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WO2025174248A1 - Trans-cyclooctenes with "or gate" release - Google Patents

Trans-cyclooctenes with "or gate" release

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Publication number
WO2025174248A1
WO2025174248A1 PCT/NL2025/050076 NL2025050076W WO2025174248A1 WO 2025174248 A1 WO2025174248 A1 WO 2025174248A1 NL 2025050076 W NL2025050076 W NL 2025050076W WO 2025174248 A1 WO2025174248 A1 WO 2025174248A1
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WO
WIPO (PCT)
Prior art keywords
compound
group
formula
hetero
moiety
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/NL2025/050076
Other languages
French (fr)
Inventor
Bing Liu
Marc Stefan Robillard
Ronny Mathieu Versteegen
Lieke Willemijn Maria WOUTERS
Raffaella Rossin
Philip Wilson Howard
Lucas Hendricus Maria ZIJLMANS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tagworks Pharmaceuticals BV
Original Assignee
Tagworks Pharmaceuticals BV
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Filing date
Publication date
Application filed by Tagworks Pharmaceuticals BV filed Critical Tagworks Pharmaceuticals BV
Publication of WO2025174248A1 publication Critical patent/WO2025174248A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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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
    • A61K47/6801—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
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • 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
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
    • 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
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin
    • 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
    • A61K47/6853—Carcino-embryonic antigens
    • 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
    • A61K47/6889—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the disclosure relates to compounds comprising an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene, and also if said compound is present in a tumor or in a tumor microenvironment, in particular also if said compound is present in a tumor or in a tumor microenvironment in the absence of a diene; preferably the diene is a tetrazine.
  • the compound comprises an (E)-cyclooctene moiety, wherein at least one non-vinylic carbon of said moiety is substituted with at least one group according to Formula (1) as disclosed herein.
  • compositions comprising a compound according to the first aspect; preferably the composition is a pharmaceutical composition.
  • the disclosure pertains to combinations of (A1) a compound according to the first aspect; or (A2) a composition according to the second aspect; with (B) a diene; preferably the diene is a tetrazine.
  • the disclosure relates to compounds according to the first aspect; the compositions according to the second aspect; or the combinations according to the third aspect; for use as a medicament.
  • the compound of the disclosure comprises an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene and also if said compound is present in a tumor, in particular in a tumor cell, or in a tumor microenvironment.
  • the at least one payload is linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene, and that said payload is also released under the conditions present in a tumor, in particular a tumor cell, or in a tumor microenvironment in the absence of a diene, in particular in the absence of a tetrazine, and that the at least one payload is linked to said (E)-cyclooctene moiety in such a way that at most a relatively low amount, more preferably no substantial amount, of said at least one payload is released from said (E)-cyclooctene moiety if said compound is present in an in vivo environment other than a tumor cell or a tumor microenvironment.
  • T C is a group that is separable from L C due to conditions present in a tumor and/or due to conditions in a tumor microenvironment.
  • T C is a group that is separable from L C by an enzyme expressed by a tumor cell; an enzyme overexpressed in a tumor microenvironment; by thiols; and/or the reducing potential in a tumor or a tumor microenvironment.
  • the enzyme expressed by a tumor cell is an enzyme overexpressed by a tumor cell.
  • Such standard techniques include nuclear magnetic resonance, mass spectrometry, isothermal titration calorimetry, and competitive binding studies using for example fluorogenic or chromogenic substrates and measurements using a spectrophotometer, such as a fluorescence spectrophotometer, an UV-vis spectrophotometer, and/or an infrared spectrophotometer.
  • a spectrophotometer such as a fluorescence spectrophotometer, an UV-vis spectrophotometer, and/or an infrared spectrophotometer.
  • T C is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, disulfide, phosphate, sulphate, heparan sulphate, and combinations thereof. More preferably, T C is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, disuflide, phosphate, and combinations thereof.
  • T C is selected from the group consisting of glucuronidyl, polyglucuronidyl, a peptide, a disulfide, and combinations thereof. Even more preferably, T C is glucuronidyl or a peptide. When T C is a peptide, it is preferred that T C is a dipeptide or a tripeptide. If T C is a tripeptide, it is preferred that T C is alanine- tryptophan-lysine. More preferably, T C is a dipeptide, even more preferably an N-terminally protected dipeptide that is connected to L C via a peptide bond at the C-terminus of said N- terminally protected dipeptide.
  • the peptide is R P -C(O)-P 1 -P 2 -, wherein P 1 and P 2 are independently amino acid residues.
  • P 1 is valine, phenylalanine, or arginine.
  • P 2 is alanine, citrulline, lysine, or arginine; more preferably P 2 is citrulline, lysine, or arginine.
  • R P is selected from the group consisting of -H, -NH 2 , -CF 3 , - CF2H, -CFH 2 , -(S P ) i -C B , (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)cycloalkyl, (hetero)cycloalkenyl, (hetero)cycloalkynyl, (hetero)aryl, and combinations thereof.
  • S P is a spacer as defined herein
  • C B is Construct B as defined herein
  • i is an integer in a range of from 0 to 4, preferably i is 0 or 1.
  • R P is selected from the group consisting of -NH 2 , C 1-4 alkyl, -(S P ) i -C B , and -O-benzyl.
  • C B is a targeting agent, preferably an antibody, most preferably an antibody targeting a tumor.
  • R P is selected from the group consisting of -NH 2 , and C 1-4 alkyl.
  • the C 1-4 alkyl is methyl.
  • T C is glucuronidyl
  • x is 0 or 1, preferably x is 1.
  • Y 8 and Y 10 are independently O or S; Y 9 is independently O, S, a secondary amine, a tertiary amine, or -C(Y 9A ) 2 -; preferably Y 9 is O, S, a secondary amine, or a tertiary amine; Y 9A is hydrogen or methyl, preferably Y 9A is hydrogen; and Y 11 is hydrogen or methyl.
  • Y 11 is hydrogen or methyl
  • Y 12 is N or CH
  • Y 13 and Y 14 are independently O or S.
  • the asterisk indicates a bond to - (T C ) y in Formula (1) or a bond to a further self-immolative unit;
  • the wiggly line indicates a bond to - (S P ) x - in Formula (1) or a bond to a preceding self-immolative unit;
  • the double dashed line indicates a bond to C A or a bond to a further self-immolative unit.
  • the self-immolative linker consists of one or two self-immolative units according to Formula (2A) and/or one or two self-immolative units Formula (2B). Even more preferably, the self-immolative linker consists of one self- immolative unit according to Formula (2A) and/or one self-immolative unit Formula (2B). In some preferred embodiments, the self-immolative linker consists of one self-immolative unit according to Formula (2B), and one self-immolative unit according to Formula (2A). In other preferred embodiments, the self-immolative linker consists of one self-immolative unit according to Formula (2A) or Formula (2B).
  • a “preceding self-immolative unit” is a self-immolative unit that is closer to the E-cyclooctene moiety
  • a “further self-immolative unit” is a self-immolative unit that is further away from the A’-cyclooctene moiety.
  • second amine preferably refers to an -NH- group. It will be understood that as used herein, “tertiary amine” preferably refers to an -N(CH 3 )- group.
  • A is a 5-membered or 6-membered (hetero)aromatic ring, preferably a 6-membered (hetero)aromatic ring, most preferably A is a phenyl ring.
  • Y 3 and Y 5 are independently O, S, a secondary amine, or a tertiary amine; more preferably a secondary amine or a tertiary amine. Most preferably, Y 3 is a tertiary amine. Most preferably, Y 5 is a secondary amine.
  • Y 4 , Y 6 , and Y 7 are independently O or S. Most preferably, Y 4 is O.
  • Y 6 is O.
  • Y 7 is O.
  • e is 0, 1 or 2; preferably 1 or 2; most preferably e is 1.
  • f is 0 or 1; preferably f is 0.
  • A1 is 1 or 2; most preferably A1 is 1.
  • A2 is 0, 1, 2, or 3; preferably A2 is 0, 1, or 2; more preferably A2 is 0, or 1; and most preferably A2 is 0.
  • Cl is preferably a group according to RG1 or RG5, more preferably RG1, as disclosed herein.
  • Cl is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid.
  • each B1 is an optionally substituted carbon; preferably the substituent on the carbon is selected from the group of radicals according to RG1 and RG5; most preferably each B1 is -CH 2 -.
  • R 2A is -Y 5 -, with Y 5 preferably being a secondary or tertiary amine, so that R 2A and the self-immolative linker together form a peptide bond.
  • Formula (2A) is according to any one of Formulae (2A-i), (2A-ii), or (2A- iii): wherein J1 and J2 are independently -CH- or -N-; preferably J1 is -CH-; preferably J2 is - CH-; wherein each of BL 1 , BL 2 , and BL 3 independently is hydrogen, -Y 3 - or -((R 2A )f-*) e ; provided that at least one of BL 1 , BL 2 , and BL 3 is -Y 3 - wherein J1, J2, and J3 are independently -CH- or -N-; preferably J1 is -CH-; preferably J2 is
  • each of BL 1 , and BL 2 independently is hydrogen, -Y 3 - or -((R 2A )f-*) e ; provided that at least one of BL 1 , and BL 2 is -Y 3 - wherein J1, J2, and J3 are independently -CH- or -N-; preferably J1 is -CH-; preferably J2 is -CH-; preferably J3 is -CH-; wherein each of BL 1 , and BL 2 , independently is hydrogen, -Y 3 - or -((R 2A )f-*) e ; provided that at least one of BL 1 , and BL 2 is -Y 3 -
  • g is 0 or 1, preferably g is 1.
  • Y 8 and Y 10 are independently O or S.
  • Y 8 is O.
  • Y 10 is O.
  • Y 9 is independently O, S, a secondary amine, or a tertiary amine; preferably Y 9 is -NH- or -N(CH 3 ).
  • Y 11 is hydrogen or methyl; preferably Y 11 is methyl.
  • the compound of the disclosure has a structure according to Formula (3 A), Formula (3B), or Formula (3C):
  • Y 9 is O, S, a secondary amine, or a tertiary amine; preferably for Formula (3B) and Formula (3C) Y 9 is O.
  • Y 11 is hydrogen or methyl, preferably hydrogen.
  • each moiety R L is independently selected from the group consisting of hydrogen, halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, carboxylic acid, R L1 , and R L2 .
  • at least one moiety R L is R L1 and at least one moiety R L is R L2 .
  • R L is independently selected from the group consisting of R L1 , and R L2 .
  • R L1 is therein, each R LC is independently hydrogen or methyl; preferably R LC is methyl. In R L1 , each R LC is independently hydrogen or methyl; preferably methyl. In R L1 , R NM is 0 or 1; preferably 1.
  • X 2 , X 3 , X 4 , X 5 , and X 6 are optionally substituted carbon atoms;
  • one of X 2 , X 3 , X 4 , X 5 , and X 6 is -C(R 47 ) 2 - and the others are -CH 2 -.
  • R L1 is
  • R LC , X 2 , X 3 , X 5 , and X 6 are disclosed herein; preferably T 1 is -CH 3 or -OH, and preferably R 47 is hydrogen or -(S P ) j -C B , wherein j is 0 or 1, preferably j is 1; S P is a spacer as disclosed herein; C B is a Construct B as disclosed herein, most preferably C B is AVP0458; most preferably R LC is methyl; and most preferably X 2 , X 3 , X 5 , and X 6 are -CH 2 -.
  • R L1 is
  • R LC , X 2 , X 3 , X 5 , and X 6 are disclosed herein; preferably R 47 is hydrogen or -(S P ) j -C B , wherein j is 0 or 1, preferably j is 1; S P is a spacer as disclosed herein; C B is a Construct B as disclosed herein, most preferably C B is AVP0458; most preferably R LC is methyl; and most preferably X 2 , X 3 , X 5 , and X 6 are -CH 2 -.
  • each R L2 is independently selected from the group consisting of
  • R Q is hydrogen or acetyl; preferably R Q is hydrogen.
  • each R L2 is independently selected from the group consisting of Most preferably, R L2 is
  • X 2 , X 3 , X 4 , X 5 , and X 6 are optionally substituted carbon atoms.
  • X 2 , X 3 , X 4 , X 5 , and X 6 are -C(R 47 ) 2 -, wherein R 47 is a group T 1 or a group -(S P ) j -C B , wherein j is 0 or 1, preferably j is 1.
  • X 2 is -CHR 47 -, more preferably -CH 2 -.
  • X 3 is - CHR 47 -, more preferably -CH 2 -.
  • X 5 is -CHR 47 -, more preferably -CH 2 -.
  • X 6 is -CHR 47 -, more preferably -CH 2 -.
  • X 4 is -C(R 47 ) 2 -, wherein one R 47 is -OH, and the other R 47 is preferably hydrogen or -(S P ) j -C B , wherein j is 0 or 1, preferably j is 1.
  • the other R 47 is -(S P ) j - C B .
  • C B is according to Radical Group 4 or Radical Group 5, as defined herein.
  • C B is selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof.
  • C B is a protein. Most preferably, C B is an antibody.
  • antibody preferably includes antibodies, antibody variations, antibody fragments, antibody derivatives, antibody fusions, and antibody analogs. As such, when C B is an antibody, C B is preferably selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, minibodies, Fabs, CH2-deleted antibodies, VHH (aka nanobodies or sdAb/ single domain antibodies), VHH-Fc fusions, VHH multimers, and diabodies.
  • the antibody may be a diabody.
  • a preferred diabody is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1.
  • C B is linked to the remainder of the compound of the disclosure or the conjugate of the disclosure via S or N that is part of C B . More preferably, C B is linked to the remainder of the compound of the disclosure or the conjugate of the disclosure via S that is part of C B .
  • AVP0458 refers to a TAG72-binding diabody derived from the CC49 antibody.
  • AVP0458 is a diabody consisting of two monomers, each monomer having an amino acid sequence according to SEQ ID NO: 1 :
  • SEQ ID NO:1 amino acid sequence of AVP0458 diabody monomer
  • L 1 contains of from 1 to 100 atoms, preferably of from 2 to 75 atoms, more preferably of from 3 to 60 atoms, even more preferably of from 4 to 50 atoms, more preferably still of from 5 to 40 atoms, yet more preferably of from 6 to 35 atoms, even more preferably of from 7 to 30 atoms, more preferably still of from 8 to 25 atoms, even more preferably of from 9 to 22 atoms, and most preferably of from 10 to 20 atoms.
  • L 1 contains about 15 atoms.
  • L 1 is selected from the group consisting of linear or branched C 1 -C 12 (hetero)alkylene, C 3 -C 8 (hetero)cycloalkylene, C 6 -C 12 arylene, and C 4 -C 11 heteroarylene. More preferably than the foregoing, L 1 is selected from the group consisting of linear or branched C 1 -C 12 alkylene, C 3 -C 8 (hetero)cycloalkylene, C 6 -C 12 arylene, and C 4 -C 11 heteroarylene.
  • L 2 is:
  • L 2 has the following structure: .
  • L 2a , L 2b , L 2c , and L 2d are each independently a linker.
  • L 2a , L 2b , L 2c , and L 2d are each independently according to Radical Group 2 as defined herein.
  • L 2a is a linker.
  • L 2a is according to Radical Group 2 as defined herein. More preferably, L 2a is a linker containing at most twenty atoms. More preferably than the foregoing, L 2a is a linker containing at most fifteen atoms. More preferably than the foregoing, L 2a is a linker containing at most ten atoms. More preferably than the foregoing, L 2a is a linker containing at most five atoms.
  • L 2b is a linker.
  • L 2b is according to Radical Group 2 as defined herein. More preferably, L 2b is a linker containing at most twenty atoms. More preferably than the foregoing, L 2b is a linker containing at most fifteen atoms. More preferably than the foregoing, L 2b is a linker containing at most ten atoms. More preferably than the foregoing, L 2b is a linker containing at most five atoms.
  • L 2c is selected from the group consisting of C 1 -C 8 (hetero)alkanetriyl, C 5 -C 6 (hetero)arenetriyl. C 3 -C 7 cycloalkanetriyl, and C 2 -C 7 heterocycloalkanetriyl. More preferably than the foregoing, L 2c is C 1 -C 8 (hetero)alkanetriyl. More preferably than the foregoing, L 2c is C 1 -C 8 alkanetriyl. More preferably than the foregoing, L 2c is C 2 -C 7 alkanetriyl.
  • L 2d is a linker.
  • L 2d is according to Radical Group 2 as defined herein. More preferably than the foregoing, L 2d is a linker containing at most twenty atoms. More preferably than the foregoing, L 2d is a linker containing at most fifteen atoms. More preferably than the foregoing, L 2d is a linker containing at most ten atoms. More preferably than the foregoing, L 2d is a linker containing at most five atoms.
  • T 1 is according to Radical Group 1, Radical Group 3, Radical Group 4, or Radical Group 5, as defined herein.
  • each T 1 is independently according to Radical Group 1 as defined herein.
  • each T 1 is independently selected from the group consisting of - OT 1A , hydrogen, C 1 -C 12 (hetero)alkyl, C 6 aryl, C 4 -C 5 heteroaryl, C 3 -C 6 (hetero)cycloalkyl, C 5 - C 12 alkyl(hetero)aryl, C 5 -C 12 (hetero)arylalkyl, C 4 -C 12 alkylcycloalkyl, -N(T 1A ) 2 , -ST 1A , - SO 3 H, -C(O)T 1A , -C(O)OT 1A , -O-C(O)T 1A -C(O)N(T 1A ) 2 , -N(T 1A ) 2 -CO-T 1A , and -Si(T 1A ) 3 .
  • each T 1 is independently selected from the group consisting of -OT 1A , hydrogen, C 2 -C 6 alkyl, C 6 aryl, C 4 -C 5 heteroaryl, C 3 -C 6 cycloalkyl, C 5 -C 12 alkyl(hetero)aryl, C 5 -Ci2 (hetero)arylalkyl, C 4 -C 12 alkylcycloalkyl, -N(T 1A ) 2 , -ST 1A , -SO 3 H, -C(O)T 1A , - C(O)OT 1A , -O-C(O)T 1A -C(O)N(T 1A ) 2 , -N(T 1A ) 2 -CO-T 1A , and -Si(T 1A ) 3 .
  • each T 1 is independently selected from the group consisting of -OT 1A , C 2 -C 6 alkyl, C 6 aryl, C 4 -C 5 heteroaryl, C 3 -C 6 cycloalkyl, C 5 -C 12 alkyl(hetero)aryl, C 5 -C 12 (hetero)arylalkyl, C 4 -C 12 alkylcycloalkyl, -N(T 1A ) 2 , -ST 1A , -SO 3 H, -C(O)T 1A , -C(O)OT 1A , -O-C(O)T 1A -C(O)N(T 1A ) 2 , - N(T 1A ) 2 -CO-T 1A , and -Si(T 1A ) 3 . More preferably still, T 1 is -OT 1A .
  • T 1 is -OH.
  • each T 1A is independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue. Most preferably, T 1A is hydrogen.
  • T 1 is in an axial position.
  • R 48 is a releasable group
  • T 1 aids in releasing the payload. This results in optimal release yields and/or release kinetics.
  • T 2 is an organic moiety.
  • T 2 is according to any one of Radical Group 1, Radical Group 3, or Radical Group 5, as defined herein, or wherein T 2 is a group -L 3 -C B .
  • T 2 is a bioconjugation moiety, a residue of a bioconjugation moiety, or a group -L 3 -C B .
  • T 2 is a bioconjugation moiety, or a group -L 3 -C B .
  • T 2 is a bioconjugation moiety. These embodiments typically relate to compounds that can be coupled to e.g. a protein. More preferably, T 2 is according to Radical Group If as defined herein. Residues of these bioconjugation moieties are known in the art. More preferably, T 2 is N-maleimidyl. In these embodiments, it is most preferred that T 2 is:
  • T 2 is a residue of a bioconjugation moiety. These embodiments typically relate to conjugates of the disclosure, wherein T 2 links to e.g. a protein. Such residues are well-known to the skilled person. In these embodiments, it is most preferred that T 2 is: wherein the asterisk indicates a bond to the protein, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
  • T 2 is a group -L 3 -C B .
  • C B Construct B
  • C B is as defined herein.
  • L 3 is according to Radical Group 2.
  • L 3 is a residue of a bioconjugation moiety. More preferably, L 3 is a residue of an N-maleimidyl moiety or a residue of an N- hydroxy-succinimidyl moiety.
  • T 2 is selected from the group consisting of In these embodiments, it is most preferred that T 2 is:
  • L 3 and a sulfur atom, secondary nitrogen atom, or tertiary nitrogen atom, preferably a sulfur atom, of C B together form any one of the following structures -L 3 -C B : wherein C B1 indicates S, secondary N, or tertiary N that is part of C B , preferably S; the wiggly lines indicates a bond to moiety L 1 , and the asterisk indicates a bond to the remainder of C B , preferably AVP0458.
  • T 3 is an organic moiety.
  • T 3 is according to any one of Radical Group 1, Radical Group 3, or Radical Group 5, as defined herein. More preferably, T 3 is according to Radical Group 3, as defined herein. Even more preferably, T 3 is a polymer. More preferably still, T 3 is a polymer comprising a polyethylene glycol moiety.
  • T 3 comprises a moiety -(CH 2 CH 2 -O-) y -T 4 .
  • y is an integer in a range of from 1 to 50, preferably y is an integer in a range of from 2 to 45, more preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, even more preferably in a range of from 23 to 25, and most preferably y is 24.
  • This definition and these preferences for y also apply to compounds of Formula (2), Formula (3), Formula (G), Formula (O), Formula (P), and Formula (Q), wherein y is used as well.
  • T 4 is according to Radical Group 1, Radical Group 3, Radical Group 4, or Radical Group 5 as defined herein.
  • T 4 is according to Radical Group 1. More preferably, T 4 is according to Radical Group la. More preferably, T 4 is according to Radical Group lb. More preferably, T 4 is according to Radical Group 1c. More preferably, T 4 is according to Radical Group Id. Even more preferably, T 4 is according to Radical Group le. Most preferably, T 4 is methyl.
  • T 3 is a moiety -(CH 2 CH 2 -O-) y -T 4 . Most preferably, T 3 is a moiety -(CH 2 CH 2 -O-) 24 -CH 3 .
  • R 48 and T 1 are as defined herein.
  • yl is an integer of from 0 to 4, preferably an integer of from 1 to 2, most preferably yl is 1.
  • y2 is an integer of from 0 to 5, preferably an integer of from 1 to 4, more preferably an integer of from 1 to 3, even more preferably an integer of from 1 to 2, and most preferably y2 is 1.
  • y3 is an integer of from 1 to 5, preferably an integer of from 1 to 4, more preferably an integer of from 1 to 3, even more preferably an integer of from 1 to 2, and most preferably y3 is 1.
  • each of X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 is independently selected from the group consisting of a substituted or unsubstituted carbon atom, a nitrogen atom, or an oxygen atom, provided that if one of X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 is a nitrogen atom or an oxygen atom, an adjacent X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 is not a nitrogen atom or an oxygen atom.
  • each of X 1 , X 2 , X 3 , X 4 , X 5 , and X 6 is independently a substituted or unsubstituted carbon atom. More preferably, X 1 and/or X 6 are independently a carbon atom substituted with R 48 . Even more preferably, X 1 is a carbon atom substituted with R 48 , and most preferably, X 1 is -CHR 48 -. More preferably still, X 1 is -CHR 48 -, and X 4 is -CT 1 TL-.
  • X 2 , X 3 , X 5 , and X 6 are unsubstituted carbon atoms, more preferably -CH 2 -.
  • x is an integer in a range of from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6, and most preferably x is 5.
  • All linkers as used herein may each independently be a spacer S P .
  • a spacer S P as used herein is a moiety according to RG2, more preferably any one of the preferred and/or specific embodiments thereof.
  • a spacer S P consists of one or multiple Spacer Units S U arranged linearly and/or branched and may be connected to one or more C B moieties and/or one or more L C or T R moieties.
  • a Spacer unit does not necessarily connect two entities together, it may also be bound to only one component, e.g. the T R or L C .
  • the Spacer may comprise a Spacer Unit linking C B to T R and in addition may comprise another Spacer Unit that is only bound to the Spacer and serves to modulate the properties of the conjugate (Example F below; with reference to Formula 5a and 5b: e ⁇ 1).
  • the Spacer may also consist of two different types of S U constructs, e.g.
  • the Spacer may be bound to the Activator in similar designs such as depicted in above examples A- F.
  • Each individual spacer unit S U may be independently selected from the group of radicals according to RG2.
  • the Spacer Units include but are not limited to amino acids, nucleosides, nucleotides, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units.
  • Preferred biopolymer S U are peptides.
  • each S U comprises at most 50 carbon atoms, more preferably at most 25 carbon atoms, more preferably at most 10 carbon atoms.
  • the S U is independently selected from the group consisting of (CH 2 ) r , (C 3 -C 8 carbocyclo), O-(CH 2 ) r , arylene, (CH 2 ) r -arylene, arylene-(CH 2 ) r , (CH 2 ) r -(C 3 -C 8 carbocyclo), (C 3 -C 8 carbocyclo)-(CH 2 ) r , (C 3 -C 8 heterocyclo), (CH 2 ) r -(C 3 -C 8 heterocyclo), (C 3 -C 8 heterocyclo)-(CH 2 ) r , -(CH 2 ) r C(O)NR’(CH 2 ) r , (CH 2 CH 2 O) r , (CH 2 CH 2 O) r CH 2 ,(CH 2 ) r C(O)NR’(CH 2 CH 2 O) r , (CH 2 ) r C(O) NR’
  • each R’ is independently selected from the group consisting of radicals according to RG1.
  • R’ is hydrogen.
  • Other examples of Spacer Units S U are linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH 3 , -OCH 2 CH 3 , OCH 2 CH 2 CO 2 H.
  • polymeric Spacer Units are polymers and copolymers such as poly-(2-oxazoline), poly(A-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF).
  • polymers and copolymers such as poly-(2-oxazoline), poly(A-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF).
  • Other exemplary polymers are polysaccharides, glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides,
  • Examples of naturally occurring polysaccharides that can be used as S U are cellulose, amylose, dextran, dextrin, levan, fucoidan, carrageenan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin.
  • the polymeric S U comprises a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof.
  • Preferred polymeric S U are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides.
  • polymers used in a S U have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 Dalton to 5 kDa.
  • dendrimers such as polypropylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol-based dendrimers.
  • the S U of the disclosure expressly include but are not limited to conjugates prepared with commercially available cross-linker reagents such as BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB, DTME, BMB, BMDB, BMH, BMOE, BM(PEO) 3 and BM(PEO) 4 .
  • a branching Spacer may use a S U based on one or several natural or non-natural amino acids, amino alcohol, aminoaldehyde, or polyamine residues or combinations thereof that collectively provide the required functionality for branching.
  • serine has three functional groups, i.e. acid, amino and hydroxyl groups and may be viewed as a combined amino acid an aminoalcohol residue for purpose of acting as a branching S U .
  • Other exemplary amino acids are lysine and tyrosine.
  • the Spacer consists of one Spacer Unit, therefore in those cases S P equals S U .
  • the Spacer consists of two, three or four Spacer Units.
  • S P has a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, or from 500 Dalton to 5 kDa.
  • the S P has a mass of no more than 5000 Daltons, no more than 4000 Daltons, no more than 3000 Daltons, no more than 2000 Daltons, no more than 1000 Daltons, no more than 800 Daltons, no more than 500 Daltons, no more than 300 Daltons, no more than 200 Daltons.
  • S P comprises a moiety RG2a, RG2b, RG2c, or a residue of RG1f, as described herein.
  • said RG2a, RG2b, RG2c, or a residue of RG1f connects the S P to C B , L C , or T R .
  • xl7 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2.
  • xl8 is an integer in a range of from 0 to 50, preferably of from 1 to 30, more preferably of from 2 to 20, and most preferably 3.
  • xl9 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2.
  • the preferred example below on the left functions by means of the cascade mechanism, wherein the bond between the allylic carbon of the Trigger and the -O- or -S- attached to said carbon is cleaved, and an electron pair of Y 1 , for example an electron pair of NR 6 , shifts into the benzyl moiety resulting in an electron cascade and the formation of 4-hydroxybenzyl alcohol, CO 2 and the liberated payload.
  • Self-immolative linkers that undergo cyclization include but are not limited to substituted and unsubstituted aminobutyric acid amide, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring system, 2-aminophenylpropionic acid amides, and trimethyl lock-based linkers, see e.g. [Chem. Biol. 1995, 2, 223], [J. Am. Chem. Soc. 1972, 94, 5815], [J. Org. Chem. 1990, 55, 5867], the contents of which are hereby incorporated by reference.
  • L C can be found in W02009017394(A1), US7375078, WO2015038426 A1, W02004043493, Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509, the contents of which are hereby incorporated by reference.
  • the L C has a mass of no more than 1000 Daltons, no more than 500 Daltons, no more than 400 Daltons, no more than 300 Daltons, or from 10, 50 or 100 to 1000 Daltons, from 10, 50, 100 to 400 Daltons, from 10, 50, 100 to 300 Daltons, from 10, 50, 100 to 200 Daltons, e.g., 10-1000 Daltons, such as 50-500 Daltons, such as 100 to 400 Daltons.
  • one L C may be connected to another L C that is bound to C A , wherein upon reaction of the Activator with the Trigger T R , L C -L C -C A is released from the T R , leading to self-immolative release of both L C moi eties and the payload.
  • the L C linking the T R to the other L C then does not release the payload but an L C that is bound via Y C1 and further links to C A .
  • this principle also holds for further linkers L C linked to L C , e.g. L C -L C -L C -L C -C A .
  • the self-immolative linker is not linked to a group T C , the self- immolative linker is according to any one of Group I, Group II, Group III, and Group IV as shown below.
  • the compound of the disclosure is a conjugate of compound A-12 wherein said compound is bound to a targeting agent via the maleimide group of compound A-12; wherein the targeting agent is an antibody or a diabody.
  • the maleimide group may be coupled to the targeting agent via a thiol residue that is part of said targeting agent.
  • the maleimide group is converted to a residue of a maleimide group, viz. wherein the asterisk indicates a bond to the targeting agent, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
  • the disclosure also relates to a combination of (A1) a compound according to the disclosure, or the salt, hydrate, or solvate thereof; (A2) a conjugate according to the disclosure, or the salt, hydrate, or solvate thereof; and/or (A3) a composition according to the disclosure; with (B) a diene or a salt, solvate, or hydrate thereof.
  • a compound according to the disclosure is a dienophile and/or comprises a dienophile moiety, and may be called a “Trigger”.
  • the diene may be referred to as an “Activator”.
  • the diene is a tetrazine. More preferably, the diene is selected from the group consisting of:
  • (TZ1) for example a higher maximum tolerated dose, lower enzyme inhibition, and/or a more straightforward synthesis. Therefore, combinations with at least one of (TZ2), (TZ3), (TZ4), and (TZ5) are preferred over combinations comprising (TZ1), and combinations with (TZ5) are most preferred.
  • the disclosure pertains to a non-therapeutic method and a non-therapeutic use.
  • the dienophile used therein is as described in relation to the combination of the disclosure.
  • the compound of the disclosure (viz. (ia)), the conjugate of the disclosure (viz. (iia)), and/or the composition of the disclosure (viz. (iiia)), and the diene are further contacted with a solvent.
  • suitable solvents for a reaction between a trans-cyclooctene (TCO) and a tetrazine Preferably, the solvent comprises water, and more preferably the solvent is water.
  • the click reaction is preferably a bioorthogonal click reaction.
  • the click reaction is performed in vitro, although non-therapeutic reactions in vivo can be carried out as well.
  • the disclosure also relates to a compound of the disclosure, or the salt, hydrate, or solvate thereof; the conjugate of the disclosure, or the salt, hydrate, or solvate thereof; the composition of the disclosure; or the combination of the disclosure; for use in the treatment of a disease in a subject.
  • the disclosure also pertains to a method of treating a disease in a subject, wherein said method comprises the step of administering to said subject: (a) the compound according to the disclosure, or the salt, hydrate, or solvate thereof; (b) the conjugate according to the disclosure, or the salt, hydrate, or solvate thereof; (c) the composition according to the disclosure; and/or
  • the subject is a human.
  • the disease is cancer.
  • the disclosure also relates to a method for synthesizing a compound of the disclosure, wherein said method comprises coupling a compound of Formula (R) to a compound of Formula (S):
  • R 48 , T 1 , and y are as defined herein; wherein T 2 , and x, are as defined herein, and S 10 is -COOH or an active ester, preferably S 10 is -COOH.
  • S 10 is -COOH or an active ester, preferably S 10 is -COOH.
  • x is an integer of from 4 to 6, and most preferably x is 5.
  • the compound of Formula (S) is contacted with at least one coupling reagent, preferably in the presence of a base, preferably a non-nucleophilic base.
  • a base preferably a non-nucleophilic base.
  • Preferred non- nucleophilic bases are N,N-diisopropylethylamine (DIPEA), l,8-diazabicycloundec-7-ene (DBU), and l,5-diazabicyclo(4.3.0)non-5-ene (DBN).
  • the coupling is carried out at a temperature of from -20°C to 80°C, more preferably of from 0°C to 60°C, even more preferably of from 4°C to 50°C, more preferably still of from 10°C to 40°C, and most preferably of from 15 °C to 30°C.
  • the coupling is carried out in the presence of a solvent, wherein preferably the solvent is an organic solvent.
  • the disclosure also relates to an alternative method for synthesizing a compound of the disclosure, wherein said method comprises coupling a compound of Formula (T) to a compound of Formula (U): wherein T 1 and R 48 are as defined herein; and S 11 is -COOH or an active ester, preferably S 11 is an active ester, more preferably S 11 is selected from the group consisting of - C(O)O-A-succinimidyl, -C(O)O-pentafluorophenyl, -C(O)O-tetrafluorophenyl, -C(O)O-4- nitrophenyl, and -C(O)Cl; even more preferably, S 11 is -C(O)O-A-succinimidyl, or -C(O)O- pentafluorophenyl; and most preferably, S 11 is -C(O)O-pentafluorophenyl.
  • An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl. Preferably, an aryl group is phenyl.
  • a C 3 - C 24 alkyl(hetero)aryl group is meant to include a C 7 -C 24 alkylaryl group and a C 3 -C 24 alkylheteroaryl group
  • a C 3 -C 24 (hetero)arylalkyl is meant to include a C 7 -C 24 arylalkyl group and a C 3 -C 24 heteroarylalkyl group.
  • the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, S, P, and Si.
  • the one or more heteroatoms is selected from the group consisting of O, N, S, and P. It will be understood that for any compound containing a heteroatom, the N, S, and P atoms are optionally oxidized and the N atoms are optionally quatemized.
  • a C 1 -C 4 heteroalkyl contains at most 2 heteroatoms.
  • cycloalkylalkenylene denotes the combination of a cycloalkylene group (see the definition of the suffix -ene below) and an alkenylene group, not the combination of a cycloalkylene and a cycloalkenylene group.
  • (cyclo) when placed before a group, it refers to both the variant of the group without the prefix cyclo- as well as the group with the prefix cyclo-.
  • the suffix -ene denotes divalent groups, i.e. that the group is linked to at least two other moieties.
  • An example of an alkylene is propylene (-CH 2 -CH 2 -CH 2 -), which is linked to another moiety at both termini. It is understood that if a group with the suffix -ene is substituted at one position with -H, then this group is identical to a group without the suffix.
  • an alkylene attached to an -H is identical to an alkyl group. I.e.
  • alkylarylene is understood as a combination of an arylene group and an alkylene group.
  • An example of an alkylarylene group is -phenyl-CH 2 -
  • an example of an arylalkylene group is -CH 2 -phenyl-.
  • glucosamine GIcNH 2
  • galactosamine GalNH 2
  • GlcNAc N- acetylglucosamine
  • GalNAc N-acetylgalactosamine
  • sialic acid Sia which is also referred to as N-acetylneuraminic acid (NeuNAc)
  • N-acetylmuramic acid MurNAc
  • glucuronic acid GlcA
  • IdoA iduronic acid
  • a sugar may be without further substitution, and then it is understood to be a monosaccharide.
  • amino acids in relation to the disclosure comprise both natural and unnatural amino acids.
  • amino acids as used herein are selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, azidolysine, beta-alanine (bAla), 4-aminomethyl phenylalanine (Amf), 4- guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl phenylalanine (laf), 3 -pyridyl alanine (Pya), 4-piperidyl alanine (Ppa), 4-amino
  • An antibody is a protein that is capable of recognizing and binding to a specific antigen.
  • Antibodies can be generated by the immune system of a living organism, but can also be produced using organic synthesis methods or by protein expression in host cells, such as bacteria or yeast. Antibodies can also be designed by using for example computational methods. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this disclosure, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE.
  • Antibody mimetics include, but are not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference.
  • antibody is meant to encompass all of the antibody variations, antibody fragments, antibody derivatives, antibody fusions, antibody analogs and antibody mimetics outlined in this paragraph, unless specified otherwise.
  • peptide is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acid residues in a range of from 2 to 9.
  • an organic molecule is defined as a molecule comprising a C-H bond.
  • Organic compound and organic molecule are used synonymously.
  • an inorganic molecule is defined as any molecule not being an organic molecule, i.e. not comprising a C-H bond. It will be understood that “inorganic molecule” typically also comprises hydrogen, -COOH, etc.
  • particle is preferably defined as a microparticle or a nanoparticle.
  • salt thereof means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like.
  • salt thereof also means a compound formed when an amine is protonated.
  • the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient.
  • the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
  • salt means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter-ions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
  • “Pharmaceutically acceptable salt” refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc. It will be understood that herein, the terms “moiety” and “group” are used interchangeably when referring to a part of a molecule.
  • a “drug” refers to a pharmaceutical agent.
  • drug pharmaceutically active compounds.
  • the pharmaceutically active compound is selected from the group consisting of cytotoxins, antiproliferative/antitumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti-angiogenic factors, and enzyme inhibitors.
  • cytotoxic drug types for use as conjugates to the Trigger and to be released upon IEDDA reaction with the Activator include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercal ators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors.
  • exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HD AC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, and RNA polymerase inhibitors.
  • the drug is an auristatin.
  • Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat No. 4,986,988), dolastatin 14 (U.S. Pat No. 5,138,036), dolastatin 15 (U.S. Pat No.
  • duocarmycins and analogs examples include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin, CPI, CBI.
  • Other examples include those described in, for example, US Patent No. 5,070,092; 5,101,092; 5,187,186; 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,548,530;
  • Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin.
  • Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracy clines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines and the like.
  • Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors.
  • Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib.
  • Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure.
  • Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZDI 152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI 2536, BI6727, GSK461364, ON- 01910; and KSP inhibitors such as, for example, SB 743921, SB 715992, MK-0731, AZD8477, AZ3146 and ARRY-520.
  • CDK inhibitors such as, for example, BMS-387032 and PD0332991
  • Rho-kinase inhibitors such as, for example, AZD7762
  • aurora kinase inhibitors such as, for example, AZDI 152, MLN8054 and MLN8237
  • PLK inhibitors such as, for
  • Exemplary AKT inhibitors include, but are not limited to AT7867.
  • Exemplary MAPK signaling pathway inhibitors include MEK, Ras, INK, B-Raf and p38 MAPK inhibitors.
  • Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,944 and include GDC- 0973, GSK1120212, MSC1936369B, AS703026, RO5126766 and RO4987655, PD0325901, AZD6244, AZD8330 and GDC-0973.
  • Exemplary B-raf inhibitors include CDC-0879, PLX- 4032, and SB590885.
  • Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190.
  • Exemplary receptor tyrosine kinases inhibitors include but are not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), Gefitinib (Iressa), AP24534 (Ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (Dovitinib), Sunitinib (Sutent), Sorafenib (Nexavar), and Vatalinib.
  • Exemplary protein chaperon inhibitors include HSP90 inhibitors.
  • Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.
  • Exemplary HD AC inhibitors include Belinostat (PR 48 101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ- 26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MCI 568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA).
  • Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3 -aminobenzamide, A-966492, and AZD2461.
  • Exemplary Wnt/Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV- 939.
  • Exemplary RNA polymerase inhibitors include amatoxins.
  • amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin.
  • immunomodulators are APRIL, cytokines, including IL-2, IL-7, IL-10, IL12, IL-15, IL-21, TNF, interferon gamma, GMCSF, NDV-GMCSF, and agonists and antagonists of STING, agonists and antagonists of TLRs including TLR1/2, TLR3, TLR4, TLR7/8, TLR9, TLR12, agonists and antagonists of GITR, CD3, CD28, CD40, CD74, CTLA4, 0X40, PD1, PDL1, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A.
  • cytokines including IL-2, IL-7, IL-10, IL12, IL-15, IL-21, TNF, interferon gamma, GMCSF, NDV-GMCSF, and agonists and antagonists of STING, agonists and antagonists of TLRs including
  • exemplary drugs include puromycins, topetecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cemadotin, discodermolide, eleutherobin, mitoxantrone, pyrrolobenzimidazoles (PBI), gamma-interferon, Thialanostatin (A) and analogs, CDK11, immunotoxins, comprising e.g. ricin A, diphtheria toxin, cholera toxin.
  • the drug is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor, (e.g. P13 kinase inhibitor: GDC-0941 and PI- 103), MEK inhibitor, KSP inhibitor, RNA polymerase inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, dolastatin, calicheamicins, SN38, pyrrol Whyzodiazepines, pyridinobenzodiazepines, indolinobenzodiazepines, DNA binding drugs, maytansinoids DM1 and DM4, auristatin MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs.
  • kinase inhibitor e.g. P13 kinase inhibitor: GDC-0941 and PI- 103
  • MEK inhibitor e.g. P13 kinase inhibitor: GDC-0941 and PI- 103
  • the drug is selected from DNA binding drugs and microtubule agents, including pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines, maytansinoids, maytansines, auristatins, tubulysins, duocarmycins, anthracyclines, taxanes.
  • the drug is selected from colchinine, vinca alkaloids, tubulysins, irinotecans, an inhibitory peptide, amanitin and deBouganin.
  • the drug is a radioactive moiety, said moiety comprising a radioactive isotope for radiation therapy.
  • the radioactive moiety When the radioactive moiety is intended to comprise a metal, such as 177 LU, such radiometal is preferably provided in the form of a chelate.
  • the radioactive moiety preferably comprises a structural moiety capable of forming a coordination complex with such a metal.
  • a good example hereof are macrocylic lanthanide(III) chelates derived from l,4,7, 10-tetraazacyclododecane-l,4,7, 10-tetraacetic acid (H 4 dota).
  • the structural moiety capable of forming a coordination complex with such a metal is a chelating moiety as defined herein.
  • the radioactive moiety comprises a prosthetic group (i.e.
  • Drugs optionally include a (portion of a) membrane translocation moiety (e.g. adamantine, poly- lysine/arginine, TAT, human lactoferrin) and/or a targeting agent (against e.g. a tumor cell receptor) optionally linked through a stable or labile linker.
  • adamantine poly- lysine/arginine, TAT, human lactoferrin
  • a targeting agent e.g. a tumor cell receptor
  • Exemplary references include: Trends in Biochemical Sciences, 2015,. 40, 12, 749; J. Am. Chem. Soc. 2015, 137, 12153-12160; Pharmaceutical Research, 2007, 24, 11, 1977.
  • a targeting agent T T may optionally be attached to a drug, optionally via a spacer S P .
  • the targeting agent (or C B ) may comprise one or more additional drugs which are bound to the targeting agent by other types of linkers, e.g. cleavable by proteases, pH, thiols, or by catabolism.
  • Drugs containing a hydroxyl function group for coupling to the Trigger include etoposide, camptothecin, taxol, esperamicin, l,8-dihydroxy-bicyclo[7.3.1]trideca-4-9- diene-2,6-diyne-13-one (U.S. Pat No. 5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof.
  • Drugs containing a sulfhydryl functional group for coupling to the Trigger include esperamicin and 6-mecaptopurine, and derivatives thereof.
  • drugs in relation to the disclosure are monomethyl auristatin E (MMAE), exatecan, and exatecan derivatives.
  • exatecan and exatecan derivatives have the following structure: wherein E 1 is -H, or an optionally substituted C 1 -C 4 alkyl group. It will be understood that when E 1 is -H, said structure is exatecan.
  • E 1 is -H, -CH 3 , or -C(O)- CH 2 -OH. If E 1 is - H or -CH 3 , then the exatecan or exatecan derivative is preferably linked to the remainder of R 48 via the nitrogen atom to which E 1 is attached.
  • RG1 also refers to e.g. an alkyl group substituted with one or more -Cl and/or -OH groups.
  • RG1 also comprises radicals such as -NH-CH 2 -CO0H (a glycine residue), which is a combination of a heteroalkyl and -COOH.
  • RG1f is selected from the group consisting of hydroxyl, amine, halogens, vinyl pyridine, disulfide, pyridyl disulfide, sulfonyloxy, mercaptoacetamide, anhydride, sulfonylated hydroxyacetamido, sulfonyl chlorides, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • RG1f is a group that can be connected to another group by means of an enzyme, for example sortase or Tubulin tyrosine ligase.
  • the radical is selected from the group consisting of (hetero)alkylene, (hetero)alkenylene, (hetero)alkynylene, (hetero)cycloalkylene, (hetero)cycloalkenylene, (hetero)cycloalkynylene, (hetero)arylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
  • RG2 “combinations thereof’ in particular, but not exclusively, refers to alkyl(hetero)arylene, (hetero)aryl alkylene, (hetero)arylalkenylene, (hetero)arylalkynylene, alkenyl(hetero)arylene, and alkynyl(hetero)arylene.
  • the radical is selected from the group consisting of C 1 -C 24 (hetero)alkylene, C 2 -C 24 (hetero)alkenylene, C 2 -C 24 (hetero)alkynylene, C 3 -C 24 cycloalkylene, C 2 -C 24 heterocycloalkylene, C 5 -C 24 cycloalkenylene, C 3 -C 24 heterocycloalkenylene, C 7 -C 24 cycloalkynylene, C 5 -C 24 (hetero)cycloalkynylene, C 6 -C 24 arylene, C 2 -C 24 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
  • the radical is selected from the group consisting of C 1 -C 12 (hetero)alkylene, C 2 -C 12 (hetero)alkenylene, C 2 -C 12 (hetero)alkynylene, C 3 -C 12 cycloalkylene, C 2 -C 12 heterocycloalkylene, C 5 -C 12 cycloalkenylene, C 3 -C 12 heterocycloalkenylene, C 7 -C 12 cycloalkynylene, C 5 -C 12 (hetero)cycloalkynylene, C 6 -C 12 arylene, C 2 -C 12 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
  • the radical is selected from the group consisting of C 1 - C 6 (hetero)alkylene, C 2 -C 6 (hetero)alkenylene, C 2 -C 6 (hetero)alkynylene, C 3 -C 6 cycloalkylene, C 2 -C 6 heterocycloalkylene, C 5 -C 7 cycloalkenylene, C 3 -C 5 heterocycloalkenylene, C 8 cycloalkynylene, C 6 -C 7 (hetero)cycloalkynylene, phenylene, C 3 -C 5 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
  • the radical is selected from the group consisting of C 1 -C 3 (hetero)alkylene, C 3 -C 6 cycloalkylene, C 2 -C 5 heterocycloalkylene, phenylene, C 4 -C 5 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
  • R 4 is according to RG1, preferably R 4 is hydrogen or methyl, more preferably R 4 is hydrogen.
  • the radical is RG2b or RG2c, most preferably RG2b.
  • RG2b is selected from the group consisting of
  • R' is a radical according to RG1, preferably R’ is hydrogen or C 1-3 alkyl.
  • the dashed and wiggly lines denote bonds to the other parts of the molecule.
  • RG2c is selected from the group consisting of
  • R' is a radical according to RG1, preferably R’ is hydrogen or C 1-3 alkyl.
  • R’ is hydrogen or C 1-3 alkyl.
  • the dashed and wiggly lines denote bonds to the other parts of the molecule.
  • Radical Group 3 organic molecule
  • the radical is an organic molecule selected from the group consisting of a nucleic acid, a peptide, a protein, a carbohydrate, an aptamer, a hormone, a toxin, a steroid, a cytokine, a lipid, a small organic molecule as defined herein, a polymer, LNA, PNA, an amino acid, a peptoid, a chelating moiety, a molecule comprising a radionuclide, a fluorescent dye, a phosphorescent dye, a drug, a resin, a bead, an organic particle, a gel, an organic surface, an organometallic compound, a cell, and combinations thereof.
  • the radical is a a nucleic acid, a peptide, a protein, a carbohydrate, a lipid, a polymer, an amino acid, a chelating moiety, a drug, or a gel.
  • a nucleic acid is preferably selected from the group consisting of an oligonucleotide, a polynucleotide, DNA, and RNA.
  • a protein is preferably an antibody or a diabody.
  • a preferred antibody is CC49, and a preferred diabody is AVP0458.
  • a carbohydrate is preferably selected from the group consisting of a monosaccharide, an oligosaccharide, and a polysaccharide.
  • a resin is preferably a polystyrene resin or an agarose resin.
  • an organic particle is preferably a liposome or a polymersome.
  • a chelating moiety is preferably selected from the group consisting of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane- N,N',N",N" -tetraacetic acid), NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid), TETA (1,4,8, l l-tetraazacyclotetradecane-N,N',N",N' -tetraacetic acid), OTTA (N 1 -(p- isothiocyanatobenzyl)-diethylenetriamine-N 1 ,N 2 ,N 3 ,N 3 -tetraacetic acid), deferoxamine or DFA (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-l,4- dioxobutyl]
  • a chelating moiety is selected from the group consisting of
  • the wiggly line denotes a bond to the remaining part of the molecule, optionally bound via -C(O)NH-, wherein the chelator moieties according to said group optionally chelate a metal, wherein the metal is preferably selected from the group consisting of 44 Sc, 62 Cu, 64 Cu, 66 Ga, 67 Ga, 67 Cu, 68 Ga, 86 Y, 89 Zr, 90 Y, 99m Tc, 111 In, 166 Ho, 177 Lu, 186 Re, 188 Re, 211 Bi, 212 Bi, 212 Pb, 213 Bi, 214 Bi, and 225 Ac. Radical Group 4: inorganic molecule
  • the radical is an inorganic molecule selected from the group consisting of an inorganic surface, an inorganic particle, an allotrope of carbon, an inorganic drug, a radionuclide, and combinations thereof.
  • an inorganic surface is preferably selected from the group consisting of chips, wafers, metal such as gold, and silica-based surfaces such as glass.
  • an inorganic particle is preferably selected from the group consisting of beads, silica-based particles, polymer-based materials, and iron oxide particles.
  • a bead is a magnetic bead or a gold bead.
  • an allotrope of carbon is preferably selected from the group consisting of fullerenes such as Buckminsterfullerene; graphite, graphene, diamond, Lonsdaleite, Q- carbon, linearn acetylenic carbon, amorphous carbon, and carbon nanotubes.
  • an inorganic drug is preferably cisplatin.
  • RG5 the radical is: wherein the dashed line indicates a bond to the remaining part of the dienophile or diene.
  • each R 10 is independently selected from RG2, preferably from RG2a.
  • each R 11 is independently selected from RG2, preferably not being RG2a, RG2b, or RG2c.
  • R 12 is selected from RG1 or RG3, preferably RG3, more preferably a protein, polymer, or chelating moiety.
  • z is an integer in a range of from 0 to 12, preferably from 0 to 10, more preferably from 0 to 8, even more preferably from 1 to 6, most preferably from 2 to 4.
  • z is 0.
  • each z is independently selected.
  • h is 0 or 1.
  • each h, z, and n is independently selected.
  • each n belonging to RG5 is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3.
  • n is 1.
  • n is an integer in the range from 12 to 24.
  • z is 0, and n is 1.
  • z is 1, and n is 1.
  • the moiety RG5 has a molecular weight in a range of from 100 Da to 3000 Da, preferably, in a range of from 100 Da to 2000 Da, more preferably, in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, the moiety RG5 has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.
  • RG5 is selected from the group RG5a consisting of:
  • wiggly line denotes a bond to the remainder of the molecule.
  • -((R 10 ) h -R 11 ) n -(R 10 ) h -R 12 may be preceded by a group -(R 10 ) h -R 11 - so as to form a group -(R 10 ) h -R 11 -((R 10 ) h -R 11 ) n -(R 10 ) h -R 12 . It is understood that this follows from the definition of how to write out the repeating units, i.e.
  • the disclosure also relates to the subject-matter of any one of Clauses A1-A16. It will be understood that the embodiments, definitions, Formulae, parameters, values, and the like as disclosed for Clauses A1 -A16 that are equivalent to embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the present disclosure, will have the same preferences as said embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the disclosure, and can be combined therewith as such.
  • S P is a spacer;
  • L C is a self-immolative linker;
  • T C is a group that is separable from L C due to conditions present in a tumor and/or due to conditions in a tumor microenvironment;
  • L C or C A is connected to said allylic carbon via O or S, wherein said O or S is part of L C or C A ; e) if said compound does not comprise another group according to Formula (A1) in addition to said first group, then in said first group y is an integer of from 1 to 4; f) if said compound comprises one or more groups according to Formula (A1) in addition to said first group, then in said one or more groups y is an integer of from 1 to 4.
  • Clause A4 The compound of any one of Clauses A1 to A3, or the salt, solvate, and/or hydrate thereof, wherein T C is selected from the group consisting of glucuronidyl, polyglucuronidyl, a peptide, and combinations thereof; preferably T C is a peptide.
  • Clause A12 A composition comprising a compound according to any one of Clauses A1 to A11, or the salt, solvate, or hydrate thereof. Clause A13. A combination of
  • (A1) a compound according to any one of Clauses A1 to A11, or the salt, solvate, and/or hydrate thereof; or
  • Clause A14 The compound according to any one of Clauses A1 to A11, or the salt, solvate, and/or hydrate thereof; the composition according to Clause A12; or the combination according to Clause A13; for use as a medicament.
  • the disclosure also relates to the subject-matter of any one of Clauses B1 -B16. It will be understood that the embodiments, definitions, Formulae, parameters, values, and the like as disclosed for Clauses B1-B16 that are equivalent to embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the present disclosure, will have the same preferences as said embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the disclosure, and can be combined therewith as such.
  • Clause B1- B16 In case of a discrepancy between embodiments, definitions, Formulae, parameters, values, and the like, in Clauses B1- B16, and the rest of the disclosure, such embodiments, definitions, Formulae, parameters, values, and the like only relate to Clauses B1 -B16 and not to the rest of the present disclosure. Clause B1.
  • Y 1 and Y 2 are independently O or S; ii.
  • Clause B3 The compound of any one of Clauses B 1 to B2, wherein T C is a group that is separable from L C by: a) an enzyme expressed by a tumor cell; b) an enzyme overexpressed in a tumor microenvironment; and/or c) the reducing potential in a tumor or a tumor microenvironment.
  • Clause B4. The compound of any one of Clauses B 1 to B3, wherein T C is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, phosphate, and combinations thereof; preferably T C is a peptide.
  • T C is a dipeptide; preferably the dipeptide is an N-terminally protected dipeptide that is connected to L C via a peptide bond at the C-terminus of said N-terminally protected dipeptide; more preferably the dipeptide is R P -C(O)-P 1 -P 2 -, wherein R P is -NH 2 , C 1-4 alkyl, or -O-benzyl; and P 1 and P 2 are independently amino acid residues; preferably P 1 -P 2 is valine-citrulline or phenylalaninelysine.
  • Clause B8 The compound of any one of Clauses B 1 to B7, wherein the self-immolative linker consists of one or more self-immolative units, wherein the one or more self-immolative units are independently a group according to Formula (B2A), or a group according to Formula (B2B): wherein the asterisk indicates a bond to -(T C ) y in Formula (B1); the wiggly line indicates a bond to
  • A is a 5-membered or 6-membered (hetero)aromatic ring
  • Y 3 and Y 5 are independently O, S, a secondary amine, or a tertiary amine
  • Y 4 , Y 6 , and Y 7 are independently O or S
  • e is 0, 1 or 2
  • f is 0 or 1
  • A1 is 1 or 2
  • A2 is 0, 1, 2, or 3
  • each B1 is an optionally substituted carbon
  • each C1 is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid; provided that if A is a 6-membered (heter
  • Clause B9 The compound of any one of Clauses B 1 to B8, wherein said compound has a structure according to Formula (B3A) or Formula (B3B): wherein each moiety R L is independently selected from the group consisting of hydrogen, halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, carboxylic acid, R L1 , and R L2 , provided that at least one moiety R L is R L1 and at least one moiety R L is R L2 ; wherein Y 9 is O, S, a secondary amine, or a tertiary amine; wherein for both Formulae (B3 A) and (B3B): R L1 is wherein X 2 , X 3 , X 4 , X 5 , and X 6 are optionally substituted carbon atoms;
  • R LC is hydrogen or methyl; and each R L2 is independently selected from the group consisting
  • Clause B10 The compound of any one of Clauses B 1 to B9, wherein said compound has a structure according to any one of Formulae (B4A), (B4B), or (B4C): wherein in each of Formulae (B4A), (B4B), or (B4C): X 2 , X 3 , X 4 , X 5 , and X 6 are optionally substituted carbon atoms; R LC is hydrogen or methyl; and C A is a payload; wherein in each of Formulae (B4B) and (B4C) R P is -NH 2 , C 1-4 alkyl, or -O-benzyl.
  • Clause B 11 A compound comprising an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene and also if said compound is present in a tumor or in a tumor microenvironment; preferably the diene is a tetrazine.
  • Clause B12 A composition comprising a compound according to any one of Clauses B1 to B11; preferably the composition is a pharmaceutical composition.
  • Clause B 14 The compound according to any one of Clauses B1 to B 11 ; the composition according to Clause B 12; or the combination according to Clause B 13; for use as a medicament.
  • Clause B15 The compound according to any one of Clauses B1 to B 11 ; the composition according to Clause B 12; or the combination according to Clause B 13; for use in the treatment of a disease in a subject, preferably the subject is a human, preferably the disease is cancer.
  • HPLC-MS/PDA was performed using a Shimadzu LC-20 AD VP series HPLC coupled to a diode array detector (Shimadzu SPD- M20A) and an Ion-Trap (LCQ Fleet, Thermo Scientific) MS-detector. Size exclusion chromatography (SEC) was performed on a Shimadzu system equipped with a Superdex200 increase 10/300 column (Cytiva) eluted at 0.75 mL/min with PBS. SDS-PAGE was performed on a Mini -PROTEAN Tetra Cell system using 4-20% precast Mini -PROTEAN TGX gels and Precision Plus Protein All Blue protein standards (BioRad Laboratories). The gels were stained with Coomassie Brilliant Blue for protein detection.
  • antibody conjugate 1.13 which is a compound in line with the present claims.
  • Said compound comprises a trans-cyclooctene (TCO) moiety bearing a releasable group on one allylic carbon.
  • TCO trans-cyclooctene
  • MMAE drug monomethyl auristatin E
  • a diene such as a tetrazine (e.g. bis-(2-pyridyl)- tetrazine), but also if the glucuronidyl group of conjugate 1.13 is cleaved off by an enzyme that is expressed in tumors (e.g. glucuronidase).
  • the antibody used in conjugate 1.13 can be used to target conjugate 1.13 to a tumor and/or a tumor microenvironment.
  • Example 1.1 Synthesis of compound 1.1 (tert-butyl methyl(2- (methylamino) ethyl) carbamate) N 1 , N 2 -Dimethylethane- 1,2-diamine (2.58 mL, 23.9 mmol) was dissolved in dried dichloromethane (DCM, 25 mL). A solution of di-tert-butyl dicarbonate (1.54 g, 7.06 mmol) in DCM (5 mL) was added dropwise to the reaction mixture under stirring. The reaction mixture was stirred at room temperature overnight.
  • Triphosgene (0.237 g, 0.8 mmol) was dissolved in DCM (2 mL) and the solution was cooled to 0 °C with an ice bath.
  • Compound 1.1 (0.237 g, 1.2 mmol) was dissolved in DCM (2 mL) and pyridine (0.5 mL) to form a mixture, and said mixture was added dropwise to the triphosgene solution under a nitrogen atmosphere.
  • trastuzumab (Tmab) was functionalized via maleimide chemistry. Tmab was partially reduced with tris(2-carboxyethyl)phosphine (TCEP;
  • CEA-targeting labetuzumab was functionalized via maleimide chemistry by partially reducing with tris(2-carboxyethyl)phosphine (TCEP; 2 eq) in phosphate-buffered saline (PBS) pH 6.8 for 30 minutes at 37°C, followed by incubation with 10 eq. of compound 1.12 for 2 hours at room temperature.
  • the unreacted linker-drug was removed via preparative SEC, yielding a solution of conjugate 1.14 in PBS pH 7.4 with >95% purity as confirmed by analytical SEC and SDS-PAGE.
  • the concentration of the obtained solution was measured by UV at 280 nm (Nanodrop, Thermofisher).
  • a drug-to-antibody (DAR) ratio of 3-3.5 was measured using a tetrazine titration.
  • antibody conjugate 2.14 which is a compound in line with the present claims.
  • Said compound comprises a trans-cyclooctene (TCO) moiety bearing a releasable group on one allylic carbon.
  • TCO trans-cyclooctene
  • the drug doxorubicin can be released upon reaction of conjugate 2.14 with a diene, such as a tetrazine (e.g. bis-(2-pyridyl)-tetrazine), but also if the glucuronidyl group of conjugate 2.14 is cleaved off by an enzyme that is overexpressed in tumors (e.g. glucuronidase).
  • a diene such as a tetrazine (e.g. bis-(2-pyridyl)-tetrazine)
  • glucuronidyl group of conjugate 2.14 is cleaved off by an enzyme that is overexpressed in tumors (e.g. glu
  • the antibody used in conjugate 2.14 can be used to target conjugate 2.14 to a tumor and/or a tumor microenvironment.
  • Example 2. 7 Synthesis of compound 2. 7 ((2S,3R,4S,5S,6S)-2-(4-((((2-((2-aminoethyl)((4- (hydroxymethyl)phenoxy)carbonyl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
  • compound 3.1 Said compound comprises a TCO moiety and MMAE that can be released upon reaction with a diene such as a tetrazine (e.g. bis-(2- pyridyl)-tetrazine).
  • a diene such as a tetrazine (e.g. bis-(2- pyridyl)-tetrazine).
  • Compound 3.2 (Medchem Express) contains a MMAE drug that can be cleaved off by an enzyme that is expressed in tumors (glucuronidase).
  • Their trastuzumab conjugates 3.3 (from 3.1) and 3.4 (from 3.2) were used as controls in examples 15 and 16.
  • Compound 3.1 was prepared in several steps in situ. To a solution of MMAE (286 mg, 0.398 mmol) in 4 mL of anhydrous DMF in a glass vial was added bis-PFP TCO (213 mg, 0.379 mmol; compound 1) and DIEA (126 ⁇ L, 0.72 mmol). The mixture was stirred at room temperature in the dark for 2 days, at which point LC-MS analysis indicated complete consumption of compound 1 and formation of intermediate 3.5. To this reaction mixture was added a solution of maleimide-PEG4-amine (as mono TFA salt, 163 mg, 0.379 mmol) and DIEA (70 ⁇ L, 0.40 mmol) in 4 mL anhydrous DMF.
  • bis-PFP TCO 213 mg, 0.379 mmol; compound 1
  • DIEA 126 ⁇ L, 0.72 mmol
  • model compound 4.8 which comprises a self- immolative linker functionalized with cleavable TCO and glucuronide moiety and phenethylamine as model for a releasable drug.
  • This example details the preparation of compound 6.3, which is an analog of compound 5.8 and comprises a self-immolative linker functionalized with cleavable TCO and glucuronide moiety and doxorubicin as releasable drug.
  • This example details the preparation of compound 7.10, which is an analog of compound 4.8 and comprises a self-immolative linker functionalized with cleavable TCO and a glucuronide moiety, doxorubicin as releasable drug, and a conjugation handle to an antibody.
  • the release of MMAE from the Compound 1.12 in the presence of tetrazine activator was evaluated under physiological conditions (pH 7.3, 37 °C).
  • reaction mixture comprising 20 ⁇ L of compound 1.12 stock solution (10 mM in DMSO) and 20 ⁇ L of ⁇ -glucuronidase enzyme (GUS, 140 units) in 1000 ⁇ L of pre-warmed aqueous buffer (TEA/CH 3 COOH, pH 7.3) was incubated at 37°C. 150 ⁇ L aliquots were withdrawn from the reaction mixture at 0.3, 1, 2, 3, and 4 hours. Each aliquot was immediately subjected to centrifugal filtration using an Ami con® Centrifugal Filter (10 kDa) at 14,000 rpm for 10 minutes to remove the enzyme.
  • GUS ⁇ -glucuronidase enzyme
  • Example 14 In vitro tetrazine-activated payload release from compound 6.2
  • Example 15 In vitro cytotoxicity evaluation in tumor cells
  • the HER2 expressing BT-474 cell line (American Type Culture Collection) was cultured in RPMI-1640 medium supplemented with 2 mM glutamine and 10% heat inactivated fetal calf serum. On the day of the experiment, stock solutions of conjugates 1.13, 3.3 and 3.4, as well as unconjugated Tmab and MMAE, were serially diluted with RPMI-1640 in 48-well cell culture plates. BT-474 cells were then added to the wells at a 20,000 cells/well density, resulting in a 0.4 ml/well final volume, and the plates were incubated at 37°C with 5% CO 2 .
  • t 0 and after 24, 48, and 72 hours incubation, a serially diluted solution of tetrazine 15 (Tz; 100 eq with respect to TCO) was added to some wells. Three wells were used per condition. After 120 hours incubation, an MTT assay was performed in duplicate and the cell proliferation data were analyzed using GraphPad Prism (v. 10.4.1). The IC50 values calculated from non-linear curve fitting are reported in Table 3.
  • Table 3 In vitro cytotoxicity assay in BT-474 cells after 120 h incubation: IC 50 (half-maximal inhibitory concentration) values obtained with conjugates 1.13 and 3.3 alone or with tetrazine 15 (Tz) added at time 0, 24h, 48h or 72 h (Tmab, MMAE, Tz and conjugate 3.4 used as controls)
  • tetrazine addition results in MMAE release only from the portion of conjugate 3.3 that is still located outside the cells and therefore the achieved cell killing efficacy is lower that what can be achieved with conjugate 1.13.
  • glucuronidyl- and TCO-containing linker design of this disclosure gives an advantage in drug release over exclusively chemically-cleavable linkers when used in internalizing antibody-drug conjugates, and also can have a benefit over exclusively biologically-cleavable linkers.
  • Example 16 In vivo in-tumor MMAE release from conjugates 1.13 and 3.3
  • mice 75h post-mAb administration, the animals were euthanized. Plasma, tumor and other selected tissues were harvested and weighed. The samples from mice injected with 111 In-labeled Tmab were measured in a gamma-counter, together with standards, to calculate the % injected dose per gram (%ID/g).
  • mice injected with conjugates 1.13 and 3.3 were homogenized and MMAE was extracted using a published procedure (Rossin et al., Nat Commun 2018). Briefly, the samples were contacted with MeOH (5 mL/gr tumor) with an internal standard (d8-MMAE, MedChem Express) and were homogenized using a MagNA Lyser device (Roche; 4 ⁇ 30 sec cycles, 6500 rpm, with 1 min cooling between cycles). After eliminating the debris, MeOH was evaporated under a stream of N 2 and the residue was reconstituted in 20% ACN in water added with 0.1% formic acid.
  • the solutions were analyzed by quantitative HPLC-SIM-MS on a Shimadzu LC-10 AD VP series HPLC equipped with an electrospray ion-trap mass spectrometer (LCQ Fleet, Thermo Scientific).
  • the absolute amount of MMAE released in tumors was quantified from the ratio between MMAE and internal standard while the % release was estimated from the %ID/g 111 In-labeled Tmab, assuming similar tumor uptakes for unconjugated Tmab and conjugates 1.13 and 3.3.

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Abstract

Disclosed herein are trans-cyclooctenes (TCOs) that release a payload after a reaction with a diene, such as a tetrazine, and/or when subjected to conditions present in a tumor, in particular in a tumor cell, or the tumor microenvironment. The disclosure also pertains to in vivo and in vitro methods of using said trans-cyclooctenes, as well as medical uses thereof, and methods for making said TCOs.

Description

Title: TRANS-CYCLOOCTENES WITH “OR GATE” RELEASE
Technological field
The disclosure disclosed herein relates to compounds comprising an (E)-cyclooctene moiety. The compounds of the disclosure bear a payload that can be released upon reaction of the compound with a diene, but the payload can also be released due to the conditions in a tumor cell and/or a tumor microenvironment.
Background
In the field of bioorthogonal chemistry the ligation between (E)-cyclooctenes, often referred to as trans-cyclooctenes or TCOs, and dienes, in particular tetrazines, is well-studied. In particular, TCOs bearing a payload on the allylic position have attracted attention, since reactions of these TCOs with a diene release the payload. This occurs under both in vitro and in vivo conditions, and the payload may be virtually any molecule, including medicines. Prior to release, the activity of the payload medicine is typically sharply reduced, but the activity is restored upon release of the medicine.
As such, this technology is highly interesting from a medical perspective, especially because the reaction is bioorthogonal, viz. it proceeds rapidly and selectively in environments in which many other reactive species are present, such as in vivo. Moreover, the ring structure of TCOs allows coupling to targeting agents such as antibodies, diabodies, and peptides.
By combining these properties a conjugate can be provided that, when administered to a subject, may accumulate on the desired target site (for example a tumor) by virtue of the targeting agent, and after clearance of off-target conjugates, a diene can be administered that may release the active payload at the target site. This may result in a high local concentration of active payload at the target site, while the reduced activity of the TCO-bound payload may lower side effects at off-target sites.
Although good results have been achieved using payload-bearing TCOs, both in vitro and in vivo, it is desired that more flexibility be obtained as to how and where the payload is released, that an improved payload release be achieved, and/or that the efficacy of treatment be increased. There is thus a need for TCOs that address one or more of the abovementioned problems and/or desires.
Summary
In a first aspect, the disclosure relates to compounds comprising an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene, and also if said compound is present in a tumor or in a tumor microenvironment, in particular also if said compound is present in a tumor or in a tumor microenvironment in the absence of a diene; preferably the diene is a tetrazine. Preferably, the compound comprises an (E)-cyclooctene moiety, wherein at least one non-vinylic carbon of said moiety is substituted with at least one group according to Formula (1) as disclosed herein.
In a second aspect, the disclosure relates to compositions comprising a compound according to the first aspect; preferably the composition is a pharmaceutical composition.
In a third aspect, the disclosure pertains to combinations of (A1) a compound according to the first aspect; or (A2) a composition according to the second aspect; with (B) a diene; preferably the diene is a tetrazine.
In a fourth aspect, the disclosure relates to compounds according to the first aspect; the compositions according to the second aspect; or the combinations according to the third aspect; for use as a medicament.
In a fifth aspect, the disclosure pertains to compounds according to the first aspect; the compositions according to the second aspect; or the combinations according to the third aspect; for use in the treatment of a disease in a subject, preferably the subject is a human, preferably the disease is cancer.
In a sixth aspect, the disclosure relates to a non-therapeutic method for reacting: (ia) a compound according to the first aspect; or (iia) a composition according to the second aspect; with a diene, wherein said method comprises the step of contacting (ia) or (iia) with said diene, preferably said contacting is in vitro., and preferably said diene is a tetrazine.
Detailed Description
The disclosure, in a broad sense, is based on the judicious insight that TCOs of the disclosure meet one or more of the aforementioned desires. In particular, payload release from compounds of the disclosure may not only occur upon reaction of the compound with a diene, but also due to the conditions in a tumor, in particular a tumor cell, and/or a tumor microenvironment. This may allow, inter alia, more flexibility as to how and where the payload is released. Due to the various mechanisms by which the payload can be released, improved payload release and/or improved efficacy of treatment may be achieved.
Meanwhile, the TCOs of the disclosure typically lead to very low, if any, off-target payload release (i.e. payload release in in vivo environments other than a tumor and/or a tumor microenvironment).
As such, in general the compound of the disclosure comprises an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene and also if said compound is present in a tumor, in particular in a tumor cell, or in a tumor microenvironment. Preferably, the at least one payload is linked to said (E)-cyclooctene moiety in such a way that at most a relatively low amount, more preferably no substantial amount, of said at least one payload is released from said (E)- cyclooctene moiety if said compound is present in an in vivo environment other than a tumor, in particular a tumor cell, or a tumor microenvironment.
Consequently, it is preferred that in the compound of the disclosure the at least one payload is linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene, and that said payload is also released under the conditions present in a tumor, in particular a tumor cell, or in a tumor microenvironment in the absence of a diene, in particular in the absence of a tetrazine, and that the at least one payload is linked to said (E)-cyclooctene moiety in such a way that at most a relatively low amount, more preferably no substantial amount, of said at least one payload is released from said (E)-cyclooctene moiety if said compound is present in an in vivo environment other than a tumor cell or a tumor microenvironment.
The skilled person is aware that cleavable bonds as disclosed herein are relatively stable in extracellular in vivo environments other than a tumor or a tumor microenvironment, and that targeting strategies are available to direct a relatively large amount of the compound of the disclosure to a tumor and/or a tumor microenvironment, and particular into a tumor cell, for example via targeting an internalizing receptor that is present on a tumor cell. For example, in the compound of the disclosure one or more of the carbon atoms of the (E)- cyclooctene moiety is preferably connected, directly or via a spacer, to a targeting agent. It is also preferred that TC is connected, directly or via a spacer, to a targeting agent, in particular if TC comprises a peptide that is a substrate for an enzyme that is expressed, or overexpressed, in a tumor cell and/or a tumor microenvironment. Preferably, the targeting agent is an antibody, more preferably an antibody that targets a biomolecule that is specific for and/or is overexpressed in a tumor and/or a tumor microenvironment.
Preferred embodiments of the disclosure are further described below. All of these embodiments, regardless of whether said embodiments are disclosed in the general part of the description or in e.g. the Examples, can be combined as long as said embodiments are not mutually exclusive.
Compounds of the disclosure
Wherever herein reference is made to “compound of the disclosure” or “compounds of the disclosure”, it will be understood that also the salt, hydrate, and/or solvate of said compound(s) are included by such a statement. In general, any compound referred to herein is intended to include the salt, hydrate, and/or solvate of said compound, unless stated otherwise.
Preferably, a compound of the disclosure comprise an (E)-cyclooctene moiety, wherein at least one non-vinylic carbon of said moiety is substituted with at least one group according to Formula (1); wherein SP is a spacer; LC is a self- immolative linker; TC is a group that is separable from LC due to conditions present in a tumor and/or due to conditions in a tumor microenvironment; CA is a payload; x is 0 or 1; y is an integer of from 0 to 4; z is 0 or 1; provided that: a) at least one allylic carbon of said (E)-cyclooctene moiety is substituted with a first group according to Formula (1); i. if in said first group x is 1, then SP is -Y1-C(=Y2)-, and LC or CA is connected to SP via O, S, or N, wherein said O, S, or N is part of LC or CA; Y1 and Y2 are independently O or S; ii. if in said first group x is 0, then LC or CA is connected to said allylic carbon via O or S, wherein said O or S is part of LC or CA; b) if said compound does not comprise another group according to Formula (1) in addition to said first group, then in said first group y is an integer of from 1 to 4; c) if said compound comprises one or more groups according to Formula (1) in addition to said first group, then in said one or more groups y is an integer of from 1 to 4. Preferably, a compound of the disclosure does not comprise another group according to Formula (1) in addition to said first group.
Compounds of the disclosure typically comprise at least one group according to Formula (1). These groups contain a payload, viz. CA, that may be released under several conditions.
If the group of Formula (1) is attached to an allylic carbon of the (E)-cyclooctene moiety, then CA may be released after contacting the compound of the disclosure with a diene, preferably a tetrazine. This may occur regardless of whether spacer SP and/or self-immolative linker LC are present in said group according to Formula (1). Release of payloads from the allylic position of (E)-cyclooctenes may occur when the payload is attached directly to said allylic carbon (viz. in Formula (1) x, z, and y are 0) via an oxygen or sulfur atom. Simultaneously, release of the payload may also occur if a spacer SP and/or self-immolative linker LC are present in said group according to Formula (1). In case of a spacer SP being -Y1- C(=Y2)-, the reaction with a diene ensures that a molecule Y1=C=Y2, e.g. CO2, is released, after which either the payload is released if no self-immolative linker is present, or after which the self-immolative linker self-immolates and the payload is released.
If the group of Formula (1) contains a group TC, then the payload may also be released from said group of Formula (1) if the compound of the disclosure is present in a tumor, a tumor microenvironment, and/or in a tumor cell, also in the absence of a diene. The group TC may be separated from LC due to conditions present in a tumor and/or due to conditions in a tumor microenvironment. Thereafter, the self-immolative linker self-immolates, and payload CA is released. When a group of Formula (1) contains a group TC, this release mechanism may work regardless of the position of said group of Formula (1) on the (E)-cyclooctene moiety. Thus, a compound according to the claims may contain a group of Formula (1) being for example -SP-CA on the allylic position of the (E)-cyclooctene moiety, and a group of Formula (1) being for example -SP-LC(TC)-CA on a non-allylic and non-vinylic position of the (E)-cyclooctene moiety. However, it is preferred that the compounds of the disclosure have a first group according to Formula (1) on the allylic position of the (E)-cyclooctene moiety, wherein in said at least one group z is 1 and y is an integer of from 1 to 4. In this case, the group of Formula (1) on said allylic position allows the release of the payload upon reaction with a diene, preferably a tetrazine, and/or when the compound of the disclosure is present in a tumor, in particular a tumor cell, and/or a tumor microenvironment. This embodiment is preferred due to ease of synthesis, and/or because only one payload molecule is required to prepare the compound of the disclosure. More preferably, the compound of the disclosure does not comprise another group according to Formula (1) in addition to said first group. Again, the main advantage of having only one group according to Formula (1) in the compound of the disclosure is ease of synthesis.
Preferred embodiments of the compounds of the disclosure are further described below in relation to several Formulae and variables.
Formula (1)
In Formula (1), SP is a spacer. If in said first group x is 1, then SP in said first group is -Y1- C(=Y2)-, and LC or CA is connected to SP via O, S, or N, wherein said O, S, or N is part of LC or CA. In this context, the N atom is preferably a secondary or a tertiary amine, more preferably a tertiary amine. Y1 and Y2 are independently O or S. Preferably, Y1 and Y2 are both O. If in said first group x is 0, then LC or CA in said first group is connected to said allylic carbon via O or S, wherein said O or S is part of LC or CA.
In Formula (1), TC is a group that is separable from LC due to conditions present in a tumor and/or due to conditions in a tumor microenvironment. Preferably, TC is a group that is separable from LC by an enzyme expressed by a tumor cell; an enzyme overexpressed in a tumor microenvironment; by thiols; and/or the reducing potential in a tumor or a tumor microenvironment. Preferably, the enzyme expressed by a tumor cell is an enzyme overexpressed by a tumor cell. Examples of enzymes overexpressed in a tumor microenvironment are glycosidase, cathepsin B, alkaline phosphatase, plasmin, and sulfatase (such as sulfatase- 1 and/or sulfatase-2). Preferably, the enzyme overexpressed in a tumor microenvironment is a glycosidase. Preferred glycosidases are β-galactosidase (β-Gal), N- acetyl-β-D-glucosaminidase (β-GlcNAc), α-L-fucosidase (α-Fuc), and β-glucuronidase (β- Glu). These glycosidases are typically overexpressed in tumor microenvironments, especially solid tumors (see Zadlo-Dobrowolska et al.. Org. Biomol. Chem. 2016, 14 (38), 9146-9150). Thus, preferably is TC is a substrate for an enzyme expressed by a tumor cell and/or an enzyme overexpressed in a tumor microenvironment, and/or thiols in tumor cells or tumor microenvironment. More preferably, TC is a substrate for an enzyme selected from the group consisting of glycosidase, cathepsin B, alkaline phosphatase, plasmin, and sulfatase. More preferably, TC is a substrate for an enzyme selected from the group consisting of glycosidase, cathepsin B, alkaline phosphatase, and plasmin. More preferably, TC is a substrate for a glycosidase or cathepsin B. If TC is a substrate for a glycosidase, it is preferred that the glycosidase is selected from the group consisting of β-galactosidase (β-Gal), N-acetyl -β-D- glucosaminidase (β-GlcNAc), α-L-fucosidase (α-Fuc), and β-glucuronidase (β-Glu). Most preferably, TC is a substrate for β-glucuronidase or cathepsin B. The skilled person is well aware of such substrates, which are usually known from literature. Alternatively, the recognition sites and/or cleavage sites of said enzymes are known from literature. Additionally, simple tests are available to evaluate whether a compound is a substrate for any one of glycosidase, cathepsin B, alkaline phosphatase, plasmin, and sulfatase. In particular, a compound can be added to a solution comprising an enzyme, and standard analysis techniques may be used to determine whether the compound is bound to and/or cleaved by the enzyme. Such standard techniques include nuclear magnetic resonance, mass spectrometry, isothermal titration calorimetry, and competitive binding studies using for example fluorogenic or chromogenic substrates and measurements using a spectrophotometer, such as a fluorescence spectrophotometer, an UV-vis spectrophotometer, and/or an infrared spectrophotometer.
Preferably, TC is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, disulfide, phosphate, sulphate, heparan sulphate, and combinations thereof. More preferably, TC is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, disuflide, phosphate, and combinations thereof. More preferably still, TC is selected from the group consisting of glucuronidyl, polyglucuronidyl, a peptide, a disulfide, and combinations thereof. Even more preferably, TC is glucuronidyl or a peptide. When TC is a peptide, it is preferred that TC is a dipeptide or a tripeptide. If TC is a tripeptide, it is preferred that TC is alanine- tryptophan-lysine. More preferably, TC is a dipeptide, even more preferably an N-terminally protected dipeptide that is connected to LC via a peptide bond at the C-terminus of said N- terminally protected dipeptide. Even more preferably the peptide is RP-C(O)-P1-P2-, wherein P1 and P2 are independently amino acid residues. Preferably, P1 is valine, phenylalanine, or arginine. Preferably, P2 is alanine, citrulline, lysine, or arginine; more preferably P2 is citrulline, lysine, or arginine. More preferably, P1-P2 is valine-alanine, valine-citrulline, phenylalanine-lysine, phenylalanine-arginine, or arginine-arginine; even more preferably P1- P2 is valine-citrulline, phenylalanine-lysine, phenylalanine-arginine, or arginine-arginine; most preferably P1-P2 is valine-citrulline or valine-alanine. RP is according to RG1 as disclosed herein. Preferably, RP is selected from the group consisting of -H, -NH2, -CF3, - CF2H, -CFH2, -(SP)i-CB, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)cycloalkyl, (hetero)cycloalkenyl, (hetero)cycloalkynyl, (hetero)aryl, and combinations thereof. Herein, SP is a spacer as defined herein, CB is Construct B as defined herein, and i is an integer in a range of from 0 to 4, preferably i is 0 or 1. More preferably, RP is selected from the group consisting of -NH2, C1-4 alkyl, -(SP)i-CB, and -O-benzyl. For RP it is preferred that CB is a targeting agent, preferably an antibody, most preferably an antibody targeting a tumor. Most preferably, RP is selected from the group consisting of -NH2, and C1-4 alkyl. For RP it is preferred that the C1-4 alkyl is methyl.
In other preferred embodiments, TC is glucuronidyl.
In Formula (1), CA is a payload. Preferably, CA is according to any one of RG1, RG3, RG4, or RG5 as disclosed herein. More preferably, CA is a drug, preferably a drug selected from the group consisting of monomethyl auristatin E, doxorubicin, camptothecin, camptothecin derivatives, exatecan, exatecan derivatives, pyrrolobenzodiazepine and pyrrolobenzodiazepine derivatives; more preferably a drug selected from the group consisting of monomethyl auristatin E, doxorubicin, camptothecin, camptothecin derivatives, exatecan, and exatecan derivatives. Most preferably CA is monomethyl auristatin E.
In Formula (1), x is 0 or 1, preferably x is 1.
In Formula (1), y is an integer of from 0 to 4. If the compound of the disclosure does not comprise another group according to Formula (1) in addition to said first group, then in said first group y is an integer of from 1 to 4. If the compound of the disclosure comprises one or more groups according to Formula (1) in addition to said first group, then in said one or more groups y is an integer of from 1 to 4. It will be understood that in the latter case, in the first group y may be an integer of from 0 to 4, since in that case the compound of the disclosure will contain a further group according to Formula (1) wherein at least one TC is present. Preferably, in Formula (1) y is 1 or 2, most preferably y is 1.
In Formula (1), z is 0 or 1, preferably z is 1. It will be understood that if a group according to Formula (1) comprises a group TC (viz. y is at least 1) then in said group according to Formula (1) z is 1. In Formula (1), LC is a self-immolative linker. More preferably, the self-immolative linker comprises one or more self-immolative units selected from the group consisting of self- immolative units according to Formula (2A), self-immolative units according to Formula (2B), and self-immolative units according to Formula (2C). More preferably, the self- immolative linker consists of one or more self-immolative units according to Formula (2A), Formula (2B), and/or Formula (2C), for example one self-immolative unit according to Formula (2A) and one self-immolative unit according to Formula (2B). The one or more self- immolative units can be independently selected, as self-immolative units of different Formulae can be combined by coupling them to each other.
Self-immolative units according to Formula (2 A) are as follows: wherein A is a 5-membered or 6-membered (hetero)aromatic ring; R2A is -C(=Y4)-Y5- or -Y5-; Y3 and Y5 are independently O, S, a secondary amine, or a tertiary amine; Y4, Y6, and Y7 are independently O or S; e is 0, 1 or 2; f is 0 or 1; A1 is 1 or 2; A2 is 0, 1, 2, or 3; each B1 is an optionally substituted carbon; each Cl is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid; provided that if A is a 6-membered (hetero)aromatic ring, then -Y3- is at an ortho or para position relative to -(B 1)A1-Y6-C(=Y7)-; and if e is at least 1, then each (*-C(=Y4)-Y5)e, is at an ortho or para position relative to -(B 1)A1-Y6-C(=Y7)-; and provided that if A is a 5- membered (hetero)aromatic ring and -(B 1)A1-Y6-C(=Y7)- is at the 1-position of said 5- membered (hetero)aromatic ring, then -Y3- is at the 3- or 4-position, and if e is at least 1, then each (*-C(=Y4)-Y5)e, is at the 3- or 4-position.
Self-immolative units according to Formula (2B) are as follows:
wherein g is 0 or 1; R2B is a bond or
-C(=Y8)-; Y8 and Y10 are independently O or S; Y9 is independently O, S, a secondary amine, a tertiary amine, or -C(Y9A)2-; preferably Y9 is O, S, a secondary amine, or a tertiary amine; Y9A is hydrogen or methyl, preferably Y9A is hydrogen; and Y11 is hydrogen or methyl.
Self-immolative units according to Formula (2C) are as follows: Formula (2C); wherein R2C is a bond or
-C(=Y14)-; Y11 is hydrogen or methyl; Y12 is N or CH; and Y13 and Y14 are independently O or S.
For Formula (2A), Formula (2B), and Formula (2C), the asterisk indicates a bond to - (TC)y in Formula (1) or a bond to a further self-immolative unit; the wiggly line indicates a bond to - (SP)x- in Formula (1) or a bond to a preceding self-immolative unit; and the double dashed line indicates a bond to CA or a bond to a further self-immolative unit.
More preferably, the self-immolative linker consists of from 1 to 3 self-immolative units according to Formula (2A), Formula (2B), and/or Formula (2C). In some preferred embodiments, the self-immolative linker consists of one self-immolative unit according to Formula (2C), and two self-immolative units according to Formula (2A).
In yet other preferred embodiments, the self-immolative linker consists of one or two self-immolative units according to Formula (2A) and/or one or two self-immolative units Formula (2B). Even more preferably, the self-immolative linker consists of one self- immolative unit according to Formula (2A) and/or one self-immolative unit Formula (2B). In some preferred embodiments, the self-immolative linker consists of one self-immolative unit according to Formula (2B), and one self-immolative unit according to Formula (2A). In other preferred embodiments, the self-immolative linker consists of one self-immolative unit according to Formula (2A) or Formula (2B).
It will be understood that in the definitions of the self-immolative units of Formulae (2A), (2B), and (2C), a “preceding self-immolative unit” is a self-immolative unit that is closer to the E-cyclooctene moiety, and a “further self-immolative unit” is a self-immolative unit that is further away from the A’-cyclooctene moiety.
It will be understood that as used herein, “secondary amine” preferably refers to an -NH- group. It will be understood that as used herein, “tertiary amine” preferably refers to an -N(CH3)- group.
In Formula (2A), A is a 5-membered or 6-membered (hetero)aromatic ring, preferably a 6-membered (hetero)aromatic ring, most preferably A is a phenyl ring. In Formula (2A), Y3 and Y5 are independently O, S, a secondary amine, or a tertiary amine; more preferably a secondary amine or a tertiary amine. Most preferably, Y3 is a tertiary amine. Most preferably, Y5 is a secondary amine. In Formula (2A), Y4, Y6, and Y7 are independently O or S. Most preferably, Y4 is O. Most preferably, Y6 is O. Most preferably, Y7 is O. In Formula (2A), e is 0, 1 or 2; preferably 1 or 2; most preferably e is 1. In Formula (2A), f is 0 or 1; preferably f is 0. In Formula (2A), A1 is 1 or 2; most preferably A1 is 1. In Formula (2A), A2 is 0, 1, 2, or 3; preferably A2 is 0, 1, or 2; more preferably A2 is 0, or 1; and most preferably A2 is 0. In Formula (2A), Cl is preferably a group according to RG1 or RG5, more preferably RG1, as disclosed herein. More preferably, Cl is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid. In Formula (2A), each B1 is an optionally substituted carbon; preferably the substituent on the carbon is selected from the group of radicals according to RG1 and RG5; most preferably each B1 is -CH2-.
In Formula (2A), R2A is -C(=Y4)-Y5- or -Y5-. It will be understood that it typically depends on the nature of YC whether R2A is present, and if so, whether R2A is -C(=Y4)-Y5- or - Y5-. For example, if YC is connected to the self-immolative linker via -O- or a peptide bond, then R2A can be absent (viz. f in Formula (2A) is 0). On the other hand, if YC is for example a peptide radical ending with -C(=O)-, then it is preferred that R2A is -Y5-, with Y5 preferably being a secondary or tertiary amine, so that R2A and the self-immolative linker together form a peptide bond. Likewise, in other circumstances it is preferred that R2A is -C(=Y4)-Y5-. Preferably, Formula (2A) is according to any one of Formulae (2A-i), (2A-ii), or (2A- iii): wherein J1 and J2 are independently -CH- or -N-; preferably J1 is -CH-; preferably J2 is - CH-; wherein each of BL1, BL2, and BL3 independently is hydrogen, -Y3- or -((R2A)f-*)e; provided that at least one of BL1, BL2, and BL3 is -Y3- wherein J1, J2, and J3 are independently -CH- or -N-; preferably J1 is -CH-; preferably J2 is
-CH-; preferably J3 is -CH-; wherein each of BL1, and BL2, independently is hydrogen, -Y3- or -((R2A)f-*)e; provided that at least one of BL1, and BL2 is -Y3- wherein J1, J2, and J3 are independently -CH- or -N-; preferably J1 is -CH-; preferably J2 is -CH-; preferably J3 is -CH-; wherein each of BL1, and BL2, independently is hydrogen, -Y3- or -((R2A)f-*)e; provided that at least one of BL1, and BL2 is -Y3-
In Formulae (2A-i), (2A-ii), and (2A-iii) it is preferred that at most one of BL1, BL2, and BL3, insofar as present, is -Y3-
Preferably, Formula (2A) is according to Formula (2A-ii), wherein preferably BL1 is -Y3- and preferably BL2 is -((R2A)f-*)e.
In Formula (2B), g is 0 or 1, preferably g is 1. In Formula (2B), R2B is a bond or -C(=Y8)-. In Formula (2B), Y8 and Y10 are independently O or S. Preferably, Y8 is O. Preferably, Y10 is O. In Formula (2B), Y9 is independently O, S, a secondary amine, or a tertiary amine; preferably Y9 is -NH- or -N(CH3). In Formula (2B) Y11 is hydrogen or methyl; preferably Y11 is methyl.
In Formula (2C) R2C is a bond or -C(=Y14)-. In Formula (2C) Y11 is hydrogen or methyl; preferably Y11 is hydrogen. In Formula (2C) Y12 is N or CH; preferably Y12 is N. In formula (2C), Y13 and Y14 are independently O or S. Preferably, Y13 is O. Preferably, Y14 is O.
Formulae (3 A), (3B), (3C\ (4A), (4B), (4C\ (4D), and (4E)
Preferably, the compound of the disclosure has a structure according to Formula (3 A), Formula (3B), or Formula (3C):
For Formula (3B) and Formula (3C) Y9 is O, S, a secondary amine, or a tertiary amine; preferably for Formula (3B) and Formula (3C) Y9 is O. For Formula (3C) Y11 is hydrogen or methyl, preferably hydrogen.
In Formula (3 A), each moiety RL is independently selected from the group consisting of hydrogen, halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, carboxylic acid, RL1, and RL2. Preferably, in Formula (3 A) at least one moiety RL is RL1 and at least one moiety RL is RL2. Preferably, RL is independently selected from the group consisting of RL1, and RL2.
In Formulae (3 A), (3B), and (3C) RL1 is Therein, each RLC is independently hydrogen or methyl; preferably RLC is methyl. In RL1, each RLC is independently hydrogen or methyl; preferably methyl. In RL1, RNM is 0 or 1; preferably 1.
In RL1, X2, X3, X4, X5, and X6 are optionally substituted carbon atoms; Preferably, one of X2, X3, X4, X5, and X6 is -C(R47)2- and the others are -CH2-. More preferably, X4 is - C(R47)2- and X2, X3, X5, and X6 are -CH2-; wherein preferably one R47 is -OH or hydrogen, and preferably the other R47 is -(SP)j-CB, wherein j is 0 or 1, preferably j is 1; SP is a spacer as disclosed herein; CB is a Construct B as disclosed herein, most preferably CB is AVP0458.
Preferably, RL1 is
RLC, X2, X3, X5, and X6 are disclosed herein; preferably T1 is -CH3 or -OH, and preferably R47 is hydrogen or -(SP)j-CB, wherein j is 0 or 1, preferably j is 1; SP is a spacer as disclosed herein; CB is a Construct B as disclosed herein, most preferably CB is AVP0458; most preferably RLC is methyl; and most preferably X2, X3, X5, and X6 are -CH2-.
More preferably, RL1 is
RLC, X2, X3, X5, and X6 are disclosed herein; preferably R47 is hydrogen or -(SP)j-CB, wherein j is 0 or 1, preferably j is 1; SP is a spacer as disclosed herein; CB is a Construct B as disclosed herein, most preferably CB is AVP0458; most preferably RLC is methyl; and most preferably X2, X3, X5, and X6 are -CH2-. Preferably, in Formulae (3 A), (3B), and (3C) each RL2 is independently selected from the group consisting of
In Formulae (3 A), (3B), and (3C), RP is -NH2 or C1-4 alkyl; preferably RP is -NH2 or methyl.
In Formulae (3 A), (3B), and (3C), RQ is hydrogen or acetyl; preferably RQ is hydrogen.
More preferably, each RL2 is independently selected from the group consisting of Most preferably, RL2 is
Preferably, the compound of the disclosure has a structure according to any one of Formulae
(4 A), (4B), (4C), (4D), and (4E): wherein in each of Formulae (4 A), (4B), (4C), (4D), and (4E): X2, X3, X4, X5, and X6 are optionally substituted carbon atoms; RLC is hydrogen or methyl; and CA is a payload; and wherein in each of Formulae (4B) and (4C) RP is -NH2, C1-4 alkyl, or -O-benzyl; wherein for Formula (4D) Y11 is hydrogen or methyl. Preferably, for Formulae (4A), (4B), (4C), and (4E), RLC is methyl. Preferably, for Formula (4D), RLC is hydrogen. In each of Formulae (4B) and (4C) RP is -NH2 or C1-4 alkyl, preferably RP is -NH2 or methyl.
Preferably, the compound of the disclosure has a structure according to Formula (4D) or (4E).
As used herein (for example in each of Formulae (3 A), (3B), (4A), (4B), (4C), (4D), and (4E), X2, X3, X4, X5, and X6 are optionally substituted carbon atoms. Preferably, X2, X3, X4, X5, and X6 are -C(R47)2-, wherein R47 is a group T1 or a group -(SP)j-CB, wherein j is 0 or 1, preferably j is 1. Preferably, X2 is -CHR47-, more preferably -CH2-. Preferably, X3 is - CHR47-, more preferably -CH2-. Preferably, X5 is -CHR47-, more preferably -CH2-. Preferably, X6 is -CHR47-, more preferably -CH2-.
Preferably, X4 is -C(R47)2-, wherein one R47 is -OH, and the other R47 is preferably hydrogen or -(SP)j-CB, wherein j is 0 or 1, preferably j is 1. Preferably, the other R47 is -(SP)j- CB.
In some preferred embodiments, R47 is -C(=O)NH-(CH2)x17-(CH2-CH2-O)x18-NH- C(=O)-(CH2)x19-T47. Therein xl7 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2. Therein, xl8 is an integer in a range of from 0 to 50, preferably of from 1 to 30, more preferably of from 2 to 20, and most preferably 3. Therein, xl9 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2. Therein, T47 is a residue of a bioconjugation moiety, as disclosed herein. Preferably, T47 is: wherein the asterisk indicates a bond to e.g. CB, and the wiggly line denotes a bond to the rest of the compound of the disclosure. More preferably, X4 is -C(R47)2-, wherein one R47 is -OH, and the other R47 is R47 is -C(=O)NH-(CH2)x17-(CH2- CH2-O)x18-NH-C(=O)-(CH2)x19-T47.
In other preferred embodiments, R47 is -C(=O)NH-(CH2)x17-(CH2-CH2-O)x18-NH- C(=O)-(CH2)x19-T48. Therein x17 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2. Therein, xl8 is an integer in a range of from 0 to 50, preferably of from 1 to 30, more preferably of from 2 to 20, and most preferably 3. Therein, xl9 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2. Therein, T48 is a bioconjugation moiety, as disclosed herein; more preferably T48 is a maleimide. More preferably, X4 is - C(R47)2-, wherein one R47 is -OH, and the other R47 is -C(=O)NH-(CH2)x17-(CH2-CH2-O)x18- NH-C(=O)-(CH2)x19-T48.
CB is according to Radical Group 4 or Radical Group 5, as defined herein. Preferably, CB is selected from the group consisting of proteins, nucleic acids, peptides, carbohydrates, aptamers, lipids, small organic molecules, polymers, LNA, PNA, amino acids, peptoids, chelating moieties, fluorescent dyes, phosphorescent dyes, organic particles, gels, cells, and combinations thereof.
More preferably, CB is a moiety, preferably a protein or a peptide, capable of binding a cancer epitope. The skilled person is aware of cancer epitopes and which moieties are capable of binding said epitopes. For example, cancer epitopes can be found in public databases, such as the Cancer Epitpope Database and Analysis Resourse (cedar.iedb.org). Simple binding tests are available to test whether a moiety binds to said epitope. Preferably, the cancer epitope is selected from the group consisting of New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1), Melanoma-Associated Antigen A3 (MAGE-A3), Melanoma- Associated Antigen A4 (MAGE-A4), Wilms Tumor 1 (WT1), Preferentially Expressed Antigen in Melanoma (PRAME), Human Epidermal Growth Factor Receptor 2 (HER2), Epidermal Growth Factor Receptor Variant III (EGFRvIII), Glycoprotein 100 (gplOO), Melanoma Antigen Recognized by T Cells 1 (MART-1), Tyrosinase-Related Protein 1 (TRP- 1), Tyrosinase-Related Protein 2 (TRP-2), Carcinoembryonic Antigen (CEA), Mucin 1 (MUC1), Mesothelin (Mesothelin), Survivin (Survivin), Prostate-Specific Membrane Antigen (PSMA), Prostatic Acid Phosphatase (PAP), Receptor Tyrosine Kinase-Like Orphan Receptor 1 (R0R1), Disialoganglioside 2 (GD2), Disialoganglioside 3 (GD3), Fibroblast Activation Protein (FAP), Cluster of Differentiation 19 (CD 19), Cluster of Differentiation 20 (CD20), Cluster of Differentiation 22 (CD22), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 38 (CD38), B-Cell Maturation Antigen (BCMA), Latent Membrane Protein 1 (LMP1), Latent Membrane Protein 2 (LMP2), Human Papillomavirus Oncoprotein E6 (HPV E6), Human Papillomavirus Oncoprotein E7 (HPV E7), Human Telomerase Reverse Transcriptase (hTERT), Anaplastic Lymphoma Kinase (ALK), Kirsten Rat Sarcoma Viral Oncogene Homolog G12D (KRAS G12D), Isocitrate Dehydrogenase 1 R132H (IDH1 R132H), B-Raf Proto-Oncogene V600E (BRAF V600E), and p53 mutant peptides.
More preferably, CB is a protein. Most preferably, CB is an antibody. It will be understood that as used herein, the term “antibody” preferably includes antibodies, antibody variations, antibody fragments, antibody derivatives, antibody fusions, and antibody analogs. As such, when CB is an antibody, CB is preferably selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, minibodies, Fabs, CH2-deleted antibodies, VHH (aka nanobodies or sdAb/ single domain antibodies), VHH-Fc fusions, VHH multimers, and diabodies.
The antibody may be a diabody. A preferred diabody is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1.
Preferably, CB is linked to the remainder of the compound of the disclosure or the conjugate of the disclosure via S or N that is part of CB. More preferably, CB is linked to the remainder of the compound of the disclosure or the conjugate of the disclosure via S that is part of CB.
As used herein, AVP0458 refers to a TAG72-binding diabody derived from the CC49 antibody. AVP0458 is a diabody consisting of two monomers, each monomer having an amino acid sequence according to SEQ ID NO: 1 :
SEQ ID NO:1 (amino acid sequence of AVP0458 diabody monomer):
SVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDD FKYNERFKGKATLTADKSSSTAYLQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTV SSGGGGSDIVMTQSCSSCPVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSP KLLIYWASTRESGVPDRFTGSGSGTDFTLSISSVETEDLAVYYCQQYYSYPLTFGAGT KLVLKR
Herein, the underlining indicates the cysteines that are preferably modified with or linked to a compound of the disclosure or the remainder thereof if AVP0458 is itself part of the compound of the disclosure.
Thus, in SEQ ID NO: 1 it is preferred that at least one of the underlined cysteines, more preferably both underlined cysteines, is modified with or linked to a compound according to the disclosure. In other words: it is preferred that the sulfur atom of the underlined cysteines is coupled to a moiety T2 as defined herein, preferably T2 is the residue of an N-maleimidyl group.
Formulae (A)-(Q)
Preferably, the compound of the disclosure is according to a Formula selected from the group consisting of Formula (A1), Formula (A2), Formula (A3), Formula (B), Formula (C), Formula (D), Formula (E), Formula (F), Formula (G), Formula (H), Formula (I), Formula (J), Formula (K), Formula (L), Formula (M), Formula (N), Formula (O), Formula (P), and Formula (Q).
As used herein, R48 is a group according to Formula (1).
Preferably, the compound of the disclosure is according to Formula (B). More preferably, the compound of the disclosure is according to Formula (C). Even more preferably, the compound of the disclosure is according to Formula (A1). More preferably still, the compound of the disclosure is according to Formula (D). Even more preferably, the compound of the disclosure is according to Formula (E). Yet more preferably, the compound of the disclosure is according to Formula (F). Still more preferably, the compound of the disclosure is according to Formula (G). More preferably still, the compound of the disclosure is according to Formula (A2). Even more preferably still, the compound of the disclosure is according to Formula (H). Yet more preferably still, the compound of the disclosure is according to Formula (I). Even more preferably, the compound of the disclosure is according to Formula (J). More preferably still, the compound of the disclosure is according to Formula (K). Even more preferably still, the compound of the disclosure is according to Formula (L). Yet more preferably, the compound of the disclosure is according to Formula (M). Even more preferably still, the compound of the disclosure is according to Formula (N). Still more preferably, the compound of the disclosure is according to Formula (O). Yet more preferably, the compound of the disclosure is according to Formula (A3). Still more preferably, the compound of the disclosure is according to Formula (P). Even more preferably, the compound of the disclosure is according to Formula (Q).
Ll
L1 is a linker. Preferably, L1 is according to Radical Group 2 as defined herein.
Preferably, L1 contains of from 1 to 100 atoms, preferably of from 2 to 75 atoms, more preferably of from 3 to 60 atoms, even more preferably of from 4 to 50 atoms, more preferably still of from 5 to 40 atoms, yet more preferably of from 6 to 35 atoms, even more preferably of from 7 to 30 atoms, more preferably still of from 8 to 25 atoms, even more preferably of from 9 to 22 atoms, and most preferably of from 10 to 20 atoms. Preferably, L1 contains about 15 atoms.
More preferably, L1 is selected from the group consisting of linear or branched C1-C12 (hetero)alkylene, C3-C8 (hetero)cycloalkylene, C6-C12 arylene, and C4-C11 heteroarylene. More preferably than the foregoing, L1 is selected from the group consisting of linear or branched C1-C12 alkylene, C3-C8 (hetero)cycloalkylene, C6-C12 arylene, and C4-C11 heteroarylene. More preferably than the foregoing, L1 is selected from the group consisting of linear or branched C2-C12 alkylene, C3-C8 (hetero)cycloalkylene, C6-C12 arylene, and C4-C11 heteroarylene. More preferably than the foregoing, L1 is selected from the group consisting of linear or branched C3-C12 alkylene, C3-C8 (hetero)cycloalkylene, C6-C12 arylene, and C4-C11 heteroarylene. More preferably than the foregoing, L1 is selected from the group consisting of linear or branched C4-C12 alkylene, C3-C8 (hetero)cycloalkylene, C6-C12 arylene, and C4-C11 heteroarylene. More preferably than the foregoing, L1 is a linear or branched C1-C12 alkylene. More preferably than the foregoing, L1 is a linear or branched C2-C12 alkylene. More preferably than the foregoing, L1 is a linear or branched C3-C12 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C12 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C11 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C10 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C9 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C8 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C7 alkylene. More preferably than the foregoing, L1 is a linear or branched C4-C6 alkylene. More preferably than the foregoing, L1 is a linear or branched C5 alkylene. More preferably than the foregoing, L1 is a linear C1-C12 alkylene. More preferably than the foregoing, L1 is a linear C2-C12 alkylene. More preferably than the foregoing, L1 is a linear C3-C12 alkylene. More preferably than the foregoing, L1 is a linear C4-C12 alkylene. More preferably than the foregoing, L1 is a linear C4-C11 alkylene. More preferably than the foregoing, L1 is a linear C4-C10 alkylene. More preferably than the foregoing, L1 is a linear C4-C9 alkylene. More preferably than the foregoing, L1 is a linear C4-C8 alkylene. More preferably than the foregoing, L1 is a linear C4- C7 alkylene. More preferably than the foregoing, L1 is a linear C4-C6 alkylene. Most preferably, L1 is a linear C5 alkylene.
L1 can be substituted or unsubstituted. Preferably, L1 is unsubstituted. Most preferably, L1 is an unsubstituted, linear C5 alkylene.
L2
L2 is a linker. Preferably, L2 is according to Radical Group 2 as defined herein.
Preferably, L2 contains of from 1 to 200 atoms, preferably of from 2 to 150 atoms, more preferably of from 3 to 100 atoms, even more preferably of from 4 to 90 atoms, more preferably still of from 5 to 80 atoms, yet more preferably of from 6 to 70 atoms, even more preferably of from 7 to 60 atoms, more preferably still of from 8 to 50 atoms, even more preferably of from 9 to 45 atoms, and most preferably of from 10 to 35 atoms.
Preferably, L2 is selected from the group consisting of linear or branched C1-C12 (hetero)alkanetriyl, C3-C8 (hetero)cycloalkanetriyl, C6-C12 arenetriyl, and C4-C11 heteroarenetriyl. More preferably than the foregoing, L2 is a linear or branched C1-C12 (hetero)alkanetriyl. More preferably than the foregoing, L2 is a linear or branched C1-C12 heteroalkanetriyl. More preferably than the foregoing, L2 is a branched C1-C12 (hetero)alkanetriyl. More preferably than the foregoing, L2 is a branched C1-C12 heteroalkanetriyl. More preferably than the foregoing, L2 is a branched C3-C11 heteroalkanetriyl. More preferably than the foregoing, L2 is a branched C6-C10 heteroalkanetriyl. More preferably than the foregoing, L2 is a branched C8 heteroalkanetriyl. More preferably than the foregoing, L2 is a branched C8 heteroalkanetriyl substituted with up to five =O groups. More preferably than the foregoing, L2 is a branched C8 heteroalkanetriyl substituted with three =O groups. More preferably than the foregoing, L2 is a branched C8 heteroalkanetriyl containing up to five -NH- groups. More preferably than the foregoing, L2 is a branched C8 heteroalkanetriyl containing three -NH- groups. More preferably than the foregoing, L2 is a branched C8 heteroalkanetriyl containing three -NH- groups, and wherein the C8 heteroalkanetriyl is substituted with three =O groups.
More preferably, L2 is:
Even more preferably, L2 is: O
More preferably still, L2 is:
Most preferably, L2 is:
In preferred embodiments, L2 has the following structure: . Herein, L2a, L2b, L2c, and L2d are each independently a linker. Preferably, L2a, L2b, L2c, and L2d are each independently according to Radical Group 2 as defined herein. L2a
L2a is a linker. Preferably, L2a is according to Radical Group 2 as defined herein. More preferably, L2a is a linker containing at most twenty atoms. More preferably than the foregoing, L2a is a linker containing at most fifteen atoms. More preferably than the foregoing, L2a is a linker containing at most ten atoms. More preferably than the foregoing, L2a is a linker containing at most five atoms. More preferably than the foregoing, L2a is selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2T is hydrogen or methyl. More preferably than the foregoing, L2a is selected from the group consisting of -C(O)NL2T-, and -NL2TC(O)-. More preferably than the foregoing, L2a is selected from the group consisting of -C(O)NH-, and -NHC(O)-. Most preferably, L2a is -NHC(O)-. L2b
L2b is a linker. Preferably, L2b is according to Radical Group 2 as defined herein. More preferably, L2b is a linker containing at most twenty atoms. More preferably than the foregoing, L2b is a linker containing at most fifteen atoms. More preferably than the foregoing, L2b is a linker containing at most ten atoms. More preferably than the foregoing, L2b is a linker containing at most five atoms. More preferably than the foregoing, L2b is selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2T is hydrogen or methyl. More preferably than the foregoing, L2b is selected from the group consisting of -C(O)NL2T-, and -NL2TC(O)-. More preferably than the foregoing, L2b is selected from the group consisting of -C(O)NH-, and -NHC(O)-. Most preferably, L2b is -NHC(O)-. L2C
L2C is a linker. Preferably, L2c is according to Radical Group 2 as defined herein. More preferably than the foregoing, L2c is a linker comprising at most 50 atoms. More preferably than the foregoing, L2c is a linker comprising at most 40 atoms. More preferably than the foregoing, L2c is a linker comprising at most 30 atoms. More preferably than the foregoing, L2C is a linker comprising at most 20 atoms. More preferably than the foregoing, L2c is a linker comprising at most 15 atoms. More preferably than the foregoing, L2c is selected from the group consisting of C1-C8 (hetero)alkanetriyl, C5-C6 (hetero)arenetriyl. C3-C7 cycloalkanetriyl, and C2-C7 heterocycloalkanetriyl. More preferably than the foregoing, L2c is C1-C8 (hetero)alkanetriyl. More preferably than the foregoing, L2c is C1-C8 alkanetriyl. More preferably than the foregoing, L2c is C2-C7 alkanetriyl. More preferably than the foregoing, L2C is C3-C6 alkanetriyl. More preferably than the foregoing, L2c is C4-C5 alkanetriyl. More preferably than the foregoing, L2c is C5 alkanetriyl. Most preferably, L2c is >CH-CH2-CH2- CH2-CH2-.
L2d
L2d is a linker. Preferably, L2d is according to Radical Group 2 as defined herein. More preferably than the foregoing, L2d is a linker containing at most twenty atoms. More preferably than the foregoing, L2d is a linker containing at most fifteen atoms. More preferably than the foregoing, L2d is a linker containing at most ten atoms. More preferably than the foregoing, L2d is a linker containing at most five atoms. More preferably than the foregoing, L2d is selected from the group consisting of -C(O)NL2T-, -NL2TC(O)-, -O-, -S-, -NL2T-, -N=N-, and -C(O)-; wherein L2T is hydrogen or methyl. More preferably than the foregoing, L2d is selected from the group consisting of -C(O)NL2T-, and -NL2TC(O)-. More preferably than the foregoing, L2d is selected from the group consisting of -C(O)NH-, and -NHC(O)-. Most preferably, L2d is -C(O)NH-. l!
T1 is according to Radical Group 1, Radical Group 3, Radical Group 4, or Radical Group 5, as defined herein. Preferably, each T1 is independently according to Radical Group 1 as defined herein.
More preferably, each T1 is independently selected from the group consisting of - OT1A, hydrogen, C1-C 12 (hetero)alkyl, C6 aryl, C4-C5 heteroaryl, C3-C6 (hetero)cycloalkyl, C5- C12 alkyl(hetero)aryl, C5-C12 (hetero)arylalkyl, C4-C12 alkylcycloalkyl, -N(T1A)2, -ST1A, - SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A -C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3. Even more preferably, each T1 is independently selected from the group consisting of -OT1A, hydrogen, C2-C6 alkyl, C6 aryl, C4-C5 heteroaryl, C3-C6 cycloalkyl, C5-C12 alkyl(hetero)aryl, C5-Ci2 (hetero)arylalkyl, C4-C12 alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, - C(O)OT1A, -O-C(O)T1A -C(O)N(T1A)2, -N(T1A)2-CO-T1A, and -Si(T1A)3. Yet more preferably, each T1 is independently selected from the group consisting of -OT1A, C2-C6 alkyl, C6 aryl, C4-C5 heteroaryl, C3-C6 cycloalkyl, C5-C12 alkyl(hetero)aryl, C5-C12 (hetero)arylalkyl, C4-C12 alkylcycloalkyl, -N(T1A)2, -ST1A, -SO3H, -C(O)T1A, -C(O)OT1A, -O-C(O)T1A -C(O)N(T1A)2, - N(T1A)2-CO-T1A, and -Si(T1A)3. More preferably still, T1 is -OT1A.
Most preferably, T1 is -OH.
As used herein, each T1A is independently selected from the group consisting of hydrogen, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)aryl, and an amino acid residue. Most preferably, T1A is hydrogen.
Preferably, T1 is in an axial position. Without wishing to be bound by theory, the inventors believe that in that case and when R48 is a releasable group, when the compound of the disclosure reacts with a diene, T1 aids in releasing the payload. This results in optimal release yields and/or release kinetics.
T2
T2 is an organic moiety. Preferably, T2 is according to any one of Radical Group 1, Radical Group 3, or Radical Group 5, as defined herein, or wherein T2 is a group -L3-CB. More preferably, T2 is a bioconjugation moiety, a residue of a bioconjugation moiety, or a group -L3-CB. More preferably, T2 is a bioconjugation moiety, or a group -L3-CB.
In preferred embodiments, T2 is a bioconjugation moiety. These embodiments typically relate to compounds that can be coupled to e.g. a protein. More preferably, T2 is according to Radical Group If as defined herein. Residues of these bioconjugation moieties are known in the art. More preferably, T2 is N-maleimidyl. In these embodiments, it is most preferred that T2 is:
In other preferred embodiments, T2 is a residue of a bioconjugation moiety. These embodiments typically relate to conjugates of the disclosure, wherein T2 links to e.g. a protein. Such residues are well-known to the skilled person. In these embodiments, it is most preferred that T2 is: wherein the asterisk indicates a bond to the protein, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
In other preferred embodiments, T2 is a group -L3-CB. These embodiments relate to when T2 itself comprises a Construct B (CB), which is usually a protein. CB is as defined herein. L3 is according to Radical Group 2. Preferably, L3 is a residue of a bioconjugation moiety. More preferably, L3 is a residue of an N-maleimidyl moiety or a residue of an N- hydroxy-succinimidyl moiety. In these embodiments, it is preferred that T2 is selected from the group consisting of In these embodiments, it is most preferred that T2 is:
For the moiety -L3-CB, it is preferred that L3 and a sulfur atom, secondary nitrogen atom, or tertiary nitrogen atom, preferably a sulfur atom, of CB together form any one of the following structures -L3-CB: wherein CB1 indicates S, secondary N, or tertiary N that is part of CB, preferably S; the wiggly lines indicates a bond to moiety L1, and the asterisk indicates a bond to the remainder of CB, preferably AVP0458.
T3
T3 is an organic moiety. Preferably, T3 is according to any one of Radical Group 1, Radical Group 3, or Radical Group 5, as defined herein. More preferably, T3 is according to Radical Group 3, as defined herein. Even more preferably, T3 is a polymer. More preferably still, T3 is a polymer comprising a polyethylene glycol moiety.
More preferably, T3 comprises a moiety -(CH2CH2-O-)y-T4. Herein, y is an integer in a range of from 1 to 50, preferably y is an integer in a range of from 2 to 45, more preferably y is an integer in a range of from 10 to 40, more preferably in a range of from 12 to 37, even more preferably in a range of from 15 to 35, more preferably still in a range of from 20 to 30, even more preferably in a range of from 23 to 25, and most preferably y is 24. This definition and these preferences for y also apply to compounds of Formula (2), Formula (3), Formula (G), Formula (O), Formula (P), and Formula (Q), wherein y is used as well.
T4 is according to Radical Group 1, Radical Group 3, Radical Group 4, or Radical Group 5 as defined herein. Preferably, T4 is according to Radical Group 1. More preferably, T4 is according to Radical Group la. More preferably, T4 is according to Radical Group lb. More preferably, T4 is according to Radical Group 1c. More preferably, T4 is according to Radical Group Id. Even more preferably, T4 is according to Radical Group le. Most preferably, T4 is methyl. Even more preferably, T3 is a moiety -(CH2CH2-O-)y-T4. Most preferably, T3 is a moiety -(CH2CH2-O-)24-CH3.
Variables of Formula (B)
In Formula (B), R48 and T1 are as defined herein. In Formula (B), yl is an integer of from 0 to 4, preferably an integer of from 1 to 2, most preferably yl is 1. In Formula (B), y2 is an integer of from 0 to 5, preferably an integer of from 1 to 4, more preferably an integer of from 1 to 3, even more preferably an integer of from 1 to 2, and most preferably y2 is 1. In Formula (B), y3 is an integer of from 1 to 5, preferably an integer of from 1 to 4, more preferably an integer of from 1 to 3, even more preferably an integer of from 1 to 2, and most preferably y3 is 1. In Formula (B), each of X1, X2, X3, X4, X5, and X6 is independently selected from the group consisting of a substituted or unsubstituted carbon atom, a nitrogen atom, or an oxygen atom, provided that if one of X1, X2, X3, X4, X5, and X6 is a nitrogen atom or an oxygen atom, an adjacent X1, X2, X3, X4, X5, and X6 is not a nitrogen atom or an oxygen atom. Preferably, each of X1, X2, X3, X4, X5, and X6 is independently a substituted or unsubstituted carbon atom. More preferably, X1 and/or X6 are independently a carbon atom substituted with R48. Even more preferably, X1 is a carbon atom substituted with R48, and most preferably, X1 is -CHR48-. More preferably still, X1 is -CHR48-, and X4 is -CT1TL-. Preferably, X2, X3, X5, and X6 are unsubstituted carbon atoms, more preferably -CH2-.
Variable x in Formulae (A3), (O), (P), and (Q)
In Formula (A3), Formula (O), Formula (P), and Formula (Q), x is an integer in a range of from 4 to 12; preferably x is an integer in a range of from 4 to 8, more preferably x is an integer in a range of from 4 to 6, and most preferably x is 5.
Spacers SP
All linkers as used herein may each independently be a spacer SP.
As the skilled person is aware, the specific structure of a spacer used in either a dienophile or diene as described herein does not typically influence whether the payload is released.
However, in some cases specific spacers are preferred. Below, first spacers in general are discussed, and thereafter the more specific self-immolative linkers.
In general, a spacer SP as used herein is a moiety according to RG2, more preferably any one of the preferred and/or specific embodiments thereof.
Preferably, a spacer SP consists of one or multiple Spacer Units SU arranged linearly and/or branched and may be connected to one or more CB moieties and/or one or more LC or TR moieties. The Spacer may be used to connect CB to one TR (Example A below; with reference to Formula 5a and 5b: f, e, a = 1) or more TR (Example B and C below; with reference to Formula 5a and 5b: f, e = 1, a ≥ 1), but it can also be used to modulate the properties, e.g. pharmacokinetic properties, of the CB-TR-CA conjugate (Example D below; with reference to Formula 5a and 5b: one or more of c,e,g,h ≥ 1). Thus a Spacer unit does not necessarily connect two entities together, it may also be bound to only one component, e.g. the TR or LC. Alternatively, the Spacer may comprise a Spacer Unit linking CB to TR and in addition may comprise another Spacer Unit that is only bound to the Spacer and serves to modulate the properties of the conjugate (Example F below; with reference to Formula 5a and 5b: e ≥ 1). The Spacer may also consist of two different types of SU constructs, e.g. a PEG linked to a peptide, or a PEG linked to an alkylene moiety (Example E below; with reference to Formula 5a and 5b: e ≥ 1). For the sake of clarity, Example B depicts a SU that is branched by using a multivalent branched SU. Example C depicts a SU that is branched by using a linear SU polymer, such as a peptide, whose side chain residues serve as conjugation groups.
The Spacer may be bound to the Activator in similar designs such as depicted in above examples A- F.
Each individual spacer unit SU may be independently selected from the group of radicals according to RG2. The Spacer Units include but are not limited to amino acids, nucleosides, nucleotides, and biopolymer fragments, such as oligo- or polypeptides, oligo- or polypeptoids, or oligo- or polylactides, or oligo- or poly-carbohydrates, varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. Preferred biopolymer SU are peptides. Preferably each SU comprises at most 50 carbon atoms, more preferably at most 25 carbon atoms, more preferably at most 10 carbon atoms. In some embodiments the SU is independently selected from the group consisting of (CH2)r, (C3-C8 carbocyclo), O-(CH2)r, arylene, (CH2)r-arylene, arylene-(CH2)r, (CH2)r -(C3-C8 carbocyclo), (C3-C8 carbocyclo)-(CH2)r, (C3-C8 heterocyclo), (CH2)r -(C3-C8 heterocyclo), (C3-C8 heterocyclo)-(CH2)r, -(CH2)rC(O)NR’(CH2)r, (CH2CH2O)r, (CH2CH2O)rCH2,(CH2)rC(O)NR’(CH2 CH2O)r, (CH2)rC(O)NR’(CH2CH2O)rCH2, (CH2CH2O)r C(O)NR’(CH2CH2O)r, (CH2CH2O)r C(O)NR’(CH2CH2O)rCH2, (CH2CH2O)rC(O)NR’CH2; wherein r is independently an integer from 1 -10. As used herein, each R’is independently selected from the group consisting of radicals according to RG1. Preferably, R’is hydrogen. Other examples of Spacer Units SU are linear or branched polyalkylene glycols such as polyethylene glycol (PEG) or polypropylene glycol (PPG) chains varying from 2 to 200, particularly 2 to 113, preferably 2 to 50, more preferably 2 to 24 and more preferably 2 to 12 repeating units. It is preferred that when polyalkylene glycols such as PEG and PPG polymers are only bound via one end of the polymer chain, that the other end is terminated with -OCH3, -OCH2CH3, OCH2CH2CO2H. Other polymeric Spacer Units are polymers and copolymers such as poly-(2-oxazoline), poly(A-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), dextran, polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF). Other exemplary polymers are polysaccharides, glycopolysaccharides, glycolipids, polyglycoside, polyacetals, polyketals, polyamides, polyethers, polyesters. Examples of naturally occurring polysaccharides that can be used as SU are cellulose, amylose, dextran, dextrin, levan, fucoidan, carrageenan, inulin, pectin, amylopectin, glycogen, lixenan, agarose, hyaluronan, chondroitinsulfate, dermatansulfate, keratansulfate, alginic acid and heparin. In yet other exemplary embodiments, the polymeric SU comprises a copolymer of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof. Preferred polymeric SU are PEG, HPMA, PLA, PLGA, PVP, PHF, dextran, oligopeptides, and polypeptides. In some embodiments, polymers used in a SU have a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, from 500 Dalton to 5 kDa. Other exemplary SU are dendrimers, such as polypropylene imine) (PPI) dendrimers, PAMAM dendrimers, and glycol-based dendrimers. The SU of the disclosure expressly include but are not limited to conjugates prepared with commercially available cross-linker reagents such as BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo- KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB, DTME, BMB, BMDB, BMH, BMOE, BM(PEO)3 and BM(PEO)4. To construct a branching Spacer one may use a SU based on one or several natural or non-natural amino acids, amino alcohol, aminoaldehyde, or polyamine residues or combinations thereof that collectively provide the required functionality for branching. For example serine has three functional groups, i.e. acid, amino and hydroxyl groups and may be viewed as a combined amino acid an aminoalcohol residue for purpose of acting as a branching SU. Other exemplary amino acids are lysine and tyrosine. In some embodiments, the Spacer consists of one Spacer Unit, therefore in those cases SP equals SU. Preferably the Spacer consists of two, three or four Spacer Units. In some embodiments, SP has a molecular weight ranging from 2 to 200 kDa, from 2 to 100 kDa, from 2 to 80 kDa, from 2 to 60 kDa, from 2 to 40 kDa, from 2 to 20 kDa, from 3 to 15 kDa, from 5 to 10 kDa, or from 500 Dalton to 5 kDa. In some embodiments, the SP has a mass of no more than 5000 Daltons, no more than 4000 Daltons, no more than 3000 Daltons, no more than 2000 Daltons, no more than 1000 Daltons, no more than 800 Daltons, no more than 500 Daltons, no more than 300 Daltons, no more than 200 Daltons. In some aspects the SP has a mass from 100 Daltons, from 200 Daltons, from 300 Daltons to 5000 Daltons. In some aspects of the SP has a mass from 30, 50, or 100 Daltons to 1000 Daltons, from about 30, 50, or 100 Daltons to 500 Daltons.
Preferably, SP comprises a moiety RG2a, RG2b, RG2c, or a residue of RG1f, as described herein. Preferably, said RG2a, RG2b, RG2c, or a residue of RG1f connects the SP to CB, LC, or TR.
Preferably, the spacer is -C(=O)NH-(CH2)x17-(CH2-CH2-O)x18-NH-C(=O)-(CH2)x19- T47. Therein xl7 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2. Therein, xl8 is an integer in a range of from 0 to 50, preferably of from 1 to 30, more preferably of from 2 to 20, and most preferably 3. Therein, xl9 is an integer in a range of from 1 to 6, preferably of from 1 to 4, more preferably of from 1 to 3, and most preferably 2. Therein, T47 is a residue of a bioconjugation moiety, as disclosed herein. Preferably, T47 is: wherein the asterisk indicates a bond to e.g. CB, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
Self-immolative linkers Lc
LC is a self-immolative linker, which may consist of multiple units arranged linearly and/or branched. The possible LC structures, their use, position and ways of attachment of linkers LC, CA and the TR (the Trigger, i.e. the trans-cyclooctene moiety) are known to the skilled person, see for example [Papot et al., Anticancer Agents Med. Chem., 2008, 8, 618- 637], Nevertheless, preferred but non-limiting examples of self-immolative linkers LC are benzyl-derivatives, such as those drawn below. There are two main self-immolation mechanisms: electron cascade elimination and cyclization-mediated elimination. The preferred example below on the left functions by means of the cascade mechanism, wherein the bond between the allylic carbon of the Trigger and the -O- or -S- attached to said carbon is cleaved, and an electron pair of Y1, for example an electron pair of NR6, shifts into the benzyl moiety resulting in an electron cascade and the formation of 4-hydroxybenzyl alcohol, CO2 and the liberated payload. The preferred example in the middle functions by means of the cyclization mechanism, wherein cleavage of the bond to the NR6 on the side of the Trigger leads to nucleophilic attack of the amine on the carbonyl, forming a 5-ring 1,3- dimethylimidazolidin-2-one and liberating the payload. The preferred example on the right combines both mechanisms. This linker will degrade not only into CO2 and one unit of 4- hydroxybenzyl alcohol (when Y1 is O), but also into one l,3-dimethylimidazolidin-2-one unit. wherein the wiggly line indicates a bond to -O- or -S- on the allylic position of the transcyclooctene, and the double dashed line indicates a bond to CA.
By substituting the benzyl groups of aforementioned self-immolative linkers LC, it is possible to tune the rate of release of the payload, caused by either steric and/or electronic effects on the cyclization and/or cascade release. Synthetic procedures to prepare such substituted benzyl-derivatives are known to the skilled person (see for example [Greenwald et al, J. Med. Chem., 1999, 42, 3657-3667] and [Thornthwaite et al, Polym. Chem., 2011, 2, 773-790], Some preferred substituted benzyl-derivatives with different release rates are drawn below.
Self-immolative linkers that undergo cyclization include but are not limited to substituted and unsubstituted aminobutyric acid amide, appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring system, 2-aminophenylpropionic acid amides, and trimethyl lock-based linkers, see e.g. [Chem. Biol. 1995, 2, 223], [J. Am. Chem. Soc. 1972, 94, 5815], [J. Org. Chem. 1990, 55, 5867], the contents of which are hereby incorporated by reference. Further preferred examples of LC can be found in W02009017394(A1), US7375078, WO2015038426 A1, W02004043493, Angew. Chem. Int. Ed. 2015, 54, 7492 - 7509, the contents of which are hereby incorporated by reference.
Preferably the LC has a mass of no more than 1000 Daltons, no more than 500 Daltons, no more than 400 Daltons, no more than 300 Daltons, or from 10, 50 or 100 to 1000 Daltons, from 10, 50, 100 to 400 Daltons, from 10, 50, 100 to 300 Daltons, from 10, 50, 100 to 200 Daltons, e.g., 10-1000 Daltons, such as 50-500 Daltons, such as 100 to 400 Daltons.
A person skilled in the art will know that one LC may be connected to another LC that is bound to CA, wherein upon reaction of the Activator with the Trigger TR, LC-LC-CA is released from the TR, leading to self-immolative release of both LC moi eties and the payload. With respect to the LC formulas disclosed herein, the LC linking the TR to the other LC then does not release the payload but an LC that is bound via YC1 and further links to CA. The skilled person will acknowledge that this principle also holds for further linkers LC linked to LC, e.g. LC-LC-LC-LC-CA.
Preferably, if the self-immolative linker is not linked to a group TC, the self- immolative linker is according to any one of Group I, Group II, Group III, and Group IV as shown below.
Self-immolative linkers according to Group I are
, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene, wherein U, V, W, Z are each independently selected from the group consisting of -CR7-, and -N-, wherein e is 0 or 1, wherein X is selected from the group consisting of -O-, -S- and -NR6-, wherein preferably each R8 and R9 are independently selected from the group consisting of hydrogen, C1-C4 (hetero)alkyl, C2-C4 (hetero)alkenyl, and C4-6 (hetero)aryl; wherein for R8 and R9 the (hetero)alkyl, (hetero)alkenyl, and (hetero)aryl are optionally substituted with a moiety selected from the group consisting of -Cl, -F, -Br, -I, -OH, -NH2, =O, -SH, -SO3H, -PO3H -PO4H2 and -NO2 and preferably contain at most two heteroatoms selected from the group consisting of -O-, -S-, -NH-, -P-, and -Si-, wherein the N, S, and P atoms are optionally oxidized. Preferably, for releasable groups of Group I both R8 and R9 are hydrogen.
The self-immolative linker according to Group II is
, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene, wherein m is an integer between 0 and 2, preferably m is 0, wherein e is 0 or 1. Preferably, for self-immolative linkers of Group II both R8 and R9 are hydrogen. Preferably, for self-immolative linkers of Group II R7 is methyl or isopropyl. Optionally, R6, R7, R8, R9 comprised in said Group I, and II, are -(SP)i-CB.
For all self-immolative linkers according to Group I and Group II YC1 is selected from the group consisting of -O-, -S-, and -NR6-, preferably -NR6-. For all linkers according to Group I, and Group II, YC2 is selected from the group consisting of O and S, preferably O.
Self-immolative linkers according to Group III are
, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene.
Self-immolative linkers according to Group IV are
, wherein the wiggly line may also indicate a bond to -S- on the allylic position of the transcyclooctene.
Preferably, R6, R7, R8, R9 are according to RG1 or any preferred embodiment thereof. Preferably, R6, R7, R8, R9 as used herein are not substituted. Most preferably, R6, R7, R8, R9 as used herein are hydrogen.
Further preferred embodiments
In some preferred embodiments, the compound of the disclosure is compound A-12:
In other preferred embodiments, the compound of the disclosure is compound A-28:
In yet other preferred embodiments, the compound of the disclosure is a conjugate of compound A-12 wherein said compound is bound to a targeting agent via the maleimide group of compound A-12; wherein the targeting agent is an antibody or a diabody. The maleimide group may be coupled to the targeting agent via a thiol residue that is part of said targeting agent. In that case, the maleimide group is converted to a residue of a maleimide group, viz. wherein the asterisk indicates a bond to the targeting agent, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
In yet other preferred embodiments, the compound of the disclosure is a conjugate of compound A-28 wherein said compound is bound to a targeting agent via the maleimide group of compound A-28; wherein the targeting agent is an antibody or a diabody. The maleimide group may be coupled to the targeting agent via a thiol residue that is part of said targeting agent. In that case, the maleimide group is converted to a residue of a maleimide group, viz. wherein the asterisk indicates a bond to the targeting agent, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
Conjugates of the disclosure
The disclosure also relates to a conjugate, or a salt, hydrate, or solvate thereof, wherein the conjugate comprises a protein or peptide, preferably a protein, wherein the protein or peptide is conjugated to at least one compound according to the disclosure wherein T2 is a residue of a bioconjugation moiety, and said protein or said peptide is conjugated to said compound via T2. Thus, the conjugate of the disclosure is to be understood as a compound of the disclosure (wherein T2 was originally a bioconjugation moiety) linked to a protein or peptide, preferably a protein, via T2, wherein due to the coupling of said compound and said protein or said peptide, T2 in the conjugate of the disclosure is the residue of a bioconjugation moiety, preferably the residue of an N-maleimidyl group, viz. : wherein the asterisk indicates a bond to the protein or peptide, and the wiggly line denotes a bond to the rest of the compound of the disclosure.
In the conjugate of the disclosure, the protein or peptide is preferably capable of binding a cancer epitope. Suitable cancer epitopes are disclosed herein.
In the conjugate of the disclosure, the protein is preferably an antibody,. More preferably, in the conjugate of the disclosure the protein is an antibody, which preferably includes antibodies, antibody variations, antibody fragments, antibody derivatives, antibody fusions, and antibody analogs. As such, in the conjugate of the disclosure the protein is preferably selected from the group consisting of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, minibodies, Fabs, CH2-deleted antibodies, VHH (aka nanobodies or sdAb/ single domain antibodies), VHH-Fc fusions, VHH multimers, and diabodies. The antibody may be a diabody, preferably the diabody is AVP0458 consisting of two monomers, wherein each of the two monomers has an amino acid sequence according to SEQ ID NO: 1.
Preferably, in the conjugate of the disclosure the protein and the compound of the disclosure are conjugated via T2 and a residue of a sulfhydryl of said protein or said peptide, a residue of a hydroxyl of said protein or said peptide, or a residue of an amine of said protein or said peptide; more preferably via T2 and a residue of a sulfhydryl of said protein or said peptide. Preferably, the residue of the sulfhydryl group of said protein or said peptide is part of a cysteine residue of said protein or said peptide. Compositions of the disclosure
The disclosure also pertains to a composition comprising a compound according to the disclosure, or the salt, hydrate, or solvate thereof. Preferably, the composition is a pharmaceutical composition. Preferably, the composition of the disclosure further comprises a pharmaceutically acceptable carrier. It is also preferred that if a salt of a compound of the disclosure is included in the composition of the disclosure, a pharmaceutically acceptable salt is used.
Combinations of the disclosure
The disclosure also relates to a combination of (A1) a compound according to the disclosure, or the salt, hydrate, or solvate thereof; (A2) a conjugate according to the disclosure, or the salt, hydrate, or solvate thereof; and/or (A3) a composition according to the disclosure; with (B) a diene or a salt, solvate, or hydrate thereof. It will be understood that herein, a compound according to the disclosure is a dienophile and/or comprises a dienophile moiety, and may be called a “Trigger”. The diene may be referred to as an “Activator”.
Preferably, the combination is of (A1) and (B). Preferably, the combination is of (A2) and (B). Preferably, the combination is of (A3) and (B). Preferably, the combination is of (A1), (A2), and (B). Preferably, the combination is of (A1), (A3), and (B). Preferably, the combination is of (A2), (A3), and (B). Preferably, the combination is of (A1), (A2), (A3), and (B).
Preferably, the combination of the disclosure is a kit. More preferably, the combination of the disclosure is a kit wherein (A1), (A2), and/or (A3) is/are physically separated from (B).
Preferably the diene is a tetrazine. More preferably, the diene is selected from the group consisting of:
or a salt, hydrate, and/or solvate thereof. Preferably, the diene is (TZ1) or a salt, hydrate, and/or solvate thereof. More preferably, the diene is (TZ2) or a salt, hydrate, and/or solvate thereof. More preferably, the diene is (TZ3) or a salt, hydrate, and/or solvate thereof. More preferably, the diene is (TZ4) or a salt, hydrate, and/or solvate thereof. Most preferably, the diene is (TZ5) or a salt, hydrate, and/or solvate thereof. It appears that (TZ2), (TZ3), (TZ4), and (TZ5) have one or more advantages over
(TZ1), for example a higher maximum tolerated dose, lower enzyme inhibition, and/or a more straightforward synthesis. Therefore, combinations with at least one of (TZ2), (TZ3), (TZ4), and (TZ5) are preferred over combinations comprising (TZ1), and combinations with (TZ5) are most preferred.
Non-therapeutic methods using and uses for using compounds of the disclosure
The disclosure pertains to a non-therapeutic method and a non-therapeutic use. Preferably, the dienophile used therein is as described in relation to the combination of the disclosure. For the non-therapeutic method of the disclosure it is preferred that the compound of the disclosure (viz. (ia)), the conjugate of the disclosure (viz. (iia)), and/or the composition of the disclosure (viz. (iiia)), and the diene are further contacted with a solvent. The skilled person is aware of suitable solvents for a reaction between a trans-cyclooctene (TCO) and a tetrazine. Preferably, the solvent comprises water, and more preferably the solvent is water.
For the non-therapeutic use, the click reaction is preferably a bioorthogonal click reaction. Preferably, the click reaction is performed in vitro, although non-therapeutic reactions in vivo can be carried out as well.
Medical use
The disclosure also relates to a compound of the disclosure, or the salt, hydrate, or solvate thereof; the conjugate of the disclosure, or the salt, hydrate, or solvate thereof; the composition of the disclosure; or the combination of the disclosure; for use in the treatment of a disease in a subject.
The disclosure also pertains to a method of treating a disease in a subject, wherein said method comprises the step of administering to said subject: (a) the compound according to the disclosure, or the salt, hydrate, or solvate thereof; (b) the conjugate according to the disclosure, or the salt, hydrate, or solvate thereof; (c) the composition according to the disclosure; and/or
(d) the combination according to the disclosure.
Use of (a) a compound according to the disclosure, or the salt, hydrate, or solvate thereof; (b) a conjugate according to the disclosure, or the salt, hydrate, or solvate thereof; (c) a composition according to the disclosure; and/or (d) a combination according to the disclosure; for the manufacture of a medicament for the treatment of a disease in a subject.
In relation to the medical use, preferably the subject is a human. Preferably, the disease is cancer.
Methods of synthesizing compounds of the disclosure
The disclosure also relates to a method for synthesizing a compound of the disclosure, wherein said method comprises coupling a compound of Formula (R) to a compound of Formula (S):
wherein R48, T1, and y are as defined herein; wherein T2, and x, are as defined herein, and S10 is -COOH or an active ester, preferably S10 is -COOH. Preferably, in Formula (S) x is an integer of from 4 to 6, and most preferably x is 5.
In the method for synthesizing a compound of the disclosure, when S10 is -COOH, it is preferred that the compound of Formula (S) is contacted with at least one coupling reagent, preferably in the presence of a base, preferably a non-nucleophilic base. Preferred non- nucleophilic bases are N,N-diisopropylethylamine (DIPEA), l,8-diazabicycloundec-7-ene (DBU), and l,5-diazabicyclo(4.3.0)non-5-ene (DBN).
The skilled person is aware of suitable conditions to carry out a coupling reaction between a compound of Formula (R) and a compound of Formula (S).
Preferably, the coupling is carried out at a temperature of from -20°C to 80°C, more preferably of from 0°C to 60°C, even more preferably of from 4°C to 50°C, more preferably still of from 10°C to 40°C, and most preferably of from 15 °C to 30°C.
Preferably, the coupling is carried out in the presence of a solvent, wherein preferably the solvent is an organic solvent.
The disclosure also relates to an alternative method for synthesizing a compound of the disclosure, wherein said method comprises coupling a compound of Formula (T) to a compound of Formula (U): wherein T1 and R48 are as defined herein; and S11 is -COOH or an active ester, preferably S11 is an active ester, more preferably S11 is selected from the group consisting of - C(O)O-A-succinimidyl, -C(O)O-pentafluorophenyl, -C(O)O-tetrafluorophenyl, -C(O)O-4- nitrophenyl, and -C(O)Cl; even more preferably, S11 is -C(O)O-A-succinimidyl, or -C(O)O- pentafluorophenyl; and most preferably, S11 is -C(O)O-pentafluorophenyl. wherein T2, x, and y, are as defined herein.
Preferably, in Formula (U) x is an integer of from 4 to 6, and most preferably x is 5.
In the alternative method for synthesizing a compound of the disclosure, when S11 is - COOH, it is preferred that the compound of Formula (S) is contacted with at least one coupling reagent, preferably in the presence of a base, preferably a non-nucleophilic base. Preferred non-nucleophilic bases are N,N-diisopropylethylamine (DIPEA), 1,8- diazabicycloundec-7-ene (DBU), and l,5-diazabicyclo(4.3.0)non-5-ene (DBN).
The skilled person is aware of suitable conditions to carry out a coupling reaction between a compound of Formula (T) and a compound of Formula (U). Preferably, the coupling is carried out at a temperature of from -20°C to 80°C, more preferably of from 0°C to 60°C, even more preferably of from 4°C to 50°C, more preferably still of from 10°C to 40°C, and most preferably of from 15°C to 30°C. Preferably, the coupling is carried out in the presence of a solvent, wherein preferably the solvent is an organic solvent.
Methods of synthesizing conjugates of the disclosure
The disclosure also pertains to a method for synthesizing a conjugate of the disclosure, wherein said method comprises the step of coupling a protein to a compound of the disclosure, or a salt, hydrate, or solvate thereof; wherein in said compound T2 is a bioconjugation moiety; wherein preferably in said protein disulfide bonds have been reduced.
As T2 in the compound of the disclosure is preferably a bioconjugation moiety that can react with a sulfhydryl group, such as an N-maleimidyl group, it is preferred that the protein contains free sulfhydryl groups. Typically, such sulfhydryl groups can be obtained by reducing disulfide bonds present in the protein. To that end, it is preferred that the protein has been contacted with a reducing agent prior to the coupling. Preferably, the reducing agent is selected from the group consisting of dithiothreitol (DTT), and tris-2-carboxyethylphosphine hydrochloride (TCEP). If the protein is contacted with a reducing agent prior to the coupling, the reducing agent is preferably DTT. Additionally or alternatively, the formation of free sulfhydryl groups on the protein can also be performed in situ. To that end, preferably the coupling is carried out in the presence of a reducing agent. In that case, it is preferred to use a reducing agent that does not contain free sulfhydryl groups itself. Thus, if the coupling is carried out in the presence of a reducing agent, it is preferred that the reducing agent is TCEP.
The skilled person is aware of suitable conditions to carry out the method of synthesizing a conjugate of the disclosure.
Preferably, the coupling is carried out at a temperature of from 0°C to 40°C, more preferably of from 1°C to 30°C, more preferably still of from 2°C to 20°C, even more preferably of from 4°C to 10°C, and most preferably at about 4°C.
Preferably, the coupling is carried out in an aqueous solution, preferably the aqueous solution is an aqueous buffer solution.
Preferably the coupling is carried out at a pH of from 6.0 to 8.5, preferably of from 6.2 to 8.0, more preferably of from 6.4 to 7.8, even more preferably of from 6.5 to 7.4, more preferably still of from 6.6 to 7.0, and most preferably at a pH of about 6.8.
The present disclosure is herein described with respect to particular embodiments, but the disclosure is not limited thereto but only by the claims. Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun unless something else is specifically stated.
The verb "to comprise", and its conjugations, as used in this description and in the claims is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
Thus, the scope of the expression "a device comprising means A and B" should not be limited to devices consisting only of components A and B. It means that with respect to the present disclosure, the only relevant components of the device are A and B.
The compounds herein may occur in different tautomeric forms. The compounds according to the disclosure are meant to include all tautomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific tautomer, it is to be understood that the disclosure of the present application is not limited to that specific tautomer, unless stated otherwise.
The compounds herein may occur in different enantiomeric forms. The compounds according to the disclosure are meant to include all enantiomeric forms, unless stated otherwise. When the structure of a compound is depicted as a specific enantiomer, it is to be understood that the disclosure of the present application is not limited to that specific enantiomer, unless stated otherwise.
Unless stated otherwise, the compounds of the disclosure and/or groups thereof may be protonated or deprotonated. It will be understood that it is possible that a compound may bear multiple charges which may be of opposite sign. For example, in a compound containing an amine and a carboxylic acid, the amine may be protonated while simultaneously the carboxylic acid is deprotonated.
In several formulae, groups or substituents are indicated with reference to letters such as “A”, “B”, “X”, “Y”, and various (numbered) “R” groups. In addition, the number of repeating units may be referred to with a letter, e.g. n in -(CH2)n-. The definitions of these letters are to be read with reference to each formula, i.e. in different formulae these letters, each independently, can have different meanings unless indicated otherwise.
Herein, reference is made to "alkyl", and the like. The number of carbon atoms that these groups have, excluding the carbon atoms comprised in any optional substituents according to Radical Group 1, can be indicated by a designation preceding such terms (e.g. “C1-C8 alkyl” means that said alkyl may have from 1 to 8 carbon atoms). For the avoidance of doubt, a butyl group substituted with a -OCH3 group is designated as a C4 alkyl, because the carbon atom in the substituent is not included in the carbon count.
A cycloalkyl group is a cyclic alkyl group. Unsubstituted cycloalkyl groups comprise at least three carbon atoms and have the general formula CnH2n-1. Optionally, the cycloalkyl groups are substituted by one or more substituents further specified in this document. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
An alkenyl group comprises one or more carbon-carbon double bonds, and may be linear or branched. Unsubstituted alkenyl groups comprising one C-C double bond have the general formula CnH2n-1. Unsubstituted alkenyl groups comprising two C-C double bonds have the general formula CnH2n-3. An alkenyl group may comprise a terminal carbon-carbon double bond and/or an internal carbon-carbon double bond. A terminal alkenyl group is an alkenyl group wherein a carbon-carbon double bond is located at a terminal position of a carbon chain. An alkenyl group may also comprise two or more carbon-carbon double bonds. Examples of an alkenyl group include ethenyl, propenyl, isopropenyl, t-butenyl, 1,3- butadienyl, 1,3-pentadienyl, etc. Unless stated otherwise, an alkenyl group may optionally be substituted with one or more, independently selected, substituents according to Radical Group 1.
A cycloalkenyl group is a cyclic alkenyl group. An unsubstituted cycloalkenyl group comprising one double bond has the general formula CnH2n-3. Optionally, a cycloalkenyl group is substituted by one or more substituents further specified in this document. An example of a cycloalkenyl group is cyclopentenyl.
An alkynyl group comprises one or more carbon-carbon triple bonds, and may be linear or branched. Unsubstituted alkynyl groups comprising one C-C triple bond have the general formula CnH2n-3. An alkynyl group may comprise a terminal carbon-carbon triple bond and/or an internal carbon-carbon triple bond. A terminal alkynyl group is an alkynyl group wherein a carbon-carbon triple bond is located at a terminal position of a carbon chain. An alkynyl group may also comprise two or more carbon-carbon triple bonds. Unless stated otherwise, an alkynyl group may optionally be substituted with one or more, independently selected, substituents according to Radical Group 1. Examples of an alkynyl group include ethynyl, propynyl, isopropynyl, t-butynyl, etc.
A cycloalkynyl group is a cyclic alkynyl group. An unsubstituted cycloalkynyl group comprising one triple bond has the general formula CnH2n-5. Optionally, a cycloalkynyl group is substituted by one or more substituents further specified in this document. An example of a cycloalkynyl group is cyclooctynyl.
An aryl group refers to an aromatic hydrocarbon ring system that comprises six to twenty-four carbon atoms, more preferably six to twelve carbon atoms, and may include monocyclic and polycyclic structures. When the aryl group is a polycyclic structure, it is preferably a bicyclic structure. Optionally, the aryl group may be substituted by one or more substituents further specified in this document. Examples of aryl groups are phenyl and naphthyl. Preferably, an aryl group is phenyl.
Arylalkyl groups and alkylaryl groups comprise at least seven carbon atoms and may include monocyclic and bicyclic structures. Optionally, the arylalkyl groups and alkylaryl may be substituted by one or more substituents further specified in this document. An arylalkyl group is for example benzyl. An alkylaryl group is for example 4-tert-butyl phenyl.
Preferably, heteroaryl groups comprise five to sixteen carbon atoms and contain between one to five heteroatoms. Heteroaryl groups comprise at least two carbon atoms (i.e. at least C2) and one or more heteroatoms N, O, P or S. A heteroaryl group may have a monocyclic or a bicyclic structure. Optionally, the heteroaryl group may be substituted by one or more substituents further specified in this document. Examples of suitable heteroaryl groups include pyridinyl, quinolinyl, pyrimidinyl, pyrazinyl, pyrazolyl, imidazolyl, thiazolyl, pyrrolyl, furanyl, triazolyl, benzofuranyl, indolyl, purinyl, benzoxazolyl, thienyl, phospholyl and oxazolyl.
Heteroarylalkyl groups and alkylheteroaryl groups comprise at least three carbon atoms (i.e. at least C3) and may include monocyclic and bicyclic structures. Optionally, the heteroaryl groups may be substituted by one or more substituents further specified in this document.
Where an aryl group is denoted as a (hetero)aryl group, the notation is meant to include an aryl group and a heteroaryl group. Similarly, an alkyl(hetero)aryl group is meant to include an alkylaryl group and an alkylheteroaryl group, and (hetero)arylalkyl is meant to include an arylalkyl group and a heteroaryl alkyl group. A C2-C24 (hetero)aryl group is thus to be interpreted as including a C2-C24 heteroaryl group and a C6-C24 aryl group. Similarly, a C3- C24 alkyl(hetero)aryl group is meant to include a C7-C24 alkylaryl group and a C3-C24 alkylheteroaryl group, and a C3-C24 (hetero)arylalkyl is meant to include a C7-C24 arylalkyl group and a C3-C24 heteroarylalkyl group.
In general, when (hetero) is placed before a group, it refers to both the variant of the group without the prefix hetero- as well as the group with the prefix hetero-. Herein, the prefix hetero- denotes that the group contains one or more heteroatoms selected from the group consisting of O, N, S, P, and Si. Preferably, the one or more heteroatoms is selected from the group consisting of O, N, S, and P. It will be understood that for any compound containing a heteroatom, the N, S, and P atoms are optionally oxidized and the N atoms are optionally quatemized. Preferably, up to two heteroatoms are consecutive, such as in for example -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. More preferably, however, the heteroatoms are not directly bound to one another.
Examples of heteroalkyls include -CH2CH2-O-CH3, -CH2CH2-NH-CH3, -CH2CH2- S(O)-CH3, -CH=CH-O-CH3, CH2CH2-NH2, CH2CH2-SH, -CH2CH2-OH, -CH2CH2-COOH, - CH2C(O)H, -C(O)HCH3, and -Si(CH3)3. Preferably, a C1-C4 heteroalkyl contains at most 2 heteroatoms.
Herein, it will be understood that when the prefix hetero- is used for combinations of groups, the prefix hetero- only refers to the one group before it is directly placed. For example, heteroarylalkyl denotes the combination of a heteroaryl group and an alkyl group, not the combination of a heteroaryl and a heteroalkyl group. Herein, the prefix cyclo- denotes that groups are cyclic. It will be understood that when the prefix cyclo- is used for combinations of groups, the prefix cyclo- only refers to the one group before it is directly placed. For example, cycloalkylalkenylene denotes the combination of a cycloalkylene group (see the definition of the suffix -ene below) and an alkenylene group, not the combination of a cycloalkylene and a cycloalkenylene group. In general, when (cyclo) is placed before a group, it refers to both the variant of the group without the prefix cyclo- as well as the group with the prefix cyclo-.
Herein, the suffix -ene denotes divalent groups, i.e. that the group is linked to at least two other moieties. An example of an alkylene is propylene (-CH2-CH2-CH2-), which is linked to another moiety at both termini. It is understood that if a group with the suffix -ene is substituted at one position with -H, then this group is identical to a group without the suffix. For example, an alkylene attached to an -H is identical to an alkyl group. I.e. propylene, - CH2-CH2-CH2-, attached to an -H at one terminus, -CH2-CH2-CH2-H, is logically identical to propyl, -CH2-CH2-CH3.
Herein, when combinations of groups are listed with the suffix -ene, it refers to a divalent group, i.e. that the group is linked to at least two other moieties, wherein each group of the combination contains one linkage to one of these two moieties. As such, for example alkylarylene is understood as a combination of an arylene group and an alkylene group. An example of an alkylarylene group is -phenyl-CH2-, and an example of an arylalkylene group is -CH2-phenyl-.
Herein, the suffix -triyl denotes trivalent groups, i.e. that the group is linked to at least three other moieties. An example of an arenetriyl is depicted below: wherein the wiggly lines denote bonds to different groups of the main compound.
It is understood that if a group with the suffix -triyl is substituted at one position with - H, then this group is identical to a divalent group with the suffix -ene. For example, an arenetriyl substituted with -H is identical to an arylene group. Similarly, it is understood that if a group with the suffix -triyl is substituted at two positions with -H, then this group is identical to a monovalent group. For example, an arenetriyl substituted with two -H is identical to an aryl group. Unless indicated otherwise, a hetero group may contain a heteroatom at non-terminal positions or at one or more terminal positions. In this case, “terminal” refers to the terminal position within the group, and not necessarily to the terminal position of the entire compound. For example, C2 heteroalkylene may refer to -NH-CH2-CH2-, -CH2-NH-CH2-, and -CH2-CH2- NH-. For example, C2 heteroalkyl may refer to -NH-CH2-CH3, -CH2-NH-CH3, and -CH2- CH2-NH2.
Herein, it is understood that cyclic compounds (i.e. aryl, cycloalkyl, cycloalkenyl, etc.) are understood to be monocyclic, polycyclic or branched. It is understood that the number of carbon atoms for cyclic compounds not only refers to the number of carbon atoms in one ring, but that the carbon atoms may be comprised in multiple rings. These rings may be fused to the main ring or substituted onto the main ring. For example, C10 aryl optionally containing heteroatoms may refer to inter alia a naphthyl group (fused rings) or to e.g. a bipyridyl group (substituted rings, both containing an N atom).
Unless stated otherwise, any group disclosed herein that is not cyclic is understood to be linear or branched. In particular, (hetero)alkyl groups, (hetero)alkenyl groups, (hetero)alkynyl groups, (hetero)alkylene groups, (hetero)alkenylene groups, (hetero)alkynylene groups, and the like are linear or branched, unless stated otherwise.
As used herein, unless stated otherwise all of the following groups: (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)cycloalkyl, (hetero)cycloalkenyl, (hetero)cycloalkynyl, (hetero)aryl, (hetero)alkylene, (hetero)alkenylene, (hetero)alkynylene, (hetero)cycloalkylene, (hetero)cycloalkenylene, (hetero)cycloalkynylene, (hetero)arylene, (hetero)alkanetriyl, (hetero)cycloalkanetriyl, arenetriyl, heteroarenetriyl, combinations thereof, and the like, can be substituted or unsubstituted; preferably these groups are unsubstituted. If said groups are substituted, said groups preferably contain up to 4, more preferably up to 3, more preferably still up to 2, and most preferably 1 substituent according to Radical Group 1 as defined herein.
The general term "sugar" is herein used to indicate a monosaccharide, for example glucose (Glc), galactose (Gal), mannose (Man) and fucose (Fuc). The term "sugar derivative" is herein used to indicate a derivative of a monosaccharide sugar, i.e. a monosaccharide sugar comprising substituents and/or functional groups. Examples of a sugar derivative include amino sugars and sugar acids, e.g. glucosamine (GIcNH2), galactosamine (GalNH2) N- acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), sialic acid (Sia) which is also referred to as N-acetylneuraminic acid (NeuNAc), and N-acetylmuramic acid (MurNAc), glucuronic acid (GlcA) and iduronic acid (IdoA). A sugar may be without further substitution, and then it is understood to be a monosaccharide. A sugar may be further substituted with at one or more of its hydroxyl groups, and then it is understood to be a disaccharide or an oligosaccharide. A disaccharide contains two monosaccharide moieties linked together. An oligosaccharide chain may be linear or branched, and may contain from 3 to 10 monosaccharide moieties.
The term “amino acid” is used herein in its normal scientific meaning. In particular, amino acids in relation to the disclosure comprise both natural and unnatural amino acids. Preferably, amino acids as used herein are selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, azidolysine, beta-alanine (bAla), 4-aminomethyl phenylalanine (Amf), 4- guanidine phenylalanine (Gnf), 4-aminomethyl-N-isopropyl phenylalanine (laf), 3 -pyridyl alanine (Pya), 4-piperidyl alanine (Ppa), 4-aminomethyl cyclohexyl alanine (Ama), 4- aminocyclohexyl alanine (Aca), ornithine (Om), citrulline, hydroxylysine (Hyl), allohydroxylysine (aHyl), 6-N-methyllysine (MeLys), desmosine (Des), isodesmosine (Ide), 2- aminoadipic acid (Aad), 3 -aminoadipic acid (bAad), 2-aminobutyric acid (Abu), 4- aminobutyric acid (4Abu), 6-aminohexonic acid (Acp), 2-aminoheptanoic acid (Ahe), 2- aminoisobutyric acid (Aib), 3-aminoisobutyric acid (bAib), 2-aminopimelic acid (Apm), 2,4- diaminobutyric acid (Dbu), 2,2’-diaminopimelic acid (Dpm), 2-3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), 3-hydroxyproline (3 Hyp), 4- hydroxyproline (4Hyp), allo-isoleucine (Alle), sarcosine (MeGly), N-methylisoleucine (Melle), N-methylvaline (MeVal), norvaline (Nva), and norleucine (Nle).
The term “amino acid residue” as used herein refers to an amino acid that is part of a peptide, viz. an amino acid of which the N-terminus and/or the C-terminus is part of a peptide bond. As such, “amino acid residue” does not refer to the side chain of an amino acid.
The term "protein" is herein used in its normal scientific meaning. Herein, polypeptides comprising about 10 or more amino acids are considered proteins. A protein may comprise natural, but also unnatural amino acids. The term “protein” herein is understood to comprise antibodies and antibody fragments.
An antibody is a protein that is capable of recognizing and binding to a specific antigen. Antibodies can be generated by the immune system of a living organism, but can also be produced using organic synthesis methods or by protein expression in host cells, such as bacteria or yeast. Antibodies can also be designed by using for example computational methods. While antibodies or immunoglobulins derived from IgG antibodies are particularly well-suited for use in this disclosure, immunoglobulins from any of the classes or subclasses may be selected, e.g. IgG, IgA, IgM, IgD and IgE. Suitably, the immunoglobulin is of the class IgG including but not limited to IgG subclasses (IgGl, 2, 3 and 4) or class IgM which is able to specifically bind to a specific epitope on an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelized single domain antibodies (sdAb), recombinant antibodies, anti-idiotype antibodies, multispecific antibodies, antibody fragments, such as, Fv, VHH, Fab, F(ab)2, Fab', Fab'-SH, F(ab')2, single chain variable fragment antibodies (scFv), tandem/bis-scFv, Fc, pFc', scFv-Fc, disulfide Fv (dsFv), bispecific antibodies and derivatives (bc-scFv) such as BiTE antibodies, trispecific antibody derivatives such as tribodies, camelid antibodies, minibodies, CH2-deleted antibodies, nanobodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single domain antibodies (sdAb, also known as VHH or Nanobody™), chimeric antibodies, chimeric antibodies comprising at least one human constant region, dual-affinity antibodies such as dual-affinity retargeting proteins (DART™), and multimers, fusions and derivatives thereof, such as divalent or multivalent single-chain variable fragments (e.g. di-scFvs, tri- scFvs), nanobody fusions and multimers, Fc-VHH fusions, and including but not limited to minibodies, diabodies, triabodies, tribodies, tetrabodies, and the like, and multivalent antibodies. Reference is made to [Trends in Biotechnology 2015, 33, 2, 65], [Trends Biotechnol. 2012, 30, 575-582], and [Cane. Gen. Prot. 2013 10, 1-18], and [BioDrugs 2014, 28, 331-343], the contents of which are hereby incorporated by reference. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, i.e. the antigen-binding region. Antibody mimetics include, but are not limited to Affimers, Anticalins, Avimers, Alphabodies, Affibodies, DARPins, and multimers and derivatives thereof; reference is made to [Trends in Biotechnology 2015, 33, 2, 65], the contents of which is hereby incorporated by reference. For the avoidance of doubt, in the context of this disclosure the term "antibody" is meant to encompass all of the antibody variations, antibody fragments, antibody derivatives, antibody fusions, antibody analogs and antibody mimetics outlined in this paragraph, unless specified otherwise.
Preferably, an antibody is selected from the group consisting of AVP0458, CC49, 3F8, abagovomab, abeiximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, alacizumab pegol, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, amivantamab, anatumomab mafenatox, andecaliximab, anetumab ravtansine, anifrolumab, ansuvimab, anrukinzumab, apolizumab, aprutumab ixadotin, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atidortoxumab, atinumab, atoltivimab, atoltivimab, maftivimab, odesivimab, atorolimumab, avelumab, azintuxizumab vedotin, bamlanivimab, bapineuzumab, basiliximab, bavituximab, BCD-100, bebtelovimab, bectumomab, bedinvetmab, begelomab, belantamab mafodotin, belimumab, bemarituzumab, benralizumab, berlimatoxumab, bermekimab, bersanlimab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bimekizumab, birtamimab, bivatuzumab, bleselumab, blinatumomab, blontuvetmab, blosozumab, bococizumab, brazikumab, brentuximab vedotin, briakinumab, brodalumab, brolucizumab, brontictuzumab, burosumab, cabiralizumab, camidanlumab tesirine, camrelizumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, casirivimab, capromab, carlumab, carotuximab, catumaxomab, cBR96-doxorubicin immunoconjugate, cedelizumab, cemiplimab, cergutuzumab amunaleukin, certolizumab pegol, cetrelimab, cetuximab, cibisatamab, cilgavimab, cirmtuzumab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, codrituzumab, cofetuzumab pelidotin, coltuximab ravtansine, conatumumab, concizumab, cosfroviximab, crenezumab, crizanlizumab, crotedumab, CR6261, cusatuzumab, dacetuzumab, daclizumab, dalotuzumab, dapirolizumab pegol, daratumumab, dectrekumab, demcizumab, denintuzumab mafodotin, denosumab, depatuxizumab mafodotin, derlotuximab biotin, detumomab, dezamizumab, dinutuximab, dinutuximab beta, diridavumab, divozilimab, domagrozumab, donanemab, dorlimomab aritox, dostarlimab, drozitumab, DS-8201, duligotuzumab, dupilumab, durvalumab, dusigitumab, duvortuxizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, eldelumab, elezanumab, elgemtumab, elotuzumab, elsilimomab, emactuzumab, emapalumab, emibetuzumab, emicizumab, enapotamab vedotin, enavatuzumab, enfortumab vedotin, enlimomab pegol, enoblituzumab, enokizumab, enoticumab, ensituximab, epcoritamab, epitumomab cituxetan, epratuzumab, eptinezumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etesevimab, etigilimab, etrolizumab, evinacumab, evolocumab, exbivirumab, fanolesomab, faralimomab, faricimab, farletuzumab, fasinumab, FBTA05, felvizumab, fezakinumab, fibatuzumab, ficlatuzumab, figitumumab, firivumab, flanvotumab, fletikumab, flotetuzumab, fontolizumab, foralumab, foravirumab, fremanezumab, fresolimumab, frovocimab, frunevetmab, fulranumab, futuximab, galcanezumab, galiximab, gancotamab, ganitumab, gantenerumab, gatipotuzumab, gavilimomab, gedivumab, gemtuzumab ozogamicin, gevokizumab, gilvetmab, gimsilumab, girentuximab, glembatumumab vedotin, glofitamab, golimumab, gomiliximab, gosuranemab, guselkumab, ianalumab, ibalizumab, sintilimab, ibritumomab tiuxetan, icrucumab, idarucizumab, ifabotuzumab, igovomab, iladatuzumab vedotin, imalumab, imaprelimab, imciromab, imdevimab, imgatuzumab, inclacumab, indatuximab ravtansine, indusatumab vedotin, inebilizumab, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iomab-B, iratumumab, isatuximab, iscalimab, istiratumab, itolizumab, ixekizumab, keliximab, labetuzumab, lacnotuzumab, ladiratuzumab vedotin, lampalizumab, lanadelumab, landogrozumab, laprituximab emtansine, larcaviximab, lebrikizumab, lecanemab, lemalesomab, lendalizumab, lenvervimab, lenzilumab, lerdelimumab, leronlimab, lesofavumab, letolizumab, lexatumumab, libivirumab, lifastuzumab vedotin, ligelizumab, loncastuximab tesirine, losatuxizumab vedotin, lilotomab satetraxetan, lintuzumab, lirilumab, lodelcizumab, lokivetmab, lorvotuzumab mertansine, lucatumumab, lulizumab pegol, lumiliximab, lumretuzumab, lupartumab, lupartumab amadotin, lutikizumab, maftivimab, mapatumumab, margetuximab, marstacimab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mirikizumab, mirvetuximab soravtansine, mitumomab, modotuximab, mogamulizumab, monalizumab, morolimumab, mosunetuzumab, motavizumab, moxetumomab pasudotox, muromonab-CD3, nacolomab tafenatox, namilumab, naptumomab estafenatox, naratuximab emtansine, namatumab, natalizumab, navicixizumab, navivumab, naxitamab, nebacumab, necitumumab, nemolizumab, NEOD001, nerelimomab, nesvacumab, netakimab, nimotuzumab, nirsevimab, nivolumab, nofetumomab merpentan, obiltoxaximab, obinutuzumab, ocaratuzumab, ocrelizumab, odesivimab, odulimomab, ofatumumab, olaratumab, oleclumab, olendalizumab, olokizumab, omalizumab, omburtamab, OMS721, onartuzumab, ontuxizumab, onvatilimab, opicinumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otilimab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, pamrevlumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pasotuxizumab, pateclizumab, patritumab, PDR001, pembrolizumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, pozelimab, prezalumab, plozalizumab, pogalizumab, polatuzumab vedotin, ponezumab, porgaviximab, prasinezumab, prezalizumab, priliximab, pritoxaximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ralpancizumab, ramucirumab, ranevetmab, ranibizumab, raxibacumab, ravagalimab, ravulizumab, refanezumab, regavirumab, regdanvimab, relatlimab, remtolumab, reslizumab, retifanlimab, rilotumumab, rinucumab, risankizumab, rituximab, rivabazumab pegol, robatumumab, Rmab, roledumab, romilkimab, romosozumab, rontalizumab, rosmantuzumab, rovalpituzumab tesirine, rovelizumab, rozanolixizumab, ruplizumab, SA237, sacituzumab govitecan, samalizumab, samrotamab vedotin, sarilumab, satralizumab, satumomab pendetide, secukinumab, selicrelumab, seribantumab, setoxaximab, setrusumab, sevirumab, sibrotuzumab, SGN-CD19A, SHP647, sifalimumab, siltuximab, simtuzumab, siplizumab, sirtratumab vedotin, sirukumab, sofituzumab vedotin, solanezumab, solitomab, sonepcizumab, sontuzumab, sotrovimab, spartalizumab, spesolimab, stamulumab, sulesomab, suptavumab, sutimlimab, suvizumab, suvratoxumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, tafasitamab, talacotuzumab, talizumab, talquetamab, tamtuvetmab, tanezumab, taplitumomab paptox, tarextumab, tavolimab, teclistamab, tefibazumab, telimomab aritox, telisotuzumab, telisotuzumab vedotin, tenatumomab, teneliximab, teplizumab, tepoditamab, teprotumumab, tesidolumab, tetulomab, tezepelumab, TGN1412, tibulizumab, tildrakizumab, tigatuzumab, timigutuzumab, timolumab, tiragol, umab, tiragotumab, tislelizumab, tisotumab vedotin, tixagevimab, TNX-650, tocilizumab, tomuzotuximab, toralizumab, tosatoxumab, tositumomab, tovetumab, tralokinumab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, TRBS07, tregalizumab, tremelimumab, trevogrumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, ulocuplumab, urelumab, urtoxazumab, ustekinumab, utomilumab, vadastuximab talirine, vanalimab, vandortuzumab vedotin, vantictumab, vanucizumab, vapaliximab, varisacumab, varlilumab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, vilobelimab, visilizumab, vobarilizumab, volociximab, vonlerolizumab, vopratelimab, vorsetuzumab mafodotin, votumumab, vunakizumab, xentuzumab, XMAB-5574, zalutumumab, zanolimumab, zatuximab, zenocutuzumab, ziralimumab, zolbetuximab, and zolimomab aritox.
The term “peptide” is herein used in its normal scientific meaning. Herein, peptides are considered to comprise a number of amino acid residues in a range of from 2 to 9.
The term “peptoid” is herein used in its normal scientific meaning.
A spacer is herein defined as a moiety that connects two or more elements of a compound. The terms “spacer” and “linker” are used herein interchangeably. Typically, a spacer is herein denoted as SP, and the more specific self-immolative linkers as LC. It will be understood that when herein, it is stated that “each individual SP is linked at all ends to the remainder of the structure” this refers to the fact that the spacer SP connects multiple moieties within a structure, and therefore the spacer has multiple ends by definition. The spacer SP may be linked to each individual moiety via different or identical moieties that may be each individually selected. Typically, these linking moieties are to be seen to be part of spacer SP itself. In case the spacer SP links two moieties within a structure, “all ends” should be interpreted as “both ends”. As an example, if the spacer connects a trans-cylooctene moiety to a Construct B, then “the remainder of the molecule” refers to the trans-cylooctene moiety and Construct B, while the connecting moieties between the spacer and the trans-cyclooctene moiety and Construct B (i.e. at both ends) may be individually selected.
As used herein, an organic molecule is defined as a molecule comprising a C-H bond. Organic compound and organic molecule are used synonymously.
As used herein, an inorganic molecule is defined as any molecule not being an organic molecule, i.e. not comprising a C-H bond. It will be understood that “inorganic molecule” typically also comprises hydrogen, -COOH, etc.
As used herein, a “small molecule” is preferably a small organic molecule. In general, a small molecule has a molecular weight of at most 2 kDa, more preferably at most 1 kDa, more preferably at most 750 Da, more preferably at most 500 Da, and most preferably at most 300 Da. Preferably, a small molecule has a molecular weight of at least 15 Da, more preferably at least 50 Da, more preferably at least 75 Da, and most preferably at least 100 Da.
As used herein, “particle” is preferably defined as a microparticle or a nanoparticle.
The term "salt thereof’ means a compound formed when an acidic proton, typically a proton of an acid, is replaced by a cation, such as a metal cation or an organic cation and the like. The term "salt thereof’ also means a compound formed when an amine is protonated. Where applicable, the salt is a pharmaceutically acceptable salt, although this is not required for salts that are not intended for administration to a patient. For example, in a salt of a compound the compound may be protonated by an inorganic or organic acid to form a cation, with the conjugate base of the inorganic or organic acid as the anionic component of the salt.
The term "pharmaceutically accepted salt” means a salt that is acceptable for administration to a patient, such as a mammal (salts with counter-ions having acceptable mammalian safety for a given dosage regime). Such salts may be derived from pharmaceutically acceptable inorganic or organic bases and from pharmaceutically acceptable inorganic or organic acids.
"Pharmaceutically acceptable salt" refers to pharmaceutically acceptable salts of a compound, which salts are derived from a variety of organic and inorganic counter ions known in the art and include, for example, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, etc., and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, formate, tartrate, besylate, mesylate, acetate, maleate, oxalate, etc. It will be understood that herein, the terms “moiety” and “group” are used interchangeably when referring to a part of a molecule.
It will be understood that when a heteroatom is denoted as -X(R’)2-, wherein X is the heteroatom and R’ is a certain moiety, then this denotes that two moieties R’ are attached to the heteroatom.
It will be understood that when a group is denoted as, for example, -((R51)2-R52)2- or a similar notation, in which R51 and R52 are certain moieties, then this denotes that first, it should be written as -R51-R51-R52-R51-R51-R52- before the individual R51 and R52 moieties are selected, rather than first selecting moieties R51 and R52 and then writing out the formula.
As used herein, “activated carboxylic acid” and “active ester” may be used interchangeably. As the skilled person is aware, an “activated carboxylic acid” or an “active ester” is a derivative of a carboxylic acid (-C(O)OH) of which the -OH moiety has been exchanged for a better leaving group. Preferred activated carboxylic acids or active esters are selected from the group consisting of -C(O)O-A-succinimidyl, -C(O)O-pentafluorophenyl, - C(O)O-tetrafluorophenyl, -C(O)O-4-nitrophenyl, and -C(O)Cl. More preferably, the activated carboxylic acid or active ester is -C(O)O-A-succinimidyl, or -C(O)O-pentafluorophenyl.
As the skilled person is aware, an “active carbonate” is a derivative of a carbonate (-0- C(O)-OH) of which the -OH moiety has been exchanged for a better leaving group. Preferred active carbonates are -OC(O)O-A-succinimidyl, -OC(O)O-pentafluorophenyl, -OC(O)O- tetrafluorophenyl, -OC(O)O-4-nitrophenyl, and -OC(O)Cl. More preferably, the active carbonate is -OC(O)O-A-succinimidyl, or -OC(O)O-pentafluorophenyl.
As used herein, a “drug” refers to a pharmaceutical agent. As such, “drug”, “pharmaceutical agent”, “therapeutic agent”, and “medicine” can typically be used interchangeably. Drugs that can be used in a compound of the disclosure are pharmaceutically active compounds. Preferably the pharmaceutically active compound is selected from the group consisting of cytotoxins, antiproliferative/antitumor agents, antiviral agents, antibiotics, anti-inflammatory agents, chemosensitizing agents, radiosensitizing agents, immunomodulators, immunosuppressants, immunostimulants, anti-angiogenic factors, and enzyme inhibitors. Preferably these pharmaceutically active compounds are selected from the group consisting of antibodies, antibody derivatives, antibody fragments, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, carbohydrates, as well as peptides, peptoids, steroids, toxins, hormones, cytokines, and chemokines. Most preferably, the drug is a protein, a toxin, a chelating moiety, monomethyl auristatin E, or doxorubicin; wherein preferably the chelating moiety comprises a radionuclide. Preferably these drugs are low to medium molecular weight compounds, preferably organic compounds (e.g. about 200 to about 2500 Da, preferably about 300 to about 1750 Da, more preferably about 300 to about 1000 Da). Exemplary cytotoxic drug types for use as conjugates to the Trigger and to be released upon IEDDA reaction with the Activator, for example for use in cancer therapy, include but are not limited to DNA damaging agents, DNA crosslinkers, DNA binders, DNA alkylators, DNA intercal ators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors, radiation sensitizers, anti-metabolites, natural products and their analogs, peptides, oligonucleotides, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors. Examples include but are not limited to colchinine, vinca alkaloids, anthracyclines (e.g. doxorubicin, epirubicin, idarubicin, daunorubicin), camptothecins, taxanes, taxols, vinblastine, vincristine, vindesine, calicheamycins, tubulysins, tubulysin M, cryptophycins, methotrexate, methopterin, aminopterin, dichloromethotrexate, irinotecans, enediynes, amanitins, deBouganin, dactinomycines, CC1065 and its analogs, duocarmycins, maytansines, maytansinoids, dolastatins, auristatins, pyrrolobenzodiazepines and dimers (PBDs), indolinobenzodiazepines and dimers, pyridinobenzodiazepines and dimers, mitomycins (e.g. mitomycin C, mitomycin A, caminomycin), melphalan, leurosine, leurosideine, actinomycin, tallysomycin, lexitropsins, bleomycins, podophyllotoxins, etoposide, etoposide phosphate, staurosporin, esperamicin, the pteridine family of drugs, SN-38 and its analogs, platinum -based drugs, cytotoxic nucleosides. Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K/m-TOR/AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HD AC inhibitors, PARP inhibitors, Wnt/Hedgehog signaling pathway inhibitors, and RNA polymerase inhibitors. In some embodiments, the drug is an auristatin. Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HP A), auristatin F phenylene diamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ. MMAE is a preferred auristatin. Suitable auristatins are also described in U.S. Publication Nos. 2003/0083263, 2011/0020343, and 2011/0070248; PCT Application Publication Nos. WO09/117531, W02005/081711, WO04/010957; W002/088172 and WOOl/24763, and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; 5,767,237; 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety. Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat No. 4,986,988), dolastatin 14 (U.S. Pat No. 5,138,036), dolastatin 15 (U.S. Pat No. 4,879,278), dolastatin 16 (U.S. Pat No. 6,239,104), dolastatin 17 (U.S. Pat No. 6,239,104), and dolastatin 18 (U.S. Pat No. 6,239,104), each patent incorporated herein by reference in their entirety. Exemplary maytansines, maytansinoids, such as DM4 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Patent Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371,533; 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441,163; 6,716,821 and 7,276,497. Other examples include mertansine and ansamitocin. Pyrrolobenzodiazepines (PBDs), which expressly include dimers and analogs, include but are not limited to those described in [Denny, Exp. Opin. Ther. Patents, 10(4):459-474 (2000)], [Hartley et al., Expert Opin Investig Drugs. 2011, 20(6):733-44], Antonow et al., Chem Rev. 2011, 111(4), 2815-64], Calicheamicins include, e.g. enediynes, esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116. Examples of duocarmycins and analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin, CPI, CBI. Other examples include those described in, for example, US Patent No. 5,070,092; 5,101,092; 5,187,186; 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,548,530;
6,586,618; 6,660,742; 6,756,397; 7,049,316; 7,553,816; 8,815,226; US20150104407; 61/988,011 filed may 2, 2014 and 62/010,972 filed June 11, 2014; the disclosure of each of which is incorporated herein in its entirety. Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Publication Nos.
2002/0103136 and 2010/0305149, and in U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entirety. Exemplary epothilone compounds include epothilone A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO97/19086; WO98/08849; WO98/22461; WO98/25929; WO98/38192; WO99/01124; WO99/02514; WO99/03848; WO99/07692; WO99/27890; and WO99/28324; the disclosures of which are incorporated herein by reference in their entirety. Exemplary cryptophycin compounds are described in U.S. Patent Nos. 6,680,311 and 6,747,021; the disclosures of which are incorporated herein by reference in their entirety. Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin. Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracy clines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines and the like. Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones and cryptophycins. Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT- 11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, diflometotecan, belotecan, lurtotecan and S39625. Other camptothecin compounds that can be used in the present disclosure include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med Chem., 30: 1774 (1987). Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure. Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZDI 152, MLN8054 and MLN8237; PLK inhibitors such as, for example, BI 2536, BI6727, GSK461364, ON- 01910; and KSP inhibitors such as, for example, SB 743921, SB 715992, MK-0731, AZD8477, AZ3146 and ARRY-520. Exemplary P13K/m-T0R/AKT signalling pathway inhibitors include phosphoinositide 3 -kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors. Exemplary P13 kinases are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, perifosine, PF- 04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765. Exemplary AKT inhibitors include, but are not limited to AT7867. Exemplary MAPK signaling pathway inhibitors include MEK, Ras, INK, B-Raf and p38 MAPK inhibitors. Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,944 and include GDC- 0973, GSK1120212, MSC1936369B, AS703026, RO5126766 and RO4987655, PD0325901, AZD6244, AZD8330 and GDC-0973. Exemplary B-raf inhibitors include CDC-0879, PLX- 4032, and SB590885. Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190. Exemplary receptor tyrosine kinases inhibitors include but are not limited to AEE788 (NVP-AEE 788), BIBW2992 (Afatinib), Lapatinib, Erlotinib (Tarceva), Gefitinib (Iressa), AP24534 (Ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (Dovitinib), Sunitinib (Sutent), Sorafenib (Nexavar), and Vatalinib. Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922 and KW-2478. Exemplary HD AC inhibitors include Belinostat (PR48101), CUDC-101, Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ- 26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MCI 568, MGCD0103 (Mocetinostat), MS-275 (Entinostat), PCI-24781, Pyroxamide (NSC 696085), SB939, Trichostatin A and Vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (Veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3 -aminobenzamide, A-966492, and AZD2461. Exemplary Wnt/Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV- 939. Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin. Exemplary immunomodulators are APRIL, cytokines, including IL-2, IL-7, IL-10, IL12, IL-15, IL-21, TNF, interferon gamma, GMCSF, NDV-GMCSF, and agonists and antagonists of STING, agonists and antagonists of TLRs including TLR1/2, TLR3, TLR4, TLR7/8, TLR9, TLR12, agonists and antagonists of GITR, CD3, CD28, CD40, CD74, CTLA4, 0X40, PD1, PDL1, RIG, MDA-5, NLRP1, NLRP3, AIM2, IDO, MEK, cGAS, and CD25, NKG2A. Other exemplary drugs include puromycins, topetecan, rhizoxin, echinomycin, combretastatin, netropsin, estramustine, cemadotin, discodermolide, eleutherobin, mitoxantrone, pyrrolobenzimidazoles (PBI), gamma-interferon, Thialanostatin (A) and analogs, CDK11, immunotoxins, comprising e.g. ricin A, diphtheria toxin, cholera toxin. In exemplary embodiments of the disclosure, the drug moiety is a mytomycin compound, a vinca alkaloid compound, taxol or an analogue, an anthracycline compound, a calicheamicin compound, a maytansinoid compound, an auristatin compound, a duocarmycin compound, SN38 or an analogue, a pyrrol obenzodiazepine compound, a indolinobenzodiazepine compound, a pyridinobenzodiazepine compound, a tubulysin compound, a non-natural camptothecin compound, a DNA binding drug, a kinase inhibitor, a MEK inhibitor, a KSP inhibitor, a P13 kinase inhibitor, a topoisomerase inhibitor, or analogues thereof. In one preferred embodiment the drug is a non-natural camptothecin compound, vinca alkaloid, kinase inhibitor, (e.g. P13 kinase inhibitor: GDC-0941 and PI- 103), MEK inhibitor, KSP inhibitor, RNA polymerase inhibitor, PARP inhibitor, docetaxel, paclitaxel, doxorubicin, dolastatin, calicheamicins, SN38, pyrrol obenzodiazepines, pyridinobenzodiazepines, indolinobenzodiazepines, DNA binding drugs, maytansinoids DM1 and DM4, auristatin MMAE, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs. In another preferred embodiment the drug is selected from DNA binding drugs and microtubule agents, including pyrrolobenzodiazepines, indolinobenzodiazepines, pyridinobenzodiazepines, maytansinoids, maytansines, auristatins, tubulysins, duocarmycins, anthracyclines, taxanes. In another preferred embodiment the drug is selected from colchinine, vinca alkaloids, tubulysins, irinotecans, an inhibitory peptide, amanitin and deBouganin. In another preferred embodiment the drug is a radioactive moiety, said moiety comprising a radioactive isotope for radiation therapy. A radionuclide used for therapy is preferably an isotope selected from the group consisting of 24Na, 32P, 33P, 47Sc, 59Fe, 67Cu, 76 As, 77 As, 80Br, 82Br, 89Sr, 90Nb, 90Y, 103Ru, 105Rh, 109Pd, 111Ag, 111In, 121Sn, 127Te, 131I, 140La, 141Ce, 142Pr, 143Pr, 144Pr, 149Pm, 149Tb, 151Pm, 153Sm, 159Gd, 161Tb, 165Dy, 166Dy, 166Ho, 169Er, 172Tm, 175Yb, 177LU, 186Re, 188Re, 198 Au, 199 Au, 211At, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, 223Ra, 224Ra, 225 Ac, and 227Th. When the radioactive moiety is intended to comprise a metal, such as 177LU, such radiometal is preferably provided in the form of a chelate. In such a case the radioactive moiety preferably comprises a structural moiety capable of forming a coordination complex with such a metal. A good example hereof are macrocylic lanthanide(III) chelates derived from l,4,7, 10-tetraazacyclododecane-l,4,7, 10-tetraacetic acid (H4dota). Preferably, the structural moiety capable of forming a coordination complex with such a metal is a chelating moiety as defined herein. In other embodiments the radioactive moiety comprises a prosthetic group (i.e. a phenol) that is bound by a non-metal radionuclide, such as 131I. Drugs optionally include a (portion of a) membrane translocation moiety (e.g. adamantine, poly- lysine/arginine, TAT, human lactoferrin) and/or a targeting agent (against e.g. a tumor cell receptor) optionally linked through a stable or labile linker. Exemplary references include: Trends in Biochemical Sciences, 2015,. 40, 12, 749; J. Am. Chem. Soc. 2015, 137, 12153-12160; Pharmaceutical Research, 2007, 24, 11, 1977.
It will further be understood that, in addition to one or more targeting agents (or CB) that may be attached to the Trigger or Linker LC a targeting agent TT may optionally be attached to a drug, optionally via a spacer SP. Alternatively, it will be further understood that the targeting agent (or CB) may comprise one or more additional drugs which are bound to the targeting agent by other types of linkers, e.g. cleavable by proteases, pH, thiols, or by catabolism.
It will be understood that chemical modifications may also be made to the desired compound in order to make reactions of that compound more convenient for purposes of preparing conjugates of the disclosure.
Drugs containing an amine functional group for coupling to the Trigger include mitomycin-C, mitomycin-A, daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, 9-amino camptothecin, N8-acetyl spermidine, l-(2 chloroethyl) 1,2- dimethanesulfonyl hydrazide, tallysomycin, cytarabine, dolastatins (including auristatins) and derivatives thereof. Drugs containing a hydroxyl function group for coupling to the Trigger include etoposide, camptothecin, taxol, esperamicin, l,8-dihydroxy-bicyclo[7.3.1]trideca-4-9- diene-2,6-diyne-13-one (U.S. Pat No. 5,198,560), podophyllotoxin, anguidine, vincristine, vinblastine, morpholine-doxorubicin, n-(5,5-diacetoxy-pentyl)doxorubicin, and derivatives thereof. Drugs containing a sulfhydryl functional group for coupling to the Trigger include esperamicin and 6-mecaptopurine, and derivatives thereof.
Most preferably, drugs in relation to the disclosure are monomethyl auristatin E (MMAE), exatecan, and exatecan derivatives. Preferably, exatecan and exatecan derivatives have the following structure: wherein E1 is -H, or an optionally substituted C1-C4 alkyl group. It will be understood that when E1 is -H, said structure is exatecan. Preferably, E1 is -H, -CH3, or -C(O)- CH2-OH. If E1 is - H or -CH3, then the exatecan or exatecan derivative is preferably linked to the remainder of R48 via the nitrogen atom to which E1 is attached. If E1 is C(O)-CH2-OH, then the exatecan derivative is preferably linked to the remainder of R48 via the oxygen atom that is part of the hydroxyl group of E1. More preferably, E1 is -H or -CH3. Most preferably, E1 is -H. Most preferably, the drug is monomethyl auristatin E (MMAE). Radical groups (RG)
Radical Group 1: terminal groups
For Radical Group 1 (RG1), the radical is selected from the group consisting of -H, - Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -CN, -N3, -NCS, -SCN, -SO3H, -PO3H -PO4H2, -NO2, -CF3, -CF2H, -CFH2, =O, =NH, -SH, -SO2H, -(SP)i-CB, (hetero)alkyl, (hetero)alkenyl, (hetero)alkynyl, (hetero)cycloalkyl, (hetero)cycloalkenyl, (hetero)cycloalkynyl, (hetero)aryl, and combinations thereof. Herein, SP is a spacer as defined herein, CB is Construct B as defined herein, and i is an integer in a range of from 0 to 4, preferably i is 0 or 1.
For RG1, “combinations thereof’ in particular refers to (hetero)alkylcycloalkyl, (hetero)alkylcycloalkenyl, (hetero)alkylcycloalkynyl, (hetero)cycloalkylalkyl, (hetero)cycloalkenylalkyl, (hetero)cycloalkynylalkyl, (hetero)alkenylcycloalkyl, (hetero)alkenylcycloalkenyl, (hetero)alkenylcycloalkynyl, (hetero)cycloalkylalkenyl, (hetero)cycloalkenylalkenyl, (hetero)cycloalkynylalkenyl, (hetero)alkynylcycloalkyl, (hetero)alkynylcycloalkenyl, (hetero)alkynylcycloalkynyl, (hetero)cycloalkylalkynyl, (hetero)cycloalkenylalkynyl, (hetero)cycloalkynylalkynyl, (hetero)aryl alkyl, (hetero)arylalkenyl, (hetero)arylalkynyl, alkyl(hetero)aryl, alkenyl(hetero)aryl, alkynyl(hetero)aryl, cycloalkyl(hetero)aryl, cycloalkenyl(hetero)aryl, cycloalkynyl(hetero)aryl, (hetero)arylcycloalkyl, (hetero)arylcycloalkenyl, and (hetero)arylcycloalkynyl. In addition, “combinations thereof’ in relation to RG1 also refers to e.g. an alkyl group substituted with one or more -Cl and/or -OH groups. As such, RG1 also comprises radicals such as -NH-CH2-CO0H (a glycine residue), which is a combination of a heteroalkyl and -COOH.
Preferably, for RG1 the radical is selected from the group RGla consisting of -H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C24 (hetero)alkyl, C2-C24 (hetero)alkenyl, C2-C24 (hetero)alkynyl, C3-C24 cycloalkyl, C2-C24 heterocycloalkyl, C5-C24 cycloalkenyl, C3-C24 heterocycloalkenyl, C7-C24 cycloalkynyl, C5-C24 (hetero)cycloalkynyl, C6-C24 aryl, C2-C24 heteroaryl, and combinations thereof.
More preferably, for RG1 the radical is selected from the group RGlb consisting of - H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -P03H -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C12 (hetero)alkyl, C2-C42 (hetero)alkenyl, C2-C42 (hetero)alkynyl, C3- C12 cycloalkyl, C2-C42 heterocycloalkyl, C5-C12 cycloalkenyl, C3-C42 heterocycloalkenyl, C7- C12 cycloalkynyl, C5-C12 (hetero)cycloalkynyl, C6-C12 aryl, C2-C12 heteroaryl, and combinations thereof.
Even more preferably, for RG1 the radical is selected from the group RGlc consisting of -H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C8 (hetero)alkyl, C2-C8 (hetero)alkenyl, C2-C8 (hetero)alkynyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, C5-C8 cycloalkenyl, C3-C8 heterocycloalkenyl, C7-C8 cycloalkynyl, C5-C8 (hetero)cycloalkynyl, C6-C8 aryl, C2-C8 heteroaryl, and combinations thereof.
More preferably still, for RG1 the radical is selected from the group RGld consisting of -H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C6 (hetero)alkyl, C2-C6 (hetero)alkenyl, C2-C6 (hetero)alkynyl, C3-C6 cycloalkyl, C2-C6 heterocycloalkyl, C5-C7 cycloalkenyl, C3-C5 heterocycloalkenyl, C8 cycloalkynyl, C6-C7 (hetero)cycloalkynyl, phenyl, C3-C5 heteroaryl, and combinations thereof.
Most preferably, for RG1 the radical is selected from the group RGle consisting of -H, -Cl, -F, -Br, -I, -OH, -NH2, -COOH, -CONH2, -SO3H, -PO3H -PO4H2, -NO2, -CF3, =O, =NH, -SH, -(SP)i-CB, C1-C3 (hetero)alkyl, C3-C6 cycloalkyl, C2-C5 heterocycloalkyl, phenyl, C4-C5 heteroaryl, and combinations thereof.
In some embodiments, for RG1 the radical is a conjugation moiety, which is a chemical group that can be used for binding, conjugation or coupling of a Construct, such as Construct-B, or a Spacer, or another molecule or construct of interest. The person skilled in the art is aware of the myriad of strategies that are available for the chemoselective or -unselective or enzymatic coupling or conjugation of one molecule or construct to another.
In some embodiments, RG1 is a moiety that allows conjugation to a protein comprising natural and/or non-natural amino acids. Moieties suitable for conjugation are known to the skilled person. Conjugation strategies are for example found in [O. Boutureira, G.J.L. Bernardes, Chem. Rev., 2015, 115, 2174-2195],
If RG1 is a conjugation moiety, it is preferably selected from the group RG1f consisting of N-maleimidyl, halogenated N-alkylamido, sulfonyloxy N-alkylamido, vinyl sulfone, (activated) carboxylic acids, active ester, benzenesulfonyl halides, ester, carbonate, sulfonyl halide, thiol or derivatives thereof, C2-6 alkenyl, C2-6 alkynyl, C7-18 cycloalkynyl, C5- 18 heterocycloalkynyl, bicyclo[6.1.0]non-4-yn-9-yl], C3-12 cycloalkenyl, azido, phosphine, nitrile oxide, nitrone, nitrile imine, isonitrile, diazo, ketone, (O-alkyl)hydroxylamino, hydrazine, halogenated N-maleimidyl, aryloxymaleimides, dithiophenolmaleimides, bromo- and dibromopyridazinediones, 2, 5 -dibromohexanedi ami de, alkynone, 3-arylpropiolonitrile, l,l-bis(sulfonylmethyl)-methylcarbonyl or elimination derivatives thereof, carbonyl halide, allenamide, 1,2-quinone, isothiocyanate, isocyanate, aldehyde, triazine, squaric acids, 2- imino-2-methoxyethyl, (oxa)norbomene, (oxa)norbornadiene, (imino)sydnones, methyl sulfonyl phenyl oxadi azole, aminooxy, 2-amino benzamidoxime, ethynylphosphonamidates, reactive in the Pictet-Spengler ligation and hydrazine- Pictet-Spengler (HIPS) ligation, DNA intercal ators, tetrazine, trans-cyclooctene, and photocrosslinkers. More preferably, RG1f is N-maleimidyl.
In other embodiments RG1f is selected from the group consisting of hydroxyl, amine, halogens, vinyl pyridine, disulfide, pyridyl disulfide, sulfonyloxy, mercaptoacetamide, anhydride, sulfonylated hydroxyacetamido, sulfonyl chlorides, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. In yet other embodiments RG1f is a group that can be connected to another group by means of an enzyme, for example sortase or Tubulin tyrosine ligase.
Radical Group 2: connecting groups
For Radical Group 2 (RG2), the radical is selected from the group consisting of (hetero)alkylene, (hetero)alkenylene, (hetero)alkynylene, (hetero)cycloalkylene, (hetero)cycloalkenylene, (hetero)cycloalkynylene, (hetero)arylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
The radicals from RG2 are optionally attached to one or more radicals according to RG1. Thus, RG2 also covers e.g. -NH-CH(CH2OH)-C(O)- (i.e. a serine residue), which is a heteroalkylene attached to -OH and =O.
For RG2, “combinations thereof’ in particular, but not exclusively, refers to alkyl(hetero)arylene, (hetero)aryl alkylene, (hetero)arylalkenylene, (hetero)arylalkynylene, alkenyl(hetero)arylene, and alkynyl(hetero)arylene.
Preferably, for RG2 the radical is selected from the group consisting of C1-C24 (hetero)alkylene, C2-C24 (hetero)alkenylene, C2-C24 (hetero)alkynylene, C3-C24 cycloalkylene, C2-C24 heterocycloalkylene, C5-C24 cycloalkenylene, C3-C24 heterocycloalkenylene, C7-C24 cycloalkynylene, C5-C24 (hetero)cycloalkynylene, C6-C24 arylene, C2-C24 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof. More preferably, for RG2 the radical is selected from the group consisting of C1-C12 (hetero)alkylene, C2-C12 (hetero)alkenylene, C2-C12 (hetero)alkynylene, C3-C12 cycloalkylene, C2-C12 heterocycloalkylene, C5-C12 cycloalkenylene, C3-C12 heterocycloalkenylene, C7-C12 cycloalkynylene, C5-C12 (hetero)cycloalkynylene, C6-C12 arylene, C2-C12 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
Even more preferably, for RG2 the radical is selected from the group consisting of C1- C8 (hetero)alkylene, C2-C8 (hetero)alkenylene, C2-C8 (hetero)alkynylene, C3-C8 cycloalkylene, C2-C8 heterocycloalkylene, C5-C8 cycloalkenylene, C3-C8 heterocycloalkenylene, C7-C8 cycloalkynylene, C5-C8 (hetero)cycloalkynylene, C6-C8 arylene, C2-C8 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
More preferably still, for RG2 the radical is selected from the group consisting of C1- C6 (hetero)alkylene, C2-C6 (hetero)alkenylene, C2-C6 (hetero)alkynylene, C3-C6 cycloalkylene, C2-C6 heterocycloalkylene, C5-C7 cycloalkenylene, C3-C5 heterocycloalkenylene, C8 cycloalkynylene, C6-C7 (hetero)cycloalkynylene, phenylene, C3-C5 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
Even more preferably still, for RG2 the radical is selected from the group consisting of C1-C3 (hetero)alkylene, C3-C6 cycloalkylene, C2-C5 heterocycloalkylene, phenylene, C4-C5 heteroarylene, amino acid, peptide, protein, polymer, oligonucleotide, nucleotide, carbohydrate, RG2a, RG2b, RG2c, and combinations thereof.
RG2a is selected from the group consisting of -O-, -S-, -SS-, -NR4-, -N=N-, -C(O)-, - C(O)NR4-, -OC(O)-, -C(O)O-, -OC(O)O-, -OC(O)NR4-, -NR4C(O)-, -NR4C(O)O-, - NR4C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR4-, -NR4C(O)S-, -S(O)-, - S(O)2-, -OS(O)2-, -S(O2)O-, -OS(O)2O-, -OS(O)2NR4-, -NR4S(O)2O-, -C(O)NR4S(O)2NR4-, - OC(O)NR4S(O)2NR4-, -OS(O)-, -OS(O)O-, -OS(O)NR4-, -ONR4C(O)-, -ONR4C(O)O-, - ONR4C(O)NR4-, -NR4OC(O)-, -NR4OC(O)O-, -NR4OC(O)NR4-, -ONR4C(S)-, -ONR4C(S)O- , -ONR4C(S)NR4-, -NR4OC(S)-, -NR4OC(S)O-, -NR4OC(S)NR4-, -OC(S)-, -C(S)O-, - OC(S)O-, -OC(S)NR4-, -NR4C(S)-, -NR4C(S)O-, -SS(O)2-, -S(O)2S-, -OS(O2)S-, -SS(O)2O-, - NR4OS(O)-, -NR4OS(O)O-, -NR4OS(O)NR4-, -NR4OS(O)2-, -NR4OS(O)2O-, - NR4OS(O)2NR4-, -ONR4S(O)-, -ONR4S(O)O-, -ONR4S(O)NR4-, -ONR4S(O)2O-, - ONR4S(O)2NR4-, -ONR4S(O)2-, -OP(O)(R4)2-, -SP(O)(R4)2-, and -NR4P(O)(R4)2-.
Herein, R4 is according to RG1, preferably R4 is hydrogen or methyl, more preferably R4 is hydrogen.
Preferably, RG2a is selected from the group consisting of -O-, -S-, -SS-, -NR4-, -N= :N- , -C(O)-, -C(O)NR4-, -OC(O)-, -C(O)O-, -OC(O)NR4-, -NR4C(O)-, -NR4C(O)O-, - NR4C(O)NR4-, -SC(O)-, -C(O)S-, -SC(O)O-, -OC(O)S-, -SC(O)NR4-, -NR4C(O)S-, -S(O) S(O)2-, -C(O)NR4S(O)2NR4-, -OC(O)NR4S(O)2NR4-, -OC(S)-, -C(S)O-, -OC(S)O-, -
OC(S)NR4-, -NR4C(S)-, -NR4C(S)O-, and -SS(O)2-.
More preferably, for RG2 the radical is RG2b or RG2c, most preferably RG2b.
RG2b is selected from the group consisting of
Therein, R' is a radical according to RG1, preferably R’ is hydrogen or C1-3 alkyl. The dashed and wiggly lines denote bonds to the other parts of the molecule.
RG2c is selected from the group consisting of
Therein, R' is a radical according to RG1, preferably R’ is hydrogen or C1-3 alkyl. The dashed and wiggly lines denote bonds to the other parts of the molecule. Radical Group 3: organic molecule
For Radical Group 3 (RG3) the radical is an organic molecule selected from the group consisting of a nucleic acid, a peptide, a protein, a carbohydrate, an aptamer, a hormone, a toxin, a steroid, a cytokine, a lipid, a small organic molecule as defined herein, a polymer, LNA, PNA, an amino acid, a peptoid, a chelating moiety, a molecule comprising a radionuclide, a fluorescent dye, a phosphorescent dye, a drug, a resin, a bead, an organic particle, a gel, an organic surface, an organometallic compound, a cell, and combinations thereof.
Preferably, for RG3 the radical is a a nucleic acid, a peptide, a protein, a carbohydrate, a lipid, a polymer, an amino acid, a chelating moiety, a drug, or a gel.
As used herein, a nucleic acid is preferably selected from the group consisting of an oligonucleotide, a polynucleotide, DNA, and RNA.
As used herein, a protein is preferably an antibody or a diabody. A preferred antibody is CC49, and a preferred diabody is AVP0458.
As used herein, a carbohydrate is preferably selected from the group consisting of a monosaccharide, an oligosaccharide, and a polysaccharide.
As used herein, a polymer is typically selected from the group consisting of polyethyleneglycol (PEG), poly(A-(2-hydroxypropyl)methacrylamide) (HPMA), polylactic acid (PLA), polylactic-glycolic acid (PLGA), polyglutamic acid (PG), polyvinylpyrrolidone (PVP), poly(l -hydroxymethylethylene hydroxymethyl-formal (PHF), copolymers of a polyacetal/polyketal and a hydrophilic polymer selected from the group consisting of polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, oligopeptides, polypeptides and derivatives thereof, oligopeptides, polypeptides, glycopolysaccharides, and polysaccharides such as dextran and hyaluronan. Preferably, a polymer as used herein is polyethylene glycol (PEG).
As used herein, a resin is preferably a polystyrene resin or an agarose resin.
As used herein, an organic particle is preferably a liposome or a polymersome.
As used herein, a chelating moiety is preferably selected from the group consisting of DTPA (diethylenetriaminepentaacetic acid), DOTA (1,4,7,10- tetraazacyclododecane- N,N',N",N" -tetraacetic acid), NOTA (l,4,7-triazacyclononane-N,N',N"-triacetic acid), TETA (1,4,8, l l-tetraazacyclotetradecane-N,N',N",N' -tetraacetic acid), OTTA (N 1 -(p- isothiocyanatobenzyl)-diethylenetriamine-N1,N2,N3,N3-tetraacetic acid), deferoxamine or DFA (N'-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-l,4- dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide) or HYNIC (hydrazinonicotinamide), EDTA (ethylenediaminetetraacetic acid), OTAM, TACN, sarcophagine, and 3,4-HOPO-based chelators.
More preferably, herein a chelating moiety is selected from the group consisting of
wherein the wiggly line denotes a bond to the remaining part of the molecule, optionally bound via -C(O)NH-, wherein the chelator moieties according to said group optionally chelate a metal, wherein the metal is preferably selected from the group consisting of 44Sc,62Cu, 64Cu, 66Ga, 67Ga, 67Cu, 68Ga, 86Y, 89Zr, 90Y, 99mTc, 111In, 166Ho, 177Lu, 186Re, 188Re, 211Bi, 212Bi, 212Pb, 213Bi, 214Bi, and 225 Ac. Radical Group 4: inorganic molecule
For Radical Group 4 (RG4), the radical is an inorganic molecule selected from the group consisting of an inorganic surface, an inorganic particle, an allotrope of carbon, an inorganic drug, a radionuclide, and combinations thereof.
As used herein, an inorganic surface is preferably selected from the group consisting of chips, wafers, metal such as gold, and silica-based surfaces such as glass.
As used herein, an inorganic particle is preferably selected from the group consisting of beads, silica-based particles, polymer-based materials, and iron oxide particles. Preferably, a bead is a magnetic bead or a gold bead.
As used herein, an allotrope of carbon is preferably selected from the group consisting of fullerenes such as Buckminsterfullerene; graphite, graphene, diamond, Lonsdaleite, Q- carbon, linearn acetylenic carbon, amorphous carbon, and carbon nanotubes.
As used herein, an inorganic drug is preferably cisplatin.
Radical group 5: further terminal groups
For RG5 the radical is: wherein the dashed line indicates a bond to the remaining part of the dienophile or diene.
For RG5, each R10 is independently selected from RG2, preferably from RG2a.
For RG5, each R11 is independently selected from RG2, preferably not being RG2a, RG2b, or RG2c.
For RG5, R12 is selected from RG1 or RG3, preferably RG3, more preferably a protein, polymer, or chelating moiety.
Preferably, z is an integer in a range of from 0 to 12, preferably from 0 to 10, more preferably from 0 to 8, even more preferably from 1 to 6, most preferably from 2 to 4. Preferably, z is 0. In case the compound according to the disclosure comprises more than one moiety RG5, each z is independently selected.
Preferably, h is 0 or 1. In case the compound according to the disclosure comprises more than one moiety RG5, each h, z, and n is independently selected. Preferably, each n belonging to RG5 is an integer independently selected from a range of from 0 to 24, preferably from 1 to 12, more preferably from 1 to 6, even more preferably from 1 to 3. Preferably, n is 1. In other preferred embodiments n is an integer in the range from 12 to 24. Preferably, z is 0, and n is 1. In other embodiments, z is 1, and n is 1. Preferably, the moiety RG5 has a molecular weight in a range of from 100 Da to 3000 Da, preferably, in a range of from 100 Da to 2000 Da, more preferably, in a range of from 100 Da to 1500 Da, even more preferably in a range of from 150 Da to 1500 Da. Even more preferably still, the moiety RG5 has a molecular weight in a range of from 150 Da to 1000 Da, most preferably in a range of from 200 Da to 1000 Da.
Preferably, RG5 is selected from the group RG5a consisting of:
, wherein the wiggly line denotes a bond to the remainder of the molecule.
It is understood that when n is more than 1, -((R10)h-R11)n-(R10)h-R12 may be preceded by a group -(R10)h-R11- so as to form a group -(R10)h-R11-((R10)h-R11)n-(R10)h-R12 . It is understood that this follows from the definition of how to write out the repeating units, i.e. -((R10)h-R11)2- would first be written as -(R10)h-R11-(R10)h-R11- before R10, h, and R11 are independently selected.
List of Clauses A1 -A16 The disclosure also relates to the subject-matter of any one of Clauses A1-A16. It will be understood that the embodiments, definitions, Formulae, parameters, values, and the like as disclosed for Clauses A1 -A16 that are equivalent to embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the present disclosure, will have the same preferences as said embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the disclosure, and can be combined therewith as such. In case of a discrepancy between embodiments, definitions, Formulae, parameters, values, and the like, in Clauses A1- A16, and the rest of the disclosure, such embodiments, definitions, Formulae, parameters, values, and the like only relate to Clauses A1 -A16 and not to the rest of the present disclosure.
Clause A1. A compound, or a salt, solvate, and/or hydrate thereof, wherein the compound comprises an (E)-cyclooctene moiety, wherein at least one non-vinylic carbon of said moiety is substituted with at least one group according to Formula (A1); wherein
SP is a spacer; LC is a self-immolative linker; TC is a group that is separable from LC due to conditions present in a tumor and/or due to conditions in a tumor microenvironment;
CA is a payload; x is 0 or 1; y is an integer of from 0 or 4; z is 0 or 1; provided that: d) at least one allylic carbon of said (E)-cyclooctene moiety is substituted with a first group according to Formula (A1); i. if in said first group x is 1, then SP is -Y1-C(=Y2)-, and LC or CA is connected to SP via O, S, or N, wherein said O, S, or N is part of LC or CA; Y1 and Y2 are independently O or S; ii. if in said first group x is 0, then LC or CA is connected to said allylic carbon via O or S, wherein said O or S is part of LC or CA; e) if said compound does not comprise another group according to Formula (A1) in addition to said first group, then in said first group y is an integer of from 1 to 4; f) if said compound comprises one or more groups according to Formula (A1) in addition to said first group, then in said one or more groups y is an integer of from 1 to 4.
Clause A2. The compound of Clause A1, or the salt, solvate, and/or hydrate thereof, wherein said compound does not comprise another group according to Formula (A1) in addition to said first group.
Clause A3. The compound of any one of Clauses A1 to A2, or the salt, solvate, and/or hydrate thereof, wherein TC is a group that is separable from LC by: a) an enzyme expressed by a tumor cell; b) an enzyme overexpressed in a tumor microenvironment; and/or c) the reducing potential in a tumor or a tumor microenvironment.
Clause A4. The compound of any one of Clauses A1 to A3, or the salt, solvate, and/or hydrate thereof, wherein TC is selected from the group consisting of glucuronidyl, polyglucuronidyl, a peptide, and combinations thereof; preferably TC is a peptide.
Clause A5. The compound of any one of Clauses A1 to A4, or the salt, solvate, and/or hydrate thereof, wherein TC is a dipeptide; preferably the peptide is an N-terminally protected dipeptide that is connected to LC via a peptide bond at the C-terminus of said N-terminally protected dipeptide; more preferably the peptide is RP -C(O)-P1-P2-, wherein RP is -NH2 or C1- 4 alkyl, and P1 and P2 are independently amino acid residues; preferably P1-P2 is valinecitrulline or phenylalanine-lysine.
Clause A6. The compound of any one of Clause A1 to A3, or the salt, solvate, and/or hydrate thereof, wherein TC is glucuronidyl.
Clause A7. The compound of any one of Clauses A1 to A6, or the salt, solvate, and/or hydrate thereof, wherein CA is a drug; preferably CA is selected from the group consisting of monomethyl auristatin E, doxorubicin, camptotecin, camptotecin derivatives, exatecan, and exatecan derivatives; most preferably CA is monomethyl auristatin E.
Clause A8. The compound of any one of Clauses A1 to A7, or the salt, solvate, and/or hydrate thereof, wherein the self-immolative linker consists of one or more self-immolative units, wherein the one or more self-immolative units are independently a group according to
Formula (A2A), or a group according to Formula (A2B): Formula (A2A); wherein the asterisk indicates a bond to -(TC)y in Formula (A1); the wiggly line indicates a bond to -(SP)x- in Formula (A1) or a preceding self-immolative unit; the double dashed line indicates a bond to CA or a further self-immolative unit; A is a 5- membered or 6-membered (hetero)aromatic ring; Y3 and Y5 are independently O, S, a secondary amine, or a tertiary amine; Y4, Y6, and Y7 are independently O or S; e is 0, 1 or 2; f is 0 or 1; A1 is 1 or 2; A2 is 0, 1, 2, or 3; each B1 is an optionally substituted carbon; each Cl is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid; provided that if A is a 6-membered (hetero)aromatic ring, then -Y3- is at an ortho or para position relative to - (B 1)A1-Y6-C(=Y7)-; and if e is at least 1, then (*-C(=Y4)-Y5)e, is at an ortho or para position relative to -(B 1)A1-Y6-C(=Y7)-; and provided that if A is a 5-membered (hetero)aromatic ring and -(B 1)A1-Y6-C(=Y7)- is at the 1-position of said 5-membered (hetero)aromatic ring, then - Y3- is at the 3- or 4-position, and if e is at least 1, then (*-C(=Y4)-Y5)e, is at the 3- or 4- position; wherein the asterisk indicates a bond to -(TC)y in
Formula (A1); the wiggly line indicates a bond to -(SP)x- in Formula (A1) or a preceding self- immolative unit; the double dashed line indicates a bond to CA or a further self-immolative unit; Y8 and Y10 are independently O or S; and Y9 is independently O, S, a secondary amine, or a tertiary amine.
Clause A9. The compound of any one of Clauses A1 to A8, or the salt, solvate, and/or hydrate thereof, wherein said compound has a structure according to Formula (A3): wherein each moiety RL is independently selected from the group consisting of hydrogen, halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, carboxylic acid, RL1, and RL2; provided that at least one moiety RL is RL1 and at least one moiety RL is RL2; wherein RL1 is wherein X2, X3, X4, X5, and X6 are optionally substituted carbon atoms;
RLC is hydrogen or methyl; and each RL2 is independently selected from the group consisting of wherein RP is -NH2 or C1-4 alkyl.
Clause A10. The compound of any one of Clauses A1 to A9, or the salt, solvate, and/or hydrate thereof, wherein said compound has a structure according to any one of Formulae (A4 A), (A4B), or (A4C) :
wherein in each of Formulae (A4A), (A4B), or (A4C): X2, X3, X4, X5, and X6 are optionally substituted carbon atoms; RLC is hydrogen or methyl; and CA is a payload; wherein in each of Formulae (A4B) and (A4C) RP is -NH2 or C1-4 alkyl.
Clause A11. A compound, or a salt, solvate, and/or hydrate thereof, wherein the compound comprises an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene and also if said compound is present in a tumor or in a tumor microenvironment.
Clause A12. A composition comprising a compound according to any one of Clauses A1 to A11, or the salt, solvate, or hydrate thereof. Clause A13. A combination of
(A1) a compound according to any one of Clauses A1 to A11, or the salt, solvate, and/or hydrate thereof; or
(A2) a composition according to Clause A12; with
(B) a diene or a salt, solvate, or hydrate thereof; preferably the diene is a tetrazine.
Clause A14. The compound according to any one of Clauses A1 to A11, or the salt, solvate, and/or hydrate thereof; the composition according to Clause A12; or the combination according to Clause A13; for use as a medicament.
Clause A15. The compound according to any one of Clause A1 to A11, or the salt, solvate, and/or hydrate thereof; the composition according to Clause A12; or the combination according to Clause A13; for use in the treatment of a disease in a subject, preferably the subject is a human, preferably the disease is cancer.
Clause A16. A non-therapeutic method for reacting:
(ia) a compound according to any one of Clause A1 to A11, or a salt, solvate, and/or hydrate thereof; or
(iia) a composition according to Clause A12; with a diene or a salt, solvate, or hydrate thereof, wherein said method comprises the step of contacting (ia) or (iia) with said diene or salt, solvate, or hydrate thereof, preferably said contacting is in vitro., and preferably said diene is a tetrazine.
List of Clauses B1 -B16
The disclosure also relates to the subject-matter of any one of Clauses B1 -B16. It will be understood that the embodiments, definitions, Formulae, parameters, values, and the like as disclosed for Clauses B1-B16 that are equivalent to embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the present disclosure, will have the same preferences as said embodiments, definitions, Formulae, parameters, values, and the like, of the rest of the disclosure, and can be combined therewith as such. In case of a discrepancy between embodiments, definitions, Formulae, parameters, values, and the like, in Clauses B1- B16, and the rest of the disclosure, such embodiments, definitions, Formulae, parameters, values, and the like only relate to Clauses B1 -B16 and not to the rest of the present disclosure. Clause B1. A compound comprising an (E)-cyclooctene moiety, wherein at least one non- vinylic carbon of said moiety is substituted with at least one group according to Formula (B1); wherein SP is a spacer; LC is a self- immolative linker; TC is a group that is separable from LC due to conditions present in a tumor and/or due to conditions in a tumor microenvironment; CA is a payload; x is 0 or 1; y is an integer of from 0 or 4; z is 0 or 1; provided that: a) at least one allylic carbon of said (E)-cyclooctene moiety is substituted with a first group according to Formula (B1); i. if in said first group x is 1, then SP is -Y1-C(=Y2)-, and LC or CA is connected to SP via O, S, or N, wherein said O, S, or N is part of LC or CA; Y1 and Y2 are independently O or S; ii. if in said first group x is 0, then LC or CA is connected to said allylic carbon via O or S, wherein said O or S is part of LC or CA; b) if said compound does not comprise another group according to Formula (B1) in addition to said first group, then in said first group y is an integer of from 1 to 4; c) if said compound comprises one or more groups according to Formula (B 1) in addition to said first group, then in said one or more groups y is an integer of from 1 to 4.
Clause B2. The compound of Clause B1, wherein said compound does not comprise another group according to Formula (B1) in addition to said first group.
Clause B3. The compound of any one of Clauses B 1 to B2, wherein TC is a group that is separable from LC by: a) an enzyme expressed by a tumor cell; b) an enzyme overexpressed in a tumor microenvironment; and/or c) the reducing potential in a tumor or a tumor microenvironment. Clause B4. The compound of any one of Clauses B 1 to B3, wherein TC is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, phosphate, and combinations thereof; preferably TC is a peptide.
Clause B5. The compound of any one of Clauses B 1 to B4, wherein TC is a dipeptide; preferably the dipeptide is an N-terminally protected dipeptide that is connected to LC via a peptide bond at the C-terminus of said N-terminally protected dipeptide; more preferably the dipeptide is RP-C(O)-P1-P2-, wherein RP is -NH2, C1-4 alkyl, or -O-benzyl; and P1 and P2 are independently amino acid residues; preferably P1-P2 is valine-citrulline or phenylalaninelysine.
Clause B6. The compound of any one of Clauses B 1 to B3, wherein TC is glucuronidyl.
Clause B7. The compound of any one of Clauses B 1 to B6, wherein CA is a drug; preferably CA is selected from the group consisting of monomethyl auristatin E, doxorubicin, camptotecin, camptotecin derivatives, exatecan, and exatecan derivatives; most preferably CA is monomethyl auristatin E.
Clause B8. The compound of any one of Clauses B 1 to B7, wherein the self-immolative linker consists of one or more self-immolative units, wherein the one or more self-immolative units are independently a group according to Formula (B2A), or a group according to Formula (B2B): wherein the asterisk indicates a bond to -(TC)y in Formula (B1); the wiggly line indicates a bond to
- (SP)x- in Formula (B1) or a preceding self-immolative unit; the double dashed line indicates a bond to CA or a further self-immolative unit; A is a 5-membered or 6-membered (hetero)aromatic ring; R2A is -C(=Y4)-Y5- or -Y5-; Y3 and Y5 are independently O, S, a secondary amine, or a tertiary amine; Y4, Y6, and Y7 are independently O or S; e is 0, 1 or 2; f is 0 or 1; A1 is 1 or 2; A2 is 0, 1, 2, or 3; each B1 is an optionally substituted carbon; each C1 is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid; provided that if A is a 6-membered (hetero)aromatic ring, then -Y3- is at an ortho or para position relative to - (B 1)A1-Y6-C(=Y7)-; and if e is at least 1, then each (*-C(=Y4)-Y5)e, is at an ortho or para position relative to -(B 1)A1-Y6-C(=Y7)-; and provided that if A is a 5-membered (hetero)aromatic ring and -(B 1)A1-Y6-C(=Y7)- is at the 1-position of said 5-membered (hetero)aromatic ring, then -Y3- is at the 3- or 4-position, and if e is at least 1, then each (*-C(=Y4)-Y5)e, is at the 3- or 4-position; wherein the asterisk indicates a bond to - (TC)y in Formula (B1); the wiggly line indicates a bond to -(SP)x- in Formula (B1) or a preceding self-immolative unit; the double dashed line indicates a bond to CA or a further self-immolative unit; g is 0 or 1; R2B is a bond or -C(=Y8)-; Y8 and Y10 are independently O or S; and Y9 is independently O, S, a secondary amine, or a tertiary amine; preferably the self- immolative linker consists of one self-immolative unit; more preferably, the self-immolative linker consists of one self-immolative unit according to Formula (B2A); more preferably, the self-immolative linker consists of one self-immolative unit according to Formula (B2B).
Clause B9. The compound of any one of Clauses B 1 to B8, wherein said compound has a structure according to Formula (B3A) or Formula (B3B): wherein each moiety RL is independently selected from the group consisting of hydrogen, halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, carboxylic acid, RL1, and RL2, provided that at least one moiety RL is RL1 and at least one moiety RL is RL2; wherein Y9 is O, S, a secondary amine, or a tertiary amine; wherein for both Formulae (B3 A) and (B3B): RL1 is wherein X2, X3, X4, X5, and X6 are optionally substituted carbon atoms;
RLC is hydrogen or methyl; and each RL2 is independently selected from the group consisting
C1-4 alkyl, or -O-benzyl; and RQ is hydrogen or acetyl. Clause B10. The compound of any one of Clauses B 1 to B9, wherein said compound has a structure according to any one of Formulae (B4A), (B4B), or (B4C): wherein in each of Formulae (B4A), (B4B), or (B4C): X2, X3, X4, X5, and X6 are optionally substituted carbon atoms; RLC is hydrogen or methyl; and CA is a payload; wherein in each of Formulae (B4B) and (B4C) RP is -NH2, C1-4 alkyl, or -O-benzyl. Clause B 11. A compound comprising an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene and also if said compound is present in a tumor or in a tumor microenvironment; preferably the diene is a tetrazine.
Clause B12. A composition comprising a compound according to any one of Clauses B1 to B11; preferably the composition is a pharmaceutical composition.
Clause B 13. A combination of
(A1) a compound according to any one of Clauses B1 to B11; or (A2) a composition according to Clause B 12; with
(B) a diene; preferably the diene is a tetrazine.
Clause B 14. The compound according to any one of Clauses B1 to B 11 ; the composition according to Clause B 12; or the combination according to Clause B 13; for use as a medicament.
Clause B15. The compound according to any one of Clauses B1 to B 11 ; the composition according to Clause B 12; or the combination according to Clause B 13; for use in the treatment of a disease in a subject, preferably the subject is a human, preferably the disease is cancer.
Clause B16. A non-therapeutic method for reacting:
(ia) a compound according to any one of Clauses B1 to B11; or (iia) a composition according to Clause B12; with a diene, wherein said method comprises the step of contacting (ia) or (iia) with said diene, preferably said contacting is in vitro., and preferably said diene is a tetrazine.
Examples
General methods
All reagents, chemicals, materials and solvents were obtained from commercial sources and were used as received. All solvents were of AR quality. Compounds 1, 2 and 4 were prepared as described in patent application WO 2022/197182. 3-(2-(2-(3-Oxo-3-((6-(6-(Pyridin-2-yl)- l,2,4,5-tetrazin-3-yl)pyridin-3-yl)amino)propoxy)ethoxy)ethoxy)propanoic acid (compound 15) was prepared as described in patent application WO 2024/172652. In the synthetic procedures, equivalents (eq) are molar equivalents. Analytical thin layer chromatography was performed on Kieselgel F-254 precoated silica plates. Column chromatography was carried out on Screening Devices B.V. silica gel (40-63 μm mesh) or on Buchi FlashPure Ecoflex silica 50 μm irregular. 1H NMR and 13C NMR spectra were recorded on a Bruker Avance III HD (400 MHz for 1H NMR and 100 MHz for 13C NMR) spectrometer or a JEOL (500 MHz for 1H NMR) at 298 K. Chemical shifts are reported in ppm downfield from TMS. Abbreviations used for splitting patterns are s = singlet, d = doublet, dd = double doublet, t = triplet, q = quartet, m = multiplet and br = broad. HPLC-MS/PDA was performed using a Shimadzu LC-20 AD VP series HPLC coupled to a diode array detector (Shimadzu SPD- M20A) and an Ion-Trap (LCQ Fleet, Thermo Scientific) MS-detector. Size exclusion chromatography (SEC) was performed on a Shimadzu system equipped with a Superdex200 increase 10/300 column (Cytiva) eluted at 0.75 mL/min with PBS. SDS-PAGE was performed on a Mini -PROTEAN Tetra Cell system using 4-20% precast Mini -PROTEAN TGX gels and Precision Plus Protein All Blue protein standards (BioRad Laboratories). The gels were stained with Coomassie Brilliant Blue for protein detection.
Example 1: Synthesis of antibody conjugate 1.13
Below, the synthesis is described of antibody conjugate 1.13, which is a compound in line with the present claims. Said compound comprises a trans-cyclooctene (TCO) moiety bearing a releasable group on one allylic carbon. The drug monomethyl auristatin E (MMAE) can be released upon reaction of conjugate 1.13 with a diene, such as a tetrazine (e.g. bis-(2-pyridyl)- tetrazine), but also if the glucuronidyl group of conjugate 1.13 is cleaved off by an enzyme that is expressed in tumors (e.g. glucuronidase).
The antibody used in conjugate 1.13 (i.e. trastuzumab) can be used to target conjugate 1.13 to a tumor and/or a tumor microenvironment. Example 1.1 Synthesis of compound 1.1 (tert-butyl methyl(2- (methylamino) ethyl) carbamate) N1 , N2 -Dimethylethane- 1,2-diamine (2.58 mL, 23.9 mmol) was dissolved in dried dichloromethane (DCM, 25 mL). A solution of di-tert-butyl dicarbonate (1.54 g, 7.06 mmol) in DCM (5 mL) was added dropwise to the reaction mixture under stirring. The reaction mixture was stirred at room temperature overnight. The mixture was then washed with saturated aqueous NaHCO3. The aqueous layer was extracted with DCM (2 x 20 mL). The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel using 10% methanol in DCM as the eluent to afford compound 1.1 as a yellow liquid (0.75 g, 3.98 mmol, 57% yield). 1H NMR (500 MHz, CDCl3) δ 3.35 (s, 2H), 2.88 (s, 3H), 2.84 - 2.69 (m, 2H), 2.47 (s, 3H), 1.48 (s, 9H). ESI-MS: m/z Calc, for C9H20N2O2: 196.11 Da; Obs. [M+H]+ 197.12 Da.
Example 1.2 Synthesis of compound 1.2 (tert-butyl (2-((chlorocarbonyl)(methyl)amino)- ethyl) (methyl) carbamate)
Triphosgene (0.237 g, 0.8 mmol) was dissolved in DCM (2 mL) and the solution was cooled to 0 °C with an ice bath. Compound 1.1 (0.237 g, 1.2 mmol) was dissolved in DCM (2 mL) and pyridine (0.5 mL) to form a mixture, and said mixture was added dropwise to the triphosgene solution under a nitrogen atmosphere. The reaction mixture was allowed to reach room temperature and was stirred for 2 hours. After monitoring the reaction completion by thin-layer chromatography (TLC; eluent: MeOH: DCM = 1 : 10, Rf= 0.6), the reaction mixture was diluted with 0.1 N HCl (5 mL) and extracted with DCM (10 mL). The organic layer was washed with HCl (0.1 N, 10 mL) and brine (10 mL), dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure to afford the crude product, compound 1.2, as a yellow oil (0.15 g, 0.60 mmol, 48% yield). The crude product was used directly in the next step without further purification. Example 1.3 Synthesis of compound 1.3 ((2S,3R,4S,5S,6S)-2-(4-formyl-3-hydroxyphenoxy)- 6-(methoxycarbonyl)tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (2.9 g, 7.3 mmol), 2,4- dihydroxybenzaldehyde (2 g, 14.18 mmol) and Ag2O (3.3 g, 14.24 mmol) were dissolved in dried acetonitrile (ACN; 75 mL) under nitrogen atmosphere. The reaction mixture was stirred in the dark at room temperature for 17 h. The solvent was removed under reduced pressure and the residue was dissolved in ethyl acetate (80 mL) and filtered through a Celite cake. The filtrate was washed with IM HCl (50 mL), aqueous NaHCO3 saturated solution (3 x 50 mL) and brine (50 mL) and dried over anhydrous Na2SO4. The mixture was filtered, and the solvent was removed under reduced pressure to yield a dark brown oil, which was purified by column chromatography (eluent: petroleum ether: ethyl acetate (EtOAc) = 2: 1) to afford compound 1.3 as a white solid (1.4 g, 61% yield). 1H NMR (500 MHz, CDCl3) δ 11.33 (s, 1H, -OH), 9.74 (s, 1H), 7.46 (d, J= 8.5 Hz, 1H), 6.59 (dd, J= 8.6, 2.3 Hz, 1H), 6.52 (d, J= 2.3 Hz, 1H), 5.36 - 5.31 (m, 2H), 5.29 - 5.25 (m, 2H), 4.30 - 4.20 (m, 1H), 3.71 (s, 3H), 2.03 (s, 9H).
Example 1.4 Synthesis of compound 1.4 (( 2S, 3R, 4S, 5S, 6S)-2-(3-( ((2-(( tert- butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-4-formylphenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 1.3 (0.06 g, 0.13 mmol) was dissolved in dried DMF (2 mL). Anhydrous K2CO3 (0.07 g, 0.50 mmol) and compound 1.2 (crude product; 0.064 g, 0.25 mmol) were added. The reaction mixture was stirred at room temperature overnight, and then diluted with water (10 mL) and extracted with DCM (2 x 10 mL). The combined organic layers were washed with saturated aqueous NaHCO3, dried over anhydrous Na2SO4, filtered, and concentrated. The crude product was purified by column chromatography (eluent: petroleum ether: EtOAc = 3: 1) to afford compound 1.4 (0.071 g, 80% yield). 1H NMR (500 MHz, CDCl3) δ 10.00 (s, 1H), 7.87 - 7.75 (m, 1H), 6.93 (dq, J= 8.7, 3.3 Hz, 1H), 6.88 - 6.81 (m, 1H), 5.51 - 5.21 (m, 4H), 4.30 - 4.20 (m, 1H), 3.71 (s, 3H), 3.70 (s, 3H), 3.69 (s, 3H), 3.47 - 3.43 (m, 4H), 2.04 (s, 9H), 1.44 (s, 9H).
Example 1.5 Synthesis of compound 1.5 (( 2S, 3R, 4S, 5S, 6S)-2-(3-(((2-(( tert- butoxycarbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-4-(hydroxymethyl)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 1.4 (60 mg, 0.09 mmol) was dissolved in DCM (1 mL) and isopropanol (0.25 mL). 4Å molecular sieves were added to the solution. The mixture was cooled to 0 °C with an ice bath, and then NaBH4 (10 mg, 0.26 mmol) was added. The reaction mixture was maintained at 0 °C for 45 minutes. After dilution with DCM and filtration, the filtrate was washed with brine (2 × 5 mL). The organic layer was concentrated under reduced pressure and purified by column chromatography (eluent: from 30% EtOAc in petroleum ether to 40% EtOAc in petroleum ether) to afford compound 1.5 (35 mg, 0.052 mmol) in 58% yield. 1H NMR (500 MHz, CDCl3) δ 7.36 (m, 1H), 6.86 (ddd, J= 8.2, 4.9, 2.5 Hz, 1H), 6.82 - 6.68 (m, 1H), 5.44 - 5.14 (m, 4H), 4.56 - 4.37 (m, 2H), 4.28 - 4.17 (m, 1H), 3.70 (s, 3H), 3.63 - 3.40 (m, 4H), 3.14 (s, 3H), 3.04 (s, 3H), 2.91 (d, J= 9.9 Hz, 3H), 2.03 (s, 9H), 1.43 (s, 9H).
Example 1.6 Synthesis of compound 1.6 ((2S,3R4S,5S,6S)-2-(4-(hydroxymethyl)-3- ((methyl(2-(methylamino)ethyl)carbamoyl)oxy)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H- pyran-3, 4, 5-triyl triacetate) Compound 1.5 (30 mg, 0.045 mmol) was dissolved in DCM (1 mL) and trifluoroacetic acid (TFA; 0.1 mL) was added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure to give compound 1.6. The crude product was used directly in the next step without further purification.
Example 1. 7 Synthesis of compound 1. 7
To a reaction vial, compound 1.6 (80 mg, 0.14 mmol) and bis-PFP TCO (0.095 g, 0.1067 mmol; compound 1) were added. Dimethylformamide (DMF; 2 mL) was added to the vial to dissolve the solids. 4-Methylmorpholine (0.14 mL, 0.50 mmol) was added to the solution. The reaction mixture was stirred at room temperature overnight. To the reaction mixture, TFA (10 μL) and water (1.3 mL) were added. The mixture was filtered and the filtrate was purified by preparative RP-HPLC (10% to 60% ACN in water, with 0.1% TFA), and subsequently freeze- dried, to yield the product compound 1.7 as a white powder (50 mg, 38% yield over 2 steps). 1H NMR (500 MHz, CDCl3) δ 7.45 - 7.30 (m, 1H), 6.88 - 6.82 (m, 1H), 6.82 - 6.70 (m, 1H), 5.92 - 5.77 (m, 2H), 5.42 - 5.29 (m, 3H), 5.26 - 5.12 (m, 1H), 4.45 (m, 2H), 4.28 - 4.14 (m, 1H), 3.70 (s, 3H), 3.63 - 3.47 (m, 4H), 3.15 (s , 3H), 3.04 (s, 3H), 2.65 - 2.49 (m, 1H), 2.38 - 2.02 (m, 3H), 2.02 (s , 9H), 2.01 - 1.97 (m, 3H), 1.91 - 1.84 (m, 1H).
Example 1.8 Synthesis of compound 1.8 (N-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)- 2, 2, 2-trifluoroacetamide)
Amino-PEGs-amine (0.5 mL, 2.60 mmol) was dissolved in THF (5 mL). The solution was cooled to -18 °C using a salt ice bath. Ethyl trifluoroacetate (0.2 mL, 1.5 mmol) was dissolved in THF (5 mL) and added dropwise to the solution. The reaction mixture was allowed to warm to room temperature and stirred at room temperature overnight. The reaction mixture was evaporated, and the residue was purified by preparative RP-HPLC (5% to 50% ACN in water, with 0.1% TFA), and subsequently freeze-dried, to yield the product compound 1.8 as a colourless oil (0.3 g, 1.03 mmol, 69% yield). 1H NMR (500 MHz, MeOD-d4) δ 3.76 - 3.71 (m, 2H), 3.69 (s, 4H), 3.68 - 3.65 (m, 4H), 3.63 (dd, J= 5.9, 5.2 Hz, 2H), 3.50 (t, J= 5.5 Hz, 2H), 3.19 - 3.12 (m, 2H).
Example 1.9 Synthesis of compound 1.9 ((2S,3R,4S,5S,6S)-2-(3-(((2-(((((lR,6S,E)-6- hydroxy-6-(( 1, 1, l-trifluoro-2-oxo-6, 9, 12-trioxa-3-azatetradecan-14-yl)carbamoyl)cyclooct-2- en-l-yl)oxy)carbonyl)(methyl)amino)ethyl)(methyl)carbamoyl)oxy)-4- (hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
To a solution of compound 1.7 (0.015 g, 0.0158 mol) in DMF (2 mL) were added 4- methylmorpholine (0.046 mL, 0.049 mmol) and compound 1.8 (0.06 g, 20.82 mmol). The reaction mixture was stirred at room temperature overnight. The mixture was diluted with MilliQ water (4 mL, with 0.1% TFA), and (ACN, 2 mL with 0.1% TFA). The resulting mixture was filtered through a 0.45 μm membrane filter and the filtrate was purified by preparative RP-HPLC (30% to 70% ACN in water, with 0.1% TFA), and subsequently freeze- dried, to yield the product compound 1.9 as a colourless oil (13 mg, 64% yield). 1H NMR (500 MHz,CDCl3) δ 7.42 - 7.33 (m, 1H), 6.97 - 6.69 (m, 2H), 5.90 - 5.59 (m, 2H), 5.45 - 5.14 (m, 3H), 4.69 - 4.46 (m, 2H), 4.44 - 4.15 (m, 1H), 3.71 (d, J= 7.2 Hz, 3H), 3.80 - 3.36 (m, 20H), 3.17 - 2.98 (m, 6H), 2.60 - 2.36 (m, 2H), 2.36 - 2.18 (m, 2H), 2.04 (s, 9H), 2.02 - 1.63 (m, 4H).
Example 1.10 Synthesis of compound 1.10
Compound 1.9 (20 mg, 0.019 mmol) was dissolved in anhydrous DMF (1 mL), and to this solution was added bis(4-nitrophenyl)carbonate (12 mg, 0.030 mmol), followed by N,N- diisopropylethylamine (DIEA; 4 μL, 0.023 mmol). The mixture was stirred at room temperature. After 16 h, monomethyl auristatin E (MMAE; free base, 21 mg, 0.029 mmol) was added, followed by DIEA (10 μL, 0.057 mmol). The reaction was stirred at room temperature for 72 h. The crude product was purified by RP-HPLC to afford compound 1.10 (28 mg, 0.016 mmol, yield 84%) as a white powder after lyophilization. ESI-MS: m/z Calc. for C85H128F3N9O29: 1795.88 Da; Obs. [M+H]+: 1797.2 Da.
Example 1.11 _ Synthesis of compound 1.11
To a cooled (ice bath) solution of compound 1.10 (28 mg, 0.016 mmol) in MeOH/ACN/water (1.5 mL, 1 : 1 : 1, v/v/v) was added LiOH (1 N, 0.13 mL, 8 eq.) and the reaction was stirred at room temperature for 4 h. Hydrochloric acid (1 N, 0.07 mL) was added and the mixture was purified directly by RP-HPLC to give compound 1.11 as a white powder after lyophilization (16 mg, TFA salt, 0.01 mmol, yield 63%). ESI-MS: m/z Calc, for C76H121N9O25: 1559.85 Da;
Obs. [M+H]+: 1561.1 Da.
Example 1.12 Synthesis o f compound 1.12
To a stirred solution of compound 1.11 (16 mg, 0.01 mmol) was added 3-maleimidopropanoic acid-pentafluorophenyl ester (4 mg, 0.011 mmol), followed by DIEA (7 μL). The mixture was stirred at room temperature for 10 min, and the crude product was purified by RP-HPLC to give compound 1.12 (16 mg, 0.09 mmol, yield 90%) as a white powder after lyophilization. ESI-MS: m/z Calc, for C83H126N10O28: 1710.87 Da; Obs. [M+H]+ 1712.1 Da.
Example 1.13 Synthesis of conjugate 1.13 (viz, compound 1.12 coupled to HER2- targeting trastuzumab)
To afford antibody-drug conjugate 1.13, trastuzumab (Tmab) was functionalized via maleimide chemistry. Tmab was partially reduced with tris(2-carboxyethyl)phosphine (TCEP;
2 eq) in phosphate-buffered saline (PBS) pH 6.8 for 30 minutes at 37°C, followed by incubation with 10 eq. of compound 1.12 for 2 hours at room temperature. The unreacted linker-drug was removed via preparative SEC, yielding a solution of conjugate 1.13 in PBS pH 7.4 with >95% purity as confirmed by analytical SEC and SDS-PAGE. The concentration of the obtained solution was measured by UV at 280 nm (Nanodrop, Thermofisher). A drug- to-antibody (DAR) ratio of 3-3.5 was measured using a tetrazine titration.
Example 1.14 Synthesis of conjugate 1.14 (viz, compound 1.12 coupled to CEA- targeting labetuzumab)
To afford antibody-drug conjugate 1.14, CEA-targeting labetuzumab was functionalized via maleimide chemistry by partially reducing with tris(2-carboxyethyl)phosphine (TCEP; 2 eq) in phosphate-buffered saline (PBS) pH 6.8 for 30 minutes at 37°C, followed by incubation with 10 eq. of compound 1.12 for 2 hours at room temperature. The unreacted linker-drug was removed via preparative SEC, yielding a solution of conjugate 1.14 in PBS pH 7.4 with >95% purity as confirmed by analytical SEC and SDS-PAGE. The concentration of the obtained solution was measured by UV at 280 nm (Nanodrop, Thermofisher). A drug-to-antibody (DAR) ratio of 3-3.5 was measured using a tetrazine titration.
Example 2: Synthesis of antibody conjugate 2.14
Below, the synthesis is described of antibody conjugate 2.14, which is a compound in line with the present claims. Said compound comprises a trans-cyclooctene (TCO) moiety bearing a releasable group on one allylic carbon. The drug doxorubicin can be released upon reaction of conjugate 2.14 with a diene, such as a tetrazine (e.g. bis-(2-pyridyl)-tetrazine), but also if the glucuronidyl group of conjugate 2.14 is cleaved off by an enzyme that is overexpressed in tumors (e.g. glucuronidase).
The antibody used in conjugate 2.14 (trastuzumab) can be used to target conjugate 2.14 to a tumor and/or a tumor microenvironment.
Example 2.1 Synthesis of compound 2.1 ((2S,3R,4S,5S,6S)-2-(4-formylphenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
2,3,4,6-Tetra-O-acetyl-α-D-glucopyranosyl bromide (0.5 g, 1.26 mmol), 4-hydroxy- benzaldehyde (0.3 g, 2.46 mmol) and Ag2O (0.45 g, 1.94 mmol) were dissolved in dried ACN (75 mL) under nitrogen atmosphere. The reaction mixture was stirred in the dark at room temperature for 16 h. The reaction mixture was filtered through a Celite cake. The filtrate was concentrated, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 2: 1) to afford compound 2.1 (0.18 g, 0.41 mmol, yield 32%) as a white solid. 1H NMR (500 MHz, CDCl3) δ 9.93 (s, 1H), 8.36 - 7.74 (m, 2H), 7.18 - 7.06 (m, 2H), 5.45 - 5.26 (m, 4H), 4.30 - 4.22 (m, 1H), 3.71 (s, 3H), 2.08 - 2.05 (m, 9H). Example 2.2 Synthesis of compound 2.2 ((2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)phenoxy)-6-
(methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 2.1 (180 mg, 0.4 mmol) was dissolved in DCM (2 mL) and isopropanol (0.5 mL). 4Å molecular sieves were added to the solution. The mixture was cooled to 0 °C with an ice bath, and then NaBH4 (40 mg, 1.04 mmol) was added. The reaction mixture was stirred at 0 °C for 45 minutes. After dilution with DCM and filtration, the filtrate was washed with brine (2 x 10 mL). The organic layer was concentrated under reduced pressure and purified by silica gel column chromatography (eluent: from 30% EtOAc in petroleum ether to 40% EtOAc in petroleum ether) to afford compound 2.2 (60 mg, 0.136 mmol, yield 34%). 1H NMR (500 MHz, CDCl3) δ 7.35 - 7.28 (m, 2H), 7.02 - 6.94 (m, 2H), 5.34 (dd, J= 6.9, 2.7 Hz, 2H), 5.27 (td, J = 7.1, 2.8 Hz, 1H), 5.13 (d, J = 7.4 Hz, 1H), 4.64 (s, 2H), 4.22 - 4.15 (m, 1H), 3.73 (s, 3H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H).
Example 2.3 Synthesis of compound 2.3 (tert-butyl (2-((2-aminoethyl)amino)ethyl)- carbamate)
Diethylenetriamine (1.5 mL, 13.8 mmol) was dissolved in dioxane (10 mL). Boc2O (0.69 g, 3.2 mmol) was dissolved in dioxane (15 mL) and added dropwise over 1 hour, while maintaining the reaction mixture in a salt-ice bath at -18°C. The reaction mixture was then stirred overnight at room temperature. The solvent was evaporated under reduced pressure. The residue was dissolved in water, filtered, and the filtrate was extracted with DCM (3 x 15 mL). The combined organic layers were dried over anhydrous MgSO4 , filtered, and concentrated under reduced pressure. The residue was purified using silica gel column chromatography (eluent: methanol with 1% ammonia in DCM from 0% to 20%) to give compound 2.3 (0.29 g, 1.42 mmol, 45% yield). 1H NMR (500 MHz, CDCl3) δ 5.23 (br-s, 1H),
3.38 - 3.09 (m, 2H), 2.77 (q, J= 7.1 Hz, 2H), 2.65 (dt, J= 18.5, 5.7 Hz, 4H), 2.33 (br-s, 3H),
1.38 (s, 9H). Example 2.4 Synthesis of compound 2.4 ((2S,3R,4S,5S,6S)-2-(4-(13,13-dimethyl-3,l 1-dioxo- 2, 12-dioxa-4, 7,10-triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3, 4,5- triyl triacetate)
Compound 2.2 (60 mg, 0.136 mmol) was dissolved in DMF (2 mL). Carbonyldiimidazole (CDI, 24 mg, 0.148 mmol) was added to the solution, and the reaction mixture was stirred at room temperature for 2 hours. A solution of compound 2.3 (22.5 mg, 0.11 mmol) in DMF (0.3 mL) was added to the reaction mixture, and the reaction mixture was then stirred overnight at room temperature. The reaction mixture was diluted with water (with 0.1% TFA) and ACN (with 0.1% TFA) and filtered. The filtrate was purified by preparative RP-HPLC (10% to 60% ACN in water, with 0.1% TFA), and subsequently freeze-dried, to yield the product compound 2.4 (25 mg, 0.037 mmol, 34% yield). 1H NMR (500 MHz, CDCl3) δ 7.34 - 7.27 (m, 2H), 7.01 - 6.91 (m, 2H), 5.35 - 5.29 (m, 2H), 5.27 - 5.22 (m, 1H), 5.13 (d, J= 7.5 Hz, 1H), 5.01 (s, 2H), 4.18 (d, J = 9.3 Hz, 1H), 3.71 (s, 3H), 3.41 - 3.33 (m, 2H), 3.33 - 3.21 (m, 2H), 2.90 - 2.81 (m, 4H), 2.10 - 1.97 (m, 9H), 1.41 (s, 9H). ESI-MS: m/z Calc, for C30H43N3O14: 669.27 Da; Obs. [M+H]+: 670.08 Da.
Example 2.5 Synthesis of compound 2.5 (4-(hydroxymethyl) phenyl (perfluorophenyl) carbonate)
4-Hydroxybenzyl alcohol (0.5 g, 4.02 mmol) was dissolved in DCM (20 mL). Triethylamine (1.5 mL, 10.77 mmol) was added to the solution, followed by the addition of bis(pentafluoro- phenyl) carbonate (1 g, 2.68 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was washed successively with HCl (2N, aqueous) and saturated aqueous NaHCO3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (eluent: EtOAc in petroleum ether from 20% to 80%). Compound 2.5 was obtained as a white solid (0.39 g, 1.17 mmol, 38% yield). 1H NMR (500 MHz, CDCl3) δ 7.47 - 7.35 (m, 2H), 7.29 - 7.20 (m, 2H), 4.71 (s, 2H).
Example 2.6 Synthesis of compound 2.6 (( 2S, 3R, 4S, 5S, 6S)-2-(4-(7-( (4- (hydroxymethyl)phenoxy)carbonyl)-13, 13 -dimethyl- 3, 1 l-dioxo-2, 12-dioxa-4, 7, 10- triazatetradecyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate)
Compound 2.4 (0.1 g, 0.15 mmol) was dissolved in DMF (5 mL). Tri ethylamine (0.11 mL, 0.78 mmol) was added, followed by the addition of compound 2.5 (0.1 g, 0.30 mmol) and 4- dimethylaminopyridine (DMAP; 3 mg, 0.024 mmol). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (with 0.1% TFA) and ACN (with 0.1% TFA) and filtered, and the filtrate was purified by preparative RP-HPLC (10% to 60% ACN in water, with 0.1% TFA), and subsequently freeze-dried, to yield compound 2.6 (50 mg, 0.061 mmol, 41% yield). 1H NMR (500 MHz, CDCl3) δ 7.50 - 7.23 (m, 6H), 7.06 - 6.88 (m, 2H), 5.39 - 5.23 (m, 3H), 5.13 (dd, J= 16.6, 7.4 Hz, 1H), 5.03 (s, 2H), 4.69 (s, 2H), 4.21 - 4.14 (m, 1H), 3.74 - 3.70 (m, 3H), 3.60 - 3.10 (m, 6H), 2.60 - 2.47 (m, 2H), 2.12 - 1.83 (m, 9H), 1.41 (s, 9H). ESI-MS: m/z Calc, for C38H49N3O17: 819.31 Da; Obs. [M+H]+: 820.08 Da.
Example 2. 7 Synthesis of compound 2. 7 ((2S,3R,4S,5S,6S)-2-(4-((((2-((2-aminoethyl)((4- (hydroxymethyl)phenoxy)carbonyl)amino)ethyl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 2.6 (50 mg, 0.061 mmol) was dissolved in DCM (2 mL) and TFA (0.2 mL) was added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated on a rotary evaporator to give compound 2.7. The crude product was used directly in the next step without further purification. ESI-MS: m/z Calc, for C33H41N3O15: 719.25 Da; Obs. [M+H]+: 720.28 Da.
Example 2.8 Synthesis of compound 2.8
Compounds 1.8 (0.2 g, 0.49 mmol) and 2 (0.12 g, 0.38 mmol) were dissolved in ACN (10 mL). Benzotriazol- 1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP; 0.4 g, 0.86 mmol) and diisopropylethylamine (0.36 mL, 2.08 mmol) were added to the solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in DCM and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated. The residue was purified by flash column chromatography (eluent: MeOH in DCM from 0% to 20%), to give compound 2.8 (0.14 g, 0.31 mmol) in a yield of 48%. 1H NMR (500 MHz, CDCl3) δ 5.97 (ddd, J= 15.6, 11.4, 3.4 Hz, 1H), 5.69 (dd, J= 16.5, 2.4 Hz, 1H), 4.50 - 4.26 (m, 1H), 3.67 - 3.52 (m, 8H), 3.10 - 2.97 (m, 8H), 2.40 (qd, J= 12.0, 4.6 Hz, 1H), 2.22 - 2.14 (m, 1H), 2.07 - 1.97 (m, 2H), 1.87 (ddt, J= 14.0, 4.4, 2.0 Hz, 1H), 1.55 (dd, J= 14.0, 6.1 Hz, 1H). Example 2.9 Synthesis o f compound 2.9
To a solution of compound 2.8 (0.14 g, 0.31 mmol) in ACN (5 mL), bis(pentafluorophenyl) carbonate (0.4 g, 1.0 mmol) and diisopropylethylamine (0.16 mL, 0.9 mmol) were added. The reaction mixture was stirred at room temperature for 48 hours. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in DCM and washed with brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by preparative RP-HPLC (40% to 90% ACN in water, with 0.1% TFA), and subsequently freeze-dried, to yield compound 2.9 (0.16 g, 0.24 mmol, 80% yield). 1H NMR (500 MHz, CDCl3) δ 6.00 (dd, J= 17.2, 11.3 Hz, 1H), 5.75 (d, J = 16.2 Hz, 1H), 5.32 (s, 1H), 3.72 - 3.07 (m, 16H), 2.69 - 2.37 (m, 1H), 2.26 - 2.09 (m, 1H), 2.08 - 1.94 (m, 3H), 1.54 - 1.35 (m, 1H). ESI-MS: m/z Calc, for C26H30F8N2O9: 666.18 Da; Obs. [M+H]+: 666.92 Da.
Example 2.10 Synthesis of compound 2.10
Compound 2.6 (30 mg, 0.042 mmol) was dissolved in DMF (1 mL). Compound 2.9 (30 mg, 0.045 mmol) and triethylamine (0.05 mL, 0.35 mmol) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with water (with 0.1% TFA) and ACN (with 0.1% TFA) and filtered, and the filtrate was purified by preparative RP-HPLC (10% to 90% ACN in water, with 0.1% TFA), and subsequently freeze- dried, to yield compound 2.10 (7.5 mg, 0.0062 mmol, 14% yield). 1H NMR (500 MHz, CDCl3) δ 7.67 - 7.20 (m, 4H), 7.02 (d, J= 8.2 Hz, 2H), 6.84 (d, J= 8.2 Hz, 2H), 5.98 - 5.79 (m, 1H), 5.68 (d, J= 16.6 Hz, 1H), 5.50 - 5.34 (m, 2H), 5.19 (td, J= 9.6, 4.7 Hz, 2H), 5.13 (s, 1H), 5.06 (s, 2H), 4.72 - 4.64 (m, 2H), 4.50 - 4.44 (m, 1H), 4.41 - 4.30 (m, 2H), 4.29 - 4.14 (m, 2H), 3.71 (s, 3H), 3.65 - 3.43 (m, 16H), 3.24 - 3.06 (m, 4H), 2.49 - 2.38 (m, 1H), 2.33 - 2.10 (m, 2H), 2.09 - 1.98 (m, 9H), 1.85 - 1.75 (m, 1H), 1.71 - 1.59 (m, 1H), 1.57 - 1.48 (m, 1H). ESI-MS: m/z Calc, for C53H70F3N5O23: 1201.44 Da; Obs. [M-H2O]+: 1183.2 Da, [M- Ac]+ 1159.4 Da. Example 2.11 Synthesis of compound 2.11
Compound 2.10 (2 mg, 0.0017 mmol) was dissolved in anhydrous DMF (0.5 mL) and to this solution 4-nitrophenyl chloroformate (2 mg, 0.0099 mmol) and DIPEA (2 μL) were added. The reaction mixture was stirred at room temperature for 16 hours. Doxorubicin hydrochloride (1.3 mg, 0.0022 mmol) was added, followed by adding DIPEA (2 μL). The reaction mixture was stirred at room temperature for 72 hours. The crude product was purified by RP-HPLC to give compound 2.11 (2 mg, 68% yield) as a light orange powder after lyophilization. ESI-MS: m/z Calc, for C81H97F3N6O35: 1770.59 Da; Obs. [M-Ac]+: 1728.5 Da, [M+Na]+ 1792.9 Da. Example 2.12 Synthesis o f compound 2.12
The synthesis of compound 2.12 follows the procedure described for compound 1.11 in Example 1.11, but starting with compound 2.11. Example 2.13 Synthesis of compound 2.13 and 2.14
The synthesis of compound 2.13 follows the procedure described for compound 1.12 in Example 1.12, but starting with compound 2.12. Compound 2.13 is coupled to trastuzumab, in line with the procedure presented in Example 1.13, to afford antibody doxorubicin conjugate 2.14.
Example 3. Synthesis of other antibody-drug conjugates, used as controls
Below, the synthesis is described of compound 3.1. Said compound comprises a TCO moiety and MMAE that can be released upon reaction with a diene such as a tetrazine (e.g. bis-(2- pyridyl)-tetrazine). Compound 3.2 (Medchem Express) contains a MMAE drug that can be cleaved off by an enzyme that is expressed in tumors (glucuronidase). Their trastuzumab conjugates 3.3 (from 3.1) and 3.4 (from 3.2) were used as controls in examples 15 and 16. Example 3.1 -synthesis of compound 3.1 ((E)-6-((14-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-
3, 6, 9, 12-tetraoxatetradecyl)carbamoyl)-6-hydroxycyclooct-2-en-l-yl ( (S)-l-(( (S)-l-
((( 3R, 4S, 5S)-l-( (S)-2-( (1R, 2R)-3-( ((IS, 2R)-l-hydroxy-l-phenylpropan-2-yl)amino)-l- methoxy-2-methyl-3-oxopropyl)pyrrolidin-l-yl)-3-methoxy-5-methyl-l-oxoheptan-4- yl)(methyl)amino)-3-methyl-l-oxobutan-2-yl)amino)-3-methyl-l-oxobutan-2- yl) (methyl) carbamate)
Compound 3.1 was prepared in several steps in situ. To a solution of MMAE (286 mg, 0.398 mmol) in 4 mL of anhydrous DMF in a glass vial was added bis-PFP TCO (213 mg, 0.379 mmol; compound 1) and DIEA (126 μL, 0.72 mmol). The mixture was stirred at room temperature in the dark for 2 days, at which point LC-MS analysis indicated complete consumption of compound 1 and formation of intermediate 3.5. To this reaction mixture was added a solution of maleimide-PEG4-amine (as mono TFA salt, 163 mg, 0.379 mmol) and DIEA (70 μL, 0.40 mmol) in 4 mL anhydrous DMF. The reaction mixture was stirred at room temperature in the dark for 3 h. The mixture was purified by preparative RP-HPLC (solvent A (0.05% TFA in water), solvent B (ACN), 15 to 75% of B over 30 min at a rate of 50 mL/min, elution time 25 min). The collected fractions were analyzed by LC-MS. The pure fractions were lyophilized in the dark to give compound 3.1. (232 mg, 50 % yield from compound 1). ESIMS: m/z Calc, for C63H101N7O17: 1227.73 Da; Obs. [M+H]+ 1228.9 Da.
Example 3.2 -synthesis of conjugate 3.3
Compound 3.1 was reacted with trastuzumab using the procedure described in Example 1.13, to afford corresponding conjugate 3.3. Example 3.3 -synthesis of conjugate 3.4
Compound 3.2 was reacted with trastuzumab using the procedure described in Example 1.13, to afford corresponding conjugate 3.4.
Example 4. Synthesis of Compound 4.8
This example details the preparation of model compound 4.8, which comprises a self- immolative linker functionalized with cleavable TCO and glucuronide moiety and phenethylamine as model for a releasable drug.
Example 4.1 - Synthesis of compound 4.1 ((2S,3R,4S,5S,6S)-2-(2-formyl-5-nitrophenoxy)-6-
(methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
(2S,3R,4S,5S,6S)-2-Bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3, 4, 5-triyl triacetate (2.18 g, 5.5 mmol) and 2-hydroxy-4-nitrobenzaldehyde (0.84 g, 5.0 mmol) were dissolved in ACN (25 mL), and silver(I)oxide (3.48 g, 15.0 mmol) was added. The orange suspension was stirred at room temperature overnight, and then diluted with EtOAc (25 mL). The brown mixture was filtered over celite and the dark filtrate was evaporated to dryness. The crude product was purified with silica gel column chromatography (eluent: 20% EtOAc in chloroform -> 50% EtOAc in chloroform) and gave compound 4.1 as a white solid (1.43 g, 2.9 mmol, 59% yield). 1H NMR (400 MHz, CDCl3) δ 10.39 (s, 1H), 8.03 (m, 3H), 5.41 (m, 4H), 4.37 (s, 1H), 3.74 (s, 3H), 2.08 (m, 9H) ppm. 13C NMR (100 MHz, CDCl3) δ 187.56, 169.84, 169.31, 169.12, 166.40, 158.17, 151.86, 129.86, 129.65, 118.31, 111.47, 98.45, 72.62, 70.99, 70.48, 68.55, 53.17, 20.57, 20.51 ppm. ESI-MS: m/z Calc. for C20H21NO13483.10 Da; Obs. [M+Na]+ 506.67 Da. Example 4.2 - Synthesis of compound 4.2 ((2S,3R,4S,5S,6S)-2-(2-(hydroxymethyl)-5- nitrophenoxy)-6-(methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 4.1 (0.20 g, 0.41 mmol) was dissolved in chloroform (5 mL) and 2-propanol (1.5 mL). Silica (150 mg) was added and the mixture was cooled to 0°C. NaBH4 (0.024 mg, 0.62 mmol) was added, and the mixture was stirred at 0°C for 30 min, and subsequently filtered over celite. The yellowish solution was washed with brine (5 mL), and the organic layer was dried over Na2SO4, and evaporated to dryness to yield compound 4.2 as a white solid (0.151 g, 0.31 mmol, 76% yield). 1H NMR (400 MHz, CDCl3) δ 8.00 (dd, J= 8.4, 2.1 Hz, 1H), 7.87 (d, J = 2.2 Hz, 1H), 7.60 (d, J= 8.4 Hz, 1H), 5.46 - 5.22 (m, 4H), 4.70 (m, 2H), 4.25 (d, J = 92 Hz, 1H), 3.72 (s, 3H), 2.65 (d, J = 2.2 Hz, 1H), 2.12 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H) ppm. 13C NMR (100 MHz, CDCl3) δ 169.86, 169.58, 169.41, 166.67, 153.88, 148.17, 138.93, 129.71, 119.05, 110.89, 99.18, 72.33, 71.18, 70.76, 68.70, 59.94, 53.18, 20.68, 20.58, 20.50 ppm. ESIMS: m/z Calc, for C20H23NO13 485.12 Da; Obs. [M+Na]+ 508.75 Da.
Example 4.3 - Synthesis of compound 4.3 ((2S,3R,4S,5S,6S)-2-(2-(((tert- butyldimethylsilyl)oxy)methyl)-5-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-
3,4,5-triyl triacetate)
Compound 4.2 (50 mg, 0.103 mmol) was dissolved in DCM (2 mL). Imidazole (14 mg, 0.206 mmol) was added, followed by tert-butyldimethylsilyl chloride (31 mg, 0.206 mmol). The turbid mixture was stirred at room temperature for 1 h, and then diluted with chloroform (3 mL), and washed with, subsequently, saturated sodium bicarbonate solution (4 mL) and brine (4 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified with silica gel column chromatography (eluent: 20% EtOAc in heptane -> 60% EtOAc in heptane) and gave compound 4.3 as a white solid (47 mg, 0.078 mmol, 76% yield). 1H NMR (400 MHz, CDCl3) δ 8.01 (dd, J= 8.4, 2.1 Hz, 1H), 7.82 (d, J= 2.1 Hz, 1H), 7.70 (d, J= 8.4 Hz, 1H), 5.47 - 5.17 (m, 4H), 4.72 (m, 2H), 4.28 (m, 1H), 3.75 (s, 3H), 2.07 (d, J= 5.0 Hz, 9H), 0.96 (s, 9H), 0.13 (s, 6H) ppm. ESI-MS: m/z Calc, for C26H37NO13Si 599.20 Da; Obs. [M+Na]+ 622.75 Da.
Example 4.4 - Synthesis of compound 4.4 ((2S,3R,4S,5S,6S)-2-(5-amino-2-(((tert- butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate)
Compound 4.3 (47 mg, 0.078 mmol) was dissolved in MeOH (3 mL), and triethylamine (3 mg, 0.03 mmol) was added, followed by palladium on activated charcoal (10%) (17 mg). The mixture was vigorously stirred under an atmosphere of hydrogen for 30 min, and then filtered over celite. The filtrate was evaporated to dryness and dried in vacuo to give compound 4.4 as a white solid (44 mg, 0.076 mmol, 98% yield). 1H NMR (400 MHz, CDCl3) δ 7.23 (d, J= 8.4 Hz, 1H), 6.44 (dd, J= 8.1, 2.3 Hz, 1H), 6.34 (d, J= 2.2 Hz, 1H), 5.31 (m, 3H), 5.07 (d, J= 6.6 Hz, 1H), 4.59 (m, 2H), 4.15 (dt, J= 9.6, 4.5 Hz, 1H), 3.73 (s, 3H), 2.05 (m, 9H), 0.93 (s, 9H), 0.08 (s, 6H) ppm. ESI-MS: m/z Calc, for C26H39NO11Si 569.23 Da; Obs. [M+Na]+ 592.50 Da.
Example 4.5 Synthesis o f compound 4.5 (methyl (E)-l-hydroxy-6-(((perfluorophenoxy)carbonyl)oxy)cycloocl-4-ene-l -carboxylate)
Compound 4 (0.1 g, 0.5 mmol) was dissolved in dried DCM (5 mL). Bis(pentafluorophenyl) carbonate (0.2 g, 0.5 mmol) and DIPEA (0.2 mL, 1.15 mmol) were added. The reaction mixture was stirred at room temperature overnight. The reaction mixture was washed successively with citric acid (0.1M, aqueous) and saturated aqueous NaHCO3. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (eluent: EtOAc in petroleum ether from 20% to 80%). Compound 4.5 was obtained as a white solid (0.15 g, 0.37 mmol, 75% yield).1H NMR (500 MHz, CDCl3) δ 6.14 - 5.94 (m, 1H), 5.80 (dd, J= 16.6, 2.3 Hz, 1H), 5.34 (td, J= 2.6, 1.2 Hz, 1H), 2.64 - 2.49 (m, 1H), 2.33 - 2.23 (m, 2H), 2.13 - 2.01 (m, 2H), 2.00 - 1.92 (m, 2H), 1.74 (ddd, J= 16.1, 6.1, 1.6 Hz, 1H).
Example 4.6 - Synthesis of compound 4.6 ((2S,3R,4S,5S,6S)-2-(2-(((tert- butyldimethylsilyl)oxy)methyl)-5-( 6(( Ed-6-hydroxy-6-( methoxy carbonyl)cyclooct-2-en-l- yl)oxy)carbonyl)amino)phenoxy)-6-(methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 4.4 (30 mg, 0.053 mmol) was dissolved in DCM (0.5 mL) and compound 4.5 (32 mg, 0.078 mmol) was added, followed by DIPEA (14 mg, 0.11 mmol) and DMAP (1.6 mg, 0.013 mmol). The mixture was stirred at room temperature overnight and then diluted with chloroform (3 mL) and washed with, subsequently, 0.5 M aqueous citric acid solution (2 mL), saturated sodium bicarbonate solution (2 mL), and brine (2 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 20% EtOAc in heptane -> 80% EtOAc in heptane) and gave compound 4.6 as a white solid (31 mg, 0.039 mmol, 74% yield). 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J = 8.3 Hz, 1H), 7.31 (br.s, 1H), 6.97 (ddd, J= 10.2, 8.3, 2.0 Hz, 1H), 6.75 (s, 1H), 5.91 (m, 1H), 5.82 (m, 1H), 5.43 - 5.24 (m, 4H), 5.17 (m, 1H), 4.66 (m, 2H), 4.21 (m, 1H), 3.77 (s, 3H), 3.72 (s, 3H), 3.06 (s, 1H), 2.53 (qd, J= 11.8, 4.9 Hz, 1H), 2.35 - 2.12 (m, 2H), 2.05 (m, 10H), 2.00 - 1.77 (m, 3H), 1.69 (m, 1H), 0.94 (s, 9H), 0.10 (s, 6H) ppm. ESI-MS: m/z Calc, for C37H53NO16Si 795.31 Da; Obs. [M+Na]+ 819.33 Da. Example 4.7 - Synthesis of compound 4. 7 ((2S,3R,4S,5S,6S)-2-(5-(((((E)-6-hydroxy-6- (methoxycarbonyl)cyclooct-2-en-l-yl)oxy)carbonyl)amino)-2-(hydroxymethyl)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 4.6 (31 mg, 0.039 mmol) was dissolved in THF (0.3 mL), and water (0.7 mL) and formic acid (10 μL) were added. The turbid mixture was stirred at room temperature overnight. The clear solution was evaporated to dryness and the residue was dissolved in chloroform (2 mL), dried over Na2SO4, filtered, and concentrated in vacuo. The residue was redissolved in chloroform (0.2 mL) and precipitated in hexane (2 mL). The precipitate was filtered and dried in vacuo to give compound 4.7 as a white solid (24 mg, 0.035 mmol, 91% yield). 1H NMR (400 MHz, CDCl3) δ 7.46 (s, 1H), 7.26 (s, 1H), 6.89 (d, J= 8.1 Hz, 1H), 6.78 (s, 1H), 5.84 (m, 2H), 5.48 - 5.23 (m, 4H), 5.18 (m, 1H), 4.73 (dd, J= 12.3, 4.5 Hz, 1H), 4.41 (m, 1H), 4.14 (dd, J= 9.4, 5.4 Hz, 1H), 3.77 (s, 3H), 3.70 (s, 3H), 3.05 (s, 1H), 2.78 (m, 1H), 2.53 (m, 1H), 2.24 (m, 2H), 2.11 (s, 3H), 2.06 (s, 3H),. 2.05 (s, 3H), 1.96 (m, 3H), 1.72 (m, 1H) ppm. 13C NMR (100 MHz, CDCl3) δ 179.93, 169.91, 169.62, 169.47, 166.93, 154.99, 152.42, 139.04, 133.38, 130.69, 130.64, 128.62, 127.53, 127.36, 113.97, 100.13, 99.97, 73.23, 72.94, 72.19, 71.53, 70.84, 68.96, 60.19, 53.15, 53.06, 45.46, 32.90, 31.81, 31.59, 30.23, 22.66, 20.70, 20.60, 20.50, 14.12 ppm. ESI-MS: m/z Calc, for C31H39NO16 681.23 Da; Obs. [M+Na]+ 704.83 Da.
Example 4.8 - Synthesis of compound 4.8 ((2S,3R,4S,5S,6S)-2-(5-(((((E)-6-hydroxy-6- (methoxycarbonyl)cyclooct-2-en-l-yl)oxy)carbonyl)amino)-2-
( 77phenethylcarbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl) tetrahydro-2H-pyran- 3, 4, 5-triyl triacetate)
Compound 4.7 (9.0 mg, 0.013 mmol) was dissolved in chloroform (0.6 mL), and phenethyl isocyanate (3.9 mg, 0.026 mmol) and dibutyltin dilaurate (0.41 mg, 0.65 μmol) were added. The clear solution was stirred at 40°C for 2 days, and then evaporated to dryness. The residue was dissolved in chloroform (0.15 mL) and precipitated in hexane (1.5 mL). The precipitate was allowed to settle, and the supernatant was carefully decanted. The residue was washed with hexane and dried in vacuo to give compound 4.8 as a white solid (8.8 mg, 0.011 mmol, 82% yield).. 1H NMR (400 MHz, CDCl3) δ 7.40 (d, J= 13.4 Hz, 1H), 7.34 - 7.06 (m, 5H), 6.96 (t, J= 9.9 Hz, 1H), 6.76 (s, 1H), 5.89 (m, 1H), 5.81 (d, J= 16.6 Hz, 1H), 5.45 - 5.15 (m, 5H), 5.02 (m, 2H), 4.21 (dd, J = 9.4, 3.8 Hz, 2H), 3.77 (s, 3H), 3.65 (s, 3H), 3.41 (m, 2H), 3.04 (s, 1H), 2.80 (m, 2H), 2.53 (dd, J= 11.8, 4.8 Hz, 1H), 2.22 (m, 2H), 2.07 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 1.96 (m, 2H), 1.69 (dd, J = 15.7, 5.8 Hz, 1H) ppm. ESI-MS: m/z Calc, for C40H48N2O17 828.30 Da; Obs. [M+Na]+ 851.75 Da.
Example 4.9 - Synthesis of compound 4.9 ((2S,3S,4S,5R,6S)-6-(5-(((((E)-6-carboxy-6- hydroxycyclooct-2-en-l-yl)oxy)carbonyl) amino)-2-(((phene thy lcarbam()yl)()xy)melhyl)phen()xy)-3, 4, 5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid)
Compound 4.8 (1 eq.) is dissolved in MeOH, and water and lithium hydroxide monohydrate (5 eq.) are added. The mixture is stirred at room temperature overnight, and then neutralized by addition of Amberlite IR120 (H+ form). Product 4.9 is isolated by lyophilization which gives a white powder.
Example 5. Synthesis of Compound 5.8
This example details the preparation of compound 5.8, which comprises a self-immolative linker functionalized with cleavable TCO and glucuronide moiety and phenethylamine as model for a releasable drug. Example 5.1 - Synthesis of compound 5.1 ((2S,3R,4S,5S,6S)-2-(4-formyl-3-nitrophenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
(2S,3R,4S,5S,6S)-2-Bromo-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2.00 g, 5.0 mmol) and 4-hydroxy-2-nitrobenzaldehyde (1.01 g, 6.0 mmol) were dissolved ACN (25 mL), and silver(I)oxide (3.50 g, 15.1 mmol) was added. The orange suspension was stirred at room temperature overnight, and then diluted with EtOAc (25 mL). The brown mixture was filtered over silica and the dark filtrate was evaporated to dryness. The crude product was purified with silica gel column chromatography (eluent: 10% EtOAc in chloroform -> 50% EtOAc in chloroform) and gave compound 5.1 as a white solid (1.51 g, 3.13 mmol, 63% yield). 1H NMR (400 MHz, CDCl3) δ 10.33 (d, J= 0.7 Hz, 1H), 7.99 (d, J= 8.6 Hz, 1H), 7.68 (d, J = 2.4 Hz, 1H), 7.36 (ddd, J = 8.7, 2.5, 0.7 Hz, 1H), 5.52 - 5.11 (m, 4H), 4.30 (d, J= 8.9 Hz, 1H), 3.72 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H) ppm. ESI-MS: m/z Calc, for C20H21NO13 483.10 Da; Obs. [M+Na]+ 506.50 Da.
Example 5.2 - Synthesis of compound 5.2 ((2S,3R,4S,5S,6S)-2-(4-(hydroxymethyl)-3- nitrophenoxy)-6-(methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 5.1 (0.20 g, 0.41 mmol) was dissolved in chloroform (5 mL) and 2-propanol (1 mL). Silica (120 mg) was added, and the mixture was cooled to 0°C. NaBH4 (0.024 mg, 0.62 mmol) was added, and the mixture was stirred at 0°C for 60 min, and subsequently filtered over celite. The yellowish solution was washed with brine (5 mL), and the organic layer was dried over
Na2SO4 and evaporated to dryness to yield compound 5.2 as a white solid (0.196 g, 0.40 mmol, 98% yield). 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J= 2.6 Hz, 1H), 7.68 (d, J = 8.6 Hz, 1H), 7.31 (dd, J= 8.6, 2.6 Hz, 1H), 5.45 - 5.15 (m, 4H), 4.92 (s, 2H), 4.25 (d, J= 9.4 Hz, 1H), 3.74 (s, 3H), 2.53 (br.s, 1H), 2.09 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H) ppm. ESI-MS: m/z Calc, for C20H23NO13 485.12 Da; Obs. [M+Na]+ 508.42 Da. Example 5.3 - Synthesis of compound 5.3 ((2S,3R,4S,5S,6S)-2-(4-(((tert- butyldimethylsilyl)oxy)methyl)-3-nitrophenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran- 3,4,5-triyl triacetate)
Compound 5.2 (174 mg, 0.358 mmol) was dissolved in DCM (5 mL). Imidazole (49 mg, 0.717 mmol) was added, followed by tert-butyldimethylsilyl chloride (108 mg, 0.717 mmol). The turbid mixture was stirred at room temperature for 30 min, and then diluted with chloroform (10 mL), and washed with, subsequently, saturated sodium bicarbonate solution (5 mL) and brine (5 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to dryness. The crude product was purified with silica gel column chromatography (eluent: 20% EtOAc in heptane -> 60% EtOAc in heptane). The product was dissolved in chloroform (0.8 mL) and precipitated in cold heptane. The precipitate was filtered and dried in vacuo to give compound 5.3 as a white solid (164 mg, 0.273 mmol, 76% yield). 1H NMR (400 MHz, CDCl3) δ 7.84 (d, J= 8.7 Hz, 1H), 7.75 (d, J= 2.6 Hz, 1H), 7.32 (dd, J= 8.7, 2.6 Hz, 1H), 5.43 - 5.15 (m, 4H), 5.04 (s, 2H), 4.23 (d, J= 9.5 Hz, 1H), 3.74 (s, 3H), 2.08 (s, 3H), 2.07 (s, 3H), 2.06 (s, 3H), 0.96 (s, 9H), 0.13 (s, 6H) ppm. ESI-MS: m/z Calc, for C26H37NO13Si 599.20 Da; Obs. [M+Na]+ 623.17 Da.
Example 5.4 Synthesis of compound 5.4 ((2S,3R,4S,5S,6S)-2-(3-amino-4-(((tert- butyldimethylsilyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5-triyl triacetate) Compound 5.3 (51 mg, 0.085 mmol) was dissolved in MeOH (2 mL), and triethylamine (2 mg, 0.025 mmol) was added, followed by palladium on activated charcoal (10%) (22 mg). The mixture was vigorously stirred under an atmosphere of hydrogen for 30 min, and then filtered over celite. The filtrate was evaporated to dryness and dried in vacuo to give compound 5.4 as a white solid (48 mg, 0.084 mmol, 99% yield). 1H NMR (400 MHz, CDCl3) δ 6.91 (d, J= 8.0 Hz, 1H), 6.37 - 6.23 (m, 2H), 5.38 - 5.18 (m, 3H), 5.09 (d, J= 7.4 Hz, 1H), 4.62 (s, 2H), 4.26 (br.s, 2H), 4.16 (d, J= 9.6 Hz, 1H), 3.73 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.03 (s, 3H), 0.89 (s, 9H), 0.06 (s, 6H) ppm. ESI-MS: m/z Calc, for C26H39NO11Si 569.23 Da; Obs. [M+Na]+ 592.92 Da.
Example 5.5 - Synthesis of compound 5.5 ((2S,3R,4S,5S,6S)-2-(4-(((tert- butyldimethylsilyl)oxy)methyl)-3-(((((E)-6-hydroxy-6-(methoxycarbonyl)cyclooct-2-en-l- yl)oxy)carbonyl)amino)phenoxy) -6-(methoxycarbonyl) tetrahydro-2H-pyran-3,4,5-triyl triacetate)
Compound 5.4 (48 mg, 0.084 mmol) was dissolved in DCM (0.8 mL) and compound 4.5 (52 mg, 0.127 mmol) was added, followed by DIPEA (22 mg, 0.17 mmol) and DMAP (2.6 mg, 0.021 mmol). The mixture was stirred at room temperature overnight and then diluted with chloroform (5 mL) and washed with, subsequently, 0.5 M aqueous citric acid solution (3 mL), saturated sodium bicarbonate solution (3 mL), and brine (3 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 20% EtOAc in heptane -> 80% EtOAc in heptane) and gave compound 5.5 as a white solid (49 mg, 0.062 mmol, 73% yield). 1H NMR (400 MHz, CDCl3) δ 8.55 (s, 1H), 7.86 (s, 1H), 6.97 (d, J= 8.3 Hz, 1H), 6.61 (dd, J= 8.3, 2.5 Hz, 1H), 5.83 (m, 2H), 5.40 - 5.13 (m, 5H), 4.70 (s, 2H), 4.20 (m, 1H), 3.78 (s, 3H), 3.72 (s, 3H), 3.03 (br.s, 1H), 2.51 (m, 1H), 2.23 (m, 2H), 2.06 (s, 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.01 - 1.75 (m, 4H), 1.68 (m, 1H), 0.96 (s, 9H), 0.12 (s, 6H) ppm. ESI-MS: m/z Calc, for C37H53NO16Si 795.31 Da; Obs. [M+Na]+ 818.83 Da.
Example 5.6 Synthesis o f compound 5.6 ( (2S, 3R, 4S, 5S, 6S)-2-( 3-( ((((E(()-6-hydroxy-6- (methoxycarbonyl)cyclooct-2-en-l-yl)oxy)carbonyl)amino)-4-(hydroxymethyl)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 5.5 (48 mg, 0.060 mmol) was dissolved in ACN (1 mL), and water (1 mL) and formic acid (20 μL) were added. The turbid mixture was stirred at room temperature overnight. The clear solution was lyophilized and the residue was dissolved in chloroform (0.3 mL) and precipitated in hexane (3 mL). The precipitate was filtered and dried in vacuo to give compound 5.6 as a white solid (32 mg, 0.047 mmol, 78% yield). 1H NMR (400 MHz, CDCl3) δ 8.03 (s, 1H), 7.77 (s, 1H), 7.07 (d, J = 8.3 Hz, 1H), 6.66 (dd, J = 8.3, 1.4 Hz, 1H), 5.90 (dd, J= 11.1, 3.5 Hz, 1H), 5.83 (m, 1H), 5.41 - 5.10 (m, 5H), 4.69 (d, J = 4.2 Hz, 2H), 4.20 (d, J = 6.7 Hz, 1H), 3.77 (s, 3H), 3.72 (s, 3H), 3.04 (s, 1H), 2.53 (dd, J= 11.8, 4.7 Hz, 1H), 2.24 (m, 2H), 2.10 (m, 1H), 2.06 (s, 3H), 2.04 (s, 6H), 2.00 - 1.86 (m, 4H), 1.69 (dd, J= 15.8, 6.0 Hz, 1H) ppm. 13C NMR (101 MHz, CDCl3) δ 180.03, 170.07, 169.39, 169.30, 166.96, 157.33, 152.89, 139.23, 133.50, 129.75, 128.57, 123.31, 111.67, 111.60, 98.86, 98.82, 72.99, 72.62, 71.96, 70.97, 69.15, 63.99, 53.12, 52.94, 45.40, 32.89, 31.83, 30.23, 20.63, 20.52 ppm. ESI-MS: m/z Calc, for C31H39NO16 681.23 Da; Obs. [M+Na]+ 704.67 Da. Example 5.7 Synthesis of compound 5.7 ( (2S, 3R, 4S, 5S, 6S)-2-( 3-(((((E)-6-hydroxy-6- (methoxycarbonyl)cyclooct-2-en-l-yl)oxy)carbonyl)amino)-4-
(((phenethylcarbamoyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl) tetrahydro-2H-pyran- 3,4,5-triyl triacetate)
Compound 5.6 (5.1 mg, 7.5 μmol) was dissolved in chloroform (1.2 mL), and phenethyl isocyanate (2.2 mg, 15 μmol) and dibutyltin dilaurate (0.24 mg, 0.38 μmol) were added. The clear solution was stirred at 40°C for 2 days, and then evaporated to dryness. The residue was dissolved in chloroform (0.15 mL) and precipitated in hexane (1.5 mL). The precipitate was allowed to settle, and the supernatant was carefully decanted. The residue was washed with hexane and dried in vacuo to give compound 5.7 as a white solid (6.1 mg, 7.4 μmol, 99% yield). 1H NMR (400 MHz, CDCl3) δ 8.59 (s, 1H), 7.73 (s, 1H), 7.40 - 7.05 (m, 5H), 6.70 (d, J= 8.4 Hz, 1H), 6.00 (t, J= 14.1 Hz, 1H), 5.82 (d, J= 16.3 Hz, 1H), 5.42 - 5.16 (m, 5H), 5.04 (m, 2H), 4.77 (t, J= 6.0 Hz, 1H), 4.20 (d, J= 6.8 Hz, 1H), 3.75 (s, 3H), 3.71 (s, 3H), 3.44 (q, J= 6.7 Hz, 2H), 3.03 (s, 1H), 2.80 (q, J= 6.7 Hz, 2H), 2.54 (dd, J= 12.2, 4.6 Hz, 1H), 2.23 (m, 2H), 2.05 (s, 3H), 2.04 (s, 6H), 1.96 (m, 3H), 1.70 (dd, J= 15.6, 5.8 Hz, 1H) ppm. ESI-MS: m/z Calc, for C40H48N2O17 828.30 Da; Obs. [M+Na]+ 851.83 Da.
Example 5.8 Synthesis of compound 5.8 ((2S,3S,4S,5R,6S)-6-(3-(((((E)-6-carboxy-6- hydroxycyclooct-2-en-l-yl)oxy)carbonyl) amino)-4-
(((phenethylcarbamoyl)oxy)methyl)phenoxy)-3, 4, 5-trihydroxytetrahydro-2H-pyran-2- carboxylic acid)
Compound 5.7 (5.0 mg, 6.0 μmol) was dissolved in methanol (0.3 mL), and water (0.3 mL) and lithium hydroxide monohydrate (1.3 mg, 30 μmol) were added. The clear mixture was stirred at room temperature overnight, and then neutralized by addition of Amberlite IR120 (H+ form) (40 mg). Product 5.8 was isolated by lyophilization which gave a white powder (4 mg, 5.9 μmol, 99% yield). 1H NMR (400 MHz, MeOD-d4) δ 7.40 - 7.05 (m, 5H), 6.99 (d, J= 7.9 Hz, 1H), 5.97 (m, 1H), 5.75 (d, J= 16.2 Hz, 1H), 5.25 - 4.95 (m, 4H), 3.90 (m, 1H), 3.60 (m, 3H), 3.37 (q, J = 7.1 Hz, 2H), 2.77 (q, J = 7.1 Hz, 2H), 2.39 (m, 1H), 2.23 (m, 1H), 2.12 (m, 2H), 1.97 (m, 3H), 1.75 (m, 1H) ppm. ESI-MS: m/z Calc, for C32H38N2O14 674.23 Da; Obs. [M+Na]+ 697.50 Da, [M-H]- 673.75 Da.
Example 6. Synthesis of Compound 6.3
This example details the preparation of compound 6.3, which is an analog of compound 5.8 and comprises a self-immolative linker functionalized with cleavable TCO and glucuronide moiety and doxorubicin as releasable drug.
Example 6.1 - Synthesis of compound 6.1 ((2S,3R,4S,5S,6S)-2-(3-(((((E)-6-hydroxy-6- (methoxycarbonyl)cyclooct-2-en-l-yl)oxy)carbonyl)amino)-4-( (((4- nitrophenoxy)carbonyl)oxy)methyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H-pyran-3,4,5- triyl triacetate)
Compound 5.6 (22 mg, 0.032 mmol) was dissolved in dichloromethane (1 mL). 4-Nitrophenyl chloroformate (10 mg, 0.048 mmol) and DIPEA (6.3 mg, 0.048 mmol) were added, and the mixture was stirred at room temperature overnight and subsequently washed with 0.5 M aqueous citric acid solution (1 mL), saturated sodium bicarbonate solution (1 mL), and brine (1 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 20% EtOAc in heptane -> 80% EtOAc in heptane) and gave compound 6.1 as a white solid (12 mg, 0.014 mmol, 45% yield). 1H NMR (400 MHz, CDCl3) δ 8.29 (d, J= 9.0 Hz, 2H), 7.66 (m, 1H), 7.55 (m, 1H), 7.39 (d, J= 9.0 Hz, 2H), 7.09 (d, J= 9.1 Hz, 1H), 6.79 (d, J= 8.0 Hz, 1H), 5.90 (m, 1H), 5.83 (m, 2H), 5.40 - 5.05 (m, 6H), 4.22 (m, 1H), 3.78 (s, 3H), 3.73 (s, 3H), 3.05 (s, 1H), 2.53 (m, 1H), 2.22 (m, 2H), 2.10 (m, 1H), 2.06 (s, 3H), 2.05 (s, 6H), 2.00 - 1.77 (m, 4H), 1.68 (m, 1H) ppm. ESI-MS: m/z Calc, for C38H42N2O20 846.23 Da; Obs. [M+Na]+ 869.58 Da.
Example 6.2 - Synthesis of compound 6.2 ((2S,3R,4S,5S,6S)-2-(4-(((((2S,3S,4S,6R)-3- hydroxy-2-methyl-6-(((lS,3S)-3,5,12-trihvdroxy-3-(2-hvdroxyacetyl)-10-methoxy-6,ll-dioxo-
1,2,3,4,6,1 l-hexahydrotetracen-l-yl)oxy) tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)methyl)- 3-(((((E)-6-hydroxy-6-(methoxycarbonyl) cyclooct-2-en-l-yl)oxy)carbonyl)amino)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 6.1 (9.2 mg, 0.011 mmol) was dissolved in DMF (0.200 mL) and doxorubicin.HCl (12.6 mg, 0.021 mmol) and DIPEA (7 mg, 0.054 mmol) were added. The mixture was stirred at room temperature for 4 h, and then evaporated to dryness. The residue was dissolved in chloroform (2 mL) and washed with 0.5 M aqueous citric acid solution (1 mL), saturated sodium bicarbonate solution (1 mL), and brine (1 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 10% MeOH in chloroform) to give compound 6.2 as an orange solid (10 mg, 0.008 mmol, 75%). ESI-MS: m/z Calc, for C59H66N2O28 1250.38 Da; Obs. [M+Na]+ 1273.75 Da.
Example 6.3 - Synthesis of compound 6.3 ((2S,3S,4S,5R,6S)-6-(3-(((((E)-6-carboxy-6- hydroxycyclooct-2-en-l-yl)oxy) carbonyl)amino)-4-( f((2S, 3S, 4S, 6R)-3-hydroxy-2-methyl-6- (((1S, 3S)-3, 5, 12-trihydroxy-3-( 2-hydroxyacetyl)-l O-methoxy-6, 11 -dioxo- 1, 2, 3,4,6,11- hexahydrotetracen-l-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid)
Compound 6.2 (1 eq.) is dissolved in MeOH and water and lithium hydroxide monohydrate (5 eq.) are added. The mixture is stirred at room temperature for 1 h, and then neutralized by addition of Amberlite IR120 (H+ form). Compound 6.3 is isolated by lyophilization to give an orange solid.
Example 7. Synthesis of conjugate 7.10
This example details the preparation of compound 7.10, which is an analog of compound 4.8 and comprises a self-immolative linker functionalized with cleavable TCO and a glucuronide moiety, doxorubicin as releasable drug, and a conjugation handle to an antibody.
Example 7.1 - Synthesis of compound 7.1 (tert-butyl (E)-(l-(l ,6-dihydroxycyclooct-4-en-l - yl)-l-oxo-5,8, 1 l-trioxa-2 -azatridecan- 13-yl)carbamate) (E)-l,6-Dihydroxycyclooct-4-ene-l -carboxylic acid (100 mg, 0.538 mmol; compound 2) and tert-butyl (2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)carbamate (165 mg, 0.564 mmol) were dissolved in DMF (2 mL) and DIPEA (347 mg, 2.69 mmol) and PyBOP (307 mg, 0.591 mmol) were added. The mixture was stirred at room temperature overnight and then diluted with chloroform (10 mL) and washed with 0.5 M aqueous citric acid solution (5 mL), saturated sodium bicarbonate solution (5 mL), and brine (5 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 7% MeOH in chloroform) and gave compound 7.1 as a colorless viscous oil. (200 mg, 0.435 mmol, 81% yield). 1H NMR (400 MHz, CDCl3) δ 6.49 (br.s, 1H), 6.03 (t, J= 13.9 Hz, 1H), 5.78 (dd, J= 16.5, 2.4 Hz, 1H), 5.16 (br.s, 1H), 4.52 (s, 1H), 3.63 (m, 8H), 3.57 (m, 4H), 3.45 (m, 2H), 3.3 (m, 2H), 2.48 (qd, J = 11.9, 4.8 Hz, 1H), 2.27 (m, 1H), 2.15 - 1.85 (m, 4H), 1.78 (m, 1H), 1.64 (m, 1H), 1.45 (s, 9H) ppm. ESI-MS: m/z Calc, for C22H40N2O8 460.28 Da; Obs. [M+Na]+ 483.50 Da.
Example 7.2 - Synthesis of compound 7.2 (tert-butyl (E)-(l-(l-hydroxy-6- (((l)erfluoroi)henoxy)carbonyl)oxy)cycloocl-4-en-l-yl)-l-oxo-5,8, 11-lrioxa-2-azalridecan-l 3- yl)carbamate)
Compound 7.1 (50 mg, 0.109 mmol) was dissolved in chloroform (4 mL) and DIPEA (107 mg, 0.271 mmol), DMAP (2.7 mg, 0.022 mmol), and bis(pentafluorophenyl) carbonate (64 mg, 0.163 mmol) were added. The mixture was stirred at room temperature overnight and then washed with 0.5 M aqueous citric acid solution (2 × 5 mL) and saturated sodium bicarbonate solution (2 x 5 mL), subsequently. The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 5% MeOH in chloroform) and gave compound 7.2 as a colorless viscous oil (59 mg, 0.088 mmol, 81% yield). 1H NMR (400 MHz, CDCh) δ 6.50 (br. s, 1H), 6.01 (ddd, J= 15.6, 11.3, 3.5 Hz, 1H), 5.79 (dd, J= 16.5, 2.4 Hz, 1H), 5.34 (s, 1H), 5.11 (br. s, 1H), 3.64 (s, 8H), 3.58 (t, J = 5.1 Hz, 4H), 3.46 (m, 2H), 3.32 (m, 2H), 2.54 (dd, J= 11.9, 4.8 Hz, 1H), 2.42 - 1.88 (m, 6H), 1.79 (dd, J = 15.6, 5.8 Hz, 1H), 1.44 (s, 9H) ppm. ESI-MS: m/z Calc, for C29H39F5N2O10 670.25 Da; Obs. [M+Na]+ 693.70 Da. Example 7.3 - Synthesis of compound 7.3 ((2S,3R,4S,5S,6S)-2-(2-(((tert- butyldimethylsilyl)oxy)methyl)-5-(((((E)-6-( (2, 2-dimethyl-4-oxo-3, 8,11, 14-tetraoxa-5- azahexadecan-16-yl)carbamoyl)-6-hydroxycyclooct-2-en-l-yl)oxy)carbonyl)amino)phenoxy)- 6-(methoxycarbonyl)tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 7.2 (59 mg, 0.088 mmol) was dissolved in dichloromethane (2 mL) and compound 5.4 (33 mg, 0.059 mmol) was added, followed by DIPEA (15 mg, 0.12 mmol) and DMAP (1.8 mg, 0.015 mmol). The mixture was stirred at room temperature overnight, and then diluted with di chloromethane (2 mL), and washed with 0.5 M aqueous citric acid solution (2 mL), saturated sodium bicarbonate solution (2 mL), and brine (2 mL), subsequently. The organic layer was dried over Na2SO4, filtered and concentrated to dryness. The crude product was purified with silica gel column chromatography (eluent: 5% MeOH in chloroform) and gave compound 7.3 as a colorless viscous oil (22 mg, 0.021 mmol, 24% yield). 1H NMR (400 MHz, CDCl3) δ 7.38 (m, 2H), 7.00 (m, 1H), 6.55 (br. s, 1H), 5.90 (m, 1H), 5.79 (d, J= 16.5 Hz, 1H), 5.40- 5.24 (m, 4H), 5.17 (m, 1H), 4.67 (m, 2H), 4.21 (m, 1H), 3.72 (s, 3H), 3.69 - 3.61 (m, 11H), 3.58 (dt, J = 6.1, 3.6 Hz, 4H), 3.46 (m 2H), 3.33 (m, 3H), 2.50 (dd, J= 12.0, 4.9 Hz, 1H), 2.35 - 2.10 (m, 2H), 2.06 (s, 3H), 2.04 (s, 6H), 1.95 (dd, J = 27.4, 15.0 Hz, 3H), 1.73 (dd, J= 15.5, 6.1 Hz, 1H), 1.46 (s, 9H), 0.94 (s, 9H), 0.09 (s, 6H) ppm. ESI-MS: m/z Calc, for C49H77N3O20Si 1055.49 Da; Obs. [M+Na]+ 1078.92 Da.
Example 7.4 - Synthesis of compound 7.4 ((2S,3R,4S,5S,6S)-2-(5-(((((E)-6-((2,2-dimethyl-4- oxo-3, 8,11, 14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-6-hydroxycyclooct-2-en-l- yl)oxy)carbonyl)amino)-2-(hydroxymethyl)phenoxy)-6-(methoxycarbonyl)tetrahydro-2H- pyran-3,4,5-triyl triacetate
Compound 7.3 (22 mg, 0.021 mmol) was dissolved in ACN (0.5 mL) and water (0.5 mL), and formic acid (0.010 mL) were added. The turbid mixture was stirred at room temperature overnight, resulting in a clear solution that was concentrated in vacuo. The residue was dissolved in chloroform (3 mL) and washed with brine (1 mL). The organic layer was dried over Na2SO4, filtered and concentrated to dryness to give compound 7.4 as a white solid (14 mg, 0.015 mmol, 71%). ESI-MS: m/z Calc, for C43H63N3O20 941.40 Da; Obs. [M+Na]+ 964.75 Da.
Example 7.5 - Synthesis of compound 7.5 ((2S,3R,4S,5S,6S)-2-(5-(((((E)-6-((2,2-dimethyl-4- oxo-3, 8,11, 14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-6-hydroxycyclooct-2-en-l- yl)oxv)carbonyl)amino)-2-( (((4-nitrophenoxy) carbonyl)oxy)methyl)phenoxy) -6-
(methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate)
Compound 7.4 (1 eq.) is dissolved in dichloromethane. 4-Nitrophenyl chloroformate (1.2 eq.) and DIPEA (5 eq.) are added, and the mixture is stirred at room temperature overnight and subsequently washed with 0.5 M aqueous citric acid solution, saturated sodium bicarbonate solution, and brine. The organic layer is dried over Na2SO4, filtered and concentrated to dryness. The crude product is purified with silica gel column chromatography to give compound 7.5.
Example 7.6 - Synthesis of compound 7.6 ((2S,3R,4S,5S,6S)-2-(5-(((((E)-6-((2,2-dimethyl-4- oxo-3, 8,11, 14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-6-hydroxycyclooct-2-en-l- yl)oxy)carbonyl)amino)-2-(((((2S, 3S, 4S, 6R)-3-hydroxy-2-methyl-6-(((1S, 3S)-3, 5, 12- trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6, 11 -dioxo- 1, 2, 3, 4, 6, 11 -hexahydrotetr acen-1 - yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)methyl)phenoxy)-6- (methoxycarbonyl) tetrahydro-2H-pyran-3, 4, 5-triyl triacetate) Compound 7.5 (1 eq.) is dissolved in DMF and doxorubicin.HCl (1.1 eq.) and DIPEA (5 eq.) are added. The mixture is stirred at room temperature for 4 h, and then evaporated to dryness. The residue is dissolved in chloroform and washed with 0.5 M aqueous citric acid solution, saturated sodium bicarbonate solution, and brine. The organic layer is dried over Na2SO4, filtered and concentrated to dryness. The crude product is purified with silica gel column chromatography to give compound 7.6 as an orange solid.
Example 7.7 - Synthesis of compound 7. 7 ((2S,3S,4S,5R,6S)-6-(5-(((((E)-6-((2,2-dimethyl-4- oxo-3, 8,11, 14-tetraoxa-5-azahexadecan-16-yl)carbamoyl)-6-hydroxycyclooct-2-en-l- yl)oxy)carbonyl)amino)-2-(((((2S, 3S, 4S, 6R)-3-hydroxy-2-methyl-6-(((1S, 3S)-3, 5, 12- trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6, 11 -dioxo- 1, 2, 3, 4, 6, 11 -hexahydrotetr acen-1- yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid)
Compound 7.6 (1 eq.) is dissolved in MeOH, and water and lithium hydroxide monohydrate (4 eq.) are added. The mixture is stirred at room temperature overnight, and then neutralized by addition of Amberlite IR120 (H+ form). Product 7.7 is isolated by lyophilization, which gives an orange powder.
Example 7.8 - Synthesis of compound 7.8 ((2S,3S,4S,5R,6S)-6-(5-(((((E)-6-((2-(2-(2-(2- aminoethoxy) ethoxy) ethoxy) ethyl) carbamoyl)-6-hydroxycyclooct-2-en-l- yl)oxy)carbonyl)amino)-2-(((((2S,3S, 4S,6R)-3-hydroxy-2-methyl-6-(((1S,3S)-3,5,12- trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6, 11 -dioxo- 1, 2, 3, 4, 6, 11-hexahydrotetracen-l- yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid)
Compound 7.7 (1 eq.) is dissolved in dichloromethane and TFA is added. The mixture is stirred for 2 h, and then evaporated to dryness, redissolved in chloroform, and precipitated in diethyl ether. The orange precipitate is dried in vacuo to give compound 7.8 as the TFA salt.
Example 7.9 - Synthesis of compound 7.9 ((2S,3S,4S,5R,6S)-6-(5-(((((E)-6-((l-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)-2-oxo-6,9,12-trioxa-3-azatetradecan-14-yl)carbamoyl)-6- hydroxycyclooct-2-en-l-yl)oxy)carbonyl)amino)-2-(((((2S, 3S, 4S, 6R)-3-hydroxy-2-methyl-6- (((1S, 3S)-3, 5, 12-trihydroxy-3-( 2-hydroxyacetyl)-10-methoxy-6, 11 -dioxo- 1, 2, 3,4,6,11- hexahydrotetracen-l-yl)oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)oxy)methyl)phenoxy)-3,4,5- trihydroxytetrahydro-2H-pyran-2-carboxylic acid)
Compound 7.8 (1 eq.) is dissolved in DMF, and DIPEA (5 eq.) is added, followed by maleimidoacetic acid N-hydroxysuccinimide ester (1.2 eq.). The mixture is stirred at room temperature for 2 h, and then evaporated to dryness. The residue is dissolved in chloroform and washed with 0.5 M aqueous citric acid solution, saturated sodium bicarbonate solution, and brine. The organic layer is dried over Na2SO4, filtered and concentrated to dryness. The crude product is purified with silica gel column chromatography to give compound 7.9 as an orange solid.
Example 7.10: Synthesis of conjugate 7.10
Compound 7.9 is coupled to the antibody trastuzumab, in line with the procedure presented in Example 1.13, to afford conjugate 7.10.
Example 8. In vitro tetrazine-activated MMAE release from compound 1.12
The release of MMAE from the Compound 1.12 in the presence of tetrazine activator was evaluated under physiological conditions (pH 7.3, 37 °C). The reaction mixture was prepared by adding 10 μL of Compound 1.12 stock solution (10 mM in DMSO) and 8 μL of 3,6- di(pyridin-2-yl)-l,2,4,5-tetrazine stock solution (12.5 mM in DMSO) to 500 μL of pre-warmed aqueous buffer (TEA/CH3COOH; pH = 7.3). Aliquots (50 μL) were collected at 0.1, 1, 2, and 3 hours, quenched with 10 μL of 1 M HCl, and analyzed by HPLC-MS (gradient: 10% → 90% acetonitrile in 0.1% formic acid over 10 min; UV detection at 215 nm). Upon the addition of tetrazine activator, compound 1.12 rapidly undergoes a quantitative inverse electron-demand Diels-Alder (IEDDA) reaction, leading to the formation of the intermediate compound 8.1, as shown in Scheme 1. The relative peak areas of free MMAE (m/z = +718 [M+H]+) and compound 8.1 (m/z = +1174 [M+H]+) were quantified. Time-dependent release data are summarized in Table 1. Within 3 hours, >99% of the MMAE payload had been released. Scheme 1 : Generation of MMAE-linker intermediate (compound 8.1) after tetrazine activation
Table 1 : Tetrazine-activated release of free MMAE from compound 1.12 at pH 7.3 and 37°C
Example 9. In vitro β-Glucuronidase-activated MMAE release from compound 1.12
A reaction mixture comprising 20 μL of compound 1.12 stock solution (10 mM in DMSO) and 20 μL of β-glucuronidase enzyme (GUS, 140 units) in 1000 μL of pre-warmed aqueous buffer (TEA/CH3COOH, pH 7.3) was incubated at 37°C. 150 μL aliquots were withdrawn from the reaction mixture at 0.3, 1, 2, 3, and 4 hours. Each aliquot was immediately subjected to centrifugal filtration using an Ami con® Centrifugal Filter (10 kDa) at 14,000 rpm for 10 minutes to remove the enzyme. The filtrate was analyzed by HPLC-MS (gradient: 10% → 90% acetonitrile in 0.1% formic acid over 10 min; UV detection at 215 nm). The relative peak areas of free MMAE (m/z = +718 [M+H]+) and compound 1.12 (m/z = +1711 [M+H]+, +1729 [M+H2O+H]+) were quantified. Time-dependent release data are summarized in Table 2 and show 77.5% payload release in 4 hours.
Table 2: Glucuronidase-activated MMAE release from Compound 1.12 at pH 7.3 and 37 °C
Example 10. In vitro stability of compound 1.12
The stability of compound 1.12 under specified conditions was monitored by HPLC-MS (gradient: 10% → 90% acetonitrile in 0.1% formic acid over 10 min). Example 10.1 - Short-term stability at 37 °C:
Aliquots of compound 1.12 (2 μL, 10 mM in DMSO) were diluted with 100 μL of pre-warmed aqueous buffer (TEA/CH3COOH; pH 7.3) and incubated at 37 °C for 4 h. HPLC-MS analysis showed no detectable MMAE release (m/z = +718 [M+H]+). The same procedure was repeated for a 24 h incubation period at 37 °C, and similarly, no MMAE release was observed.
Example 10.2 - Long-term storage stability at -20 °C:
Compound 1.12 stock solution (10 mM in DMSO) was stored at -20 °C for 4 months. 2 μL aliquots were diluted with 100 μL aqueous buffer (TEA/CH3COOH; pH 7.3). HPLC-MS analysis confirmed no MMAE release (m/z = +718 [M+H]+).
Example 11. In vitro tetrazine-activated payload release from compound 5.7
Compound 5.7 (0.17 mg, 0.20 μmol) was dissolved in ACN (0.300 mL) and water (0.700 mL).
3,6-Di(pyridin-2-yl)-l,2,4,5-tetrazine (0.059 mg, 0.25 μmol) was added. The solution was homogenized and immediately analyzed by HPLC-MS/PDA to reveal the complete conversion of 5.7, and formation of the elimination product (m/z = +391 [M+H]+) and the payload-linker construct (m/z = +625 [M+Na]+). The sample was incubated at 37°C for 3 h, and then analyzed once again by HPLC-MS/PDA to show the decrease of the payload-linker construct and formation of the free payload phenethylamine (m/z = +122 [M+H]+) and linker-glucuronide (m/z = +478 [M+Na]+). Example 12. In vitro tetrazine-activated payload release from compound 5.8
Compound 5.8 (0.12 mg, 0.18 μmol) was dissolved in ACN (0.100 mL) and water (0.900 mL). 3,6-Di(pyridin-2-yl)-l,2,4,5-tetrazine (0.042 mg, 0.18 pmol) was added. The solution was homogenized and immediately analyzed by HPLC-MS/PDA to reveal the complete conversion of 5.8, and formation of the elimination product (m/z = +377 [M+H]+) and the payload-linker construct (m/z = -461 [M-H]'). The sample was incubated at 37°C for 1 h, and then analyzed once again by HPLC-MS/PDA to show the complete disappearance of the payload-linker construct and formation of the free payload phenethylamine (m/z = +122 [M+H]+).
Example 13. In vitro enzyme-activated payload release and stability of compound 5.8
Example 13.1. In vitro enzyme-activated payload release from compound 5.8
A reaction mixture comprising 20 μL of compound 5.8 stock solution (50 mM in DMSO) and 2 μL of β-glucuronidase enzyme (GUS, 140 units) in 500 μL of pre-warmed aqueous buffer (TEA/CH3COOH, pH 7.3) was incubated at 37°C. 150 μL aliquots were withdrawn from the reaction mixture at 1 min, 10 min, 30 min and 1 hour. Each aliquot was immediately subjected to centrifugal filtration using an Amicon® Centrifugal Filter (10 kDa) at 14,000 rpm for 10 minutes to remove the enzyme. The filtrate was analyzed by HPLC-MS to show the complete disappearance of the payload-linker construct and formation of the free payload phenethylamine (m/z = +122 [M+H]+) within 1 hour. Example 13.2 Short-term stability at 37 °C of compound 5.8
Aliquots of compound 5.8 (3 μL, 50 mM in DMSO) were diluted with 75 μL of pre-warmed aqueous buffer (TEA/CH3COOH; pH 7.3) and incubated at 37°C for 2 h. HPLC-MS analysis showed no detectable payload (m/z = +122 [M+H]+).
Example 14. In vitro tetrazine-activated payload release from compound 6.2
Compound 6.2 (1 eq.) was dissolved in 30% ACN in water. 3,6-Di(pyridin-2-yl)-l,2,4,5- tetrazine (1 eq.) was added and the solution was homogenized and immediately analyzed by HPLC-MS/PDA to reveal the complete conversion of 6.2, and formation of the elimination product (m/z = +391 [M+H]+) and the payload-linker construct (m/z = +1047 [M+Na]+). Incubation at 37°C for 3 h, and analysis by HPLC-MS/PDA showed the complete disappearance of the payload-linker construct and formation of free doxorubicin (m/z = +544 [M+H]+).
Example 15. In vitro cytotoxicity evaluation in tumor cells
The HER2 expressing BT-474 cell line (American Type Culture Collection) was cultured in RPMI-1640 medium supplemented with 2 mM glutamine and 10% heat inactivated fetal calf serum. On the day of the experiment, stock solutions of conjugates 1.13, 3.3 and 3.4, as well as unconjugated Tmab and MMAE, were serially diluted with RPMI-1640 in 48-well cell culture plates. BT-474 cells were then added to the wells at a 20,000 cells/well density, resulting in a 0.4 ml/well final volume, and the plates were incubated at 37°C with 5% CO2. At t = 0 and after 24, 48, and 72 hours incubation, a serially diluted solution of tetrazine 15 (Tz; 100 eq with respect to TCO) was added to some wells. Three wells were used per condition. After 120 hours incubation, an MTT assay was performed in duplicate and the cell proliferation data were analyzed using GraphPad Prism (v. 10.4.1). The IC50 values calculated from non-linear curve fitting are reported in Table 3.
Table 3: In vitro cytotoxicity assay in BT-474 cells after 120 h incubation: IC50 (half-maximal inhibitory concentration) values obtained with conjugates 1.13 and 3.3 alone or with tetrazine 15 (Tz) added at time 0, 24h, 48h or 72 h (Tmab, MMAE, Tz and conjugate 3.4 used as controls)
The results in Table 3 show that internalizing conjugate 1.13 is approximately 80-fold more toxic than unconjugated Tmab and approximately as toxic towards HER2 positive BT-474 cells as conjugate 3.4 (enzymatically-cleavable control), proving that enzymatic cleavage of the linker after conjugate 1.13 internalization in cells is effective. The toxicity of free MMAE is completely restored when conjugate 1.13 is treated with tetrazine at various times, thus releasing extracellularly the MMAE conjugated to ADC that has not yet internalized.
On the contrary, conjugate 3.3, in which MMAE is linked to Tmab via TCO, is only as toxic as unconjugated Tmab unless a tetrazine is administered when the ADC is still located outside the cells (t=0). After 24, 48 or 72 h, when some conjugate 3.3 has already internalized and is out of reach of the cell-impermeable tetrazine 15, tetrazine addition results in MMAE release only from the portion of conjugate 3.3 that is still located outside the cells and therefore the achieved cell killing efficacy is lower that what can be achieved with conjugate 1.13. This proves that the glucuronidyl- and TCO-containing linker design of this disclosure gives an advantage in drug release over exclusively chemically-cleavable linkers when used in internalizing antibody-drug conjugates, and also can have a benefit over exclusively biologically-cleavable linkers.
Example 16. In vivo in-tumor MMAE release from conjugates 1.13 and 3.3
The animal studies were performed in accordance with the principles established by the revised Dutch Act on Animal Experimentation (1997) and were approved by the institutional Animal Welfare Committee of the Radboud University Nijmegen. A 17-β estradiol pellet (0.18 mg/60-day release) was placed subcutaneously in the neck of female BALB/c nude mice (7-9-week-old, 20-25 g body weight) under anesthesia and the mice were then subcutaneously inoculated with BT-474 cells (5 million cells in 100 μL 1 : 1 medium: matrigel) in the right flank. When the tumors reached an approximately 100 mm3 size, the animals were injected with 111In-labeled Tmab, conjugate 1.13 or conjugate 3.3 (5 mg/kg in 100 μL). 75h post-mAb administration, the animals were euthanized. Plasma, tumor and other selected tissues were harvested and weighed. The samples from mice injected with 111In-labeled Tmab were measured in a gamma-counter, together with standards, to calculate the % injected dose per gram (%ID/g).
The tumors of mice injected with conjugates 1.13 and 3.3 were homogenized and MMAE was extracted using a published procedure (Rossin et al., Nat Commun 2018). Briefly, the samples were contacted with MeOH (5 mL/gr tumor) with an internal standard (d8-MMAE, MedChem Express) and were homogenized using a MagNA Lyser device (Roche; 4×30 sec cycles, 6500 rpm, with 1 min cooling between cycles). After eliminating the debris, MeOH was evaporated under a stream of N2 and the residue was reconstituted in 20% ACN in water added with 0.1% formic acid. The solutions were analyzed by quantitative HPLC-SIM-MS on a Shimadzu LC-10 AD VP series HPLC equipped with an electrospray ion-trap mass spectrometer (LCQ Fleet, Thermo Scientific). The two analytes, dO-MMAE and d8-MMAE, were detected in SIM mode at m/z=718.4 Da and 726.4 Da, respectively. The absolute amount of MMAE released in tumors was quantified from the ratio between MMAE and internal standard while the % release was estimated from the %ID/g 111In-labeled Tmab, assuming similar tumor uptakes for unconjugated Tmab and conjugates 1.13 and 3.3.
Table 4: Concentration of free MMAE and % MMAE release (±SD) in BT-474 xenografts in mice administered conjugate 1.13 (n=4) and conjugate 3.3 (n=3), 75 hours post-administration
The results in Table 4 show that at 3 days post-injection the linker containing the glucuronidyl group in conjugate 1.13 is activated enzymatically in mouse xenografts in vivo producing a high concentration of free MMAE. On the contrary, the administration of conjugate 3.3 containing the TCO linker, if not followed by a tetrazine, produces only a low amount of drug in tumor upon conjugate catabolism.

Claims

Claims
1. A compound comprising an (E)-cyclooctene moiety, wherein at least one non-vinylic carbon of said moiety is substituted with at least one group according to Formula (1); wherein
SP is a spacer;
LC is a self-immolative linker;
TC is a group that is separable from LC due to conditions present in a tumor and/or due to conditions in a tumor microenvironment;
CA is a payload; x is 0 or 1; y is an integer of from 0 to 4; z is 0 or 1; provided that: a) at least one allylic carbon of said (E)-cyclooctene moiety is substituted with a first group according to Formula (1); i. if in said first group x is 1, then SP is -Y1-C(=Y2)-, and LC or CA is connected to SP via O, S, or N, wherein said O, S, or N is part of LC or CA; Y1 and Y2 are independently O or S; ii. if in said first group x is 0, then LC or CA is connected to said allylic carbon via O or S, wherein said O or S is part of LC or CA; b) if said compound does not comprise another group according to Formula (1) in addition to said first group, then in said first group y is an integer of from 1 to 4; c) if said compound comprises one or more groups according to Formula (1) in addition to said first group, then in said one or more groups y is an integer of from 1 to 4.
2. The compound of claim 1, wherein said compound does not comprise another group according to Formula (1) in addition to said first group.
3. The compound of any one of the preceding claims, wherein TC is a group that is separable from LC by: a) an enzyme expressed by a tumor cell; b) an enzyme overexpressed in a tumor microenvironment; and/or c) the reducing potential in a tumor or a tumor microenvironment.
4. The compound of any one of the preceding claims, wherein TC is selected from the group consisting of glucuronidyl, polyglucuronidyl, N-acetylglucosamidyl, galactosyl, a peptide, phosphate, and combinations thereof; preferably TC is a peptide.
5. The compound of any one of the preceding claims, wherein TC is a dipeptide; preferably the dipeptide is an N-terminally protected dipeptide that is connected to LC via a peptide bond at the C-terminus of said N-terminally protected dipeptide; more preferably the dipeptide is RP-C(O)-P1-P2-, wherein RP is
-NH2, C1-4 alkyl, or -O-benzyl; and P1 and P2 are independently amino acid residues; preferably P1-P2 is valine-citrulline or phenylalanine-lysine.
6. The compound of any one of claims 1 to 4, wherein TC is glucuronidyl.
7. The compound of any one of the preceding claims, wherein CA is a drug; preferably CA is selected from the group consisting of monomethyl auristatin E, doxorubicin, camptotecin, camptotecin derivatives, exatecan, and exatecan derivatives; most preferably CA is monomethyl auristatin E.
8. The compound of any one of the preceding claims, wherein the self-immolative linker consists of one or more self-immolative units, wherein the one or more self-immolative units are independently a group according to Formula (2A), a group according to Formula (2B), or a group according to Formula (2C): wherein
A is a 5-membered or 6-membered (hetero)aromatic ring;
R2A is -C(=Y4)-Y5- or -Y5-;
Y3 and Y5 are independently O, S, a secondary amine, or a tertiary amine;
Y4, Y6, and Y7 are independently O or S; e is 0, 1 or 2; f is 0 or 1; A1 is 1 or 2;
A2 is 0, 1, 2, or 3; each B1 is an optionally substituted carbon; each Cl is selected from the group consisting of halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, and carboxylic acid; provided that if A is a 6-membered (hetero)aromatic ring, then -Y3- is at an ortho or para position relative to -(B 1)A1-Y6-C(=Y7)-; and if e is at least 1, then each (*-C(=Y4)-Y5)e, is at an ortho or para position relative to -(B 1)A1-Y6-C(=Y7)-; and provided that if A is a 5-membered (hetero)aromatic ring and -(B 1)A1-Y6-C(=Y7)- is at the 1-position of said 5-membered (hetero)aromatic ring, then -Y3- is at the 3- or 4-position, and if e is at least 1, then each (*-C(=Y4)-Y5)e, is at the 3- or 4-position; wherein g is 0 or 1;
R2B is a bond or -C(=Y8)-; Y8 and Y10 are independently O or S;
Y9 is independently O, S, a secondary amine, a tertiary amine, or -C(Y9A)2-;
Y9A is hydrogen or methyl;
Y11 is hydrogen or methyl; wherein
R2C is a bond or -C(=Y14)-;
Y11 is hydrogen or methyl;
Y12 is N or CH; and
Y13 and Y14 are independently O or S; and wherein for Formula (2A), Formula (2B), and Formula (2C), the asterisk indicates a bond to - (TC)y in Formula (1) or a bond to a further self-immolative unit; the wiggly line indicates a bond to - (SP)x- in Formula (1) or a bond to a preceding self-immolative unit; and the double dashed line indicates a bond to CA or a bond to a further self-immolative unit; preferably Y9 is O, S, a secondary amine, or a tertiary amine; and preferably Y9A is hydrogen.
9. The compound of any one of the preceding claims, wherein said compound has a structure according to Formula (3 A), Formula (3B), or Formula (3C): wherein each moiety RL is independently selected from the group consisting of hydrogen, halogen, an optionally substituted carbon, an optionally substituted nitrogen, hydroxyl, ester, ether, carboxylic acid, RL1, and RL2, provided that at least one moiety RL is RL1 and at least one moiety RL is RL2;
wherein for Formula (3B) and Formula (3C) Y9 is O, S, a secondary amine, or a tertiary amine; wherein Y11 is hydrogen or methyl; wherein for Formula (3 A), Formula (3B), and Formula (3C):
RL1 is wherein X2, X3, X4, X5, and X6 are optionally substituted carbon atoms; each RLC is independently hydrogen or methyl;
RNM is 0 or 1; and each RL2 is independently selected from the group consisting of
RP is -NH2, C1-4 alkyl, or -O-benzyl; and RQ is hydrogen or acetyl.
10. The compound of any one of the preceding claims, wherein said compound has a structure according to any one of Formulae (4A), (4B), (4C), (4D), or (4E):
wherein in each of Formulae (4 A), (4B), (4C), (4D), and (4E):
X2, X3, X4, X5, and X6 are optionally substituted carbon atoms;
RLC is hydrogen or methyl; and CA is a payload; and wherein in each of Formulae (4B) and (4C) RP is -NH2, C1-4 alkyl, or -O-benzyl; wherein for Formula (4D) Y11 is hydrogen or methyl.
11. A compound according to any one of the preceding claims, wherein the compound is compound A-12 or compound A-28: wherein compound A-12 is: and compound A-28 is:
; or a conjugate of compound A-12 or compound A-28 wherein said compound is bound to a targeting agent via the maleimide group of compound A-12 or compound A-28; wherein the targeting agent is an antibody or a diabody.
12. A compound comprising an (E)-cyclooctene moiety and at least one payload linked to said (E)-cyclooctene moiety in such a way that said at least one payload is released from said (E)-cyclooctene moiety if said compound is contacted with a diene and also if said compound is present in a tumor or in a tumor microenvironment; preferably the diene is a tetrazine.
13. A composition comprising a compound according to any one of the preceding claims; preferably the composition is a pharmaceutical composition.
14. A combination of
(A1) a compound according to any one of claims 1 to 12; or
(A2) a composition according to claim 13; with
(B) a diene; preferably the diene is a tetrazine.
15. The compound according to any one of claims 1 to 12; the composition according to claim 13; or the combination according to claim 14; for use as a medicament.
16. The compound according to any one of claims 1 to 12; the composition according to claim 13; or the combination according to claim 14; for use in the treatment of a disease in a subject, preferably the subject is a human, preferably the disease is cancer.
17. A non-therapeutic method for reacting:
(ia) a compound according to any one of claims 1 to 12; or (iia) a composition according to claim 13; with a diene, wherein said method comprises the step of contacting (ia) or (iia) with said diene, preferably said contacting is in vitro., and preferably said diene is a tetrazine.
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