WO2022103983A2 - Composés de fluorénylméthyloxycarbonyle et de fluorénylméthylaminocarbonyle, conjugués protéines associés et méthodes d'utilisation - Google Patents

Composés de fluorénylméthyloxycarbonyle et de fluorénylméthylaminocarbonyle, conjugués protéines associés et méthodes d'utilisation Download PDF

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WO2022103983A2
WO2022103983A2 PCT/US2021/059014 US2021059014W WO2022103983A2 WO 2022103983 A2 WO2022103983 A2 WO 2022103983A2 US 2021059014 W US2021059014 W US 2021059014W WO 2022103983 A2 WO2022103983 A2 WO 2022103983A2
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independently
compound
conjugate
group
certain embodiments
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WO2022103983A3 (fr
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Krishna BAJJURI
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Sutro Biopharma, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/331—Polymers modified by chemical after-treatment with organic compounds containing oxygen
    • C08G65/332—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof
    • C08G65/3324—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof cyclic
    • C08G65/3326—Polymers modified by chemical after-treatment with organic compounds containing oxygen containing carboxyl groups, or halides, or esters thereof cyclic aromatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33303—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group
    • C08G65/3331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group cyclic
    • C08G65/33313—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group cyclic aromatic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen

Definitions

  • FIELD FIELD
  • fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds, and macromolecule conjugates thereof are provided herein; pharmaceutical compositions comprising the compounds and/or conjugates; methods of producing the compounds and/or conjugates; and methods of using the compounds, conjugates, and compositions for therapy.
  • the compounds, conjugates, and compositions are useful, for instance, in methods of treatment and prevention of cell proliferation and cancer, methods of detection of cell proliferation and cancer, and methods of diagnosis of cell proliferation and cancer.
  • the compounds, conjugates, and compositions are also useful in methods of treatment, prevention, detection, and diagnosis of inflammatory diseases or conditions.
  • BACKGROUND [0003] Biotherapeutics provide a wealth of treatment and diagnostic potential for patients worldwide.
  • fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds of Formulas (I), (X), and sub-formulas thereof compositions comprising the compounds, methods of producing the compounds, and methods of using the compounds, conjugates, and compositions in treatment and diagnosis.
  • the compounds of Formula (I), (X), and sub-formulas and embodiments thereof are useful for modulating the bioavailability and ADME of macromolecular compounds.
  • the compounds can be used to prepare prodrug conjugates of macromolecular compounds for use in vivo or elsewhere.
  • the compounds and conjugates feature electron withdrawing groups on fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl cores. These electron withdrawing groups can be varied to tune the plasma stability of the conjugates. This provides a platform for modulating the bioavailability and ADME of a macromolecule in vivo.
  • a compound of Formula (I) or (X) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein RG is a reactive group; POLY is a water soluble polymer; X is a bond, -O-, or -N(R 2 )-; each of L 1 , L 2 , and L 3 is, independently, a linker; each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group; wherein either L 1 comprises a carbonyl carbon covalently bound to the fluorene, or R 1 comprises an electron withdrawing group, or both; R 4 is hydrogen or an electron withdrawing group; R 2 is hydrogen or lower alkyl; n is an integer selected from one to four; n1 is an integer selected from one to four; m is an integer selected from zero to four; and m2 is an integer selected from one to four.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein RG is a reactive group; POLY is a water soluble polymer; X is a bond, -O-, or -N(R 2 )-; each of L 1 and L 2 is, independently, a linker wherein L 1 comprises a carbonyl carbon covalently bound to the fluorene; each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group; R 2 is hydrogen or lower alkyl; and n is an integer selected from one to four.
  • a compound of Formula (X) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof; wherein RG is a reactive group; POLY is a water soluble polymer; X is a bond, -O-, or -N(R 2 )-; each of L 1 , L 2 , and L 3 is, independently, a linker wherein L 1 comprises a carbonyl carbon covalently bound to the fluorene; each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group; R 4 is hydrogen or an electron withdrawing group; R 2 is hydrogen or lower alkyl; and m is an integer selected from zero to four.
  • conjugates comprising residues of compounds of Formula (I) or (X), and sub formulas and embodiments thereof.
  • the conjugates are according to or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof, regioisomer, and/or mixture of regioisomers thereof; wherein each RG′ is independently a divalent residue of a reactive group; each POLY is independently a water soluble polymer; each X is independently a bond, -O-, or -N(R 2 )-; each L 1 , L 2 , and L 3 is, independently, a linker; each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group; wherein either L 1 comprises a carbonyl carbon covalently bound to the fluorene, or R 1 comprises an electron withdrawing group, or both R 4 is hydrogen or an electron withdrawing group; each R 2 is independently hydrogen or lower alkyl; each
  • conjugates comprising residues of compounds of Formula (I) or (X), and sub formulas and embodiments thereof.
  • the conjugate is according to Formula (I′), wherein each RG′ is independently a divalent residue of a reactive group; each POLY is independently a water soluble polymer; each X is independently a bond, -O-, or -N(R 2 )-; each of L 1 and L 2 is, independently, a linker wherein L 1 comprises a carbonyl carbon covalently bound to the fluorene; each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group; each R 2 is independently hydrogen or lower alkyl; each n is independently an integer selected from one to four; PRO is a macromolecular moiety; and w is an integer selected from one to ten; or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, and/or mixture of regioisomers thereof.
  • the conjugate is according to Formula (X′), wherein each RG′ is independently a divalent residue of a reactive group; each POLY is independently a water soluble polymer; each X is independently a bond, -O-, or -N(R 2 )-; each of L 1 , L 2 , and L 3 is, independently, a linker wherein L 1 comprises a carbonyl carbon covalently bound to the fluorene; each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group; each R 4 is hydrogen or an electron withdrawing group; each R 2 is independently hydrogen or lower alkyl; each m is independently an integer selected from zero to four; PRO is a macromolecular moiety; and w is an integer selected from one to ten; or a pharmaceutically acceptable salt, solvate, stereoisomer, tautomer, and/or mixture of regioisomers thereof.
  • each RG′ is independently a divalent residue of a reactive group
  • each POLY is independently a
  • the conjugates are useful in methods of treatment and prevention of cell proliferation and cancer, methods of detection of cell proliferation and cancer, and methods of diagnosis of cell proliferation and cancer. In certain aspects, the conjugates are also useful in methods of treatment and prevention of inflammatory diseases and conditions.
  • compositions comprising a compound of Formula (I) or (X), or certain embodiments thereof, or a conjugate of Formula (I′) or (X′), or certain embodiments thereof. In some embodiments, the compositions are pharmaceutical compositions. Any suitable pharmaceutical composition may be used.
  • kits comprising the compound of Formula (I) or (X), or embodiments thereof, or a conjugate of Formula (I′) or (X′), or a pharmaceutical composition thereof.
  • methods of using the compounds or the conjugates described herein are for delivering one or more macromolecules to a target cell or tissue.
  • the methods are for treatment.
  • the methods are diagnostic methods.
  • the methods are analytical methods.
  • the compounds or conjugates described herein are used to treat a disease or condition.
  • the disease or condition is selected from a cancer, and/or an inflammatory disease or condition.
  • FIGS. 1 and 2 show protein conjugation and subsequent release of functionalized fluorenylmethyloxycarbonyl compounds
  • FIG. 3 is an SDS-PAGE gel showing release of the PEG from the compound A conjugate.
  • FIG.4 is an SDS-PAGE gel showing conjugation efficiency.
  • the term “about” indicates and encompasses an indicated value and a range above and below that value. In certain embodiments, the term “about” indicates the designated value ⁇ 10%, ⁇ 5%, or ⁇ 1%. In certain embodiments, the term “about” indicates the designated value ⁇ one standard deviation of that value. In certain embodiments, for example, logarithmic scales (e.g., pH), the term “about” indicates the designated value ⁇ 0.3, ⁇ 0.2, or ⁇ 0.1. [0022] When referring to the compounds provided herein, the following terms have the following meanings unless indicated otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art.
  • Alkoxy and alkoxyl refer to the group –OR′′ where R′′ is alkyl or cycloalkyl. Alkoxy groups include, in certain embodiments, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like.
  • alkoxyamine refers to the group -alkylene-O-NH 2 , wherein alkylene is as defined herein.
  • alkoxyamine groups can react with aldehydes to form oxime residues.
  • alkoxyamine groups include -CH 2 CH 2 -O-NH 2 and -CH 2 -O-NH 2 .
  • alkyl refers to a saturated straight or branched hydrocarbon.
  • the alkyl group is a primary, secondary, or tertiary hydrocarbon.
  • the alkyl group includes one to ten carbon atoms (i.e., C 1 to C 10 alkyl).
  • the alkyl is a lower alkyl , for example, C1-6alkyl, and the like.
  • the alkyl group is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, 3-methylpentyl, 2,2-dimethylbutyl, and 2,3- dimethylbutyl.
  • substituted alkyl refers to an alkyl substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy. In some embodiments, alkyl is unsubstituted.
  • halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • alkyl haloalkyl
  • hydroxyl amino, alkylamino
  • alkoxy alkyl
  • alkyl is unsubstituted.
  • alkylene refers to a divalent alkyl group, as defined herein. “Substituted alkylene” refers to an alkylene group substituted as described herein for alkyl.
  • alkylene is unsubstituted.
  • Alkenyl refers to an olefinically unsaturated hydrocarbon group, in certain embodiments, having up to about eleven carbon atoms or from two to six carbon atoms (e.g., “lower alkenyl”), which can be straight-chained or branched, and having at least one or from one to two sites of olefinic unsaturation.
  • Substituted alkenyl refers to an alkenyl group substituted as described herein for alkyl.
  • Alkenylene refers to a divalent alkenyl as defined herein.
  • Lower alkenylene is, for example, C 2 -C 6 -alkenylene.
  • Alkynyl refers to acetylenically unsaturated hydrocarbon groups, in certain embodiments, having up to about eleven carbon atoms or from two to six carbon atoms (e.g., “lower alkynyl”), which can be straight-chained or branched, and having at least one or from one to two sites of acetylenic unsaturation.
  • alkynyl groups include acetylene (-C ⁇ CH), propargyl (-CH2C ⁇ CH), and the like.
  • Substituted alkynyl refers to an alkynyl group substituted as described herein for alkyl.
  • Alkynylene refers to a divalent alkynyl as defined herein. Lower alkynylene is, for example, C 2 -C 6 -alkynylene.
  • Amino refers to -NH2.
  • alkylamino refers to the group –NHR′′ where R′′ is, for example, C1-10alkyl, as defined herein. In certain embodiments, alkylamino is C 1-6 alkylamino.
  • dialkylamino refers to the group –NR′′R′′ where each R′′ is independently C1-10alkyl, as defined herein. In certain embodiments, dialkylamino is di-C 1-6 alkylamino.
  • aryl refers to phenyl, biphenyl, or naphthyl. The term includes both substituted and unsubstituted moieties.
  • An aryl group can be substituted with any described moiety including, but not limited to, one or more moieties (e.g., in some embodiments one, two, or three moieties) selected from the group consisting of halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid, phosphate, and phosphonate, wherein each moiety is independently either unprotected, or protected as necessary, as would be appreciated by those skilled in the art (e.g., Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, 1991); and wherein the aryl in the arylamino and aryloxy substituents are not further
  • arylamino refers to an - NR′R′′ group where R′ is hydrogen or C1-C6-alkyl; and R′′ is aryl, as defined herein.
  • arylene refers to a divalent aryl group, as defined herein.
  • aryloxy refers to an -OR group where R is aryl, as defined herein.
  • Alkarylene refers to an arylene group, as defined herein, wherein the aryl ring is substituted with one or two alkyl groups.
  • Substituted alkarylene refers to an alkarylene, as defined herein, where the arylene group is further substituted, as defined herein for aryl.
  • “Aralkylene” refers to an -CH 2 -arylene-, -arylene-CH 2 -, or -CH 2 -arylene-CH 2 - group, where arylene is as defined herein.
  • “Substituted aralkylene” refers to an aralkylene, as defined herein, where the aralkylene group is substituted, as defined herein for aryl.
  • Carboxyl or “carboxy” refers to -C(O)OH or -COOH.
  • cycloalkyl refers to a saturated cyclic hydrocarbon.
  • the cycloalkyl group may be a saturated, and/or bridged, and/or non-bridged, and/or a fused bicyclic group.
  • the cycloalkyl group includes three to ten carbon atoms (i.e., C3 to C10 cycloalkyl).
  • the cycloalkyl has from three to fifteen carbons (C 3-15 ), from three to ten carbons (C3-10), from three to seven carbons (C3-7), or from three to six carbons (C3-C6) (i.e., “lower cycloalkyl”).
  • the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexylmethyl, cycloheptyl, bicyclo[2.1.1]hexyl, bicyclo[2.2.1]heptyl, decalinyl, or adamantyl.
  • cycloalkylene refers to a divalent cycloalkyl group, as defined herein.
  • the cycloalkylene group is cyclopropylene , cyclobutylene , cyclopentylene , cyclohexylene , cycloheptylene , and the like.
  • Lower cycloalkylene refers to a C 3 -C 6 -cycloalkylene.
  • cycloalkylalkyl refers to an alkyl group, as defined herein, substituted with one or two cycloalkyl, as defined herein.
  • ester refers to -C(O)OR or -COOR where R is alkyl, as defined herein.
  • fluorene refers to , wherein any one or more carbons bearing one or more hydrogens can be substituted with a chemical functional group as described herein.
  • haloalkyl refers to an alkyl group, as defined herein, substituted with one or more halogen atoms (e.g., in some embodiments one, two, three, four, or five) which are independently selected.
  • heteroalkyl refers to an alkyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms.
  • heteroalkenyl refers to an alkenyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms.
  • heteroalkynyl refers to an alkynyl, as defined herein, in which one or more carbon atoms are replaced by heteroatoms. Suitable heteroatoms include, but are not limited to, nitrogen (N), oxygen (O), and sulfur (S) atoms.
  • heteroalkyl, heteroalkenyl, and heteroalkynyl are optionally substituted.
  • heteroalkyl moieties include, but are not limited to, aminoalkyl, sulfonylalkyl, and sulfinylalkyl.
  • heteroalkyl moieties also include, but are not limited to, methylamino, methylsulfonyl, and methylsulfinyl.
  • “Substituted heteroalkyl” refers to heteroalkyl substituted with one, two, or three groups independently selected from halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a heteroalkyl group may comprise one, two, three, or four heteroatoms.
  • a 4- membered heteroalkyl may generally comprise one or two heteroatoms
  • a 5- or 6-membered heteroalkyl may generally comprise one, two, or three heteroatoms
  • a 7- to 10-membered heteroalkyl may generally comprise one, two, three, or four heteroatoms.
  • heteroalkylene refers to a divalent heteroalkyl, as defined herein.
  • “Substituted heteroalkylene” refers to a divalent heteroalkyl, as defined herein, substituted as described for heteroalkyl.
  • heterocycloalkyl refers to a monovalent, monocyclic, or multicyclic non- aromatic ring system, wherein one or more of the ring atoms are heteroatoms independently selected from oxygen (O), sulfur (S), and nitrogen (N) (e.g., where the nitrogen or sulfur atoms may be optionally oxidized, and the nitrogen atoms may be optionally quaternized) and the remaining ring atoms of the non-aromatic ring are carbon atoms.
  • heterocycloalkyl is a monovalent, monocyclic, or multicyclic fully-saturated ring system.
  • the heterocycloalkyl group has from three to twenty, from three to fifteen, from three to ten, from three to eight, from four to seven, from four to eleven, or from five to six ring atoms.
  • the heterocycloalkyl may be attached to a core structure at any heteroatom or carbon atom which results in the creation of a stable compound.
  • the heterocycloalkyl is a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused or bridged ring system and in which the nitrogen or sulfur atoms may be optionally oxidized, and/or the nitrogen atoms may be optionally quaternized.
  • heterocycloalkyl radicals include, but are not limited to, 2,5- diazabicyclo[2.2.2]octanyl, decahydroisoquinolinyl, dihydrobenzisoxazinyl, dihydrofuryl, dihydroisoindolyl, dihydropyranyl, dihydropyrazolyl, dihydropyrazinyl, dihydropyridinyl, dihydropyrimidinyl, dihydropyrrolyl, dioxolanyl, 1,4-dithianyl, furanonyl, imidazolidinyl, imidazolinyl, indolinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, oxazolidinonyl, oxazolidinyl, oxiranyl, piperazin
  • heterocycloalkyl may also be optionally substituted as described herein.
  • heterocycloalkyl is substituted with one, two, or three groups independently selected from halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a heterocycloalkyl group may comprise one, two, three, or four heteroatoms.
  • heterocycloalkylene refers to a divalent heterocycloalkyl as defined herein.
  • heteroaryl refers to a monovalent monocyclic aromatic group and/or multicyclic aromatic group, wherein at least one aromatic ring contains one or more heteroatoms independently selected from oxygen, sulfur, and nitrogen in the ring.
  • Each ring of a heteroaryl group can contain one or two oxygen atoms, one or two sulfur atoms, and/or one to four nitrogen atoms, provided that the total number of heteroatoms in each ring is four or less and each ring contains at least one carbon atom.
  • the heteroaryl has from five to twenty, from five to fifteen, or from five to ten ring atoms.
  • a heteroaryl may be attached to the rest of the molecule via a nitrogen or a carbon atom.
  • monocyclic heteroaryl groups include, but are not limited to, furanyl, imidazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl, triazolyl, thiadiazolyl, thiazolyl, thienyl, tetrazolyl, and triazinyl.
  • bicyclic heteroaryl groups include, but are not limited to, benzofuranyl, benzimidazolyl, benzoisoxazolyl, benzopyranyl, benzothiadiazolyl, benzothiazolyl, benzothienyl, benzotriazolyl, benzoxazolyl, furopyridyl, imidazopyridinyl, imidazothiazolyl, indolizinyl, indolyl, indazolyl, isobenzofuranyl, isobenzothienyl, isoindolyl, isoquinolinyl, naphthyridinyl, oxazolopyridinyl, phthalazinyl, pteridinyl, purinyl, pyridopyridyl, pyrrolopyridyl, quinolinyl, quinoxalinyl, quinazolinyl, thiadiazolopyrimidyl, and thi
  • tricyclic heteroaryl groups include, but are not limited to, acridinyl, benzindolyl, carbazolyl, dibenzofuranyl, perimidinyl, phenanthrolinyl, phenanthridinyl, phenarsazinyl, phenazinyl, phenothiazinyl, phenoxazinyl, and xanthenyl.
  • heteroaryl may also be optionally substituted as described herein.
  • “Substituted heteroaryl” is a heteroaryl substituted as defined for aryl.
  • heteroarylene refers to a divalent heteroaryl group, as defined herein.
  • “Substituted heteroarylene” is a heteroarylene substituted as defined for aryl.
  • the term “protecting group,” as used herein, and unless otherwise specified, refers to a group that is added to an oxygen, nitrogen, or phosphorus atom to prevent its further reaction, or for other purposes. A wide variety of oxygen and nitrogen protecting groups are known to those skilled in the art of organic synthesis.
  • “Pharmaceutically acceptable salt” refers to any salt of a compound provided herein which retains its biological properties and which is not toxic or otherwise undesirable for pharmaceutical use. Such salts may be derived from a variety of organic and inorganic counter- ions well known in the art.
  • Such salts include, but are not limited to (1) acid addition salts formed with organic or inorganic acids such as hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, sulfamic, acetic, trifluoroacetic, trichloroacetic, propionic, hexanoic, cyclopentylpropionic, glycolic, glutaric, pyruvic, lactic, malonic, succinic, sorbic, ascorbic, malic, maleic, fumaric, tartaric, citric, benzoic, 3-(4-hydroxybenzoyl)benzoic, picric, cinnamic, mandelic, phthalic, lauric, methanesulfonic, ethanesulfonic, 1,2-ethane-disulfonic, 2-hydroxyethanesulfonic, benzenesulfonic, 4-chlorobenzenesulfonic, 2-naphthalenesulfonic, 4-tol
  • Pharmaceutically acceptable salts further include, by way of example and without limitation, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium salts, and the like, and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as hydrohalides, for example, hydrochloride and hydrobromide, sulfate, phosphate, sulfamate, nitrate, acetate, trifluoroacetate, trichloroacetate, propionate, hexanoate, cyclopentylpropionate, glycolate, glutarate, pyruvate, lactate, malonate, succinate, sorbate, ascorbate, malate, maleate, fumarate, tartarate, citrate, benzoate, 3-(4-hydroxybenzoyl)benzoate, picrate, cinnamate, mandelate, phthalate, laurate, methanesulfonate (mesylate),
  • compositions that includes at least 85% or 90% by weight, in certain embodiments 95%, 98 %, 99%, or 100% by weight; or in certain embodiments, 95%, 98%, 99%, or 100% of the designated enantiomer or diastereomer of a compound.
  • the compounds are substantially free of one of two enantiomers. In certain embodiments, in the methods and compounds provided herein, the compounds are substantially free of one of two diastereomers.
  • the compounds are substantially free of enantiomers (i.e., a racemic or 50:50 mixture of compounds).
  • isolated refers to a composition that includes at least 85%, 90%, 95%, 98%, or 99% to 100% by weight, of the compound, the remainder comprising other chemical species, enantiomers or diastereomers.
  • Solvate refers to a compound provided herein, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces.
  • the solvate is a hydrate.
  • “Isotopic composition” refers to the amount of each isotope present for a given atom
  • “natural isotopic composition” refers to the naturally occurring isotopic composition or abundance for a given atom.
  • Atoms containing their natural isotopic composition may also be referred to herein as “non-enriched” atoms. Unless otherwise designated, the atoms of the compounds recited herein are meant to represent any stable isotope of that atom. For example, unless otherwise stated, when a position is designated specifically as hydrogen (H), the position is understood to have hydrogen at its natural isotopic composition.
  • H hydrogen
  • Isotopic enrichment refers to the percentage of incorporation of an amount of a specific isotope at a given atom in a molecule in the place of that atom’s natural isotopic abundance.
  • deuterium (D) enrichment of 1% at a given position means that 1% of the molecules in a given sample contain deuterium at the specified position. Because the naturally occurring distribution of deuterium is about 0.0156%, deuterium enrichment at any position in a compound synthesized using non-enriched starting materials is about 0.0156%.
  • the isotopic enrichment of the compounds provided herein can be determined using conventional analytical methods known to one of ordinary skill in the art, including mass spectrometry and nuclear magnetic resonance spectroscopy.
  • “Isotopically enriched” refers to an atom having an isotopic composition other than the natural isotopic composition of that atom. “Isotopically enriched” may also refer to a compound containing at least one atom having an isotopic composition other than the natural isotopic composition of that atom.
  • alkyl, alkylene, alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “arylene,” “alkoxy,” “amino,” “carboxyl,” “heterocycloalkyl,” “heteroaryl,” “heteroarylene,” “carboxyl,” and “amino acid” groups optionally comprise deuterium (D) at one or more positions where hydrogen (H) atoms are present, and wherein the deuterium composition of the atom or atoms is other than the natural isotopic composition.
  • alkyl alkylene, alkylamino,” “dialkylamino,” “cycloalkyl,” “aryl,” “arylene,” “alkoxy,” “amino,” “carboxyl,” “heterocycloalkyl,” “heteroaryl,” “heteroarylene,” “carboxyl,” and “amino acid” groups optionally comprise carbon-13 ( 13 C) at an amount other than the natural isotopic composition.
  • the term “macromolecule” or “macromolecular moiety” refers to a protein, peptide, antibody, nucleic acid, carbohydrate, or other large molecule composed of polymerized monomers.
  • a macromolecule is at least 1000 Da in mass. In certain embodiments, a macromolecule has at least 1000 atoms. In certain embodiments, a macromolecule can be modified. For instance, a protein, peptide, or antibody can be modified with one or more carbohydrates.
  • immunoglobulin refers to a class of structurally related proteins generally comprising two pairs of polypeptide chains: one pair of light (L) chains, and one pair of heavy (H) chains. In an “intact immunoglobulin,” all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized.
  • each heavy chain typically comprises a heavy chain variable region (VH or VH) and a heavy chain constant region (C H or CH).
  • the heavy chain constant region typically comprises three domains, abbreviated C H 1 (or CH1), C H 2 (or CH2), and C H 3 (or CH3).
  • Each light chain typically comprises a light chain variable region (VL or VL) and a light chain constant region.
  • the light chain constant region typically comprises one domain, abbreviated CL or CL.
  • An antibody includes intact antibodies (e.g., intact immunoglobulins), and antibody fragments (e.g., antigen binding fragments or antigen-binding fragments of antibodies).
  • Antibodies comprise at least one antigen-binding domain.
  • an antigen-binding domain is an antigen binding domain formed by a V H -V L dimer.
  • amino acid refers to the twenty common naturally occurring amino acids.
  • Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), Glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V), and the less common pyrrolysine and selenocysteine.
  • Natural amino acids also include citrulline.
  • Naturally encoded amino acids include post-translational variants of the twenty-two naturally occurring amino acids such as prenylated amino acids, isoprenylated amino acids, myrisoylated amino acids, palmitoylated amino acids, N-linked glycosylated amino acids, O-linked glycosylated amino acids, phosphorylated amino acids, and acylated amino acids.
  • amino acid also includes non-natural (or unnatural) or synthetic ⁇ -, ⁇ -, ⁇ -, or ⁇ - amino acids, and includes, but is not limited to, amino acids found in proteins, i.e.
  • amino acid is in the L-configuration. In certain embodiments, the amino acid is in the D-configuration.
  • the amino acid can be a derivative of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, argininyl, histidinyl, ⁇ -alanyl, ⁇ -valinyl, ⁇ -leucinyl, ⁇ -isoleuccinyl, ⁇ -prolinyl, ⁇ - phenylalaninyl, ⁇ -tryptophanyl, ⁇ -methioninyl, ⁇ -glycinyl, ⁇ -serinyl, ⁇ -threoninyl, ⁇ - cysteinyl
  • Unnatural amino acids are not proteinogenic amino acids, or post- translationally modified variants thereof.
  • the term unnatural amino acid refers to an amino acid that is not one of the twenty common amino acids or pyrrolysine or selenocysteine, or post-translationally modified variants thereof.
  • conjugate refers to a fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound described herein linked to one or more macromolecular moieties.
  • the macromolecular moiety is as defined herein or is any macromolecule deemed suitable to the person of skill in the art.
  • the compound can be any compound described herein.
  • the fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound can be directly linked to the macromolecular moiety via a covalent bond, or the fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound can be linked to the macromolecular moiety indirectly via a linker.
  • the linker is covalently bonded to the macromolecular moiety and also covalently bonded to the fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound.
  • linker refers to a molecular moiety that is capable of forming at least two covalent bonds. Typically, a linker is capable of forming at least one covalent bond to a macromolecular moiety and at least another covalent bond to a fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound. In certain embodiments, a linker can form more than one covalent bond to a macromolecular moiety.
  • a linker can form more than one covalent bond to a fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound or can form covalent bonds to more than one fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound.
  • a linker forms a bond to a macromolecular moiety, or a fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound, or both, the remaining structure(i.e. the residue of the linker (“linker residue”) after one or more covalent bonds are formed) may still be referred to as a “linker” herein.
  • linker precursor refers to a linker having one or more reactive groups capable of forming a covalent bond with a macromolecule, or fluorenylmethoxycarbonyl, or fluorenylaminocarbonyl compound, or both.
  • linker means linker precursor with one reactive group, a linker precursor with more than one reactive groups, a linker residue which is covalently bonded to the macromolecule, a linker residue which is covalently bonded to a fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound, and/or a linker residue which is covalently bonded to the macromolecule and is covalently bonded to a fluorenylmethyloxycarbonyl or fluorenylmethylaminocarbonyl compound.
  • the linker is a cleavable linker.
  • a cleavable linker can be one that is released by a bio-labile or enzymatic function, which may or may not be engineered.
  • the linker is a non- cleavable linker.
  • a non-cleavable linker can be one that is released upon degradation of the macromolecular moiety.
  • term “EC 50 ” refers to a dosage, concentration, or amount of a particular test compound that elicits a dose-dependent response at 50% of maximal expression of a particular response that is induced, provoked, or potentiated by the particular test compound.
  • the term “IC50” refers to an amount, concentration, or dosage of a particular test compound that achieves a 50% inhibition of a maximal response in an assay that measures such response.
  • the terms “subject” and “patient” are used interchangeably.
  • the terms “subject” and “subjects” refer to an animal, such as a mammal including a non-primate (e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., a monkey, such as a cynomolgous monkey, a chimpanzee, and a human), and in certain embodiments, a human.
  • the subject is a farm animal (e.g., a horse, a cow, a pig, etc.) or a pet (e.g., a dog or a cat). In certain embodiments, the subject is a human.
  • the terms “therapeutic agent” and “therapeutic agents” refer to any agent(s) which can be used in the treatment or prevention of a disorder or one or more symptoms thereof.
  • the term “therapeutic agent” includes a compound or conjugate provided herein.
  • a therapeutic agent is an agent which is known to be useful for, or has been or is currently being used for the treatment or prevention of a disorder or one or more symptoms thereof.
  • “Therapeutically effective amount” refers to an amount of a compound or composition that, when administered to a subject for treating a condition, is sufficient to effect such treatment for the condition.
  • a “therapeutically effective amount” can vary depending on, inter alia, the compound, the disease or disorder and its severity, and the age, weight, etc., of the subject to be treated.
  • “Treating” or “treatment” of any disease or disorder refers, in certain embodiments, to ameliorating a disease or disorder that exists in a subject. In another embodiment, “treating” or “treatment” includes ameliorating at least one physical parameter, which may be indiscernible by the subject.
  • “treating” or “treatment” includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both.
  • “treating” or “treatment” includes delaying or preventing the onset of the disease or disorder, or delaying or preventing recurrence of the disease or disorder.
  • “treating” or “treatment” includes the reduction or elimination of either the disease or disorder, or retarding the progression of the disease or disorder or of one or more symptoms of the disease or disorder, or reducing the severity of the disease or disorder or of one or more symptoms of the disease or disorder.
  • the term “inhibits growth” is intended to include any measurable decrease in cell growth (e.g., tumor cell growth) when contacted with a compound or conjugate herein, as compared to the growth of the same cells not in contact with the compound or conjugate herein.
  • growth may be inhibited by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%.
  • the decrease in cell growth can occur via a variety of mechanisms, including but not limited to, conjugate or compound internalization, apoptosis, necrosis, and/or effector function- mediated activity.
  • prophylactic agent and “prophylactic agents” as used refer to any agent(s) which can be used in the prevention of a disorder or one or more symptoms thereof.
  • the term “prophylactic agent” includes a compound or conjugate provided herein.
  • the term “prophylactic agent” does not refer a compound or conjugate provided herein.
  • a prophylactic agent is an agent which is known to be useful for, or has been or is currently being used to prevent or impede the onset, development, progression, and/or severity of a disorder.
  • prophylactically effective amount refers to the amount of a therapy (e.g., prophylactic agent) which is sufficient to result in the prevention or reduction of the development, recurrence, or onset of one or more symptoms associated with a disorder or to enhance or improve the prophylactic effect(s) of another therapy (e.g., another prophylactic agent).
  • a therapy e.g., prophylactic agent
  • another therapy e.g., another prophylactic agent
  • this curvy/wavy/wiggly line indicates the atoms in the backbone of a conjugate, linker-fluorenylmethoxycarbonyl, or linker-fluorenylmethaminocarbonyl compound structure to which the illustrated chemical entity is bonded.
  • this curvy/wavy/wiggly line indicates the atoms in the macromolecule as well as the atoms in the backbone of a conjugate, linker- fluorenylmethoxycarbonyl, or linker-fluorenylmethoxycarbonyl compound structure to which the illustrated chemical entity is bonded.
  • the g wherein subscript q is an integer from zero to four and in which the positions of substituent R 1 are described generically, i.e., not directly attached to any vertex of the bond line structure, i.e., specific ring carbon atom, includes the following, non-limiting examples of groups in which the substituent R 1 is bonded to a specific ring carbon [0082]
  • site-specific refers to a modification of a polypeptide at a predetermined sequence location in the polypeptide. The modification is at a single, predictable residue of the polypeptide with little or no variation. In particular embodiments, a modified amino acid is introduced at that sequence location, for instance recombinantly or synthetically.
  • a moiety can be “site-specifically” linked to a residue at a particular sequence location in the polypeptide.
  • a polypeptide can comprise more than one site-specific modification.
  • Compounds of Formulae (I), (II), (III), (IV), (X), (XII), (XIII) and (XIV) [0083] Provided herein are fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds useful for modulating one or more properties of a macromolecule.
  • the fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds can be formed as described herein and used for forming a conjugate with one or more macromolecules.
  • the conjugates can be useful for therapy or diagnosis.
  • therapy is the treatment of a cancer or an inflammatory disease or condition.
  • the embodiments described herein include the recited compounds as well as a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, and/or mixture thereof.
  • a compound of Formula (I) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein RG is a reactive group; POLY is a water soluble polymer; X is a bond, -O-, or -N(R 2 )-; each L 1 and L 2 is, independently, a linker; each R 1 is an electron withdrawing group; R 2 is H or lower alkyl; and n is an integer selected from one to four.
  • X is -O- or -N(R 2 )- .
  • X is -O-.
  • X is -N(R 2 )-.
  • n is one. In certain embodiments, n is two. In certain embodiments, n is three. In certain embodiments, n is four. In certain embodiments, n1 is one. In certain embodiments, n1 is two. In certain embodiments, n1 is three. In certain embodiments, n1 is four. [0086] In certain embodiments, provided is a compound of Formula (I) or (X), wherein either L 1 comprises a carbonyl carbon covalently bound to the fluorene, or R 1 comprises an electron withdrawing group, or both.
  • R 1 when L 1 comprises a carbonyl carbon covalently bound to the fluorine, then R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group. By way of further example, in one embodiment, when R 1 comprises an electron withdrawing group, then L 1 is a linker. By way of further example, in one embodiment, when L 1 comprises a carbonyl carbon covalently bound to the fluorine, then R 1 comprises an electron withdrawing group. [0087] In certain embodiments, the compound of Formula (I) is according to Formula (II) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof
  • the compound of Formula (I) is according to Formula (III) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein POLY and R 1 are as defined in the Summary, or in any embodiment herein, and p is an integer selected from one to eight. In certain embodiments, p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five. In certain embodiments, p is six. In certain embodiments, p is seven.
  • the compound of Formula (I) is according to Formula (IV) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein POLY and R 1 are as defined in the Summary, or in any embodiment herein, and p is an integer selected from one to eight.
  • p is one.
  • p is two.
  • p is three.
  • p is four.
  • p is five.
  • p is six.
  • p is seven.
  • p is eight.
  • the compound of Formula (I) is according to Formula (IV) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein POLY and R 1 are as defined in the Summary, or in any embodiment herein, and p is an integer selected from one to eight. In certain embodiments, p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five. In certain embodiments, p is six. In certain embodiments, p is seven. In certain embodiments, p is eight.
  • a compound of Formula (X) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein RG is a reactive group; POLY is a water soluble polymer; X is a bond, -O-, or -N(R 2 )-; each of L 1 , L 2 , and L 3 is, independently, a linker; each R 1 is an electron withdrawing group; R 2 is H or lower alkyl; m is an integer selected from zero to four.
  • X is -O- or -N(R 2 )-.
  • X is -O-.
  • X is -N(R 2 )-.
  • m is zero. In certain embodiments, m is one. In certain embodiments, m is two. In certain embodiments, m is three. In certain embodiments, m is four. In certain embodiments, m1 is one. In certain embodiments, m1 is two. In certain embodiments, m1 is three. In certain embodiments, m1 is four. In certain embodiments, m2 is one. In certain embodiments, m2 is two. In certain embodiments, m2 is three. In certain embodiments, m2 is four.
  • the compound of Formula (X) is according to Formula (XII) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein RG, POLY, X, L 2 , L 3 , and R 4 are as defined in the Summary, or in any embodiment herein.
  • the compound of Formula (X) is according to Formula (XIII) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof wherein POLY and R 4 are as defined in the Summary, or in any embodiment herein, and p is an integer selected from one to eight. In certain embodiments, p is one.
  • p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five. In certain embodiments, p is six. In certain embodiments, p is seven. In certain embodiments, p is eight. [0094] In certain embodiments, the compound of Formula (X) is according to Formula (XIV) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers thereof
  • each linker L 1 , L 2 , and L 3 can be any linker deemed suitable by a person of skill in the art.
  • L 1 includes an ammonium where the positively charged nitrogen of the ammonium functional group is covalently bound to the fluorene.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is selected from the group consisting of -O-C 1-6 alkylene-S-C(O)-, -O-C 1-6 alkylene-NH-C(O)-, -S-C1-6alkylene-O-C(O)-, -S-C1-6alkylene-NH-C(O)-, -NH-C1-6alkylene-O-C(O)-, -NH-C 1-6 alkylene-S-C(O)-, -O-C 1-6 alkylene-N(Me)-C(O)-, -S-C 1-6 alkylene-N(Me)-C(O)-, -N(Me)-C1-6alkylene-O-C(O)-, -N(Me)-C1-6alkylene-S-C(O)-, -O-C1-6alkylene-N(Et)-C(O)-, -S-C 1-6 alkylene-N(Et)-C(O)
  • L 1 is -O-C 1-6 alkylene-S-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -O-C1-6alkylene-NH-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C 1-6 alkylene-O-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C1-6alkylene-NH-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -NH-C1-6alkylene-O-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -NH-C 1-6 alkylene-S-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -O-C1-6alkylene-N(Me)-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C 1-6 alkylene-N(Me)-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Me)-C1-6alkylene-O-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Me)-C 1-6 alkylene-S-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -O-C 1-6 alkylene-N(Et)-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C1-6alkylene-N(Et)-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Et)-C 1-6 alkylene-O-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Et)-C1-6alkylene-S-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • alkyl haloalkyl
  • hydroxyl amino, alkylamino, and alkoxy.
  • provided is a compound of Formula (I) or (X), wherein L 1 is -O-CH2-CH2-N(H)-C(O)- wherein the carbonyl carbon is covalently bound to the fluorene.
  • L 2 is -CH2- optionally substituted with a group selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH 2 CH 2 - independently optionally substituted with one or two groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH 2 CH 2 CH 2 - independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH2CH2CH2CH2- independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH2CH2CH2CH2CH2- independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH2CH2CH2CH2CH2CH2- independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a compound of Formula (I), (II), or (X) wherein L 2 is -CH 2 -.
  • RG is any reactive group deemed suitable by the person of skill.
  • a compound of Formula (I), (II), (X), or (XII) wherein RG comprises an azide, alkyne, hydrazide, aldehyde, alkoxyamine, amine or -NR 2 , carboxyl, ester, or maleimide.
  • a compound of Formula (I), (II), (X), or (XII) wherein RG comprises an azide.
  • provided is a compound of Formula (I), (II), (X), or (XII), wherein RG comprises a hydrazide.
  • POLY can be any water-soluble polymer deemed useful by a person of skill in the art. Useful polymers are described here, and in the sections below.
  • POLY is uncapped (e.g., terminates with hydroxyl).
  • POLY is methoxy-PEG (i.e., terminates with methyl or methoxy).
  • PEG is linear.
  • PEG is branched.
  • each R 1 is hydrogen, an electron donating group, or an electron withdrawing group. In one embodiment, each R 1 is hydrogen. In one embodiment, each R 1 is an electron donating group. In one embodiment, each R 1 is an electron withdrawing group.
  • the electron donating group can be any electron donating group deemed suitable to the person of skill in the art.
  • the electron withdrawing group can be any electron withdrawing group deemed suitable to the person of skill in the art.
  • each R 1 is independently selected from the group consisting of hydrogen, haloalkyl, halogen, -CN, -SO3H, -C(O)R 3 , -C(O)OR 3 , -OR 3 , -N(H)C(O)R 3 , - N(H)CO 2 R 3 , and -N(H)C(O)C(H)(R 3 )CO 2 H wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • each R 1 is independently selected from the group consisting of haloalkyl, halogen, -CN, - SO 3 H, -C(O)R 3 , -C(O)OR 3 , -OR 3 , -N(H)C(O)R 3 , -N(H)CO 2 R 3 , and - N(H)C(O)C(H)(R 3 )CO 2 H wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • each R 1 is independently selected from the group consisting of -H, -CF3, -Br, -Cl, -F, -CN, -SO3H, - C(O)Me, -CO 2 Me, -OMe, -N(H)C(O)Me, -N(H)CO 2 Me, and -N(H)C(O)C(H)(Me)CO 2 H.
  • each R 1 is independently selected from the group consisting of -CF3, -Br, -Cl, -F, -CN, -SO3H, -C(O)Me, -CO2Me, -OMe, -N(H)C(O)Me, -N(H)CO2Me, and -N(H)C(O)C(H)(Me)CO 2 H.
  • each R 4 is hydrogen or an electron withdrawing group. In one embodiment, each R 4 is hydrogen. In one embodiment, each R 4 is an electron withdrawing group.
  • the electron withdrawing group can be any electron withdrawing group deemed suitable to the person of skill in the art.
  • each R 4 is independently selected from the group consisting of -C(O)R 3 , -C(O)OR 3 , and -S(O)2R 3 , wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • each R 4 is independently selected from the group consisting of -C(O)R 3 , -C(O)OR 3 , and -S(O)2R 3 wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • Optically Active Compounds [00103]
  • compounds provided herein may have several chiral centers and may exist in and be isolated in optically active and racemic forms. In certain embodiments, some compounds may exhibit polymorphism.
  • This technique can be used if crystals of the separate enantiomers exist (i.e., the material is a conglomerate, and the crystals are visually distinct); ii) simultaneous crystallization - a technique whereby the individual enantiomers are separately crystallized from a solution of the racemate, only if the latter is a conglomerate in the solid state; iii) enzymatic resolutions - a technique wherein partial or complete separation of a racemate is accomplished by virtue of different rates of reaction of the enantiomers in the presence of an enzyme; iv) enzymatic asymmetric synthesis - a synthetic technique wherein at least one step of the synthesis uses an enzymatic reaction to obtain an enantiomerically pure or enriched synthetic precursor of the desired enantiomer; v) chemical asymmetric synthesis - a synthetic technique wherein the desired enantiomer is synthesized from an achiral precursor using chiral catalysts or chiral auxiliaries to produce a
  • the desired enantiomer is then derived from the diastereomer; viii) kinetic resolutions - this technique refers to the achievement of partial or complete resolution of a racemate (or of a further resolution of a partially resolved compound) by virtue of unequal reaction rates of the enantiomers with a chiral or non-racemic reagent or catalyst under kinetic conditions; ix) enantiospecific synthesis from non-racemic precursors - a synthetic technique wherein the desired enantiomer is obtained from chiral starting materials and where the stereochemical integrity is not or is only minimally compromised over the course of the synthesis; x) chiral liquid chromatography - a technique wherein the enantiomers of a racemate are separated in a liquid mobile phase by virtue of their different interactions with a stationary phase.
  • the stationary phase can be made of chiral material or the mobile phase can contain an additional chiral material to provoke the different interactions; xi) chiral gas chromatography - a technique wherein the racemate is volatilized and enantiomers are separated by virtue of their different interactions in the gaseous mobile phase with a column containing a fixed non-racemic adsorbent phase; xii) extraction with chiral solvents - a technique wherein the enantiomers are separated by virtue of kinetic or thermodynamic dissolution of one enantiomer into a particular chiral solvent; xiii) transport across chiral membranes - a technique wherein a racemate is placed in contact with a thin membrane barrier.
  • the compounds are substantially free of other stereoisomers.
  • the composition includes a compound that is at least 85%, 90%, 95%, 98%, or 99% to 100% by weight of the compound, the remainder comprising other chemical species or enantiomers.
  • provided herein are compositions of compounds of Formula (I)-(IV), (X), (XII), (XIII), (XIV), or compounds 101-106, that are substantially free of a designated enantiomer of that compound.
  • the compounds are substantially free of other enantiomers.
  • the composition includes a compound that is at least 85%, 90%, 95%, 98%, or 99% to 100% by weight of the compound, the remainder comprising other chemical species or enantiomers.
  • isotopically enriched compounds including, but not limited to, isotopically enriched compounds of Formula (I)-(IV), (X), (XII), (XIII), (XIV), or compounds 101-106.
  • Isotopic enrichment for example, deuteration
  • PK pharmacokinetics
  • PD pharmacodynamics
  • toxicity profiles has been previously demonstrated within some classes of drugs. See, for example, Lijinsky et al., Food Cosmet. Toxicol., 20: 393 (1982); Lijinsky et al., J. Nat.
  • Isotopic enrichment of a drug can be used, for example, to (1) reduce or eliminate unwanted metabolites; (2) increase the half-life of the parent drug; (3) decrease the number of doses needed to achieve a desired effect; (4) decrease the amount of a dose necessary to achieve a desired effect; (5) increase the formation of active metabolites, if any are formed; and/or (6) decrease the production of deleterious metabolites in specific tissues.
  • Isotopic enrichment of a drug can also be used to create a more effective and/or safer drug for combination therapy, whether the combination therapy is intentional or not.
  • Replacement of an atom for one of its isotopes often will result in a change in the reaction rate of a chemical reaction.
  • KIE Kinetic Isotope Effect
  • DKIE Deuterium Kinetic Isotope Effect
  • the magnitude of the DKIE can be expressed as the ratio between the rates of a given reaction in which a C–H bond is broken, and the same reaction where deuterium is substituted for hydrogen and the C–D bond is broken.
  • the DKIE can range from about one (no isotope effect) to very large numbers, such as 50 or more, meaning that the reaction can be fifty, or more, times slower when deuterium has been substituted for hydrogen.
  • Substitution of tritium (“T”) for hydrogen results in yet a stronger bond than deuterium and gives numerically larger isotope effects.
  • the animal body expresses a variety of enzymes for the purpose of eliminating foreign substances, such as therapeutic agents, from its circulation system.
  • enzymes include the cytochrome P450 enzymes (“CYPs”), esterases, proteases, reductases, dehydrogenases, and monoamine oxidases to react with and convert these foreign substances to more polar intermediates or metabolites for renal excretion.
  • C–H carbon-hydrogen
  • C—O carbon-oxygen
  • the resultant metabolites may be stable or unstable under physiological conditions, and can have substantially different PK/PD, and acute and long-term toxicity profiles relative to the parent compounds. For many drugs, such oxidations are rapid. Therefore, these drugs often require the administration of multiple or high daily doses.
  • the conjugates are covalently linked directly or indirectly, via a linker.
  • a conjugate comprises a macromolecule conjugated to one or more fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds described herein. In certain embodiments, a conjugate comprises more than one fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl group. In certain embodiments, conjugate comprises more than one macromolecule. In certain embodiments, the macromolecule is linked to one fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl group. In further embodiments, the macromolecule is linked to more than one fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds.
  • the macromolecule is linked to two, three, four, five, six, seven, eight, or more fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl groups.
  • the linker can be any linker capable of forming at least one bond to the macromolecule and at least one bond to a fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compound. Useful linkers are described in the sections and examples herein and in particular, below.
  • the macromolecule can be any macromolecule deemed suitable by the person of skill in the art.
  • the macromolecule is a protein, peptide, antibody or antigen-binding fragment thereof, nucleic acid, carbohydrate, or other large molecule composed of polymerized monomers. In certain embodiments, the macromolecule is a peptide of two or more residues. In certain embodiments, the macromolecule is a peptide of ten or more residues. In certain embodiments, the macromolecule is at least 1000 Da in mass. In certain embodiments, the macromolecule comprises at least 1000 atoms. Useful macromolecules are described in the sections below.
  • a conjugate of Formula (I′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO is a macromolecule; w is an integer selected from one to ten; each RG′ is independently a divalent residue of a reactive group; each POLY is independently a water soluble polymer; each X is independently a bond, -O-, or -N(R 2 )-; each L 1 and L 2 is, independently, a linker; each R 1 is independently an electron withdrawing group; each R 2 is independently H or lower alkyl; and each n is independently an integer selected from one to four.
  • PRO is a macromolecule
  • w is an integer selected from one to ten
  • each RG′ is independently a divalent residue of a reactive group
  • each POLY is independently a water soluble polymer
  • each X is independently a bond, -O-,
  • each X is independently -O- or -N(R 2 )-. In certain embodiments, each X is independently -O-. In certain embodiments, each X is independently -N(R 2 )-. In certain embodiments, each n is one. In certain embodiments, each n is two. In certain embodiments, each n is three. In certain embodiments, each n is four. In certain embodiments, n1 is one. In certain embodiments, n1 is two. In certain embodiments, n1 is three. In certain embodiments, n1 is four. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four.
  • w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine. In certain embodiments, w is ten.
  • L 1 comprises a carbonyl carbon covalently bound to the fluorine
  • R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group.
  • the conjugate of Formula (I′) is according to Formula (II′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO, w, RG′, POLY, X, and R 1 are as defined in the Summary, or in any embodiment herein.
  • w is one.
  • w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine. In certain embodiments, w is ten. [00120] In certain embodiments, the conjugate of Formula (I′) is according to Formula (III′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof
  • PRO, w, POLY, and R 1 are as defined in the Summary, or in any embodiment herein, and each p is independently an integer selected from one to eight.
  • PRO is bound to a nitrogen of the triazole that is other than the middle nitrogen of the triazole. Accordingly, in certain embodiments, the triazole formation provides one of two possible regioisomeric products. In one regioisomeric embodiment, PRO is bound to a nitrogen shown below . In one regioisomeric embodiment, PRO is bound to a nitrogen shown below .
  • p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five.
  • p is six. In certain embodiments, p is seven. In certain embodiments, p is eight. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine. In certain embodiments, w is ten.
  • the conjugate of Formula (I′) is according to Formula (IV′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO, w, POLY, and R 1 are as defined in the Summary, or in any embodiment herein, and each p is independently an integer selected from one to eight.
  • PRO is bound to a nitrogen of the triazole that is other than the middle nitrogen of the triazole. Accordingly, in certain embodiments, the triazole formation provides one of two possible regioisomeric products. In one regioisomeric embodiment, PRO is bound to a nitrogen shown below . In one regioisomeric embodiment, PRO is bound to a nitrogen shown below
  • p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five. In certain embodiments, p is six. In certain embodiments, p is seven. In certain embodiments, p is eight. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine. In certain embodiments, w is ten.
  • the conjugate of Formula (I′) is according to Formula (V′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO, w, POLY, and R 1 are as defined in the Summary, or in any embodiment herein, and each p is independently an integer selected from one to eight.
  • p is one.
  • p is two.
  • p is three.
  • p is four.
  • p is five.
  • p is six.
  • p is seven.
  • p is eight. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine. In certain embodiments, w is ten.
  • a conjugate of Formula (X′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO is a macromolecule; w is an integer selected from one to ten; each RG′ is independently a divalent residue of a reactive group; each POLY is independently a water soluble polymer; each X is independently a bond, -O-, or -N(R 2 )-; each L 1 , L 2 , and L 3 is, independently, a linker; each R 1 is independently an electron withdrawing group; each R 2 is independently H or lower alkyl; and each m is independently an integer selected from one to four.
  • each X is independently -O- or -N(R 2 )-. In certain embodiments, each X is independently -O-. In certain embodiments, each X is independently -N(R 2 )-. In certain embodiments, m is one. In certain embodiments, m is two. In certain embodiments, m is three. In certain embodiments, m is four. In certain embodiments, m1 is one. In certain embodiments, m1 is two. In certain embodiments, m1 is three. In certain embodiments, m1 is four. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four.
  • the conjugate of Formula (X′) is according to Formula (XII′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof
  • PRO, w, RG′, POLY, X, L 2 , L 3 , and R 4 are as defined in the Summary, or in any embodiment herein.
  • w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine. In certain embodiments, w is ten.
  • the conjugate of Formula (X′) is according to Formula (XIII′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO, w, POLY, and R 4 are as defined in the Summary, or in any embodiment herein, and p is independently an integer selected from one to eight.
  • PRO is bound to a nitrogen of the triazole that is other than the middle nitrogen of the triazole. Accordingly, in certain embodiments, the triazole formation provides one of two possible regioisomeric products. In one regioisomeric embodiment, PRO is bound to a nitrogen shown below .
  • PRO is bound to a nitrogen shown below .
  • p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five. In certain embodiments, p is six. In certain embodiments, p is seven. In certain embodiments, p is eight. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight. In certain embodiments, w is nine.
  • the conjugate of Formula (X′) is according to Formula (XIV′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof wherein PRO, w, POLY, and R 4 are as defined in the Summary, or in any embodiment herein, and p is independently an integer selected from one to eight.
  • PRO is bound to a nitrogen of the triazole that is other than the middle nitrogen of the triazole. Accordingly, in certain embodiments, the triazole formation provides one of two possible regioisomeric products. In one regioisomeric embodiment, PRO is bound to a nitrogen shown below
  • PRO is bound to a nitrogen shown below .
  • p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five. In certain embodiments, p is six. In certain embodiments, p is seven. In certain embodiments, p is eight. In certain embodiments, w is one. In certain embodiments, w is two. In certain embodiments, w is three. In certain embodiments, w is four. In certain embodiments, w is five. In certain embodiments, w is six. In certain embodiments, w is seven. In certain embodiments, w is eight.
  • the conjugate of Formula (X′) is according to Formula (XV′) or a pharmaceutically acceptable salt, tautomer, stereoisomer, and/or mixture of stereoisomers, regioisomer, and/or mixture of regioisomers thereof (XV′) wherein PRO, w, POLY, and R 4 are as defined in the Summary, or in any embodiment herein, and p is independently an integer selected from one to eight. In certain embodiments, p is one. In certain embodiments, p is two. In certain embodiments, p is three. In certain embodiments, p is four. In certain embodiments, p is five.
  • each linker L 1 , L 2 , and L 3 can be any linker deemed suitable by the practitioner of skill in the art.
  • L 1 includes an ammonium where the positively charged nitrogen of the ammonium functional group is covalently bound to the fluorene.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is selected from the group consisting of -O-C 1-6 alkylene-S-C(O)-, -O-C 1-6 alkylene-NH-C(O)-, -S-C1-6alkylene-O-C(O)-, -S-C1-6alkylene-NH-C(O)-, -NH-C1-6alkylene-O-C(O)-, -NH-C 1-6 alkylene-S-C(O)-, -O-C 1-6 alkylene-N(Me)-C(O)-, -S-C 1-6 alkylene-N(Me)-C(O)-, -N(Me)-C1-6alkylene-O-C(O)-, -N(Me)-C1-6alkylene-S-C(O)-, -O-C1-6alkylene-N(Et)-C(O)-, -S-C 1-6 alkylene-N(Et)-C(O)
  • L 1 is -O-C1-6alkylene-S-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -O-C 1-6 alkylene-NH-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C1-6alkylene-O-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C 1-6 alkylene-NH-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -NH-C1-6alkylene-O-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -NH-C1-6alkylene-S-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -O-C 1-6 alkylene-N(Me)-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C1-6alkylene-N(Me)-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Me)-C 1-6 alkylene-O-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Me)-C1-6alkylene-S-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -O-C 1-6 alkylene-N(Et)-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -S-C 1-6 alkylene-N(Et)-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Et)-C1-6alkylene-O-C(O)- and -C1-6alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a halogen e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)
  • L 1 is -N(Et)-C 1-6 alkylene-S-C(O)- and -C 1-6 alkylene- is optionally substituted with one, two, or three groups independently selected from a halogen (e.g., fluoro (F), chloro (Cl), bromo (Br), or iodo (I)), alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • a conjugate of Formula (I′) or (X′) wherein each L 1 is independently -O-CH 2 -CH 2 -N(H)-C(O)- wherein the carbonyl carbon is covalently bound to the fluorene.
  • each L 2 is independently lower alkylene, -CH2-, -CH 2 CH 2 OCH 2 CH 2 OCH 2 CH 2 -, or -CH 2 -CH 2 -C(O)-.
  • each L 2 is independently lower alkylene selected from the group consisting of -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2- , -CH 2 CH 2 CH 2 CH 2 -, and -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - wherein each are independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH 2 - optionally substituted with a group selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH 2 CH 2 - independently optionally substituted with one or two groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH 2 CH 2 CH 2 - independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH2CH2CH2CH2- independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH2CH2CH2CH2CH2- independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • L 2 is -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - independently optionally substituted with one, two, or three groups independently selected from a halogen, alkyl, haloalkyl, hydroxyl, amino, alkylamino, and alkoxy.
  • provided is a conjugate of Formula (I′), (II′), (X′), or (XII′), wherein each L 2 is -CH 2 -.
  • provided is a conjugate of Formula (I′), (II′), (X′), or (XII′), wherein each L 2 is -CH2CH2OCH2CH2OCH2CH2-.
  • each RG′ is independently a divalent residue of any reactive group RG deemed suitable by the person of skill in the art.
  • provided is a conjugate of Formula (I′), (II′), (X′), or (XII′), wherein each RG′ is independently a divalent residue of RG where RG comprises an azide, alkyne, hydrazide, aldehyde, alkoxyamine, amine, carboxyl, ester, or maleimide.
  • each RG′ is independently a divalent residue of RG where each RG is independently selected from diment, provided is a conjugate of Formula (I′), (II′), (X′), or (XII′), w is independently a divalent residue of RG where each RG is one embodiment, provided is a conjugate of Formula (I′), (II′), (X′), or (XII′), wherein each RG′ is independently a divalent residue of RG where each RG is one embodiment, provided is a conjugate of Formula (I′), (II′), (X′), or (XII′), wherein each RG′ is independently a divalent residue of RG where each RG is one embodiment, provided is a conjugate of Formula (I′), (II′), (X′), or ( RG′ is independently a divalent residue of RG where each RG is bodiment, provided is a conjugate of Formula (I′) ′ ′ ′ wherein each RG′ is independently a divalent residue of
  • each POLY is polyethylene glycol (PEG).
  • each POLY is PEG having a molecular weight of about 20 kDa.
  • each POLY is uncapped (i.e., terminates with a hydroxyl).
  • each POLY is methoxy-PEG (i.e., terminates with a methoxy).
  • each PEG is linear.
  • each PEG is branched.
  • each R 1 is independently hydrogen, an electron donating group, or an electron withdrawing group. In one embodiment, each R 1 is hydrogen. In one embodiment, each R 1 is an electron donating group. In one embodiment, each R 1 is an electron withdrawing group. The electron donating group can be any electron donating group deemed suitable to the person of skill in the art. The electron withdrawing group can be any electron withdrawing group deemed suitable to the person of skill in the art.
  • each R 1 is independently selected from the group consisting of hydrogen, haloalkyl, halogen, -CN, -SO 3 H, -C(O)R 3 , -C(O)OR 3 , -OR 3 , -N(H)C(O)R 3 , -N(H)CO 2 R 3 , and -N(H)C(O)C(H)(R 3 )CO2H wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • each R 1 is independently selected from the group consisting of hydrogen, -CF 3 , -Br, -Cl, -F, -CN, - SO3H, -C(O)Me, -CO2Me, -OMe, -N(H)C(O)Me, -N(H)CO2Me, and - N(H)C(O)C(H)(Me)CO 2 H.
  • each R 1 is independently selected from the group consisting of haloalkyl, halogen, -CN, -SO 3 H, -C(O)R 3 , -C(O)OR 3 , -OR 3 , -N(H)C(O)R 3 , -N(H)CO2R 3 , and -N(H)C(O)C(H)(R 3 )CO2H wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • each R 1 is independently selected from the group consisting of -CF3, -Br, -Cl, -F, -CN, -SO3H, -C(O)Me, -CO2Me, -OMe, -N(H)C(O)Me, - N(H)CO 2 Me, and -N(H)C(O)C(H)(Me)CO 2 H.
  • each R 1 is independently selected from the group consisting of -CF 3 , -Br, -Cl, -F, - CN, -SO3H, -C(O)Me, -CO2Me, -OMe, -N(H)C(O)Me, -N(H)CO2Me, and -N(H)C(O)C(H)(Me)CO 2 H.
  • each R 4 is independently hydrogen or an electron withdrawing group. In one embodiment, each R 4 is hydrogen. In one embodiment, each R 4 is an electron withdrawing group.
  • the electron withdrawing group can be any electron withdrawing group deemed suitable to the person of skill in the art.
  • each R 4 is independently selected from the group consisting of hydrogen, haloalkyl, - C(O)R 3 , -C(O)OR 3 , and -S(O) 2 CH 3 wherein each R 3 is independently alkyl, cycloalkyl, heteroalkyl, heterocycloalkyl, aryl, or heteroaryl.
  • R 4 is -S(O)2CH3.
  • the macromolecule (PRO) can be any macromolecule deemed suitable by the person of skill in the art.
  • the macromolecule is a protein, peptide, antibody or antigen binding fragment thereof, nucleic acid, carbohydrate, or other large molecule composed of polymerized monomers.
  • the macromolecule is a protein.
  • the macromolecule is an antibody, or an antigen binding fragment thereof.
  • the macromolecule is selected from the group consisting of bone morphogenic protein, erythropoietin, G-CSF, GM-CSF, interferon alpha, interferon beta, interferon gamma, IL-2, and IL-11.
  • the macromolecule is a cytokine.
  • the macromolecule is an interleukin.
  • the macromolecule is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, or IL-36.
  • the macromolecule is IL-2.
  • the macromolecule is an interferon.
  • the macromolecule is interferon alpha. In one embodiment, the macromolecule is interferon beta. In one embodiment, the macromolecule is interferon gamma. In one embodiment, the macromolecule is a tumor necrosis factor. In one embodiment, the macromolecule is TNF alpha. In one embodiment, the macromolecule is TNF beta. In one embodiment, the macromolecule is a transforming growth factor. In one embodiment, the macromolecule is a chemokine. In one embodiment, the macromolecule is G-CSF. In one embodiment, the macromolecule is GM-CSF. In one embodiment, the macromolecule is erythropoietin. In one embodiment, the macromolecule is alpha- galactosidase A.
  • the macromolecule is tissue plasminogen activator. In one embodiment, the macromolecule is insulin. In one embodiment, the macromolecule is insulin-like growth factor. [00138] In one embodiment, the macromolecule is an antibody or an antigen binding fragment thereof.
  • Useful antibodies include, but are not limited to, rituximab (Rituxan®, IDEC/Genentech/Roche) (see, e.g., U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently being developed by Genmab, an anti-CD20 antibody described in U.S. Pat. No.
  • trastuzumab Herceptin®, Genentech
  • trastuzumab Herceptin®, Genentech
  • U.S. Pat. No.5,677,171 a humanized anti-Her2/neu antibody approved to treat breast cancer
  • pertuzumab rhuMab-2C4, Omnitarg®
  • cetuximab Erbitux®, Imclone
  • the therapeutics include KRN330 (Kirin); huA33 antibody (A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V integrins, Centocor); MEDI-522 (alpha V ⁇ 3integrin, Medimmune); volociximab (alpha V ⁇ 1 integrin, Biogen/PDL); Human mAb 216 (B cell glycosolated epitope, NCl); BiTE MT103 (bispecific CD19 ⁇ CD3, Medimmune); 4G7 ⁇ H22 (Bispecific Bcell ⁇ FcgammaR1, Medarex/Merck KGa); rM28 (Bispecific CD28 ⁇ MAPG, EP Patent No.
  • EP1444268 MDX447 (EMD 82633) (Bispecific CD64 ⁇ EGFR, Medarex); Catumaxomab (removab) (Bispecific EpCAM ⁇ anti- CD3, Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech); oregovomab (OvaRex) (CA-125, ViRexx); Rencarex® (WX G250) (carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105 (CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Bristol Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507) (CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab (Rituxan) (CD20, Genentech); veltuzumab
  • bispecific antibodies include, but are not limited to, those with one antibody directed against a tumor cell antigen and the other antibody directed against a cytotoxic trigger molecule such as anti-Fc ⁇ RI/anti-CD 15, anti-p185 HER2 /Fc ⁇ RIII (CD16), anti-CD3/anti-malignant B-cell (1D10), anti-CD3/anti-p185 HER2 , anti-CD3/anti-p97, anti- CD3/anti-renal cell carcinoma, anti-CD3/anti-OVCAR-3, anti-CD3/L-D1 (anti-colon carcinoma), anti-CD3/anti-melanocyte stimulating hormone analog, anti-EGF receptor/anti- CD3, anti-CD3/anti-CAMA1, anti-CD3/anti-CD19, anti-CD3/MoV18, anti-neural cell adhesion molecule (NCAM
  • the conjugate can be formed from a macromolecule that comprises one or more reactive groups. In certain embodiments, the conjugate can be formed from a macromolecule comprising all naturally encoded amino acids.
  • the conjugate can comprise a fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound or linker linked to the residue of an antibody reactive group.
  • the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound precursor or linker precursor comprises a reactive group capable of forming a bond with an antibody or antigen binding fragment thereof reactive group.
  • Typical reactive groups include maleimide groups, activated carbonates (including, but not limited to, p-nitrophenyl ester), activated esters (including, but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester, and aldehydes).
  • Particularly useful reactive groups include maleimide and succinimide, for instance N- hydroxysuccinimide, for forming bonds to cysteine and lysine side chains. Further reactive groups are described in the sections and examples below.
  • the macromolecule comprises one or more modified amino acids having a reactive group, as described herein.
  • the modified amino acid is not a naturally encoded amino acid.
  • These modified amino acids can comprise a reactive group useful for forming a covalent bond to a linker precursor or to a fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound precursor.
  • One of skill in the art can use the reactive group to link the macromolecule to any molecular entity capable of forming a covalent bond to the modified amino acid.
  • conjugates comprising a macromolecule comprising a modified amino acid residue linked to a macromolecule directly or indirectly via a linker. Exemplary modified amino acids are described in the sections below.
  • the modified amino acids have reactive groups capable of forming bonds to linkers or fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compounds with complementary reactive groups.
  • the non-natural amino acids are positioned at select locations in a polypeptide chain of the macromolecule. These locations were identified as providing optimum sites for substitution with the non-natural amino acids. Each site is capable of bearing a non-natural amino acid with optimum structure, function, and/or methods for producing the macromolecule.
  • a site-specific position for substitution provides a macromolecule that is stable. Stability can be measured by any technique apparent to those of skill in the art.
  • a site-specific position for substitution provides a macromolecule that has optimal functional properties.
  • the macromolecules comprise one or more non-natural amino acids suitable for conjugation.
  • the macromolecules provided herein comprise one, or more than one, non-natural amino acids at site-specific positions.
  • at least one of the non-natural amino acid residues is a pAMF residue.
  • the macromolecules provided herein comprise two non- natural amino acids at site-specific positions.
  • the macromolecule, such as an antibody or an antigen binding fragment thereof includes a Y180 pAMF mutation, a F404 pAMF mutation, or both.
  • the macromolecules provided herein comprise three non-natural amino acids at site-specific positions. In certain embodiments, the macromolecules provided herein comprise more than three non-natural amino acids at site-specific positions. [00146] In certain embodiments, the macromolecules provided herein are antibodies or antigen binding fragments thereof comprising one or more non-natural amino acids each at a position independently selected from the group consisting of heavy chain or light chain residues HC-F404, HC-K121, HC-Y180, HC-F241, HC-221, LC-T22, LC-S7, LC-N152, LC- K42, LC-E161, LC-D170, HC-S136, HC-S25, HC-A40, HC-S119, HC-S190, HC-K222, HC- R19, HC-Y52, or HC-S70, according to the Kabat, Chothia, or EU numbering scheme, or a post-translationally modified variant thereof.
  • the antibodies provided herein comprise one or more non-natural amino acids each at a position independently selected from the group consisting of HC-180, HC-222, LC-7, and LC-42, according to the Kabat, Chothia, or EU numbering scheme, or a post-translationally modified variant thereof.
  • HC indicates a heavy chain residue
  • LC indicates a light chain residue.
  • the non-natural amino acids are at HC-F404.
  • the non-natural amino acids are at HC-Y180.
  • the non-natural amino acids are at HC-F404 and HC-Y180.
  • the non-natural amino acids are at HC-K222.
  • the non-natural amino acids are at LC-S7. In certain embodiments, the non-natural amino acids are at LC-K42. In certain embodiments, the non- natural amino acids are at HC-Y180, HC-K222, LC-S7, and LC-K42. In certain embodiments, the non-natural amino acids are the same. In certain embodiments, the non-natural amino acids are different. In certain embodiments, the non-natural amino acids are residues of Formula (30), herein. [00147] In some embodiments, the macromolecule sequence may encompass a Q-tag sequence that is compatible with transglutaminase conjugation.
  • the one or more glutamine residues are in Q-tags independently selected from the group consisting of LLQGG, YAHQAHY, YRYRQ, PNPQLPF, PKPQQFM, GQQQLG, WALQRPH, WELQRPY, YPMQGWF, LSLSQG, GGGLLQGG, GLLQG, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, LLQGR, LLQGPA, LLQGPP, or GGLLQGPP.
  • the acyl donor glutamine-containing tag comprises at least one glutamine (Gln, or Q).
  • the acyl donor glutamine-containing tag comprises an amino acid sequence XXQX, wherein X is any amino acid (e.g., conventional amino acid Leu, Ala, Gly, Ser, Val, Phe, Tyr, His, Arg, Asn, Glu, Asp, Cys, Gln, Ile, Met, Pro, Thr, Lys, or Trp, or nonconventional amino acid).
  • the acyl donor glutamine-containing tag comprises an amino acid sequence selected from the group consisting of LLQGG, LLQG, LSLSQG, GGGLLQGG, GLLQG, GSPLAQSHGG, GLLQGGG, GLLQGG, GLLQ, LLQLLQGA, LLQGA, LLQYQGA, LLQGSG, LLQYQG, LLQLLQG, SLLQG, LLQLQ, LLQLLQ, and LLQGR.
  • the acyl donor glutamine-containing tag (Q-tag) comprises an amino acid sequence selected from the group consisting of LLQGPA, LLQGPP, and GGLLQGPP. In some embodiments, the acyl donor glutamine-containing tag (Q-tag) comprises an amino acid sequence selected from the group consisting of LLQGG and LLQGA. In such embodiments, a linker-fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound bearing an amino group can be conjugated to the side chain of one or more glutamine (Q) residues in the macromolecule in the presence of transglutaminase.
  • Reactive groups facilitate conjugation of the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compounds described herein to a second compound, such as an macromolecule described herein.
  • the reactive group is designated RG herein.
  • Reactive groups can react via any suitable reaction mechanism known to those of skill in the art.
  • a reactive group reacts through a [3+2] alkyne-azide cycloaddition reaction, inverse-electron demand Diels-Alder ligation reaction, thiol- electrophile reaction, or carbonyl-oxyamine reaction, as described in detail herein.
  • the reactive group comprises an alkyne, strained alkyne, tetrazine, thiol, para- acetyl-phenylalanine residue, oxyamine, maleimide, or azide. In certain embodiments, the reactive group is:
  • R 201 is methyl, ethyl, or propyl. In one embodiment, R 201 is methyl. In one embodiment, R 201 is ethyl. In one embodiment, R 201 is propyl. Additional reactive groups are described in, for example, U.S. Patent Application Publication No. 2014/0356385, U.S. Patent Application Publication No. 2013/0189287, U.S. Patent Application Publication No.2013/0251783, U.S. Patent No. 8,703,936, U.S. Patent No. 9,145,361, U.S. Patent No.9,222,940, and U.S. Patent No.8,431,558.
  • a divalent residue of the reactive group (viz., RG′) is formed and is bonded to the residue of a second compound.
  • the structure of the divalent residue is determined by the type of conjugation reaction employed to form the conjugate.
  • the divalent residue of the reactive group e.g., RG′
  • the divalent residue of the reactive group comprises a triazole ring or fused cyclic group comprising a triazole ring.
  • the divalent residue of the reactive group is:
  • the divalent residue of the reactive group comprises a fused bicyclic ring having at least two adjacent nitrogen atoms in the ring.
  • the divalent residue of the reactive group (e.g., RG′) is in certain embodiments, when a conjugate is formed through a thiol-maleimide reaction, the divalent residue of the reactive group comprises sulfur linkage. In certain embodiments, when a conjugate is formed through a thiol-maleimide, , reaction the divalent residue of the reactive group ( certain embodiments when a conjugate is formed through an oxime conjugation reaction, the divalent residue of the reactive group comprises a divalent residue of a non-natural amino acid.
  • the divalent residue of the reactive group when a conjugate is formed through an oxime conjugation reaction, the divalent residue of the reactive group ( In certain embodiments when a conjugate is formed through an oxime conjugation reaction, the divalent residue of the reactive group comprises an oxime linkage. In certain embodiments when a conjugate is formed through an oxime conjugation reaction, the divalent residue of the reactive group ( [00152] In particular embodiments, provided herein are conjugates wherein the macromolecule comprises a residue of a non-natural amino acid according to Formula (30), below.
  • conjugates wherein the macromolecule is an antibody or antigen binding fragment thereof comprising a residue of a non-natural amino acid according to Formula (30), below, at heavy chain position 404 according to the EU numbering system (30).
  • amino acids such as Formula (30) are incorporated into macromolecules as residues.
  • a residue of Formula (30) can be according to the following Formula (30′) Further modification, for instance at -N 3 is also contemplated and encompassed within the term “residue” herein.
  • conjugates wherein the macromolecule comprises a residue of a non-natural amino acid according to Formula (56), below.
  • conjugates wherein the macromolecule is an antibody or antigen binding fragment thereof comprising a residue of the non-natural amino acid according to Formula (56), below, at heavy chain position 404 according to the EU numbering system.
  • the non-natural amino acid according to Formula (56) is [00155]
  • conjugates wherein the macromolecule comprises a non-natural amino acid residue of para-azidomethyl-L- phenylalanine.
  • conjugates wherein the macromolecule is an antibody or antigen binding fragment thereof comprising a residue of the non-natural amino acid residue para-azidomethyl-L-phenylalanine at heavy chain position 404 according to the EU numbering system [00156]
  • conjugates wherein the macromolecule comprises a non-natural amino acid residue of para-acetyl-L-phenylalanine.
  • conjugates wherein the macromolecule is an antibody or antigen binding fragment thereof comprising a residue of the non-natural amino acid residue para-acetyl-L-phenylalanine at heavy chain position 404 according to the EU numbering system..
  • the conjugate comprises one or more water soluble polymers.
  • a wide variety of macromolecular polymers and other molecules can be linked to the polypeptides described herein to modulate biological properties of the polypeptide, and/or provide new biological properties to the polypeptide.
  • These macromolecular polymers can be linked to the polypeptide via a naturally encoded amino acid, via a non-naturally encoded amino acid, or any functional substituent of a natural or modified amino acid, or any substituent or functional group added to a natural or modified amino acid.
  • the molecular weight of the polymer may include a wide range including, but not limited to, between about 100 Da and about 100,000 Da or more.
  • the polymer selected may be water soluble so that a protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer may be branched or unbranched.
  • the polymer will be pharmaceutically acceptable.
  • the proportion of polyethylene glycol molecules to polypeptide molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is minimal excess unreacted protein or polymer) may be determined by the molecular weight of the polyethylene glycol selected and on the number of available reactive groups available.
  • the water soluble polymer may be any structural form including, but not limited to, linear, forked, or branched.
  • the water soluble polymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG), but other water soluble polymers can also be employed.
  • PEG poly(ethylene glycol)
  • PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene oxide according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).
  • PEG is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of a PEG, and can be represented as linked to a polypeptide by the formula: X′O–(CH 2 CH 2 O) n –CH 2 CH 2 –Y where n is an integer selected from 2 to 10,000, X′ is hydrogen or a terminal modification including, but not limited to, C 1-4 alkyl, and Y is the attachment point to the polypeptide. [00162] In some cases, a PEG terminates on one end with hydroxy or methoxy, i.e., X′ is hydrogen or CH 3 (aka “methoxy PEG”).
  • the PEG can terminate with a PEG reactive group, thereby forming a bifunctional polymer.
  • Typical PEG reactive groups can include those reactive groups that are commonly used to react with the functional groups found in the twenty common amino acids (including, but not limited to, maleimide groups, activated carbonates (including, but not limited to, p-nitrophenyl ester), activated esters (including, but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester, and aldehydes) as well as functional groups that are inert to the twenty common amino acids, but that react specifically with complementary functional groups present in non-naturally encoded amino acids (including, but not limited to, azide groups and/or alkyne groups).
  • Y may be an amide, carbamate, or urea linkage to an amine group (including, but not limited to, the epsilon amine of lysine or the N-terminus) of the polypeptide.
  • Y may be a maleimide linkage to a thiol group (including, but not limited to, the thiol group of cysteine).
  • Y may be a linkage to a residue not commonly accessible via the twenty common amino acids.
  • an azide group on the PEG can be reacted with an alkyne group on the polypeptide to form a Huisgen [3+2] cycloaddition product.
  • an alkyne group on the PEG can be reacted with an azide group present in a non-naturally encoded amino acid, such as the modified amino acids described herein, to form a similar product.
  • a strong nucleophile (including, but not limited to, hydrazine, hydrazide, hydroxylamine, or semicarbazide) can be reacted with an aldehyde or ketone group present in a non-naturally encoded amino acid to form a hydrazone, oxime, or semicarbazone, as applicable, which in some cases can be further reduced by treatment with an appropriate reducing agent.
  • the strong nucleophile can be incorporated into the polypeptide via a non-naturally encoded amino acid and used to react preferentially with a ketone or aldehyde group present in the water soluble polymer.
  • Any molecular mass for a PEG can be used as practically desired including, but not limited to, from about 100 Daltons (Da) to 100,000 Da or more as desired (including, but not limited to, in certain embodiments 0.1-50 kDa or 10-40 kDa).
  • Branched chain PEGs including, but not limited to, PEG molecules with each chain having a molecular weight (MW) ranging from 1-100 kDa (including, but not limited to, 1-50 kDa or 5-20 kDa) can also be used.
  • MW molecular weight
  • a wide range of PEG molecules are described in the Shearwater Polymers, Inc. catalog, and the Nektar Therapeutics catalog, each incorporated herein by reference.
  • the PEG molecule is available for reaction with the remainder of the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound.
  • PEG derivatives bearing alkyne and azide moieties for reaction with amino acid side chains can be used to attach PEG to non-naturally encoded amino acids as described herein. If the non-naturally encoded amino acid comprises an azide, then the PEG will typically contain either an alkyne moiety to effect formation of the [3+2] cycloaddition product or an activated PEG species (i.e., ester, carbonate) containing a phosphine group to effect formation of the amide linkage.
  • the PEG will typically contain an azide moiety to effect formation of the [3+2] Huisgen cycloaddition product.
  • the PEG will typically comprise a nucleophile (including, but not limited to, a hydrazide, hydrazine, hydroxylamine, or semicarbazide functionality) in order to effect formation of corresponding hydrazone, oxime, and semicarbazone linkages, respectively.
  • the polypeptide variant with a PEG derivative contains a chemical functionality that is reactive with the chemical functionality present on the side chain of the non-naturally encoded amino acid.
  • POLY is an azide- or acetylene-containing polymer comprising a water soluble polymer backbone having an average molecular weight from about 800 Da to about 100,000 Da.
  • the polymer backbone of the water-soluble polymer can be poly(ethylene glycol).
  • PEG water soluble polymers
  • PEG poly(ethylene)glycol and other related polymers, including poly(dextran) and poly(propylene glycol)
  • PEG poly(ethylene glycol)
  • PEG poly(ethylene glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG, pendent PEG (i.e., PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein.
  • the polymer backbone can be linear or branched.
  • Branched polymer backbones are generally known in the art.
  • a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to the central branch core.
  • PEG is commonly used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol, and sorbitol.
  • the central branch moiety can also be derived from several amino acids, such as lysine.
  • the branched poly(ethylene glycol) can be represented in general form as R-(-PEG-OH) m in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms.
  • R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol
  • m represents the number of arms.
  • Multi-armed PEG molecules such as those described in U.S. Pat. Nos. 5,932,462; 5,643,575; 5,229,490; and 4,289,872; U.S. Pat. Appl. No. 2003/0143596; and WO 96/21469 and WO 93/21259, each of which is incorporated by reference herein in its entirety, can also be used as the polymer backbone.
  • Branched PEG can also be in the form of a forked PEG represented by PEG(-YCHZ 2 ) n , where Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length.
  • the pendant PEG has PEG reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.
  • the polymer can also be prepared with weak or degradable linkages in the backbone.
  • PEG can be prepared with ester linkages in the polymer backbone that are subject to hydrolysis.
  • poly(ethylene glycol) or “PEG” represents or includes all the forms known in the art including, but not limited to, those disclosed herein.
  • polymer backbones that are water-soluble, with from two to about three hundred termini, are particularly suitable.
  • suitable polymers include, but are not limited to, other poly(alkylene glycols), such as poly(propylene glycol) (“PPG”), copolymers thereof (including, but not limited to, copolymers of ethylene glycol and propylene glycol), terpolymers thereof, mixtures thereof, and the like.
  • PPG poly(propylene glycol)
  • copolymers thereof including, but not limited to, copolymers of ethylene glycol and propylene glycol
  • terpolymers thereof mixtures thereof, and the like.
  • the molecular weight of each chain of the polymer backbone can vary, it is typically in the range of from about 800 Da to about 100,000 Da, often from about 6,000 Da to about 80,000 Da.
  • substantially water-soluble backbones is by no means exhaustive and is merely exemplary, and that all polymeric materials having the qualities described herein are contemplated as being suitable for use.
  • the polymer derivatives are "multi-functional," meaning that the polymer backbone has at least two termini, and possibly as many as about 300 termini, functionalized or activated with a functional group.
  • Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each terminus being bonded to a functional group which may be the same or different.
  • Linkers [00174]
  • the macromolecules can be linked to the fluorenylmethyloxycarbonyl and/or fluorenylmethylaminocarbonyl compounds with one or more linkers capable of reacting with a macromolecule amino acid and with a fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound.
  • the one or more linkers can be any linkers apparent to those of skill in the art.
  • linker is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent linkages as defined above.
  • Useful linkers include those described herein.
  • the linker is any divalent or multivalent linker known to those of skill in the art.
  • Useful divalent linkers include, carbonyl, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarylene, and substituted heteroarylene.
  • the linker is C1-10 alkylene or C1-10 heteroalkylene.
  • the C 1-10 heteroalkylene is PEG.
  • the linker is hydrolytically stable. Hydrolytically stable linkers or linkages means that the linkers or linkages are substantially stable in water (i.e., do not react with water at useful pH values) including, but not limited to, physiological conditions for an extended period of time, perhaps even indefinitely in certain embodiments.
  • the linker is hydrolytically unstable. Hydrolytically unstable or degradable linkers or linkages mean that the linkers or linkages are degradable in water or in aqueous solutions including, for example, blood. Enzymatically unstable or degradable linkers or linkages mean that the linkers or linkage can be degraded by one or more enzymes.
  • PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule.
  • ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent.
  • hydrolytically degradable linkers or linkages include, but are not limited to, carbonate linkages; imine linkages resulting from reaction of an amine and an aldehyde or ketone; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are the reaction product of a hydrazide and an aldehyde or ketone; acetal linkages that are the reaction product of an aldehyde or ketone and an alcohol; orthoester linkages that are the reaction product of a nitrile and an alcohol; peptide linkages formed by an amine group including, but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite groupat the end of a polymer, and a 5'-hydroxyl group of an oligonucleotide.
  • cleavable linkers are known to those of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225; and 4,625,014.
  • the mechanisms for release of an agent from these linker groups include, for example, irradiation of a photolabile bond and acid- catalyzed hydrolysis.
  • U.S. Pat. No. 4,671,958, for example includes a description of immunoconjugates comprising linkers that are cleaved at the target site in vivo by the proteolytic enzymes of the patient's complement system.
  • the length of the linker may be predetermined or selected depending upon a desired spatial relationship between the polypeptide and the molecule linked to it.
  • the linker may have a wide range of molecular weight or molecular length. Larger or smaller molecular weight linkers may be used to provide a desired spatial relationship or conformation between the polypeptide and the linked entity. Linkers having longer or shorter molecular length may also be used to provide a desired space or flexibility between the polypeptide and the linked entity.
  • a linker having a particular shape or conformation may be utilized to impart a particular shape or conformation to the polypeptide or the linked entity, either before or after the polypeptide reaches its target.
  • the functional groups present on each end of the linker may be selected to modulate the release of a macromolecule under desired conditions. This optimization of the spatial relationship between the polypeptide and the linked entity may provide new, modulated, or desired properties to the molecule.
  • water-soluble bifunctional linkers that have a dumbbell structure that includes: a) an azide, an alkyne, a hydrazine, a hydrazide, a hydroxylamine, or a carbonyl-containing moiety on at least a first end of a polymer backbone; and b) at least a second functional group on a second end of the polymer backbone.
  • the second functional group can be the same or different as the first functional group.
  • the second functional group in some embodiments, is not reactive with the first functional group.
  • water-soluble compounds that comprise at least one arm of a branched molecular structure are provided.
  • the branched molecular structure can be a dendritic structure.
  • the linker is derived from a linker precursor selected from the group consisting of N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(2-pyridyldithio)pentanoate (SPP), N-succinimidyl 4-(2- pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB), N-succinimidyl iodoacetate (SIA), N-succinimidyl(4- iodoacetyl)aminobenzoate (SIAB), maleimide PEG NHS, N-succinimidyl 4-(maleimide PEG NHS, N-succ
  • the linker is derived from the linker precursor N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC). In one embodiment, the linker is derived from the linker precursor N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP). In one embodiment, the linker is derived from the linker precursor N-succinimidyl 4- (2-pyridyldithio)pentanoate (SPP). In one embodiment, the linker is derived from the linker precursor N-succinimidyl 4-(2-pyridyldithio)butanoate (SPDB).
  • SCC N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate
  • SPDP N-succinimidyl-3-(2-pyridyldithio)propionate
  • SPP (2-pyridyldithio
  • the linker is derived from the linker precursor N-succinimidyl-4-(2-pyridyldithio)-2-sulfo-butanoate (sulfo-SPDB). In one embodiment, the linker is derived from the linker precursor N- succinimidyl iodoacetate (SIA). In one embodiment, the linker is derived from the linker precursor N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB). In one embodiment, the linker is derived from the linker precursor maleimide PEG NHS.
  • the linker is derived from the linker precursor N-sulfosuccinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (sulfo-SMCC).
  • the linker is derived from the linker precursor 2,5-dioxopyrrolidin-1-yl 17-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)-5,8,11,14-tetraoxo-4,7,10,13-tetraazaheptadecan-1-oate (CX1-1).
  • the linker is derived from a linker precursor selected from the group consisting of dipeptides, tripeptides, tetrapeptides, and pentapeptides.
  • the linker can be cleaved by a protease.
  • Exemplary dipeptides include, but are not limited to, valine-citrulline (VC or val-cit), alanine-phenylalanine (AF or ala-phe); phenylalanine-lysine (FK or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit).
  • exemplary tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit), glycine-glycine-glycine (gly-gly-gly), glycine- (methoxyethoxyethyl)serine-valine, and O-MeSerValAla).
  • a linker comprises a self-immolative spacer.
  • the self-immolative spacer comprises p-aminobenzyl.
  • a p-aminobenzyl alcohol is attached to an amino acid unit via an amide bond, and a carbamate, methylcarbamate, or a carbonate is made between the benzyl alcohol and the payload (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087-1103).
  • the linker comprises p-aminobenzyloxycarbonyl (PABC).
  • PABC p-aminobenzyloxycarbonyl
  • Other examples of self-immolative spacers include, but are not limited to, aromatic compounds that are electronically similar to the PABC group, such as 2-aminoimidazol-5-methanol derivatives (U.S. Pat. No. 7,375,078; Hay et al. (1999) Bioorg. Med.
  • spacers can be used that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ring systems (Storm et al. (1972) J. Amer. Chem. Soc.94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al. (1990) J. Org. Chem.55:5867).
  • Linkage of a drug to the ⁇ -carbon of a glycine residue is another example of a self-immolative spacer that may be useful in conjugates (Kingsbury et al. (1984) J. Med. Chem.27:1447).
  • linker precursors can be combined to form larger linkers.
  • linkers comprise the dipeptide valine-citrulline and p-aminobenzyloxycarbonyl. These are also referenced as ValCit-PABC- linkers.
  • the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compounds can be linked to the linkers, referred to herein as a linker-fluorenylmethoxycarbonyl and/or linker-fluorenylmethylaminocarbonyl compounds, with one or more linker groups capable of reacting with an macromolecule amino acid group.
  • the one or more linkers can be any linkers apparent to those of skill in the art or those set forth herein.
  • Linker precursors can be prepared as described herein in the Examples section, and/or by standard techniques (e.g., WO 2019/055931, WO 2019/055909, WO 2017/132617, WO 2017/132615, each incorporated by reference in their entirety), or obtained from commercial sources.
  • Compositions and Uses Pharmaceutical Compositions and Methods of Administration [00189] The conjugates provided herein can be formulated into pharmaceutical compositions using methods available in the art and those disclosed herein. Any of the conjugates provided herein can be provided in the appropriate pharmaceutical composition and be administered by a suitable route of administration. [00190] The methods provided herein encompass administering pharmaceutical compositions comprising at least one conjugate provided herein and one or more compatible and pharmaceutically acceptable carriers.
  • the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and in certain embodiments in humans.
  • carrier includes a diluent, adjuvant (e.g., Freund’s adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered.
  • adjuvant e.g., Freund’s adjuvant (complete and incomplete)
  • excipient e.g., complete and incomplete
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils including petroleum, animal, vegetable, or oils of synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water can be used as a carrier when the pharmaceutical composition is administered intravenously.
  • compositions or conjugates provided herein may be administered by any route known in the art.
  • routes of administration include, but are not limited to, inhalation, intraarterial, intradermal, intramuscular, intraperitoneal, intravenous, nasal, parenteral, pulmonary, and subcutaneous routes.
  • a pharmaceutical composition or conjugate provided herein is administered parenterally.
  • the compositions for parenteral administration can be emulsions or sterile solutions.
  • Parenteral compositions may include, for example, propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters (e.g., ethyl oleate). These compositions can also contain wetting, isotonizing, emulsifying, dispersing, and stabilizing agents. Sterilization can be carried out in several ways, for example, using a bacteriological filter, via radiation, or via heating. Parenteral compositions can also be prepared in the form of sterile solid compositions which can be dissolved at the time of use in sterile water or any other injectable sterile medium. [00193] In some embodiments, a composition provided herein is a pharmaceutical composition or a single unit dosage form.
  • compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic conjugates.
  • the pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, wherein a person of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients. Non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
  • composition or dosage form Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a subject and the specific conjugate in the dosage form.
  • the composition or single unit dosage form if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. Accordingly, the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference herein in its entirety.
  • the pharmaceutical composition comprises an anti- foaming agent.
  • Any suitable anti-foaming agent may be used.
  • the anti- foaming agent is selected from an alcohol, an ether, an oil, a wax, a silicone, a surfactant, and combinations thereof.
  • the anti-foaming agent is selected from a mineral oil, a vegetable oil, ethylene bis stearamide, a paraffin wax, an ester wax, a fatty alcohol wax, a long- chain fatty alcohol, a fatty acid soap, a fatty acid ester, a silicon glycol, a fluorosilicone, a polyethylene glycol-polypropylene glycol copolymer, polydimethylsiloxane-silicon dioxide, ether, octyl alcohol, capryl alcohol, sorbitan trioleate, ethyl alcohol, 2-ethyl-hexanol, dimethicone, oleyl alcohol, simethicone, and combinations thereof.
  • the pharmaceutical composition comprises a co-solvent.
  • co-solvents include ethanol, poly(ethylene) glycol, butylene glycol, dimethylacetamide, glycerin, and propylene glycol.
  • the pharmaceutical composition comprises a buffer.
  • buffers include acetate, borate, carbonate, lactate, malate, phosphate, citrate, hydroxide, diethanolamine, monoethanolamine, glycine, methionine, guar gum, and monosodium glutamate.
  • the pharmaceutical composition comprises a carrier or filler.
  • the pharmaceutical composition comprises a surfactant.
  • surfactants include d-alpha tocopherol, benzalkonium chloride, benzethonium chloride, cetrimide, cetylpyridinium chloride, docusate sodium, glyceryl behenate, glyceryl monooleate, lauric acid, macrogol 15 hydroxystearate, myristyl alcohol, phospholipids, polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, polyoxylglycerides, sodium lauryl sulfate, sorbitan esters, and vitamin E polyethylene(glycol) succinate.
  • the pharmaceutical composition comprises an anti- caking agent.
  • anti-caking agents include calcium phosphate (tribasic), hydroxymethyl cellulose, hydroxypropyl cellulose, and magnesium oxide.
  • Other excipients that may be used with the pharmaceutical compositions include, for example, albumin, antioxidants, antibacterial agents, antifungal agents, bioabsorbable polymers, chelating agents, controlled release agents, diluents, dispersing agents, dissolution enhancers, emulsifying agents, gelling agents, ointment bases, penetration enhancers, preservatives, solubilizing agents, solvents, stabilizing agents, and sugars. Specific examples of each of these agents are described, for example, in the Handbook of Pharmaceutical Excipients, Rowe et al.
  • the pharmaceutical composition comprises a solvent.
  • the solvent is saline solution, such as a sterile isotonic saline solution or dextrose solution.
  • the solvent is water for injection.
  • the pharmaceutical compositions are in a particulate form, such as a microparticle or a nanoparticle. Microparticles and nanoparticles may be formed from any suitable material, such as a polymer or a lipid. In some aspects, the microparticles or nanoparticles are micelles, liposomes, or polymersomes.
  • anhydrous pharmaceutical compositions and dosage forms comprising a conjugate, since, in some embodiments, water can facilitate the degradation of some antibodies or antigen binding fragments thereof.
  • Anhydrous pharmaceutical compositions and dosage forms provided herein can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine can be anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
  • An anhydrous pharmaceutical composition can be prepared and stored such that its anhydrous nature is maintained.
  • anhydrous compositions can be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits.
  • suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.
  • Lactose-free compositions provided herein can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmocopeia (USP) SP (XXI)/NF (XVI).
  • lactose-free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts.
  • Exemplary lactose-free dosage forms comprise an active ingredient, microcrystalline cellulose, pre gelatinized starch, and magnesium stearate.
  • pharmaceutical compositions and dosage forms that comprise one or more excipients that reduce the rate by which a conjugate will decompose.
  • excipients which are referred to herein as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
  • Parenteral Dosage Forms [00209] In certain embodiments, provided are parenteral dosage forms. Parenteral dosage forms can be administered to subjects by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial.
  • parenteral dosage forms are typically sterile or capable of being sterilized prior to administration to a subject.
  • parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
  • Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art.
  • Examples include, but are not limited to Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer’s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer’s Injection; water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer’s Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer’s Injection
  • water miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropy
  • compositions provided herein is a pharmaceutical composition or a single unit dosage form.
  • Pharmaceutical compositions and single unit dosage forms provided herein comprise a prophylactically or therapeutically effective amount of one or more prophylactic or therapeutic antibodies or antigen binding fragments thereof.
  • the amount of the conjugate or composition which will be effective in the prevention or treatment of a disorder or one or more symptoms thereof will vary with the nature and severity of the disease or condition, and the route by which the conjugate is administered.
  • the frequency and dosage will also vary according to factors specific for each subject depending on the specific therapy (e.g., therapeutic or prophylactic agents) administered, the severity of the disorder, disease, or condition, the route of administration, as well as age, body, weight, response, and the past medical history of the subject.
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • exemplary doses of a composition include milligram or microgram amounts of the conjugate per kilogram of subject or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 microgram per kilogram to about 10 milligrams per kilogram).
  • the dosage of the conjugate provided herein, based on weight of the conjugate, administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 10 mg/kg, or 15 mg/kg or more of a subject’s body weight.
  • the dosage of the composition or a composition provided herein administered to prevent, treat, manage, or ameliorate a disorder, or one or more symptoms thereof in a subject is 0.1 mg to 200 mg, 0.1 mg to 100 mg, 0.1 mg to 50 mg, 0.1 mg to 25 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 10 mg, 0.1 mg to 7.5 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 20 mg, 0.25 to 15 mg, 0.25 to 12 mg, 0.25 to 10 mg, 0.25 mg to 7.5 mg, 0.25 mg to 5 mg, 0.25 mg to 2.5 mg, 0.5 mg to 20 mg, 0.5 to 15 mg, 0.5 to 12 mg, 0.5 to 10 mg, 0.5 mg to 7.5 mg, 0.5 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 7.5 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.
  • the dose can be administered according to a suitable schedule, for example, once, two times, three times, or for times weekly. It may be necessary to use dosages of the conjugate outside the ranges disclosed herein in some cases, as will be apparent to those of ordinary skill in the art. Furthermore, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with subject response. [00217] Different therapeutically effective amounts may be applicable for different diseases and conditions, as will be readily known by those of ordinary skill in the art.
  • amounts sufficient to prevent, manage, treat, or ameliorate such disorders, but insufficient to cause, or sufficient to reduce, adverse effects associated with the antibodies or antigen binding fragments thereof provided herein are also encompassed by the described dosage amounts and dose frequency schedules herein.
  • the dosage administered to the subject may be increased to improve the prophylactic or therapeutic effect of the composition or it may be decreased to reduce one or more side effects that a particular subject is experiencing.
  • treatment or prevention can be initiated with one or more loading doses of a conjugate or composition provided herein followed by one or more maintenance doses.
  • a dose of a conjugate or composition provided herein can be administered to achieve a steady-state concentration of the conjugate in blood or serum of the subject.
  • the steady-state concentration can be determined by measurement according to techniques available to those of skill or can be based on the physical characteristics of the subject such as height, weight, and age.
  • administration of the same composition may be repeated and the administrations may be separated by at least one day, two days, three days, five days, ten days, fifteen days, thirty days, forty-five days, two months, seventy-five days, three months, or six months.
  • administration of the same prophylactic or therapeutic agent may be repeated and the administration may be separated by at least one day, two days, three days, five days, ten days, fifteen days, thirty days, forty-five days, two months, seventy-five days, three months, or six months.
  • Therapeutic Applications the conjugates are administered to a mammal, in certain embodiments, a human, in a pharmaceutically acceptable dosage form such as those known in the art and those discussed herein.
  • the conjugates of this disclosure may be administered to a human intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra- articular, intrasynovial, intrathecal, or intratumoral routes.
  • the conjugates also are suitably administered by peritumoral, intralesional, or perilesional routes, to exert local as well as systemic therapeutic effects.
  • the intraperitoneal route may be particularly useful, for example, in the treatment of ovarian tumors.
  • the conjugates provided herein may be useful for the treatment of any disease or condition described herein (e.g., inflammatory and/or proliferative disease or condition).
  • the disease or condition is a disease or condition that can be diagnosed by overexpression of an antigen. In some embodiments, the disease or condition is a disease or condition that can benefit from treatment with an macromolecule. In some embodiments, the disease or condition is a cancer. [00223] Any suitable cancer may be treated with the conjugates provided herein.
  • Exemplary or suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular cancer, histiocytosis,
  • the disease to be treated with the conjugates provided herein is gastric cancer, colorectal cancer, renal cell carcinoma, cervical cancer, non-small cell lung carcinoma, ovarian cancer, uterine cancer, endometrial carcinoma, prostate cancer, breast cancer, head and neck cancer, brain carcinoma, liver cancer, pancreatic cancer, mesothelioma, and/or a cancer of epithelial origin.
  • the disease is colorectal cancer.
  • the disease is ovarian cancer.
  • the disease is breast cancer.
  • the disease is lung cancer.
  • the disease is head and neck cancer.
  • the disease is renal cell carcinoma.
  • the disease is brain carcinoma.
  • the disease is endometrial carcinoma.
  • the disease is pancreatic cancer, multiple myeloma, colorectal cancer, renal and mammary carcinomas, skin cancer, and/or cervical intraepithelial neoplasia.
  • the disease is non-Hodgkin’s lymphoma, pancreatic cancer, multiple myeloma, colorectal cancer, renal and mammary carcinomas, skin cancer, and/or cervical intraepithelial neoplasia.
  • provided herein are methods for the treatment of cancer that include the administration of an effective amount of conjugates provided herein, or a pharmaceutically acceptable salt thereof.
  • kits for treating cancer in a subject encompass the step of administering to the subject in need thereof an amount of a conjugate described herein effective for the treatment of cancer in combination with a second agent effective for the treatment or prevention of the infection.
  • the conjugate is in the form of a pharmaceutical composition or dosage form, as described elsewhere herein.
  • the subject is a treatment na ⁇ ve subject.
  • the subject has previously received therapy for a cancer. For instance, in certain embodiments, the subject has not responded to a single agent treatment regimen.
  • the subject is a subject that discontinued some other therapy because of one or more adverse events associated with the other therapy.
  • the subject has received some other anti-cancer therapy and discontinued that therapy prior to administration of a method provided herein. In further embodiments, the subject has received therapy and continues to receive that therapy along with administration of a conjugate provided herein.
  • the conjugates described herein can be co- administered with other therapy for treatment of cancer according to the judgment of one of skill in the art.
  • the methods or compositions provided herein can be co-administered with a reduced dose of the other therapy for the treatment of cancer.
  • the subject can be a subject that has responded poorly to some other anti-cancer treatment.
  • the conjugates provided herein are used in diagnostic applications. These assays may be useful, for example, in making a diagnosis and/or prognosis for a disease, such as a cancer.
  • the conjugate may be labeled with a detectable moiety. Suitable detectable moieties include, but are not limited to, radioisotopes, fluorescent labels, and enzyme-substrate labels.
  • the conjugate need not be labeled, and the presence of the conjugate can be detected using a labeled antibody or antigen binding fragment thereof which specifically binds to the conjugate.
  • kits [00233] In some embodiments, a conjugate provided herein is provided in the form of a kit (i.e., a packaged combination of reagents in predetermined amounts with instructions for performing a procedure). In some embodiments, the procedure is a diagnostic assay. In certain embodiments, the procedure is a therapeutic procedure. [00234] In some embodiments, the kit further comprises a solvent for the reconstitution of the conjugate. In some embodiments, the conjugate is provided in the form of a pharmaceutical composition. [00235] In some embodiments, the kits can include a conjugate or composition provided herein, an optional second agent or composition, and instructions providing information to a health care provider regarding usage for treating the disorder.
  • a unit dose of a conjugate or a composition provided herein, or a second agent or composition can include a dosage such that when administered to a subject, a therapeutically or prophylactically effective plasma level of the compound or composition can be maintained in the subject for at least one day.
  • a compound or composition can be included as a sterile aqueous pharmaceutical composition or dry powder (e.g., lyophilized) composition.
  • suitable packaging is provided.
  • packaging includes a solid matrix or material customarily used in a system and capable of holding within fixed limits a compound provided herein and/or a second agent suitable for administration to a subject.
  • materials include glass and plastic (e.g., polyethylene, polypropylene, and polycarbonate) bottles, vials, paper, plastic, plastic-foil laminated envelopes, and the like. If e-beam sterilization techniques are employed, the packaging should have sufficiently low density to permit sterilization of the contents.
  • the conjugates can be prepared by standard techniques.
  • a macromolecule is contacted with a fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound under conditions suitable for forming a bond from the macromolecule to the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound to form a conjugate.
  • a macromolecule is contacted with a linker precursor under conditions suitable for forming a bond from the macromolecule to the linker.
  • the resulting macromolecule-linker is contacted with a fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound under conditions suitable for forming a bond from the macromolecule-linker to the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound to form a conjugate.
  • a fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound is contacted with a linker precursor under conditions suitable for forming a bond from the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound to the linker.
  • the resulting fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound-linker is contacted with a macromolecule under conditions suitable for forming a bond from the fluorenylmethoxycarbonyl and/or fluorenylmethylaminocarbonyl compound-linker to the macromolecule to form a conjugate.
  • Suitable linkers for preparing the conjugates are disclosed herein and exemplary conditions for conjugation are described in the Examples below EXAMPLES [00239]
  • the compounds provided herein can be prepared, isolated, or obtained by any method apparent to those of skill in the art. Compounds provided herein can be prepared according to the Exemplary Preparation Schemes provided below.
  • Step 2 Final DBCO Acylation of mono-PEGylated Fmoc Compound 4
  • Fmoc PEGylated Compound 4 (11.4 g, 0.52 mmol) (azeotropically dried with 100 mL toluene removed at 50 °C under vacuum prior to use), and anhydrous DCM (70 mL).
  • the clear solution was flushed with argon and then triphosgene (231.9 mg, 0.78 mmol) and pyridine (0.06 mL, 0.73 mmol) were added sequentially.
  • the reaction mixture was stirred at room temperature for 2 h under nitrogen.
  • reaction mixture was stirred for 30 min at the same temperature.
  • a hot solution of 10% sulfuric acid 1300 mL (70 °C) was added slowly to the reaction mixture and stirred at 100 °C for 5 h. Progress of the reaction was monitored by TLC. After completion, the reaction was cooled to room temperature. A solid precipitated out and was filtered and washed with water (250 mL). The solid was dried to obtain 48.6 g of 9H-fluoren-2-ol (6), Yield: 48.6 g, 97.2%, Mass by GCMS: 182.1.
  • Example 8 Conjugation of Compound A [00297] A 5 mM stock solution of compound A was mixed with a final concentration of 1-50 mg/mL cytokine, antibody, or any protein incorporated with pAMF (nnAA) in 1xPBS at a Compound A to pAMF ratio of 2-50. The conjugation reaction mixture was incubated at 30 °C overnight. The conjugation efficiency was measured by SDS-PAGE. Unconjugated compound A is removed by cation exchange. PEGylated protein conjugates are formulated in 10 mM citric acid, pH 4.5 buffer, and stored at -80 °C.
  • PEG release To release the PEG from the compound A conjugate, the compound A conjugate was buffer exchanged into 100 mM sodium bicarbonate buffer at pH 9.0 and incubated at 30 °C overnight. The released product was analyzed on SDS-PAGE (FIG.3).

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  • Peptides Or Proteins (AREA)
  • Medicinal Preparation (AREA)
  • Polyethers (AREA)

Abstract

La présente invention concerne des composés, des produits conjugués associés, des méthodes et des compositions pharmaceutiques destinées à une utilisation thérapeutique ou diagnostique.
PCT/US2021/059014 2020-11-11 2021-11-11 Composés de fluorénylméthyloxycarbonyle et de fluorénylméthylaminocarbonyle, conjugués protéines associés et méthodes d'utilisation WO2022103983A2 (fr)

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