WO2009158668A1 - Prodrugs and drug-macromolecule conjugates having controlled drug release rates - Google Patents

Prodrugs and drug-macromolecule conjugates having controlled drug release rates Download PDF

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Publication number
WO2009158668A1
WO2009158668A1 PCT/US2009/048943 US2009048943W WO2009158668A1 WO 2009158668 A1 WO2009158668 A1 WO 2009158668A1 US 2009048943 W US2009048943 W US 2009048943W WO 2009158668 A1 WO2009158668 A1 WO 2009158668A1
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Prior art keywords
formula
compound
drug
optionally substituted
macromolecule
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PCT/US2009/048943
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French (fr)
Inventor
Daniel V. Santi
Gary Ashley
Brian Hearn
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Prolynx Llc
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Application filed by Prolynx Llc filed Critical Prolynx Llc
Priority to EP09771199.8A priority Critical patent/EP2306986B1/en
Priority to DK09771199.8T priority patent/DK2306986T3/en
Priority to ES09771199.8T priority patent/ES2672526T3/en
Priority to US12/999,287 priority patent/US8680315B2/en
Priority to CN2009801238298A priority patent/CN102076331B/en
Priority to JP2011516733A priority patent/JP6084770B2/en
Publication of WO2009158668A1 publication Critical patent/WO2009158668A1/en
Priority to US14/221,842 priority patent/US9387254B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • 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
    • 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
    • C08G65/33317—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing amino group heterocyclic
    • 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
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use

Definitions

  • the invention relates to compounds that are designed for pharmacokinetic control in delivering drugs.
  • the invention relates to prodrugs and drug-macromolecule conjugates having desired rates of drug release.
  • PEG poly(ethyleneglycol)
  • PEG-SOD PEG- superoxide dismutase
  • Monoclonal antibody and serum albumin likewise have very long resident times in the systemic and other physiological compartments, which leads to greatly extended systemic/compartmental half-lives of macromolecular conjugated drugs, largely dependent upon the molecular weight of the conjugate.
  • PEG polymer residues
  • PEGylation has resulted in such modified drugs as PEG- bovine adenosine deaminase for the treatment of X-linked severe combined immunogenicity syndrome (ADAGEN®, Enzon), PEG-alpha interferon for the treatment of hepatitis C (PEGASYS®, Hoffman-LaRoche; PEG-Intron®, Schering-Plough/Enzon), PEG-L- asparaginase from the treatment of acute lymphoblastic leukemia (Oncaspar®, Enzon), PEG- recombinant human granulocyte colony stimulating factor for the treatment of neutropenia (Neulasta®, Amgen), PEG-anti-tumor necrosis factor alpha for the treatment of Crohn's disease (Cimzia®, Enzon), PEG-growth hormone receptor antagonist for the treatment of acromegaly (Somavert®, Pfizer), and PEG-anti-TNF Fab for rheumatoid arthritis (CD870, Pfizer).
  • PEGylation has also been shown to improve delivery of nucleic acids to cells.
  • US patent publication 2008/0064863 discloses double-stranded nucleic acids, one strand of which is covalently attached to a poly(ethyleneoxide) unit, in complex with a polycation for use in the delivery of nucleic acid drugs to cells.
  • PCT publication WO2007/021142 discloses covalently PEGylated siRNA molecules.
  • PEG-anti-VEGF aptamer has been approved for intraocular treatment of age-related macular degeneration (Macugen®, OSI/Pfizer).
  • U.S. patent 6,504,005 describes prodrug molecules that release active drug under physiological conditions by virtue of beta elimination department on pH.
  • a specific embodiment of this approach is described in WO2004/089279.
  • This approach albeit limited in scope and examples, utilizing a spontaneous, first-order rate of cleavage of the drug from the PEG carrier that is initiated when the conjugate is exposed to physiological pH, is described in US Patent Publication 2006/0171920.
  • a general strategy for providing macromolecule-drug conjugates having a variety of spontaneous, first-order release rates that are predictable and controllable under physiological conditions would provide a valuable therapeutic tool for the treatment of disease. Disclosure of the Invention
  • the present invention provides prodrugs and drug-macromolecule conjugates for controlling the delivery rate of therapeutic agents ("drugs") when administered to patients requiring treatment with the therapeutic agents.
  • the prodrugs and drug-macromolecule conjugates of the invention provide a means of delivering therapeutic agents over a sustained period of time, even for therapeutic agents which are rapidly cleared from the system, thus prolonging the therapeutic effects of the therapeutic agents.
  • the invention is directed to compounds that are drug-macromolecule conjugates of the formula
  • m is an integer of 1-10;
  • Z is the residue of a macromolecule;
  • L' is the residue of a linker;
  • D is the residue of a drug or of a prodrug;
  • X is O or S;
  • A is alkenyl (C 2 ), aryl or absent; each R 1 and R 2 is independently CN; NO 2 ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R 1 and R 2 is independently COR 3 or SOR 3 or SO 2 R 3 wherein R 3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or
  • each R is independently H or optionally substituted alkyl; or each R 1 and R 2 is independently SR 4 wherein
  • R 4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R 1 and R 2 may be joined to form a 3-8 member ring; and wherein both R 1 and R 2 cannot be H; wherein R 5 is H or alkyl (C 1-6 ).
  • the linker residue is a bivalent chain having a molecular weight between 14 Da and 20 kDa which may include unsaturation, heteroatoms, ring structures, aromatic portions, and/or heteroaromatic portions that covalently links Z to the remainder of the molecule.
  • the linker may include a peptide backbone, a pseudopeptide backbone, a triazole, a phenylene, or a maleimido residue or combinations thereof.
  • the carbonyl shown in formula (3) originates in the drug itself.
  • m is 1 and/or R 5 is H. The value of m may also be any intermediate integer between 1 and 10; thus m may be 2, 5, 7, or any intervening integer.
  • the present invention provides prodrugs having the formula (2)
  • L is a linking group that can attach to a macromolecule
  • D is the residue of a drug or a prodrug.
  • m is 1 and/or R 5 is H.
  • the compounds of formula (3) and of formula (2) release the drug or prodrug at controlled rates under physiological conditions in the body, by non-enzymatic elimination.
  • the rate of drug release is tunable by the appropriate choice of groups R 1 and R 2 .
  • each of groups R 1 and R 2 may be independently substituted by electron-donating and/or electron- withdrawing substituents that alter the acidity of the intervening R -CH-R proton so that enormous flexibility and control over the rate of drug elimination can be achieved.
  • mixtures of compounds of formula (3) or of formula (2), each with a different rate of drug release under physiological conditions may be used in order to provide tailored drug delivery profile to a patient in need of treatment.
  • the invention is also directed to methods to prepare the compounds of formulas (3) and (2) as well as compounds that are intermediates in their formation.
  • the invention is directed to compounds of the formula
  • X, A, L', Z, R 1 , R 2 and R 5 are defined as above.
  • the invention is also directed to pharmaceutical compositions containing compounds of formulas (2) or (3) and to methods to prepare these prodrugs or macromolecular conjugates.
  • the starting point is the compound of formula (1) which may be converted directly to the prodrugs of formula (2) by suitable reaction with the drug compound.
  • the prodrug of formula (2) can then, in turn, be converted to the macromolecular conjugate of formula (3) by virtue of reaction of L with the macromolecule, Z.
  • the compound of formula (3) may be prepared by converting the compound of formula (1) to the compound of formula (4) which is then reacted with the drug.
  • FIGURE 1 shows the base-catalyzed elimination of drug from the macromolecular carrier-drug conjugate of the invention.
  • FIGURE 2 shows a variety of functional groups for R 1 and R 2 and their effects on the acidity of adjacent protons. As the acidity increases (lower pKa), the rate of deprotonation and hence the rate of drug release from the prodrug or drug-macromolecular conjugate is expected to increase.
  • FIGURE 3 shows the effect of substitution on the acidity of the 9-proton in various 2-substituted fluorenes. A shown, the effect follows the expected Hammett linear free energy correlation, such that the acidity of any given 2-substituted fluorene can be closely estimated based on the sigma-parameter of the substituent.
  • FIGURE 4 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as the fraction of total drug administered.
  • Relatively fast release rates for example, a release rate of 5 per day
  • Relatively slow release rates for example, a release rate of 0.1 per day
  • FIGURE 5 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as percent of the maximal concentration (% Cmax) versus time. As the rate of drug release from the prodrug or drug-macromolecular conjugate slows, the period of time after administration wherein the free drug concentration is above a particular percentage of the maximal concentration is extended.
  • the compounds of formulas (2) and (3) are designed to control the pharmacokinetics of the drug or prodrug defined as "D".
  • the mechanism whereby the drug or prodrug, D, is released is shown in Figure 1 using formula (3) for illustration.
  • the rate is controlled according to a pH dependent elimination mechanism.
  • the groups R and R are selected to provide the appropriate reactivity of the intervening proton R'-CH— R 2 , as illustrated in part in Figure 2, and thus providing control over the rate of drug or prodrug release from the prodrug or conjugate.
  • the nature of the linking group and the group A also influence this process, providing multiple points of control over the rate of drug release from the resulting conjugate.
  • the properties of R 1 and R 2 may be modulated by the optional addition of electron-donating or electron- withdrawing substituents, as illustrated in Figure 3.
  • a particular advantage of the invention is the position of attachment of the linking group. While it is possible to attach a linking group to one of groups R 1 or R 2 , doing so complicates any further substitution chemistry of R 1 or R 2 , and may result in electronic interactions between the linking group and the electron-donating or electron- withdrawing substituents, thus limiting the tunability of the release rates.
  • the active form of the drug is directly released from the conjugates of the invention, in some cases, it is possible to release the active drug in the form of a prodrug thereof. On example of such a system is shown below:
  • the compounds of formulas (2) and (3) provide tunable release rates for drugs or prodrugs so as to control the pharmacokinetics of these drugs.
  • various intermediates are of the formulas (1), (2) and (4); as noted, the compounds of formula (2) are also prodrugs with tunable release rates.
  • the compounds of formula (1) have neither the drug/prodrug nor the macromolecule attached and can be regarded as starting materials.
  • electron-donating group is meant a substituent resulting in a decrease in the acidity of the benzylic H group.
  • suitable electron-donating substituents include but are not limited to lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, and dialkylamino.
  • electron-withdrawing group is meant a substituent resulting in an increase in the acidity of the benzylic H group.
  • Non-hydrogen electron-donating or electron-withdrawing substituents may be present in multiple positions on rings to which they are bound. While, for convenience, in most examples, only a single occurrence of a non-hydrogen substituent on a single ring is shown, multiple substituents may also be present and are within the scope of the invention. The substituents may be the same or different.
  • alkyl is meant to include linear, branched, or cyclic saturated hydrocarbon groups of 1-8 carbons, or in some embodiments 1-6 or 1-4 carbon atoms.
  • alkenyl is meant to include non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds.
  • alkenyl (C 2 ) is meant a mono-, di-, tri-, or tetra- substituted carbon-carbon double bond of any geometric configuration.
  • alkynyl is meant to include non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds.
  • alkynyl (C 2 ) is meant a mono- or di-substituted carbon-carbon triple bond.
  • alkoxy is meant to include alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.
  • aryl is meant to include aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl.
  • heteroaryl is meant to include aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.
  • halogen includes bromo, fluoro, chloro and iodo.
  • maleimido is meant a group of the formula
  • protein and “peptide” are used herein interchangeably regardless of chain length, and these terms further include pseudopeptides which comprise linkages other than amide linkages, such as CH 2 NH 2 linkages as well as peptidomimetics.
  • nucleic acids and “oligonucleotides” are also used interchangeably regardless of chain length.
  • the nucleic acids or oligonucleotides may be single-chain or duplexed or may be DNA, RNA, or modified forms thereof with altered linkages, such as phosphodiesters, phosphoramidates, and the like.
  • these terms also include those with side chains not found in nature in the case of proteins and bases not found in nature in the case of nucleic acids.
  • Small molecules in the context of drugs is a term well understood in the art, and is meant to include compounds other than proteins and nucleic acids that either are synthesized or are isolated from nature and in general do not resemble proteins or nucleic acids. Typically, they have molecular weights ⁇ 1,000, although there is no specific cutoff recognized. Nevertheless, the term is well understood in the fields of pharmacology and medicine.
  • macromolecule a macromolecule having a molecular weight of between about 10,000 and 100,000, which is itself essentially devoid of cytotoxic, hormonal, or cell signaling activity but is capable of conjugation to an active drug molecule so as to serve to carry the active drug molecule in the systemic circulation and provides a reservoir of active drug which is released over time.
  • R 1 and R 2 are joined to form cyclic structures, this includes groups wherein the R'-CH-R 2 moiety forms a substructure such as, for example,
  • aryl groups typically contain only one substituent. This is for simplicity only, it will be understood that embodiments of R 1 and R 2 wherein ring systems contain multiple non-hydrogen substituents are included. Preferably the number of substituents on a single ring is 1, 2 or 3.
  • n 1-6.
  • the molecules of formula (1) have the more specific formula (Ia)
  • R 7 and R 8 are each independently H, an electron-donating group, or an electron- withdrawing group, and wherein electron donating and/or electron withdrawing forms of R 7 and
  • R are present at 1-5 positions preferably 3 or less, on the phenyl moieties;
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent; and
  • L is a linking group capable of being attached to a macromolecule,
  • R 5 is H or alkyl (C 1-6 ), for example,
  • the molecules of formula (1) have the more specific structure (Ib)
  • R 7 and R 8 are each independently H, an electron-donating group, or an electron- withdrawing group, and wherein electron donating and/or electron withdrawing forms of R 7 and R 8 are present at 1-5 positions on the phenyl moieties;
  • the molecules of formula (1) have the more specific formula (Ic)
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent;
  • L is a linking group capable of being attached to a macromolecule; and wherein R 5 is H or alkyl (C 1-6 ), R 10 and R 11 are each independently H, an electron-donating group, or an electron- withdrawing group and wherein R 10 and R 11 may occur as non-hydrogen substituents at 1-4 or 1-2 positions in their respective rings. Examples include
  • the molecules of formula (Ic) include
  • the molecules of formula (1) have the more specific formula (Id)
  • the molecules of the invention have the formula (Ie)
  • R 10 and R 11 are each independently H, an electron-donating group, or an electron- withdrawing group, wherein R 10 and R 11 may be non-hydrogen substituents at 1-4 positions of their respective rings;
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent;
  • L is a linking group capable of being attached to a macromolecule; and
  • Examples include
  • the molecules of formula (1) have the more specific formula (If)
  • A is alkenyl (C 2 ), aryl, or absent;
  • L is a linking group capable of being attached to a macromolecule;
  • R 9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl, and R 5 is H or alkyl (C 1-6 ).
  • R 2 is H. In another embodiment, R 2 is aryl, substituted aryl, heteroaryl, or substituted heteroaryl. In another embodiment, R 2 is CN.
  • the molecules of formula (If) include
  • R 10 as an electron- withdrawing or an electron-donating substituent may occur at 1-5 positions of the phenyl rings to which it is attached.
  • the molecules of formulas (If) wherein R 10 and R 11 is each independently H or an electron withdrawing or electron donating group, wherein R 10 and R 11 as non-hydrogen substituents may be present at 1-5 positions of their respective phenyl rings have formula
  • the compounds of the invention have the formula
  • the molecules of formula (1) have the more specific formula (Ig)
  • R 2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO 2 , C(O)-R 3 , SOR 3 , SO 2 R 3 , or SR 4 ;
  • R 9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl;
  • n 1-6
  • R 5 is H or alkyl (C 1-6 ).
  • Molecules of formula (1 g) wherein R 10 is H or an electron withdrawing or electron donating group include
  • R 10 as a non-hydrogen substituent has been shown in only one position, such non-hydrogen substituent can be present at 1-5 positions on the phenyl moiety shown.
  • the molecules of formula (1) have the more specific formula (Ih)
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent;
  • L is a linking group capable of being attached to a macromolecule, and R 5 is H or alkyl (C 1-6 ).
  • R 2 is H. In another embodiment, R is aryl, substituted aryl, heteroaryl, or substituted heteroaryl.
  • Molecules of formula ( 1 h) include
  • R 10 is H, an electron-withdrawing group, or an electron-donating group.
  • R 10 is specifically shown at only one position in the phenyl rings above, a non- hydrogen embodiment of R 10 may be present in 1-5 positions, preferably 3 or less positions on the phenyl ring.
  • Molecules of formula (Ih) also include
  • the molecules of formula (1) have the more specific formula (Ik)
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent; and
  • L is a linking group capable of being attached to a macromolecule, and R 5 is H or alkyl (C 1-6 ).
  • Molecules of formula (Im) include
  • Molecules of formula (In) include
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent;
  • L is a linking group capable of being attached to a macromolecule;
  • R 10 and R 11 are each independently H, an electron- withdrawing group, or an electron-donating group, wherein a non-hydrogen form of R 10 and R 11 may be present at 1-5 positions, preferably 3 or less positions of the phenyl moiety, and R 5 is H or alkyl (C 1-6 ).
  • Molecules of formula ( 1 p) include
  • the molecules of formula (1) have the more specific formula (Iq)
  • X is O or S;
  • A is alkenyl (C 2 ), aryl, or absent;
  • L is a linking group capable of being attached to a macromolecule;
  • R 10 is H, an electron- withdrawing group, or an electron- donating group, wherein a non-hydrogen form of R 10 may be present at 1-4 positions, preferably 2 or less positions of the ring to which it is bound, and
  • R 5 is H or alkyl (C 1-6 ).
  • Molecules of formula (1 q) also include
  • L is a linking group capable of being attached to a macromolecule.
  • linking group capable of being attached to a macromolecule is meant a group comprising a functionality through which L can be chemically bonded to a macromolecule.
  • suitable functionality include but are not limited to amines, alcohols, thiols, chlorides, iodides, bromides, azides, maleimides, alkenes, alkynes, aldehydes, carboxylates, and phosphates.
  • L is (CH 2 ) n R 12 ,
  • L is (CH 2 ) n CCH, giving compounds of the invention having the formula
  • the molecules of formula (1) include
  • non-hydrogen forms of substituents on ring systems may be present in multiple locations, preferably at no more than 2 or 3 locations.
  • the non-hydrogen substituents on a single ring may be the same or different.
  • L is
  • L is a group of the formula
  • n 1-6.
  • a condensing agent for example a carbodiimide (DCC, EDCI) or a uronium reagent (e.g., HATC, HBTA, TATU).
  • L' is determined by the nature of the foregoing embodiments of L, and thus these embodiments are illustrative of formulas (3) and (4).
  • a strong base for example butyllithium, NaH, lithium diisopropylamide, lithium bis(trimethylsilylamide), or similar
  • a suitable reducing agent for example NaBH 4 or NaBH 3 CN
  • R 1 R 2 CH 2 fluorene is reacted with a strong base, for example butyllithium, NaH or lithium diisopropylamide, to form a fluorenyl carbanion, which is then reacted with L-CHO, the reaction is as follows:
  • thiosulfate is used to form an intermediate that is hydrolyzed by acid treatment to form the thiol.
  • A alkenylene (C 2 ).
  • the starting materials of formula (1) are then derivatized either first to the drug or first to the macromolecule to obtain an intermediate in the formation of the compound of formula (3).
  • all of the embodiments which result from the many illustrated forms of formula (1), and specifically embodiments of L, A, X, R 1 , R 2 and R 5 are retained in the intermediates and in the final product of formula (3), except that L, when reacted with the macromolecule, becomes L' as a result of this reaction.
  • n is an integer of 1-10;
  • Z is the residue of a macromolecule
  • L is a linking moiety that can react to couple a macromolecule
  • D is the residue of a drug or of a prodrug
  • X is O or S;
  • A is alkenyl (C 2 ), aryl or absent;
  • each R 1 and R 2 is independently CN, NO 2 ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R 1 and R 2 is independently COR 3 or SOR 3 or SO 2 R 3 wherein R 3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or
  • each R is independently H or optionally substituted alkyl; or each R 1 and R 2 is independently SR 4 wherein
  • R 4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R 1 and R 2 may be joined to form a 3-8 member ring; and wherein both R 1 and R 2 cannot be H; wherein R 5 is H or alkyl (C 1-6 ).
  • the compounds of formula (2) can themselves release the therapeutic agent at a controlled rate or they can be used as intermediates to connect the therapeutic agent with a macromolecule. Both formula (2) and (3) release the therapeutic agent at a controlled rate according to a pH-dependent elimination mechanism.
  • Compounds of formula (2) may be generally prepared by condensation of a molecule of formula (1), described above, with a drug molecule D.
  • a suitable reagent for example phosgene or triphosgene
  • the activating reagent may be a substituted phenyl chloroformate, for example, 4-nitrophenyl chloroformate, 2,4-dinitrophenyl chloroformate, or pentafluorophenyl chloroformate, resulting in formation of an intermediate substituted phenyl carbonate.
  • D is a peptide drug, wherein D is conjugated to the molecule of formula (1) to produce a molecule of formula (2).
  • D is a peptide drug, wherein the molecule of formula (2) is prepared by a method in which the molecule of formula (1) is attached during the synthesis of D.
  • the final step in the synthesis of D by solid-phase peptide synthesis methods well-known in the art involves attachment of the N-terminal amino acid of the sequence of peptide D in protected form. In the present embodiment, this final step uses the N-terminal amino acid in a form using a compound of formula (1) as the protecting group.
  • R is the side chain of an amino acid
  • This embodiment is advantageous in that the position and stoichiometry of derivitization is completely controlled.
  • the drug is conjugated via the XH group, which is OH, or SH.
  • the drug will comprise a compatible functional group, for example carboxyl, OH, NH 2 , alkyl- NH such as MeNH, EtNH, and 1 PrNH, aryl-NH such as phenyl-NH or substituted phenyl-NH, and SH.
  • the drugs of interest are peptides or proteins or nucleic acids, such as aptamers or antisense oligomers, or small molecules.
  • suitable drugs include those for human or veterinary use including, but not limited to, antidiabetic drugs (e.g., insulin); growth promoters (e.g., human or bovine growth hormone); antibacterials including aminoglycosides (e.g., gentamicin, neomycin and streptomycin), penicillins, (amoxicillin, ampicillin, piperacillin), cephalosporins, (e.g., cefaclor, cefminox and cephalexin), macrolides (e.g., carbomycin, erythromycin, telithromycin) and peptides (e.g., bacitracin, gramicidins, and polymyxins), trimethoprim, piromidic acid, and sulfamethazine;
  • antidiabetic drugs e
  • peptide drugs suitable for use in the invention include but are not limited to: glucagon-like peptide 1 (GLP-I), exendin-2, exendin-3, exendin-4, atrial natriuretic factor (ANF), ghrellin, vasopressin, growth hormone, growth hormone-releasing hormone (GHRH), RC-3095, somatostatin, bombesin, PCK-3145, Phe-His-Ser-Cys-Asn (PHSCN), IGFl, B-type natriuretic peptide, peptide YY (PYY), interferons, thrombospondin, angiopoietin, calcitonin, gonadotropin-releasing hormone, hirudin, glucagon, anti-TNF-alpha, fibroblast growth factor, granulocyte colony stimulating factor, obinepitide, pit
  • protein drugs suitable for use in the invention include but are not limited to: immunotoxin SSlP, adenosine deaminase, argininase, and others.
  • nucleic acid-based drugs suitable for use in the invention include, but are not limited to the sense strand and antisense strand of any gene from an animal, and particularly from a mammal.
  • genes can be those that are already the subjects of antisense DNAs that have been provided with the purpose of treating various diseases, for example genes for protein kinase C-alpha (Aprinocarsen, non-small cell lung cancer), BCL-2 (Oblimersen, malignant melanoma, lung cancer), ICAM-I (ISIS-3082, Crohn's disease, HCV-related hepatitis C, ischemic/reperfusion injury in transplant), tumor necrosis factor alpha (rheumatoid arthritis, SARS, and psoriasis), adenosine Al receptor (asthma), c-raf kinase (ovarian cancer), H-ras (pancreatic cancer), c-myc (coronary artery disease), protein kina
  • D is an oligonucleotide drug.
  • the compounds of formula (2) wherein D is an oligonucleotide may be prepared by chemical synthesis of the oligonucleotide drug comprising a 5 '-terminal modification that allows for conjugation with a molecule of formula (1).
  • the oligonucleotide may be chemically synthesized such that the 5 '-terminal nucleotide unit, added at the last round of synthesis, comprises a phosphate group modified to contain an amino-alkyl group.
  • oligonucleotide is then conjugated to a molecule of formula (1) to form a molecule of formula (2) wherein D is an oligonucleotide drug.
  • D is an oligonucleotide drug.
  • Figure 4 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as the fraction of total drug administered, assuming slow clearance of the prodrug.
  • Relatively fast release rates for example, a release rate of 5 per day
  • Relatively slow release rates for example, a release rate of 0.1 per day
  • Figure 5 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as percent of the maximal concentration (% Cmax) versus time. As the rate of drug release from the prodrug or drug-macromolecular conjugate slows, the period of time after administration wherein the free drug concentration is above a given percentage of the maximal concentration is extended.
  • the invention also includes compositions containing mixtures comprising two or more prodrugs of formula (2) where the drugs, D, are the same or different.
  • each prodrug of formula (2) has a different rate of drug release under physiological conditions.
  • the pharmacokinetics are also further controlled by including a macromolecule as on compounds of the formula
  • m is an integer of 1-10;
  • Z is the residue of a macromolecule;
  • L' is the residue of a linker;
  • D is the residue of a drug or of a prodrug;
  • X is O or S;
  • A is alkenyl (C 2 ), aryl or absent; each R 1 and R 2 is independently CN; NO 2 ; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R 1 and R 2 is independently COR 3 or SOR 3 or SO 2 R 3 wherein R 3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or OR or NR 2 wherein each R is independently H or optionally substituted alkyl; or each R 1 and R 2 is independently SR 4 wherein
  • R 4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R 1 and R 2 may be joined to form a 3-8 member ring; and wherein both R 1 and R 2 cannot be H; and wherein R 5 is H or alkyl (C 1-6 ).
  • R 5 is H.
  • Z is a protein, oligosaccharide, or synthetic polymer having a molecular weight of between 10,000 and 250,000.
  • the macromolecule Z is a synthetic polymer having a molecular weight of between 10,000 and 250,000. In more specific embodiments, the macromolecule Z is a synthetic polymer having a molecular weight of between 10,000 and 100,000. In certain embodiments of the invention, Z is a derivatized linear, branched, or dendrimeric poly(ethyleneglycol) (PEG), monomethoxy-PEG (mPEG), poly(ethyleneimine) (PEI), or PEG-PEI copolymer.
  • PEG poly(ethyleneglycol)
  • mPEG monomethoxy-PEG
  • PEI poly(ethyleneimine)
  • PEG-PEI copolymer PEG-PEI copolymer
  • the macromolecule Z is a protein of molecular weight between 10,000 and 250,000.
  • Z is an antibody or antibody fragment, either monoclonal or polyclonal.
  • various reactive functional groups such as amines and thiols will be present.
  • the protein can be chemically derivatized using methods known in the art to add groups such as thiols and maleimides.
  • Z is an oligosaccharide having a molecular weight of between 10,000 and 250,000.
  • Z is a dextran having a molecular weight of between 10,000 and 250,000.
  • Z is a dextran having a molecular weight of between 10,000 and 100,000.
  • the macromolecule Z will comprise at least one functional group R 13 suitable for reaction with a molecule of formula (1) or (2).
  • suitable R 13 groups include but are not limited to: hydroxyl, amine, azide, carboxyl, aldehyde, N-hydroxysuccinimidyl ester, imidazolylcarboxamino, imidazolylcarboxy, nitrophenyl carbonate, isocyanate, maleimide, thiol, epoxide, CCH (terminal alkyne), and guanidino groups.
  • Z is the antibody m38c2 or a humanized version thereof (US patent application 2006/0205670, incorporated herein by reference).
  • Z is selected from the group consisting of an antibody; an albumin; a linear, branched, or dendrimeric polyethylene glycol (PEG); a linear, branched, or dendrimeric monomethoxypolyethylene glycol (mPEG); a linear, branched, or dendrimeric polyethylene imine (PEI); a linear, branched, or dendrimeric PEG-PEI copolymer; a linear, branched, or dendrimeric dextran; and a nanoparticle.
  • the drug-macromolecule conjugates have the formula (3 a) wherein Z is an antibody.
  • the drug-macromolecule conjugates have the formula (3a) wherein Z is a linear, branched, or dendrimeric polyethylene glycol (PEG). In other particular embodiments of the invention, the drug-macromolecule conjugates have the formula (3a) wherein Z is a linear, branched, or dendrimeric monomethoxypolyethylene glycol (mPEG).
  • L' can be (CH 2 ) n NHCO, (CH 2 ) n NHCH 2 , (CH 2 ) n NHCONH, (CH 2 ) n triazolyl, (CH 2 ) n thiosuccinimidoyl, (CH 2 ) n (hydroxyethylthio), (CH 2 ) n succinimidoylthio, (CH 2 ) n CONH,
  • the compounds of formula (4) are prepared as described below wherein the reactant to be coupled to the macromolecule Z is the compound of formula (1); if the compound of formula (2) is employed, the compounds of formula (3) result. Similarly, the compounds of formula (4) may be converted as described above into compounds of formula (3) by reaction with appropriate drugs as described above.
  • the linking group L' is formed by the reaction of a functional group R 13 on the macromolecule Z with a functional group R on the linker group L of the molecules of formula (1) or (2) during the conjugation process.
  • L is (CH 2 ) n R 12 ,
  • R 12 is NH 2
  • R is SH
  • R 12 is COOH
  • L is a group having the formula
  • conjugation of the prodrug and macromolecule provides an enamino-ketone linkage:
  • the drug-macromolecule conjugates of formula (3) have the formula:
  • the conjugates of formula (3) have the more specific formula (3 a)
  • Z is the residue of a macromolecule
  • D is the residue of a drug molecule
  • X is O or S
  • L' is the residue of a linking group
  • A is alkenyl (C 2 ), aryl, or absent;
  • R 7 and R 8 are each independently H, an electron-donating group, or an electron- withdrawing group, wherein R and R in the form of non-hydrogen substituents may be present at 1-5 positions, preferably 3 or less positions on the rings to which they are bound, and R 5 is H or alkyl (C 1-6 ).
  • Z and L' are as described above, and/or X is O, and/or A is absent or is alkenyl (C 2 ).
  • the drag-macromolecule conjugates of formula (3) have the more specific formula (3b)
  • Z is the residue of a macromolecule
  • D is the residue of a drug molecule
  • X is of O or S
  • L' is the residue of a linking group
  • A is alkenyl (C 2 ), aryl, or absent;
  • R 10 and R 11 are each independently H, an electron-donating group, or an electron- withdrawing group, wherein each of R 10 and R 11 may be present at 1-4 positions, preferably 2 or fewer positions on the rings to which they are bound.
  • the drug-macromolecule conjugates have the formula (3b) wherein Z and L' are as exemplified above.
  • X may be O, and/or wherein A is absent, or is alkenyl (C 2 ).
  • the drug-macromolecule conjugates of formula (3b) have the more specific formula:
  • the drug-macromolecule conjugates of formula (3b) include as well as
  • conjugates of formula (3) have the formula (3 c)
  • R 2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO 2 , COR 3 , SOR 3 or SO 2 R 3 : and
  • R 9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl, and wherein R 2 and R 9 may be connected to form a cyclic structure.
  • the conjugates of formula (3c) may comprise embodiments of Z and L' illustrated above.
  • X is O, and/or A is absent, or is alkenyl (C 2 ).
  • the drug-macromolecule conjugates of formula (3c) may include
  • a non-hydrogen substituent may be present at 1-5 positions, preferably 3 or less positions on the phenyl ring shown.
  • conjugates of formula (3) have the more specific formula (3d)
  • Z is the residue of a macromolecule
  • D is the residue of a drug molecule
  • X is O or S
  • L' is the residue of a linking group
  • A is alkenyl (C 2 ), aryl, or absent
  • R 2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO 2 , COR 3 , SOR 3 or SO 2 R 3 .
  • the drug-macromolecule conjugates have the formula (3d) may include embodiments of Z and L' as listed above.
  • Formula (3d) includes embodiments wherein X is O, and/or wherein A is absent, or is alkenyl (C 2 ).
  • the drug-macromolecule conjugates of formula (3d) includes
  • conjugates of formula (3) have the more specific formula (3e)
  • Z is the residue of a macromolecule
  • D is the residue of a drug molecule
  • X is O or S
  • L' is the residue of a linking group
  • A is alkenyl (C 2 ), aryl, or absent;
  • R 2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO 2 COR 3 , SOR 3 or SO 2 R 3 ;
  • R 9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl, and wherein R 2 and R 9 may be connected to form a cyclic structure.
  • the conjugates of formula (3e) contain embodiments of Z and L' as listed above.
  • X may be O, and/or A is absent, or is alkenyl (C 2 ).
  • the drug-macromolecule conjugates of formula (3e) include
  • the invention provides pharmaceutical and veterinary compositions comprising compounds of formulas (2) or (3) or pharmaceutically acceptable salts thereof or mixtures thereof and a pharmaceutically acceptable carrier.
  • Any suitable route of administration of drugs to humans and animals is envisaged by the invention, for example via conventional injectable, implantable, oral, intraocular, intrathecal, rectal or topical administration.
  • These preparations can be prepared by conventional methods known to those skilled in the art, for example as described in Remington's Pharmaceutical Science, A. R. Gennaro, ed., 17th edition, 1985, Mack Publishing Company, Easton, Penn., USA.
  • Subjects that may be treated with the compounds of formula (2) or (3) and compositions thereof include humans, veterinary animals, livestock and laboratory animals such as mice and rats.
  • compositions may contain mixtures comprising two or more compounds of formula (3) or of formula (2).
  • the mixture comprises two or more compounds of formula (2) or (3), wherein each compound of formula (2) or (3) has the same drug D but a different rate of drug release under physiological conditions, thus providing a tailored drug release profile for drug D.
  • the mixture comprises two or more compounds of formula (2) or (3), wherein each compound of formula (2) or (3) has a different drug D, and optionally a different rate of drug release for each drug D under physiological conditions, thus providing tailored combination therapy.
  • the invention can thus be used to control the release rates - and thus steady state concentrations and durations of action - of two or more drugs. Hence, for combinations of two drugs one can optimize the concentration and duration of both drugs.
  • an optimal conjugate for a first drug A is experimentally determined. Such an optimal conjugate is characterized as having an optimal drug concentration versus time profile (i.e., optimal drug concentrations and length of exposure).
  • each conjugate of drug B having a different drug release profile, the most effective combination is then determined experimentally.
  • a converse experiment may then be undertaken, using the optimal conjugate of drug B together with multiple conjugates of the second drug A in order to verify that the optimal mixture has been determined.
  • each of drugs A and B are made into conjugates that have a set of half-lives for release of the drugs from the conjugates (for example: 1, 2, 4, and 8 hr half-lives). Combinations of these conjugates that span all the possible permutations of conjugates of drug A and B are then tested.
  • GLP-I or an analogue thereof, for example an exendin is conjugated to form a first drug-macromolecule conjugate of formula (3), and gastrin is conjugated to form a second drug-macromolecule conjugate of formula (3).
  • insulin is conjugated to form a first drug- macromolecule conjugate of formula (3), and insulin C-peptide is conjugated to form a second drug-macromolecule conjugate of formula (3).
  • the resulting crude tosylate is dissolved in isopropanol and reacted with aqueous sodium thiosulfate at 5O 0 C to form the Bunte salt, which is hydrolyzed by treatment with aqueous HCl.
  • the mercaptan product is purified by chromatography on silica gel.
  • Example 5
  • a solution of the compound of formula (1) (1.0 equivalent) in THF is added to a 1.0 M solution of LiHMDS (1.0 equivalent) in THF at -78 0 C. After 15 minutes, a solution of bis(4-nitrophenyl) carbonate (1.5 equivalent) is added. The mixture is allowed to warm slowly to ambient temperature, quenched by addition of 1 N HCl, and extracted with ether. The extract is washed sequentially with 1 N HCl, water, and brine, then dried over MgSO 4 , filtered, and evaporated. The product 4-nitrophenyl carbonate is purified by chromatography on silica gel.
  • the peptide is synthesized using standard conditions known in the art, for example using FMOC chemistry. Upon coupling the final amino acid, the terminal FMOC protecting group is removed by treatment under standard conditions. The resin-bound peptide is then reacted with excess compound of Example 5 or Example 6 to cap the N-terminus. The peptide is then deprotected and cleaved from the resin using TFA/triethylsilane, and purified by reversed phase HPLC.
  • a solution of fiuorene (1.0 equivalent) in tetrahydrofuran (THF) is added to a solution of lithium diisopropylamide (LDA) at -78 0 C (1.0 equivalent).
  • LDA lithium diisopropylamide
  • the mixture is allowed to warm slowly to O 0 C, then recooled to -78 0 C prior to the addition of 4-ethynyl-benzaldehyde (1.0 equivalent). After 30 minutes, the mixture is allowed to warm slowly to ambient temperature, quenched by addition of saturated aqueous NH 4 Cl, and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO 3 , and brine, then dried over MgSO 4 , filtered, and evaporated. The product is purified by chromatography on silica gel.
  • a solution of fiuorene (1.0 equivalent) in tetrahydrofuran (THF) is added to a solution of lithium diisopropylamide (LDA) at -78°C (1.0 equivalent).
  • LDA lithium diisopropylamide
  • the mixture is allowed to warm slowly to O 0 C, then recooled to -78°C prior to the addition of methyl 3-(dimethylarnino)propenoate (1.0 equivalent).
  • the mixture is allowed to warm slowly to ambient temperature, quenched by addition of 1 N HCl, and extracted with ether.
  • the extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO 3 , and brine, then dried over MgSO 4 , filtered, and evaporated.
  • the product ester is purified by chromatography on silica gel.
  • a mixture comprising the above-described aldehyde (1.0 equivalent), 3-(N-BOC- amino)phenylboronic acid (2.0 equivalents), Cs 2 CO 3 (2.0 equivalents), Pd 2 (dba) 3 »CHCl 3 (0.025 equivalent), and Ph 3 P (0.05 equivalent) in toluene is heated at 8O 0 C for 24 hours. After cooling to ambient temperature, the mixture is concentrated and the product is purified by chromatography on silica gel.
  • the peptide sequence of exendin-4 ( ⁇ GEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS) is synthesized using standard FMOC chemistry. Upon coupling the final histidine amino acid, the terminal FMOC protecting group is removed by treatment under standard conditions. The resin-bound peptide is then reacted with excess compound of Example 13 to cap the N-terminus. The peptide is then deprotected and cleaved from the resin using TFA/triethylsilane, and purified by reversed phase HPLC using standard methodology.
  • the peptide sequence of exendin-4 (HGEGTFTSDLSKQMEEEAVRL- FIEWLKNGGPSSGAPPPS) is synthesized using standard FMOC chemistry. Upon coupling the final histidine amino acid, the terminal FMOC protecting group is removed by treatment under standard conditions. The resin-bound peptide is then reacted with excess compound of Example 14 to cap the N-terminus. The peptide is then deprotected and cleaved from the resin using TFA/triethylsilane, and purified by reversed phase HPLC using standard methodology.
  • Example 19 The compound of Example 19 is dissolved in TFA/triethylsilane to remove the tert- butoxycarbonyl group, then evaporated to dryness. The resulting compound is dissolved in acetonitrile and reacted with mPEG-carboxylate N-hydroxysuccinimide ester in the presence of p ⁇ 6 buffer. The mixture is stirred at ambient temperature overnight, then dried. The product is purified by reversed-phase ⁇ PLC.
  • the reaction was quenched by addition of a solution OfNaHSO 4 (1 g in 10 mL of water), and the reaction mixture was warmed to room temperature.
  • the aqueous layer was extracted with ethyl acetate (EtOAc) (15mLX 2), and the combined organic solution was dried over MgSO 4 and concentrated under reduced pressure.
  • EtOAc ethyl acetate
  • n-butyllithium (2.5M in hexanes, 1.0 mL, 3.0mmol) was added to a solution of N,N-diisopropylamine (0.42mL, 3.0 mmol) in anhydrous tetrahydrofuran (THF) (6 mL) at O 0 C.
  • THF tetrahydrofuran
  • the resulting reaction mixture was stirred at the same temperature for 15 min, then cooled to -78 0 C.
  • 5-Chloro-2,3-dihydroinden-l-one (0.5 g, 3.0 mmol) in THF (3 mL) was added. After stirring at -78 0 C for 30 min, a solution of 5-hexynal was added.
  • reaction mixture was then stirred at -78 0 C for 2 hours, quenched by addition of sat. aq. NH 4 Cl, and warmed to room temperature.
  • the aqueous phase was extracted with ethyl acetate (EtOAc), and the combined organic solution was washed with brine, dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure.
  • the residue was purified by column chromatography on silica gel to obtain the desired product as pale yellow solid (0.29 g, yield 41.3%).
  • n-butyllithium (2.5 M in hexanes, 0.47 mL, 1.35 mmol) was added into the solution of fluorene (0.24 g, 1.48 mmol) in anhydrous THF (2 mL) at -78 0 C, and the reaction mixture was stirred at he same temperature for 2.5h. A solution of 5-hexynal was added and the reaction was stirred at -78 0 C for 3 hours, quenched by addition of sat. aq. NH 4 Cl, and warmed to room temperature.
  • n-butyllithium (2.5 M in hexanes, 0.47 mL, 1.35 mmol) is added to a solution of fluorene (0.24 g, 1.48 mmol) in anhydrous THF (2 mL) at 78 0 C, and the reaction mixture is stirred at he same temperature for 2.5 h.
  • a solution of 6- azidohexanal is added and the reaction is stirred at -78 0 C for 3 hours, quenched by addition of sat. aq. NH 4 Cl, and warmed to room temperature.
  • the mixture is extracted with EtOAc, and then the combined organic phase is washed with brine, dried over anhydrous MgSO 4 , filtered, and concentrated under reduced pressure.
  • the crude product is purified by column chromatography on silica gel to obtain the desired product.
  • Antibody for example, h38C2 IgGl or bl2 IgGl ; see US Patent Publication 2006/0205670 Al
  • Antibody is added to a solution of a compound of Example 30 to a final concentration of 25 nM antibody binding site and 125 nM (2). This mixture is incubated at room temperature for 10 minutes. The formation of the conjugate is determined by the formation of UV absorbance at 318 nm.
  • an optimal drug-macromolecule conjugate for a first drug A is experimentally determined.
  • Such an optimal drug- macromolecule conjugate is characterized as having an optimal drug concentration versus time profile (i.e., optimal drug concentrations and length of exposure).
  • each conjugate of drug B having a different drug release profile the most effective combination is then determined experimentally.
  • a converse experiment may then be undertaken, using the optimal conjugate of drug b together with multiple drug-macromolecule conjugates of the second drug a in order to verify that the optimal mixture has been determined.
  • each of drugs A and B are made into conjugates that have a set of half-lives for release of the drugs from the drug-macromolecule conjugates (for example: 1, 2, 4, and 8 hr half-lives). Combinations of these conjugates that span all the possible permutations of conjugates of drug A and B are then tested. (Table 1).
  • Table 1 Exemplary permutations of drug combination conjugates to be tested to determine optimal combination.
  • Half-lives of release of first drug A from conjugate w, x, y, and z hrs.
  • Half-lives of release of second drug B from conjugate a, b, c, and d hrs.
  • Sulfone linkers may be generally prepared by first reacting an appropriate thiol with a bromoketone to generate an beta-ketosulfide, which is subsequently oxidized to the beta- ketosulfone; reduction of the carbonyl then yields a beta-hydroxysulfone.
  • Step 1 4-methoxythiophenol (615 ⁇ L) was added to a mixture of 2-bromo-3'-nitroacetophenone (1.22 g) in 10 mL of 1:1 acetonitrile/water with sodium bicarbonate (0.84 g). After 1 hour, the resulting mixture was diluted with water and extracted with ethyl acetate. The extract was dried with MgSO 4 , filtered, and evaporated to an orange oil, which crystallized upon addition of 3:1 hexane/ethyl acetate. The orange crystals were collected and dried to provide the beta-ketosulfide, 1.1 g.
  • Step 2 Wet 3-chloroperoxybenzoic acid (2.0 g, -50%) was added carefully in small portions to a solution of the ketosulfide (670 mg) in 10 mL of dichloromethane. The mixture warmed. After stirring 2 hrs, the suspension was diluted with ethyl acetate and washed carefully with sat. NaHCO 3 , water, and brine, then dried with MgSO 4 , filtered, and evaporated to give 630 mg of solid ketosulfone, which was crystallized from ethyl acetate.
  • Step 3 Sodium borohydride (100 mg) was added to a suspension of ketosulfone (400 mg) in 5 mL of methanol. After 30 min, sat. aq. NH 4 Cl was added and the mixture was concentrated. The residue was partitioned between ethyl acetate and water, then the organic phase was washed with brine, dried with MgSO 4 , filtered, and evaporated to give the crude product, which was crystallized from ethyl acetate/hexane to give 360 mg of the nitro alcohol.
  • Step 4 Solid ammonium formate (200 mg) was added to a mixture of the nitro alcohol (217 mg) and 10% palladium on carbon (50 mg) in 5 mL of methanol. The mixture was stirred vigorously for 1 hr, then an additional 50 mg of catalyst was added. After an additional 30 minutes, the mixture was filtered and evaporated. The residue was dissolved in ethyl acetate and washed carefully with sat. NaHCO 3 , water, and brine, then dried with MgSO 4 , filtered through a plug of silica gel, and evaporated to give 180 mg of the amino alcohol as a clear glass.
  • the amino alcohol may be acylated on the amine to provide a more facile means of attachment to the macromolecular carrier, using methods known in the art. For example, a solution of the amino alcohol can be reacted with 3-maleimidopropionic acid anhydride as described for 2-aminofiuorenes (Tsubery, H., et al., J. Biological Chem. (2004) 279:38118-38124).
  • the amino alcohol may be acylated on the amine using an azido-acid derivative, for example 6-azidohexanoyl chloride or 6-azidohexanoic anhydride.
  • the amino alcohol may be acylated on the amine using an alkynyl-acid derivative, for example 5-hexynoyl chloride or 5-hexynoic anhydride.
  • Step 6 The acylated amino alcohol is activated for attachment of the drug as the N-hydroxysuccinimdyl carbonate, using the methods described above or known in the art.
  • nitrile linkers may be prepared according to the tin-mediated reaction of bromoacetonitrile with aldehydes according to the method of Sun and Shi, J. Chem. Research (S) (1999), 318-319, which is incorporated herein by reference.
  • nitrile linkers may be prepared by reduction of beta-ketonitriles, which in turn may be prepared from suitable ketones by reaction of a beta-ketoaldehyde enolate with hydroxylamine hydrochloride:
  • the extract was dried with MgSO 4 , filtered, and evaporated to provide 510 mg of a cloudy oil, which was filtered through silica gel using 1 :1 ethyl acetate/hexane to provide the product (496 mg) as a thick oil.
  • n-butyllithium (1.8 mL, 5.2 mmol) was added to a solution of N,N-diisopropylamine (0.75 mL, 5.2 mmol) in anhydrous THF (10 mL) at O 0 C. The reaction mixture was stirred for 30 min and then was chilled to -78 0 C. 2-(4-methoxyphenyl)acetonitrile (0.6 mL, 4.34 mmol) was added into the above mixture via syringe. After stirring at the same temperature for 30 min, a solution of 5-hexynal (ca.
  • a mixture of the compound prepared as in Example 33 (1 mmol), N,N'-disuccinimidyl carbonate (2 mmol), and 4-(dimethylamino)pyridine (0.1 mmol) in 2 mL of dry acetonitrile is allowed to stir for 1 hour (for primary alcohols) or for 16 hours (for secondary alcohols), then is diluted with 5 mL of water containing 0.2 mL of 1 N HCl and extracted with ethyl acetate. The organic phase is washed with water and brine, then dried over MgSO 4 , filtered, and concentrated to provide the crude mixed carbonate, suitable for further use.
  • the mixed carbonate is optionally further purified by silica gel chromatography using ethyl acetate/hexane.
  • N ⁇ -(2,4-dinitrophenyl)-L-lysine hydrochloride 35 mg
  • 200 ⁇ L of 1.0 N NaOH is treated successively with 200 ⁇ L of 1.0 M NaHCO 3 and a solution of 0.1 mmol of the N-hydroxysuccinimide carbonate in 1.0 mL of acetonitrile.
  • the resulting yellow solution is stirred at ambient temperature for 1 hour, then is diluted by addition of 10 mL of water and is loaded onto a Cl 8 solid phase extraction column (Varian BondElut, 1 g).
  • the column is washed with 3 mL of water, 1 mL of 1% CF 3 CO 2 H in water, 3 mL of water, then 3 mL of 50% aqueous methanol to elute any unreacted lysine analog.
  • the product is eluted with 100% methanol, and the yellow solution is evaporated to dryness.
  • the rates of release of N ⁇ -(2,4-dinitrophenyl)-L-lysine, "H-Lys(DNP)-OH" from various compounds were determined as follows. Reaction mixtures were prepared containing 0.1 M buffer, 0.05% NaN 3 , and approximately 0.1 mg/mL of the starting compound, and were kept at 37 0 C. Aliquots were removed periodically and analyzed by injection onto a Varian Polaris 3 ⁇ m C18-A reversed-phase HPLC column (150 x 4.6 mm), equilibrated in 50:50 water/methanol (each containing 0.5% acetic acid) at a flow rate of 0.8 mL/min.
  • Step 1 Pyridine (1.5 mmol) is added to a mixture of the linker (1.0 mmol) and the 4-formylphenyl chloroformate (1.2 mmol) in dry tetrahydrofuran, and the reaction is monitored by thin-layer chromatography. Upon completion, the mixture is diluted with ethyl acetate, washed with water and brine, dried and concentrated. The crude aldehyde product is purified by chromatography on silica gel.
  • Step 2 Sodium cyanoborohydride (2 mmol) is added to a mixture of the aldehyde from Step 1 (1 mmol) in 5% acetic acid and ethanol (2 mL). The mixture is warmed to -75 0 C for 2 hours, then cooled and concentrated. The residue is dissolved in ethyl acetate, washed with water and brine, then dried and evaporated to provide the crude benzylic alcohol, which is purified by chromatography on silica gel.
  • Step 3 Pyridine (2 mmol) is added dropwise to a mixture of the benzylic alcohol of Step 2 (1 mmol) and triphosgene (3 mmol) in dry tetrahydrofuran. After stirring for 1 hour, the mixture is filtered and evaporated to dryness to provide the crude chloroformate, which is used without further purification.
  • Step 4. Pyridine (2 mmol) is added to a mixture of the crude chloroformate from Step 3 and N-hydroxysuccinimide (5 mmol). After stirring for 30 minutes, the mixture is filtered and concentrated. The residue is dissolved in ethyl acetate, washed with water and brine, then dried and evaporated to provide the crude succinimidyl carbonate, which is purified by chromatography on silica gel.
  • Step 5 A solution of the succinimidyl carbonate from Step 4 in acetonitrile (1 equivalent) is added to a solution of the amine-containing drug in 0.1 M NaHCO 3 . After 1 hour, the mixture is diluted with water and loaded onto a Cl 8 solid phase extraction cartridge, The cartridge is washed with water, then eluted with a step gradient of water and acetonitrile. The product-containing fractions are pooled and evaporated to dryness, and optionally further purified using preparative HPLC.
  • Step 1 Pyridine (1.5 mmol) is added to a mixture of the linker (1.0 mmol) and triphosgene (3 mmol) in dry tetrahydrofuran. After stirring for 1 hour, the mixture is filtered and evaporated to dryness to provide the crude chloroformate, which is used without further purification.
  • Step 2 A mixture of the crude chloroformate from Step 1, the 4-aminobenzyl alcohol (1 mmol), and pyridine (2 mmol) in dry tetrahydrofuran (5 mL) is stirred at ambient temperature. When complete, the mix is evaporated, and the residue is dissolved in ethyl acetate and washed with water and brine, then dried and evaporated to provide the crude carbamate, which is purified by chromatography on silica gel.
  • Step 3 Pyridine (2 mmol) is added dropwise to a mixture of the carbamate of Step 2 (1 mmol) and triphosgene (3 mmol) in dry tetrahydrofuran. After stirring for 1 hour, the mixture is filtered and evaporated to dryness to provide the crude chloroformate, which is used without further purification.
  • Step 4 Pyridine (2 mmol) is added to a mixture of the crude chloroformate from Step 3 and N-hydroxysuccinimide (5 mmol). After stirring for 30 minutes, the mixture is filtered and concentrated. The residue is dissolved in ethyl acetate, washed with water and brine, then dried and evaporated to provide the crude succinimidyl carbonate, which is purified by chromatography on silica gel.
  • Step 5 A solution of the succinimidyl carbonate from Step 4 in acetonitrile (1 equivalent) is added to a solution of the amine-containing drug in 0.1 M NaHCO 3 . After 1 hour, the mixture is diluted with water and loaded onto a Cl 8 solid phase extraction cartridge, The cartridge is washed with water, then eluted with a step gradient of water and acetonitrile. The product-containing fractions are pooled and evaporated to dryness, and optionally further purified using preparative HPLC.

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Abstract

The present invention provides methods and compositions that permit controlled and prolonged drug release in vivo. The compounds are either prodrugs with tunable rates of release, or conjugates of the drug with macromolecules which exhibit tunable controlled rates of release.

Description

PRODRUGS AND DRUG-MACROMOLECULE CONJUGATES HAVING CONTROLLED DRUG RELEASE RATES
Cross-Reference to Related Applications
[0001] This application claims benefit of U.S. provisional application Serial Nos. 61/192,050 filed 15 September 2008, and 61/133,148 filed 26 June 2008, which are incorporated herein by reference in their entirety.
Technical Field
[0002] The invention relates to compounds that are designed for pharmacokinetic control in delivering drugs. In particular, the invention relates to prodrugs and drug-macromolecule conjugates having desired rates of drug release.
Background Art
[0003] Many drugs suffer from unfavorable pharmacokinetic parameters that limit their effectiveness. Rapid clearance of such drugs from physiological compartments, either via metabolism or excretion, results in short lifetimes and reduced exposure to targets. For example, the therapeutic potential of peptide- and protein-based drugs is enormous, yet peptide- and protein-based drugs often suffer rapid systemic clearance due to metabolic instability and renal clearance. Similarly, nucleic acids such as antisense DNA and small interfering RNAs (siRNAs) have great therapeutic potential, yet suffer from metabolic instability and cell impermeability. Finally, many small organic molecules also suffer from rapid clearance that limits their therapeutic effectiveness.
[0004] It would thus be highly desirable to have methods to prolong the half-life in the systemic circulation and/or other physiological compartments and improve the availability and cell uptake of small molecule, peptide-, protein-, and nucleic acid-based therapeutics in order to provide improved drug- and gene-based therapies in the treatment of disease.
[0005] One method for increasing the physiological half-life of drugs is to increase their hydrodynamic size by attaching them to macromolecules. Removal of large molecules, for example high-molecular weight proteins, antibodies, polymers, poly(ethyleneglycol) (PEG), from the systemic circulation can be extremely slow. Metabolism of PEG having mw >5000 is insignificant, and both glomerular filtration and biliary excretion of PEG having mw -50 kDa is minimally effective. For example, the plasma half-lives in rates or mice of several PEG- superoxide dismutase (PEG-SOD) conjugates have been reported to vary from 1.5 hours for a conjugate using PEG of molecular weight 1900 to 36 hours for a conjugate using PEG of molecular weight 72,000, while the half-life of unconjugated SOD was 0.08 hour. See Veronese, "Peptide and protein PEGylation: a review of problems and solutions," Biomaterials (2001) 22:405-417. Monoclonal antibody and serum albumin likewise have very long resident times in the systemic and other physiological compartments, which leads to greatly extended systemic/compartmental half-lives of macromolecular conjugated drugs, largely dependent upon the molecular weight of the conjugate.
[0006] For peptide- and protein-based drugs, covalent attachment of polymer residues, for example PEG (known as "PEGylation"), has been effective at improving the pharmacokinetic parameters and can also mask the drug agent from metabolism and from the immune system, leading to reduced immunogenicity. PEGylation has resulted in such modified drugs as PEG- bovine adenosine deaminase for the treatment of X-linked severe combined immunogenicity syndrome (ADAGEN®, Enzon), PEG-alpha interferon for the treatment of hepatitis C (PEGASYS®, Hoffman-LaRoche; PEG-Intron®, Schering-Plough/Enzon), PEG-L- asparaginase from the treatment of acute lymphoblastic leukemia (Oncaspar®, Enzon), PEG- recombinant human granulocyte colony stimulating factor for the treatment of neutropenia (Neulasta®, Amgen), PEG-anti-tumor necrosis factor alpha for the treatment of Crohn's disease (Cimzia®, Enzon), PEG-growth hormone receptor antagonist for the treatment of acromegaly (Somavert®, Pfizer), and PEG-anti-TNF Fab for rheumatoid arthritis (CD870, Pfizer).
[0007] PEGylation has also been shown to improve delivery of nucleic acids to cells. For example, US patent publication 2008/0064863 discloses double-stranded nucleic acids, one strand of which is covalently attached to a poly(ethyleneoxide) unit, in complex with a polycation for use in the delivery of nucleic acid drugs to cells. PCT publication WO2007/021142 discloses covalently PEGylated siRNA molecules. PEG-anti-VEGF aptamer has been approved for intraocular treatment of age-related macular degeneration (Macugen®, OSI/Pfizer).
[0008] PEGylation of small molecules has also been reported. EZN-2208, a PEG conjugate of SN-38, the active metabolite of irinotecan, has been shown to be active in preclinical tumor models. In this instance, PEGylation improves the solubility of the small molecule drug.
[0009] Covalent attachment of peptide and protein drugs to macromolecules other than PEG has been disclosed. For example, conjugates of various peptide drugs, such as thrombospondin-1 mimetic peptides, angiopoietin-2 antagonist, glucagon-like peptide- 1 (GLP-I), and exendins, with a monoclonal antibody have been reported. [0010] Covalent modification of peptides, proteins and small molecules with PEG, or other macromolecules, often causes deleterious loss of the biological activity of the parent drug, however. Thus, some recent activity has focused on development of reversible, or transient, PEGylation, in which the polymer chains are conjugated to the drug through a cleavable linker unit. The final PEGylated conjugate is of sufficient molecular size to have favorable systemic retention. Under physiological conditions, cleavage of the linker unit by enzyme or chemical action leads to release of a drug or prodrug that is rapidly converted to the active drug. Depending upon the rate of cleavage of the linker unit relative to the clearance rate of the prodrug or free drug, sufficient steady-state concentrations of active drug for biological activity may be realized using this approach.
[0011] Success has been reported using this approach, for example, using the immunotoxin SSlP reversibly-conjugated to PEG through lysine residues. See Filpula, et at, "Releasable PEGylation of Mesothelin Targeted Immunotoxin SSlP Achieves Single Dosage Complete regression of a Human Carcinoma in Mice," Bioconjugate Chemistry (2007) 18:773-784. Whereas unmodified SSlP was eliminated from mouse plasma with a half-life of about 26 minutes, reversible PEGylation extended the half-life to 2.5-5 hours. Reversible PEGylation of atrial natriuretic peptide (having a plasma half-life of 2-5 minutes) has been shown to result in prolonged protracted effects on blood pressure in adrenaline-treated rats (Nesher, et al. , Bioconjugate Chem (2008) 19:342-348).
[0012] Most methods for reversible PEGylated or other macromolecular conjugated drugs suffer potential drawbacks. For example, some require enzyme hydrolysis by serum proteases or esterases, others need a reducing environment to cleave a disulfide linker, and most release a "self-immolative" prodrug that undergoes spontaneous cleavage to the active drug and a small, potentially toxic alkylating agent. It would be beneficial to design versions of reversible PEGylation or macromolecular drug attachment that do not require and are unaffected by difficult-to-control entities such as enzymes, and redox environments.
[0013] U.S. patent 6,504,005 describes prodrug molecules that release active drug under physiological conditions by virtue of beta elimination department on pH. A specific embodiment of this approach is described in WO2004/089279. This approach, albeit limited in scope and examples, utilizing a spontaneous, first-order rate of cleavage of the drug from the PEG carrier that is initiated when the conjugate is exposed to physiological pH, is described in US Patent Publication 2006/0171920. A general strategy for providing macromolecule-drug conjugates having a variety of spontaneous, first-order release rates that are predictable and controllable under physiological conditions would provide a valuable therapeutic tool for the treatment of disease. Disclosure of the Invention
[0014] The present invention provides prodrugs and drug-macromolecule conjugates for controlling the delivery rate of therapeutic agents ("drugs") when administered to patients requiring treatment with the therapeutic agents. The prodrugs and drug-macromolecule conjugates of the invention provide a means of delivering therapeutic agents over a sustained period of time, even for therapeutic agents which are rapidly cleared from the system, thus prolonging the therapeutic effects of the therapeutic agents.
[0015] In one aspect, the invention is directed to compounds that are drug-macromolecule conjugates of the formula
Figure imgf000005_0001
wherein m is an integer of 1-10; Z is the residue of a macromolecule; L' is the residue of a linker; D is the residue of a drug or of a prodrug; X is O or S;
A is alkenyl (C2), aryl or absent; each R1 and R2 is independently CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R1 and R2 is independently COR3 or SOR3 or SO2R3 wherein R3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or
OR or NR2 wherein each R is independently H or optionally substituted alkyl; or each R1 and R2 is independently SR4 wherein
R4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R1 and R2 may be joined to form a 3-8 member ring; and wherein both R1 and R2 cannot be H; wherein R5 is H or alkyl (C1-6).
[0016] Typically, the linker residue is a bivalent chain having a molecular weight between 14 Da and 20 kDa which may include unsaturation, heteroatoms, ring structures, aromatic portions, and/or heteroaromatic portions that covalently links Z to the remainder of the molecule. The linker may include a peptide backbone, a pseudopeptide backbone, a triazole, a phenylene, or a maleimido residue or combinations thereof. In some embodiments, the carbonyl shown in formula (3) originates in the drug itself. In some embodiments, m is 1 and/or R5 is H. The value of m may also be any intermediate integer between 1 and 10; thus m may be 2, 5, 7, or any intervening integer.
[0017] In another aspect, the present invention provides prodrugs having the formula (2)
Figure imgf000006_0001
wherein m, X, A, R1, R2 and R5 are defined as above, L is a linking group that can attach to a macromolecule, and
D is the residue of a drug or a prodrug.
In some embodiments, m is 1 and/or R5 is H.
[0018] The compounds of formula (3) and of formula (2) release the drug or prodrug at controlled rates under physiological conditions in the body, by non-enzymatic elimination. The rate of drug release is tunable by the appropriate choice of groups R1 and R2. In some embodiments, each of groups R1 and R2 may be independently substituted by electron-donating and/or electron- withdrawing substituents that alter the acidity of the intervening R -CH-R proton so that enormous flexibility and control over the rate of drug elimination can be achieved. In certain embodiments of the invention, mixtures of compounds of formula (3) or of formula (2), each with a different rate of drug release under physiological conditions, may be used in order to provide tailored drug delivery profile to a patient in need of treatment.
[0019] The invention is also directed to methods to prepare the compounds of formulas (3) and (2) as well as compounds that are intermediates in their formation. Thus, in another aspect, the invention is directed to compounds of the formula
Figure imgf000007_0001
wherein A, X, L, R1, R2 and R5 are as above defined. [0020] The invention also provides compounds of the formula
Figure imgf000007_0002
wherein
X, A, L', Z, R1, R2 and R5 are defined as above.
[0021] The invention is also directed to pharmaceutical compositions containing compounds of formulas (2) or (3) and to methods to prepare these prodrugs or macromolecular conjugates. In general, the starting point is the compound of formula (1) which may be converted directly to the prodrugs of formula (2) by suitable reaction with the drug compound. The prodrug of formula (2) can then, in turn, be converted to the macromolecular conjugate of formula (3) by virtue of reaction of L with the macromolecule, Z. Alternatively, the compound of formula (3) may be prepared by converting the compound of formula (1) to the compound of formula (4) which is then reacted with the drug.
Brief Description of the Drawings
[0022] FIGURE 1 shows the base-catalyzed elimination of drug from the macromolecular carrier-drug conjugate of the invention.
[0023] FIGURE 2 shows a variety of functional groups for R1 and R2 and their effects on the acidity of adjacent protons. As the acidity increases (lower pKa), the rate of deprotonation and hence the rate of drug release from the prodrug or drug-macromolecular conjugate is expected to increase. [0024] FIGURE 3 shows the effect of substitution on the acidity of the 9-proton in various 2-substituted fluorenes. A shown, the effect follows the expected Hammett linear free energy correlation, such that the acidity of any given 2-substituted fluorene can be closely estimated based on the sigma-parameter of the substituent.
[0025] FIGURE 4 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as the fraction of total drug administered. Relatively fast release rates (for example, a release rate of 5 per day) gives a higher maximum concentration of released drug over a shorter duration. Relatively slow release rates (for example, a release rate of 0.1 per day) give a lower maximum concentration of released drug over a longer duration.
[0026] FIGURE 5 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as percent of the maximal concentration (% Cmax) versus time. As the rate of drug release from the prodrug or drug-macromolecular conjugate slows, the period of time after administration wherein the free drug concentration is above a particular percentage of the maximal concentration is extended.
Modes of Carrying Out the Invention
[0027] The compounds of formulas (2) and (3) are designed to control the pharmacokinetics of the drug or prodrug defined as "D". The mechanism whereby the drug or prodrug, D, is released is shown in Figure 1 using formula (3) for illustration. The rate is controlled according to a pH dependent elimination mechanism. The groups R and R are selected to provide the appropriate reactivity of the intervening proton R'-CH— R2, as illustrated in part in Figure 2, and thus providing control over the rate of drug or prodrug release from the prodrug or conjugate. The nature of the linking group and the group A also influence this process, providing multiple points of control over the rate of drug release from the resulting conjugate. The properties of R1 and R2 may be modulated by the optional addition of electron-donating or electron- withdrawing substituents, as illustrated in Figure 3.
[0028] A particular advantage of the invention is the position of attachment of the linking group. While it is possible to attach a linking group to one of groups R1 or R2, doing so complicates any further substitution chemistry of R1 or R2, and may result in electronic interactions between the linking group and the electron-donating or electron- withdrawing substituents, thus limiting the tunability of the release rates. [0029] While typically, the active form of the drug is directly released from the conjugates of the invention, in some cases, it is possible to release the active drug in the form of a prodrug thereof. On example of such a system is shown below:
Figure imgf000009_0001
wherein Q = O or NH, D' is the active form of a drug, M = usual aryl substitutions.
[0030] As noted above, the compounds of formulas (2) and (3) provide tunable release rates for drugs or prodrugs so as to control the pharmacokinetics of these drugs. In order to prepare these compounds, various intermediates are of the formulas (1), (2) and (4); as noted, the compounds of formula (2) are also prodrugs with tunable release rates. The compounds of formula (1) have neither the drug/prodrug nor the macromolecule attached and can be regarded as starting materials.
[0031] Because the substituents R , R , R , A, and X are shared by all of these compounds, the various embodiments of these substituents as presented in the alternative set forth below in connection with the compounds of formula (1) may be extrapolated to compounds of formulas (2), (3) and (4). In addition, the nature of L in formulas (1) and (2) determines the nature of L' in formulas (4) and (3). Thus, the alternatives described below for formula (1) are hereby imported as alternatives into formulas (2), (3) and (4).
Definitions
[0032] By the term "electron-donating group" is meant a substituent resulting in a decrease in the acidity of the benzylic H group. Examples of suitable electron-donating substituents include but are not limited to lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, and dialkylamino. By the term "electron-withdrawing group" is meant a substituent resulting in an increase in the acidity of the benzylic H group. Examples of suitable electron- withdrawing substituents include but are not limited to halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C(=O)-R, wherein R is H, lower alkyl, lower alkoxy, or amino, or SOR or SO2R, where R is lower alkyl, aryl, or heteroaryl. Non-hydrogen electron-donating or electron-withdrawing substituents may be present in multiple positions on rings to which they are bound. While, for convenience, in most examples, only a single occurrence of a non-hydrogen substituent on a single ring is shown, multiple substituents may also be present and are within the scope of the invention. The substituents may be the same or different.
[0033] The term "alkyl" is meant to include linear, branched, or cyclic saturated hydrocarbon groups of 1-8 carbons, or in some embodiments 1-6 or 1-4 carbon atoms.
[0034] The term "alkenyl" is meant to include non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds. By the term "alkenyl (C2)" is meant a mono-, di-, tri-, or tetra- substituted carbon-carbon double bond of any geometric configuration.
[0035] The term "alkynyl" is meant to include non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds. By the term "alkynyl (C2)" is meant a mono- or di-substituted carbon-carbon triple bond.
[0036] The term "alkoxy" is meant to include alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.
[0037] The term "aryl" is meant to include aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term "heteroaryl" is meant to include aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.
[0038] The term "halogen" includes bromo, fluoro, chloro and iodo.
[0039] By the term "maleimido" is meant a group of the formula
Figure imgf000010_0001
[0040] The terms "protein" and "peptide" are used herein interchangeably regardless of chain length, and these terms further include pseudopeptides which comprise linkages other than amide linkages, such as CH2NH2 linkages as well as peptidomimetics.
[0041] The terms "nucleic acids" and "oligonucleotides" are also used interchangeably regardless of chain length. The nucleic acids or oligonucleotides may be single-chain or duplexed or may be DNA, RNA, or modified forms thereof with altered linkages, such as phosphodiesters, phosphoramidates, and the like. For both the proteins and nucleic acids useful as drugs in the invention, these terms also include those with side chains not found in nature in the case of proteins and bases not found in nature in the case of nucleic acids.
[0042] Small molecules in the context of drugs is a term well understood in the art, and is meant to include compounds other than proteins and nucleic acids that either are synthesized or are isolated from nature and in general do not resemble proteins or nucleic acids. Typically, they have molecular weights < 1,000, although there is no specific cutoff recognized. Nevertheless, the term is well understood in the fields of pharmacology and medicine.
[0043] By the term "macromolecule" is meant a macromolecule having a molecular weight of between about 10,000 and 100,000, which is itself essentially devoid of cytotoxic, hormonal, or cell signaling activity but is capable of conjugation to an active drug molecule so as to serve to carry the active drug molecule in the systemic circulation and provides a reservoir of active drug which is released over time.
[0044] When R1 and R2 are joined to form cyclic structures, this includes groups wherein the R'-CH-R2 moiety forms a substructure such as, for example,
Figure imgf000011_0001
and forms thereof optionally substituted with electron- withdrawing and/or electron- donating groups as described above, wherein G is a bond; C=O; SO, SO2, CX2, or CX2CX2 wherein each X independently is H or Cl.
Variants of Formula (I)
[0045] In the examples set forth below, it will be noted that aryl groups, for example, typically contain only one substituent. This is for simplicity only, it will be understood that embodiments of R1 and R2 wherein ring systems contain multiple non-hydrogen substituents are included. Preferably the number of substituents on a single ring is 1, 2 or 3.
[0046] In some embodiments of the invention, compounds of formula (1) have more specific formula:
Figure imgf000011_0002
or the formula:
Figure imgf000012_0001
wherein n = 1-6.
[0047] In some embodiments, the molecules of formula (1) have the more specific formula (Ia)
Figure imgf000012_0002
wherein R7 and R8 are each independently H, an electron-donating group, or an electron- withdrawing group, and wherein electron donating and/or electron withdrawing forms of R7 and
R are present at 1-5 positions preferably 3 or less, on the phenyl moieties; X is O or S; A is alkenyl (C2), aryl, or absent; and L is a linking group capable of being attached to a macromolecule, R5 is H or alkyl (C1-6), for example,
Figure imgf000012_0003
for example
Figure imgf000013_0001
or example
Figure imgf000013_0002
Figure imgf000014_0001
[0048] In a more particular embodiment of the invention, the molecules of formula (1) have the more specific structure (Ib)
Figure imgf000014_0002
wherein, as above, R7 and R8 are each independently H, an electron-donating group, or an electron- withdrawing group, and wherein electron donating and/or electron withdrawing forms of R7 and R8 are present at 1-5 positions on the phenyl moieties; R5 is H or alkyl (C1-6), and n = 1-6. [0049] Examples include
Figure imgf000015_0001
as well as
Figure imgf000016_0001
[0050] In certain embodiments of the invention, the molecules of formula (1) have the more specific formula (Ic)
Figure imgf000016_0003
wherein X is O or S; A is alkenyl (C2), aryl, or absent; L is a linking group capable of being attached to a macromolecule; and wherein R5 is H or alkyl (C1-6), R10 and R11 are each independently H, an electron-donating group, or an electron- withdrawing group and wherein R10 and R11 may occur as non-hydrogen substituents at 1-4 or 1-2 positions in their respective rings. Examples include
Figure imgf000016_0002
Figure imgf000017_0001
[0051] In more particular embodiments of the invention, the molecules of formula (Ic) include
Figure imgf000017_0002
[0052] In a particular embodiment of the invention, the molecules of formula (1) have the more specific formula (Id)
Figure imgf000017_0003
wherein R , 10 a „n. d R , 11 are each independently H, an electron-donating group, or an electron- withdrawing group and wherein R10 and R11 may occur as non-hydrogen substituents at 1-4 or 1-2 positions in their respective rings, n = 1-6, and R5 is H or alkyl (C1-6), for example,
Figure imgf000018_0001
as well as
Figure imgf000019_0001
[0053] In certain embodiments, the molecules of the invention have the formula (Ie)
Figure imgf000019_0002
wherein R10 and R11 are each independently H, an electron-donating group, or an electron- withdrawing group, wherein R10 and R11 may be non-hydrogen substituents at 1-4 positions of their respective rings; X is O or S; A is alkenyl (C2), aryl, or absent; L is a linking group capable of being attached to a macromolecule; and G is a bond, C=O, O, SO, SO2, CX2, or CX2CX2, wherein each X is independently H or Cl, and R5 is H or alkyl (C1-6). [0054] Examples include
Figure imgf000020_0001
as well as
Figure imgf000021_0001
including
Figure imgf000021_0002
[0055] In certain embodiments of the invention, the molecules of formula (1) have the more specific formula (If)
Figure imgf000022_0001
wherein R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2, C(=O)-R3, SOR3; SO2R3, SR4, X is O or S; A is alkenyl (C2), aryl, or absent; L is a linking group capable of being attached to a macromolecule; R9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl, and R5 is H or alkyl (C1-6).
[0056] In one embodiment, R2 is H. In another embodiment, R2 is aryl, substituted aryl, heteroaryl, or substituted heteroaryl. In another embodiment, R2 is CN.
[0057] Examples of formula (If) wherein R10 is H or an electron withdrawing or electron donating group include
Figure imgf000022_0002
as well as
Figure imgf000023_0001
[0058] In even more particular embodiments of the invention, the molecules of formula (If) include
Figure imgf000023_0002
Figure imgf000023_0003
[0059] Although only one instance of substitution by R10 is shown, R10 as an electron- withdrawing or an electron-donating substituent may occur at 1-5 positions of the phenyl rings to which it is attached. [0060] In one particular embodiment of the invention, the molecules of formulas (If) wherein R10 and R11 is each independently H or an electron withdrawing or electron donating group, wherein R10 and R11 as non-hydrogen substituents may be present at 1-5 positions of their respective phenyl rings have formula
Figure imgf000024_0001
including
Figure imgf000024_0002
and
Figure imgf000025_0001
[0061] In another more particular embodiment, the compounds of the invention have the formula
Figure imgf000025_0002
[0062] In certain embodiments of the invention, the molecules of formula (1) have the more specific formula (Ig)
Figure imgf000026_0001
wherein R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2, C(O)-R3, SOR3, SO2R3, or SR4; R9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl; n = 1-6, and R5 is H or alkyl (C1-6).
[0063] Molecules of formula (1 g) wherein R10 is H or an electron withdrawing or electron donating group include
Figure imgf000026_0002
as well as
Figure imgf000027_0001
and
Figure imgf000027_0002
and
Figure imgf000027_0003
[0064] Although R10 as a non-hydrogen substituent has been shown in only one position, such non-hydrogen substituent can be present at 1-5 positions on the phenyl moiety shown.
[0065] In certain embodiments of the invention, the molecules of formula (1) have the more specific formula (Ih)
Figure imgf000027_0004
wherein R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2, C(=0)-R3, SOR3, SO2R3 or SR4; X is O or S; A is alkenyl (C2), aryl, or absent; L is a linking group capable of being attached to a macromolecule, and R5 is H or alkyl (C1-6).
[0066] In one embodiment, R2 is H. In another embodiment, R is aryl, substituted aryl, heteroaryl, or substituted heteroaryl.
[0067] Molecules of formula ( 1 h) include
Figure imgf000028_0001
wherein R10 is H, an electron-withdrawing group, or an electron-donating group. Although R10 is specifically shown at only one position in the phenyl rings above, a non- hydrogen embodiment of R10 may be present in 1-5 positions, preferably 3 or less positions on the phenyl ring.
[0068] Molecules of formula (Ih) also include
Figure imgf000028_0002
and
Figure imgf000029_0001
as well as
Figure imgf000029_0002
wherein n= 1-6. [0069] In certain embodiments of the invention, the molecules of formula (1) have the more specific formula (Ik)
Figure imgf000030_0003
wherein R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2, C(=O)-R3, SOR3, SO2R3 or SR4; X is O or S; A is alkenyl (C2), aryl, or absent; and L is a linking group capable of being attached to a macromolecule, and R5 is H or alkyl (C1-6).
[0070] In a particular embodiment of the invention, the molecules of formula (Ik) have R2 = H, giving molecules of formula (Im), wherein R11 is H or an electron withdrawing or electron donating group, wherein R1 as a non-hydrogen substituent may be present at 1-5 positions, preferably less than 3 positions on the phenyl moiety.
Figure imgf000030_0001
[0071] Molecules of formula (Im) include
Figure imgf000030_0002
as well as
Figure imgf000031_0001
[0072] In a particular embodiment of the invention, the molecules of formula (Im) have the formula (In)
Figure imgf000032_0001
wherein n = 1-6; R11 is H, an electron- withdrawing group, or an electron-donating group wherein R11 as a non-hydrogen substituent may be present at 1-5 positions, preferably less than 3 positions on the phenyl moiety, and R5 is H or alkyl (C1-6).
[0073] Molecules of formula (In) include
Figure imgf000033_0001
[0074] In a particular embodiment of the invention, the molecules of formula (Ik) have R2 = aryl, and have the formula (Ip)
Figure imgf000033_0002
wherein X is O or S; A is alkenyl (C2), aryl, or absent; L is a linking group capable of being attached to a macromolecule; R10 and R11 are each independently H, an electron- withdrawing group, or an electron-donating group, wherein a non-hydrogen form of R10 and R11 may be present at 1-5 positions, preferably 3 or less positions of the phenyl moiety, and R5 is H or alkyl (C1-6).
[0075] Molecules of formula ( 1 p) include
Figure imgf000034_0001
Figure imgf000034_0002
as well as
Figure imgf000034_0003
and
Figure imgf000034_0004
and
Figure imgf000035_0001
[0076] In a particular embodiment of the invention, the molecules of formula (1) have the more specific formula (Iq)
Figure imgf000035_0002
wherein X is O or S; A is alkenyl (C2), aryl, or absent; L is a linking group capable of being attached to a macromolecule; R10 is H, an electron- withdrawing group, or an electron- donating group, wherein a non-hydrogen form of R10 may be present at 1-4 positions, preferably 2 or less positions of the ring to which it is bound, and R5 is H or alkyl (C1-6). [0077] Molecules of formula ( 1 q) include
Figure imgf000035_0003
[0078] Molecules of formula ( 1 q) include
Figure imgf000035_0004
and
Figure imgf000036_0001
and
Figure imgf000036_0002
9] Molecules of formula (1 q) also include
Figure imgf000036_0003
Figure imgf000037_0001
Figure imgf000038_0001
[0080] L is a linking group capable of being attached to a macromolecule. By the term "linking group capable of being attached to a macromolecule" is meant a group comprising a functionality through which L can be chemically bonded to a macromolecule. Examples of suitable functionality include but are not limited to amines, alcohols, thiols, chlorides, iodides, bromides, azides, maleimides, alkenes, alkynes, aldehydes, carboxylates, and phosphates. Thus, in one embodiment, L is (CH2)nR12,
Figure imgf000038_0002
wherein n = 1-6 and R . 112Z is NH2, N3, Cl, Br, I, SH, COOH, CHO, CH=CH2, CCH, or maleimido.
[0081] In a particular embodiment, L is (CH2)nR12, wherein n = 1-6, and R12 is NH2, N3, 1, SH, COOH, CHO, CCH, or maleimido, or R12 is N3, SH, CHO, CCH, or maleimido, or R12 is N3 or CCH.
[0082] In certain embodiments, L is (CH2)nCCH, giving compounds of the invention having the formula
Figure imgf000038_0003
wherein X, R | 1 , t R)2 , τ R>5 , A and X are as defined above. [0083] Exemplary molecules of formula (1) thus have the formula
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000039_0003
Figure imgf000040_0001
Figure imgf000040_0002
Figure imgf000040_0003
[0084] In more particular embodiments, the molecules of formula (1) include
Figure imgf000040_0004
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000042_0002
Figure imgf000042_0003
Figure imgf000042_0004
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000046_0002
wherein n = 1-6. In more particular embodiments, n = 3.
[0085] In all of the foregoing examples of compounds (Ia)-(Iq), non-hydrogen forms of substituents on ring systems may be present in multiple locations, preferably at no more than 2 or 3 locations. The non-hydrogen substituents on a single ring may be the same or different.
[0086] In another embodiment, L is
Figure imgf000046_0003
wherein R12 is NH2, N3, Cl, Br, I, SH, OH, COOH, CHO, CH=CH2, CCH, or maleimido. [0087] In another embodiment, L is a group of the formula
Figure imgf000046_0004
wherein n = 1-6. These compounds may be prepared starting from compounds wherein L = (CH2)nNH2, by reaction with 4-[4-(3,5-Dioxo-hexyl)-phenylcarbamoyl]-butyric acid in the presence of a condensing agent, for example a carbodiimide (DCC, EDCI) or a uronium reagent (e.g., HATC, HBTA, TATU).
[0088] The nature of L' is determined by the nature of the foregoing embodiments of L, and thus these embodiments are illustrative of formulas (3) and (4).
Preparation of Compounds of Formula (1)
[0089] The compounds of formula (1) wherein A is absent may be prepared by the addition of a carbanion R1R2CH* formed by reacting R1R2CH2 with a strong base, for example butyllithium, NaH, lithium diisopropylamide, lithium bis(trimethylsilylamide), or similar, with a molecule L-C(=X)R5 to produce a compound of formula (Ix)
Figure imgf000047_0001
[0090] Alternatively, compounds of formula (Ix) wherein A is absent and X = O may be prepared by a two-step process. In the first step, the addition of a carbanion R R CH" formed by reacting R1R2CH2 with a strong base, for example butyllithium, NaH, lithium diisopropylamide, lithium bis(trimethylsilylamide), or similar, with an ester L-C(=O)OR*, wherein R* is lower alkyl, produces an intermediate ketone R1R2CH-C(^O)-L, which may in the second step be reacted with a suitable reducing agent, for example NaBH4 or NaBH3CN, to provide the compound of formula (1) wherein X=O.
[0091] For example, when L-CHO is 5-hexenal, a compound of formula (1 a) wherein X = O and L is (CH2)3CH=CH2 is obtained. When L-CHO is 5-hexynal, a compound of formula (Ia) wherein X = O and L is (CH2)3CCH is obtained. When L is 6-azidohexanal, a compound of formula (Ia) wherein X = O and L is (CH2)5N3 is obtained. When L is 3-azidobenzaldehyde, a compound of formula (Ia) is obtained wherein X = O and L is 3-azidophenyl. When L = 4-bromobenzaldehyde, a compound of formula (Ia) wherein X = O and L is 4-bromophenyl is obtained.
[0092] For example, when R1R2CH2 = fluorene is reacted with a strong base, for example butyllithium, NaH or lithium diisopropylamide, to form a fluorenyl carbanion, which is then reacted with L-CHO, the reaction is as follows:
Figure imgf000048_0001
[0093] Corresponding compounds wherein X = S may be similarly prepared using the appropriate analogue Z-C(=S)R5, or may alternatively be prepared by subsequent chemical transformation of compounds (Ia) using methods known in the art, for example activation of the alcohol group in (Ia), for example by conversion to a bromide using PBr3 or Ph3PBr2, or by conversion to the tosylate or triflate, and displacement by a suitable nucleophilic group such as thiourea or thiosulfate to form a compound of formula (Ib). In a preferred embodiment, thiosulfate is used to form an intermediate that is hydrolyzed by acid treatment to form the thiol.
[0094] In certain embodiments of the invention, A = alkenylene (C2). Compounds wherein A = alkenyl (C2) may be prepared by addition of the carbanion derived by lithiation of R1R2CH2, for example using a strong base such as NaH, butyllithium, lithium bis(trimethyl- silylamide), or similar, to an unsaturated compound such as methyl 3-(dimethylamino)-acrylate to provide an intermediate ester, which may be reduced, either via one step or through multiple steps, to the corresponding unsaturated aldehyde:
Figure imgf000048_0002
[0095] Reaction of the unsaturated aldehyde with an arylboronic acid R12-aryl-B(OH)2 in the presence of a palladium catalyst, for example as described in Org. Letts. (2005) 7:4153-5, provides a compound of formula (Ic), wherein A = alkenyl (C2), L = substituted aryl, and X = O.
Figure imgf000048_0003
[0096] Alternatively, reaction of the unsaturated aldehyde with an allylborane according to the method of Soderquist provides compounds of formula (Id), wherein A = alkenyl (C2), X = O and L = CH2CH=CH2, and formula (Ie), wherein A = alkenyl (C2), X = O and L = CH2CCH. See Burgos, C. H., et al., J. Am. Chem. Soc. (2005) 127:8044.
Figure imgf000049_0001
Preparation of Compounds of Formulas (2), (3) and (4)
[0097] The starting materials of formula (1) are then derivatized either first to the drug or first to the macromolecule to obtain an intermediate in the formation of the compound of formula (3). In these intermediates and in the product of formula (3), all of the embodiments which result from the many illustrated forms of formula (1), and specifically embodiments of L, A, X, R1, R2 and R5 are retained in the intermediates and in the final product of formula (3), except that L, when reacted with the macromolecule, becomes L' as a result of this reaction.
[0098] Thus, in the following illustrations of the intermediate wherein the drug is first added, all of the embodiments of R1, R2, R5, A, X and L set forth above for formula (1) are included as illustrations of the compounds of formula (2):
Figure imgf000049_0002
wherein m is an integer of 1-10;
Z is the residue of a macromolecule;
L is a linking moiety that can react to couple a macromolecule;
D is the residue of a drug or of a prodrug;
X is O or S; A is alkenyl (C2), aryl or absent; each R1 and R2 is independently CN, NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R1 and R2 is independently COR3 or SOR3 or SO2R3 wherein R3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or
OR or NR2 wherein each R is independently H or optionally substituted alkyl; or each R1 and R2 is independently SR4 wherein
R4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R1 and R2 may be joined to form a 3-8 member ring; and wherein both R1 and R2 cannot be H; wherein R5 is H or alkyl (C1-6).
[0099] Thus, L, A, R1, R2 and R5 as well as X are generically defined as above for the compound of formula (1), and the specific embodiments of these substituents illustrated above for formula (1) are hereby incorporated by reference as examples of the compounds of formula (2).
[0100] The compounds of formula (2) can themselves release the therapeutic agent at a controlled rate or they can be used as intermediates to connect the therapeutic agent with a macromolecule. Both formula (2) and (3) release the therapeutic agent at a controlled rate according to a pH-dependent elimination mechanism.
[0101] As set forth above, the nature of R1, R2, A and L all influence the rate of release. [0102] Compounds of formula (2) may be generally prepared by condensation of a molecule of formula (1), described above, with a drug molecule D. In one embodiment of the invention, a compound of formula (1) is first activated for condensation by reaction with a suitable reagent, for example phosgene or triphosgene, optionally in the presence of N-hydroxysuccinimide; 1,1-carbonyldiimidazole; 1,1-carbonylditriazole; or similar reagents for the conversion of a compound of formula (1) into an activated compounds of formula (1*), wherein W = F, Cl, imidazolyl, triazolyl, or O-succinimidyl:
Figure imgf000051_0001
[0103] For example, reaction of a compound of formula (1) wherein X = O with triphosgene and N-hydroxysuccinimide yields a compounds of formula (1*) wherein X = O and W = O-succinimidyl.
Figure imgf000051_0002
[0104] Compounds of formula (1 *) wherein X = O and W = O-succinimidyl are particularly preferred when the drug molecule to be conjugated, D-H, has an amino group. In this case, the resulting prodrug of formula (2) comprises a carbamate linkage. For cases wherein D-H is a peptide or protein drug, the amino group that reacts with the compounds of formula (1*) may be a terminal alpha-amino group or the amino group of a side-chain, for example of a lysine, ornithine, or unnatural amino acid residue.
[0105] Alternatively, the activating reagent may be a substituted phenyl chloroformate, for example, 4-nitrophenyl chloroformate, 2,4-dinitrophenyl chloroformate, or pentafluorophenyl chloroformate, resulting in formation of an intermediate substituted phenyl carbonate.
[0106] Compounds of formula (1 *) wherein X = O and W = F or Cl are particularly preferred when the drug molecule to be conjugated, D-H, has no amino group but instead has a hydroxy group, for example when D-H is a peptide or protein drug from a side-chain tyrosine, serine, or threonine residue, or when D-H is a nucleic acid-based drug such as a deoxynucleic acid or ribonucleic acid.
[0107] In the case of peptide-, protein-, or nucleic acid-based drugs, multiple reactive groups may be present leading to multiple reactions with the compound of formula (1 *). The extent of this multiple reaction may be controlled using standard conditions known in the art, for example by varying the reaction temperature, concentrations, and stoichiometrics in order to obtain the desired reaction product.
[0108] In one embodiment of the invention, D is a peptide drug, wherein D is conjugated to the molecule of formula (1) to produce a molecule of formula (2). In another embodiment of the invention, D is a peptide drug, wherein the molecule of formula (2) is prepared by a method in which the molecule of formula (1) is attached during the synthesis of D. For example, the final step in the synthesis of D by solid-phase peptide synthesis methods well-known in the art involves attachment of the N-terminal amino acid of the sequence of peptide D in protected form. In the present embodiment, this final step uses the N-terminal amino acid in a form using a compound of formula (1) as the protecting group.
Figure imgf000052_0001
wherein R is the side chain of an amino acid.
[0109] This embodiment is advantageous in that the position and stoichiometry of derivitization is completely controlled.
[0110] As described above, the drug is conjugated via the XH group, which is OH, or SH. The drug will comprise a compatible functional group, for example carboxyl, OH, NH2, alkyl- NH such as MeNH, EtNH, and 1PrNH, aryl-NH such as phenyl-NH or substituted phenyl-NH, and SH. The functional groups on the carrier and on the drug are conjugated using a carbonyl (C=O) group.
[0111] In general, the drugs of interest are peptides or proteins or nucleic acids, such as aptamers or antisense oligomers, or small molecules. [0112] Examples of suitable drugs include those for human or veterinary use including, but not limited to, antidiabetic drugs (e.g., insulin); growth promoters (e.g., human or bovine growth hormone); antibacterials including aminoglycosides (e.g., gentamicin, neomycin and streptomycin), penicillins, (amoxicillin, ampicillin, piperacillin), cephalosporins, (e.g., cefaclor, cefminox and cephalexin), macrolides (e.g., carbomycin, erythromycin, telithromycin) and peptides (e.g., bacitracin, gramicidins, and polymyxins), trimethoprim, piromidic acid, and sulfamethazine; analgesic and anti-inflammatory drugs (e.g., acetaminophen, aspirin, ibufenac, indomethacin), antiallergic and antiasthmatic drugs (e.g., amlexanox and cromolyn), antihypercholesterolemic drugs (e.g., clofibric acid, oxiniacic acid and triparanol), beta- adrenergic blockers and antihypertensive drugs (e.g., bupranolol, captopril, indenolol, propranolol and 4-aminobutanoic acid), antineoplastic drugs (e.g., daunorubicin, azacytidine, 6-mercaptopurine, interferons, interleukin-2, methotrexate, taxol 5-fluorouridine, 5-fluorouracil, capcitibine, and vinblastine), antiviral drugs (e.g., acyclovir, ganciclovir, amantadine, interferons, AZT and ribavirin, etc).
[0113] Particularly preferred are peptide, protein, and nucleic acid drugs. Examples of peptide drugs suitable for use in the invention include but are not limited to: glucagon-like peptide 1 (GLP-I), exendin-2, exendin-3, exendin-4, atrial natriuretic factor (ANF), ghrellin, vasopressin, growth hormone, growth hormone-releasing hormone (GHRH), RC-3095, somatostatin, bombesin, PCK-3145, Phe-His-Ser-Cys-Asn (PHSCN), IGFl, B-type natriuretic peptide, peptide YY (PYY), interferons, thrombospondin, angiopoietin, calcitonin, gonadotropin-releasing hormone, hirudin, glucagon, anti-TNF-alpha, fibroblast growth factor, granulocyte colony stimulating factor, obinepitide, pituitary thyroid hormone (PTH), leuprolide, sermorelin, pramorelin, nesiritide, rotigaptide, cilengitide, MBP-8298, AL- 108, enfuvirtide, thymalfasin, daptamycin, HLFl-1 1, Lactoferrin, Delmitide, glutathione, T-cell epitope PRl, Protease-3 peptides 1-11, B-cell epitope P3, lutenizing hormone-releasing hormone (LHRH), substance P, neurokinin A, neurokinin B, CCK-8, enkephalins, including leucine enkephalin and methionine enkephalin, dermaseptin, [des-Ala20, Gln34]-dermaseptin, surfactant-associated antimicrobial anionic peptide, Apidaecin IA; Apidaecin IB; OV-2 ; 1025, Acetyl- Adhesin Peptide (1025 - 1044) amide; Theromacin (49 - 63); Pexiganan (MSI-78); Indolicidin; Apelin - 15 (63-77); CFPlO (71-85); Lethal Factor (LF) Inhibitor Anthrax related; Bactenecin; Hepatitis Virus C NS3 Protease Inhibitor 2; Hepatitis Virus C NS3 Protease Inhibitor 3; Hepatitis Virus NS3 Protease Inhibitor 4; NS4A-NS4B Hepatitis Virus C (NS3 Protease Inhibitor 1); HIV-I, HIV-2 Protease Substrate; Anti-Fltl Peptide; Bak - BH3; Bax BH3 peptide (55-74) (wild type); Bid BH3 - r8; CTT (Gelatinase Inhibitor); E75 (Her -2/neu) (369 - 377); GRP78 Binding Chimeric Peptide Motif; p53(17-26); EGFR2/KDR Antagonist; Colivelin AGA-(C8R) HNGl 7 (Humanin derivative); Activity - Dependent Neurotrophic Factor (ADNF); Beta - Secretase Inhibitor 1; Beta - Secretase Inhibitor 2; ch[beta] - Amyloid (30-16); Humanun (HN) sHNG, [GIyW] - HN, [Glyl 4] -Humanin; Angiotensin Converting Enzyme Inhibitor (BPP); Renin Inhibitor III; Annexin 1 (ANXA - 1; Ac2-12); Anti-Inflammatory Peptide 1 ; Anti-Inflammatory Peptide 2; Anti-Inflammatory Apelin 12; [D - Phel2, Leul4]-Bombesin; Antennapedia Peptide (acid) (penetratin); Antennepedia Leader Peptide (CT); Mastoparan; [Thr28, Nle31]- Cholecystokinin (25-33) sulfated; Nociceptin (1-13) (amide); Fibrinolysis Inhibiting Factor; Gamma - Fibrinogen (377-395); Xenin; Obestatin (human); [Hisl, Lys6]-GHRP (GHRP-6); [Ala5, [beta]-Ala8] -Neurokinin A (4-10); Neuromedin B; Neuromedin C; Neuromedin N; Activity-Dependent Neurotrophic Factor (ADNF- 14); Acetalin 1 (Opioid Receptor Antagonist 1); Acetalin 2 (Opioid Receptor Antagonist 2); Acetalin 3 (Opioid Receptor Antagonist 3); ACTH (1-39) (human); ACTH (7-38) (human); Sauvagine; Adipokinetic Hormone (Locusta Migratoria); Myristoylated ADP-Ribosylation Factor 6, myr-ARF6 (2-13); PAMP (1-20) (Proadrenomedullin (1-20) human); AGRP (25-51); Amylin (8-37) (human); Angiotensin I (human); Angiotensin II (human); Apstatin (Aminopeptidase P Inhibitor); Brevinin - 1; Magainin 1; RL-37; LL-37 (Antimicrobial Peptide) (human); Cecropin A; Antioxidant peptide A; Antioxidant peptide B; L-Carnosine; BcI 9-2; NPVF; Neuropeptide AF (hNPAF) (Human); Bax BH3 peptide (55-74); bFGF Inhibitory Peptide; bFGF inhibitory Peptide II; Bradykinin; [Des-Argl O]-HOE 140; Caspase 1 Inhibitor II; Caspase 1 Inhibitor VIII; Smac N7 Protein (; MEKl Derived Peptide Inhibitor 1; hBD-1 ([beta]-Defensin-l) (human); hBD-3 ([beta]-Defensin-3) (human); hBD-4 ([beta]-Defensin-4) (human); HNP-I (Defensin Human Neutrophil Peptide 1); HNP-2 (Defensin Human neutrophil Peptide -2 Dynorphin A (1-17)); Endomorphin- 1; [beta] -Endorphin (human porcine); Endothelin 2 (human); Fibrinogen Binding Inhibitor Peptide; Cyclo(-GRGDSP); TP508 (Thrombin-derived Peptide); Galanin (human); GIP (human); Gastrin Releasing Peptide (human); Gastrin - 1 (human); Ghrelin (human); PDGF-BB peptide; [D-Lys3] - GHRP - 6; HCV Core Protein (1-20); a3Bl Integrin Peptide Fragment (325) (amide); Laminin Pentapeptide (amide) Melanotropin - Potentiating Factor (MPF); VA- [beta] -MSH, Lipotropin - Y (Proopiomelanocortin - derived); Atrial Natriuretic Peptide (1-28) (human); Vasonatrin Peptide (1-27); [Ala5, B - Ala8] -Neurokinin A (4-10); Neuromedin L (NKA ); Ac-(Leu28, 31)-Neuropeptide Y (24-26); Alytesin; Brain Neuropeptide II; [D-tyrll] -Neurotensin; IKKy NEMO Binding Domain (NBD) Inhibitory Peptide; PTD-p50 (NLS) Inhibitory Peptide; Orexin A (bovine, human, mouse, rat); Orexin B (human); Aquaporin - 2(254-267) (human Pancreastatin )(37-52); Pancreatic Polypeptide (human); Neuropeptide; Peptide YY (3-36) (human); Hydroxymethyl-Phytochelatin 2; PACAP (1-27) (amide, human, bovine, rat); Prolactin Releasing Peptide (1-31) (human); Salusin-alpha; Salusin-beta; Saposin C22; Secretin (human); L-Selectin; Endokinin A/B; Endokinin C (Human); Endokinin D (Human); Thrombin Receptor (42-48) Agonist (human); LSKL (Inhibitor of Thrombospondin); Thyrotropin Releasing Hormone (TRH); P55-TNFR Fragment; Urotensin II (human); VIP (human, porcine, rat); VIP Antagonist; Helodermin; Exenatide; ZPlO (AVEOOlOO); Pramlinitide; AC 162352 (PYY)(3-36); PYY; Obinepitide; Glucagon; GRP; Ghrelin (GHRP6); Leuprolide; Histrelin; Oxytocin; Atosiban (RWJ22164); Sermorelin; Nesiritide; bivalirudin (Hirulog); Icatibant; Aviptadin; Rotigaptide (ZP123, GAP486); Cilengitide (EMD-121924, RGD Peptides); AlbuBNP; BN-054; Angiotensin II; MBP-8298; Peptide Leucine Arginine; Ziconotide; AL-208; AL-108; Carbeticon; Tripeptide; SAL; Coliven; Humanin; ADNF-14; VIP (Vasoactive Intestinal Peptide); Thymalfasin; Bacitracin; Gramidicin; Pexiganan (MSI-78); Pl 13; PAC-113; SCV- 07; HLFl-1 1 (Lactoferrin); DAPTA; TRI-1144; Tritrpticin; Antiflammin 2; Gattex (Teduglutide, ALX-0600); Stimuvax (L-BLP25); Chrysalin (TP508); Melanonan II; Spantide II; Ceruletide; Sincalide; Pentagastin; Secretin; Endostatin peptide; E-selectin; HER2; IL-6; IL-8; IL-IO; PDGF; Thrombospondin; uPA (1); uPA (2); VEGF; VEGF (2); Pentapeptide-3; XXLRR; Beta - Amyloid Fibrillogenesis; Endomorphin - 2; TIP 39 (Tuberoinfundibular Neuropeptide); PACAP (1-38) (amide, human, bovine, rat); TGFB activating peptide; Insulin sensitizing factor (ISF402); Transforming Growth Factor Bl Peptide (TGF-Bl); Caerulein Releasing Factor; IELLQAR (8- branch MAPS); Tigapotide PK3145; Goserelin; Abarelix; Cetrorelix; Ganirelix; Degarelix (Triptorelin); Barusiban (FE 200440); Pralmorelin; Octreotide; Eptifibatide; Netamiftide (INN-00835); Daptamycin; Spantide II; Delmitide (RDP-58); AL-209; Enfuvirtide; IDR-I; Hexapeptide-6; Insulin- A chain; Lanreotide; Hexa[rho]eptide-3; Insulin B-chain; Glargine- A chain; Glargine- B chain; Insulin- LisPro B- chain analog; Insulin- Aspart B- chain analog; Insulin- Glulisine B chain analog; Insulin- Determir B chain analog; Somatostatin Tumor Inhibiting Analog; Pancreastatin (37-52); Vasoactive Intestinal Peptide fragment (KKYL-NH2); and Dynorphin A. These and other peptide and protein drugs listed, for example, in PCT publication WO2008/058016 Al (incorporated herein by reference for identity and sequence) are suitable for use with the present invention.
[0114] Examples of protein drugs suitable for use in the invention include but are not limited to: immunotoxin SSlP, adenosine deaminase, argininase, and others.
[0115] Examples of nucleic acid-based drugs suitable for use in the invention include, but are not limited to the sense strand and antisense strand of any gene from an animal, and particularly from a mammal. Such genes can be those that are already the subjects of antisense DNAs that have been provided with the purpose of treating various diseases, for example genes for protein kinase C-alpha (Aprinocarsen, non-small cell lung cancer), BCL-2 (Oblimersen, malignant melanoma, lung cancer), ICAM-I (ISIS-3082, Crohn's disease, HCV-related hepatitis C, ischemic/reperfusion injury in transplant), tumor necrosis factor alpha (rheumatoid arthritis, SARS, and psoriasis), adenosine Al receptor (asthma), c-raf kinase (ovarian cancer), H-ras (pancreatic cancer), c-myc (coronary artery disease), protein kinase A RI alpha (colon cancer, AIDS), DNA methyl-transferase (solid cancers), VEGF receptor (cancer), ribonucleotide reductase (kidney cancer), cytomegalovirus IE2 (CMV retinitis), matrix metalloproteinase-9 (prostate cancer), TGF beta 2 (malignant glioma), CD49d (multiple sclerosis), PTP-IB (diabetes), c-myb (cancer), EGFR (breast cancer), mdrl (cancer), autotaxin (cancer), phosphatidylinositol glycan anchor class F (PIGF, cancer), and GLUT-I (cancer). The nucleic acid drugs may be DNA, modified DNA such as phosphorothioate-DNA, RNA, modified RNA such as 2'-OMe-RNA, locked nucleic acids, peptide nucleic acids, or hybrids.
[0116] In one embodiment of the invention, D is an oligonucleotide drug. The compounds of formula (2) wherein D is an oligonucleotide may be prepared by chemical synthesis of the oligonucleotide drug comprising a 5 '-terminal modification that allows for conjugation with a molecule of formula (1). For example, the oligonucleotide may be chemically synthesized such that the 5 '-terminal nucleotide unit, added at the last round of synthesis, comprises a phosphate group modified to contain an amino-alkyl group. The resulting amine-modified oligonucleotide is then conjugated to a molecule of formula (1) to form a molecule of formula (2) wherein D is an oligonucleotide drug. See, for example, Zhao, et al. , Bioconjugate Chemistry (2005) 16(4):758-766.
[0117] The predicted effects of changing the release rate of the drug from the prodrug of formula (2) are illustrated in Figures 4 and 5. Figure 4 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as the fraction of total drug administered, assuming slow clearance of the prodrug. Relatively fast release rates (for example, a release rate of 5 per day) gives a higher maximum concentration of released drug over a shorter duration. Relatively slow release rates (for example, a release rate of 0.1 per day) gives a lower maximum concentration of released drug over a longer duration.
[0118] As the total amount of drug administered can be varied according to need, it is useful to consider the release of drug from the compound of formula (2) in terms relative to the maximal level of free drug achieved. Figure 5 shows calculated drug release profiles for a series of prodrugs having different release rates, with a fixed rate of clearance from the system of 0.5 per day, expressed as percent of the maximal concentration (% Cmax) versus time. As the rate of drug release from the prodrug or drug-macromolecular conjugate slows, the period of time after administration wherein the free drug concentration is above a given percentage of the maximal concentration is extended.
[0119] The models illustrated in Figures 4 and 5 serve to demonstrate that various drug time-concentration profiles may be achieved by selecting the appropriate drug release rate from the prodrug of formula (2) relative to the clearance rate of the drug from the system.
[0120] The invention also includes compositions containing mixtures comprising two or more prodrugs of formula (2) where the drugs, D, are the same or different. In one embodiment, of such a mixture each prodrug of formula (2) has a different rate of drug release under physiological conditions.
[0121] The pharmacokinetics are also further controlled by including a macromolecule as on compounds of the formula
Figure imgf000057_0001
wherein m is an integer of 1-10; Z is the residue of a macromolecule; L' is the residue of a linker; D is the residue of a drug or of a prodrug; X is O or S;
A is alkenyl (C2), aryl or absent; each R1 and R2 is independently CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R1 and R2 is independently COR3 or SOR3 or SO2R3 wherein R3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or OR or NR2 wherein each R is independently H or optionally substituted alkyl; or each R1 and R2 is independently SR4 wherein
R4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R1 and R2 may be joined to form a 3-8 member ring; and wherein both R1 and R2 cannot be H; and wherein R5 is H or alkyl (C1-6).
[0122] When m=l, the drug-macromolecule conjugates of formula (3) have the more specific formula:
Figure imgf000058_0001
[0123] In many embodiments, R5 is H.
[0124] As was the case for compounds of formula (2), the embodiments set forth for R1, R2, A, X, R5 and L illustrated in the case of formula (1) apply and these embodiments are hereby incorporated by reference as appropriate substituents of formula (3). In the case of L', the nature of L determines its structure, and thus the variants described in connection with formulas (1) and (2) apply here as well.
[0125] The drugs illustrated and listed above in connection with formula (2) apply to formula (3) as well.
[0126] Upon release of the drug from formula (3) by non-enzymatic elimination, the linking group remains attached to the macromolecule. It has been demonstrated that clearance rate of molecules from the body is dependent upon their hydrodynamic radius, and hence their molecular weight. See, for example, Veronese, Biomaterials (2001) 22:405-417, which shows that the half-life of superoxide dismutase (SOD) in mouse increases from 0.08 hours for free SOD, to 1.5 hours for SOD conjugated to PEG of molecular weight 1,900 Da, to 36 hours for SOD conjugated to PEG of molecular weight 72,000 Da. Thus, conjugation of the prodrugs of formula (2) to a macromolecule to give a molecule of formula (3) can be used to increase the circulating lifetime of the prodrug. [0127] Macromolecular carriers for the delivery of drugs are also alternative intermediates in the formation of the compounds of formula (3).
Figure imgf000059_0001
[0128] As was the case for the compounds of formulas (2) and (3), all of the variations specifically described for R1, R2, R5, X and L described for formula (1) are incorporated into specific embodiments of compounds of formula (4).
[0129] In some embodiments, Z is a protein, oligosaccharide, or synthetic polymer having a molecular weight of between 10,000 and 250,000.
[0130] In certain embodiments, the macromolecule Z is a synthetic polymer having a molecular weight of between 10,000 and 250,000. In more specific embodiments, the macromolecule Z is a synthetic polymer having a molecular weight of between 10,000 and 100,000. In certain embodiments of the invention, Z is a derivatized linear, branched, or dendrimeric poly(ethyleneglycol) (PEG), monomethoxy-PEG (mPEG), poly(ethyleneimine) (PEI), or PEG-PEI copolymer. Various sizes of derivatized synthetic polymers like PEG and mPEG are commercially available, having a variety of terminal functional groups resulting in molecules comprising hydroxyl, amine, azide, carboxyl, aldehyde, N-hydroxysuccinimidyl ester, imidazolylcarboxamino, imidazolylcarboxy, nitrophenyl carbonate, isocyanate, maleimide, thiol, or epoxide functional groups. Other synthetic polymer derivatives may be prepared using methods known in the art, for example by derivitization using a bromomethyl- substituted aromatic or heteroaromatic aldehyde or an allyl or propargyl halide.
[0131] In another embodiment, the macromolecule Z is a protein of molecular weight between 10,000 and 250,000. In a more specific embodiment of the invention, Z is an antibody or antibody fragment, either monoclonal or polyclonal. Depending upon the sequence of the protein, various reactive functional groups such as amines and thiols will be present. Alternatively, the protein can be chemically derivatized using methods known in the art to add groups such as thiols and maleimides.
[0132] In another embodiment, Z is an oligosaccharide having a molecular weight of between 10,000 and 250,000. In certain embodiments, Z is a dextran having a molecular weight of between 10,000 and 250,000. In more specific embodiments, Z is a dextran having a molecular weight of between 10,000 and 100,000.
[0133] The macromolecule Z will comprise at least one functional group R13 suitable for reaction with a molecule of formula (1) or (2). For compounds of the invention, suitable R13 groups include but are not limited to: hydroxyl, amine, azide, carboxyl, aldehyde, N-hydroxysuccinimidyl ester, imidazolylcarboxamino, imidazolylcarboxy, nitrophenyl carbonate, isocyanate, maleimide, thiol, epoxide, CCH (terminal alkyne), and guanidino groups.
[0134] In a particular embodiment of the invention, Z is the antibody m38c2 or a humanized version thereof (US patent application 2006/0205670, incorporated herein by reference).
[0135] In certain embodiments in the compounds of the formula (3) or (4) Z is selected from the group consisting of an antibody; an albumin; a linear, branched, or dendrimeric polyethylene glycol (PEG); a linear, branched, or dendrimeric monomethoxypolyethylene glycol (mPEG); a linear, branched, or dendrimeric polyethylene imine (PEI); a linear, branched, or dendrimeric PEG-PEI copolymer; a linear, branched, or dendrimeric dextran; and a nanoparticle. In particular embodiments of the invention, the drug-macromolecule conjugates have the formula (3 a) wherein Z is an antibody. In other particular embodiments of the invention, the drug-macromolecule conjugates have the formula (3a) wherein Z is a linear, branched, or dendrimeric polyethylene glycol (PEG). In other particular embodiments of the invention, the drug-macromolecule conjugates have the formula (3a) wherein Z is a linear, branched, or dendrimeric monomethoxypolyethylene glycol (mPEG).
[0136] In compounds of formulas (3) and (4) of the invention, L' can be (CH2)nNHCO, (CH2)nNHCH2, (CH2)nNHCONH, (CH2)ntriazolyl, (CH2)nthiosuccinimidoyl, (CH2)n(hydroxyethylthio), (CH2)nsuccinimidoylthio, (CH2)nCONH,
Figure imgf000060_0001
wherein n ^ 1-6.
Coupling to Macromolecules
[0137] The compounds of formula (4) are prepared as described below wherein the reactant to be coupled to the macromolecule Z is the compound of formula (1); if the compound of formula (2) is employed, the compounds of formula (3) result. Similarly, the compounds of formula (4) may be converted as described above into compounds of formula (3) by reaction with appropriate drugs as described above.
[0138] In each case, the linking group L' is formed by the reaction of a functional group R13 on the macromolecule Z with a functional group R on the linker group L of the molecules of formula (1) or (2) during the conjugation process. As described above, in compounds of formulas (1) and (2), L is (CH2)nR12,
Figure imgf000061_0001
wherein n = 1-6 and R12 is NH2, N3, Cl, Br, I, SH, COOH, CHO, CH=CH2, CCH, or maleimido.
[0139] Compounds of formula (3) or (4) may be prepared by any of several routes, the most appropriate of which will be dependent primarily upon the nature of the group R on group L. The appropriate method and nature of the resulting linking group L' is determined by the choice of functional groups as follows, using reaction with the compound of formula (2) when m = 1 as illustrated.
[0140] When R12 is NH2, the derivatized macromolecule Z-R13 is conjugated to the compounds of formula (1) or (2) through R13 = COOH, using a condensing agent such as a carbodiimide, or directly using R13 = CO-O-succinimidoyl (N-hydroxysuccinimidoyl ester), CO-imidazolyl, or CO-O-(nitrophenyl), to give the amide linkage L' = (CH2)nNHCO
Figure imgf000061_0002
Figure imgf000061_0003
Figure imgf000062_0001
wherein Z, X, A, D, R1, and R2 are as defined above.
[0141] Alternately, when R12 is NH2, the derivatized macromolecule Z-R13 is conjugated to the compounds of formula (1) or (4) through R13 = CHO, using reductive animation, for
Figure imgf000062_0002
wherein Z, X, A, D, R1, and R2 are as defined above.
[0142] Alternately, when R12 is NH2, the derivatized macromolecule Z-R13 is conjugated to the compounds of formula (1) or (2) through R13 = isocyanate, to give the urea linkage
Figure imgf000062_0003
wherein Z, X, A, D, R1, and R2 are as defined above. [0143] When R12 is N3, the derivatized macromolecule Z-R13 may be conjugated to the compounds of formula (2) through R13 = CCH, using conditions for "click chemistry", a 1,3-dipolarcyclo addition reaction, to form a 1,2,3-triazole linkage in W = (CH2)n-triazolyl
Figure imgf000063_0001
Figure imgf000063_0002
wherein Z, X, A, D, R1, and R2 are as defined above.
[0144] When R is SH, the derivatized macromolecule Z-R is conjugated to the compounds of formula (1) or (2) through R13 = maleimide or epoxide to form
5 (CH2)n-(succinimidoylthio),
Figure imgf000063_0003
Figure imgf000063_0004
Figure imgf000064_0001
wherein Z, X, A, D, R1, and R are as defined above.
[0145] When R12 is COOH, the derivatized macromolecule Z-R13 is conjugated to the compounds of formula (1) or (2) through R13 = amine using a condensing agent such as a
carbodiimide to give L'
Figure imgf000064_0002
Figure imgf000064_0003
wherein Z, X, A, D, R1, and R2 are as defined above.
[0146] When R is CCH, the derivatized macromolecule Z-R is conjugated to the compounds of formula (1) or (2) through R13 = N3 using "click-chemistry" to form
a 1,2,3-triazole linkage
Figure imgf000064_0004
Figure imgf000064_0005
wherein Z, X, A, D, R1, and R are as defined above. [0147] When R12 is maleimido, the derivatized macromolecule Z-R13 is conjugated to the compounds of formula (2) through R13 = SH to give L' = (CH2)n-succinimidoylthio
Figure imgf000065_0001
wherein Z, X, A, D, R1, and R2 are as defined above.
[0148] In one embodiment of the invention, L is a group having the formula
Figure imgf000065_0002
wherein n = 1-6 and Z is an antibody comprising a reactive lysine residue such that R13 = NH2. In this embodiment, conjugation of the prodrug and macromolecule provides an enamino-ketone linkage:
Figure imgf000065_0003
[0149] In some embodiments, the drug-macromolecule conjugates of formula (3) have the formula:
Figure imgf000065_0004
or
Figure imgf000066_0003
where R1, R2, Z, A and D are as above-defined, and n is 1-6. [0150] In one embodiment, the conjugates of formula (3) have the more specific formula (3 a)
Figure imgf000066_0001
wherein
Z is the residue of a macromolecule;
D is the residue of a drug molecule;
X is O or S;
L' is the residue of a linking group;
A is alkenyl (C2), aryl, or absent; and
R7 and R8 are each independently H, an electron-donating group, or an electron- withdrawing group, wherein R and R in the form of non-hydrogen substituents may be present at 1-5 positions, preferably 3 or less positions on the rings to which they are bound, and R5 is H or alkyl (C1-6).
[0151] In some embodiments of formula (3a), Z and L' are as described above, and/or X is O, and/or A is absent or is alkenyl (C2).
[0152] The compounds of formula (3 a) are illustrated by:
Figure imgf000066_0002
including examples that include:
Figure imgf000067_0001
[0153] In another embodiment, the drag-macromolecule conjugates of formula (3) have the more specific formula (3b)
Figure imgf000067_0002
wherein
Z is the residue of a macromolecule;
D is the residue of a drug molecule;
X is of O or S;
L' is the residue of a linking group;
A is alkenyl (C2), aryl, or absent;
G is a bond C=O, O, S, SO, SO2, CX2 or CX2CX2, wherein each X is independently H or Cl and
R10 and R11 are each independently H, an electron-donating group, or an electron- withdrawing group, wherein each of R10 and R11 may be present at 1-4 positions, preferably 2 or fewer positions on the rings to which they are bound.
[0154] In certain embodiments of the invention, the drug-macromolecule conjugates have the formula (3b) wherein Z and L' are as exemplified above.
[0155] In the formula (3b) X may be O, and/or wherein A is absent, or is alkenyl (C2). [0156] In particular embodiments of the invention, the drug-macromolecule conjugates of formula (3b) have the more specific formula:
Figure imgf000068_0001
or
Figure imgf000068_0002
[0157] Illustrative embodiments of the invention, the drug-macromolecule conjugates of formula (3b) include
Figure imgf000069_0001
as well as
Figure imgf000070_0001
[0158] In another embodiment of the invention, conjugates of formula (3) have the formula (3 c)
Figure imgf000070_0002
wherein, as above
Z is the residue of a macromolecule; D is the residue of a drug molecule; X is O or S; L' is the residue of a linking group; A is alkenyl (C2), aryl, or absent;
R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2, COR3, SOR3 or SO2R3: and
R9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl, and wherein R2 and R9 may be connected to form a cyclic structure.
[0159] The conjugates of formula (3c) may comprise embodiments of Z and L' illustrated above.
[0160] In some embodiments, of formula (3c) X is O, and/or A is absent, or is alkenyl (C2).
[0161] The drug-macromolecule conjugates of formula (3c) may include
Figure imgf000072_0001
[0162] In each of the exemplified molecules, a non-hydrogen substituent may be present at 1-5 positions, preferably 3 or less positions on the phenyl ring shown.
[0163] In another embodiment, the conjugates of formula (3) have the more specific formula (3d)
Figure imgf000072_0002
wherein
Z is the residue of a macromolecule; D is the residue of a drug molecule; X is O or S;
L' is the residue of a linking group; A is alkenyl (C2), aryl, or absent; and
R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2, COR3, SOR3 or SO2R3.
[0164] The drug-macromolecule conjugates have the formula (3d) may include embodiments of Z and L' as listed above.
[0165] Formula (3d) includes embodiments wherein X is O, and/or wherein A is absent, or is alkenyl (C2).
[0166] In certain particular embodiments of the invention, the drug-macromolecule conjugates of formula (3d) includes
Figure imgf000073_0001
[0167] In another embodiment, the conjugates of formula (3) have the more specific formula (3e)
Figure imgf000073_0002
wherein Z is the residue of a macromolecule;
D is the residue of a drug molecule;
X is O or S;
L' is the residue of a linking group;
A is alkenyl (C2), aryl, or absent;
R2 is H, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, alkynyl, CN, NO2 COR3, SOR3 or SO2R3; and
R9 is alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkenyl, or alkynyl, and wherein R2 and R9 may be connected to form a cyclic structure. [0168] The conjugates of formula (3e) contain embodiments of Z and L' as listed above. [0169] In formula (3e) X may be O, and/or A is absent, or is alkenyl (C2).
[0170] In certain particular embodiments of the invention, the drug-macromolecule conjugates of formula (3e) include
Figure imgf000075_0001
Figure imgf000075_0002
Figure imgf000075_0003
[0171] The foregoing illustrations of formulas (3a)-(3e) further include embodiments wherein m formula (3) is 2-10, including all intervening integers, and any rings showing non- hydrogen substituents may be substituted at multiple positions thereon. The substituents may be the same or different.
Pharmaceutical Compositions
[0172] The invention provides pharmaceutical and veterinary compositions comprising compounds of formulas (2) or (3) or pharmaceutically acceptable salts thereof or mixtures thereof and a pharmaceutically acceptable carrier. Any suitable route of administration of drugs to humans and animals is envisaged by the invention, for example via conventional injectable, implantable, oral, intraocular, intrathecal, rectal or topical administration. These preparations can be prepared by conventional methods known to those skilled in the art, for example as described in Remington's Pharmaceutical Science, A. R. Gennaro, ed., 17th edition, 1985, Mack Publishing Company, Easton, Penn., USA.
[0173] Subjects that may be treated with the compounds of formula (2) or (3) and compositions thereof include humans, veterinary animals, livestock and laboratory animals such as mice and rats.
[0174] The compositions may contain mixtures comprising two or more compounds of formula (3) or of formula (2). In one embodiment, the mixture comprises two or more compounds of formula (2) or (3), wherein each compound of formula (2) or (3) has the same drug D but a different rate of drug release under physiological conditions, thus providing a tailored drug release profile for drug D.
[0175] In another embodiment, the mixture comprises two or more compounds of formula (2) or (3), wherein each compound of formula (2) or (3) has a different drug D, and optionally a different rate of drug release for each drug D under physiological conditions, thus providing tailored combination therapy. In addition to controlling the release of a single drug, the invention can thus be used to control the release rates - and thus steady state concentrations and durations of action - of two or more drugs. Hence, for combinations of two drugs one can optimize the concentration and duration of both drugs. In one embodiment, an optimal conjugate for a first drug A is experimentally determined. Such an optimal conjugate is characterized as having an optimal drug concentration versus time profile (i.e., optimal drug concentrations and length of exposure). Using the optimized conjugate of the first drug A together with conjugates of the second drug B, each conjugate of drug B having a different drug release profile, the most effective combination is then determined experimentally. A converse experiment may then be undertaken, using the optimal conjugate of drug B together with multiple conjugates of the second drug A in order to verify that the optimal mixture has been determined. [0176] In another embodiment, each of drugs A and B are made into conjugates that have a set of half-lives for release of the drugs from the conjugates (for example: 1, 2, 4, and 8 hr half-lives). Combinations of these conjugates that span all the possible permutations of conjugates of drug A and B are then tested.
[0177] In one particular embodiment of the invention, GLP-I or an analogue thereof, for example an exendin, is conjugated to form a first drug-macromolecule conjugate of formula (3), and gastrin is conjugated to form a second drug-macromolecule conjugate of formula (3). In another particular embodiment of the invention, insulin is conjugated to form a first drug- macromolecule conjugate of formula (3), and insulin C-peptide is conjugated to form a second drug-macromolecule conjugate of formula (3).
[0178] The invention is illustrated but not limited by the examples outlined below.
Example 1 General Procedure, Formula d*), A = Absent, X = O
[0179] A solution OfR1R2CH2 (1.0 equivalent) in tetrahydrofuran (THF) is added to a solution of lithium diisopropylamide (LDA) or butyllithium at -780C (1.0 equivalent). The mixture is allowed to warm slowly to O0C, then recooled to -780C prior to the addition of aldehyde L-CHO (1.0 equivalent). After 30 minutes, the mixture is allowed to warm slowly to ambient temperature, quenched by addition of saturated aqueous NH4Cl, and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product is purified if necessary by chromatography on silica gel.
Example 2 General Procedure, Formula (1), A = Absent, X = S
[0180] A solution of the compound of formula (1) wherein A is absent and X = O (1.0 equivalent) in THF is added to a solution of 1 M lithium bis(trimethylsilylamide) (LiHMDS) (1.0 equivalent) at -780C. After 15 minutes, a solution of p-toluenesulfonyl chloride (1.0 equivalent) is added, and the mixture is allowed to warm slowly to ambient temperature, quenched by addition of saturated aqueous NH4Cl, and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The resulting crude tosylate is dissolved in isopropanol and reacted with aqueous sodium thiosulfate at 5O0C to form the Bunte salt, which is hydrolyzed by treatment with aqueous HCl. The mercaptan product is purified by chromatography on silica gel.
Example 3 General Procedure, Formula (1), A = Alkenyl (CA X = Q, L = Substituted Aryl
[0181] A solution of R1R2CH2 (1.0 equivalent) in tetrahydrofuran (THF) is added to a solution of lithium diisopropylamide (LDA) or butyllithium at -780C (1.0 equivalent). The mixture is allowed to warm slowly to O0C, then recooled to -780C prior to the addition of methyl 3-(dimethylamino)propenoate (1.0 equivalent). The mixture is allowed to warm slowly to ambient temperature, quenched by addition of 1 N HCl, and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product ester is purified by chromatography on silica gel.
[0182] A solution of the ester (1.0 equivalent) in THF is treated with excess lithium aluminum hydride, then quenched by addition of oxalic acid and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product alcohol is purified by chromatography on silica gel.
[0183] The alcohol from above (1.0 equivalent) is oxidized to the aldehyde by reaction with Dess-Martin periodinane (1.5 equivalent) in dichloromethane solution. The solution is filtered and washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product aldehyde is purified by chromatography on silica gel.
[0184] The aldehyde from above is reacted with the substituted arylboronic acid as described in Organic Letters (2005) 7:4153-4155 (incorporated herein by reference). Thus, a mixture comprising the aldehyde (1.0 equivalent), arylboronic acid (2.0 equivalents), Cs2CO3 (2.0 equivalents), Pd2(dba)3 »CHCl3 (0.025 equivalent), and Ph3P (0.05 equivalent) in toluene is heated at 8O0C for 24 hours. After cooling to ambient temperature, the mixture is concentrated and the product is purified by chromatography on silica gel.
Example 4 General Procedure, Formula (1), A = alkenyl (CA X = S, L = substituted aryl
[0185] The product of Example 3 is converted to the corresponding compound wherein X = S using the procedure of Example 2. Example 5
General Procedures, Activation of Compounds of Formula (1), X = O as the 4-Nitrophenylcarbonates
[0186] A solution of the compound of formula (1) (1.0 equivalent) in THF is added to a 1.0 M solution of LiHMDS (1.0 equivalent) in THF at -780C. After 15 minutes, a solution of bis(4-nitrophenyl) carbonate (1.5 equivalent) is added. The mixture is allowed to warm slowly to ambient temperature, quenched by addition of 1 N HCl, and extracted with ether. The extract is washed sequentially with 1 N HCl, water, and brine, then dried over MgSO4, filtered, and evaporated. The product 4-nitrophenyl carbonate is purified by chromatography on silica gel.
Example 6
General Procedures, Activation of Compounds of Formula (D, X = O as the N-Hvdroxysuccinimidoyl Carbonates via Intermediate Chloroformates
[0187] A solution of the compound of formula (1) (1 equivalent) in chloroform is treated with triphosgene (5 equivalents) for 24 hours at ambient temperature. The solvent is removed by evaporation to provide the crude chloroformate.
[0188] The chloroformate is dissolved in THF and treated with N-hydroxysuccinimide (4 equivalents) and 2.6-lutidine (6 equivalents) at ambient temperature. When complete as judged by HPLC analysis, the mixture is diluted with ethyl acetate and washed sequentially with 1 N HCl, water, and brine, then dried over MgSO4, filtered, and evaporated. The product N-hydroxysuccinimoyl carbonate is purified by HPLC chromatography.
Example 7 General Procedure, Formula (2), X = O, D = Peptide by Conjugation
[0189] A solution of the peptide in aqueous buffer, pH 7.5, is treated with a solution of the activated compound of Example 5 or Example 6 (1 equivalent) in DMSO. The pH is maintained as necessary by the addition of 1 N NaOH. The reaction progress is monitored by HPLC. When judged complete, the mixture is purified by preparative HPLC.
Example 8 General procedure. Formula (2), X = O, D = Nucleic Acid by Conjugation
[0190] A solution of the nucleic acid in aqueous buffer, pH 7.5, is treated with a solution of the activated compound of Example 5 or Example 6 (1 equivalent) in DMSO. The pH is maintained as necessary by the addition of 1 N NaOH. The reaction progress is monitored by HPLC. When judged complete, the mixture is purified by preparative HPLC.
Example 9 General Procedure, Formula (2), X = O, D = peptide by synthesis
[0191] The peptide is synthesized using standard conditions known in the art, for example using FMOC chemistry. Upon coupling the final amino acid, the terminal FMOC protecting group is removed by treatment under standard conditions. The resin-bound peptide is then reacted with excess compound of Example 5 or Example 6 to cap the N-terminus. The peptide is then deprotected and cleaved from the resin using TFA/triethylsilane, and purified by reversed phase HPLC.
Example 10 General Procedure, Formula (3), X = OH, D = peptide, Z = mPEG
[0192] A compound of formula (1) wherein X = O, A is absent, and L = HCC-(CH2)n wherein n = 0-6 is prepared according to the method of Example 1 using the appropriate terminal alkynyl aldehyde HCC-(CH2)nCHO. This compound is activated according to the method of Example 5 or Example 6, and coupled to peptide D according to the method of Example 7 or Example 9 to provide a compound of formula (2) wherein X = O, D = peptide, and L = HCC-(CH2)n.
[0193] Alternatively, a compound of formula (1) wherein X = O, A is absent, and L = ethynylphenyl is prepared according to the method of Example 1 using the appropriate alkynylbenzaldehyde. This compound is activated according to the method of Example 5 or Example 6, and coupled to peptide D according to the method of Example 7 or Example 9 to provide a compound of formula (2) wherein X = O, D = peptide, and L = alkynylphenyl.
[0194] A THF solution of mPEG-N3 (1 equivalent) and the above-described alkynyl compound of formula (2) (1 equivalent) is treated with aqueous CuSO4 »5H2O (0.1 equivalent) and sodium ascorbate (0.5 equivalent) is stirred 24 hours at ambient temperature. The mixture is lyophilized, then purified by preparative reversed-phase HPLC. Example 11
(4-ethynylphenyiy9H-fluoren-9-yl)methanol Formula (1): A = absent; X = O; R1R2CH = 9-fluorenyl; L = 4-ethynylρhenyl
Figure imgf000081_0001
[0195] A solution of fiuorene (1.0 equivalent) in tetrahydrofuran (THF) is added to a solution of lithium diisopropylamide (LDA) at -780C (1.0 equivalent). The mixture is allowed to warm slowly to O0C, then recooled to -780C prior to the addition of 4-ethynyl-benzaldehyde (1.0 equivalent). After 30 minutes, the mixture is allowed to warm slowly to ambient temperature, quenched by addition of saturated aqueous NH4Cl, and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product is purified by chromatography on silica gel.
Example 12
1 -(3 -f tert-butoxycarbonylamino)phenyl-3 -(9H-fhxoren-9- vDallyl alcohol Formula (1): A = CH=CH; X = O; R1R2CH = 9-fluorenyl; L = 3-(N-BOC-amino)phenyl
Figure imgf000081_0002
[0196] A solution of fiuorene (1.0 equivalent) in tetrahydrofuran (THF) is added to a solution of lithium diisopropylamide (LDA) at -78°C (1.0 equivalent). The mixture is allowed to warm slowly to O0C, then recooled to -78°C prior to the addition of methyl 3-(dimethylarnino)propenoate (1.0 equivalent). The mixture is allowed to warm slowly to ambient temperature, quenched by addition of 1 N HCl, and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product ester is purified by chromatography on silica gel.
[0197] A solution of the ester (1.0 equivalent) in THF is treated with excess lithium aluminum hydride, then quenched by addition of oxalic acid and extracted with ether. The extract is washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product alcohol is purified by chromatography on silica gel.
[0198] The alcohol from above (1.0 equivalent) is oxidized to the aldehyde by reaction with Dess-Martin periodinane (1.5 equivalent) in dichloromethane solution. The solution is filtered and washed sequentially with 1 N HCl, saturated aqueous NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated. The product aldehyde is purified by chromatography on silica gel.
[0199] A mixture comprising the above-described aldehyde (1.0 equivalent), 3-(N-BOC- amino)phenylboronic acid (2.0 equivalents), Cs2CO3 (2.0 equivalents), Pd2(dba)3»CHCl3 (0.025 equivalent), and Ph3P (0.05 equivalent) in toluene is heated at 8O0C for 24 hours. After cooling to ambient temperature, the mixture is concentrated and the product is purified by chromatography on silica gel.
Example 13 (4-ethynylphenyl)(9H-fluoren-9-yl)methyl 4-nitrophenyl carbonate
Figure imgf000082_0001
[0200] A solution of (4-ethynylphenyl)(9H-fluoren-9-yl)methanol (1.0 equivalent) in TΗF is added to a 1.0 M solution of LiΗMDS (1.0 equivalent) in TΗF at -780C. After 15 minutes, a solution of bis(4-nitrophenyl) carbonate (1.5 equivalent) is added. The mixture is allowed to warm slowly to ambient temperature, quenched by addition of 1 N HCl, and extracted with ether. The extract is washed sequentially with 1 N HCl, water, and brine, then dried over MgSO4, filtered, and evaporated. The product 4-nitrophenyl carbonate is purified by chromatography on silica gel. Example 14
1 -(3 -(Vert-butoxycarbonylamino*)phenylV3 -(ffH-fluoren-9- vDally 1 N-hydroxysuccinimidoyl carbonate
Figure imgf000083_0001
[0201] A solution of l-(3-(tert-butoxycarbonylamino)phenyl)-3-(PH-fluoren-9-yl)allyl alcohol (1 equivalent) in chloroform is treated with triphosgene (5 equivalents) and pyridine (2 equivalents) for 24 hours at ambient temperature. The solvent is removed by evaporation to provide the crude chloroformate.
[0202] The chloroformate is dissolved in TΗF and treated with N-hydroxysuccinimide (4 equivalents) and 2,6-lutidine (6 equivalents) at ambient temperature. When complete as judged by ΗPLC analysis, the mixture is diluted with ethyl acetate and washed sequentially with 1 N HCl, water, and brine, then dried over MgSO4, filtered, and evaporated. The product N-hydroxysuccinimidoyl carbonate is purified by ΗPLC chromatography.
Example 15 [(4-ethynylphenylX9H-fluoren-9-yl')methoxycarbonyll-derivatized exendin-4
[0203] A solution of exendin-4 (ΗGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS ) in aqueous buffer, pΗ 7.5, is treated with a solution of the activated compound of Example 13 (1 equivalent) in DMSO. The pΗ is maintained as necessary by the addition of 1 N NaOH. The reaction progress is monitored by ΗPLC. When judged complete, the mixture is purified by preparative ΗPLC.
Example 16 N-[(4-ethynylphenvD(9H-fluoren-9-vDmethoxycarbonyl1-exendin-4
[0204] The peptide sequence of exendin-4 (ΗGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS) is synthesized using standard FMOC chemistry. Upon coupling the final histidine amino acid, the terminal FMOC protecting group is removed by treatment under standard conditions. The resin-bound peptide is then reacted with excess compound of Example 13 to cap the N-terminus. The peptide is then deprotected and cleaved from the resin using TFA/triethylsilane, and purified by reversed phase HPLC using standard methodology.
Example 17 N-r(4-(mPEG-triazolyl)phenyl)(9H-fluoren-9-yl)methoxycarbonyll-exendin-4
Figure imgf000084_0001
[0205] A THF solution of mPEG-N3 (1 equivalent) and the alkynyl compound of Example 16 (1 equivalent) is treated with aqueous CuSO4»5H2O (0.1 equivalent) and sodium ascorbate (0.5 equivalent) is stirred 24 hours at ambient temperature. The mixture is dried, then purified by preparative reversed-phase HPLC.
Example 18
1 -(3 -(tgrt-butoxycarbonylammo^phenvP-S -CPH-fluoren-9- vDallyloxycarbonyl-derivatized exendin-4
[0206] A solution of exendin-4 (ΗGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSS- GAPPPS ) in aqueous buffer, pΗ 7.5, is treated with a solution of the activated compound of Example 14 (1 equivalent) in DMSO. The pΗ is maintained as necessary by the addition of 1 N NaOH. The reaction progress is monitored by ΗPLC. When judged complete, the mixture is purified by preparative ΗPLC.
Example 19 N-ri-(3-('tert-butoxycarbonylamino')phenyl)-3-(PH-fluoren-9-yl)allyloxycarbonyll-exendin-4
Figure imgf000084_0002
[0207] The peptide sequence of exendin-4 (HGEGTFTSDLSKQMEEEAVRL- FIEWLKNGGPSSGAPPPS) is synthesized using standard FMOC chemistry. Upon coupling the final histidine amino acid, the terminal FMOC protecting group is removed by treatment under standard conditions. The resin-bound peptide is then reacted with excess compound of Example 14 to cap the N-terminus. The peptide is then deprotected and cleaved from the resin using TFA/triethylsilane, and purified by reversed phase HPLC using standard methodology.
Example 20 N-Fl-O -dnPEG-carboxamido*)phenvD- 3 -(PH-fluoren-9- vDallyloxycarbonyll -exendin-4
Figure imgf000085_0001
[0208] The compound of Example 19 is dissolved in TFA/triethylsilane to remove the tert- butoxycarbonyl group, then evaporated to dryness. The resulting compound is dissolved in acetonitrile and reacted with mPEG-carboxylate N-hydroxysuccinimide ester in the presence of pΗ 6 buffer. The mixture is stirred at ambient temperature overnight, then dried. The product is purified by reversed-phase ΗPLC.
Example 21 5-hexynal
[0209] Under a nitrogen atmosphere, 5-hexyn-l-ol (3 mL, 27.5 mmol, 1.0 Eq) was added to a O0C mixture of sodium acetate (4.5 g, 27.4 mmol, 2.0 equiv.) and MgSO4 (1.48 g) in dry CH2Cl2 (50 mL), followed by pyridinium chlorochromate (PCC) (11.85 g, 27.5 mmol, 2.0 equiv.). The reaction mixture was stirred at room temperature for 3 hours, and ethyl ether (Et2O) (20 mL) was added. The resulting mixture was filtered through a short pad of silica gel, and the residue was washed with 40 mL of Et2O/petroleum ether (1:1). The clear filtrate was concentrated to half its original volume, then dried over molecular sieves and stored in refrigerator for the next step.
Figure imgf000085_0002
[0210] Under the protection of N2, n-Butyllithium (2.7 mL, 7.8 mmol) was added into the solution of N,N-diisopropylamine(l.l mL, 7.8 mmol) in 20 ml of anhydrous tetrahydrofuran (THF) at 550C, the reaction mixture was stirred for 15 min, and chilled to -780C. Indanone (0.95 g, 7.2 mmol) in THF (10 mL) was added into the above mixture via cannula. After stirring at the same temperature 30 min, a solution of 5-hexynal was added into the flask and stirred for 3 hours. The reaction was quenched by addition of a solution OfNaHSO4 (1 g in 10 mL of water), and the reaction mixture was warmed to room temperature. The aqueous layer was extracted with ethyl acetate (EtOAc) (15mLX 2), and the combined organic solution was dried over MgSO4 and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel to obtain the product as a red solid (0.52 g, yield 38.3%).
Figure imgf000086_0001
[0211] Under a nitrogen atmosphere, n-butyllithium (2.5M in hexanes, 1.0 mL, 3.0mmol) was added to a solution of N,N-diisopropylamine (0.42mL, 3.0 mmol) in anhydrous tetrahydrofuran (THF) (6 mL) at O0C. The resulting reaction mixture was stirred at the same temperature for 15 min, then cooled to -780C. 5-Chloro-2,3-dihydroinden-l-one (0.5 g, 3.0 mmol) in THF (3 mL) was added. After stirring at -780C for 30 min, a solution of 5-hexynal was added. The reaction mixture was then stirred at -780C for 2 hours, quenched by addition of sat. aq. NH4Cl, and warmed to room temperature. The aqueous phase was extracted with ethyl acetate (EtOAc), and the combined organic solution was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography on silica gel to obtain the desired product as pale yellow solid (0.29 g, yield 41.3%).
Figure imgf000086_0002
[0212] Under the protection of N2, n-butyllithium (2.5 M in hexanes, 2.7 mL, 7.8 mmol) was added to a solution of N,N-diisopropylamine (1.1 mL, 7.8 mmol) in anhydrous THF (20 mL) at O0C. The reaction mixture was stirred for 15 min and then was chilled to -78°C. 5- Methoxy-2,3-dihydroinden-l-one (1.17 g, 7.2 mmol) in THF (10 mL) was added into the above mixture via cannula. After stirring at the same temperature for 30 min, a solution of 5-hexynal (ca. 6 mmol) was added into the flask and the reaction mixture was stirred at -780C for 3 hours. The reaction was quenched by addition of a saturated solution OfNH4Cl (10 mL), and then warmed up to room temperature. The mixture was extracted with EtOAc (15 mLX 2), and the combined organic solution was dried over MgSO4, and concentrated under reduced pressure. The residue was purified by flash chromatography on silica gel to obtain the product as a white solid (0.58 g, yield 31.2%).
Figure imgf000087_0001
[0213] Under the protection of N2, n-butyllithium (2.5 M in hexanes, 0.39 mL, 1.13 mmol) was added to a solution of N,N-diisopropylamine (0.17 mL, 1.13 mmol) in anhydrous THF (4 mL) at O0C. The reaction mixture was stirred at O0C for 20 min, chilled to -78°C, and 5-nitro- 2,3-dihydroinden-l-one (0.2 g, 1.13 mmol) in THF (4 mL) was added by needle. After stirring at the same temperature for 30 min, a solution of 5-hexynal (ca. 1 mmol) was added, and then stirred for at -780C for 3 hours. The reaction was quenched by addition OfNaHSO4 (0.24 g in 5 mL of water) and warmed up to room temperature. The aqueous layer was extracted with EtOAc (15 mL x 2), and the combined organic solution was dried over anhydrous MgSO4, filtered, concentrated, and the residue was purified with column chromatography on silica gel to obtain the product as a light yellow solid (0.117 g Yield 38%).
Example 26
Figure imgf000087_0002
[0214] Under a nitrogen atmosphere, n-butyllithium (2.5 M in hexanes, 0.47 mL, 1.35 mmol) was added into the solution of fluorene (0.24 g, 1.48 mmol) in anhydrous THF (2 mL) at -780C, and the reaction mixture was stirred at he same temperature for 2.5h. A solution of 5-hexynal was added and the reaction was stirred at -780C for 3 hours, quenched by addition of sat. aq. NH4Cl, and warmed to room temperature. The mixture was extracted with EtOAc, and then the combined organic phase was washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel to obtain the desired product as a pale yellow solid (0.04O g, yield 11.9%).
Example 27
Figure imgf000088_0001
[0215] Under a nitrogen atmosphere, n-butyllithium (2.5 M in hexanes, 0.47 mL, 1.35 mmol) is added to a solution of fluorene (0.24 g, 1.48 mmol) in anhydrous THF (2 mL) at 780C, and the reaction mixture is stirred at he same temperature for 2.5 h. A solution of 6- azidohexanal is added and the reaction is stirred at -780C for 3 hours, quenched by addition of sat. aq. NH4Cl, and warmed to room temperature. The mixture is extracted with EtOAc, and then the combined organic phase is washed with brine, dried over anhydrous MgSO4, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography on silica gel to obtain the desired product.
Example 28
Figure imgf000088_0002
[0216] A solution of 9-(6-azido-l-hydroxyhexyl)fluorene (1 Eq) in 9:1 dimethylformamide/water is treated with trimethylphosphine (10 Eq) for 1 hr at ambient temperature. To the resulting solution is added 4-[4-(3,5-Dioxo-hexyl)-phenylcarbamoyl]- butyric acid (1 Eq) and dimethylaminopropyl-ethyl-carbodiimide (EDCI) (5 Eq). After stirring overnight, the mixture is diluted with ethyl acetate and washed sequentially with water, IN HCl, sat. aq. NaHCO3, and brine, then dried over MgSO4, filtered, and concentrated. The product is isolated by chromatography on silica gel. Example 29
Figure imgf000089_0001
[0217] A solution of the compound of Example 28 (1 Eq) in chloroform is treated with triphosgene (5 equivalents) for 24 hours at ambient temperature. The solvent is removed by evaporation to provide the crude chloroformate.
[0218] The chloroformate is dissolved in THF and treated with N-hydroxysuccinimide (4 equivalents) and 2.6-lutidine (6 equivalents) at ambient temperature. When complete as judged by HPLC analysis, the mixture is diluted with ethyl acetate and washed sequentially with 1 N HCl, water, and brine, then dried over MgSO4, filtered, and evaporated. The product N-hydroxysuccinimidoyl carbonate is purified by HPLC chromatography.
Example 30
Figure imgf000089_0002
[0219] A solution of exendin-4 (HGEGTFTSDL SKQMEEEAVR LFIEWLKNGG PSSGAPPPS ) in aqueous buffer, pH 7.5, is treated with a solution of the activated compound of Example 29 (1 equivalent) in DMSO. The pH is maintained as necessary by the addition of 1 N NaOH. The reaction progress is monitored by HPLC. When judged complete, the mixture is purified by preparative HPLC.
Example 31 Formation of an Antibody-Exendin Conjugate
[0220] Antibody (for example, h38C2 IgGl or bl2 IgGl ; see US Patent Publication 2006/0205670 Al) is added to a solution of a compound of Example 30 to a final concentration of 25 nM antibody binding site and 125 nM (2). This mixture is incubated at room temperature for 10 minutes. The formation of the conjugate is determined by the formation of UV absorbance at 318 nm.
Example 32 Use of Conjugate Mixtures
[0221] In addition to controlling the release of a single drug, the technology of the invention can be used to control the release rates - and thus steady state concentrations and durations of action - of two or more drugs. Hence, for combinations of two drugs one can optimize the concentration and duration of both drugs. In one embodiment, an optimal drug-macromolecule conjugate for a first drug A is experimentally determined. Such an optimal drug- macromolecule conjugate is characterized as having an optimal drug concentration versus time profile (i.e., optimal drug concentrations and length of exposure). Using the optimized drug- macromolecule conjugate of the first drug A together with multiple drug-macromolecule conjugates of the second drug B, each conjugate of drug B having a different drug release profile, the most effective combination is then determined experimentally. A converse experiment may then be undertaken, using the optimal conjugate of drug b together with multiple drug-macromolecule conjugates of the second drug a in order to verify that the optimal mixture has been determined.
[0222] In another embodiment of the invention, each of drugs A and B are made into conjugates that have a set of half-lives for release of the drugs from the drug-macromolecule conjugates (for example: 1, 2, 4, and 8 hr half-lives). Combinations of these conjugates that span all the possible permutations of conjugates of drug A and B are then tested. (Table 1).
Table 1 Exemplary permutations of drug combination conjugates to be tested to determine optimal combination. Half-lives of release of first drug A from conjugate = w, x, y, and z hrs. Half-lives of release of second drug B from conjugate = a, b, c, and d hrs.
Figure imgf000090_0001
Example 33 General Procedure for Preparation of Sulfone Linkers
[0223] Sulfone linkers may be generally prepared by first reacting an appropriate thiol with a bromoketone to generate an beta-ketosulfide, which is subsequently oxidized to the beta- ketosulfone; reduction of the carbonyl then yields a beta-hydroxysulfone.
Figure imgf000091_0001
[0224] In one example, this is illustrated by the reaction of a thiophenol and a bromo- acetophenone as follows.
Figure imgf000091_0002
Figure imgf000091_0003
[0225] To a solution of the appropriate thiophenol (1 eq) and appropriate 2-bromoacetophenone (1 eq) in tetrahydrofuran (THF) is added triethylamine (Et3N) (1.2 eq). The reaction is stirred at ambient temperature for 1 hour. After diluting with ethyl acetate (EtOAc), the reaction is quenched with sat. NH4Cl (aq). The layers are separated, and the aqueous phase is extracted with EtOAc (2x). The combined organic layers are dried over MgSO4, filtered, and concentrated by rotary evaporation to provide crude mercaptoketone.
[0226] To a solution of the mercaptoketone (1 eq) is added 3-chloroperoxybenzoic acid (mCPBA) (3 eq) portionwise. For sequences aimed at generating the sulfoxides, use of 1 eq of mCPBA suffices. The reaction is stirred at ambient temperature until TLC analysis indicated that reaction progress was complete. After diluting with EtOAc, the reaction is washed with NaHCO3 (aq). The layers are separated, and the aqueous phase is extracted with EtOAc (2x). The combined organic layers are dried over MgSO4, filtered, and concentrated to provide crude ketosulfone. Purification by silica gel chromatography (eluting with EtOAc in hexanes) provides the desired ketosulfone. [0227] To a suspension of ketosulfone (1 eq) in MeOH is added solid NaBH4 (1 eq) portionwise. The reaction is stirred at ambient temperature until TLC analysis indicates that reaction progress is complete, typically 30 minutes. Careful quench with NH4Cl (aq) is followed by dilution with EtOAc. The layers are separated, and the aqueous phase is extracted with EtOAc (2x). The combined organic layers are dried over MgSO4, filtered, and concentrated to provide crude hydroxysulfone, which can be purified either by silica gel chromatography (eluting with EtOAc in hexanes) or crystallization (EtOAc/hexane).
[0228] Compounds prepared using this method:
Figure imgf000092_0001
[0229] Replacement of the bromoacetophenone with an appropriate aliphatic bromoketone allows for preparation of linkers with the corresponding aliphatic segments
Example 34 Preparation of a Bifunctional Sulfone Linker
Figure imgf000093_0001
[0230] Step 1. 4-methoxythiophenol (615 μL) was added to a mixture of 2-bromo-3'-nitroacetophenone (1.22 g) in 10 mL of 1:1 acetonitrile/water with sodium bicarbonate (0.84 g). After 1 hour, the resulting mixture was diluted with water and extracted with ethyl acetate. The extract was dried with MgSO4, filtered, and evaporated to an orange oil, which crystallized upon addition of 3:1 hexane/ethyl acetate. The orange crystals were collected and dried to provide the beta-ketosulfide, 1.1 g.
[0231] Step 2. Wet 3-chloroperoxybenzoic acid (2.0 g, -50%) was added carefully in small portions to a solution of the ketosulfide (670 mg) in 10 mL of dichloromethane. The mixture warmed. After stirring 2 hrs, the suspension was diluted with ethyl acetate and washed carefully with sat. NaHCO3, water, and brine, then dried with MgSO4, filtered, and evaporated to give 630 mg of solid ketosulfone, which was crystallized from ethyl acetate.
[0232] Step 3. Sodium borohydride (100 mg) was added to a suspension of ketosulfone (400 mg) in 5 mL of methanol. After 30 min, sat. aq. NH4Cl was added and the mixture was concentrated. The residue was partitioned between ethyl acetate and water, then the organic phase was washed with brine, dried with MgSO4, filtered, and evaporated to give the crude product, which was crystallized from ethyl acetate/hexane to give 360 mg of the nitro alcohol.
[0233] Step 4. Solid ammonium formate (200 mg) was added to a mixture of the nitro alcohol (217 mg) and 10% palladium on carbon (50 mg) in 5 mL of methanol. The mixture was stirred vigorously for 1 hr, then an additional 50 mg of catalyst was added. After an additional 30 minutes, the mixture was filtered and evaporated. The residue was dissolved in ethyl acetate and washed carefully with sat. NaHCO3, water, and brine, then dried with MgSO4, filtered through a plug of silica gel, and evaporated to give 180 mg of the amino alcohol as a clear glass.
[0234] Step 5. The amino alcohol may be acylated on the amine to provide a more facile means of attachment to the macromolecular carrier, using methods known in the art. For example, a solution of the amino alcohol can be reacted with 3-maleimidopropionic acid anhydride as described for 2-aminofiuorenes (Tsubery, H., et al., J. Biological Chem. (2004) 279:38118-38124). Alternatively, the amino alcohol may be acylated on the amine using an azido-acid derivative, for example 6-azidohexanoyl chloride or 6-azidohexanoic anhydride. Alternatively, the amino alcohol may be acylated on the amine using an alkynyl-acid derivative, for example 5-hexynoyl chloride or 5-hexynoic anhydride.
[0235] Step 6. The acylated amino alcohol is activated for attachment of the drug as the N-hydroxysuccinimdyl carbonate, using the methods described above or known in the art.
Example 35 General Procedure for Preparation of Nitrite Linkers
Figure imgf000094_0001
[0236] In one method, nitrile linkers may be prepared according to the tin-mediated reaction of bromoacetonitrile with aldehydes according to the method of Sun and Shi, J. Chem. Research (S) (1999), 318-319, which is incorporated herein by reference. [0237] In another method, nitrile linkers may be prepared by reduction of beta-ketonitriles, which in turn may be prepared from suitable ketones by reaction of a beta-ketoaldehyde enolate with hydroxylamine hydrochloride:
Figure imgf000095_0001
Figure imgf000095_0002
[0238] For example, the preparation of benzoylacetonitrile is provided in US Patent 6,861,162.
Example 36 Preparation of 3-(4-bromophenylV3-hvdroxypropanenitrile
Figure imgf000095_0003
[0239] A suspension of 4-bromobenzoylacetonitrile (500 mg) in ethanol (5 mL) and acetic acid (300 μL) was heated with sodium cyanoborohydride (280 mg) using an 8O0C hot plate for 2 hours. After cooling to ambient temperature, the mixture was diluted with water and concentrated to a syrup, which was diluted with ethyl acetate and washed with water, sat. NaHCO3, and brine. The extract was dried with MgSO4, filtered, and evaporated to provide 510 mg of a cloudy oil, which was filtered through silica gel using 1 :1 ethyl acetate/hexane to provide the product (496 mg) as a thick oil.
Example 37
Figure imgf000095_0004
[0240] Under the protection of N2> n-butyllithium (1.8 mL, 5.2 mmol) was added to a solution of N,N-diisopropylamine (0.75 mL, 5.2 mmol) in anhydrous THF (10 mL) at O0C. The reaction mixture was stirred for 30 min and then was chilled to -780C. 2-(4-methoxyphenyl)acetonitrile (0.6 mL, 4.34 mmol) was added into the above mixture via syringe. After stirring at the same temperature for 30 min, a solution of 5-hexynal (ca. 4 mmol) was added into the flask by syringe and the reaction mixture was stirred at -780C for 1.5 hours. The reaction was quenched by addition of a saturated solution OfNH4Cl, and then warmed to room temperature. The mixture was extracted with EtOAc (3x 30 mL), and the combined organic solution was dried over MgSO4, and concentrated under reduced pressure. The product was purified by silica gel chromatography. 1H-NMR (CDCl3): δ 7.18 (d, 2H, J = 5.4 Hz), 6.83 (d, 2H, J = 5.4 Hz), 3.72-3.76 (s+m, 5H), 2.47 (IH, s), 2.13 (m, 2H), 1.88 (t, IH)5 1.51-1.68 (m, 4H).
Figure imgf000096_0001
[0241] This product was prepared according to the method of Example 33, substituting 2-(4-nitrophenyl)acetonitrile in place of 2-(4-methoxyphenyl)acetonitrile. 1H-NMR (CDCl3): δ 8.20 (d, 2H, J = 5.6 Hz), 7.51 (d, 2H, J = 5.6 Hz), 3.99 (m, 2H), 2.17 (m, 2H), 1.89 (br s, IH), 1.65 (m, 2H), 1.55 (m, 2H).
Example 39
Figure imgf000096_0002
[0242] This product was prepared according to the method of Example 37, substituting 2-phenylacetonitrile in place of 2-(4-methoxyphenyl)acetonitrile. 1H-NMR (CDCl3): δ 7.3 (m, 5H), 3.9 (m, 2H), 2.23 (m, 2H), 1.96 (br s, IH), 1.77 (m, 2H), 1.60 (m, 2H).
Figure imgf000096_0003
[0243] This product was prepared according to the method of Example 37, substituting 2-(4-chlorophenyl)-acetonitrile in place of 2-(4-methoxyphenyl)acetonitrile. 1H-NMR (CDCl3): δ 7.2-7.3 (m, 4H), 3.8 (m, 2H), 2.23 (m, 2H), 1.96 (br s, IH), 1.8-1.5 (m, 4H).
Example 41 General Procedure for Activation Using N,N'-disuccinimidyl Carbonate
[0244] A mixture of the compound prepared as in Example 33 (1 mmol), N,N'-disuccinimidyl carbonate (2 mmol), and 4-(dimethylamino)pyridine (0.1 mmol) in 2 mL of dry acetonitrile is allowed to stir for 1 hour (for primary alcohols) or for 16 hours (for secondary alcohols), then is diluted with 5 mL of water containing 0.2 mL of 1 N HCl and extracted with ethyl acetate. The organic phase is washed with water and brine, then dried over MgSO4, filtered, and concentrated to provide the crude mixed carbonate, suitable for further use. The mixed carbonate is optionally further purified by silica gel chromatography using ethyl acetate/hexane.
5] Compounds prepared according to this method include:
Figure imgf000098_0001
Figure imgf000099_0001
Example 42 General Procedure for Activation Using Triphosgene and N-Hydroxysuccinimide
[0246] A solution of an alcohol of formula (1) (0.5 mmol) and triphosgene (0.72 mmol) in 5 mL of anhydrous tetrahydrofuran (THF) is stirred under inert atmosphere, and pyridine (84 μL) is added dropwise to give a white precipitate. After 10 minutes, the mixture is filtered using nitrogen pressure and concentrated to remove excess phosgene. The residue is redissolved in 5 mL of THF and treated with N-hydroxysuccinimide (2.65 mmol) and pyridine (130 μL) for 20 minutes, then evaporated to dryness. The residue is dissolved in ethyl acetate, washed successively with water, 0.1 N HCl, sat. NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated to provide the crude carbonate. Purification by chromatography on silica gel (ethyl acetate/hexane) provides the product.
Example 43
Activation of (4-bromophenvD-(9-fluorenvDmethanol using triphosgene and
N-hydroxysuccinimide
Figure imgf000100_0001
[0247] A solution of (4-bromophenyl)-(9-fluorenyl)methanol (175 mg, 0.5 mmol) and triphosgene (212 mg, 0.72 mmol) in 5 mL of anhydrous THF was treated with pyridine (84 μL) for 10 minutes under inert atmosphere, then filtered and evaporated. The residue was dissolved in 5 mL of THF and treated with N-hydroxysuccinimide (310 mg, 2.65 mmol) and pyridine (130 μL) for 209 minutes, then evaporated to dryness. The residue was dissolved in ethyl acetate, washed successively with water, 0.1 N HCl, sat. NaHCO3, and brine, then dried over MgSO4, filtered, and evaporated to provide the crude carbonate. The crude product was dissolved in 1 mL of dichloromethane and loaded onto a 5-mL column of silica gel equilibrated in hexane. Initial elution using hexane removed some colored material. The column was then eluted with 3 : 1 hexane/ethyl acetate, and finally 1 : 1 hexane/ethyl acetate which eluted the purified product (206 mg, 92%).
Example 44
Figure imgf000100_0002
[0248] The crude mixed carbonate of Example was dissolved in DMSO (0.5 mL) and mixed with sodium 4-(aminomethyl)benzoate (0.1 mL of a 1.0 M solution in water). After 5 minutes, the mixture was diluted with 5 mL of water, and the milky solution was washed three times with dichloromethane. The aqueous phase was acidified using 1 N HCl, then extracted three times with ethyl acetate. The ethyl acetate extracts were combined, washed with water, dried over MgSO4, filtered, and evaporated to dryness. The residue is washed once with 1 : 1 EtOAc/hexanes, then dissolved in EtOAc and reconcentrated to provide the purified product (J mg).
Example 45 General Method for Preparation of N£-(2,4-dinitrophenyl)-L-lysine derivatives
Figure imgf000101_0001
[0249] A solution of Nε-(2,4-dinitrophenyl)-L-lysine hydrochloride (35 mg) in 600 μL of water and 200 μL of 1.0 N NaOH is treated successively with 200 μL of 1.0 M NaHCO3 and a solution of 0.1 mmol of the N-hydroxysuccinimide carbonate in 1.0 mL of acetonitrile. The resulting yellow solution is stirred at ambient temperature for 1 hour, then is diluted by addition of 10 mL of water and is loaded onto a Cl 8 solid phase extraction column (Varian BondElut, 1 g). The column is washed with 3 mL of water, 1 mL of 1% CF3CO2H in water, 3 mL of water, then 3 mL of 50% aqueous methanol to elute any unreacted lysine analog. The product is eluted with 100% methanol, and the yellow solution is evaporated to dryness.
Example 46 General Method for Measurement of Drug Release Rates from Compounds of Formula (2)
[0250] This illustrates one example of a method to measure the rate of release of a drug from a compound of formula (2). Stock solutions of the compound of formula (2) are prepared by dissolving the compound in a suitable water-miscible solvent, for example DMSO or acetonitrile. A suitable volume of this stock solution is then diluted into aqueous buffer optionally containing an internal standard for HPLC analysis, for example a sodium benzoate, to provide a clear solution, which is maintained at a set temperature. Poorly soluble compounds may require addition of a cosolvent such as DMSO. Aliquots are periodically removed and either immediately analyzed by HPLC or quenched by addition of an equal volume of 1% trifluoroacetic acid (TFA) in acetonitrile and stored for later analysis. A portion of the aliquot is injected onto an HPLC column for analysis. The areas of the peaks for the remaining compound of formula (2) and, if possible, the drag itself, are then measured and compared to the area of the peak for the internal standard. In certain cases, for example using drags that do not have sufficiently strong UV light absorbance to detect by HPLC, the concentration of free drug may be measured by mass spectral analysis.
[0251] As one example, the rates of release of Nε-(2,4-dinitrophenyl)-L-lysine, "H-Lys(DNP)-OH", from various compounds were determined as follows. Reaction mixtures were prepared containing 0.1 M buffer, 0.05% NaN3, and approximately 0.1 mg/mL of the starting compound, and were kept at 370C. Aliquots were removed periodically and analyzed by injection onto a Varian Polaris 3 μm C18-A reversed-phase HPLC column (150 x 4.6 mm), equilibrated in 50:50 water/methanol (each containing 0.5% acetic acid) at a flow rate of 0.8 mL/min. Compounds were eluted from the column using a gradient to 100% methanol + 0.5% acetic acid, and detected by absorbance at 350 nm. Peak integration gave areas for remaining starting material (As) and for released H-Lys(DNP)-OH (Ap), and the percent reaction was calculated according to
%reaction = AP/(AP + As) * 100
[0252] Release rates were then calculated from the slope of a plot of In(IOO - %reaction) versus time, and half-lives were calculated as Ty2 = ln(2)/rate.
[0253] Half-lives for release of Nε-(2,4-dinitrophenyl)-L-lysine ( H-Lys(DNP)-OH) as a model for drug release were determined in PBS, pH 7.4, 370C for the following compounds with the results as shown. It is evident that the nature of R and R and the substituents thereon provide a wide range of release rates.
Figure imgf000103_0001
[0254] Half-lives for release of Nε-(2,4-dinitrophenyl)-L-lysine ( H-Lys(DNP)-OH) from sulfonyl linkers in PBS, pH 7.4, 37 0C.
Figure imgf000104_0001
[0255] Half-lives for release of Nε-(2,4-dinitrophenyl)-L-lysine ( H-Lys(DNP)-OH) from nitrile linkers in PBS, pH 7.4, or in 0.1 M TrisΗCl, pH 8.3, 37 0C.
Figure imgf000105_0001
[0256] Half-lives for release of Nε-(2,4-dinitrophenyl)-L-lysine ( H-Lys(DNP)-OH) from fluorenyl linkers in PBS, pH 7.4, or in 0.1 M Tris'HCl, pH 8.3, 37 0C.
Example 47 Preparation of Linked 4-Hydroxybenzyl Prodrugs
Figure imgf000106_0001
[0257] Step 1. Pyridine (1.5 mmol) is added to a mixture of the linker (1.0 mmol) and the 4-formylphenyl chloroformate (1.2 mmol) in dry tetrahydrofuran, and the reaction is monitored by thin-layer chromatography. Upon completion, the mixture is diluted with ethyl acetate, washed with water and brine, dried and concentrated. The crude aldehyde product is purified by chromatography on silica gel.
[0258] Step 2. Sodium cyanoborohydride (2 mmol) is added to a mixture of the aldehyde from Step 1 (1 mmol) in 5% acetic acid and ethanol (2 mL). The mixture is warmed to -750C for 2 hours, then cooled and concentrated. The residue is dissolved in ethyl acetate, washed with water and brine, then dried and evaporated to provide the crude benzylic alcohol, which is purified by chromatography on silica gel.
[0259] Step 3. Pyridine (2 mmol) is added dropwise to a mixture of the benzylic alcohol of Step 2 (1 mmol) and triphosgene (3 mmol) in dry tetrahydrofuran. After stirring for 1 hour, the mixture is filtered and evaporated to dryness to provide the crude chloroformate, which is used without further purification. [0260] Step 4. Pyridine (2 mmol) is added to a mixture of the crude chloroformate from Step 3 and N-hydroxysuccinimide (5 mmol). After stirring for 30 minutes, the mixture is filtered and concentrated. The residue is dissolved in ethyl acetate, washed with water and brine, then dried and evaporated to provide the crude succinimidyl carbonate, which is purified by chromatography on silica gel.
[0261] Step 5. A solution of the succinimidyl carbonate from Step 4 in acetonitrile (1 equivalent) is added to a solution of the amine-containing drug in 0.1 M NaHCO3. After 1 hour, the mixture is diluted with water and loaded onto a Cl 8 solid phase extraction cartridge, The cartridge is washed with water, then eluted with a step gradient of water and acetonitrile. The product-containing fractions are pooled and evaporated to dryness, and optionally further purified using preparative HPLC.
Example 48 Preparation of Linked 4-Aminobenzyl Prodrugs
Figure imgf000107_0001
[0262] Step 1. Pyridine (1.5 mmol) is added to a mixture of the linker (1.0 mmol) and triphosgene (3 mmol) in dry tetrahydrofuran. After stirring for 1 hour, the mixture is filtered and evaporated to dryness to provide the crude chloroformate, which is used without further purification.
[0263] Step 2. A mixture of the crude chloroformate from Step 1, the 4-aminobenzyl alcohol (1 mmol), and pyridine (2 mmol) in dry tetrahydrofuran (5 mL) is stirred at ambient temperature. When complete, the mix is evaporated, and the residue is dissolved in ethyl acetate and washed with water and brine, then dried and evaporated to provide the crude carbamate, which is purified by chromatography on silica gel.
[0264] Step 3. Pyridine (2 mmol) is added dropwise to a mixture of the carbamate of Step 2 (1 mmol) and triphosgene (3 mmol) in dry tetrahydrofuran. After stirring for 1 hour, the mixture is filtered and evaporated to dryness to provide the crude chloroformate, which is used without further purification.
[0265] Step 4. Pyridine (2 mmol) is added to a mixture of the crude chloroformate from Step 3 and N-hydroxysuccinimide (5 mmol). After stirring for 30 minutes, the mixture is filtered and concentrated. The residue is dissolved in ethyl acetate, washed with water and brine, then dried and evaporated to provide the crude succinimidyl carbonate, which is purified by chromatography on silica gel.
[0266] Step 5. A solution of the succinimidyl carbonate from Step 4 in acetonitrile (1 equivalent) is added to a solution of the amine-containing drug in 0.1 M NaHCO3. After 1 hour, the mixture is diluted with water and loaded onto a Cl 8 solid phase extraction cartridge, The cartridge is washed with water, then eluted with a step gradient of water and acetonitrile. The product-containing fractions are pooled and evaporated to dryness, and optionally further purified using preparative HPLC.
[0267] All references cited above if not otherwise specified are hereby incorporated by reference in their entireties.

Claims

Claims
1. A compound of the formula
Figure imgf000109_0001
wherein m is an integer of 1-10; Z is the residue of a macromolecule; L' is the residue of a linker; D is the residue of a drug or of a prodrug; X is O or S;
A is alkenyl (C2), aryl or absent; each R1 and R2 is independently CN; NO2; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or each R1 and R2 is independently COR3 or SOR3 or SO2R3 wherein R3 is H or optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; optionally substituted alkynyl; or
OR or NR2 wherein each R is independently H or optionally substituted alkyl; or each R1 and R2 is independently SR4 wherein
R4 is optionally substituted alkyl; optionally substituted aryl; optionally substituted heteroaryl; optionally substituted alkenyl; or optionally substituted alkynyl; wherein R1 and R2 may be joined to form a 3-8 member ring; and wherein both R1 and R2 cannot be H; wherein R5 is H or alkyl (C1-6).
2. The compound of claim 1 wherein m is 1.
3. The compound of claim 1 wherein R5 is H.
4. The compound of claim 1 wherein the linker residue is a bivalent chain having a molecular weight between 14 Da and 20 kDa which may include unsaturation, heteroatoms, ring structures, aromatic portions, and/or heteroaromatic portions that covalently links Z to the remainder of the molecule.
5. The compound of claim 4 wherein the linker is a peptide or a pseudopeptide and/or comprises triazole and/or phenylene, and/or a residue of maleimido.
6. The compound of claim 1 wherein the macromolecule is an antibody, a polysaccharide, an albumin, or a synthetic polymer.
7. The compound of claim 6 wherein the synthetic polymer is polyethylene glycol (PEG).
8. The compound of claim 1 wherein the drug is a peptide, a nucleic acid or a small molecule.
9. The compound of claim 1 wherein one of R1 and R is CN.
10. The compound of claim 1 wherein at least of one R1 and R2 comprises phenyl or phenylene.
11. The compound of claim 1 wherein one of R1 and R2 is SO2R3 and the other is H, alkyl or phenyl.
12. The compound of claim 1 wherein A is absent.
13. A compound of the formula
Figure imgf000111_0001
wherein m, R1, R2, R5, A, X and D are defined as in claim 1, and wherein L is a linking group capable of binding to a macromolecule.
14. A compound of the formula
Figure imgf000111_0002
wherein R1, R2, R5, A, X, Z and L' are defined as in claim 1.
15. A compound of the formula
Figure imgf000111_0003
wherein R1, R2, R5, A and X are defined as in claim 1, and where L is a linking group capable of binding to a macromolecule.
16. A pharmaceutical composition which comprises the compound of any of claims 1-13.
17. The compound of any of claims 1 - 13 for use as a medicament.
18. A method to prepare a compound of formula (3) which method comprises reacting a compound of formula (2) with a macromolecule.
19. A method to prepare a compound of formula (3) which method comprises reacting a compound of formula (4) with a drug under conditions whereby said drug is coupled to the compound of formula (4).
20. A method to prepare a compound of formula (2) which method comprises reacting a compound of formula (1) with a drug under conditions whereby said drug is coupled to the compound of formula (1).
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CN102076331B (en) 2013-12-18
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US20110263502A1 (en) 2011-10-27
EP2306986B1 (en) 2018-03-21
US20140296476A1 (en) 2014-10-02
EP2306986A1 (en) 2011-04-13
ES2672526T3 (en) 2018-06-14
CN102076331A (en) 2011-05-25
DK2306986T3 (en) 2018-06-18
JP6084770B2 (en) 2017-02-22
JP2015172078A (en) 2015-10-01
US8680315B2 (en) 2014-03-25
US9387254B2 (en) 2016-07-12

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