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WO2013036857A1 - Sulfone linkers - Google Patents

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Publication number
WO2013036857A1
WO2013036857A1 PCT/US2012/054293 US2012054293W WO2013036857A1 WO 2013036857 A1 WO2013036857 A1 WO 2013036857A1 US 2012054293 W US2012054293 W US 2012054293W WO 2013036857 A1 WO2013036857 A1 WO 2013036857A1
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Prior art keywords
optionally substituted
alkyl
compound
heteroarylene
arylene
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PCT/US2012/054293
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French (fr)
Inventor
Gary W. Ashley
Daniel V. Santi
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Prolynx Llc
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Publication of WO2013036857A1 publication Critical patent/WO2013036857A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/46—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with hetero atoms directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the invention is in the field of drug delivery systems.
  • the invention relates to conjugates of drugs with macromolecules that release the drug in a controlled manner over time without requiring enzymatic action.
  • a releasable conjugate consists of a drug conjugated to a macromolecular carrier through a linker that degrades over time to release the drug from the carrier.
  • the active species is the free drug, thus overcoming the above-mentioned limitations of permanent conjugation.
  • linker degradation mechanism determines the rate of drug release as well as the sites where release can occur; further, the rate of drug release determines the concentration of free drug (both maximum and minimum) that can be achieved during therapy with a conjugate.
  • the nature of the linker is thus a critical factor to the success of therapy using releasable conjugates.
  • the present invention discloses conjugation linkers that provide for predictable drug release from macromolecular carriers which do not rely on enzymatic catalysis for their action while providing a range of release rates appropriate to support once-a-week to once-a-month administration using a minimum dose of conjugate.
  • These linkers are useful for the conjugation of small molecule, peptide, protein, nucleic acid, and other pharmaceuticals or diagnostic agents to both circulating and non-circulating macromolecular carriers.
  • the carriers are typically macromolecular polymers, but may also be proteins such as albumin and targeting agents such as antibodies.
  • the linkers of the invention may couple these two agents to solid supports or dendrimers.
  • the invention provides compounds of the formula (1)
  • R 1 is optionally substituted C]_-C linear, branched, or cyclic alkyl, optionally substituted aryl; optionally substituted heteroaryl; alkoxy; NR 5 2 , wherein each R 5 is independently H, optionally substituted CrC 6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, or when taken together two R 5 can be cycloalkyl or cycloheteroalkyl; or
  • R 4 is H, optionally substituted Ci-Ce alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • Y is N 3 , alkynyl or cycloalkynyl, optionally protected NH 2 , optionally protected C0 2 H,
  • X is F, CI, O-succinimidyl, O-sulfosuccinimidyl, optionally substituted phenoxy, or NB-CH 2 W, wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted, and W is CI, alkoxy, or S(R 8 ) 2 , wherein each R 8 is independently alkyl or aryl; and wherein R 2 is other than R u -Y and R 3 is other than R 12 -Y and R 1 is R 10 -Y; or R 1 is other than R 10 -Y and R 3 is other than R 12 -Y and R 2 is R u -Y; or
  • R 1 is other than R 10 -Y and R 2 is other than R u -Y and R 3 is R 12 -Y; or
  • R 1 , R 2 , R 3 and/or R 4 is (CH 2 )l(OCH 2 CH 2 )-0-alkyl(Cl-C6) wherein 1 is 1 or 2 and n is 200-1000.
  • the invention also provides methods for the preparation of compounds of formula (1) and for their use as conjugation linkers and as degradable crosslinkers in polymer formation.
  • the invention also includes the products of the compounds of the invention when reacted with drugs and/or macromolecules including solid supports and dendrimers.
  • Figures 1A-1B show the pharmacokinetics of releasably- linked mPEG-(5- aminoacetamido-fluorescein) conjugates in rat.
  • Figure 2 shows the linear free energy relationship of release rate with sulfonyl substituents.
  • a plot of log(release rate) versus substituent sigma (calculated as ApKa for the substituted phenol) is linear, allowing prediction of release rates for any substituted
  • O FT R. : - are designed as conjugate linkers for release of drugs coupled through the linker to
  • the rate of release of the drug is controlled by the acidity of the proton shown as H in formulas (1) and (2) which is in turn affected by the nature of R 1 and R 2.
  • the presence of the sulfone along with hydrogen as R on the beta-carbon has been found particularly advantageous in controlling rates of release.
  • the macromolecule to which the drug is ultimately attached may be coupled to R 1 ,
  • R 2 or R 4 may be part of these substituents.
  • one of R 1 , R2 and R 3 must contain the substituent R 10 -Y, R u -Y or R 12 -Y or R 1 , R 2 and/or R 3 must contain PEG of 200, 300, 400, etc. -1000 CH 2 CH 2 ) units as noted above.
  • Y is a substituent that is capable of reacting with a macromolecule or a macromolecular conjugate or is able to be deprotected to have such a function.
  • the macromolecule can ultimately be coupled either to the alpha or beta carbon or to the sulfur of the sulfone.
  • R 10 -Y, R u -Y or R 12 -Y must be present in formula (1) or R 1 , R2 and/or R 3 must itself be a macromolecule density linked to the sulfone or to the alpha or beta carbon.
  • Alkyl include linear, branched or cyclic hydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4 carbons wherein alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon— carbon double bonds and alkynyl includes one or more carbon— carbon triple bonds. Unless otherwise specified these contain 1-6C.
  • Aryl includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl.
  • Heteroaryl includes 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.
  • substituted is meant an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group comprising one or more substituent groups in place of one or more hydrogen atoms.
  • Substituent groups may generally be selected from halogen including F, CI, Br, and I; lower alkyl including linear, branched, and cyclic; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy including linear, branched, and cyclic; SH; lower alkylthio including linear, branched, and cyclic; amino, alkylamino, dialkylamino, silyl including alkylsilyl, alkoxysilyl, and arylsilyl; nitro; cyano; carbonyl; carboxylic acid, carboxylic ester, carboxylic amide; aminocarbonyl; aminoacyl;
  • heteroaryl including 5-member heteroaryls including as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole, and tetrazole, 6- member heteroaryls including pyridine, pyrimidine, pyrazine, and fused heteroaryls including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole, and benzisothiazole.
  • R 1 and R 2 may be modulated by the optional addition of electron- donating or electron- withdrawing substituents.
  • electron-donating group is meant a substituent resulting in a decrease in the acidity of the R 1 S0 2 R 2 CH group; electron-donating groups are typically associated with negative Hammett ⁇ or Taft ⁇ * constants and are well- known in the art of physical organic chemistry. Hammett ⁇ constants are associated with constituents on aromatic systems and Taft ⁇ * constants are associated with nonaromatic substituents. Examples of suitable electron-donating substituents include but are not limited to lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl.
  • electron- withdrawing group is meant a substituent resulting in an increase in the acidity of the relevant H; electron- withdrawing groups are typically associated with positive Hammett ⁇ or Taft ⁇ * constants and are well-known in the art of physical organic chemistry.
  • an alkoxy substituent on the ortho- or para-position of an aryl ring is electron-donating, and is characterized by a negative Hammett ⁇ constant
  • an alkoxy substituent on the meta-position of an aryl ring is electron- withdrawing and is characterized by a positive Hammett ⁇ constant.
  • a table of Hammett ⁇ and Taft ⁇ * constants values is given below.
  • H 2 C CH 0.05 -0.02 0.56
  • Halogen includes fluoro, chloro, bromo and iodo.
  • protein and “peptide” are used 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.
  • Micromolecule means a molecule 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.
  • Typical macromolecules include polymers, especially polyethylene glycol polymers as these are well established as pharmaceutical carriers.
  • Other carriers are not necessarily macromolecules, but include antibodies and albumin.
  • R 2 is H, optionally substituted CrC 6 alkyl, or
  • R 2 is H.
  • the invention provides compounds of formula (la) wherein
  • R 10 is (CH 2 ) k , arylene, heteroarylene, (CH 2 ) k NR 5 , arylene-NR 5 , heteroarylene-NR 5 , (CH 2 ) k O, arylene-O, heteroarylene-O, or (CH 2 CH 2 0) n CH 2 CH 2 where R 5 is H, optionally substituted C -C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • each k is independently 1-8;
  • n 1-1000;
  • R 2", R 3 J , and R 4" are each independently H, optionally substituted CrC 6 alkyl, optionally substituted aryl, optionally substituted heteroaryl;
  • X and Y are as defined above.
  • the invention provides compounds of formula (lb)
  • R 1 is optionally substituted C]_-C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 3 , and R 4 are each independently H, optionally substituted C -C alkyl, optionally substituted aryl, optionally substituted heteroaryl;
  • X and Y are as defined above.
  • the invention provides compounds of formula (lc)
  • R 1 is optionally substituted C]_-C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
  • R 2 , and R 4 are each independently H, optionally substituted C -C alkyl, optionally substituted aryl, optionally substituted heteroaryl;
  • R 12 is of varying lengths.
  • the invention provides compounds of formula (lc) wherein
  • R 12 is arylene or heteroarylene; Y is N 3 , alkynyl or cycloalkynyl, optionally protected NH 2 , optionally protected C0 2 H, optionally protected SH, or maleimido.
  • R 1 is phenyl, 4-(trifluoromethyl)phenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl,
  • R 2 is H;
  • R 3 is (CH 2 ) 6 N 3 , or (CH 2 ) 6 NH-CO-0- C(CH 3 ) 3 ;
  • R 4 is H; and either X is CI, O-succinimidyl, O-sulfosuccinimidyl, or X is NB-CH 2 W, wherein B is optionally substituted aryl and W is CI, alkoxy, or S(R 8 ) 2 , wherein each R 8 is independently alkyl or aryl.
  • Precursor R 1 S0 2 CH 2 R 2 is deprotonated with a strong base to produce the carbanion.
  • suitable bases include ones such as butyllithium, NaH, lithium diisopropylamide (LDA), lithium bis(trimethylsilyl)amide (LiHMDS), sodium bis(trimethylsilyl)amide (NaHMDS), or potassium tert-butoxide (KO £ Bu), in an inert solvent such as tetrahydrofuran (THF) or a mixture of THF and hexanes.
  • LDA lithium diisopropylamide
  • LiHMDS lithium bis(trimethylsilyl)amide
  • NaHMDS sodium bis(trimethylsilyl)amide
  • K £ Bu potassium tert-butoxide
  • Carbanion generation is typically performed at temperatures below 0°C, often at -78°C or at temperatures in between.
  • the chloroformate is optionally further transformed, for example by reaction with N-hydroxy-succinimide, N-hydroxy-sulfosuccinimide, or an activated phenol such as 4-nitrophenol, pentafluorophenol, or pentachlorophenol, and a weak base such as pyridine.
  • the alcohol can be more directly activated to the succinimidyl carbonate through treatment with ⁇ , ⁇ '-disuccinimidyl carbonate and
  • the starting sulfones, sulfonates, and sulfonamides R 1 S0 2 CH 2 R 2 may be prepared using methods well-known in the art.
  • R 10 -Y-SO 2 -CH 2 -R 2 when R 10 is (CH 2 ) n , arylene, heteroarylene, or (CH 2 CH 2 0) n CH 2 CH 2 , the sulfone may be produced through reaction of precursor R -CH 2 -L, wherein L is halide or tosylate, with sodium sulfinate
  • This reaction may be advantageously performed under microwave irradiation as described by Ju, et ah, J. Org. Chem. (2006) 71:6697-6700 (incorporated herein by reference).
  • the sulfone may be produced through reaction of a sodium sulfinate of the formula R -CH 2 -S0 2 Na with Y-(CH 2 ) n -L or Y-(CH 2 CH 2 0) n CH 2 CH 2 -L to produce the sulfone.
  • the sulfonates may be produced by reaction of a sulfonyl chloride R -CH 2 -SO 2 CI with an alcohol Y-(CH 2 ) n -OH, Y-arylene-OH, or Y-heteroarylene-OH in the presence of a mild base, and the sulfonamides can be similarly prepared by reaction of a sulfonyl chloride R -CH 2 -S0 2 C1 with an amine
  • Y-(CH 2 ) n NHR 5 , Y-arylene-NHR 5 , or Y-heteroarylene-NHR 5 a gain in the presence of a mild base.
  • Other methods of preparation of sulfones, sulfonates, and sulfonamides are well-known in the art.
  • R 1 -S0 2 -CH 3 is first converted to the carbanion using a strong base using conditions as described above. This carbanion is then alkylated using Y-R u -L to produce R ⁇ SOa-CHa-R 11 ⁇ as a precursor for compounds of formula (lb).
  • the sulfone linkers of the invention are employed for controlled release of drugs over time.
  • the linkers are therefore intermediates in the formation of the prodrug wherein the drug is linked to a carrier from which it will be released and thus the invention is also directed to the end product prodrug wherein the linkers have been coupled both to a macromolecular carrier and to a pharmaceutical as well as to the further intermediates where only the macromolecular carrier or the pharmaceutical have been coupled.
  • the pharmaceutical is coupled to the sulfone carrier by displacement of the substituent "X" through reaction with an amino group contained in the drug.
  • Most small molecule drugs contain amino groups, or can be easily modified to contain them.
  • Peptides, of course, and oligonucleotides contain amino groups. If multiple amino groups are present, protection of all but one of such groups may be needed to effect reaction with the linker.
  • the invention includes the product of reacting the sulfone linkers of the invention with pharmaceuticals through displacement of X.
  • the macromolecule is already included in an embodiment of R 1 , R 2 or R 3 , coupling to a macromolecule will be necessary. This is generally through interaction with Y either through displacement or direct coupling, as is the case, for example, for alkynyl embodiments.
  • the macromolecule can be coupled to the linker either before or after the drug has been coupled.
  • the invention also includes products of coupling the linkers with macromolecular carriers.
  • Typical macromolecular carriers include solid supports, dendrimers, and polymers in general. Polymers comprising polyethylene glycol are particularly convenient as polyethylene glycol has been used as a carrier for pharmaceuticals over many decades and is known to be safe.
  • the invention also includes the finished prodrug— i.e., the compounds wherein the linkers of the invention are coupled both to macro molecular carriers and to the drugs.
  • compositions of the invention which result from coupling both to macromolecules and drugs are useful in a variety of indications depending on the nature of the drug. Such applications are understood by practitioners in the art, and as will be described below, the desired rate of release can also be calibrated according to known techniques.
  • the compounds of formula (1) are thus useful as linkers in the reversible conjugation of molecules, for example of drugs to macromolecular carriers as described in PCT publications WO2009/158668, WO2011/140393, WO2011/140392 and WO2011/140376, incorporated herein by reference.
  • These linkers are especially useful as they degrade under physiological conditions of pH and temperature by an enzyme-independent beta-elimination mechanism to release the conjugated drug from its carrier as described above.
  • FIG. 1A and IB An illustration of this is shown in Figures 1A and IB using releasably linked mPEG- (5-aminoacetoamidofluorescein), i.e., mPEG-5-AAF conjugates in the rat.
  • Conjugates of 5-AAF and 4-branched mPEG (40 kDa) connected via sulfone linkers of the invention were injected into rats, and the serum concentrations of the conjugates were measured over time by fluorescence HPLC.
  • Panel A shows concentration versus time profiles for the conjugates.
  • Panel B replots the data of Panel A where the data for the non-releasable conjugate have been subtracted, revealing the rates of release from the releasable conjugates.
  • the linkers must release drug at a rate determined by a combination of factors, including the concentration of free drug needed for efficacy, the time period required between drug administrations, and the clearance rates of the conjugate and drug from the body.
  • One goal in administering a conjugate or prodrug form of a drug is to maintain the free drug concentration at or above a threshold concentration [D] th during the time between conjugate administrations, i.e. , at desired t, as a drug of a fixed potency requires a certain concentration in the system for efficacy.
  • a threshold concentration [D] th a threshold concentration
  • the concentration of drug at time t [D] T can be raised or lowered by adjustment of the conjugate dose administered, i. e. , [CD] 0 .
  • Equation (a) can be rearranged to predict the initial concentration of conjugate required to achieve this goal as shown in equation (b):
  • the optimal release rate depends primarily upon the drug elimination rate k 2 and the time between conjugate administrations T, although in practice T is the primary determinant. Many drugs that benefit from conjugation do so because they have an
  • linkers with in vitro release t 1/2 values of between 10 and 1000 hours are expected to optimally support controlled drug release from macromolecular conjugates.
  • linkers wherein one of the modulating groups is R SC ⁇ provide particularly useful drug release rates under physiological conditions so as to support optimal drug levels while reducing the amount of conjugate that must be administered. Results of measuring the release rates from such linkers are provided in the working examples below.
  • 6-azido-l-hexanol a mixture of 6-chloro-l-hexanol (25 g, 183 mmol) and sodium azide (32.5 g, 500 mmol) in 200 mL of water was heated at reflux for 20 h, then cooled to ambient temperature and extracted 3x with ethyl acetate. The combined extracts were washed with brine, dried over MgS0 4 , filtered, and concentrated to yield the product as a pale yellow oil (28.3 g).
  • Compounds prepared according to this method include: l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptanol: from
  • Compounds prepared according to this method include:
  • Compounds prepared according to this method include:
  • succinimidyl carbonates may be prepared according to this general method.
  • Compounds prepared according to this method include:
  • Compounds prepared according to this method include:
  • Compounds prepared according to this method include:
  • p-Toluenesulfonyl chloride (1 mmol) is added to a solution of 6-azido-l-hexanol (Example 1, 1 mmol) in pyridine (2 mL) cooled on ice. After 30 min, the mixture is allowed to warm to ambient temperature and treated with R ⁇ -SH (1 mmol) for an additional 1 hr. The mixture is diluted with ethyl acetate, washed sequentially with water, 1 N HC1, water, sat. aq. NaHCC"3, and brine, then dried over MgS0 4 , filtered, and evaporated.
  • a solution of an amino-thiol heterobifunctional PEG in THF is treated with excess di-iert-butyl dicarbonate until the reaction is complete, and the di-BOC product is isolated by chromatography.
  • the thiocarbonate is cleaved by treatment with one equivalent of NaOMe in methanol, and 2-bromoethanol is added to form the hydroxyethyl thioether, which is oxidized with peracetic acid to form the product.
  • a solution of the mesylate in water is treated with 2-mercaptoethanol and 2 N NaOH to form the hydroxyethyl sulfide.
  • the sulfide is subsequently oxidized to the sulfone, for example using hydrogen peroxide in the presence of a tungstic acid catalyst or alternatively using peracetic acid.
  • the hydroxyethyl sulfone is then activated as the succinimidyl carbonate according to the methods described in the examples above.
  • a DMSO solution of the compound of Example 21 (2.5 ⁇ ) was added to a solution of 4-branched mPEG-DBCO (50 mg, 1.25 ⁇ ) in 0.5 mL of THF and the mixture was kept at ambient temperature for 24 h. The mixture was diluted with 1 mL of water, then dialyzed overnight against methanol using a SpectraPor® (Spectrum Laboratories) 2 membrane (mw cutoff 12-14 kDa).
  • PEGylated linked AAF compounds (Example 22) were prepared at 250 uM in 10 mM acetate pH 5.0. Samples were administered by i.v. injection to male cd-1 mice at 2 uL/g body weight and to cannulated male Sprague Dawley rats at 100 uL/100 g body weight. For the mouse studies, four animals were used for a complete time course with each time course repeated in duplicate. Serum samples were collected from the orbital sinus using the following schedule: mouse 1: 1, 16 and 72 hr; mouse 2: 2 and 24 hr; mouse 3: 4 and 32 hr; mouse 4: 8 and 48 hr. For the rat studies, single animal was used for a complete time course with each time course repeated in duplicate. Serum samples were collected at 0, 1, 2, 4, 8, 12, 24, 48, 60, 120 hr.
  • Fluorescein standards were prepared from serial dilution of a 20 uM solution of fluorescein-PEG in mouse serum.
  • 50 of the standards and samples were loaded into black 96 well plates (Greiner-Bio) and diluted with 50 of 50 mM HEPES buffer pH 7.5.
  • ⁇ of the standards and samples were loaded into black 96 well plates (Greiner-Bio). The fluorescence signals were read using 485 nm excitation and 535 nm emission on a GENiosTM plate reader.
  • Figures 1A-1B show the pharmacokinetics of releasably- linked 40-kDa 4-branch mPEG-(5-aminoacetamido)fluorescein (5-AAF) conjugates of Example 22 in rat.
  • Panel A shows the concentration of the remaining conjugates in rat serum as a function of time, resulting in clearance half-lives of (a) 34 h; (b) 27 h; (c) 26 h; (d) 24 h; (e) 21 h; (f) 16 h; (g) 9 h, and (h) 5 h.
  • Panel B shows the result of subtracting the concentration versus time curve for the stable conjugate (a) from each of the releasable conjugates (b)-(h) of Panel A.
  • Figure 2 shows the linear free energy relationship of release rate with the substituent when R 1 is substituted aryl, where the substituents are as defined for Figure 1.
  • a plot of log(release rate) versus Hammett sigma constant is linear, both for data from in vitro and in vivo (mouse and rat) measurements. This allows prediction of release rates for any substituted aryl R 1 where the Hammett sigma can be estimated.
  • 6-Azidohexanoic anhydride (1.0 g) was added to a mixture of the aniline (486 mg) in 40 mL of dichloromethane. After 2 h, the solution was washed with sat. aq. NaHC0 3 and brine, dried over MgS0 4 , filtered, and evaporated. The crude amide was purified by chromatography on silica gel using a step gradient of hexane, 60:40 hexane/ethyl acetate, and 1:2 hexane/ethyl acetate, to provide the purified amide alcohol (0.50 g) as a pale yellow oil.
  • N,N-diisopropylethylamine (18 uL, 130 umol) in 1 mL of acetonitrile was kept for 30 min, then quenched by addition of 1 mL of 1.0 M NaHC0 3 .
  • the mixture was diluted with ethyl acetate, washed successively with 0.5 M citric acid, water, sat. aq. NaHC0 3 , water, and brine, then dried over MgS0 4 , filtered and evaporated.
  • the crude product was purified by chromatography on silica gel using a step gradient of hexane, 2: 1 hexane/acetone, 1: 1 hexane/acetone, and 1:2 hexane/acetone.

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Abstract

Sulfone linkers which couple drugs to carriers of various types are described. These linkers permit the release of the drug over time in a controlled manner. The release occurs through beta-elimination, and does not require enzymatic cleavage. Products of the linkers with both drugs and macromolecules and methods of using them are described as well.

Description

SULFONE LINKERS
Related Application
[0001] This application claims benefit of U.S. application Serial Number 61/531,999 filed 7 September 2011 which is incorporated herein by reference in its entirety.
Technical Field
[0002] The invention is in the field of drug delivery systems. In particular, the invention relates to conjugates of drugs with macromolecules that release the drug in a controlled manner over time without requiring enzymatic action.
Background Art
[0003] Conjugation of macromolecular carriers is a demonstrated method for improving the pharmacokinetics of drugs. Permanent conjugation suffers several major limitations, such as a reduction in the potency of the drug and sequestering of the drug in the blood compartment. Much research has therefor been focused on the development of releasable conjugates. A releasable conjugate consists of a drug conjugated to a macromolecular carrier through a linker that degrades over time to release the drug from the carrier. The active species is the free drug, thus overcoming the above-mentioned limitations of permanent conjugation. The choice of linker degradation mechanism determines the rate of drug release as well as the sites where release can occur; further, the rate of drug release determines the concentration of free drug (both maximum and minimum) that can be achieved during therapy with a conjugate. The nature of the linker is thus a critical factor to the success of therapy using releasable conjugates.
[0004] Most existing linkers rely on hydrolytic mechanisms for drug release. Ester linkages have been demonstrated to be cleaved by esterase enzymes and thereby release drugs from carriers such as poly(ethylene glycol) (PEG). Enzymatic release mechanisms are undesirable, however, as they provide inherently unpredictable release rates due to variations in the nature and levels of serum esterases between individuals. Conjugation linkers based on drug release through a non-hydrolytic elimination reaction have been disclosed, for example in U.S.
application US2006/0171920 and in WO2009/158668, WO2011/140393, WO2011/140392 and WO2011/140376. The elimination reaction relies on a modulating group to control the acidity of a proton; ionization of this proton results in release of the drug. [0005] It has now unexpectedly been found that a specific subset of these linkers, wherein the modulating group is RS02, provides distinct advantages in permitting a range of release rates appropriate to support once-a-week to once-a-month administration of drug conjugates.
Disclosure of the Invention
[0006] The present invention discloses conjugation linkers that provide for predictable drug release from macromolecular carriers which do not rely on enzymatic catalysis for their action while providing a range of release rates appropriate to support once-a-week to once-a-month administration using a minimum dose of conjugate. These linkers are useful for the conjugation of small molecule, peptide, protein, nucleic acid, and other pharmaceuticals or diagnostic agents to both circulating and non-circulating macromolecular carriers.
[0007] The carriers are typically macromolecular polymers, but may also be proteins such as albumin and targeting agents such as antibodies. In addition, the linkers of the invention may couple these two agents to solid supports or dendrimers.
[0008] Thus, in one aspect, the invention provides compounds of the formula (1)
9 i ψ ®
R - S - C ' C O ' C ' - X (1)
■¥ ■
R1 is optionally substituted C]_-C linear, branched, or cyclic alkyl, optionally substituted aryl; optionally substituted heteroaryl; alkoxy; NR5 2, wherein each R5 is independently H, optionally substituted CrC6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, or when taken together two R5 can be cycloalkyl or cycloheteroalkyl; or
R1 is (CH2)2(OCH2CH2)nO-alkyl, wherein n = 1-1000 or is R10-Y, wherein R10 is (CH2)k, arylene, heteroarylene, (CH2)kNR5, arylene-NR5, heteroarylene-NR5, (CH2)kO, arylene-O, heteroarylene-O, or (CH2CH20)nCH2CH2, wherein each k is independently 1-8 and n = 1-1000;
R is H, optionally substituted CrC6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or CH2(OCH2CH2)nO-alkyl, wherein n = 1- 1000, or Rn-Y, wherein R11 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1- 1000;
R is H, optionally substituted CrC6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or CH2(OCH2CH2)nO-alkyl, wherein n = 1- 1000, or R12-Y, wherein R12 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1- 1000;
R4 is H, optionally substituted Ci-Ce alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
Y is N3, alkynyl or cycloalkynyl, optionally protected NH2, optionally protected C02H,
7 7
optionally protected SH, maleimido, or NH-CO-(CH2)mR , wherein m = 1-6 and R is N3, alkynyl, cycloalkynyl, optionally protected NH2, optionally protected C02H, optionally protected SH, or maleimido; and
X is F, CI, O-succinimidyl, O-sulfosuccinimidyl, optionally substituted phenoxy, or NB-CH2W, wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally substituted, and W is CI, alkoxy, or S(R 8 )2, wherein each R 8 is independently alkyl or aryl; and wherein R2 is other than Ru-Y and R3 is other than R12-Y and R1 is R10-Y; or R1 is other than R10-Y and R3 is other than R12-Y and R2 is Ru-Y; or
R1 is other than R10-Y and R2 is other than Ru-Y and R3 is R12-Y; or
at least one of R1, R2, R3 and/or R4 is (CH2)l(OCH2CH2)-0-alkyl(Cl-C6) wherein 1 is 1 or 2 and n is 200-1000.
[0009] The invention also provides methods for the preparation of compounds of formula (1) and for their use as conjugation linkers and as degradable crosslinkers in polymer formation.
[0010] The invention also includes the products of the compounds of the invention when reacted with drugs and/or macromolecules including solid supports and dendrimers.
Brief Description of the Drawings
[0011] Figures 1A-1B show the pharmacokinetics of releasably- linked mPEG-(5- aminoacetamido-fluorescein) conjugates in rat.
[0012] Figure 2 shows the linear free energy relationship of release rate with sulfonyl substituents. A plot of log(release rate) versus substituent sigma (calculated as ApKa for the substituted phenol) is linear, allowing prediction of release rates for any substituted
phenylsulfone linker where sigma can be estimated.
Modes of Carrying Out the Invention
[0013] The compounds of formula (1)
O H FL' O
^ S-C - C -O-C™ (l)
O FT R.: - are designed as conjugate linkers for release of drugs coupled through the linker to
macromolecules, solid supports, dendrimers or other carriers that may be useful in stabilizing or targeting the drugs, extending their half-lives, and the like. The release of the drug, which will be coupled to the linker as shown in formula (2), is through the non-enzymatically mediated process of beta-elimination as shown.
Figure imgf000005_0001
[0014] The rate of release of the drug is controlled by the acidity of the proton shown as H in formulas (1) and (2) which is in turn affected by the nature of R 1 and R 2. The presence of the sulfone along with hydrogen as R on the beta-carbon has been found particularly advantageous in controlling rates of release.
[0015] The macromolecule to which the drug is ultimately attached may be coupled to R1,
R 2 or R 4 or may be part of these substituents. Thus, one of R 1 , R2 and R 3 must contain the substituent R10-Y, Ru-Y or R12-Y or R1, R2 and/or R3 must contain PEG of 200, 300, 400, etc. -1000 CH2CH2) units as noted above. Y is a substituent that is capable of reacting with a macromolecule or a macromolecular conjugate or is able to be deprotected to have such a function. Thus, the macromolecule can ultimately be coupled either to the alpha or beta carbon or to the sulfur of the sulfone. Thus, at least one of R10-Y, Ru-Y or R12-Y must be present in formula (1) or R 1 , R2 and/or R 3 must itself be a macromolecule density linked to the sulfone or to the alpha or beta carbon.
[0016] When ranges are given, e.g., 1-1000 all integers in the range are to be considered disclosed as if explicitly set forth.
[0017] "Alkyl", "alkenyl", and "alkynyl" include linear, branched or cyclic hydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4 carbons wherein alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon— carbon double bonds and alkynyl includes one or more carbon— carbon triple bonds. Unless otherwise specified these contain 1-6C.
[0018] "Aryl" includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. "Heteroaryl" includes 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.
[0019] By the term "substituted" is meant an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group comprising one or more substituent groups in place of one or more hydrogen atoms. Substituent groups may generally be selected from halogen including F, CI, Br, and I; lower alkyl including linear, branched, and cyclic; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy including linear, branched, and cyclic; SH; lower alkylthio including linear, branched, and cyclic; amino, alkylamino, dialkylamino, silyl including alkylsilyl, alkoxysilyl, and arylsilyl; nitro; cyano; carbonyl; carboxylic acid, carboxylic ester, carboxylic amide; aminocarbonyl; aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketone; sulfone; sulfonamide; aryl including phenyl, naphthyl, and anthracenyl;
heteroaryl including 5-member heteroaryls including as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole, and tetrazole, 6- member heteroaryls including pyridine, pyrimidine, pyrazine, and fused heteroaryls including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole, and benzisothiazole.
[0020] The properties of R 1 and R 2 may be modulated by the optional addition of electron- donating or electron- withdrawing substituents. By the term "electron-donating group" is meant a substituent resulting in a decrease in the acidity of the R 1 S02R 2 CH group; electron-donating groups are typically associated with negative Hammett σ or Taft σ* constants and are well- known in the art of physical organic chemistry. Hammett σ constants are associated with constituents on aromatic systems and Taft σ* constants are associated with nonaromatic substituents. Examples of suitable electron-donating substituents include but are not limited to lower alkyl, lower alkoxy, lower alkylthio, amino, alkylamino, dialkylamino, and silyl.
Similarly, by "electron- withdrawing group" is meant a substituent resulting in an increase in the acidity of the relevant H; electron- withdrawing groups are typically associated with positive Hammett σ or Taft σ* constants and are well-known in the art of physical organic chemistry. Examples of suitable electron-withdrawing substituents include but are not limited to halogen, difluoromethyl, trifluoromethyl, nitro, cyano, C(=0)-R Y , wherein R Y is H, lower alkyl, lower alkoxy, or amino, or S(0)mR X , wherein m = 1-2 and R X is lower alkyl, aryl, or heteroaryl. The art, the electronic influence of a substituent group may depend upon the position of the substituent. For example, an alkoxy substituent on the ortho- or para-position of an aryl ring is electron-donating, and is characterized by a negative Hammett σ constant, while an alkoxy substituent on the meta-position of an aryl ring is electron- withdrawing and is characterized by a positive Hammett σ constant. A table of Hammett σ and Taft σ* constants values is given below.
Substituent a(meta) a(para) σ*
H 0.00 0.00 0.49
CH3 -0.07 -0.17 0
C2H5 -0.07 -0.15 -0.10
n-C3H7 -0.07 -0.13 -0.115
i-C3H7 -0.07 -0.15 -0.19 Substituent a(meta) a(para) σ* n-C4H9 -0.08 -0.16 -0.13 t-C4l¾ -0.10 -0.20 -0.30
H2C=CH 0.05 -0.02 0.56
CeH5 0.06 -0.01 0.60
CH2C1 0.11 0.12 1.05
CF3 0.43 0.54 2.61
CN 0.56 0.66 3.30
CHO 0.35 0.42
Figure imgf000007_0001
C02H 0.37 0.45 2.08
Si(CH3)3 -0.04 -0.07 -0.81
CH2Si(CH3)4 -0.16 -0.22 -0.25
F 0.34 0.06 3.21
CI 0.37 0.23 2.96
Br 0.39 0.23 2.84
I 0.35 0.18 2.46
OH 0.12 -0.37 1.34
OCH3 0.12 -0.27 1.81
OCH2CH3 0.10 -0.24 1.68
OCF3 0.40 0.35
SH 0.25 0.15 1.68
SCH3 0.15 0.00 1.56
N02 0.71 0.78 4.0
NO 0.62 0.91
NH2 -0.16 -0.66 0.62
NHCHO 0.19 0.00
NHCOCH3 0.07 -0.15 1.40
N(CH3)2 -0.15 -0.83 0.32
N(CH3)+ 0.88 0.82 4.55
CC13 0.47 2.65
C02CH3 0.32 0.39 2.00
CH2N02 1.40
CH2CF3 0.92
CH2OCH3 0.52
CH2Ph 0.46 0.26
Ph 0.06 -0.01 0.60 [0021] Halogen" includes fluoro, chloro, bromo and iodo.
[0022] Maleimido" is a group of the formula
Figure imgf000008_0001
[0023] The terms "protein" and "peptide" are used 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.
[0024] 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.
[0025] 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.
[0026] "Macromolecule" means a molecule 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. Typical macromolecules include polymers, especially polyethylene glycol polymers as these are well established as pharmaceutical carriers. Other carriers are not necessarily macromolecules, but include antibodies and albumin.
[0027] In some embodiments, R2 is H, optionally substituted CrC6 alkyl, or
CH2(OCH2CH2)nO-alkyl, wherein n = 1-1000, or Rn-Y, wherein R11 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1-1000.
[0028] In some embodiments, R2 is H.
[0029] In some embodiments, the invention provides compounds of formula (la)
Figure imgf000008_0002
wherein
R10 is (CH2)k, arylene, heteroarylene, (CH2)kNR5, arylene-NR5, heteroarylene-NR5, (CH2)kO, arylene-O, heteroarylene-O, or (CH2CH20)nCH2CH2 where R5 is H, optionally substituted C -C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
each k is independently 1-8;
n = 1-1000;
R 2", R 3J, and R 4" are each independently H, optionally substituted CrC6 alkyl, optionally substituted aryl, optionally substituted heteroaryl;
X and Y are as defined above.
[0030] In another embodiment, the invention provides compounds of formula (lb)
0- H FT O
O R R* wherein
R1 is optionally substituted C]_-C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
R11 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1-1000;
R3, and R4 are each independently H, optionally substituted C -C alkyl, optionally substituted aryl, optionally substituted heteroaryl; and
X and Y are as defined above.
[0031] In another embodiment, the invention provides compounds of formula (lc)
O H FT Q PJ- S-C— C -D - C - X ( L C)
6 Fi~ ; --
V
wherein
R1 is optionally substituted C]_-C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
R 12 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1-1000;
R 2 , and R 4 are each independently H, optionally substituted C -C alkyl, optionally substituted aryl, optionally substituted heteroaryl; and
X and Y are as defined above. [0032] In several specific embodiments of formula (lc), listed below, any PEG included in
R 12 is of varying lengths. Thus, in these embodiments, the invention provides compounds of formula (lc) wherein R12 is (CH2)k or CH2(OCH2CH2)n, Y is N3, alkynyl or cycloalkynyl, optionally protected NH2, optionally protected C02H, optionally protected SH, or maleimido, wherein k is 1-8 and n = 1-1000, or n = 1-100, or n = 1-25, or n = 1-10 or n = 1-6.
[0033] In another embodiment, the invention provides compounds of formula (lc) wherein R12 is (CH2)k, wherein k = 1-8; and Y is N3.
[0034] In another embodiment, the invention provides compounds of formula (lc) wherein
R 12 is arylene or heteroarylene; Y is N3, alkynyl or cycloalkynyl, optionally protected NH2, optionally protected C02H, optionally protected SH, or maleimido.
[0035] In another embodiment, the invention provides compounds of formula (lc) wherein R12 is CH2(OCH2CH2)n, wherein n = 1-1000; and Y is N3.
[0036] Other embodiments include compounds of formula (lc) wherein R1 is phenyl, 4-(trifluoromethyl)phenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl,
2,4,6-trimethylphenyl, methyl, or 4-morpholino; R2 is H; R3 is (CH2)6N3, or (CH2)6NH-CO-0- C(CH3)3; R4 is H; and either X is CI, O-succinimidyl, O-sulfosuccinimidyl, or X is NB-CH2W, wherein B is optionally substituted aryl and W is CI, alkoxy, or S(R 8 )2, wherein each R 8 is independently alkyl or aryl.
Preparation Methods
[0037] Methods for preparation of compounds of formula (1) are also part of the invention. Generally, compounds of formula (1) may be prepared as illustrated in Scheme 1.
Scheme 1
Figure imgf000010_0001
: a rts a man H O Hx O H * O
R · S - C - C - O '- C '- CI R - S - G - C - O - C - K i s
O Fr R-; pyridine R ' Rf
[0038] Precursor R 1 S02CH2R 2 is deprotonated with a strong base to produce the carbanion. Typically, suitable bases include ones such as butyllithium, NaH, lithium diisopropylamide (LDA), lithium bis(trimethylsilyl)amide (LiHMDS), sodium bis(trimethylsilyl)amide (NaHMDS), or potassium tert-butoxide (KO£Bu), in an inert solvent such as tetrahydrofuran (THF) or a mixture of THF and hexanes. Carbanion generation is typically performed at temperatures below 0°C, often at -78°C or at temperatures in between. One of skill in the art can readily determine the appropriate combination of base, solvent, and temperature for carbanion formation. Once formed, the carbanion is reacted with carbonyl compound R3-CO-R4, again typically at temperatures below 0°C and most typically at -78°C, to produce an isolable alcohol product, R1S02CH(R2)CR3(R4)OH. The alcohol is then activated to the chloroformate, for example by treatment with phosgene or a phosgene equivalent, for example triphosgene, in the presence of a weak base such as pyridine. The chloroformate is optionally further transformed, for example by reaction with N-hydroxy-succinimide, N-hydroxy-sulfosuccinimide, or an activated phenol such as 4-nitrophenol, pentafluorophenol, or pentachlorophenol, and a weak base such as pyridine. Alternatively, the alcohol can be more directly activated to the succinimidyl carbonate through treatment with Ν,Ν'-disuccinimidyl carbonate and
4-(dimethylamino)pyridine, or to an activated phenyl carbonate through reaction with the appropriate activated phenyl chloroformate, for example 4-nitrophenyl chloroformate, and pyridine.
[0039] When X = NB-CH2-W, the compounds of the invention may be prepared by first reacting a compound of the invention wherein X = CI with BNH2 for form a carbamate. This carbamate is then reacted with paraformaldehyde and chlorotrimethylsilane to produce a compound of the invention wherein X = NB-CH2-W and W = CI. This compound may be further transformed, for example through reaction with an alcohol to yield W = alkoxy or with a thioether S(R )2 to produce the sulfonium salt.
[0040] The starting sulfones, sulfonates, and sulfonamides R 1 S02CH2R 2 may be prepared using methods well-known in the art.
[0041] For precursors of compounds of formula (la) R10-Y-SO2-CH2-R2, when R10 is (CH2)n, arylene, heteroarylene, or (CH2CH20)nCH2CH2, the sulfone may be produced through reaction of precursor R -CH2-L, wherein L is halide or tosylate, with sodium sulfinate
Y-(CH2)nS02Na , Y-arylene-S02Na, Y-heteroarylene-S02Na, or Y-(CH2CH20)nCH2CH2- S02Na. This reaction may be advantageously performed under microwave irradiation as described by Ju, et ah, J. Org. Chem. (2006) 71:6697-6700 (incorporated herein by reference). Alternatively, when R10 is (CH2)n or (CH2CH20)nCH2CH2, the sulfone may be produced through reaction of a sodium sulfinate of the formula R -CH2-S02Na with Y-(CH2)n-L or Y-(CH2CH20)nCH2CH2-L to produce the sulfone. In another method, when R10 is (CH2)nNR5, arylene-NR5, heteroarylene-NR5, (CH2)nO, arylene-O, or heteroarylene-O, the sulfonates may be produced by reaction of a sulfonyl chloride R -CH2-SO2CI with an alcohol Y-(CH2)n-OH, Y-arylene-OH, or Y-heteroarylene-OH in the presence of a mild base, and the sulfonamides can be similarly prepared by reaction of a sulfonyl chloride R -CH2-S02C1 with an amine
Y-(CH2)nNHR5, Y-arylene-NHR5, or Y-heteroarylene-NHR5, a gain in the presence of a mild base. Other methods of preparation of sulfones, sulfonates, and sulfonamides are well-known in the art.
[0042] In other methods, R1-S02-CH3 is first converted to the carbanion using a strong base using conditions as described above. This carbanion is then alkylated using Y-Ru-L to produce R^SOa-CHa-R11^ as a precursor for compounds of formula (lb).
[0043] 1 2
Precursors of compounds of formula (lc), R -S02-CH2-R , may be prepared
10 2
according to the methods described above for the precursors Y-R -SO2-CH2-R , using the appropriate substitutions of R1 in place of R10-Y.
Applications
[0044] As is clear from the discussion above, the sulfone linkers of the invention are employed for controlled release of drugs over time. The linkers are therefore intermediates in the formation of the prodrug wherein the drug is linked to a carrier from which it will be released and thus the invention is also directed to the end product prodrug wherein the linkers have been coupled both to a macromolecular carrier and to a pharmaceutical as well as to the further intermediates where only the macromolecular carrier or the pharmaceutical have been coupled.
[0045] Generally, the pharmaceutical is coupled to the sulfone carrier by displacement of the substituent "X" through reaction with an amino group contained in the drug. Most small molecule drugs contain amino groups, or can be easily modified to contain them. Peptides, of course, and oligonucleotides contain amino groups. If multiple amino groups are present, protection of all but one of such groups may be needed to effect reaction with the linker. Thus, the invention includes the product of reacting the sulfone linkers of the invention with pharmaceuticals through displacement of X.
[0046] Unless the macromolecule is already included in an embodiment of R1, R2 or R3, coupling to a macromolecule will be necessary. This is generally through interaction with Y either through displacement or direct coupling, as is the case, for example, for alkynyl embodiments. The macromolecule can be coupled to the linker either before or after the drug has been coupled. Thus, the invention also includes products of coupling the linkers with macromolecular carriers. Typical macromolecular carriers include solid supports, dendrimers, and polymers in general. Polymers comprising polyethylene glycol are particularly convenient as polyethylene glycol has been used as a carrier for pharmaceuticals over many decades and is known to be safe.
[0047] The invention also includes the finished prodrug— i.e., the compounds wherein the linkers of the invention are coupled both to macro molecular carriers and to the drugs.
[0048] The drug delivery compositions of the invention which result from coupling both to macromolecules and drugs are useful in a variety of indications depending on the nature of the drug. Such applications are understood by practitioners in the art, and as will be described below, the desired rate of release can also be calibrated according to known techniques.
[0049] The compounds of formula (1) are thus useful as linkers in the reversible conjugation of molecules, for example of drugs to macromolecular carriers as described in PCT publications WO2009/158668, WO2011/140393, WO2011/140392 and WO2011/140376, incorporated herein by reference. These linkers are especially useful as they degrade under physiological conditions of pH and temperature by an enzyme-independent beta-elimination mechanism to release the conjugated drug from its carrier as described above.
[0050] An illustration of this is shown in Figures 1A and IB using releasably linked mPEG- (5-aminoacetoamidofluorescein), i.e., mPEG-5-AAF conjugates in the rat. Conjugates of 5-AAF and 4-branched mPEG (40 kDa) connected via sulfone linkers of the invention were injected into rats, and the serum concentrations of the conjugates were measured over time by fluorescence HPLC. Panel A shows concentration versus time profiles for the conjugates. Panel B replots the data of Panel A where the data for the non-releasable conjugate have been subtracted, revealing the rates of release from the releasable conjugates.
[0051] The enzyme-independence of drug release predicts that the release rates will be predictable between in vitro measurements and in vivo environments, and also predicts that there will be less inter-patient variability in drug exposure that might result from variation in enzyme levels.
[0052] To be most useful, the linkers must release drug at a rate determined by a combination of factors, including the concentration of free drug needed for efficacy, the time period required between drug administrations, and the clearance rates of the conjugate and drug from the body. Scheme 2
c sfn.^cs
[0053] For a one-compartment model (Scheme 2) it can be shown, where C-D is the conjugate of carrier (C) to drug (D), that the concentration of free drug D at time t is determined by the initial concentration of conjugate [CD]o and the rates of drug release from the conjugate (k , drug elimination (k2) from the body and conjugate and carrier elimination (k3), according to equation (a):
Figure imgf000014_0001
[D]T e-(kj + k3)t _ e-(k2)t (a) k2 - (k1 + k3)
[0054] In the absence of metabolism, C-D and C are expected both to be predominantly cleared by renal filtration and thus share the same elimination rate as they are of approximately the same size.
[0055] One goal in administering a conjugate or prodrug form of a drug is to maintain the free drug concentration at or above a threshold concentration [D]th during the time between conjugate administrations, i.e. , at desired t, as a drug of a fixed potency requires a certain concentration in the system for efficacy. From equation (a) it can be seen that for a given set of rate constants k1; k2, and k3, the concentration of drug at time t [D]T can be raised or lowered by adjustment of the conjugate dose administered, i. e. , [CD]0. Equation (a) can be rearranged to predict the initial concentration of conjugate required to achieve this goal as shown in equation (b):
k2 - (k1 + k3) · [D]TH
[CD]C (b)
ki e-(k, + k3)t _ e-(k2)t
[0056] As the elimination rate constants k2 and k3 are fixed by the chemical properties of the macromolecular carrier and drug that make up the conjugate, it can be appreciated that there is an optimal value for the release rate ki that will allow for the minimum initial concentrations of conjugate [CD]o required to be administered so as to provide the threshold drug concentration [D]th at any time t. If the linker releases the drug too quickly, the level of drug will fall below the threshold concentration before time t is reached due to conjugate depletion resulting from drug release (k , while if the linker releases the drug too slowly, the level of drug will fail to reach the threshold concentration due to conjugate depletion resulting from conjugate clearance (k3).
[0057] Based on the above equations, it is found that the optimal value of ki that maximizes [D]t while minimizing [CD]0 can be approximated according equation (c)
Figure imgf000015_0001
[0058] Thus, the optimal release rate depends primarily upon the drug elimination rate k2 and the time between conjugate administrations T, although in practice T is the primary determinant. Many drugs that benefit from conjugation do so because they have an
unacceptably rapid elimination from the system, that is, they have elimination half-lives that range from about 0.5 to about 12 hours and so in their native form must be administered once to several times daily for maximal effectiveness. For conjugates of such drugs to be optimally administered (i.e., the lowest possible dose of conjugate to provide the highest free drug concentration), different linkers having different release rates are required (Table 1).
Table 1 Approximate optimal release rates (expressed as half-lives) for various frequencies of drug administration
Administration Optimal Release
Frequency t1/2
Daily 15 h
Every 2d 30 h
Weekly 115 h
Every 2 weeks 230 h
Monthly 500 h
[0059] Some uncertainty is introduced by small variations in rate due to the nature of the drug being released and potential species differences, such that linkers not having precisely one of the release rates in Table 1 are still expected to be useful for supporting these administration regimes. We have found for example that the release rate for drugs in the rodent is
approximately three-fold faster than those observed in vitro at pH 7.4, 37°C. Thus, linkers with in vitro release t1/2 values of between 10 and 1000 hours are expected to optimally support controlled drug release from macromolecular conjugates.
[0060] Thus while the published applications cited above disclose a wide array of potential linker structures suitable for use in conjugation, it has been unexpectedly found that linkers wherein one of the modulating groups is R SC^ provide particularly useful drug release rates under physiological conditions so as to support optimal drug levels while reducing the amount of conjugate that must be administered. Results of measuring the release rates from such linkers are provided in the working examples below.
[0061] It has also been found that the electronic properties of the
Figure imgf000016_0001
modulating group are readily adjusted so as to provide a predictable range of release rates. As shown in Figure 2, the release rates of a series of linkers of formula (lc) wherein R1 is phenyl or substituted phenyl follow a linear free energy relationship where the release rate is a function of the electronic nature of the substituents on the phenyl ring, i.e., the sigma values of these substituents. This relationship holds where R is either alkyl or aryl, and holds for both in vitro and in vivo situations. Thus, the release rates of new linkers in this series may be predicted based on this relationship and the sigma value of any substituent on the phenylsulfone.
[0062] All references cited above are incorporated herein by reference.
[0063] The following examples illustrate but do not limit the invention.
Example 1
Preparation of 6-azidohexanal
Figure imgf000016_0002
[0064] (1) 6-azido-l-hexanol: a mixture of 6-chloro-l-hexanol (25 g, 183 mmol) and sodium azide (32.5 g, 500 mmol) in 200 mL of water was heated at reflux for 20 h, then cooled to ambient temperature and extracted 3x with ethyl acetate. The combined extracts were washed with brine, dried over MgS04, filtered, and concentrated to yield the product as a pale yellow oil (28.3 g).
[0065] (2) 6-azidohexanal: Solid trichloroisocyanuric acid (4.3 g) was added in small portions to a vigorously stirred mixture of 6-azido-l-hexanol (7.15 g), TEMPO (50 mg), and sodium bicarbonate (5.0 g) in dichloromethane (100 mL) and water (10 mL). The mixture was stirred for an additional 30 minutes after addition, then filtered through a pad of celite. The organic phase was separated and washed successively with sat. aq. NaHC03 and brine, then dried over MgS04, filtered, and concentrated to provide the product (5.8 g), which was used without further purification. Example 2
Preparation of ω-azido-PEG-aldehvdes
TCI A
Figure imgf000017_0001
[0066] Solid trichloroisocyanuric acid (60 mg) was added to a vigorously stirred mixture of 0-(2-azidoethyl)heptaethylene glycol (n = 7; 250 mg), 1 mg of TEMPO, 100 mg of NaHC03, 2 mL of CH2CI2, and 0.2 mL of water. The mixture turned orange, and after approximately 30 minutes a white suspension was formed. TLC analysis (1: 1 acetone/hexane) indicated formation of a product that stained with phosphomolybdic acid. The mixture was diluted with 10 mL of CH2CI2, dried by stirring with MgS04, filtered, and evaporated to yield the crude product. This was dissolved in CH2CI2 and loaded onto a 4-gm column of silica gel equilibrated in hexane, which was eluted sequentially with 25 mL each of hexane, 75:25 hexane/acetone, 50:50 hexane/acetone, and 25:75 hexane/acetone. Product-containing fractions were combined and evaporated to provide the purified product.
Example 3
Preparation of azidoalcohols
¾:L: ¾!CH,kCHD OH
THF-'hsx&ne
0* 5.
6 R
[0067] A 1.6 M solution of n-butyllithium (3.1 mL, 5.0 mmol) in hexane was added dropwise to a stirred solution of R-S02CH3 (5.0 mmol) in anhydrous tetrahydrofuran (THF) (15 mL) cooled to -78°C. After addition, the cooling bath was removed and the mixture was allowed to warm slowly to 0°C over approximately 30 min. The mixture was then cooled back to -78°C, and 6-azidohexanal (5.5 mmol) was added. After stirring for 15 minutes, the cooling bath was removed and the mixture was allowed to warm. At the point where the mixture became clear, 5 mL of saturated aq. NH4C1 was added and the mixture was allowed to continue warming to ambient temperature. The mixture was diluted with ethyl acetate and washed successively with water and brine, and then dried over MgS04, filtered, and evaporated to provide the crude product as an oil. Chromatography on silica gel using a gradient of ethyl acetate in hexane provided the purified products.
[0068] Compounds prepared according to this method include: l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptanol: from
4-(trifluoromethyl)phenyl methyl sulfone (1.73 g, 94%); 1H-NMR (400 MHz, CDC13): δ 8.08 (2H, d, J = 8.4 Hz), 7.87 (2H, d, J = 8.4 Hz), 4.21 (IH, m), 3.25 (2H, t, J = 6.8 Hz), 3.28 (IH, dd, J = 8.8, 14.4 Hz), 3.20 (IH, dd, J = 2.0, 14.4 Hz), 3.12 (IH, d, J = 2.8 Hz), 1.58 (2H, m), 1.5-1.3 (6H, m);
l-(4-chlorophenylsulfonyl)-7-azido-2-heptanol; from 4-chlorophenyl methyl sulfone; colorless oil (1.49 g, 90% yield) 1H-NMR (400 MHz, d6-DMSO): δ 7.90 (2H, d, J=8.8 Hz), 7.70 (2H, d, J=8.8 Hz), 4.83 (IH, d, J=6 Hz), 3.86 (IH, m), 3.39 (2H, m), 3.29 (2H, t, J=6.8 Hz), 1.2-1.5 (8H, m);
l-(phenylsulfonyl)-7-azido-2-heptanol; from phenyl methyl sulfone; pale yellow oil (1.25 g, 85%). 1H-NMR (400 MHz, d6-DMSO): δ 7.89 (2H,m), 7.72 (lH,m), 7.63 (2H,m), 4.84 (IH, d J=6 Hz), 3.86 (IH, m), 3.33 (2H, m), 3.28 (2H, t, J=6.8 Hz), 1.47 (2H, m),
1.2-1.4 (6H, m);
l-(4-methylphenylsulfonyl)-7-azido-2-heptanol; from 4-(methylsulfonyl)toluene;
colorless oil (1.39 g, 85% yield): 1H-NMR (400 MHz, d6-DMSO): δ 7.76 (2H, d, J=6.4 Hz), 7.43 (2H, d, J=6.4 Hz), 4.82 (IH, d, J=6 Hz), 3.85 (IH, m), 3.31 (2H, m), 3.28 (2H, t, J=6.8 Hz), 2.41 (3H, s), 1.4-1.5 (2H, m), 1.2-1.4 (6H, m);
l-(4-methoxyphenylsulfonyl)-7-azido-2-heptanol; from 4-methoxyphenyl methyl sulfone (1.53 g, 94%; 1H-NMR (400 MHz, CDC13): δ 7.85 (2H, d, J=8.8 Hz), 7.04 (2H, d, J=8.8 Hz), 4.13 (IH, m), 3.90 (3H, s), 3.24 (2H, t, J = 6.8 Hz), 3.20 (IH, dd, J = 8.8, 14.4 Hz), 3.14 (IH, dd, J = 2.4, 14.4 Hz), 2.47 (3H, s), 1.57 (2H, m), 1.5-1.3 (6H, m);
l-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptanol; from (2,4,6-trimethyl)phenyl methyl sulfone (1.30 g from 4.0 mmol reaction; 96%); 1H-NMR (400 MHz, CDC13): δ 6.99 (2H, s), 4.30 (IH, m), 3.49 (IH, d, J = 2 Hz), 3.25 (2H, t, J = 6.8 Hz), 3.18 (IH, d, J = 1 Hz), 3.17 (IH, s), 2.66 (6H, s), 2.31 (3H, s), 1.59 (2H, m), 1.5-1.3 (6H, m);
l-(morpholinosulfonyl)-7-azido-2-heptanol; from 1-morpholino methylsulfonamide (1.36 g from 10 mmol reaction, 89%); 1H-NMR (400 MHz, d6-DMSO): δ 4.99 (IH, d,
J = 6.4Hz), 3.88 (IH, m), 3.62 (4H, t, J = 4.8 Hz), 3.32 (2H, t, J = 6.8 Hz), 3.20-3.15 (6H, overlap), 1.53 (2H, m), 1.46-1.25 (6H,m);
l-(methylsulfonyl)-7-azido-2-heptanol; from dimethylsulfone; colorless oil
(880 mg, 75%);
l-(N,N-diethylaminosulfonyl)-7-azido-2-heptanol;
l-(N-benzyl-N-ethylaminosulfonyl)-7-azido-2-heptanol; l-(N,N-benzylaminosulfonyl)-7-azido-2-heptanol;
l-(N,N-allylaminosulfonyl)-7-azido-2-heptanol;
l-(N-(3-methoxypropyl)-N-methylaminosulfonyl)-7-azido-2-heptanol;
l-(N-(2-methoxyethyl)-N-methylaminosulfonyl)-7-azido-2-heptanol;
1 - (N-furfuryl-N-methylamino sulf onyl) -7 - azido-2-heptanol ; and
l-cyano-7-azido-2-heptanol.
Example 4
Preparation of azidoalcohols
Figure imgf000019_0001
[0069] A 1.6 M solution of n-butyllithium (3.1 mL, 5.0 mmol) in hexane is added drop wise to a stirred solution of R-SO2CH3 (5.0 mmol) in anhydrous tetrahydrofuran (THF) (15 mL) cooled to -78°C. After addition, the cooling bath is removed and the mixture is allowed to warm slowly to 0°C over approximately 30 min. The mixture is then cooled back to -78°C, and CO-azido-heptaethylene glycol aldehyde (n=7, 1.2 g) is added. After stirring for 15 minutes, the cooling bath is removed and the mixture is allowed to warm. At the point where the mixture becomes clear, 5 mL of saturated aq. NH4C1 is added and the mixture is allowed to continue warming to ambient temperature. The mixture is diluted with ethyl acetate and washed successively with water and brine, and then dried over MgS04, filtered, and evaporated to provide the crude product. Chromatography on silica gel provides the purified products.
[0070] The results are shown in Figures 1A and IB.
Example 5
Preparation of azido-linker chloroformates
Figure imgf000019_0002
[0071] Pyridine (160 μί) was added dropwise to a stirred solution of the azidoalcohol of Example 3 (1.0 mmol) and triphosgene (500 mg) in 15 mL of anhydrous THF. The resulting suspension was stirred for 10 minutes, then filtered and concentrated to provide the crude chloroformate as an oil.
[0072] Compounds prepared according to this method include:
l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptyl chloroformate;
l-(4-chlorophenylsulfonyl)-7-azido-2-heptyl chloroformate;
l-(phenylsulfonyl)-7-azido-2-heptyl chloroformate;
l-(4-methylphenylsulfonyl)-7-azido-2-heptyl chloroformate;
l-(4-methoxyphenylsulfonyl)-7-azido-2-heptyl chloroformate;
l-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptyl chloroformate;
l-(4-morpholinosulfonyl)-7-azido-2-heptyl chloroformate;
1 - (methanesulf onyl) -7 - azido-2-heptyl chloroformate ;
l-(N,N-diethylaminosulfonyl)-7-azido-2-heptyl chloroformate;
l-(N-benzyl-N-ethylaminosulfonyl)-7-azido-2-heptyl chloroformate;
l-(N,N-benzylaminosulfonyl)-7-azido-2-heptyl chloroformate;
l-(N,N-allylaminosulfonyl)-7-azido-2-heptyl chloroformate;
l-(N-(2-methoxyethyl)-N-methylaminosulfonyl)-7-azido-2-heptyl chloroformate;
1 - (N-furfuryl-N-methylamino sulf onyl) -7 - azido-2-heptyl chloroformate ;
l-cyano-7-azido-2-heptyl chloroformate.
[0073] Other chloroformates may be prepared according to this general method.
Example 6
Preparation of azido-linker chloroformates
Figure imgf000020_0001
[0074] Pyridine (160 μί) is added dropwise to a stirred solution of the azidoalcohol of Example 4 (1.0 mmol) and triphosgene (500 mg) in 15 mL of anhydrous THF. The resulting suspension is stirred for 10 minutes, then filtered and concentrated to provide the crude chloroformate. Example 7
Preparation of azido-linker succinimidyl carbonates
Figure imgf000021_0001
[0075] Pyridine (300 μί) was added dropwise to a stirred solution of the chloroformate of Example 5 (1.0 mmol) and N-hydroxysuccinimide (350 mg) in 15 mL of anhydrous THF. The resulting suspension was stirred for 10 minutes, then filtered and concentrated to provide the crude succinimidyl carbonate. Purification by silica gel chromatography provided the purified product as an oil which spontaneously crystallized. Recrystallization could be effected using ethyl acetate/hexane.
[0076] Compounds prepared according to this method include:
0-[l-(4-(trifluoromethyl)phenylsulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate: crystals from 40:60 ethyl acetate/hexane (280 mg, 55%). 1H-NMR (400 MHz, d6-DMSO): δ 8.12 (2H, m), 8.04 (2H, m), 5.18 (1H, m), 4.15 (1H, dd, J=9.2,15.2), 3.96 (1H, dd,
J=2.4,15.2), 3.29 (2H, t, J=6.8), 2.80 (4H,s), 1.68 (2H,m), 1.47 (2H, m), 1.27 (4H, m);
0-[l-(4-chlorophenylsulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate: crystals from 40:60 ethyl acetate/hexane (392 mg, 83%). 1H-NMR (400 MHz, d6-DMSO): δ 7.85 (2H,m), 7.72 (2H,m), 5.14 (lH,m), 4.04 (lH,dd, J=9.6,15.6), 3.87 (lH,dd, J=2.4,15.6), 3.29 (2H,t,J=6.8), 2.81 (4H,s), 1.68 (2H,m), 1.47 (2H, m), 1.27 (4H, m);
0-[l-(phenylsulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate: crystals from 40:60 ethyl acetate/hexanes (391 mg, 89%). 1H-NMR (400 MHz, d6-DMSO): δ 7.91 (2H,m), 7.76 (lH,m), 7.66 (2H,m), 5.12 (lH,m), 3.96 (1H, dd, J=8.8,15.2), 3.83 (1H, dd, J=2.8,15.2), 3.29 (2H, t, J=6.8), 2.81 (4H, s), 1.69 (2H, m), 1.47 (2H, m), 1.27 (4H, m);
0-[l-(4-methylphenylsulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate: crystals upon standing after chromatography (402 mg, 89%). 1H-NMR (400 MHz, d6-DMSO): δ 7.77 (2H,d,J=8.0); 7.45 (2H,d,J=8.0); 5.11 (lH,m), 3.90 (lH,dd,J=8.8,15.2), 3.79 (lH,dd,J=1.8,15.2), 3.28 (2H,t,J=6.8), 2.81 (4H,s), 2.41 (3H,s), 1.68 (2H,m), 1.47 (2H, m), 1.27 (4H, m);
0-[l-(4-methoxyphenylsulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate: crystals from 60:40 ethyl acetate/hexane (320 mg, 68%). 1H-NMR (400 MHz, d6-DMSO): δ 7.81 (2H,d,J=8.8); 7.15 (2H,d,J=8.8); 5.11 (lH,m), 3.87 (lH,dd,J=8.8,15.2), 3.86 (3H,s), 3.76 (lH,dd,J=2.8,15.2), 3.29 (2H,t,J=6.8), 2.80 (4H,s), 1.68 (2H,m), 1.47 (2H, m), 1.27 (4H, m); 0-[l-(2,4,6-trimethylphenylsulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate: colorless oil (458 mg, 95%). 1H-NMR (400 MHz, d6-DMSO): δ 7.09 (2H,s), 5.20 (lH,m), 3.82 (1H, dd, J = 8.4,15.2 Hz), 3.67 (1H, dd, J = 3.2, 15.2 Hz), 3.30 (2H, t, J = 6.8 Hz), 2.79 (4H, s), 2.58 (6H, s), 2.28 (3H, s), 1.75 (2H, m), 1.49 (2H, m), 1.30 (4H, m);
0-[l-(morpholinosulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate: crystals upon standing after chromatography (430 mg, 95%). (400 MHz, CDC13): δ 5.23 (1H, m), 3.77 (4H, dd, J = 4.0, 5,6 Hz), 3.39 (1H, dd, J = 6.4, 14.4 Hz), 3.31 (6H, overlap), 3.17 (1H, dd, J = 4.8, 14.4 Hz), 2.85 (4H, s), 1.88 (2H, m), 1.61 (2H, m), 1.45 (4H, m);
0-[l-methylsulfonyl-7-azido-2-heptyl]-0'-succinimidyl carbonate: crystals upon standing after chromatography (360 mg, 95%). (400 MHz, CDC13): δ 5.32 (1H, m),
3.50 (1H, dd, J = 7.2, 14.8 Hz), 3.29 (2H, t, J = 6.8 Hz), 3.21 (1H, ddd, J = 0.8, 4.0, 14.8 Hz), 3.02 (3H, s), 2.85 (4H, s), 1.90 (2H, m), 1.62 (2H, m), 1.46 (4H, m);
0-[l-(N,N-diethylaminosulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate.
1H NMR (CDC13) δ 1.21 (6H, t, J = 7.0 Hz), 1.39 (4H, m), 1.60 (2H, m), 1.87 (2H, m),
2.84 (4H, s), 3.15 (1H, dd, J = 5.3 Hz, J = 14.6 Hz), 3.24 (7H, m), 5.21 (1H, m);
0-[l-(N-benzyl-N-ethylaminosulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate. (400 MHz, CDC13): δ 7.35 (5H, m), 5.279 (1H, m), 4.517 (1H, d, J = 15.2 Hz), 4.359 (1H, d, J = 15.2 Hz), 3.17-3.41 (6H, overlap), 2.856 (4H, s), 1.87 (2H, m), 1.62 (2H, m), 1.44 (4H, m), 1.111 (3H, t, J = 6.8 Hz);
0-[l-(N,N-benzylaminosulfonyl)-7-azido-2-heptyl]-0' -succinimidyl carbonate.
(400 MHz, CDC13): δ 7.32 (10H, m), 5.259 (1H, m), 4.442 (2H, d, J = 15.2 Hz), 4.359 (2H, d, J = 15.2 Hz), 3.279 (2H, t, J = 6.4 Hz), 3.196 (1H, dd, J = 6.8, 14.4 Hz), 3.0445 (1H, dd, J = 5.2, 14.4 Hz), 2.844 (4H, s), 1.80 (2H, m), 1.603 (2H, m), 1.40 (4H, m);
0-[l-(N-(3-methoxypropyl)-N-methylaminosulfonyl)-7-azido-2-heptyl]-0'- succinimidyl carbonate;
O- [ 1 - (N- (2-methoxyethyl)-N-methylaminosulfonyl)-7 -azido-2-heptyl] -O' - succinimidyl carbonate;
0-[l-(N-furfuryl-N-methylaminosulfonyl)-7-azido-2-heptyl]-0'-succinimidyl carbonate. (400 MHz, CDC13): δ 7.434 (1H, t, J = 1.6 Hz), 6.358 (2H, d, J = 1.6 Hz), 5.230 (1H, m), 4.483 (1H, d, J = 16 Hz), 4.336 (1H, d, J = 16 Hz), 3.384 (1H, dd, J = 9.6, 14.8 Hz), 3.286 (2H, t, J = 6.8 Hz), 3.158 (1H, dd, J = 5.2, 14.8 Hz), 2.894 (3H, s), 2.845 (4H, s), 1.87 (2H, m), 1.603 (2H, m), 1.45 (4H, m); and 0-[l-cyano-7-azido-2-heptyl]-0'-succinimidyl carbonate. (400 MHz, CDC13): d 4.99 (1H, m), 3.30 (2H, t, J = 6.8 Hz), 2.85 (4H, s), 2.85 (1H, dd, J = 4.8, 16.8 Hz), 2.78 (1H, dd, J = 5.2, 16.8 Hz), 1.8-2.0 (2H, m), 1.63 (2H, m), 1.4-1.5 (4H, m).
[0077] Other succinimidyl carbonates may be prepared according to this general method.
Example 8
Preparation of azido-linker succinimidyl carbonates
Q
Figure imgf000023_0001
[0078] Pyridine (300 μί) is added dropwise to a stirred solution of the chloroformate of Example 6 (1.0 mmol) and N-hydroxysuccinimide (350 mg) in 15 mL of anhydrous THF. The resulting suspension is stirred for 10 minutes, then filtered and concentrated to provide the crude succinimidyl carbonate. Purification by silica gel chromatography provides the purified product.
Example 9
Preparation of azido-linker sulfosuccinimidyl carbonates
Figure imgf000023_0002
[0079] A stirred suspension of sodium N-hydroxysuccinimide sulfonate (1 mmol) in N,N-dimethylformamide (10 mL) is treated with pyridine (3 mmol) and a chloroformate of Example 7. After the suspension clears, the mixture is diluted with ethyl acetate. Example 10
Preparation of amino-linker alcohols
Figure imgf000024_0001
[0080] A stirred solution of an azido-linker alcohol of Example 3 (R = phenyl; 1 mmol) in 1 mL of tetrahydrofuran (THF) was treated with a 1.0 M solution of trimethyl-phosphine in THF (1.2 mL) for 1 hour at ambient temperature. Water (0.1 mL) was added, and the mixture was allowed to stir for an additional 1 hour, then the mixture was evaporated to dryness using a rotary evaporator. The residue was dissolved in ethyl acetate, washed with water and brine, then was dried over MgS04, filtered, and evaporated to provide the product.
[0081] Other amino-linker alcohols may be prepared according to this general method.
Example 11
Preparation of 'BOC-amino-linker alcohols
Figure imgf000024_0002
[0082] A solution of the amino-linker alcohol of Example 10 (R=phenyl; 1.0 mmol) in 2 mL of THF was treated with di-tert-butyl dicarbonate (1.5 mmol) for 1 hour, and then evaporated to dryness. The residue was dissolved in ethyl acetate, washed with water and brine, then was dried over MgS04, filtered, and evaporated to provide the product. Chromatography on silica gel using a gradient of ethyl acetate in hexane provided the purified product.
[0083] Other 'BOC-amino-linker alcohols may be produced according to the same general method.
Example 12
Preparation of 4-(N,N-diethylcarboxamido)aniline
Figure imgf000024_0003
[0084] (1) Ν,Ν-diethyl 4-nitrobenzamide: Diethylamine (5.6 mL) was added to an ice-cold solution of 4-nitrobenzoyl chloride (5.0 g) in 100 mL of DCM. After 1 h, the mixture was washed successively with water, sat. aq. NaHC03, and brine, then dried over MgS04, filtered, and evaporated to provide a colorless liquid that crystallized on standing. Recrystallization from ethyl acetate/hexane provided the product as pale yellow crystals (4.6 g).
[0085] (2) 4-(N,N-diethylcarboxamido)aniline: A mixture of N,N-diethyl 4- nitrobenzamide (4.44 g) and 10% palladium on carbon (0.2 g) in 100 mL of methanol was treated with ammonium formate (4.0 g) for 2 h at ambient temperature. The mixture was filtered through Celite and concentrated. The residue was redissolved in DCM, washed successively with 0.5 M Na2C03, water, and brine, then dried over MgS04, filtered, and evaporated to provide a crystalline material. Recrystallization from ethyl acetate/hexane provided the product aniline.
[0086] Also prepared according to the same procedure was 4-(morpholinocarbonyl)aniline by replacing diethylamine with morpholine.
Example 13
Preparation of azidocarbamates
•% :· ···· .. ·· " ·■· .·
Figure imgf000025_0001
[0087] The crude chloroformate prepared from 2.5 mmol of azidoalcohol according to the procedure of Example 5 was dissolved in 20 mL of THF, and the aniline (2.5 mmol) and triethylamine (0.7 mL, 5.0 mmol) were added. After 1 h, the mixture was diluted with ethyl acetate, washed successively with 1 N HC1, water, sat. NaHC03, and brine, then dried over MgS04, filtered, and evaporated. The residue was chromatographed on silica gel using ethyl acetate/hexane to provide the product carbamate.
[0088] Compounds prepared according to this method include:
0-[l-(phenylsulfonyl)-7-azido-2-heptyl]- N-[4-(diethylcarboxamido)phenyl carbamate; O- [ 1 - (morpholinosulf onyl)-7-azido-2-heptyl] - N- [4- (diethylcarboxamido)phenyl carbamate;
0-[l-(methanesulfonyl)-7-azido-2-heptyl]- N-[4-(diethylcarboxamido)phenyl carbamate; 0-[l-(phenylsulfonyl)-7-azido-2-heptyl]- N-[4-(morpholinocarboxamido)phenyl carbamate; and
O- [ 1 - (phenylsulf onyl) -7 - azido-2-heptyl] - N- [4- (morpholino sulf onyl)phenyl carbamate .
Example 14
Preparation of N-chloromethyl carbamates
Figure imgf000026_0001
[0089] A mixture of the azidocarbamate of Example 13 (1.0 mmol), paraformaldehyde (45 mg), chlorotrimethylsilane (1 mL), and THF (1 mL) in a sealed 20 mL vial was heated in a 55°C bath for 17 h. After cooling to ambient temperature, the vial was opened and the mixture was concentrated on a rotary evaporator to a thick oil, which was taken up in ethyl acetate and reconcentrated. The residue was dissolved in 2: 1 ethyl acetate/hexane, filtered, and
concentrated to provide the N-chloromethyl carbamate, which was used without further purification.
[0090] Compounds prepared according to this method include:
0-[l-(phenylsulfonyl)-7-azido-2-heptyl]- N-[4-(diethylcarboxamido)phenyl]-N- chloromethyl carbamate;
0-[l-(morpholinosulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N- chloromethyl carbamate; and
0-[l-(methanesulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N- chloromethyl carbamate.
Example 15
Preparation of N-alkoxymethyl carbamates
Figure imgf000026_0002
[0091] The N-chloromethyl carbamate of Example 14 (0.4 mmol) was dissolved in 5 mL of dry methanol. After 1 h, the mixture is evaporated to dryness, and the residue was
chromatographed on silica gel (ethyl acetate/hexanes) to provide the product.
[0092] Compounds prepared according to this method include:
0-[l-(phenylsulfonyl)-7-azido-2-heptyl]- N-[4-(diethylcarboxamido)phenyl]-N- methoxymethyl carbamate;
0-[l-(morpholinosulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N- methoxymethyl carbamate; and
0-[l-(methanesulfonyl)-7-azido-2-heptyl]-N-[4-(diethylcarboxamido)phenyl]-N- methoxymethyl carbamate.
Example 16
7-(fert-butoxycarbonylamino)-2-(R1-sulfonyl)-l-heptanol
Figure imgf000027_0001
[0093] p-Toluenesulfonyl chloride (1 mmol) is added to a solution of 6-azido-l-hexanol (Example 1, 1 mmol) in pyridine (2 mL) cooled on ice. After 30 min, the mixture is allowed to warm to ambient temperature and treated with R^-SH (1 mmol) for an additional 1 hr. The mixture is diluted with ethyl acetate, washed sequentially with water, 1 N HC1, water, sat. aq. NaHCC"3, and brine, then dried over MgS04, filtered, and evaporated. The crude thioether is dissolved in ethyl acetate and treated excess peracetic acid to prepare the sulfone. After standard aqueous workup, the sulfone is purified by chromatography on silica gel. A mixture of the sulfone, ethyl formate, and 2 equivalents of sodium hydride in DMF is warmed to 50°C to provide an intermediate aldehyde, which is treated with sodium borohydride in methanol to produce the product alcohol. Example 17
Figure imgf000028_0001
[0094] A solution of an amino-thiol heterobifunctional PEG in THF is treated with excess di-iert-butyl dicarbonate until the reaction is complete, and the di-BOC product is isolated by chromatography. The thiocarbonate is cleaved by treatment with one equivalent of NaOMe in methanol, and 2-bromoethanol is added to form the hydroxyethyl thioether, which is oxidized with peracetic acid to form the product.
Example 18
HO-GH;:CHv-SH
..O H
'Π O '
Εϊ-,Ν, CH:.Ci, NisOH. H:.D
H->0-> o. o sens
"OH o' - ^"- ,. OH
iVf , i r f-
Figure imgf000028_0002
[0095] These compounds may be prepared by a method analogous to that described for methoxy-PEG-hydroxyethyl sulfone (Morpurgo, et al., Bioconjugate Chemistry (1996) 7:363-368, incorporated herein by reference). For example, a solution of 1 l-azido-3,6,9- trioxaundecan-l-ol (x = 3) (3 mmol) in toluene is dried by azeotropic distillation. After dissolution in CH2CI2, methanesulfonyl chloride is added followed by triethylamine to form the mesylate. A solution of the mesylate in water is treated with 2-mercaptoethanol and 2 N NaOH to form the hydroxyethyl sulfide. The sulfide is subsequently oxidized to the sulfone, for example using hydrogen peroxide in the presence of a tungstic acid catalyst or alternatively using peracetic acid. The hydroxyethyl sulfone is then activated as the succinimidyl carbonate according to the methods described in the examples above. Example 19
Figure imgf000029_0001
Figure imgf000029_0002
Example 20
Preparation of 4-branched mPEG-DBCO
[0096] A solution of 4-branched mPEG-amine (GLP400-PA, NOF Corporation) of mw = 40 kDa (1.0 g, 25 μιηοΐ), 6-aza-5,9-dioxo-9-(l,2-didehydrodibenzo[b,/]azocin-5(6H)- yl)nonanoic acid succinimidyl ester (DBCO-NHS, Click Chemistry Tools, Macon, GA) (25 mg, 50 μιηοΐ), and triethylamine (10 μί, 100 μιηοΐ) in 10 mL of THF was stirred for 24 h at ambient temperature, then added slowly with stirring to 50 mL of methyl tert-butyl ether (MTBE). The precipitated product was collected by vacuum filtration and washed 3x with MTBE, and dried under vacuum to provide the product. HPLC analysis indicated approximately 6 mol
DBCO-NHS, so the sample was redissolved in THF and precipitated a second time by adding to MTBE as above. The resulting product (0.9 g) was free of DBCO-NHS by HPLC analysis. The UV absorbance of a 2 mg/mL (-48 μΜ) solution in water indicated the presence of 45 μΜ DBCO (290 nm, e = 15,460 M' 1).
Example 21
Preparation of linked fluoresceins
[0097] A solution of a 25 mM DMSO solution of an NHS carbonate of Example 7
(2.5 umoles) was added to 115 uL of a 10 mg/mL solution of 5-(aminoacetamido)-fluorescein (Invitrogen) in DMSO (2.8 umoles). After 1 h at ambient temperature, the mixture was analyzed by reversed-phase HPLC, indicating complete consumption of azide-linker-HSE and formation of a single linked fluorescein product. The solution was used without purification. Example 22
Preparation of PEGylated linked AAF
[0098] A DMSO solution of the compound of Example 21 (2.5 μιηοΐ) was added to a solution of 4-branched mPEG-DBCO (50 mg, 1.25 μιηοΐ) in 0.5 mL of THF and the mixture was kept at ambient temperature for 24 h. The mixture was diluted with 1 mL of water, then dialyzed overnight against methanol using a SpectraPor® (Spectrum Laboratories) 2 membrane (mw cutoff 12-14 kDa). Evaporation followed by rinsing of the resulting residue with MTBE provided partially-purified conjugate, which was dissolved in 1 mL of 10 mM sodium acetate, pH 5, and chromatographed on a PD-10 sizing column (GE Health Care) using 10 mM sodium acetate, pH 5. The macromolecular fraction was collected, and the concentration was determined by the UV absorbance of the fluorescein.
Example 23
Linker release rates in vitro
[0099] Solutions of PEG-linker-ligands (Example 22) at about 37.5 uM in 0.1 M bicine buffer (pH 8.45 at 37°C) were incubated at 37°C. Aliquots (30 uL) were removed at intervals and quenched with 33 uL of 0.2 M HOAc, (pH 2.85). Reactions were followed for 2 to 3 half- o lives. Samples were analyzed by reversed phase HPLC (Phenomenex Jupiter 300 C4 300 A) using a linear gradient (20-100% ACN-0.1% TFA) at 1 mL/min flow rate. Rates were determined by monitoring the fractional areas of the peaks at OD260 corresponding to the initial PEG-fluorescein conjugate and the resulting fluorescein elimination product. pH-dependence experiments confirmed that release rates were first-order with respect to hydroxide
concentration, and the release rates at pH 7.4 (Table 2) were calculated accordingly.
Table 2 Rates of release from PEG lated sulfonyl linkers at pH 7.4, 37°C.
Figure imgf000030_0001
R X t M
4-(trifluoromethyl)phenyl AAF* 14
4-chlorophenyl AAF 45
phenyl AAF 79 4-methylphenyl AAF 151
4-(methoxyphenyl) AAF 176
2,4,6-trimethylphenyl AAF 315
methyl AAF 352
4-morpholino AAF 742
diethylamino AAF 10500
*AAF = 5-(aminoacetamido)fluorescein
[0100] The corresponding PEG conjugate prepared using 0-[l-cyano-7-azido-2-heptyl]-0'- succinimidyl carbonate (Example 7) released AAF with t1/2 = 2400 h.
Example 24
Pharmacokinetics of PEGylated linkers in vivo
[0101] PEGylated linked AAF compounds (Example 22) were prepared at 250 uM in 10 mM acetate pH 5.0. Samples were administered by i.v. injection to male cd-1 mice at 2 uL/g body weight and to cannulated male Sprague Dawley rats at 100 uL/100 g body weight. For the mouse studies, four animals were used for a complete time course with each time course repeated in duplicate. Serum samples were collected from the orbital sinus using the following schedule: mouse 1: 1, 16 and 72 hr; mouse 2: 2 and 24 hr; mouse 3: 4 and 32 hr; mouse 4: 8 and 48 hr. For the rat studies, single animal was used for a complete time course with each time course repeated in duplicate. Serum samples were collected at 0, 1, 2, 4, 8, 12, 24, 48, 60, 120 hr.
[0102] Fluorescein standards were prepared from serial dilution of a 20 uM solution of fluorescein-PEG in mouse serum. For the mouse studies, 50 of the standards and samples were loaded into black 96 well plates (Greiner-Bio) and diluted with 50 of 50 mM HEPES buffer pH 7.5. For the rat studies, ΙΟΟμί of the standards and samples were loaded into black 96 well plates (Greiner-Bio). The fluorescence signals were read using 485 nm excitation and 535 nm emission on a GENios™ plate reader.
[0103] Sample and standard signals were corrected for background serum fluorescence. A standard curve was created in Microsoft Excel™ and used to calculate the concentration of PEG-conjugate in each sample. PK solutions software (Summit PK) was used to calculate the PK parameters.
[0104] Figures 1A-1B show the pharmacokinetics of releasably- linked 40-kDa 4-branch mPEG-(5-aminoacetamido)fluorescein (5-AAF) conjugates of Example 22 in rat. The linkers used in the preparation of the conjugates had formula (1) wherein R 2 and R 4 = H, R 3 =
(CH2)sN3. In this experiment (a) is stable control (RxS02 is absent); and in the remaining samples, R1 is substituted aryl where the substituent in (b) is CH3; (c) 2,4,6-trimethylphenyl; (d) 4-methoxyphenyl; (e) 4-methylphenyl; (f) phenyl; (g) 4-chlorophenyl; or (h) 4- (trifluoromethyl)phenyl. Panel A shows the concentration of the remaining conjugates in rat serum as a function of time, resulting in clearance half-lives of (a) 34 h; (b) 27 h; (c) 26 h; (d) 24 h; (e) 21 h; (f) 16 h; (g) 9 h, and (h) 5 h. Panel B shows the result of subtracting the concentration versus time curve for the stable conjugate (a) from each of the releasable conjugates (b)-(h) of Panel A. The slopes of the resulting difference curves gives the rate constant for release of 5-AAF from each releasable conjugate: (a) O h; (b) 131 h; (c) 107 h; (d) 76 h; (e) 56 h; (f) 31 h; (g) 12 h; (h) 6 h.
[0105] Figure 2 shows the linear free energy relationship of release rate with the substituent when R1 is substituted aryl, where the substituents are as defined for Figure 1. A plot of log(release rate) versus Hammett sigma constant is linear, both for data from in vitro and in vivo (mouse and rat) measurements. This allows prediction of release rates for any substituted aryl R1 where the Hammett sigma can be estimated.
Example 25
Figure imgf000032_0001
[0106] A solution of 2-bromo-3'-nitroacetophenone (2.5 g) in 20 mL of acetonitrile was added slowly to a stirred mixture of thiophenol (1.0 mL) and sodium bicarbonate (1.7 g) in 20 mL of water. After 2 h, the mix was extracted with ethyl acetate, washed with brine, dried over MgS04, filtered, and evaporated to provide a yellow solid. The crude sulfide was dissolved in 100 mL of ethyl acetate, and 7.0 mL of 32% peracetic acid was added. After stirring 16 h, the solution was washed with lx water, 2x 1 M Na2C03, 2x 1 M sodium dithionite, lx water, lx sat. aq. NaHC03, and lx brine. The organics were dried over MgS04, filtered, and evaporated to provide 2.93 g of the crude nitro-sulfone. Crystallization from 1: 1 ethyl acetate/hexane provided 2.37 g of purified nitro-ketosulfone.
[0107] Sodium borohydride (0.15 g) was added to a suspension of the nitro-ketosulfone (2.0 g) in 20 mL of methanol. After initial dissolution, a precipitate formed. After 30 min, the mix was acidified with 1 N HC1 and diluted with 25 mL of water. The resulting solid was collected by vacuum filtration and dried to provide 1.85 g of product. The aqueous filtrate was extracted with ethyl acetate, and the extract was washed with brine and dried over MgS04, filtered, and evaporated. The residue was combined with the initial precipitate and the total alcohol product was crystallized from 1: 1 ethyl acetate/hexane.
[0108] A mixture of the alcohol (955 mg) and SnCl2 «2H20 (3.2 g) in 35 mL of ethanol was heated at reflux for 1 hour. The resulting clear solution was cooled and diluted with 50 mL of water and 50 mL of sat. aq. NaCl, then extracted with ethyl acetate. The organics were washed with sat. aq. NaHC03, and the resulting white emulsion was filtered through Celite. The filtrate was separated and the organics were washed with brine, dried over MgS04, filtered, and evaporated to a clear glass. The product aniline was crystallized from 2: 1 ethyl acetate hexane (690 mg).
[0109] 6-Azidohexanoic anhydride (1.0 g) was added to a mixture of the aniline (486 mg) in 40 mL of dichloromethane. After 2 h, the solution was washed with sat. aq. NaHC03 and brine, dried over MgS04, filtered, and evaporated. The crude amide was purified by chromatography on silica gel using a step gradient of hexane, 60:40 hexane/ethyl acetate, and 1:2 hexane/ethyl acetate, to provide the purified amide alcohol (0.50 g) as a pale yellow oil.
[0110] Pyridine (212 uL) was added dropwise to a solution of the amide alcohol (500 mg) and triphosgene (540 mg) in 20 mL of THF. After 30 min, the mixture was filtered and concentrated. The residue was redissolved in 20 mL of THF and treated with N- hydroxysuccinimide (450 mg) and treated with pyridine (325 uL) for 10 min. The mixture was evaporated, then redissolved in ethyl acetate and filtered. The solution was washed with water, half- saturated NaHC03, water, Nl HC1, and brine, then dried over MgS04, filtered, and evaporated. The product was purified was chromatography on silica gel using a step gradient of hexane, 1: 1 hexane/ethyl acetate, and 1:2 hexane/ethyl acetate to provide the purified product (817 mg) as a sticky glass. Example 26
Preparation of BOC -protected linker succinimidyl carbonates
Figure imgf000034_0001
[0111] The compound of Example 11 wherein R1 = phenyl (372 mg, 1.0 mmol) was dissolved in 15 mL of THF and triphosgene (500 mg, 1.7 mmol) was added. Pyridine (160 uL, 2.0 mmol) was added dropwise. After 10 min, the suspension was quickly filtered and evaporated to provide the intermediate chloroformate, which was dissolved in 15 mL of THF and treated with N-hydroxysuccinimide (350 mg, 3.0 mmol) and pyridine (250 uL, 3.0 mmol). After 10 min, the suspension was diluted with ethyl acetate was washed successively with 0.1 M citric acid, water, and brine, then dried over MgS04, filtered, and evaporated. Chromatography on silica gel using a gradient of ethyl acetate in hexane provided the intermediate product BOC succinimidyl carbonate (300 mg, 60% yield) as a sticky white foam. 1H-NMR (400 MHz, d6-DMSO): d 7.91 (2H, m), 7.75 (1H, m), 7.66 (2H, m), 5.11 (1H, m), 3.95 (1H, dd, J = 8.8, 15.2 Hz), 3.82 (1H, dd, J = 2.8, 15.2 Hz), 2.85, (2H, q, J = 6.8 Hz), 2.80 (4H, s), 1.6-1.7 (2H, m), 1.36 (9H, s), 1.4- 1.1 (6H, m).
[0112] Other compounds of the invention may be prepared according to this procedure starting with other compounds of Example 11.
Example 27
Preparation of BOC-linker-Maleimides and Amino-linker-Maleimides
Figure imgf000034_0002
Figure imgf000034_0003
r>~ 3 [0113] A solution of the compound of Example 26 wherein R1 = phenyl (410 mg,
0.8 mmol) in 5 mL of THF was added to a solution of N-(2-aminoethyl)maleimide
trifluoroacetate salt (200 mg, 0.8 mmol) in 5 mL of THF, followed by triethylamine (120 uL, 0.8 mmol). After 1 h, the mixture was diluted with ethyl acetate and washed successively with 0.5 M citric acid, water, sat. aq. NaHC03, water, and brine, then dried over MgS04, filtered and evaporated to yield a glass which spontaneously crystallized upon sitting. The crystals were rinsed with 60/40 ethyl acetate/hexane to provide the BOC-linker-maleimide product.
[0114] The BOC group was removed by treatment of the above BOC-linker-maleimide (270 mg, 0.5 mmol) with 4 mL of 1: 1 CF C02H/CH2Cl2 for 10 min at ambient temperature. The mixture was evaporated, and the residue was washed 3x with 10 mL portions of ether to provide the trifluoroacetate salt (277 mg) was a colorless oil.
[0115] Other compounds of the invention may be prepared according to this procedure starting with other compounds of Example 26.
Example 28
Preparation of Cyclooctyne-linker-Maleimides
Figure imgf000035_0001
□ BCO-i ink¾r- AL
[0116] A mixture of the amine-linker-maleimide of Example 27 wherein R1 = phenyl (30 mg, 55 umol), DBCO-NHS ester (Click Chemistry Tools, 22 mg, 50 umol), and
N,N-diisopropylethylamine (18 uL, 130 umol) in 1 mL of acetonitrile was kept for 30 min, then quenched by addition of 1 mL of 1.0 M NaHC03. The mixture was diluted with ethyl acetate, washed successively with 0.5 M citric acid, water, sat. aq. NaHC03, water, and brine, then dried over MgS04, filtered and evaporated. The crude product was purified by chromatography on silica gel using a step gradient of hexane, 2: 1 hexane/acetone, 1: 1 hexane/acetone, and 1:2 hexane/acetone. The product (27 mg, 73%) was obtained as a clear glass. [0117] Other compounds of the invention may be prepared according to this procedure starting with other compounds of Example 27, and/or using other cyclooctyne reagents such as activated fluorinated c clooctynes such as DIFO or bic clononynes such as BCN:
BCN-linker-MAL
Figure imgf000036_0001
DIFO-linker-MAL.
[0118] Similar compounds of the invention may be prepared starting from the azidoalcohols of Example 4.

Claims

670572000540
WO 2013/036857 PCT/US2012/054293
Claims
1. A compound of formula (1)
O H R"5 O
R'- S - C-C -O-C-X (!)
0 ff ^
wherein
R1 is optionally substituted C]_-C linear, branched, or cyclic alkyl, optionally substituted aryl; optionally substituted heteroaryl; alkoxy; NR5 2, wherein each R5 is independently H, optionally substituted CrC6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, or when taken together two R5 can be cycloalkyl or cycloheteroalkyl; or
R1 is (CH2)2(OCH2CH2)nO-alkyl, wherein n = 1-1000 or is R10-Y, wherein R10 is (CH2)k, arylene, heteroarylene, (CH2)kNR5, arylene-NR5, heteroarylene-NR5, (CH2)kO, arylene-O, heteroarylene-O, or (CH2CH20)nCH2CH2, wherein each k is independently 1-8 and n = 1-1000;
R is H, optionally substituted C -C alkyl, optionally substituted aryl, optionally substituted heteroaryl, or CH2(OCH2CH2)nO-alkyl, wherein n = 1- 1000, or Rn-Y, wherein R11 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1- 1000;
R is H, optionally substituted CrC6 alkyl, optionally substituted aryl, optionally substituted heteroaryl, or CH2(OCH2CH2)nO-alkyl, wherein n = 1- 1000, or R12-Y, wherein R12 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1- 1000;
R4 is H, optionally substituted Ci-Ce alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
Y is N3, alkynyl or cycloalkynyl, optionally protected NH2, optionally protected C02H,
7 7
optionally protected SH, maleimido, or NH-CO-(CH2)mR , wherein m = 1-6 and R is N3, alkynyl, cycloalkynyl, optionally protected NH2, optionally protected C02H, optionally protected SH, or maleimido; and
X is F, CI, O-succinimidyl, O-sulfosuccinimidyl, optionally substituted phenoxy, or NB-CH2W, wherein B is alkyl, aryl, arylalkyl, heteroaryl or heteroarylalkyl, each optionally
8 8
substituted, and W is CI, alkoxy, or S(R )2, wherein each R is independently alkyl or aryl; and wherein R2 is other than Ru-Y and R3 is other than R12-Y and R1 is R10-Y; or R1 is other than R10-Y and R3 is other than R12-Y and R2 is Ru-Y; or
R1 is other than R10-Y and R2 is other than Ru-Y and R3 is R12-Y; or
at least one of R1, R2, R3 and/or R4 is (CH2)l(OCH2CH2)-0-alkyl(Cl-C6) wherein 1 is 1 or 2 and n is 200- 1000. 670572000540
WO 2013/036857 PCT/US2012/054293
2. The compound of claim 1 wherein R is H, optionally substituted C -C alkyl or CH2(OCH2CH2)nO-alkyl, wherein n = 1- 1000, or Rn-Y, wherein R11 is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1-1000.
3. The compound of claim 1 wherein R is H.
4. The compound of any of claims 1-3 wherein R1 is R10-Y.
wherein
R10 is (CH2)k, arylene, heteroarylene, (CH2)kNR5, arylene-NR5, heteroarylene-NR5 wherein R5 is H, optionally substituted CrC6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl, (CH2)kO, arylene-O, heteroarylene-O, or (CH2CH20)nCH2CH2 wherein each k is independently 1-8 and n = 1- 1000; and
2 3 4
R", RJ, and R" are each independently H, optionally substituted CrC6 alkyl, optionally substituted aryl, optionally substituted heteroaryl.
5. The compound of any of claims 1-3
wherein
R1 is optionally substituted linear, branched, or cyclic CrC6 alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
R2 is Rn-Y; and
R3 and R4 are each independently H, optionally substituted C -C alkyl, optionally substituted aryl, optionally substituted heteroaryl.
6. The compound of any of claims 1-3
wherein
R1 is optionally substituted linear, branched, or cyclic C]_-C alkyl, optionally substituted aryl, or optionally substituted heteroaryl;
R3 is R12-Y; and
R , and R are each independently H, optionally substituted Ci-Ce alkyl, optionally substituted aryl, optionally substituted heteroaryl.
12
7. The compound of Claim 6 wherein R is (CH2)k, arylene, heteroarylene, or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1-1000; Y is N3, alkynyl or cycloalkynyl, optionally protected NH2, optionally protected C02H, optionally protected SH, or maleimido. 670572000540
WO 2013/036857 PCT/US2012/054293
8. The compound of claim 7 wherein R is (CH2)k, and k
9. The compound of claim 7 wherein Y is N3.
10. The compound of claim 7 wherein R is arylene or heteroarylene.
11. The compound of claim 6 wherein R is (CH2)k or CH2(OCH2CH2)n, wherein k is 1-8 and n = 1-1000; and Y is N3.
12. The compound of claim 7 wherein n = 1-100.
13. The compound of claim 12 wherein n = 1-25.
14. The compound of claim 13 wherein n = 1-10.
15. The compound of claim 6 wherein R1 is phenyl, 4-(trifluoromethyl)phenyl, 4-chlorophenyl, 4-methylphenyl, 4-methoxyphenyl, 2,4,6-trimethylphenyl, methyl, or
4-morpholino;
R2 is H;
R3 is (CH2)6N3 or (CH2)6NH-CO-0-C(CH3)3;
R4 is H; and
X is CI, O-succinimidyl, O-sulfosuccinimidyl, or NB-CH2W, wherein B is optionally substituted aryl and W is CI, alkoxy, or S(R 8 )2, wherein each R 8 is independently alkyl or aryl.
16. The compound of claim 15 wherein X is CI, O-succinimidyl or
O- sulf o succinimidyl .
17. The compound of claim 15 wherein X is NB-CH2W, wherein B is optionally substituted aryl and W is CI, alkoxy, or S(R 8 )2, wherein each R 8 is independently alkyl or aryl.
Figure imgf000040_0001
 670572000540
WO 2013/036857 PCT/US2012/054293
Figure imgf000041_0001
o
19. A compound resulting from reaction of the—c-ox group of a compound of any of claims 1-3 with a moiety that comprises an amino group.
20. The compound of claim 19 wherein said moiety is a pharmaceutical.
21. The compound of claim 20 wherein said pharmaceutical is a peptide, an oligonucleotide, or a small molecule.
22. A conjugate resulting from attachment of a compound of any of claims 1-3 to a carrier by displacement of, or reaction with, Y.
23. The conjugate of claim 22 wherein said carrier is a polymer, a solid support or a dendrimer.
24. The conjugate of claim 23 wherein the carrier is a polymer which is polyethylene glycol (PEG).
25. The conjugate of claim 23 wherein the carrier is an antibody or albumin.
26. A conjugate resulting from attachment of a compound of claim 19 to a carrier by displacement of or reaction with Y. 670572000540
WO 2013/036857 PCT/US2012/054293
27. The conjugate of claim 26 wherein said carrier is a polymer, a solid support or a dendrimer.
28. The conjugate of claim 26 wherein the carrier is an antibody or albumin.
29. The conjugate of claim 26 wherein said moiety is a pharmaceutical.
30. The conjugate of claim 27 or 28 wherein said moiety is a pharmaceutical.
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