 22 µL) were added and the yellow mixture was let to stir at room temperature for an additional 24 hours. The mixture was dried under vacuum. It was purified on silica gel column using a gradient of DCM-MeOH (0% to 2 % MeOH) to give the expected product Disulfide bond RAFT agent (15) (0.33 mmol, 144.3 mg) as a yellow oil. Yield 63%.
Figure imgf000058_0002
NMR (300 MHz, CDCl3) ^ 8.57 (ddd, J = 4.9, 1.8, 0.9 Hz, 1H), 7.63 (ddd, J = 8.1, 7.3, 1.8 Hz, 1H), 7.50 (dt, J = 8.1, 1.0 Hz, 1H), 7.18 (ddd, J = 7.3, 4.9, 1.1 Hz, 1H), 3.59 (q, J = 5.8 Hz, 2H), 3.35 (m, 2H), 2.98 – 2.90 (m, 2H), 2.55 – 2.39 (m, 4H), 1.91 (s, 3H), 1.36 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3) ^ 217.1 (Cq), 170.2 (Cq), 159.0 (Cq), 149.9 (CH), 137.0 (CH), 121.5 (CH), 121.4 (CH), 119.2 5 (Cq), 46.7 (CH2), 38.9 (Cq), 37.4 (CH2), 34.5 (CH2), 31.9 (CH2), 31.3 (CH2), 25.0 (CH3), 12.7 (CH3). LC/MS: Gradient Acetonitrile (5% to 100%), C16H21N3OS5H+ (M + H)+ = 432.0454. Retention time: 9.77 min. MEP-Boc (16): tert-butyl (2-(2-(((ethylthio)carbonothioyl)thio)propanamido)ethyl)carbamate
Figure imgf000058_0003
[0184] MEP-NHS (13) (0.78 mmol, 240.6 mg) was dissolved in anhydrous DCM (20 mL) and cooled down to 0°C. A solution of N-Boc-Ethylenediamine (0.87 mmol, 138.8 mg) dissolved in DCM anhydrous (5 mL) was added dropwise to the previous solution. The reaction was stirred overnight at room temperature. The crude was purified by column chromatography using cyclohexane/Ethyl acetate from 0 to 50 % to give the expected product MEP-Boc (16) (0.50 mmol, 175 mg) as a yellow solid. Yield 64%. 1 H NMR (300 MHz, CDC13) d 6.71 (s, 1H), 4.73 (q, / = 7.4 Hz, 2H), 3.82 - 2.98 (m, 6H), 1.60 (d, /= 6.1 Hz, 3H), 1.47 (s, 9H), 1.39 (t, / = 7.4 Hz, 3H). 13C NMR (75 MHz, CDC13) d 173.30 - 168.19, 48.23, 40.30, 33.94, 31.74, 28.37, 16.41, 12.87.
MEP-NH2 (17): l-((2-aminoethyl)amino)-l-oxopropan-2-yl ethyl carbonotrithioate
Figure imgf000059_0001
[0185] In a round bottom flask, MEP-Boc (16) (0.28 mmol, 100 mg) was dissolved in anhydrous DCM (10 mL) the mixture is cooled down to 0°C. A solution of HC1 Dioxane (16 eq, 1.05 mL) in DCM anhydrous (3 mL) was added. The mixture was stirred at room temperature for 4 hours. It was evaporated and precipitated in petroleum ether to yield MEP-NH2 (17) (0.24 mmol, 61 mg) as a yellow solid. Yield 86%. JH NMR (300 MHz, CDCb) d 6.73 (s, 1H), 4.97 - 4.57 (m, 3H), 3.57 - 3.15 (m, 6H), 1.60 (d, 7 = 7.4 Hz, 3H), 1.43 - 1.32 (m, 3H).
MEP-MCC (18): l-((2-(4-((2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)methyl)cyclohexane-l-arboxamido)ethyl)amino)-l-oxopropan-2-yl ethyl carbonotrithioate
Figure imgf000059_0002
[0186] In a round bottom flask, MEP-NH2 (17) (0.2 mmol, 51 mg) and MCC
(0.22 mmol, 53 mg) were dissolved in anhydrous DCM (2 mL). The reaction was cooled down to 0°C. A solution of EDC hydrochloride (0.1 mmol, 19 mg) in anhydrous DCM (5 mL) was added dropwise. The reaction was stirred 30 min at room temperature before adding NaiCCL (0.2 mmol, 21.4 mg) and stirred overnight. The crude was dried under vacuum before being purified by flash column chromatography using a gradient of DCM/MeOH from 0 to 2.5 % to yield MEP-MCC (18) (0.12 mmol, 56 mg) as a yellow solid. Yield 60%.
Figure imgf000060_0001
NMR (300 MHz, MeOD) d 6.82 (s, 2H), 4.89 - 4.58 (m, 4H), 3.51 - 3.15 (m, 9H), 2.24 - 1.59 (m, 6H), 1.52 (t, /= 15.1 Hz, 3H), 1.37 (dd, / = 17.3, 9.9 Hz, 3H), 1.01 (d, / = 15.7 Hz, 6H). LC-MS: RT = 8.19 min, mass: 472 m/z (M+H+), purity 97.35%.
DM1-AEMI (19): (l4S,32R,33S,10E,12E,14R)-86-chloro-l4-hydroxy-85,14- dimethoxy-33,2,7,10-tetramethyl-l2,6-dioxo-7-aza-l(6,4)-oxazinana-3(2,3)-oxirana- 8(l,3)-benzenacyclotetradecaphane-10,12-dien-4-yl /V-(3-((l-(2-aminoethyl)-2,5- dioxopyrrolidin-3-yl)thio)propanoyl)-/V-methylalaninate
Figure imgf000060_0002
[0187] In a round bottom flask AEMI (0.15 mmol, 27 mg) was dissolved in anhydrous MeOH (7 mL), NaiCCE (0.07 mmol, 7.4 mg) was added, then a solution of mertansine (DM1) (0.07 mmol, 49.5 mg) in anhydrous MeOH (3 mL) was added and the mixture was stirred for 24h at room temperature. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM-MeOH (0% to 10 % MeOH) to give the expected product DM1-AEMI (19) as a white solid. Yield 38%. LC/MS purity 87.7 %. RT = 6.44 min. m/Z = 878.3421.
1.2. SYNTHESIS OF FUNCTIONALIZED RAFT AGENTS DM1-HEMI-CDP (RAFT-1): 2-(3-((3-((l-(((l4S,32R,33S,10£,12£,14R)-86-chloro-l4- hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-l2,6-dioxo-7-aza-l(6,4)-oxazinana-
3(2,3)-oxirana-8(l,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-l- oxopropan-2-yl)(methyl)amino)-3-oxopropyl)thio)-2,5-dioxopyrrolidin-l-yl)ethyl 4- cyano-4-(((dodecylthio)carbonothioyl)thio)pentanoate
Figure imgf000061_0001
[0188] In a round bottom flask was dissolved HEMI-CDP (3) (0.16 mmol, 84 mg) in anhydrous DCM (6 mL), NaiCCL (1.35 mmol; 141.83 mg) was added, then a solution of mertansine (DM1) (0.131 mmol, 97.01 mg) in anhydrous DCM (5 mL) was added and the mixture was stirred for 48 hours at r.t. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM-MeOH (1% to 2.5 % MeOH) as eluant to give the expected product DM1-HEMI-CDP (RAFT-1) as a yellow oil that solidifies in the fridge, yield 66%.
Figure imgf000061_0002
NMR (300 MHz, CDCh) d 6.82 (dd, 7 = 6.7, 1.6 Hz, 1H), 6.65 (m, 2H), 6.42 (dd, 7 = 15.4, 11.2 Hz, 1H), 6.21 (s, 1H), 5.65 (dd, 7 = 15.0, 9.2 Hz, 1H), 5.38 (dd, 7 = 6.7, 2.6 Hz, 1H), 4.77 (dd, 7 = 12.2, 3.3 Hz, 1H), 4.39 - 4.14
(m, 3H), 3.98 (s, 3H), 3.72 (m, 3H), 3.50 (d, 7= 8.9 Hz, 1H), 3.36-3.30 (m, 5H), 3.20 (s, 3H), 3.15 - 2.97 (m, 5H), 2.85 (s, 3H), 2.68 - 2.48 (m, 5H), 2.42 - 2.26 (m, 2H), 2.18 (d, 7 = 11.5 Hz, 2H), 1.87 (s, 3H), 1.76 - 1.52 (m, 9H), 1.40 - 1.15 (m, 23H), 0.88 (t, 7= 6.7 Hz, 3H), 0.80 (s, 3H). HRMS calcd for CeoHseNsO^Na (M + Na)+, 1263.4738 ; found, 1263.4743.
DM1-HEMI-CEP (RAFT-2): 2-(3-((3-((l-(((l45,32R,335,10E,12E,14R)-86-chloro-l4- hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-l2,6-dioxo-7-aza-l(6,4)-oxazinana- 3(2,3)-oxirana-8(l,3)-benzenacyclotetradecaphane-10,12-dien-4-yl)oxy)-l- oxopropan-2-yl)(methyl)amino)-3-oxopropyl)thio)-2,5-dioxopyrrolidin-l-yl)ethyl 4- cyano-4-(((ethylthio)carbonothioyl)thio)pentanoate
Figure imgf000061_0003
[0189] In a round bottom flask was added, a solution of mertansine (DM1) (0.154 mmol, 114 mg) in anhydrous DCM (3 mL) was added to a solution of HEMI-CEP (4) (0.2 mmol, 76 mg) and Na2CO3 (1.71 mmol, 179.91 mg) in 5 ml of anhydrous DCM. The mixture was stirred for one day at r.t. After completion of the reaction, the crude was purified by column chromatography on silica gel using a gradient DCM-MeOH (0.5% to 3.5% MeOH) as eluant to give the expected product DM1-HEMI-CEP (RAFT-2) as a yellow oil, yield 73%.1H NMR (300 MHz, CDCl3) ^ 6.82 (d, J = 4.9 Hz, 1H), 6.65 (m, 2H), 6.42 (dd, J = 15.0, 11.0 Hz, 1H), 6.20 (s, 1H), 5.65 (dd, J = 14.0, 9.1 Hz, 1H), 5.38 (dd, J = 6.4, 2.8 Hz, 1H), 4.77 (dd, J = 11.7, 2.7 Hz, 1H), 4.42 – 4.14 (m, J = 9.7 Hz, 3H), 3.99 (s, 3H), 3.87 – 3.58 (m, 4H), 3.50 (d, J = 8.9 Hz, 1H), 3.40 – 3.28 (m, 5H), 3.20 (s, 3H), 3.16 – 2.96 (m, 4H), 2.85 (s, 3H), 2.71 – 2.46 (m, 5H), 2.42 – 2.25 (m, 2H), 2.26 – 2.09 (m, 1H), 1.87 (d, J = 0.8 Hz, 3H), 1.72 – 1.51 (m, 6H), 1.42 – 1.17 (m, 10H), 0.80 (s, 3H). 13C NMR (75 MHz, CDCl3) ^ 217.4, 176.1, 174.2, 171.2, 170.6, 168.6, 155.8, 154.4, 152.1, 142.1, 141.2, 139.3, 133.2, 127.6, 125.3, 122.2, 119.1, 113.1, 88.5, 80.8, 78.1, 74.0, 67.1, 61.2, 59.9, 56.6, 49.9, 46.6, 46.3, 39.8, 39.4, 38.8, 38.0, 36.1, 35.7, 35.4, 33.7, 32.4, 31.3, 29.6, 27.5, 27.1, 24.8, 15.5, 14.5, 13.3, 12.7, 12.1. DM1-AEMI-CDP (RAFT-3): (14S,32R,33S,10E,12E,14R)-86-chloro-14-hydroxy- 85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2-(4-cyano-4- (((dodecylthio)carbonothioyl)thio)pentanamido)ethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoyl)-N-methylalaninate
Figure imgf000062_0001
[0190] In a round bottom flask was dissolved AEMI-CDP (6) (0.072 mmol, 38 mg) in anhydrous DCM (2 mL), Na2CO3 (0.73 mmol, 77.05 mg) was added, then a solution of mertansine (DM1) (0.061 mmol, 44.70 mg) in anhydrous DCM (3 mL) was added and the mixture was stirred for the week end at r.t. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM-MeOH (1% to 3.5% MeOH) to give the expected product DMA-AEMI-CDP (RAFT-3) as a yellow solid yield 65%.
Figure imgf000063_0001
NMR (300 MHz, CDCl3) ^ 6.82 (d, J = 5.0 Hz, 1H), 6.69 – 6.54 (m, 2H), 6.51 – 6.34 (m, 1H), 6.26 (s, 1H), 5.74 – 5.55 (m, 1H), 5.27 (s, 1H), 4.79 (d, J = 12.0 Hz, 1H), 4.28 (s, 1H), 3.98 (s, 3H), 3.81 – 3.54 (m, 3H), 3.53 – 3.39 (m, 3H), 3.41 – 3.26 (m, 5H), 3.19 (s, 3H), 3.16 – 3.03 (m, 1H), 2.99 (dd, J = 9.2, 3.2 Hz, 1H), 2.83 (dd, J = 16.8, 4.9 Hz, 3H), 2.69 – 2.53 (m, 4H), 2.48 – 2.08 (m, 5H), 1.87 (s, 3H), 1.79 – 1.52 (m, 10H), 1.51 – 1.10 (m, 27H), 0.87 (t, J = 6.6 Hz, 3H), 0.79 (s, 3H).13C NMR (75 MHz, CDCl3) ^ 209.76 (Cq), 174.98 (Cq), 171.20 (Cq), 171.08 (Cq), 170.62 (Cq), 168.83 (Cq), 156.17 (Cq), 152.37 (Cq), 142.31 (Cq), 139.51 (Cq), 133.29 (CH), 127.92 (CH), 125.32 (CH), 122.22 (CH), 119.33 (Cq), 119.01 (Cq), 113.31 (CH), 88.69 (CH), 80.96 (Cq), 74.24 (CH), 67.05 (CH), 62.59 (CH2), 61.37 (CH2), 60.15 (Cq), 56.73 (CH3), 52.00 (CH3), 49.39 (CH2), 46.87 (Cq), 41.15 (CH), 40.03 (CH), 39.03 (CH), 37.23 (CH2), 36.34 (CH2), 35.63 (CH2), 34.53 (CH2), 33.25 (CH2), 32.61 (CH2), 32.03 (CH2), 31.66 (CH2), 31.31 (CH2), 31.27 (CH3), 29.73 (CH2), 29.67 (CH2), 29.55 (CH2), 29.45 (CH2), 29.20 (CH2), 29.08 (CH2), 27.82 (CH2), 27.06 (CH2), 25.18 (CH3), 22.80 (CH2), 15.66 (CH3), 14.71 (CH3), 14.23 (CH3), 13.59 (CH3), 12.36 (CH3). HRMS calcd for C60H87N6O13S4Na (M + Na)+, 1262.4904 ; found, 1262.4902. DM1-AEMI-CEP (RAFT-4): (14S,32R,33S,10E,12E,14R)-86-chloro-14-hydroxy- 85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2-(4-cyano-4- (((ethylthio)carbonothioyl)thio)pentanamido)ethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoyl)-N-methylalaninate
Figure imgf000063_0002
[0191] In a round bottom flask was dissolved AEMI-CEP (5) (0.36 mmol, 138 mg) in anhydrous DCM (5 mL), Na2CO3 (3.6 mmol, 378 mg) was added, then a solution of mertansine (DM1) (0.3 mmol, 221 mg) in anhydrous DCM (10 mL) was added and the mixture was stirred overnight at r.t. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM-MeOH (0% to 3 % MeOH) to give the expected product DM1-AEMI-CEP (RAFT-4) as a white-yellowish solid, yield 75%.
Figure imgf000064_0001
NMR (400 MHz, Chloroform-d) ^ 6.83 (dd, J = 9.0, 1.8 Hz, 1H), 6.67 – 6.56 (m, 2H), 6.50 – 6.33 (m, 2H), 6.30 – 6.18 (m, 1H), 5.71 – 5.52 (m, 1H), 4.79 (dd, J = 12.0, 3.0 Hz, 1H), 4.29 (qd, J = 9.7, 4.6 Hz, 1H), 3.99 (s, 3H), 3.79 – 3.29 (m, 13H), 3.19 (d, J = 1.7 Hz, 3H), 3.16 – 2.94 (m, 5H), 2.90 – 2.76 (m, 4H), 2.67 – 2.53 (m, 2H), 2.49 – 2.26 (m, 5H), 2.19-2.15 (m, 1 H), 1.88 (d, J = 0.6 Hz, 3H), 1.63 (d, J = 26.1 Hz, 4H), 1.49-1.41 (m, 1 H), 1.40 – 1.23 (m, 10H), 0.80 (s, 3H). DM1-HEMI-MEP (RAFT-5): (14S,32R,33S,10E,12E,14R)-86-chloro-14-hydroxy- 85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2-((2- (((ethylthio)carbonothioyl)thio)propanoyl)oxy)ethyl)-2,5-dioxopyrrolidin-3- yl)thio)propanoyl)-N-methylalaninate
Figure imgf000064_0002
[0192] In a round bottom flask was dissolved MEP-HEMI (14) (0.21 mmol, 68.5 mg) in anhydrous DCM (13 mL), then Na2CO3 (1.89 mmol, 199.9 mg) was added, then a solution of mertansine (DM1) (0.1 mmol, 72.83 mg) in anhydrous DCM (2 mL) was added and the mixture was stirred 24h at room temperature. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM- MeOH (0% to 3% MeOH) to give the expected product DM1-HEMI-MEP (RAFT-5) (0.09 mmol, 94.4 mg) as a yellow solid. Yield 88%.
Figure imgf000064_0003
NMR (300 MHz, CDCl3) ^ 6.84 (d, J = 6.3 Hz, 1H), 6.71 (dd, J = 16.3, 9.9 Hz, 2H), 6.51 – 6.32 (m, 1H), 6.22 (s, 1H), 5.67 (dd, J = 15.1, 8.9 Hz, 1H), 5.41 (d, J = 6.8 Hz, 1H), 5.32 (s, 1H), 4.80 (d, J = 9.3 Hz, 2H), 4.28 (dd, J = 15.1, 7.8 Hz, 3H), 4.00 (s, 3H), 3.81 – 3.61 (m, 3H), 3.52 (d, J = 9.0 Hz, 1H), 3.41– 3.34 (m, 4H), 3.23 (s, 3H), 3.09 (dd, J = 28.6, 9.5 Hz, 4H), 2.93 – 2.76 (m, 4H), 2.70 – 2.53 (m, 2H), 2.39 (ddd, J = 18.8, 7.3, 3.9 Hz, 1H), 2.20 (d, J = 10.0 Hz, 1H), 1.63 – 1.54 (m, 6H), 1.36 – 1.23 (m, 9H), 0.96 – 0.84 (m, 3H), 0.82 (s, 3H). LC/MS: Gradient Acetonitrile (5% to 100%): RT (10.48 min), m/z = 1071 m/z (M+ H2O), purity 100%. DM4-S-S-CEOEC (RAFT-6): (14S,32R,33S,10E,12E,14R)-86-chloro-14-hydroxy- 85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl 6-cyano-6,15,15,19,20- pentamethyl-9,18-dioxo-4-thioxo-3,5,13,14-tetrathia-10,19-diazahenicosan-21-oate
Figure imgf000065_0001
[0193] In a round bottom flask was dissolved Disulfide bond RAFT agent (15) (0.42 mmol, 181.3 mg) in anhydrous MeOH (17 mL), then a solution of DM4 (0.25 mmol, 192.88 mg) in anhydrous MeOH (7 mL) was added and the mixture was stirred 24h at room temperature. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM-MeOH (0% to 3 % MeOH) to give the expected product DM4-S-S-CEOEC (RAFT-6) as a yellow solid (yield 60%) 1H NMR (300 MHz, CDCl3 ^ 6.84 (d, J = 6.3 Hz, 1H), 6.71 (dd, J = 16.3, 9.9 Hz, 2H), 6.51 – 6.32 (m, 1H), 6.22 (s, 1H), 5.67 (dd, J = 15.1, 8.9 Hz, 1H), 5.41 (d, J = 6.8 Hz, 1H), 5.32 (s, 1H), 4.80 (d, J = 9.3 Hz, 2H), 4.28 (dd, J = 15.1, 7.8 Hz, 3H), 4.00 (s, 3H), 3.65 (d, J = 12.9 Hz, 1H), 3.56 – 3.47 (m, 3H), 3.41– 3.32 (m, 5H), 3.24 (s, 3H), 3.15 (d, J = 13.6 Hz, 1H), 3.03 (d, J = 9.9 Hz, 1H), 2.90 (d, J = 1.1 Hz, 3H), 2.75 (t, J = 6.2 Hz, 2H), 2.56 – 2.48 (m, 3H), 2.21 (d, J = 9.1 Hz, 2H), 1.92 (s, 3H), 1.44 – 1.05 (m, 18H), 0.87 (ddd, J = 9.7, 6.3, 4.2 Hz, 3H), 0.09 (s, 3H). LC-MS RT 10.79 min, m/Z 1100 m/z, purity 100%. DM1-MCC-MEP (RAFT-7): (14S,32R,33S,10E,12E,14R)-86-chloro-14-hydroxy- 85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana-3(2,3)- oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-((4-((2-(2- (((ethylthio)carbonothioyl)thio)propanamido)ethyl)carbamoyl)cyclohexyl)methyl)- 2,5-dioxopyrrolidin-3-yl)thio)propanoyl)-N-methylalaninate
Figure imgf000066_0001
[0194] In a round bottom flask was dissolved MEP-MCC (18) (0.13 mmol, 60 mg) in anhydrous DCM (7 mL), then Na2CO3 (1.18 mmol, 125.4 mg) was added, then a solution of mertansine (DM1) (0.08 mmol, 57.67 mg) in anhydrous DCM (3 mL) was added and the mixture was stirred 24h at room temperature. After completion of the reaction, the crude was purified by column chromatography using a gradient DCM-MeOH (0% to 4 % MeOH) to give the expected product DM1-MCC-MEP (RAFT-7) as a yellow solid (0.3 mmol, 48 mg). Yield 50%. 1H NMR (300 MHz, CDCl3) ^ 6.84 (d, J = 6.3 Hz, 1H), 6.71 (dd, J = 16.3, 9.9 Hz, 2H), 6.51 – 6.32 (m, 1H), 6.22 (s, 1H), 5.67 (dd, J = 15.1, 8.9 Hz, 1H), 5.41 (d, J = 6.8 Hz, 1H), 5.32 (s, 1H), 4.80 (d, J = 9.3 Hz, 2H), 4.28 (dd, J = 15.1, 7.8 Hz, 3H), 4.70 (d, J = 7.5 Hz, 3H), 4.01 (s, 7H), 3.65 (s, 3H), 3.51 (d, J = 9.5 Hz, 1H), 3.47 – 3.27 (m, 23H), 3.20 – 2.98 (m, 2H), 2.88 (s, 1H), 2.64 (d, J = 5.6 Hz, 1H), 2.51 – 1.80 (m, 21H), 1.83 – 1.55 (m, 7H), 1.48 – 1.19 (m, 5H), 0.83 (s, 6H). LC-MS: RT 9.31 min, m/Z: 1208 (M+H2O +H+), purity 97.3%. I.3. SYNTHESIS OF POLYMER COMPOUNDS General procedure [0195] In a 7-mL glass vial or 25 ml round bottom flask equipped with a rubber septum and a magnetic stir bar, were added AIBN, the functionalized RAFT agent, acrylamide monomer (AAm) and DMSO. The mixture was degassed with argon for 15 min under vigorous stirring before being placed in a 70 °C-preheated oil bath for 2 h under stirring. After the reaction, the polymer was precipitated in methanol. The polymer was further solubilized in milliQ water and placed in a 3.5 kDa Spectra/Por 3 dialysis bag for dialysis against de-ionized water for 3 days, with dialysis water changed twice/thrice per day. The dialysate was then freeze-dried. For 1H NMR spectroscopy of polymers, acquisition was performed in 5 mm diameter tubes in D2O at 70 °C (128 scans) on a Bruker Avance 3 HD 400 spectrometer operating at 400 MHz. NMR determination of the number-average molar mass (Mn,NMR) of the DM1 polymer derivatives was achieved by integrating the singlet at 4.03 ppm corresponding to 3 protons of methoxy-phenyl from the DM1. The ratio between this integral and the integral of the broad peak between 2.62 and 1 ppm allowed determination of the number average degree of polymerization (DPn) of AAm. Polymer compound (P1): DM1-HEMI-PAAm-C12
Figure imgf000067_0001
[0196] In a 25 ml round bottom flask, AAm (42 mmol, 2.998 g), DM1-HEMI-CDP (RAFT-1) (0.16 mmol, 202 mg), AIBN (0.03 mmol, 5.47 mg) and DMSO (10.5 mL) were reacted together. After purification, the polymer compound (P1) was isolated as white yellowish solid in 67 % yield. Mn,NMR = 35 kDa. Polymer compound (P2): DM1-HEMI-PAAm-C2
Figure imgf000067_0002
[0197] In a 25 ml round bottom flask, AAm (37 mmol, 2.629 g), DM1-HEMI-CEP (RAFT-2) (0.15 mmol, 147 mg), AIBN (0.031 mmol, 4.5 mg) and DMSO (9.25 mL) were reacted together. After purification, the polymer compound (P2) was isolated as white yellowish solid in 75 % yield. Mn,NMR = 19 kDa.
Figure imgf000068_0001
[0198] In a 25 ml round bottom flask, AAm (37 mmol, 3.909 g), DM1-AEMI-CEP (RAFT-4) (0.19 mmol, 219 mg), AIBN (0.039 mmol, 4.5 mg) and DMSO (13.75 mL) were reacted together. After purification, the polymer compound (P3) was isolated as white yellowish solid in 89 % yield. Mn,NMR = 33 kDa. Polymer compound (P4): DM1-AEMI-PAAm-C12
Figure imgf000068_0002
^ [0199] In a 7-mL glass vial, AAm (500.0 mg, 7 mmol), DM1-AEMI-CDP (RAFT-3) (33.57 mg, 0.027 mmol), AIBN (0.87 mg, 0.005 mmol) and DMSO (1.75 mL) were reacted together. After purification, the polymer compound (P4) was isolated as white yellowish solid in 56 % yield. Mn,NMR = 21 kDa.
Figure imgf000069_0001
^ [0200] In a glass vial, AAm (1065.9 mg, 15 mmol), DM1-HEMI-MEP (RAFT-5) (59.2 mg, 0.06 mmol) and AIBN (1.9 mg, 0.01 mmol) were added in DMSO (3.75 mL). The mixture was degassed under argon for 15 min, stirred for 2 hours at 70°C, then precipitated in cold acetone. After purification, the polymer compound (P5) was isolated as a yellowish solid in 75% yield. Mn,NMR = 25 kDa. SEC: Dispersity = 1.278.
Figure imgf000069_0002
^ [0201] In a glass vial, AAm (1065.9 mg, 15 mmol), DM4-S-S-CEOEC (RAFT-6) (62.1 mg, 0.06 mmol) and AIBN (2.0 mg, 0.01 mmol) were added in DMSO (3.75 mL). The mixture was degassed under argon for 15 min, stirred for 2 hours at 70°C, then precipitated in cold acetone. After purification, the polymer compound (P10) was isolated as a yellowish solid in 71% yield. Mn,NMR = 27 kDa. SEC: Dispersity = 1.224.
Figure imgf000070_0001
^ [0202] In a glass vial, AIBN (1.7 mg, 0.01 mmol) was dissolved in DMSO (0.5 mL), then AAm (933 mg, 13.1 mmol) and DM1-MCC-MEP (RAFT-7) (59 mg, 0.05 mmol) were dissolved in DMSO (2.75 mL) and then added. The mixture was degassed under argon for 15 min, stirred for 2 hours at 70°C, then precipitated in cold acetone. After purification, the polymer compound (P11) was isolated as a yellowish solid in 76% yield. Mn,NMR = 28 kDa. SEC: Dispersity = 1.435. I.4. COMPARATIVE COMPOUNDS Comparative compound: DM1-HEMI (7): (14S,32R,33S,10E,12E,14R)-86-chloro-14- hydroxy-85,14-dimethoxy-33,2,7,10-tetramethyl-12,6-dioxo-7-aza-1(6,4)-oxazinana- 3(2,3)-oxirana-8(1,3)-benzenacyclotetradecaphane-10,12-dien-4-yl N-(3-((1-(2- hydroxyethyl)-2,5-dioxopyrrolidin-3-yl)thio)propanoyl)-N-methylalaninate
Figure imgf000070_0002
[0203] In a round bottom flask HEMI (34 mmol, 4.8 mg) was dissolved in anhydrous DCM (2 mL), Na2CO3 (133 mmol, 14 mg) was added, then a solution of DM1 (27 mmol, 20 mg) in DCM (1 mL) was added under argon atmosphere and the mixture was stirred for 24 h at room temperature. DCM was evaporated under vacuum. The crude was purified by column chromatography on silica gel using a gradient of MeOH/DCM (3% to 5%) as eluant to give the expected product DM1-HEMI (7) as a white solid (yield 75%). 1H NMR (300 MHz, CDCl3) ^ 6.82 (d, J = 5.1 Hz, 1H), 6.67 (m, 2H), 6.41 (t, J = 15 Hz, 1H), 6.22 (s, 1H), 5.63 (m, 1H), 5.36 – 5.23 (m, 1H), 4.78 (dd, J = 12.0, 2.7 Hz, 1H), 4.31 (m, 1H), 4.0 (s, 3H), 3.8-3.59 (m, 5 H), 3.51 (d, J = 10 Hz, 1 H),^3.35 (s, 3H), 3.24 – 2.93 (m, 6H), 2.85 (d, J = 1.5 Hz, 3H), 2.71 – 2.51 (m, 3H), 2.41 (ddd, J = 18.7, 9.7, 3.9 Hz, 2H), 2.19 (s, J = 11.5 Hz, 1H), 1.4-1.2 (m, 8 H), 0.79 (s, 3H). HRMS calcd for C41H55ClN4O13SNa (M + Na)+, 901.3073; found, 901.3070. Comparative polymer compound: DM1-AEMI-PEG (12)
Figure imgf000071_0001
[0204] In a round bottom flask PEG-NHS [MW 23350 g/mol] (0.03 mmol, 702.6 mg) was dissolved in anhydrous DCM (3 mL), then a solution of DM1-AEMI (19) (0.03 mmol, 24 mg) in DCM (2 mL) was added, then a solution of triethylamine (0.12 mmol, 1.5 mg) in DCM (2 mL) was added dropwise and the mixture was stirred for 24 h at room temperature. PEG-NHS [MW 23350 g/mol] (0.3 eq., 0.009 mmol, 211.6 mg) was further added and the mixture was further stirred for 24 h at room temperature. MilliQ water (0.6 mL) was added then DCM was evaporated under vacuum. The crude was then purified by dialysis for 3 days, then lyophilization, to give the expected product DM1-AEMI-PEG (12) as a white solid (yield 83%). The structure of the product was confirmed by 1H NMR. ^ II. EVALUATION OF IN VITRO ANTICANCER ACTIVITY
Purpose
[0205] The MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay was used to evaluate the cytotoxicity of the polymer compounds of the invention. This colorimetric test measures the mitochondrial deshydrogenase cell activity, an indicator of cell viability. The assay is based on the reduction, by living cells, of the tetrazolium salt, MTT, which forms a blue formazan product.
Cell culture
[0206] The human breast adenocarcinoma cell line MCF7 was obtained from ATCC (catalog number HTB-22) and grown in Eagle's Minimum Essential Medium (EMEM, Sigma M-5660) supplemented with 1% non-essential amino acids (NEAA, Sigma M- 7145) and 10 % FBS. The human breast adenocarcinoma cell line MDA-MB-231 was obtained from ATCC (catalog number HTB-26) and grown in Leibovitz’s LI 5 medium (Sigma L-5520) supplemented with 1% of 200 mM L-glutamine solution (Sigma G- 7513), 20 mM sodium bicarbonate and 15 % FBS. All media were further supplemented with 50 U.mL 1 penicillin and 50 pg.mL 1 streptomycin (Sigma P-4333). Both cell lines were maintained at 37°C in a 5% CO2 humid atmosphere. Twice a week, cells were divided by trypsinization (Sigma T-3924) at 1:5 ratio for MCF7 and 1:4 ratio for MDA- MB-231. Cells were used at low passages after thawing and harvested at a maximum confluence of 70-80%.
MTT assay
[0207] Cells were seeded into 96-well plates (TPP) with 100 pL of complete medium par well at a density of 5000 cells per well for MCF7 cell lines and 10000 cells per well for MDA-MB-231. After an overnight incubation (around 24 hours), a series of increasing concentrations of tested compounds ranging from 0.1 to 5000 nM was prepared in complete growth media for each of the three cell lines. Cells were exposed to 100 pL of the treatment during 72 hours. After the end of the exposure time, 20 pL of a 5 mg.mL 1 MTT solution (Sigma T-8154) prepared in PBS (Sigma D-6429) were then added in each well and incubated at 37°C during 1 hour for MCF7 cells and 3 hours for MDA-MB-231 cells. The medium was then discarded and 200 m L of DMSO were added to dissolve the formazan crystals. The absorbance was measured at 570 nm with a microplate reader (Labsystems Multiskan MS). The percentage of viable cells was calculated as the absorbance ratio of treated to untreated cells. The inhibitory concentration 50% (IC50) was calculated as the concentration which inhibits the growth of 50% of cells relative to untreated cells. For each of the treatments, IC50 was determined from the dose-response curve using the sigmoidal fitting from GraphPad Prism Software. All measurements were performed in 6 replicates for each experiment. [0208] Tested compounds:
PTX: paclitaxel (reference compound);
DM1: mertansine (free drug);
(7): DM1-HEMI (comparative);
PI, P2, P3: polymer compounds of the invention. Results
Table 1: Inhibitory Concentration 50% (IC50 in nM) of tested compounds on MCF-7 and MDA-MB-231 breast cancer cells.
Figure imgf000073_0001
[0209] For both cell lines, DM1 and PTX have comparable IC50. The addition of a small hydrophilic group to DM1 (compound 7) increases the IC50 as the molecule is more soluble and less prone to diffuse inside the cells. For MCF-7, the addition of the very hydrophilic polyacrylamide chain (compounds PI, P2 and P3) further increases the IC50 as the proper DM1 metabolite must be cleaved from the prodrug to be able to diffuse inside the cells. For MDA-MB-231, while an IC50 value was computed for the polymer prodmgs, the cells retained at least a 40% viability even at 5000 nM (compared to less than 20% for compound (7) and 13% for DM1 and PTX). This implies that the drug was not fully cleaved from the prodrug as suggested above. Conclusion
[0210] The in vitro tests confirm that the polymer compounds of the invention allow for a decrease in toxicity compared to free DM1, while retaining its activity. The in vivo tests presented hereafter confirm the anticancer activity of the polymer compounds.
III. EVALUATION OF IN VIVO EFFICACY
III.l. TUMOR ACCUMULATION ASSAY Purpose
[0211] The tumor accumulation of the polymer compounds of the invention was assessed using fluorescent polymer compounds as model compounds. Cyanine 5.5 was used as fluorescent dye to form Cyanine-PAAm-C12 (10) and Cyanine-PAAm-C2 (11). Fluorescent polymer compounds 10 and 11 were administered to mice bearing 4T1 tumors (murine breast cancer cells) and accumulation in tumors and in other organs was measured by fluorescence. Material
[0212] The model fluorescent polymer compounds Cyanine-PAAm-C12 (10) and Cyanine-PAAm-C2 (11) were synthesized as described hereafter starting from succinimide polymer intermediates NHS-PAAm-C12 (8) and NHS-PAAm-C2 (9)
NHS-PAAm-C12 (8):
Figure imgf000074_0001
[0213] NHS-PAAm-C12 (8) was obtained using the general procedure described in example 1.3 above. In a 25 ml round bottom flask, AAm (46 mmol, 3.27 g), CDP- NHS (1) (0.166 mmol, 83.2 mg), AIBN (0.033 mmol, 5.5 mg) and DMSO (11.5 mL) were reacted together. After purification the NHS-PAAm-C12 (8) was isolated as white yellowish solid in 57 % yield. Integration of the triplet at 3.36 ppm corresponding to the two protons of the CH2-S- was used to determine the average Mn,NMR = 20.6 kDa. NHS-PAAm-C2 (9):
Figure imgf000075_0001
[0214] NHS-PAAm-C2 (9) was obtained using the general procedure described in example I.3 above. In a 25 ml round bottom flask, AAm (46 mmol, 3.27 g), CEP-NHS (2) (0.166 mmol, 59.3 mg), AIBN (0.033 mmol, 5.5 mg) and DMSO (11.5 mL) were reacted together. After purification the NHS-PAAm-C2 (9) was isolated as white yellowish solid in 66 % yield. Integration of the triplet at 3.36 ppm corresponding to the two protons of the CH2-S- was used to determine the average Mn,NMR = 19.8 kDa. Cyanine-PAAm-C12 (10):
Figure imgf000075_0002
[0215] In a 25 ml round bottom flask NHS-PAAm-C12 (8) (0.025 mmol, 503mg) was dissolved in DMSO (7.5 mL), the mixture as heated until it becomes completely soluble. Argon was bubbled inside for 5 minutes. In a separate vial, Cyanine 5.5 amine (0.029 mmol, 19.35 mg) was dissolved in DMSO (3 mL) and add triethylamine (7.64 µL), this solution was added dropwise to the solution of compound 8. The mixture was stirred 24 h at r.t. in the dark. The solution was poured dropwise into cold methanol (200 mL). The product precipitated, it was filtered and the redissolved in MilliQ water and then a 3.5 kDa Spectra/Por 3 dialysis bag for dialysis against de-ionized water for 3 days, with dialysis water changed twice/thrice per day. The dialysate was then freeze-dried. The final compound Cyanine-PAAm-C12 (10) was obtained as blue powder in 42 % yield.^The cyanine coupling was determined by integrating the peaks at 3.72 corresponding to the methyl group of CH3-N+- in the cyanine, the ratio of this methyl group with respect to the polymer size allowed to determine a cyanine mass loading of 1.93% in the polymer. Cyanine-PAAm-C2 (11):
Figure imgf000076_0001
[0216] Compound Cyanine-PAAm-C2 (1) was obtained with a procedure similar to the one used for the synthesis of compound 14, using NHS-PAAm-C2 (9) as starting material. The final product Cyanine-PAAm-C2 (11) was isolated as blue powder in 25 % yield. The cyanine loading is 1.44%. Method [0217] Nine weeks old BALB/cAnNRj female mice were orthotopically inoculated with 106 4T1 cells (ATCC, catalog number CRL-2539) in the fourth inguinal mammary fat pad (50 ^L/mouse). Tumor volume was measured with a caliper and determined based on the equation (L x l2)/2, where L is the largest tumor diameter and l the smallest. Twelve days later, mice bearing 350-550 mm3 tumors were intravenously injected with fluorescent compounds Cyanine-PAAm-C12 (10) or Cyanine-PAAm-C2 (11) or control free Cyanine 5.5 solution at an equivalent dose of Cyanine 5.5 of 1.25 mg/kg for all groups for in vivo imaging biodistribution studies (n=3 for each group). Images were recorded at 1, 4, 24, 48 hours and 3, 4, 7, 9, 11, 14 days post injection with the IVIS Lumina LT Series III system (Perkin Elmer) using 640 nm excitation filter and 695-770 nm emission filter. During imaging, mice were kept on the imaging stage under anesthesia with 2% isoflurane gas in oxygen flow (0.2 liter/min) and were imaged in supine position. Images were analyzed with the Living Imaging software. The same region of interest (ROI, same area) was selected on each tumor, and total radiant efficiency values was used for quantification. [0218] Another group of mice bearing 350-550 mm3 tumors was intravenously treated with fluorescent Cyanine-PAAm-C12 (10) or Cyanine-PAAm-C2 (11) or control free Cyanine 5.5 (n=2 for each group, at doses equivalent in Cyanine 5.5 of 1.25 mg/kg and 0.63 mg/kg) and was euthanized 48h post injection. Organs (spleen, pancreas, kidney, liver, uterus-ovary, intestine, thymus, lung, heart, bladder, brain and tumor) were removed for ex vivo imaging with the IVIS Lumina LT Series III system (Perkin Elmer) using 640 nm excitation filter and 695-770 nm emission filter. A non-treated group was also done. Results [0219] From the 14-days follow up, it can be observed in Figure 1 that free Cyanine 5.5 is rapidly eliminated from the mice in vivo with a low signal (below 3*10^10) 24h after injection and no tumor accumulation has been observed. For compounds 10 and 11 corresponding to Cyanine 5.5 coupled to PAAm-C12 and PAAm-C2 respectively, there is a preferential accumulation at the tumor site with a maximum reached after 48h followed by a slow decrease over 14 days. [0220] The same interpretation can be provided for the accumulation assay with ex vivo analysis of organs, for both doses on all groups (data not shown). The technique used is semi-quantitative. For free cyanine 5.5, there is a strong signal on the kidneys suggesting a renal clearance pathway. Little signal is detected on all other organs. For compounds 10 and 11, the signal is strongest for the tumor with a signal approximately 2 to 3 times higher than that detected in the liver. For all the other organs the signal is comparable to that obtained with free cyanine 5.5. These results confirm the observation from the 14-days follow up experiment where a specific accumulation at the tumor site was achieved. Conclusion
[0221] This accumulation assay with fluorescent polymer compounds used as models of the polymer compounds of the invention shows that the presence of the polymeric moiety enables a specific accumulation of the compound in tumors.
III.2. EFFICACY STUDY IN MICE BEARING TRIPLE NEGATIVE BREAST TUMORS
Purpose
[0222] This study aimed at determining the antitumor efficacy of the polymer compounds of the invention in mice bearing triple negative breast tumor (breast cancer model).
Experimental design
[0223] The study was performed on the human breast tumor model MDA-MB-231 implanted on SCID-CB17 mice, with 7 groups of ten 10 mice: - Vehicle - IV - qw, 4 weeks;
Mertansine (DM1) - IV - 0.5 mg/kg - qw, 4 weeks;
Paclitaxel (PTX) - IV - 15 mg/kg - qw, 4 weeks;
- DM 1 -HEMI-PAAm-C2 (P2) - IV - 107 mg/kg - qw, 4 weeks;
- DM 1 -HEMI-PAAm-C 12 (PI) - IV - 142 mg/kg - qw, 4 weeks; - DMl-AEMI-PAAm-C2 (P3) - IV - 187 mg/kg - qw, 4 weeks.
[0224] The vehicle used in the study is PBS (phosphate-buffered saline). Each dose corresponds to the maximal tolerated dose in mice. Tested compounds are administered by intravenous route (IV), once weekly (qw) for 4 weeks.
Method [0225] Tumor implantation. MDA-MB-231 (human breast cancer) cells were amplified in vitro in DMEM supplied with at least 1% Penicillin-Streptomycin, 10% of Heat Inactivation of Fetal Bovine Serum and 5mg/mL of Plasmocin prior implantation. On the day of injection, cells were harvested, counted including a trypan blue viability dye (cut-off 80%), and resuspended in serum-free medium at the appropriate concentration. The cells were orthotopically implanted in the mammary fat pad SCID mice at 5xl06cells/mice in 200m1 PBS within 30 minutes after harvesting.
[0226] Mice monitoring. Mice were randomized when the tumors reach a mean volume of 100 mm3 for the 7 groups (for a total of 70 mice). After implantation, all the mice were observed in order to detect any toxic effects of the product. The endpoints are defined by animal ethics as a tumor diameter of > 18mm, significant weight loss or alteration of animal well-being.
[0227] Treatments. The seven groups of ten mice were treated once a week for 4 weeks.
[0228] Tumor monitoring. In order to assess the effectiveness of the tested compounds on tumorigenesis, tumor volume was measured twice a week. The sizes of the primary tumors were measured using calipers and the tumor volume (TV) was extrapolated to a sphere using the formula TV= 4/3 p x r3, by calculating the mean radius from the two measurements.
Results
[0229] The survival of treated mice is reported in Figure 2. The survival rate of mice treated with the following compounds follows an increasing trend: vehicle, DM1, PI, P2, PTX, and P3. Median survival is of 77 days for the vehicle.
[0230] Median survival for compounds P2 (99 days) and P3 (112 days) at their maximal tolerated dose were significantly higher than that of compound DM1 (80 days) according to a log-rank (Mantel-Cox) test (p-values at 0.0026 and < 0.0001 respectively). There was no statistical difference in median survival between compounds PI (84 days) and DM1. [0231] Median survival for compound PTX at its maximal tolerated dose was 99 days.
Median survival for compound P3 (112 days) was significantly higher according to a log- rank (Mantel-Cox) test with a p-value of 0.0231. Conclusion
[0232] The polyacrylamide prodrug approach with DM1 allows for a higher maximal tolerated dose compared to free DM1: 3 mg/kg equivalent in DM1 for (PI) and 4.2 mg/kg equivalent in DM1 for (P2) and (P3) instead of 0.5 mg/kg for DM1 alone. It was surprisingly evidenced that compound (P3) shows the better in vivo anticancer activity while having the worst in vitro activity. Even more surprisingly, compound (P3) significantly outperforms the treatment of reference in triple negative breast cancer, paclitaxel, in terms of mice survival. III.3. EFFICACY STUDY IN IMMUNODEFICIENT MICE BEARING SUBCUTANEOUS HUMAN CAUU-6 TUMOR
Purpose
[0233] This study aimed at determining the antitumor activity of the polymer compounds of the invention in immunodeficient mice bearing subcutaneous human Calu-6 tumor (lung cancer model).
Materials and methods
[0234] Polymer prodrugs. The following polymer prodrugs were tested in the form of powders:
DM 1 - AEMI-PEG (12), as comparative compound, - DM 1 - AEMI-PAAm-C2 (P3), and
- DM4-S-S-PAAm-C2 (P10).
The vehicle used in the study was PBS (phosphate-buffered saline) IX.
[0235] Preliminary study to evaluate the recognition of the polymer by the DM1 ADC EIA kit. The DM1 ADC EIA Kit (Epitope Diagnostics, ref. KTR-756) was commercially purchased. First, the polymer was prepared in assay buffer at different concentrations and dosed against the standard of the kit. In a second step, the polymer was prepared in rat serum to ensure that quantification is possible in biological matrix. [0236] Cancer cell line. Calu-6 cell line is a human lung adenocarcinoma (American Type Culture Collection, USA) obtained from a 61-year-old Caucasian female patient, provided by Oncodesign (Dijon, France). [0237] Cell culture method. Tumor cells were grown as monolayer at 37°C in a humidified atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine supplemented with 10% fetal bovine serum. Tumor cells are adherent to plastic flasks. For experimental use, tumor cells were detached from the culture flask by a 5-minute treatment with Trypsin-Versene®, in Hanks' medium without calcium or magnesium and neutralized by addition of complete culture medium. Cells were counted and viability assessed using a 0.25% trypan blue exclusion assay. [0238] Animals. Healthy female SWISS Nude (Crl:NU(Ico)-Foxn1nu) mice, 5-7 weeks old at reception, were obtained from Charles River (Wilmington, USA). [0239] Tumor induction. Tumors were induced by subcutaneous injection of 1 x 107 Calu-6 cells in 200 µL of RPMI 1640 into the right flank of 105 female animals. Calu-6 tumor cell implantation was performed 24 to 72 hours after a whole-body irradiation with a gamma-source (2 Gy, 60Co, BioMep, France). [0240] Mice randomization. Animals were randomized based on their individual tumor volume. Randomization was performed when values reach a mean of 100-200 mm3. Animals were randomized into groups of 10 animals each. Homogeneity between groups was tested by an analysis of variance (ANOVA). [0241] Treatment. The treatment was administered by intravenous injection (IV) into the caudal vein. The recommended pH formulation for IV administration is pH 5.0 - 8.0 (min pH 3.0). The administration volume was 5 mL/kg. Treatment started on DR (day of randomization). Isoflurane gas anesthesia was used for tumor inoculation and IV injections. [0242] The treatment schedule is summarized in the table below:
Figure imgf000082_0001
[0243] Animal and clinical monitoring. All study data, including animal body weight measurements, tumor volume, clinical and mortality records, and treatment were scheduled and recorded in the Vivo Manager database (Biosystemes, France). Animal viability and behavior were observed daily. Body weights were measured a minimum of twice a week. The length and width of the tumor was measured a minimum of twice a week with calipers. Animals were euthanized ten weeks after tumor induction.
[0244] Humane endpoints. The human endpoints requiring specific action or euthanasia were defined by animal ethics and included: a body weight loss > 15% (compared to a reference day, e.g., the first day of treatment, tumor exceeding 10% of normal body weight or exceeding 2000 mm3 and > 8 mm ulcerated tumor, infection, bleeding, tissue erosion. Euthanasia of animals was performed by over dosage on gas anesthesia (isoflurane).
Results [0245] The evolution of the tumor volume overtime is reported in Figure 3.
Conclusion
[0246] These results clearly evidence that the polymer prodrugs of the present invention are also very efficient against human Calu-6 tumor, and thus relevant for use in the treatment of lung cancer. By contrast, the polyethylene-glycol based comparative polymer (DM 1 - AEMI-PEG) fails to significantly reduce the Calu-6 tumor volume. III.4. EFFICACY STUDY IN MICE BEARING GASTRIC CARCINOMA XENOGRAFTS Purpose [0247] This study aimed at determining the antitumor activity of the polymer compounds of the invention in SCID-CB17 mice bearing HER2+ gastric cancer (stomach cancer model). Materials and methods [0248] Compounds. The following compounds were tested: - Trastuzumab emtansine (T-DM1) (brand name: Kadcyla®), as comparative compound, - DM1-AEMI-PAAm-C2 (P3) in combination with Trastuzumab, and - DM4-S-S-PAAm-C2 (P10) in combination with Trastuzumab. The vehicle used in the study was PBS (phosphate-buffered saline). [0249] Animals. Healthy female SCID-CB17 mice, 5 weeks old at reception, were obtained from “Animaleries communes de Rockefeller” (Lyon, France). [0250] Tumor implantation. NCIN-87 (gastric carcinoma cancer) cells were amplified in vitro in RPMI supplied with at 1% Penicillin-Streptomycin, 10% of Heat Inactivated Fetal Bovine Serum and 5 µg/mL of Plasmocin prior implantation. On the day of injection, cells were harvested, counted including a trypan blue viability dye (cut-off 80%), and resuspended in serum-free medium at the appropriate concentration. The cells injected subcutaneously in the right flank at 5.106 cells/mouse in 200 µL PBS within 30 minutes after harvesting. A margin of 2 mice were injected with cells (total of 50 mice implanted). [0251] Mice monitoring. After implantation, mice were monitored thrice weekly for tumor uptake, and general health and behavior. After treatment onset, they were observed daily in order to detect any toxic effects of the product or deleterious effect of the tumors. They were weighted twice weekly. The endpoints are defined by animal ethics as a tumor diameter of >18mm, significant weight loss or alteration of animal well-being.
[0252] Tumor monitoring. In order to assess the effectiveness of the compounds on tumorigenesis, tumor volume was measured twice a week. The sizes of the primary tumors were measured using calipers and the tumor volume (TV) were extrapolated to a sphere using the formula TV= 4/3 p x r3, by calculating the mean radius from the two measurements.
[0253] Mice randomization. Animals were randomized based on their individual tumor volume. Randomization was performed when values reach at a median tumor volume of 226 to 242 mm3. Animals were randomized into groups of 8 animals each.
[0254] Treatment. The treatment schedule is summarized in the table below:
Figure imgf000084_0001
[0255] Euthanasia. Mice were sacrificed when the tumors reached a maximum volume of 1600 mm3 or a pre-defined end-point.
Results [0256] The evolution of the tumor volume overtime is reported in Figure 4. Conclusion
[0257] These results clearly evidence that the polymer prodrugs of the present invention used in combination with Trastuzumab also have an activity against NCIN-87 cancer line that is comparable with the standard of care, namely treatment by T-DM1 (Trastuzumab bound to DM1), and are thus relevant for use in the treatment of gastric cancer.

Claims
CLAIMS ^ 1. A polymer compound of formula (A) (A) or a pharmaceutically acceptable salt and/or solvate thereof, wherein m is 0 or 1; L is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; M is a hydrophilic polymeric moiety selected from polyacrylamide, polyacrylic acid, poly(N-(2-hydroxypropyl)methacrylamide), poly(oligo(ethylene glycol)methyl ether methacrylate), poly(2-methacryloxyethyl phosphorylcholine), and copolymers thereof; and RA is selected from halo, -S-(C=S)-S-RB, -S-(C=S)-O-RB, -S-(C=S)-NRBRC, -S-(C=S)-RD, -O-NRERF, hydrogen, -OH, -SH, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups; each of which group being optionally substituted by one or more substituent(s) selected from halo, cyano, alkoxy, haloalkoxy heterocyclyl, carboxy, -OH, oxo, amino, alkylamino, hydroxyalkylamino, and amidine; wherein RB is alkyl, aryl, or heteroaryl; RC is hydrogen, alkyl, aryl, or heteroaryl; RD is an optionally substituted aryl; and RE and RF are independently selected from alkyl, arylalkyl, and dialkylphosphorylalkyl. The polymer compound according to claim 1, of formula (I)
Figure imgf000087_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein n is an integer ranging from 10 to 1400.
3. The polymer compound according to claim 2, wherein n is ranging from 50 to 700.
4. The polymer compound according to any one of claims 1 to 3, wherein L comprises one or more group(s) selected from optionally substituted, saturated or unsaturated, linear or branched C2-C40 hydrocarbon chains; optionally substituted cycloalkyl; and optionally substituted heterocyclyl; wherein said groups are linked through single bond, -O-, -S-, -NHC(O)-, -OC(O)-, -C(0)-0-C(0)-, -NH-,
-NH-C(0)-NH-, -NH-(CS)-NH-, -C(O)-, =N-NH-, and combinations thereof.
5. The polymer compound according to any one of claims 1 to 4, of formula (1-1)
Figure imgf000087_0002
or a pharmaceutically acceptable salt and/or solvate thereof, wherein L1 is selected from moieties (i), (ii), (iii) and (iv):
Figure imgf000087_0003
wherein --- represents the points of attachment; wherein the carbonyl group of (i), (ii), (iii) or (iv) links to L2; and
L2 is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties, or L2 is absent.
6. The polymer compound according to any one of claims 1 to 5, of formula (1-3)
Figure imgf000088_0001
or a pharmaceutically acceptable salt and/or solvate thereof, wherein X is O, N or S; preferably X is O or N.
7.
Figure imgf000088_0002
Figure imgf000088_0003
or a pharmaceutically acceptable salt and/or solvate thereof, wherein RG is selected from -S-RB, -0-RB, -NRBRC and R°; preferably RG is -S-RB.
8. The polymer compound according to any one of claims 1 to 7, selected from
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
and pharmaceutically acceptable salts and/or solvates thereof.
9. A pharmaceutical composition comprising a polymer compound according to any one of claims 1 to 8 and at least one pharmaceutically acceptable carrier.
10. The polymer compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, for use as a medicament.
11. The polymer compound according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9, for use in the treatment of cancer; preferably breast cancer, pancreatic cancer, ovarian cancer or lung cancer.
12. The polymer compound or pharmaceutical composition for use according to claim 10 or claim 11, to be administered by parenteral route; preferably intravenously, subcutaneously, intramuscularly or intratumorally.
13. A method of manufacturing of a polymer compound according to any one of claims 2 to 8, comprising a step of controlled radical polymerization performed by contacting a source of radicals, acrylamide monomers and a compound of formula (B)
Figure imgf000092_0001
or a salt and/or solvate thereof, under conditions suitable to obtain the polymer compound of formula (1-4) according to claim 7; optionally followed by a step of removal or modification of the thiocarbonylthio terminal group, leading to the polymer compound of formula (I) according to any one of claims 2 to 8.
14. A compound of formula (B)
Figure imgf000092_0002
or a salt and/or solvate thereof, wherein m is 0 or 1 ;
L is a linker, which comprises up to 100 carbon atoms and is in the form of a chemical chain which optionally comprises heteroatoms and/or cyclic moieties; and RG is selected from -S-RB, -0-RB, -NRBRC, and R° wherein RB is alkyl, aryl, or heteroaryl;
Rc is hydrogen, alkyl, aryl, or heteroaryl; and RD is an optionally substituted aryl.
15. The compound according to claim 14, selected from:
Figure imgf000093_0001
Figure imgf000094_0001
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US20180264133A1 (en) * 2017-03-16 2018-09-20 Immunwork Inc. Linker units and molecular constructs comprising the same
WO2019097025A1 (en) 2017-11-17 2019-05-23 Centre National De La Recherche Scientifique Polymer prodrugs and subcutaneous and/or intramuscular administration thereof

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WO2019097025A1 (en) 2017-11-17 2019-05-23 Centre National De La Recherche Scientifique Polymer prodrugs and subcutaneous and/or intramuscular administration thereof

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