WO2023086028A2 - Allenamide linkers for antibody-drug conjugate - Google Patents

Allenamide linkers for antibody-drug conjugate Download PDF

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Publication number
WO2023086028A2
WO2023086028A2 PCT/SG2022/050818 SG2022050818W WO2023086028A2 WO 2023086028 A2 WO2023086028 A2 WO 2023086028A2 SG 2022050818 W SG2022050818 W SG 2022050818W WO 2023086028 A2 WO2023086028 A2 WO 2023086028A2
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antibody
drug conjugate
conjugate according
pharmaceutically acceptable
drug
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PCT/SG2022/050818
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French (fr)
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WO2023086028A3 (en
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Teck Peng Loh
Manikantha MARASWAMI
Min Sun Kang
Yi Xin Joycelyn KHOO
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Nanyang Technological University
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Publication of WO2023086028A3 publication Critical patent/WO2023086028A3/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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6855—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from breast cancer cell
    • 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
    • A61K47/68—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—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 antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the current invention relates to an antibody-drug conjugate suitable for the treatment of cancer. Also disclosed herein are uses of the antibody-drug conjugate and methods of synthesis.
  • Cancer is one of the major diseases that threatens the health of the aging population. Despite the rapid development of pharmaceutical and medicinal sciences, the discovery of efficient and mild methods for cancer treatment is still challenging and highly desirable.
  • chemotherapy is considered the most commonly employed method for cancer treatment by inhibiting the DNA synthesis and tubulin aggregation of the proliferating cancer cells.
  • many of the traditional chemotherapeutic drugs are incapable of differentiating oncocytes and normal rapidly dividing cells, leading to severe side effects.
  • novel, efficient and target-specific treatment for cancer as compared to the classical chemotherapy has attracted considerable attention.
  • ADCs Antibody-drug conjugates
  • FIG. 1A The general structure of an ADC is shown in FIG. 1A.
  • ADCs consist of three major components: (i) monoclonal antibodies that can specifically bind to the antigens of cancer cells; (ii) linker that joins the antibody to the payload; and (iii) payload that is usually a highly potent cytotoxic compound.
  • FIG. 1A The general structure of an ADC is shown in FIG. 1A.
  • ADCs consist of three major components: (i) monoclonal antibodies that can specifically bind to the antigens of cancer cells; (ii) linker that joins the antibody to the payload; and (iii) payload that is usually a highly potent cytotoxic compound.
  • ADCs including Brentuximab vedotin and Trastuzumab emtansine in the market approved by the U.S. Food and Drug Administration (FDA) and more than 30 ADCs in different stages of clinical trials. While the choices of the monoclonal antibodies and drugs are important, the design of the linker is also of crucial importance as it can affect the efficacy and tolerability of ADCs.
  • FDA Food and Drug Administration
  • An eligible linker should be stable during blood circulation and capable of releasing the cytotoxins after being internalized into a cancer cell.
  • most of the ADCs currently under clinical development use the maleimide linker which suffers from severe reversibility (due to retro- Michael reaction) and lability issues in blood plasma.
  • Some linkers of ADCs have been reported to get cleaved before arriving at cancer tissues, and the payloads get released prematurely in blood plasma. These issues may result in undesired side effect, inconsistent pharmacodynamics, pharmacokinetics, and quality variation during drug production.
  • Y is an antibody or a reduced antibody
  • A is S or NH
  • L is a linking moiety having a structure of formula Ila or formula lib: where the wiggly line represents the point of attachment to (X-W) m -Z and the dashed line represents the point of attachment to A and a represents from 1 to 10;
  • X is an enzyme targeting moiety
  • W is a self-immolative linker
  • Z is a drug moiety
  • E is a bond or O; m is 0 to 1 ; and n is 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, provided that when:
  • L is a linking moiety of formula Ila, then m is 1 , A is S and Y is a reduced antibody;
  • L is a linking moiety of formula lib, then m is 0, A is NH and Y is an antibody.
  • a is from 5 to 6, such as 5;
  • X is an enzyme targeting moiety that has the structure:
  • a method of treating cancer comprising the steps of administering a therapeutically effective amount of an antibody-drug conjugate according to any one of Clauses 1 to 13, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.
  • FIG. 1 depicts (A) general structure of an ADC, and (B) ADC’s mechanism of action.
  • FIG. 3 depicts (A) the structures of brentuximab vedotin and trastuzumab emtansine, and (B) our design of ADCs using allenamide linker.
  • FIG. 4 depicts the stability evaluation of ADC 1-3 in human blood plasma-MMAE release.
  • FIG. 5 depicts the stability evaluation of ADC 1-3 in blood plasma-drug to antibody rate (DAR) value change.
  • FIG. 6 depicts the in vitro cytotoxic assay of various ADCs.
  • FIG. 7 depicts the in vitro cytotoxic assay of ADC 3 and brentuximab vedotin.
  • FIG. 8 depicts the 1 H nuclear magnetic resonance (NMR) spectrum (400 MHz, MeOD) of allenamidocaproyl-VC-PABC-MMAE.
  • FIG. 10 depicts the partial reduction of brentuximab antibodies.
  • FIG. 11 depicts the synthesis of the structure analogue of brentuximab vedotin, T-Allen- MMAE (ADC 1).
  • FIG. 12 depicts the synthesis of the structure analogue of brentuximab vedotin, ADC 2.
  • FIG. 13 depicts the synthesis of the structure analogue of brentuximab vedotin, ADC 3.
  • FIG. 14 depicts the preparation of trastuzumab emtansine and its structural analogue. Description
  • changing the linker may provide an antibody-drug conjugate with superior properties, such as greater stability, better cytotoxicity and pharmacokinetic distribution.
  • an antibody-drug conjugate having a structure according to formula I:
  • Y is an antibody or a reduced antibody
  • A is S or NH
  • L is a linking moiety having a structure of formula Ila or formula lib: where the wiggly line represents the point of attachment to (X-W) m -Z and the dashed line represents the point of attachment to A and a represents from 1 to 10;
  • X is an enzyme targeting moiety
  • W is a self-immolative linker
  • Z is a drug moiety
  • E is a bond or O; m is 0 to 1 ; and n is 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, provided that when:
  • L is a linking moiety of formula Ila, then m is 1 , A is S and Y is a reduced antibody;
  • L is a linking moiety of formula lib, then m is 0, A is NH and Y is an antibody.
  • the antibody-drug conjugate having a structure according to formula I may be referred to herein as “a/the compound of formula I”.
  • A refers to a functional group present in the antibody.
  • this may be an NH 2 group, which may be used to form a covalent bond with the linking moiety L.
  • this may be a thiol group, which may be used to form a covalent bond with the linking moiety L.
  • the term “reduced antibody” refers to an antibody where one or more disulfide (e.g. di-cysteine) bridges have been broken to provide free thiol groups, which may then be reacted with the linking moiety precursor.
  • disulfide e.g. di-cysteine
  • the antibody will have two free thiol groups. If two di-sulfide bridges are broken, then the antibody will have four free thiol groups.
  • the compound of formula I may be one where n is 4.
  • the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features.
  • the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of’ or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention.
  • the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.
  • the phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present.
  • the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
  • references herein (in any aspect or embodiment of the invention) to compounds of formula I include references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
  • salts include acid addition salts and base addition salts.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g.
  • L-glutamic L-glutamic
  • a-oxoglutaric glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic
  • lactic e.g. (+)-L-lactic and ( ⁇ )-DL-lactic
  • lactobionic maleic, malic (e.g.
  • salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids
  • organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids
  • metals such as sodium, magnesium, or preferably, potassium and calcium.
  • solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent).
  • solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide.
  • Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent.
  • Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
  • TGE thermogravimetric analysis
  • DSC differential scanning calorimetry
  • X-ray crystallography X-ray crystallography
  • the solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.
  • “Pharmaceutically functional derivatives’’ of compounds of formula I as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I.
  • prodrug of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).
  • Prodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent.
  • Prodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.
  • prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. I-92, Elsevier, New York-Oxford (1985).
  • Compounds of formula I may contain double bonds and may thus exist as E (entadel) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
  • Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
  • Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation.
  • the various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or high performance liquid chromatography (HPLC), techniques.
  • HPLC high performance liquid chromatography
  • the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e.
  • a ‘chiral pool’ method by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
  • the antibody-drug conjugate may be one in which L has the structure of formula Ila.
  • a may be from 5 to 6.
  • a may be 5.
  • X may be an enzyme targeting moiety that has the structure:
  • Y may be a reduced trastuzumab with free thiol groups or a reduced brentuximab with free thiol groups.
  • the compound of formula I may be one where n is 4.
  • the compound of formula I may be one where E is a bond.
  • the drug moiety Z may be monomethyl auristatin E.
  • W may have the formula III:
  • the antibody-drug conjugate may be selected from: 18 or a pharmaceutically acceptable salt or solvate thereof.
  • L may have the structure of formula lib.
  • Y may be a reduced trastuzumab with free thiol groups.
  • n may be 4.
  • the drug moiety Z may be mertansine.
  • Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form.
  • Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.
  • Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable adjuvant diluent or carrier
  • Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
  • a pharmaceutical formulation comprising an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier.
  • the amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.
  • a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment.
  • a controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.
  • a parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • a liquid or semisolid carrier or vehicle e.g. a solvent such as water
  • one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
  • Administration may be continuous or intermittent (e.g. by bolus injection).
  • the dosage may also be determined by the timing and frequency of administration.
  • the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.
  • the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
  • the above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the compounds of formula I disclosed herein may be used in medicine.
  • a use of an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, in medicine is provided.
  • the compounds of formula I may be particularly useful in the treatment of various cancers.
  • aspects of the invention described herein may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.
  • MMAE was obtained from MedChemExpress.
  • Compound 17 was purchased from GL Biochem.
  • Compound 18 was purchased from BLDpharm.
  • Compound 24 was purchased from Combi-blocks.
  • Compound 28 was purchased from Fluorochem.
  • the payload is one of the most essential parts in ADCs.
  • the cytotoxic agent (payload) is conjugated to monoclonal antibodies through a linker. After being internalized by target cancer cells, the payload would be released to induce cell apoptosis.
  • MMAE monomethyl auristatin E
  • 3R,4S,5S)-dolaisolecine (Dil) and dolaproline (Dap) are key fragments for the total synthesis of the marine natural product dolastatin 10 isolated from Dolabella auricularia. ⁇ Ne accomplished the gram-scale preparation of these two fragments in good yields and excellent stereoselectivities.
  • Dap fragment 11 The synthesis of Dap fragment 11 proceeded smoothly over 5 steps to give the desired product with 34% overall yield.
  • the remaining white solid was dried in vacuo for at least 5h. Diethyl ether was later added to the solid and the mixture was triturated and sonicated for 2 min. The white solid was then collected by filtration. (4.97 g, quantitative).
  • N, /V-diisopropylethylamine (0.11 mL, 0.635 mmol, 1.8 eq.) was added to a solution of 22 (0.125g, 0.353 mmol, 1.0 eq.) and HATU (0.167 g, 0.440 mmol, 1.25 eq.) in anhydrous DMF (1.6 mL) and the mixture was stirred at room temperature under nitrogen atmosphere for 10 min. 21b (0.167 g, 0.440 mmol, 1.25 eq.) was added to the mixture and the reaction mixture left to stir overnight at room temperature. Upon reaction completion, dichloromethane was added to the solution to precipitate the product. The mixture was filtered and dried in vacuo to obtain an off-white solid as the product (0.251 g, quantitative).
  • MMAE (0.0144g, 0.020 mmol, 1.0 eq.), 1M HOAt (6 pL, 0.0060 mmol, 0.30 eq.) and Fmoc- Amcap-Val-Cit-PAB-PNP (0.0186 g, 0.021 mmol, 1.05 eq.) were dissolved in anhydrous DMF (1.0 mL) under nitrogen atmosphere. After 2, 6-lutidine (11.6 pL, 0.1 mmol, 5.0 eq.) was added to the solution, the resultant mixture was stirred at 45 °C overnight. LCMS analysis was used to ascertain the formation of 26 and the completion of the reaction.
  • the allenamide linker consists of three main parts: (1) allenamidocaproyl group to bind to cysteine thiolate nucleophile; (2) peptide sequence for cleavage; and (3) self-elimination group (orange) for the release of payloads (FIG. 2).
  • the second part, Val-Cit gets recognized and cleaved by Cathepsin B, a lysosomal cysteine protease that is overexpressed in malignant cells.
  • MMAE (34) was synthesized.
  • Example 4 Synthesis of succinimidyl-4-[A/-maleimidomethyl]cyclohexane-1- carboxylate (SMCC)-drug conjugate and allenamide linker-drug conjugate
  • SMCC linker 38 has been widely employed in trastuzumab emtansine as well as a number of ADCs under clinical trials. As a result, we accomplished the large-scale synthesis of trastuzumab emtansine
  • SMCC conjugate 39 in moderate yield.
  • the relatively unstable maleimide moiety could be replaced by allenamide to give the analogue of SMCC linker bearing better stability.
  • MMCC linker 2,5-dioxopyrrolidin-1-yl 4- (buta-2,3-dienamidomethyl)cyclohexane-1-carboxylate (42).
  • DM1 Mertansine
  • the drug-linker reagents 39 and 43 could be synthesized and used as reagents in the ensuing conjugation with various antibodies, which could effectively avoid the potential process impurities generated during the ADC preparation.
  • DTT dithiothreitol
  • DTPA 8.0, adjust by 50 mM borate
  • a solution of 30 (prepared in Example 2) in acetonitrile was incubated on ice and then mixed with a solution of partially reduced trastuzumab (prepared in Example 5) in PBS buffer at 4 °C. The reaction was then quenched with the addition of 20 fold excess of N-acetyl cysteine after 2 h, and the affording ADC 1 was purified through size exclusion column chromatography using G25 desalting column equilibrated in PBS buffer containing 1 mM DTPA at 4 °C.
  • ADC 2 has a maleimide linker.
  • ADC 2 was synthesized through the reaction of the partially reduced trastuzumab (prepared in Example 5) with the traditional linker-drug conjugate 34 (prepared in Example 3) by following the synthetic protocol for ADC 1 above.
  • ADC 3 was synthesized from partially reduced brentuximab (prepared in Example 5) and 30 (prepared in Example 2) by following the synthetic protocol for ADC 1 above.
  • Trastuzumab emtansine (Trade name: Kadcyla) has been employed for the treatment of HER2 positive breast cancer and was approved by FDA in 2013.
  • SMCC linker 38 which contains two reactive functional groups has been utilized as the key intermediate during the preparation of trastuzumab emtansine, wherein the succinimide group reacts with the lysine residue of trastuzumab and the maleimide moiety reacts with the free thiol group in potent microtubule-disrupting agent mertansine (DM 1).
  • the SMCC linker involves the maleimide molecule to attach the drug (DM-1/mertansine) to the lysine conjugating part.
  • the maleimide part of the SMCC linker is substituted for an allenamide group, thereby generating SACC.
  • the stability of antibody-drug conjugates plays a critical role in the design and development of ADCs.
  • the conjugation of ADCs must be relatively stable in the blood plasma before entering the target cells.
  • DARs drug to antibody rates
  • the DARs of ADC 1-3 were evaluated and examined in blood plasma. Each of ADC 1-3 with a DAR rate of 4 was incubated in blood plasma at 37 °C for 0, 24, 48, 72, 96, and 160 h.
  • Both ADC 1 and ADC 3 which employed the conjugation of thiols-allenamide linking technology showed superior stability than ADC 2 which employed the traditionally used thiols-maleimide linkage.
  • ADC 1 and ADC 3 which employed the conjugation of thiols-allenamide linking technology showed superior stability than ADC 2 which employed the traditionally used thiols-maleimide linkage.
  • FIG. 4 even after 600 h, the release of the MMAE for ADC 1 and ADC 3 was less than 12%, indicating that the linkage between allenamide and thiols was relatively more stable than thiols-maleimide linkage (18% MMAE release after 600 h, probably due to the undesired hydrolysis of succinimide or the retro-Michael reaction).
  • the potency of the newly synthesized ADC 1 (prepared in Example 6) and ADC 4 (prepared in Example 7) which employed our newly developed allenamide-thiol linking technology was evaluated in comparison with ADC 2 (prepared in Example 6) and trastuzumab emtansine (T-Mal-DM1, prepared in Example 7). Since the antibody employed for the above mentioned ADCs is trastuzumab (Herceptin) which has been considered to be effective towards metastatic human epidermal growth factor receptor 2 (HER2), the HER2-overexpressing cell line SK-BR-3 was employed. SK-BR-3 cells were plated in 96-well plates and allowed to adhere overnight at 37 °C. The cell viability and cell death assay was carried out using Cell- Titer-Glo and the cellular ATP content was measured under different doses of ADCs.
  • trastuzumab Herceptin
  • HER2-overexpressing cell line SK-BR-3 was employed.
  • SK-BR-3 cells were plated in 96-well plates and allowed
  • ADC 3 bearing Brentuximab (cAC10, specific to CD30 on hematological malignances) as an antibody was also evaluated using long-term drug exposure assay.
  • the CD30 positive anaplastic large cell lymphoma (ALCL) cell line was chosen as the target. Alamar Blue conversion was used to measure the potency of the ADC.
  • ADC 2 that employed the maleimide-thiol linking technology was 1.8-fold more potent than ADC 1 which used the newly developed allenamide-thiol linking technology, while the former had almost equal potency comparing with trastuzumab emtansine.
  • ADC 4 which used allenamide-thiol linking technology (with mertansine as the payload) showed lower potency than ADC 1 and 2.
  • the unsatisfying activity of ADC 4 might be attributed to the different stability of this ADC (the newly formed C-S bond might be too stable such that the payload could not be released to induce cell death).
  • ADC 3 bearing cAC10 as an antibody also showed promising activity towards ALCL while a better stability compared to brentuximab vedotin was observed.
  • ADCs 2.0 ADC 1 and 3
  • these novel ADCs could be bio-equivalent to the approved ADCs.
  • the novel ADCs 2.0 might offer significant potency together with greater immunological specificity due to their stability characteristics.
  • ADC 4 was also produced as an analogue of trastuzumab emtansine.
  • the stability of the newly synthesized ADCs in human blood plasma has been carefully evaluated, and it was found that ADCs 2.0 which used the allenamide-thiol linking platform exhibited greater stability than the ADCs that used maleimide-thiol linking technology.

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Abstract

Disclosed herein is an antibody-drug conjugate having a structure according to formula I: Y-(A-L-(X-W)m-Z)n, wherein Y is a reduced antibody with free thiol groups, L is a linking moiety, X is an enzyme targeting moiety, W is a self-immolative linker, and Z is a drug moiety.

Description

ALLENAMIDE LINKERS FOR ANTIBODY-DRUG CONJUGATE
Field of Invention
The current invention relates to an antibody-drug conjugate suitable for the treatment of cancer. Also disclosed herein are uses of the antibody-drug conjugate and methods of synthesis.
Background
The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.
Cancer is one of the major diseases that threatens the health of the aging population. Despite the rapid development of pharmaceutical and medicinal sciences, the discovery of efficient and mild methods for cancer treatment is still challenging and highly desirable. Currently, chemotherapy is considered the most commonly employed method for cancer treatment by inhibiting the DNA synthesis and tubulin aggregation of the proliferating cancer cells. However, many of the traditional chemotherapeutic drugs are incapable of differentiating oncocytes and normal rapidly dividing cells, leading to severe side effects. Hence, the development of novel, efficient and target-specific treatment for cancer as compared to the classical chemotherapy has attracted considerable attention.
Antibody-drug conjugates (ADCs) have emerged as a new class of clinically established anti-cancer agents. This new class of highly potent anti-cancer drugs is less toxic as it makes use of the highly specific targeting abilities of monoclonal antibodies to differentiate between healthy cells and diseased tissue. The general structure of an ADC is shown in FIG. 1A. Generally, ADCs consist of three major components: (i) monoclonal antibodies that can specifically bind to the antigens of cancer cells; (ii) linker that joins the antibody to the payload; and (iii) payload that is usually a highly potent cytotoxic compound. FIG. 1 B depicts the mechanism of action: (I) ADCs in plasma; (II) specific binding of ADCs to targeted antigen; (III) ADCs-receptor complex is internalized; (IV) release of cytotoxic agent; and (V) apoptosis (cell death).
Currently, there are nine ADCs including Brentuximab vedotin and Trastuzumab emtansine in the market approved by the U.S. Food and Drug Administration (FDA) and more than 30 ADCs in different stages of clinical trials. While the choices of the monoclonal antibodies and drugs are important, the design of the linker is also of crucial importance as it can affect the efficacy and tolerability of ADCs.
An eligible linker should be stable during blood circulation and capable of releasing the cytotoxins after being internalized into a cancer cell. Unfortunately, most of the ADCs currently under clinical development use the maleimide linker which suffers from severe reversibility (due to retro- Michael reaction) and lability issues in blood plasma. Some linkers of ADCs have been reported to get cleaved before arriving at cancer tissues, and the payloads get released prematurely in blood plasma. These issues may result in undesired side effect, inconsistent pharmacodynamics, pharmacokinetics, and quality variation during drug production.
Therefore, there is a need for the design of new linkers with better stability and chemo- and stereoselectivity for ADCs.
Summary of Invention
Aspects and embodiments of the invention are discussed by reference to the following numbered clauses.
1. An antibody-drug conjugate having a structure according to formula I:
Y-(A-L-(X-W)m-Z)n I wherein,
Y is an antibody or a reduced antibody;
A is S or NH;
L is a linking moiety having a structure of formula Ila or formula lib:
Figure imgf000003_0001
Figure imgf000004_0001
where the wiggly line represents the point of attachment to (X-W)m-Z and the dashed line represents the point of attachment to A and a represents from 1 to 10;
X is an enzyme targeting moiety;
W is a self-immolative linker;
Z is a drug moiety;
E is a bond or O; m is 0 to 1 ; and n is 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, provided that when:
L is a linking moiety of formula Ila, then m is 1 , A is S and Y is a reduced antibody; and
L is a linking moiety of formula lib, then m is 0, A is NH and Y is an antibody.
2. The antibody-drug conjugate according to Clause 1 , wherein L has the structure of formula Ila.
3. The antibody-drug conjugate according to Clause 2, wherein:
(i) a is from 5 to 6, such as 5; and/or
(ii) X is an enzyme targeting moiety that has the structure:
Figure imgf000005_0001
, where the dashed line represents the point of attachment to L and the wiggly line represents the point of attachment to W.
4. The antibody-drug conjugate according to Clause 2 or Clause 3, wherein Y is a reduced trastuzumab or a reduced brentuximab.
5. The antibody-drug conjugate according to any one of Clauses 2 to 4, wherein n is 4 and/or E is a bond. 6. The antibody-drug conjugate according to any one of Clauses 2 to 5, wherein the drug moiety Z is monomethyl auristatin E.
7. The antibody-drug conjugate according to any one of Clauses 2 to 6, wherein W has the formula III:
Figure imgf000005_0002
Ill, where the dashed line represents the point of attachment to X and the wiggly line represents the point of attachment to Z. 8. The antibody-drug conjugate according to any one of Claims 2 to 7, wherein the antibody-drug conjugate is selected from:
Figure imgf000006_0001
Figure imgf000007_0001
or a pharmaceutically acceptable salt or solvate thereof.
9. The antibody-drug conjugate according to Clause 1 , wherein L has the structure of formula lib.
10. The antibody-drug conjugate according to Clause 9, wherein Y is a reduced trastuzumab.
11 . The antibody-drug conjugate according to Clause 9 or Clause 10, wherein n is 4.
12. The antibody-drug conjugate according to any one of Clauses 9 to 11 , wherein the drug moiety Z is mertansine.
13. The antibody-drug conjugate according to any one of Clauses 9 to 12, wherein the antibody-drug conjugate is:
Figure imgf000008_0001
or a pharmaceutically acceptable salt or solvate thereof.
14. Use of an antibody-drug conjugate according to any one of Clauses 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, in medicine.
15. Use of an antibody-drug conjugate according to any one of Clauses 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament to treat cancer.
16. An antibody-drug conjugate according to any one of Clauses 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of cancer.
17. A method of treating cancer, the method comprising the steps of administering a therapeutically effective amount of an antibody-drug conjugate according to any one of Clauses 1 to 13, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.
18. The use according to Clause 15, the antibody-drug conjugate for use according to Clause 16 or the method according to Clause 17, wherein the cancer is selected from one or more of the group consisting of breast cancer, lymphoma (e.g. large cell lymphoma and Hodgkin's lymphoma), melanoma, glioma, lymphoid malignancy and cervical cancer.
19. A pharmaceutical formulation comprising an antibody-drug conjugate according to any one of Clauses 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier. Drawings
FIG. 1 depicts (A) general structure of an ADC, and (B) ADC’s mechanism of action.
FIG. 2 depicts (A) allenamide-Amcap-Val-Cit-para-amino benzyl alcohol (PABC)- monomethyl auristatin E (MMAE), and (B) mechanism of action of allenamidocaproyl- Val- Cit-PABC-MMAE (Linker-Drug Moiety) in tumor cell.
FIG. 3 depicts (A) the structures of brentuximab vedotin and trastuzumab emtansine, and (B) our design of ADCs using allenamide linker.
FIG. 4 depicts the stability evaluation of ADC 1-3 in human blood plasma-MMAE release.
FIG. 5 depicts the stability evaluation of ADC 1-3 in blood plasma-drug to antibody rate (DAR) value change.
FIG. 6 depicts the in vitro cytotoxic assay of various ADCs.
FIG. 7 depicts the in vitro cytotoxic assay of ADC 3 and brentuximab vedotin.
FIG. 8 depicts the 1H nuclear magnetic resonance (NMR) spectrum (400 MHz, MeOD) of allenamidocaproyl-VC-PABC-MMAE.
FIG. 9 depicts the partial reduction of trastuzumab antibodies.
FIG. 10 depicts the partial reduction of brentuximab antibodies.
FIG. 11 depicts the synthesis of the structure analogue of brentuximab vedotin, T-Allen- MMAE (ADC 1).
FIG. 12 depicts the synthesis of the structure analogue of brentuximab vedotin, ADC 2.
FIG. 13 depicts the synthesis of the structure analogue of brentuximab vedotin, ADC 3.
FIG. 14 depicts the preparation of trastuzumab emtansine and its structural analogue. Description
It has been surprisingly found that changing the linker may provide an antibody-drug conjugate with superior properties, such as greater stability, better cytotoxicity and pharmacokinetic distribution.
Thus, in a first aspect of the invention, there is provided an antibody-drug conjugate having a structure according to formula I:
Y-(A-L-(X-W)m-Z)n I wherein,
Y is an antibody or a reduced antibody;
A is S or NH;
L is a linking moiety having a structure of formula Ila or formula lib:
Figure imgf000010_0001
where the wiggly line represents the point of attachment to (X-W)m-Z and the dashed line represents the point of attachment to A and a represents from 1 to 10;
X is an enzyme targeting moiety;
W is a self-immolative linker; Z is a drug moiety;
E is a bond or O; m is 0 to 1 ; and n is 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, provided that when:
L is a linking moiety of formula Ila, then m is 1 , A is S and Y is a reduced antibody; and
L is a linking moiety of formula lib, then m is 0, A is NH and Y is an antibody.
The antibody-drug conjugate having a structure according to formula I may be referred to herein as “a/the compound of formula I”.
For the avoidance of doubt, A refers to a functional group present in the antibody. In a “normal” antibody, this may be an NH2 group, which may be used to form a covalent bond with the linking moiety L. In a reduced antibody, this may be a thiol group, which may be used to form a covalent bond with the linking moiety L.
When used herein, the term “reduced antibody” refers to an antibody where one or more disulfide (e.g. di-cysteine) bridges have been broken to provide free thiol groups, which may then be reacted with the linking moiety precursor. As will be appreciated, if one di-sulfide bridge is broken, then the antibody will have two free thiol groups. If two di-sulfide bridges are broken, then the antibody will have four free thiol groups. In examples that may be mentioned herein, the compound of formula I may be one where n is 4.
In embodiments herein, the word “comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word “comprising” may be replaced by the phrases “consists of’ or “consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention. In other words, the word “comprising” and synonyms thereof may be replaced by the phrase “consisting of” or the phrase “consists essentially of’ or synonyms thereof and vice versa.
The phrase, “consists essentially of” and its pseudonyms may be interpreted herein to refer to a material where minor impurities may be present. For example, the material may be greater than or equal to 90% pure, such as greater than 95% pure, such as greater than 97% pure, such as greater than 99% pure, such as greater than 99.9% pure, such as greater than 99.99% pure, such as greater than 99.999% pure, such as 100% pure.
As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a composition” includes mixtures of two or more such compositions, and the like.
References herein (in any aspect or embodiment of the invention) to compounds of formula I include references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)- (1S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)-L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.
Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
As mentioned above, also encompassed by a compound of formula I are any solvates of the compounds and their salts. Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
The solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.
For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.
“Pharmaceutically functional derivatives’’ of compounds of formula I as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of compounds of formula I.
The term “prodrug” of a relevant compound of formula I includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)).
Prodrugs of compounds of formula I may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include compounds of formula I wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.
Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N- Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. “Design of Prodrugs” p. I-92, Elsevier, New York-Oxford (1985).
Compounds of formula I, as well as pharmaceutically acceptable salts, solvates and pharmaceutically functional derivatives of such compounds are, for the sake of brevity, hereinafter referred to together as the “compounds of formula I”.
Compounds of formula I may contain double bonds and may thus exist as E (entgegen) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
Compounds of formula I may exist as regioisomers and may also exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or high performance liquid chromatography (HPLC), techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a ‘chiral pool’ method), by reaction of the appropriate starting material with a ‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e. a resolution, including a dynamic resolution), for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
In embodiments of the invention that may be mentioned herein, the antibody-drug conjugate may be one in which L has the structure of formula Ila.
In embodiments of the invention where the antibody-drug conjugate is one in which L has the structure of formula Ila, a may be from 5 to 6. For example, a may be 5.
In additional or alternative embodiments of the invention where the antibody-drug conjugate is one in which L has the structure of formula Ila, X may be an enzyme targeting moiety that has the structure:
Figure imgf000015_0001
, where the dashed line represents the point of attachment to L and the wiggly line represents the point of attachment to W.
Where the antibody-drug conjugate is one in which L has the structure of formula Ila, Y may be a reduced trastuzumab with free thiol groups or a reduced brentuximab with free thiol groups.
Where the antibody-drug conjugate is one in which L has the structure of formula Ila, the compound of formula I may be one where n is 4.
Where the antibody-drug conjugate is one in which L has the structure of formula Ila, the compound of formula I may be one where E is a bond. In embodiments of the invention where the antibody-drug conjugate is one in which L has the structure of formula Ila, the drug moiety Z may be monomethyl auristatin E.
In embodiments of the invention where the antibody-drug conjugate is one in which L has the structure of formula Ila, W may have the formula III:
Figure imgf000016_0001
III, where the dashed line represents the point of attachment to X and the wiggly line represents the point of attachment to Z.
In embodiments of the invention where the antibody-drug conjugate is one in which L has the structure of formula Ila, the antibody-drug conjugate may be selected from:
Figure imgf000016_0002
18
Figure imgf000017_0001
or a pharmaceutically acceptable salt or solvate thereof.
In alternative embodiments to those depicted above, L may have the structure of formula lib.
In embodiments where L has the structure of formula lib, Y may be a reduced trastuzumab with free thiol groups.
In embodiments where L has the structure of formula lib, n may be 4.
In embodiments where L has the structure of formula lib, the drug moiety Z may be mertansine.
In embodiments where L has the structure of formula lib, the antibody-drug conjugate may be:
Figure imgf000018_0001
or a pharmaceutically acceptable salt or solvate thereof.
Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration. Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.
Thus, in an aspect of the invention, there is provided a pharmaceutical formulation comprising an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier.
The amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.
For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.
A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
Depending on the disorder, and the patient, to be treated, as well as the route of administration, compounds of formula I may be administered at varying therapeutically effective doses to a patient in need thereof.
However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.
In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
The compounds of formula I disclosed herein may be used in medicine. Thus, in a further aspect of the invention, there is provided a use of an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, in medicine.
More particularly, the compounds of formula I may be particularly useful in the treatment of various cancers. Thus, in further aspects of the invention, there are provided:
(a) use of an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament to treat cancer; (b) an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of cancer; or
(c) a method of treating cancer, the method comprising the steps of administering a therapeutically effective amount of an antibody-drug conjugate as described herein, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.
In the above aspects, the cancer may be selected from one or more of the group consisting of breast cancer, lymphoma (e.g. large cell lymphoma and Hodgkin's lymphoma), melanoma, glioma, lymphoid malignancy and cervical cancer.
The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.
Further aspects and embodiments of the invention will now be discussed by reference to the following non-limiting examples.
Examples
Materials
MMAE was obtained from MedChemExpress. Compound 17 was purchased from GL Biochem. Compound 18 was purchased from BLDpharm. Compound 24 was purchased from Combi-blocks. Compound 28 was purchased from Fluorochem.
Example 1. Synthesis of payload MMAE
The payload is one of the most essential parts in ADCs. The cytotoxic agent (payload) is conjugated to monoclonal antibodies through a linker. After being internalized by target cancer cells, the payload would be released to induce cell apoptosis. At the outset of our study, we focused on the gram-scale preparation of the payload (monomethyl auristatin E (MMAE)) and we successfully developed an efficient synthetic route for the preparation of this drug molecule. (3R,4S,5S)-dolaisolecine (Dil) and dolaproline (Dap) are key fragments for the total synthesis of the marine natural product dolastatin 10 isolated from Dolabella auricularia. \Ne accomplished the gram-scale preparation of these two fragments in good yields and excellent stereoselectivities.
Stereoselective synthesis of Dil fragment
Figure imgf000022_0001
2.0g, 43% overall yield
Figure imgf000023_0001
66% yield
16, monomethyl auristatin E
Results and discussion
Notably, we have developed a novel and efficient method towards the synthesis of chiral /3- hydroxyl ester 4. The asymmetric hydrogen transfer using Hayashi’s Ru/TsDPEN catalytic system gave the desired product in good yield (86%) and stereoselectivity (d.r. = 89:11). The reaction proceeded smoothly at room temperature in the presence of formic acid- triethylamine azeotrope mixture, which is in sharp contrast to the previously reported asymmetric hydrogenation and/or the dynamic kinetic resolution that requires relatively harsh condition.
The synthesis of Dap fragment 11 proceeded smoothly over 5 steps to give the desired product with 34% overall yield. The Yamada coupling of Dap with Norephedrine involving the employment of diethylphosphoryl (DEPC) gave 12 in good yield.
Thus, with the key fragments Dil and Dap in hand, we successfully achieved the gram-scale preparation of the cytotoxic agent MMAE 16 in considerable yield.
Example 2. Large-scale synthesis of the allenamide linkers and the drug-linker conjugates
The choice of linkers also plays a critical role in the development of ADCs. As mentioned above, the most commonly used maleimide linker is unstable in blood circulation due to the retro-Michael reaction (that allows the exchange of maleimide with other reactive thiols in serum) and the plausible ring-opening reaction of the succinimide. In addition, the reaction of the chiral antibody molecules with the prostereogenic center of the maleimide gives complicated stereoisomers, which leads to difficulties in the analysis and purification of ADCs. We successfully accomplished the gram-scale preparation of several allenamide linkers.
Figure imgf000025_0001
Figure imgf000026_0001
19
To a solution of L-citrulline (2.63 g, 15.0 mmol) and NaHCO3 (1.26 g, 15 mmol) dissolved in deionised water (28 mL) was added a solution of 18 (4.37 g, 10.0 mmol) in 1 ,2 - di methoxyethane (28 mL). THF (14 mL) was added to increase solubility. The reaction was then left to stir for 2 days. Upon completion of reaction, aqueous citric acid (15%, 50 mL) was added to the reaction mixture and the resulting mixture was extracted with 10% isopropyl alcohol in ethyl acetate (x3). The organic layer was washed with water once and collected. After the organic solvents have been removed, the remaining white solid was dried in vacuo for at least 5h. Diethyl ether was later added to the solid and the mixture was triturated and sonicated for 2 min. The white solid was then collected by filtration. (4.97 g, quantitative).
1H NMR (400 MHz, DMSO-d6) 5 8.05 (s, 1 H), 7.89 (d, J = 7.5 Hz, 2H), 7.75 (t, J = 7.7 Hz, 2H), 5.92 (d, J = 5.8 Hz, 1 H), 5.36 (s, 2H), 4.31 - 4.18 (m, 3H), 4.07 (s, 1H), 3.89 (dd, J = 9.2, 6.8 Hz, 1 H), 2.93 (q, J = 7.0 Hz, 3H), 2.05 - 1.92 (m, 1H), 1.76 - 1.47 (m, 2H), 1.37 (t, J = 7.8 Hz, 2H), 0.86 (dd, J = 11.3, 6.7 Hz, 7H).
ESI-MS calculated for C26H33N4O6 [M + H]+ m/z 497.24, found 497.15 [M + H]+.
21
19 (0.7865 g, 1.58 mmol), 20 (0.389 g, 3.16 mmol, 2.0 eq.) and 2-ethoxy-1-ethoxycarbonyl- 1,2-dihydroquinoline (EEDQ) (0.771 g, 3.16 mmol, 2.0 eq.) were dissolved in 2:1 dichloromethane /methanol (27 mL) and the resulting mixture was stirred in the dark at room temperature for 2 days. After the reaction was completed, the solvent was then removed and the resulting white solid was triturated and sonicated for 5 min with diethyl ether. The suspension was left to stand for 30 min and later filtered and dried to provide an off-white solid as the product (0.741 g, 78%).
1H NMR (400 MHz, DMSO-d6) 5 9.99 (s, 1H), 8.12 (d, J = 7.7 Hz, 1 H), 8.00 - 7.10 (m, 13H), 5.99 (s, 1 H), 5.41 (s, 2H), 5.09 (t, J = 5.7 Hz, 1 H), 4.65 - 4.13 (m, 6H), 3.93 (dd, J = 9.0, 7.0 Hz, 1 H), 2.97 (dt, J = 27.0, 6.6 Hz, 2H), 2.10 - 1.86 (m, 1 H), 1.84 - 1.23 (m, 4H), 0.86 (dd, J = 11.0, 6.8 Hz, 6H).
ESI-MS calculated for 633^9^03 [M + H]+ m/z 602.30, found 602.04 [M + H]+.
21 b
21 (0.120 g, 0.20 mmol) was dissolved in 50% piperidine in DMF and stirred for 1 h. Upon completion of reaction, dichloromethane was added to the reaction mixture to precipitate the product. The product collected by filtration and dried in vacuo. (0.0649 g, 86%).
1H NMR (DMSO-ds) 5 10.03 (s, 1 H), 8.14 (d, J = 7.5 Hz, 1 H), 7.54 (d, J = 8.5 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 5.98 (t, J = 5.8 Hz, 1 H), 5.40 (s, 2H), 5.10 (s, 1H), 4.46-4.42 (m, 3H), 3.10 - 2.87 (m, 3H), 2.01 - 1.83 (m, 1 H), 1.77 - 1.31 (m, 4H), 0.89 (d, J = 6.9 Hz, 3H), 0.79 (d, J = 6.8 Hz, 3H).
22 6-amino-hexanoic acid (0.262 g, 2.0 mmol) was dissolved in aqueous sodium carbonate solution (10%, 6.0 mL) and 1 ,4-dioxane (4 mL). Fmoc chloride (0.569 g, 2.20 mmol, 1.1 eq.) was added to the mixture in small portions, and the reaction mixture was left to stir overnight at room temperature. Upon reaction completion, the reaction mixture was washed with diethyl ether twice before the aqueous layer was acidified using 6N HCI. The resulting suspension was filtered, and the residue was washed with water and dried to provide a white solid as the product (0.613 g, 87%).
1H NMR (400 MHz, DMSO-cfe) 5 8.02 - 7.09 (m, 9H), 4.25 (dd, J = 31 .1 , 6.8 Hz, 3H), 2.96 (q, J = 6.6 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.55 - 1.01 (m, 6H).
23
N, /V-diisopropylethylamine (0.11 mL, 0.635 mmol, 1.8 eq.) was added to a solution of 22 (0.125g, 0.353 mmol, 1.0 eq.) and HATU (0.167 g, 0.440 mmol, 1.25 eq.) in anhydrous DMF (1.6 mL) and the mixture was stirred at room temperature under nitrogen atmosphere for 10 min. 21b (0.167 g, 0.440 mmol, 1.25 eq.) was added to the mixture and the reaction mixture left to stir overnight at room temperature. Upon reaction completion, dichloromethane was added to the solution to precipitate the product. The mixture was filtered and dried in vacuo to obtain an off-white solid as the product (0.251 g, quantitative).
HRMS calculated for C39H5iN8O7 [M + H]+ m/z 715.3819, found 715.3819 [M + H]+.
25
To a solution of 23 (0.0630g, 0.088 mmol) in anhydrous DMF, 24 (0.0804 g, 0.264 mmol, 3.0 eq.) and N, /V-diisopropylethylamine (46.1 pL, 0.264 mmol, 3.0 eq.) was added to the reaction mixture under nitrogen atmosphere. The resulting mixture was stirring overnight at room temperature. Upon reaction completion, the reaction mixture was diluted with excess diethyl ether and the precipitate was collected via filtration. Purification of the precipitate by flash column chromatography using a gradient elution of 0-8% methanol in dichloromethane provided an off-white solid as the desired product. (0.0116 g, 15%)
HRMS calculated for C46H53N70iiNa [M + Na]+ m/z 902.3701 , found 902.3693 [M + Na]+.
The reaction of Fmoc-AmCap-Val-Cit-PABC-PNP linker 25 with MMAE was investigated. In the presence of HOAt and 2,6-lutidine, a mixture of 26 and 27 was obtained. After treatment with piperidine, the Fmoc-deprotection was completed to give 27 in good yield. Finally, the allenamide moiety was successfully incorporated through the reaction of 27 with but-3-ynoic acid 28 in the presence of Mukaiyama reagent 29 and diethyl amine to give the allenamide linker-drug conjugate 30 in moderate yield.
26 and 27
MMAE (0.0144g, 0.020 mmol, 1.0 eq.), 1M HOAt (6 pL, 0.0060 mmol, 0.30 eq.) and Fmoc- Amcap-Val-Cit-PAB-PNP (0.0186 g, 0.021 mmol, 1.05 eq.) were dissolved in anhydrous DMF (1.0 mL) under nitrogen atmosphere. After 2, 6-lutidine (11.6 pL, 0.1 mmol, 5.0 eq.) was added to the solution, the resultant mixture was stirred at 45 °C overnight. LCMS analysis was used to ascertain the formation of 26 and the completion of the reaction. Piperidine (70.9 pL) was then added and the reaction was stirred for another 1h at room temperature. The reaction was confirmed to be complete via LCMS analysis. The crude product (27) was precipitated using diethyl ether and used for the next reaction without further purification.
26: ESI-MS calculated for C79H115N11O15 [M + H]+ m/z 1458.87, found 1458.36 [M + H]+. 27: HRMS calculated for C64HIO6NIIOI3 [M + H]+ m/z 1236.7972, found 1236.7971 [M + H]+.
30
/V-methyl morpholine (6.3 pL) and isopropyl chloroformate (1 M in toluene, 47.8 pL, 0.0478 mmol) were successively added to a solution of 28 in anhydrous THF at 0°C. After stirring the reaction mixture for 3 min, a solution of 27 in anhydrous DMF was added to the reaction mixture at 0°C. The resulting mixture was stirred overnight and the reaction progress was tracked using LCMS.
ESI-MS calculated for CesHwsNuOu [M + H]+ m/z 1302.80, found 1302.32 [M + H]+.
While the formation of the product has been detected, the product was not isolated as the reaction did not proceed to completion and too many side products were present. As such, the synthetic route was modified (see Version 3). Synthesis ofAllenamide-Amcap-Val-Cit-PABC-MMAE (Version 3)
Figure imgf000030_0001
260 To a solution of Val-Cit-PABC-MMAE (258) (31.9 mg, 0.0284 mmol, 1.0 eq.) and compound 259 (14.8 mg, 0.0426 mmol, 1.5 eq.) in anhydrous DMF under Ar atmosphere, 2,6-lutidine (41.8 pL, 0.142 mmol, 5.0 eq.) and HOAt (1M in DMA; 8.5 pL, 0.00852 mmol, 0.30 eq.) were added and the reaction mixture was stirred overnight at 45 °C. Upon completion of reaction, the reaction was allowed to cool to room temperature. Excess diethyl ether was added to the reaction mixture to precipitate the product and the resulting slurry was stirred for 15 min before diethyl ether was decanted. The remaining residue was purified by preparative thin layer chromatography (DCM/MeOH = 14:1) to provide 260 as a white solid.
ESI-MS calculated for CegHiogNuOisNa [M + Na]+ m/z 1354.80, found 1354.84 [M + Na]+. 1H NMR is consistent with assigned structure.
Synthesis of the heterobifunctional linker
Figure imgf000031_0001
Results and discussion
The allenamide linker consists of three main parts: (1) allenamidocaproyl group to bind to cysteine thiolate nucleophile; (2) peptide sequence for cleavage; and (3) self-elimination group (orange) for the release of payloads (FIG. 2). The second part, Val-Cit, gets recognized and cleaved by Cathepsin B, a lysosomal cysteine protease that is overexpressed in malignant cells.
Example 3. Synthesis of maleimido-caproyl-Val-Cit-PABC-MMAE
In order to compare the efficacy and stability of allenamide linker-drug conjugate with the maleimide linker-drug conjugate which was employed in brentuximab vedotin, the corresponding maleimide derived linker-drug conjugate maleimido-caproyl-Val-Cit-PABC-
MMAE (34) was synthesized.
Figure imgf000031_0002
Example 4. Synthesis of succinimidyl-4-[A/-maleimidomethyl]cyclohexane-1- carboxylate (SMCC)-drug conjugate and allenamide linker-drug conjugate
SMCC linker 38 has been widely employed in trastuzumab emtansine as well as a number of ADCs under clinical trials. As a result, we accomplished the large-scale synthesis of
SMCC linker. Then, the reaction of 38 with Mertansine was carried out to afford the drug-
SMCC conjugate 39 in moderate yield.
Figure imgf000032_0001
We envisioned that the relatively unstable maleimide moiety could be replaced by allenamide to give the analogue of SMCC linker bearing better stability. Gratifyingly, we have developed a facile synthetic route towards the allenamide linker 2,5-dioxopyrrolidin-1-yl 4- (buta-2,3-dienamidomethyl)cyclohexane-1-carboxylate (42). Then, the reaction of this allenamide-based linker 42 with Mertansine (DM1) proceeded smoothly under mild reaction condition to give the drug-linker conjugate 43 in moderate yield. Notably, the drug-linker reagents 39 and 43 could be synthesized and used as reagents in the ensuing conjugation with various antibodies, which could effectively avoid the potential process impurities generated during the ADC preparation.
Example 5. Preparation of antibody-drug conjugates ADCs (2.0)
After accomplishing the synthesis of allenamide-based linkers in the previous examples, we focused on the synthesis and evaluation of the analogues of brentuximab vedotin and trastuzumab emtansine using our platform technology.
Partial reduction of trastuzumab antibodies (FIG. 9)
At the outset of our study, the partial reduction of the antibody trastuzumab was performed by the addition of a solution of tris[2-carboxyethyl]phosphine hydrochloride (TCEP, 2.0 eq) in phosphate/ethylenediaminetetraacetic acid (EDTA) buffer (pH = 7.4) at 20 °C for 90 min, giving a free thiol to antibody ratio (FTAR) of 2.0. Interestingly, the FTAR could be tuned to 4.0 by the treatment of trastuzumab with dithiothreitol (DTT, 4.2 eq) in PBS buffer containing 1 mM DTPA (pH = 8.0, adjust by 50 mM borate). The concentration of the free thiols could be determined through an Ellman assay using 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB) by checking the absorbance at 412 nm.
Partial reduction of brentuximab antibodies (FIG. 10)
Similarly, the partial reduction of the chimeric monoclonal antibody brentuximab (cAC 10) was examined as well by following the protocol for partial reduction of trastuzumab antibodies above. The reaction proceeded smoothly in PBS buffer containing 1 mM DTPA when DTT was employed as the reducing agent (FTAR = 4.0).
Example 6. Preparation of the structural analogues of brentuximab vedotin
After partial reduction of antibodies trastuzumab and brentuximab in Example 5, we next focused on the preparation of brentuximab vedotin and its structural analogues using our newly developed allenamide linking technology (FIG. 3). Initially, the reactions of the partially reduced trastuzumab (with different free thiol to antibody ratios) with linker-drug conjugate allenamide-Amcap-Val-Cit-PABC-MMAE (30) were examined.
Synthesis of the structure analogue of brentuximab vedotin, T-Allen-MMAE (ADC 1) (FIG. 11)
A solution of 30 (prepared in Example 2) in acetonitrile was incubated on ice and then mixed with a solution of partially reduced trastuzumab (prepared in Example 5) in PBS buffer at 4 °C. The reaction was then quenched with the addition of 20 fold excess of N-acetyl cysteine after 2 h, and the affording ADC 1 was purified through size exclusion column chromatography using G25 desalting column equilibrated in PBS buffer containing 1 mM DTPA at 4 °C.
In order to compare the biological activity and stability of the newly synthesized ADC 1 , the structural analogue of brentuximab vedotin ADC 2 (T-Mal-MMAE) was also synthesized. ADC 2 has a maleimide linker.
Synthesis of the structure analogue of brentuximab vedotin, ADC 2 (FIG. 12)
ADC 2 was synthesized through the reaction of the partially reduced trastuzumab (prepared in Example 5) with the traditional linker-drug conjugate 34 (prepared in Example 3) by following the synthetic protocol for ADC 1 above.
The reaction of the partially reduced brentuximab (cAC10) with 30 also proceeded smoothly to give cAC10-Allen-MMAE (ADC 3) as an analogue of brentuximab vedotin.
Synthesis of the structure analogue of brentuximab vedotin, ADC 3 (FIG. 13)
ADC 3 was synthesized from partially reduced brentuximab (prepared in Example 5) and 30 (prepared in Example 2) by following the synthetic protocol for ADC 1 above.
Example 7. Preparation of trastuzumab emtansine and its structural analogue
Trastuzumab emtansine (Trade name: Kadcyla) has been employed for the treatment of HER2 positive breast cancer and was approved by FDA in 2013. SMCC linker 38 which contains two reactive functional groups has been utilized as the key intermediate during the preparation of trastuzumab emtansine, wherein the succinimide group reacts with the lysine residue of trastuzumab and the maleimide moiety reacts with the free thiol group in potent microtubule-disrupting agent mertansine (DM 1). The SMCC linker involves the maleimide molecule to attach the drug (DM-1/mertansine) to the lysine conjugating part. The maleimide part of the SMCC linker is substituted for an allenamide group, thereby generating SACC.
We carried out the synthesis of the structure analogue of trastuzumab emtansine which may possess better activity and stability (FIG. 14). The model reaction of the lysine residue in partially reduced trastuzumab with the drug-linker conjugate allenamide-mertansine conjugate 43 was carried out in phosphate/EDTA buffer (pH = 6.5) in the presence of dimethyl sulfoxide (DMSO, v/v = 5%) to give T-Allen-DM1 (ADC 4) as an analogue of trastuzumab emtansine. The synthesis of trastuzumab emtansine also proceeded smoothly through the reaction of trastuzumab with allenamide-mertansine conjugate 39 under similar reaction conditions.
After completing the synthesis of the several antibody-drug conjugates in Examples 6 and 7, we focused on the evaluation of the stabilities, anti-tumor potencies and pharmacokinetic properties of these ADCs (2.0) (ADC 1-4), which are of key importance in determining the efficacy, tolerability and market potential of those drugs, in the following examples.
Example 8. Stability of antibody-drug conjugates ADCs (2.0) (ADC 1-3)
The stability of antibody-drug conjugates plays a critical role in the design and development of ADCs. The conjugation of ADCs must be relatively stable in the blood plasma before entering the target cells.
Stability of antibody-drug conjugates
The stability of the newly synthesized ADC 1-3 using the conjugation of cysteine residue of antibodies with Michael acceptors (Maleimide or allenamide moieties, as described in Example 6) were incubated and evaluated in the presence of human plasma at 37 °C, respectively. The release of the payload MMAE was monitored by HPLC analysis at 0, 24, 48, 72, 96, 160 and 600 h.
Determination of drug to antibody rates (DARs)
The DARs of ADC 1-3 were evaluated and examined in blood plasma. Each of ADC 1-3 with a DAR rate of 4 was incubated in blood plasma at 37 °C for 0, 24, 48, 72, 96, and 160 h.
Results and discussion
Both ADC 1 and ADC 3 which employed the conjugation of thiols-allenamide linking technology showed superior stability than ADC 2 which employed the traditionally used thiols-maleimide linkage. As shown in FIG. 4, even after 600 h, the release of the MMAE for ADC 1 and ADC 3 was less than 12%, indicating that the linkage between allenamide and thiols was relatively more stable than thiols-maleimide linkage (18% MMAE release after 600 h, probably due to the undesired hydrolysis of succinimide or the retro-Michael reaction).
For ADC 1 and ADC 3, there was no obvious evidence for the decrease of the DAR value, while for ADC 2, a slight degradation of the DAR rate was observed, indicating the loss of MMAE (FIG. 5), which is consistent with the results shown in FIG. 4. A characterization study conducted by Pfizer further supports this in-vitro study; Trastuzumab- maleimidocaproyl-VC-PABC-drug was incubated with human plasma for 144 h, and almost 100% of the DAR loss happened (Wei, C. et al., Anal. Chem. 2016, 88, 4979-4986). Intact protein mass analysis showed that at the 144 h time point, the mass of albumin in human plasma had an additional 1347 Da over the native albumin extracted from human plasma, exactly matching the mass of the maleimide linker-payload.
Therefore, our ADCs (2.0) are highly stable in blood plasma, and are stereomerically pure.
Example 9. In vitro cytotoxicity and pharmacokinetics evaluation of antibody-drug conjugates ADCs (2.0) (ADC 1-4)
The in vitro potency of the antibody-drug conjugates towards tumour cells is another key aspect of ADCs.
Potency studies
The potency of the newly synthesized ADC 1 (prepared in Example 6) and ADC 4 (prepared in Example 7) which employed our newly developed allenamide-thiol linking technology was evaluated in comparison with ADC 2 (prepared in Example 6) and trastuzumab emtansine (T-Mal-DM1, prepared in Example 7). Since the antibody employed for the above mentioned ADCs is trastuzumab (Herceptin) which has been considered to be effective towards metastatic human epidermal growth factor receptor 2 (HER2), the HER2-overexpressing cell line SK-BR-3 was employed. SK-BR-3 cells were plated in 96-well plates and allowed to adhere overnight at 37 °C. The cell viability and cell death assay was carried out using Cell- Titer-Glo and the cellular ATP content was measured under different doses of ADCs.
The in vitro potency of ADC 3 bearing Brentuximab (cAC10, specific to CD30 on hematological malignances) as an antibody was also evaluated using long-term drug exposure assay. The CD30 positive anaplastic large cell lymphoma (ALCL) cell line was chosen as the target. Alamar Blue conversion was used to measure the potency of the ADC.
Results and discussion
Gratifyingly, both the newly developed ADC 1 (IC50 = 0.23 nM), ADC 2 (IC50 = 0.13 nM) showed comparable potency to SK-BR-3 cell line in contrast with the marketed and FDA- approved drug trastuzumab emtansine (T-Mal-DM1, IC50 = 0.094 nM). As shown in FIG. 6, ADC 2 that employed the maleimide-thiol linking technology was 1.8-fold more potent than ADC 1 which used the newly developed allenamide-thiol linking technology, while the former had almost equal potency comparing with trastuzumab emtansine. Surprisingly, ADC 4 which used allenamide-thiol linking technology (with mertansine as the payload) showed lower potency than ADC 1 and 2. We postulated that the unsatisfying activity of ADC 4 might be attributed to the different stability of this ADC (the newly formed C-S bond might be too stable such that the payload could not be released to induce cell death).
As shown in FIG. 7, although ADC 3 which employed the allenamide-thiol linkage was not as efficacious as the marketed and approved brentuximab vedotin, considerable potency towards ALCL cell line was observed.
Our newly developed allenamide-thiol linking technology was readily applicable for the development of novel ADCs bearing considerable stabilities in human blood plasma and potencies towards tumor cell lines. Generally, the ADCs 2.0 using this new technology showed better stabilities than ADCs 1.0 using maleimide-thiol linking technology (ADC 1 and 3 vs ADC 2). The cytotoxicity assay of ADCs using trastuzumab indicated that the newly synthesized ADCs (ADC 1 and 2) could induce the apoptosis of HER2-overexpressing breast cancer cells, which showed comparable potency to trastuzumab emtansine. ADC 3 bearing cAC10 as an antibody also showed promising activity towards ALCL while a better stability compared to brentuximab vedotin was observed. Particularly, in light of the excellent stability of ADCs 2.0 (ADC 1 and 3) in blood plasma and the considerable potencies, we believe that these novel ADCs could be bio-equivalent to the approved ADCs. Moreover, as a number of plausible side reactions (including retro-Michael reaction and hydrolysis of succinimide) ascribe to the traditional maleimide linkers were averted, the novel ADCs 2.0 might offer significant potency together with greater immunological specificity due to their stability characteristics.
Therefore, we have accomplished the gram-scale preparation of the cytotoxic agent MMAE using a modified strategy, giving the desired molecule in good yield and stereocontrol. Then, we successfully achieved the large-scale synthesis of several allenamide-based linkers. A number of linker-drug conjugates such as 30 and 43 were attained by employing our allenamide-thiol linking technology. The linker-drug conjugates 34 and 39 were prepared as well using the traditional maleimide-thiol linking technology. The partial reduction of two antibodies (trastuzumab and brentuximab) and the ensuing conjugation of the free thiol groups with 30 and 34 were systematically examined to afford the structure analogues of brentuximab vedotin ADC 1-3. Through the reaction of the lysine residue in trastuzumab with the drug-linker conjugate allenamide-mertansine conjugate 43, ADC 4 was also produced as an analogue of trastuzumab emtansine. The stability of the newly synthesized ADCs in human blood plasma has been carefully evaluated, and it was found that ADCs 2.0 which used the allenamide-thiol linking platform exhibited greater stability than the ADCs that used maleimide-thiol linking technology. Especially, the cytotoxicity and pharmacokinetic distribution of the structural analogues of brentuximab vedotin and trastuzumab emtansine (synthesized through our allenamide-thiol linking technology) in vitro have been carefully assessed using cell lines with CD30 and HER2 antigens, respectively. To our delight, our newly developed ADC 1 and ADC 2 which have HER2 positive trastuzumab as an antibody and MMAE as a payload, showed similar or comparable potency to trastuzumab emtansine in HER2-overexpressing breast cancer cells. The better stability of these new drugs might be able to offer less toxicity and better immunological specificity to non-cancer cells, which might pave a new avenue for the development of a new class of ADCs targeting breast cancers. The development of novel ADCs with better stabilities, selectivities and anti-cancer activities will play a crucial important role in spurring the discovery of a new class of anticancer agents based on similar ADC platform and allenamide-thiol linking technology for a wide array of cancers.

Claims

38 Claims
1. An antibody-drug conjugate having a structure according to formula I:
Y-(A-L-(X-W)m-Z)n I wherein,
Y is an antibody or a reduced antibody;
A is S or NH;
L is a linking moiety having a structure of formula Ila or formula lib:
Figure imgf000039_0001
where the wiggly line represents the point of attachment to (X-W)m-Z and the dashed line represents the point of attachment to A and a represents from 1 to 10;
X is an enzyme targeting moiety;
W is a self-immolative linker;
Z is a drug moiety;
E is a bond or O; m is 0 to 1 ; and n is 1 to 6, or a pharmaceutically acceptable salt or solvate thereof, 39 provided that when:
L is a linking moiety of formula Ila, then m is 1 , A is S and Y is a reduced antibody; and
L is a linking moiety of formula lib, then m is 0, A is NH and Y is an antibody.
2. The antibody-drug conjugate according to Claim 1, wherein L has the structure of formula Ila.
3. The antibody-drug conjugate according to Claim 2, wherein:
(i) a is from 5 to 6, such as 5; and/or
(ii) X is an enzyme targeting moiety that has the structure:
Figure imgf000040_0001
, where the dashed line represents the point of attachment to L and the wiggly line represents the point of attachment to W.
4. The antibody-drug conjugate according to Claim 2 or Claim 3, wherein Y is a reduced trastuzumab or a reduced brentuximab.
5. The antibody-drug conjugate according to any one of Claims 2 to 4, wherein n is 4 and/or E is a bond.
6. The antibody-drug conjugate according to any one of Claims 2 to 5, wherein the drug moiety Z is monomethyl auristatin E.
7. The antibody-drug conjugate according to any one of Claims 2 to 6, wherein W has the formula III:
Figure imgf000041_0001
Ill, where the dashed line represents the point of attachment to X and the wiggly line represents the point of attachment to Z.
8. The antibody-drug conjugate according to any one of Claims 2 to 7, wherein the antibody-drug conjugate is selected from:
Figure imgf000041_0002
Figure imgf000042_0001
or a pharmaceutically acceptable salt or solvate thereof.
9. The antibody-drug conjugate according to Claim 1 , wherein L has the structure of formula lib.
10. The antibody-drug conjugate according to Claim 9, wherein Y is a reduced trastuzumab.
11 . The antibody-drug conjugate according to Claim 9 or Claim 10, wherein n is 4.
12. The antibody-drug conjugate according to any one of Claims 9 to 11, wherein the drug moiety Z is mertansine.
13. The antibody-drug conjugate according to any one of Claims 9 to 12, wherein the antibody-drug conjugate is:
Figure imgf000043_0001
or a pharmaceutically acceptable salt or solvate thereof.
14. Use of an antibody-drug conjugate according to any one of Claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, in medicine.
15. Use of an antibody-drug conjugate according to any one of Claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, in the preparation of a medicament to treat cancer.
16. An antibody-drug conjugate according to any one of Claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, for use in the treatment of cancer.
17. A method of treating cancer, the method comprising the steps of administering a therapeutically effective amount of an antibody-drug conjugate according to any one of 43
Claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.
18. The use according to Claim 15, the antibody-drug conjugate for use according to Claim 16 or the method according to Claim 17, wherein the cancer is selected from one or more of the group consisting of breast cancer, lymphoma (e.g. large cell lymphoma and Hodgkin's lymphoma), melanoma, glioma, lymphoid malignancy and cervical cancer.
19. A pharmaceutical formulation comprising an antibody-drug conjugate according to any one of Claims 1 to 13, or a pharmaceutically acceptable salt or solvate thereof, in combination with one or more of a pharmaceutically acceptable adjuvant, diluent or carrier.
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