EP1098659A2 - Banques de polyhydroxamates et leurs analogues - Google Patents

Banques de polyhydroxamates et leurs analogues

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Publication number
EP1098659A2
EP1098659A2 EP99937465A EP99937465A EP1098659A2 EP 1098659 A2 EP1098659 A2 EP 1098659A2 EP 99937465 A EP99937465 A EP 99937465A EP 99937465 A EP99937465 A EP 99937465A EP 1098659 A2 EP1098659 A2 EP 1098659A2
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European Patent Office
Prior art keywords
polyhydroxamate
analog
library
polyhydroxamates
group
Prior art date
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EP99937465A
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German (de)
English (en)
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EP1098659A4 (fr
Inventor
Garland R. Marshall
Leonard O. Rosik
Otto F. Schall
Urszula J. Slomczynska
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Metaphore Inc
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Metaphore Inc
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Publication of EP1098659A2 publication Critical patent/EP1098659A2/fr
Publication of EP1098659A4 publication Critical patent/EP1098659A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C259/00—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups
    • C07C259/04—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids
    • C07C259/06—Compounds containing carboxyl groups, an oxygen atom of a carboxyl group being replaced by a nitrogen atom, this nitrogen atom being further bound to an oxygen atom and not being part of nitro or nitroso groups without replacement of the other oxygen atom of the carboxyl group, e.g. hydroxamic acids having carbon atoms of hydroxamic groups bound to hydrogen atoms or to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/06—Antiabortive agents; Labour repressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/02—Antidotes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to novel hydroxamates and their analogs, methods of obtaining hydroxamates and their analogs having a specified target property such as affinity to iron, and libraries containing candidate hydroxamates and their analogs which are retrievable and analyzable for such target property.
  • Desferrioxamine B also known as deferoxamine, is a naturally-produced siderophore derived from the microorganism Streptomyces pilosus .
  • DFO is an iron chelator which has been used for decades in the clinic to treat various conditions related to acute iron poisoning or overload. For example, iron overload may be caused by the frequent transfusions required during the treatment of thalassemias and sickle cell anemia.
  • Thalassemias represent two of the most common inherited disorders, and it is estimated that over 100,000 children are born each year with forms of the disease severe enough to require treatment. Moreover, the World Health Organization estimates that each year more than 250,000 babies are born worldwide with sickle cell disease, and it is believed to affect more than 72,000 African Americans in the United States, alone.
  • HHC hereditary hemochromatosis
  • HHC is a disorder of iron metabolism that increases iron absorption and results in excessive iron accumulation. It is estimated that 24 million people worldwide carry double genes for hemochromatosis and more than 600,000,000 people carry the single gene. Hemochromatosis affects approximately one in three hundred people in the United States, and one in nine people is a carrier, making it one of the most common genetic disorders in the United States. As iron accumulates in the body, serious and sometimes fatal health problems appear, including arthritis, cirrhosis of the liver, diabetes, impotence, heart failure and liver cancer.
  • Oral iron chelators also have potential application in the treatment of infections. Many pathogens require ferric ions (the +3 form of iron) for growth, and have evolved to produce siderophores that complex and transport these ions. This ensures the continued survival of the microorganism by enabling it to compete effectively with its host for this limiting resource. In fact, man has developed an elaborate mechanism for sequestering ferric ion from pathogens as part of its natural defenses against infection. Iron chelation is potentially useful in the treatment of parasitic diseases such as malaria, and leishmaniasis, as well as in the treatment of opportunistic infections arising from Pneumocystis carnii and Histoplas a capsulatum .
  • DFO has also been reported to have utility (in complexes with Mn) as a low-molecular weight mimic of superoxide dismutase to reduce or prevent superoxide radical-induced toxicity. It is used in the treatment of conditions associated with inflamation and oxidative stress (oxyen toxicity) such as reperfusion injury, stroke, psoriasis, inflamatory bowel disease, shock, hyperbaric oxygen therapy, etc. See, e . g. , Fridovich et al . , U. S . Patent No . 5, 227, 405, which is incorporated by reference.
  • DFO is not ideal for use as a therapeutic in several significant respects.
  • DFO is not orally active, and consequently, its clinical use is often plagued by patient non-compliance.
  • DFO is cleared by the kidneys and has a short half-life in the body.
  • DFO administration is also quite costly and exhibits unwanted toxicity in some patients.
  • a superior metal chelator for treatment of iron overload, and to fill the need for a therapeutic effective for infections and related applications, is long overdue.
  • Metal chelators including DFO, also have utility as metal-binding ligands in non-therapeutic applications. For example, their affinity for ferric ions and other metals makes them useful imaging agents in the diagnosis of numerous diseases. Complexes of chelators with X-ray opaque metals such as lead, tungsten, and bismuth can be used as X-ray imaging agents, while complexes with gadolinium, iron, and other magnetically active metals are used as MRI imaging agents .
  • metal chelators Another non-therapeutic use of metal chelators is in water purification and remediation regimens.
  • Ligands attached to a polymer or other solid support sequester metals from a waste stream and in doing so, enable removal and recovery of metal pollutants.
  • DFO belongs to a class of compounds known as polyhydroxamates which utilize hydroxamic groups as ligation sites for the chelation of iron in the form of ferric ions (Fe +3 ) .
  • DFO binds Fe 3+ with an association constant, K a , of 30.4.
  • Desferrioxamine was first produced synthetically in 1962 by Prelog et al . [Helv. Chim. Acta., Vol. 75, p. 631 (1962)].
  • This lack of progress has no doubt been fueled by the perception that the synthesis of DFO or hydroxamate analogs requires a large number of reaction steps and produces only a low yield of product, making the synthesis and evaluation of this type of compound problematic and laborious.
  • the present invention is directed to a novel method of synthesizing a desired polyhydroxamate or polyhydroxamate analog.
  • the method includes linking a first component of said desired polyhydroxamate or polyhydroxamate analog to a support matrix under conditions effective to form a first matrix- bound intermediate of said desired polyhydroxamate, extending said first matrix-bound intermediate using reagents and reaction conditions effective to form one or more additional matrix-bound intermediates of said desired polyhydroxamate or polyhydroxamate analog, thereby forming a matrix-bound precursor of said desired polyhydroxamate or polyhydroxamate analog. Any protective groups used during synthesis of said precursor are removed and the matrix-bound precursor is cleaved from the support matrix, thereby synthesizing the desired polyhydroxamate or polyhydroxamate analog.
  • the present invention is further directed to a method relating to libraries of candidate polyhydroxamate or polyhydroxamate analog molecules.
  • the method includes the steps of designing a molecular scaffold or scaffolds for a prototype polyhydroxamate or polyhydroxamate analog, designing a synthetic pathway to make said prototype, obtaining a support matrix or matrices for use in construction of the library of candidate polyhydroxamate or polyhydroxamate analog molecules, and carrying out reaction steps according to the synthetic pathway so that the library is thereby created.
  • the library thus created comprises an array of at least two candidate polyhydroxamate or polyhydroxamate analog molecules substantially all of which comprise the molecular scaffold or scaffolds of the prototype linked to the support matrix or matrices .
  • a method of obtaining a polyhydroxamate or polyhydroxamate analog or mixture of polyhydroxamates or analogs of a specified target property comprises the steps of providing a library or libraries of candidate polyhydroxamates or analogs which contains at least five different candidates with each of the candidates being present in retrievable and analyzable amounts, selecting from the candidates one or more having a desired target property, and separating said polyhydroxamates or analogs having the desired target property from those not having the target property.
  • a library of polyhydroxamates or polyhydroxamate analog molecules which are candidates targeted for one or more desired properties.
  • the library includes an array of at least two different polyhydroxamate or polyhydroxamate analog molecules wherein any of the candidate molecules are retrievable and analyzable for the one or more desired target properties.
  • the invention is directed to a compound comprising a matrix-bound polyhydroxamate or polyhydroxamate analog; a compound comprising an N-nosyl intermediate of a polyhydroxamate or polyhydroxamate analog; a polyhydroxamate or polyhydroxamate analog comprising the formula:
  • R ⁇ and R 5 are independently selected and incorporate one of the following, or combinations of any of the following: hydrogen; cyclic or acyclic, branched or unbranched alkyl or heteroalkyl , aryl or heteroaryl, alkylidene or heteroalkylidene, heterocyclic, arylalkyl or heteroarylalkyl, alkylether, alkoxyalkyl, alkylpolyether, alkylthioether, alkylamino, alkylaminoalkyl, alkylpolyamino, all optionally substituted with one or more, same or different, hydroxyl, thiol , halide, alkoxy, thioalkoxy, amino, including mono-, di-, tri-, and tetrasubstituted, aminoalkyl, carboxyl, carboxamido, carboxamidoalkyl , carboxyalkyl, sulfonic and phosphonic acid groups, a support matrix, and
  • the invention is directed to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one of the polyhydroxamates or polyhydroxamate analogs first identified by selection from a library or from libraries of candidate polyhydroxamates or analogs as defined above, having the desired target property or properties, or the pharmaceutically acceptable salt or salts thereof, either with or without a complexed metal, in combination with a pharmaceutically acceptable carrier.
  • imaging agents comprising at least one of the polyhydroxamates or polyhydroxamate analogs first identified by selection from a library or from libraries of candidate polyhydroxamates or analogs as defined above having the desired target property or properties, wherein said target property or properties include the ability to provide a suitable image, complexed with a transition metal or lanthanide.
  • a radiodiagnostic agent comprising at least one of the polyhydroxamates or polyhydroxamate analogs first identified by selection from a library or from libraries of candidate polyhydroxamates or analogs as defined above having the desired target property or properties, wherein said target property or properties include the ability to serve as a suitable radiodiagnostic, complexed with a transition metal or lanthanide.
  • an X-ray contrast agent comprising at least one of the polyhydroxamates or polyhydroxamate analogs first identified by selection from a library or from libraries of candidate polyhydroxamates or analogs as defined above having the desired target property or properties, wherein said target property or properties include the ability to serve as a suitable X-ray contrast agent, complexed with a transition metal or lanthanide.
  • a system for the separation or concentration of fluid-borne metals from a fluid comprising at least one polyhydroxamate or polyhydroxamate analog and a porous container for housing the at least one polyhydroxamate or polyhydroxamate analog and for flowing the solution through, wherein the at least one polyhydroxamate or polyhydroxamate analog is first identified by selection from a library or from libraries of candidate polyhydroxamates or analogs as defined above having the desired target property or properties, wherein said target property or properties include the ability to separate or concentrate said solution-borne metals from said solution.
  • a metal chelator comprising a polyhydroxamate or polyhydroxamate analog first identified by selection from a library or libraries of candidate polyhydroxamates or analogs as defined above having the desired target property or properties, wherein said target property or properties include the ability to chelate a target metal anion.
  • compositions of the invention are also provided.
  • a method of preventing or treating a disease or disorder characterized by the presence of a cellular excess of a particular metal anion comprising administering to a subject in need of such prevention or treatment, a therapeutically, prophylactically, or resuscitatively effective amount of at least one pharmaceutical composition described above, wherein said target property or properties include the ability to bind to said particular metal anion.
  • a method of assisting in the diagnosis of a physiological condition comprising administering to a subject in need of such diagnosis, an imaging agent, a radiodiagnostic agent, or an X-ray contrast agent as described above, of a type and in an amount sufficient to aid in said diagnosis.
  • a method for the separation or concentration of fluid-borne metals from a fluid containing said metals comprising flowing said fluid through a system such as one characterized above using polyhydroxamates or polyhydroxamate analogs .
  • a method for the chelation of a target metal or metals comprising contacting the target metal or metals with a metal chelator as described above, wherein the metal chelator has an affinity for said target metal or metals is provided.
  • the method of the present invention consists of three integrated parts: i) Devising the solid phase synthesis of basic molecular scaffolds for polyhydroxamates or their analogs which are capable of selectively binding ferric ions and/or other metal ions; ii) Generating combinatorial libraries consisting of candidate polyhydroxamates or their analogs incorporating such basic molecular scaffolds; and iii) The use of high-throughput screening techniques to select those compounds with the desired target property or profile of properties.
  • a method of synthesizing polyhydroxamates comprises building a polyhydroxamate scaffold by linking a first component of a desired polyhydroxamate to a support matrix under conditions effective to form a first matrix-bound intermediate of the desired polyhydroxamate, and in subsequent steps extending this first matrix-bound intermediate using reagents and reaction conditions effective to form one or more additional matrix-bound intermediates of the desired polyhydroxamate until a matrix-bound precursor to the desired polyhydroxamate is formed.
  • the method further comprises removing any protective groups used during synthesis of the matrix- bound polyhydroxamate precursor and cleaving the matrix- bound polyhydroxamate precursor from the support matrix to form the desired polyhydroxamate.
  • a solid phase method for synthesizing polyhydroxamates comprises the following synthetic stages : a) Attachment of a suitable linker, which may or may not be an integral part of the target polyhydroxamate, onto the synthetic support (e. g. , resin matrix) . This modification affords the first support- bound intermediate. b) Incorporation of additional molecular component (s) into the growing chain prior to the introduction of the first of a series of hydroxamates or hydroxamate-analog moieties. c) Introduction of a first hydroxamate moiety as a suitably N, O-Jbis-protected hydroxylamine precursor.
  • the support matrix to be utilized for solid phase synthesis may be constructed of any suitable material to which the candidate polyhydroxamate (s) may be attached and subsequently cleaved.
  • a support matrix is defined as an insoluble solid phase (polymeric and otherwise) , such as in the form of beads, films, rods or pins; or a soluble polymeric support such as dendrimers, or bovine serum albumin, on which synthetic manipulations may be accomplished.
  • This support may in itself be of natural or synthetic origin.
  • Insoluble solid phases in the following forms as examples, but not limited to: Gel-types: polystyrene-co-divinylbenzene (0.5-2%) , polystyrene-Kel-F, polystyrene-polyethylene film (PEPS) , polystyrene-polyethyleneglycol (TentaGel, NovaSyn TG, ArgoGel), poly [styrene-co-tetraethyleneglycol diacrylate] (TEGDA-PS) .
  • Polyamides various co-polymers of
  • Miscellaneous polymers polyethylene pins grafted with various acrylates (such as the pins made by Chiron), polyolefins (ASPECT), poly [ethylene) -co-vinyl alcohol] (EVAL) , polypropylene-polyhydroxypropylacrylate (HPA-PP) , 3,6, 9-trioxadecanoic acid-PEPS (PEO-PEPS) .
  • Polymeric macroporous (rigid) solids amide-PEG based Polyhipe, polystyrene-co-divinylbenzene (8-50%) based (ArgoPore) .
  • Natural organic polymers Sephadex, cellulose, chitin.
  • Inorganics silica, glass, controlled pore glass, kiesselguhr, NovaSyn K125, and ii) Soluble polymers: polyethyleneglycol (PEG), bovine serum albumin (BSA) , Starburst dendrimers.
  • PEG polyethyleneglycol
  • BSA bovine serum albumin
  • a linker is defined as a covalent chemical linkage which facilitates the attachment of the starting material to the support matrix and the convenient and efficient removal of the product under desired conditions.
  • Linkers may be already attached to the support matrix or may be coupled to it chemically by known methods.
  • Materials suitable for use as linkers include but are not limited to: 4-alkoxybenzyl alcohol (Wang) , p-carbamoylmethyl-benzyl ester (PAM) , 2-methoxy-4-alkoxybenzyl alcohol (SASRIN) ,
  • HMBP 4-hydroxymethyl-3-methoxyphenoxybutyric
  • HMBA 4-hydroxymethylbenzoyl
  • trityl 2-chlorotrityl
  • 4-methyltrityl 4-methoxytrityl
  • 4 , 4 ' -dichlorotrityl 4-hydroxymethyl-3-methoxyphenoxybutyric (HMBP)
  • HMBP 4-hydroxymethyl-3-methoxyphenoxybutyric
  • a Wang combination solid support matrix linker is preferred, and is best described as a 4-hydroxymethylphenoxy linker covalently attached to an insoluble polymer matrix of copoly (styrene-1% divinylbenzene crosslinker) , 100-200 mesh size. See “A Practical Guide to Combinatorial Chemistry", A.W. Czarnik and S.H. DeWitt, Eds., 1997, ACS and references therein for additional support matrices and linkers.
  • polyhydroxamates explicitly excludes a polyhydroxamate which is naturally occurring or which was otherwise first discovered prior to applicants' invention thereof.
  • hydroxamate analogs or “polyhydroxamate analogs” means hydroxamate-like compounds wherein the hydroxyl group of one or more hydroxamate moieties may be replaced, e . g. , by thiol, ⁇ H 2 or R- L as defined for X in Scheme 7, and/or wherein the carbonyl oxygen of one or more of the hydroxamate moieties may be replaced, e. g. , by NH 1# NR ⁇ sulfur or selenium as defined for Z in Scheme 7.
  • An additional aspect included in the present invention is the preparation of novel O-protected-N- (nosyl) hydroxylamine derivatives, where nosyl ( ⁇ s) is 2- or 4-nitrobenzenesulfonyl .
  • PG stands for protective group, and includes but is not limited to any of tert-butyl (t-Bu), benzyl (Bn) , tetrahydropyranyl (THP) , tert-butyldimethylsilyl (TBDMS) , 4-benzyloxybenzyl (BnOBn) , 2 , 4-dimethoxybenzyl [(2,4- MeO) 2 Bn], methoxymethyl (MOM), and allyl.
  • t-Bu tert-butyl
  • Bn benzyl
  • THP tetrahydropyranyl
  • TDMS tert-butyldimethylsilyl
  • BnOBn 4-benzyloxybenzyl
  • 2 4-dimethoxybenzyl [(2,4- MeO) 2 Bn]
  • MOM methoxymethyl
  • hydroxylamine derivatives are important intermediates for the syntheses of polyhydroxamates disclosed in the present work.
  • the use of a nosyl group is advantageous from several perspectives. It is easily incorporated via commercially available nosyl chloride. It activates the ⁇ -H bond in the nosylated species to the extent that it can be deprotonated by a wide variety of organic and inorganic bases (e . g.
  • nosyl groups are conveniently and selectively removed via a Meisenheimer-type complex using a thiol nucleophile ( e . g.
  • this process includes the steps of: a) reacting a support matrix (e . g. , p- benzyloxybenzyl alcohol resin is exemplified) containing an imidazolyl-carbamate group 3.1 with 5-aminopentanol to form compound 3.2; b) activating the hydroxyl end of compound 3.2 via a sulphonate ( e . g. , with tosyl chloride) to form compound 3.3 or via an alkyl halide ( e . g .
  • PG-O- ⁇ H- ⁇ s N-nosyl -O-protected-hydroxylamine synthon
  • the protective group [PG] may be, for example, benzyl [Bn] , tetrahydropyranyl [THP] , tert-butyl [t-Bu] , 4-benzyloxybenzyl [BnOBn] , 2,4- dimethoxybenzyl [ (2, 4-MeO) 2 Bn] methoxymethyl [MOM], tert- butyldimethylsilyl [TBDMS] or allyl) in the presence of an organic or inorganic base (e.g., DBU, DIPEA, Cs 2 C0 3 , MTBD, TMG, etc.) to form compound 3.4; d) Removal of nosyl groups with a thiol
  • N-nosyl - O-protected-hydroxylamine synthon (PG-O- ⁇ H- ⁇ s, where the protective group [PG] may be, for example, benzyl [Bn] , tetrahydropyranyl [THP], tert-butyl [t-Bu] , 4-benzyloxybenzyl [BnOBn] , 2 , 4-dimethoxybenzyl [(2,4- MeO) 2 Bn] methoxymethyl [MOM], tert-butyldimethylsilyl [TBDMS] or allyl) in the presence of triphenylphosphine (Ph 3 P) and diethyl azodicarboxylate (DEAD) or diisopropyl azodicarboxylate (DIAD) for the transformation of the alcohol intermediate 3a.2 to 3a.3 [see
  • the synthesis of compound 4 comprises: a) Reacting a resin containing a hydroxyl function (e . g. , p-benzyloxybenzyl alcohol resin is exemplified) with 6-bromohexanoic acid and coupling agent to form compound 4.1; b) Displacing the halide of compound 4.1 with an N-nosyl -O-protected-hydroxylamine synthon (PG-O- ⁇ H- ⁇ s, where the protective group [PG] may be, for example, benzyl [Bn] , tetrahydropyranyl [THP] , tert-butyl [t-Bu] , 4-benzyloxybenzyl [BnOBn] , 2 , 4-dimethoxybenzyl [(2,4- MeO) 2 Bn] , methoxymethyl [MOM], tert-butyldimethylsilyl [TBDMS] or allyl) in
  • O-protected-hydroxylamine intermediate 4.3 with 6-bromohexanoic acid via a variety of coupling methods (e.g., CDI, HATU/DIPEA, acid chloride/DIPEA etc.) to form 4.4.
  • coupling methods e.g., CDI, HATU/DIPEA, acid chloride/DIPEA etc.
  • a resin containing a hydroxyl function e.g., p-benzyloxybenzyl alcohol resin is exemplified
  • 6-bromohexanoic acid and a coupling agent to form compound 5.1
  • O-protected-hydroxylamine intermediate 5.3 with 6-bromohexanoic acid via one of a variety of coupling methods (e.g., DIC/DMAP, HATU/DIPEA, acyl chloride/DIPEA, etc . ) to form 5.4.
  • DIC/DMAP DIC/DMAP
  • HATU/DIPEA HATU/DIPEA
  • acyl chloride/DIPEA etc .
  • hydroxylamine-derivatized resin 6.1 e.g., p-benzyloxybenzyl alcohol resin is exemplified, which was prepared according to the procedure of Floyd et al . , Tetrahedron Lett .
  • MeO) 2 Bn methoxymethyl [MOM], tert-butyldimethylsilyl [TBDMS] or allyl
  • organic or inorganic base e.g., DBU, MTBD, DIPEA, Cs 2 C0 3 , TMG, etc.
  • an organic or inorganic base e.g., DBU, MTBD, DIPEA, Cs 2 C0 3 , TMG, etc.
  • f) Repeating steps (c) through (e) to form compound 6.7 (nosyl group removal, acylation with 6- bromohexanoyl chloride, and nucleophilic displacement with PG-O-NH-Ns)
  • g) Repeating steps (c) through (e) and then (c) (nosyl group removal, acylation with 6-bromohexanoyl chloride, nucleophilic displacement with PG-O-NH-Ns, and nosyl group removal) followed by acetylation of the resulting secondary
  • design of a molecular scaffold for the polyhydroxamates or their analogs involves selecting and positioning ferric ion-binding and/or other metal ion-binding atoms from a set of electron-rich hetero atoms (e.g., 0, N, S, P) as donors and positioning such metal ion-binding atoms in an optimal geometric arrangement around the spherical metal ion.
  • the scaffold includes at least two hydroxamate units as ligation sites.
  • Design of the scaffold also includes the selection and placement of carbon, oxygen, phosphorus, nitrogen and/or sulfur atoms to form connective acyclic, cyclic, or branched chains which link the metal-binding atoms in the same molecule.
  • the design of an appropriate molecular scaffold may also include the utilization of a computer program in which pre-selected properties are incorporated into the design criteria.
  • properties which may be included in the design criteria are: i) Availability of viable synthetic reaction schemes to construct and integrate the design components and necessary intermediates by solid phase synthesis; ii) Avoidance of spatial coincidence of ligand and metal atoms; iii) Avoidance of van der Waals contact of ligand atoms separated by greater than two bonds; iv) Ensurance of appropriate length and angle of bonds between connecting atoms; and v) Review and incorporation of optimal geometric arrangements seen in crystal structures of preferred pre-existing and/or newly-synthesized metal-ligand complexes.
  • the candidate polyhydroxamates are preferably constructed on a support matrix which allows generation of the candidate compounds in good yield with a purity that allows identification and assaying without extensive purification.
  • a specific set of chemical reactions and reagents is employed which enables assembly of the "building blocks" containing the hydroxamate residues and/or other ligating electron-rich atoms, in accordance with design criteria.
  • fragments to be assembled on the support matrix are appropriately derivatized as part of an overall protection strategy.
  • the nosyl group is advantageously utilized to protect the amino group of hydroxylamine. This nosyl protective group may then be removed from an intermediate compound (e.g.
  • hydroxyl group of hydroxylamine may be masked with selected protective groups, such as benzyl, 2 , 4-dimethoxybenzyl, tetrahydropyranyl, and t-butyl among others already cited, and conveniently removed whenever necessary.
  • combinatorial libraries of polyhydroxamates and analogs as mixtures or individual compounds are constructed by any of a variety of means used in the field of combinatorial chemistry. These include but are not limited to methodologies such as the "tea bag” method, “pin” methods, “split and combine” methods, or spatially addressable synthesis.
  • the "tea bag” and “pin” methods are techniques which physically separate different compounds on the polymeric support.
  • the "tea-bag” method first developed by Houghten, et al . (Proc . Natl . Acad. Sci . U. S .A .
  • pin method developed by Geysen, et al . [J. Immunol . Meth . (1987) 102 : 259 -274] is an alternative to conventional resins in which rigid pins are used as a solid support.
  • Pins consist of polymer chains that are grafted at one end to a dimensionally stable plastic polymer such as polyethylene or polypropylene.
  • Pins are held in a grid referenced position (such as a 96-well microtitre format) . This grid format simplifies parallel synthesis by allowing for convenient removal of unreacted reagents, washing of the resin, and the simultaneous handling of thousands of individual compounds.
  • Compounds in combinatorial syntheses are prepared as either separate compounds, using parallel synthesis or spatially addressable synthesis, or as mixtures (e.g. a "mix and split" method).
  • Spatially addressable synthesis is a combinatorial synthesis in which the identity of a compound is ascertained by virtue of its location in the synthesis.
  • the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations of a solid support.
  • This approach enables generation of unique compounds in discrete locations, for example a specific polymeric bead, a "tea bag” of polymeric beads, a “Kan” of polymeric beads, a specific pin head in an array of pins, a specific location in a 364-well plate, a specific location in a reaction block, or a specific location of an addressable site on silicon or paper.
  • An example of light-directed, spatially addressable parallel chemical synthesis is that developed by Fodor and co- workers (Fodor et al . Science, 1991 , 251 , 767) , which combines solid phase chemistry and photolithography to generate arrays of compounds .
  • Combinatorial libraries allow development of an array of related molecules to be screened for more desirable exhibition of a target property or set of properties.
  • the present invention is directed to novel libraries of candidate polyhydroxamates or their analogs targeted for one or more desired properties.
  • the library contains at least 2 different polyhydroxamate or analog candidates and preferably 50 or more candidates. Any of the candidates are retrievable and analyzable for the one or more desired target properties.
  • R x and R 5 are independently selected and incorporate one of the following, or combinations of any of the following: hydrogen; cyclic or acyclic, branched or unbranched alkyl or heteroalkyl, aryl or heteroaryl, alkylidene or heteroalkylidene, heterocyclic, arylalkyl or heteroarylalkyl, alkylether, alkoxyalkyl, alkylpolyether, alkylthioether, alkylamino, alkylaminoalkyl, alkylpolyamino, all optionally substituted with one or more, same or different, hydroxyl, thiol, halide, alkoxy, thioalkoxy, amino (mono- , di-, tri-, and tetrasubstituted) , aminoalkyl, carboxyl, carboxamido, carboxamidoalkyl, carboxyalkyl, sulfonic and phosphonic acid groups, a support matrix, a support
  • R 2 through R 4 are independently selected and incorporate one of the following, or combinations of any of the following: no atom, all definitions of R x and R 5 .
  • R x through R 5 may be the same or different in any of their occurrences. Any pair of R x through R 5 , together with any moiety through which they are linked, may form a carbocyclic or heterocyclic ring.
  • a, b, and c are integers greater than or equal to zero, and w is an integer greater than or equal to one.
  • Each X is independently selected from the group consisting of hydroxyl, thiol, NH 2 , and NHR X .
  • Each Y is independently selected from the group consisting of no atom, oxygen, sulfur, selenium, CH 2 , CHR ⁇ R 1( NH, NOH, NNH 2 , NNHR 1# CONRi, NRjCO, CO, C0 2 , sulfonate or phosphonate ester, sulfinate or phosphinate, carboxyl, carboxamido, carboxamidoalkyl, carboxyalkyl, or any of the moieties belonging to groups R 1 and R 5 except for hydrogen.
  • Each Z is independently selected from the group consisting of oxygen, NH, NR 1# sulfur, and selenium.
  • Each X, Y, and Z can be the same or different in any of their occurrences.
  • the structural diversity of the chemical species available through applicants' methodologies described herein are extensive and multifaceted. For illustrative purposes, the following structures are provided to demonstrate the architectural variety of this approach.
  • polyhydroxamates encompassed by the invention include branched chain scaffolds, for example bifurcated and trifurcated polyhydroxamates including but not limited to those shown in Scheme 9 below:
  • R ⁇ and R 5 are independently selected and incorporate one of the following, or combinations of any of the following: hydrogen; cyclic or acyclic, branched or unbranched alkyl or heteroalkyl, aryl or heteroaryl, alkylidene or heteroalkylidene, heterocyclic, arylalkyl or heteroarylalkyl, alkylether, alkoxyalkyl, alkylpolyether, alkylthioether, alkylpolythioether, alkylamino, alkylaminoalkyl , alkylpolyamino, all optionally substituted with one or more, same or different, hydroxyl, thiol, halide, alkoxy, thioalkoxy, amino (mono-, di-, tri-, and tetra-substituted) , aminoalkyl, carboxyl, carboxamido, carboxamidoalkyl, carboxyalkyl, sulfonic and phosphonic
  • R 2 through R 4 are independently selected and incorporate one of the following or combinations of any of the following: no atom, all definitions of R 1 and R 5 .
  • R- L through R s may be the same or different in any of their occurrences. Any pair of R ⁇ through R 5 , together with any moiety through which they are linked, may form a carbocyclic or heterocyclic ring.
  • a, b, and c are integers greater than or equal to zero, and w is an integer greater than or equal to one.
  • q, r, s, t, and u are integers greater than or equal to zero.
  • Each X is independently selected from the group consisting of hydroxyl, thiol, NH 2 , and NHR X .
  • Each Y is independently selected from the group consisting of no atom, oxygen, sulfur, selenium, CH 2 , CHR 17 NR lf NH, NOH, NNH 2 , NNHR 1# CONRi, NRjCO, CO, C0 2 , sulfonate or phosphonate ester, sulfinate or phosphinate, carboxyl, carboxamido, carboxamidoalkyl, carboxyalkyl, or any of the moieties belonging to groups R x and R 5 except for hydrogen.
  • Each V is independently selected from the group consisting of no atom, oxygen, NH, NR ⁇ ; sulfur, and selenium.
  • Each Z is independently selected from the group consisting of oxygen, NH, NR 1# sulfur, and selenium.
  • Each X, Y, V and Z can be the same or different in any of their occurrences.
  • the bi- and trifurcated chains are built by substituting a bi- or tri-halo carboxylic acid for the mono-halo carboxylic acid used, for example, in the synthesis of compound 4.
  • An example would be the use of 3-bromo-2-bromomethylpropionic acid in place of 6- bromohexanoic acid (see Scheme 4) to yield a bifurcated derivative.
  • the chain building chemistry continues on in the same manner as for straight chain polyhydroxamates except that the chemistry is occurring on two or three chains simultaneously.
  • novel polyhydroxamates and libraries containing said novel polyhydroxamates and their analogs are provided. These newly discovered compounds have the general formula :
  • n, and p are independently selected from the group consisting of the integers 1 to 10.
  • This invention relates also to the complexes of such novel compounds with iron and other metals including, but not limited to, aluminum, manganese, cobalt, nickel, copper, zinc, cadmium, tungsten, platinum, gold, mercury, lead, bismuth, gadolinium, europium, technium, indium, gallium, scandium, and chromium.
  • iron and other metals including, but not limited to, aluminum, manganese, cobalt, nickel, copper, zinc, cadmium, tungsten, platinum, gold, mercury, lead, bismuth, gadolinium, europium, technium, indium, gallium, scandium, and chromium.
  • These complexes have the general formula illustrativly depicted in Scheme 11 below for a metal ion bearing a formal charge (q) of +3.
  • n, and p are independently selected from the group consisting of the integers 1 to 10; and q can be +2, +3, or +4.
  • novel matrix-bound polyhydroxamates are provided. These compounds have the general architecture/formula :
  • support matrix and linker may be independently selected from the list of support matrixes and linkers detailed above, and m, n, and p are independently selected from the group consisting of the integers 1 to 10.
  • This invention relates also to the complexes of such novel compounds with the general structure of polyhydroxamtes 4,5,6,10,13,14,15,16,17,18, and 19 with iron and other metals including, but not limited to,
  • novel matrix-bound polyhydroxamates with the general structure of polyhydroxamates 4,5,6,10,13,14,15,16,17,18, and 19 are provided.
  • the methods of the present invention can also be used to generate polyhydroxamate libraries with
  • hydroxymates which can serve as produgs and be regenerated through the gut passage by exposure to acidic and basic conditions and to esterases .
  • the hydroxyl group of -N(OH)-CO- in polyhyhydroxamate analogs can be modified with acyl , aryl-acyl, alkyl carbonate, alkyl, etc. or terminal carboxyl group could be esterified with a variety of alcohols, etc.
  • the methods of the present invention are also directed to processes for producing polyhydroxamate libraries. These processess include: a) Reacting a support matrix, in any of the variety of forms used in the field of combinatorial chemistry (tea bag, pin, split and combine, spatially addressable, etc.), with a suitable linker for those supports lacking an appropriate one; b) Reacting the matrix-bound linker, in one or more steps, to create intermediates consistent with R 1 through R 5 as defined in Scheme 7.
  • these reagents will consist of hydroxy-acids, halo-acids, or amino-alcohols, but can also include dicarboxylic acids, amino acids and other reagents which are subsequently reacted with other reagents such as amino-alcohols or halo-acids; c) Displacement of the resin-bound intermediate's terminal hydroxyl (via its sulfonate) or halide by an N-nosyl -O-protected-hydroxylamine moiety (where the O-protective group may be, e.g., benzyl [Bn] , tetrahydropyranyl [THP] , t-butyl [t-Bu] , 4-benzyloxybenzyl [BnOBn] , 2 , 4-dimethoxybenzyl [(2,4- MeO) 2 Bn], methoxymethyl [MOM], t-butyldimethylsilyl [TBDMS] or
  • these reagents will consist of hydroxy-acids, halo-acids, or amino-alcohols, but can also include dicarboxylic acids, anhydrides, dicarboxyl halides, and other reagents which are subsequently reacted with other reagents such as amino- alcohols; f) Repeating steps (c) through (e) as needed to elongate the polyhydroxamate scaffold; g) Repeating steps (c) and (d) (nucleophilic displacement with PG-O-NH-Ns and nosyl group removal) followed by treatment with an acetylating agent to form an intermediate which is ultimately consistent with R 5 as defined in Scheme 6.
  • the present invention is further directed to a novel method for identifying polyhydroxamate and analog compounds which bind metals for therapeutic or non-therapeutic use.
  • This method includes the steps of producing a library of polyhydroxamate or analog compounds on support matrices; cleaving and separating the polyhydroxamate and analog compounds from the resin- linkers; presenting each compound in the combinatorial library with a metal ion; assessing the metal binding affinity of each compound; selecting the compounds which have useful binding affinities; and determining other properties of the selected compounds which are important for therapeutic, diagnostic or other commercial uses.
  • a method of obtaining a polyhydroxamate or mixture of polyhydroxamates of a specified target property includes providing a library of candidate polyhydroxamates or analogs which contains at least ten candidates with each of the candidates being present in retrievable and analyzable amounts; selecting from the candidate polyhydroxamates or analogs one or more having a desired target property; and separating said polyhydroxamates or analogs from those not having the target property.
  • properties of potential interest for the candidate polyhydroxamates and their analogs are (1) metal affinity, (2) metal selectivity, (3) oral bioavailability, (4) absence of toxicity, (5) serum half-life, and (6) solubility.
  • the target properties to be selected from may vary depending on the projected use of the candidate hydroxamates.
  • oral bioavailability would be relevant for many therapeutic applications, but generally not in the case of a polyhydroxamate targeted for use in water purification or imaging .
  • the combinatorial libraries as delineated provide a large pool of candidate polyhydroxamates and analogs which can readily be screened to locate those having a desired target property.
  • the library is screened using a high-throughput selection protocol so that a great number of candidates are assessed simultaneously, or in rapid succession.
  • High-throughput-screening of candidates may be directed toward any target property of interest . These include, e.g., a) affinity for a desired metal, b) selectivity (one metal over another) , c) hydrophobicity, d) stability of metal-ligand complexes, and e) biological properties such as catalytic or transport activity.
  • Electrospray mass spectrometry can be used to characterize the relative affinity and specificity of ligand-metal interactions. In mixtures of ligands and metals, the spectra are dominated by the molecular ions of the complex, and their relative abundance correlates with the concentration of ligand- metal complexes present, and hence, the relative affinities and specificities of the ligand-metal binding pairs.
  • a single metal (which is incorporated in limiting concentration) is added to a mixture of ligands.
  • the ligands compete for the metal and the ligand having the highest affinity for the metal will be present in the highest concentration, and hence will show the strongest molecular ion. This method identifies the relative affinity of ligands for a given metal .
  • the relative affinities can also be determined by mass spectrometry in a competition assay.
  • a solution containing 1 equivalent of standard ligand (e.g., DFO) and 0.5 equivalent of the metal (e.g., iron) a known amount of the uncharacterized ligand is added.
  • the solution is allowed to equilibrate, and the ability of the ligand to strip metal from the standard ligand is expressed as a change in the ratio [standard ligand] / [standard ligand-metal complex] as measured by positive ion ESMS .
  • a calibration curve is generated from standard solutions which allows us to determine what ratio of [standard ligand] / [standard ligand-metal complex] corresponds to a particular amount of metal displaced.
  • This method has been tested with known ligands (EDTA, aerobactin, enterobactin) and shown to give the expected results.
  • the measurement reflects relative affinities of ligands, not absolute (K eff or K . In principle, this could be expanded to provide range information - by using ligands whose K eff is known and which represent a range of binding affinities.
  • the ligand to be displaced should be from the same class of compounds in order to avoid problems with equilibration.
  • ligand specificity a single ligand is added to a mixture of metals.
  • the metals compete for the ligand, which is in limiting concentration and the metal having the highest affinity for the ligand will show the strongest molecular ion.
  • Relative affinities can also be determined in sequential analyses, in which the tightest binding metal in a mixture of metals is first determined by ES-MS. In a subsequent analysis, this metal is eliminated from the mixture of metals and the assay repeated. A series of these assays, which eliminate one metal at each step, allows one to rank the order of affinity of a series of metals for a given ligand. Mixtures containing multiple ligands can also be analyzed by ES-MS following the addition of multiple metals. Deconvolution of the observed molecular ions enables determination of which ligand-metal complexes are of highest concentration in solution, permitting discrimination of the highest affinity ligands in the mixture .
  • HPLC methods can be adapted to analysis of ligand libraries.
  • a given ligand will show a characteristic retention time by reverse-phase HPLC. Upon complexation with a metal, the retention time will change, due to the sequestration of polar functionalities which bind to the metal and as a result are no longer exposed.
  • the relative HPLC peak areas of the ligand and the ligand-metal complex are a measure of the stability of the ligand-metal complex and the affinity of the ligand for the metal.
  • the relative retention times of ligands are a measure of their relative hydrophobicity. Using a robotic sampler, libraries of ligands can be assayed by this method.
  • HPLC can be coupled to ES-MS, allowing chromatographic peaks to be monitored by their expected masses (molecular weight of compound alone and plus metal) .
  • the affinity and selectivity of metals for members of a ligand library also can be determined by UV, visible, and fluorescence spectroscopy.
  • a candidate ligand is added to a solution of the dye complexed with iron.
  • the ligand displaces the dye to form a ligand-iron complex which no longer absorbs at 630 nm, thereby reducing the absorption of the sample by an amount proportional to the affinity of the ligand for iron.
  • This simple spectrophotometric assay is readily adapted to a standard microtiter plate format (for example, 96-well format) , enabling automated analysis of ligand libraries.
  • spectrophotometric reagents including ferron (7-iodo-8-hydroxyquinoline-5-sulfonic acid) and sulfoxine (8 -hydroxyquinoline-5-sulfonic acid) which can work at physiologically relevant pH (7.0) have been developed as tools for high throughput screening of our library.
  • test solutions are prepared in two ways: preformation of the spectrophotometric reagent :Fe complex with subsequent addition of the test ligand, and as an alternative, preformation of the ligand : Fe complex with subsequent addition of the reagent.
  • the results from both preparation methods must agree to verify that equilibrium of the complexes has been reached.
  • the method was adapted for microtiter plates, for use in a plate reader. Analysis of 96 wells requires approximately 2.5 minutes. The percentage of iron stripped by the unknown ligand is expressed as a percentage:
  • a 0 is the absorbance of the initial spectrophotometric reagent iron complex
  • A is the absorbance of the solution after addition and equilibration of uncharacterized ligand.
  • a particular biological property may be of interest . Examples could include superoxide dismutase enzymatic activity, ability of the metal-ligand complex to bind to a particular receptor, or the ability of a particular ligand to transport a metal across a cellular membrane. In these examples, specific relevant assay to quantitate each ligand would be used to guide optimization of ligand for the particular objective .
  • compositions comprising an effective amount of at least one of the polyhydroxamates or analogs selected from the library of candidate polyhydroxamates or analogs of the invention having the desired target property or properties, either with or without a complexed metal, in combination with a pharmaceutically acceptable carrier.
  • the polyhydroxamates or analogs are preferably coadministered with an agent which enhances the uptake of the polyhydroxamate or analog molecule by the cells.
  • the polyhydroxamates or analogs and the pharmaceutical compositions of the present invention may be administered by any means that achieve their intended purpose.
  • administration may be by oral, parenteral , subcutaneous, inhalable aerosol, intravenous, intramuscular, intraperitoneal, or transdermal routes, to the extent each is permitted for the particular composition and application in question.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, type of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • treatment e . g. , of iron overload, an amount sufficient to reduce ferric ion cell concentrations to acceptable levels is administered.
  • compositions within the scope of this invention include all compositions wherein the polyhydroxamate or analog is contained in an amount that is effective to achieve chelation of the target metal at desired binding levels. Although individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • polyhydroxamate or analog, or their pharmaceutically acceptable salts, e.g., the mesylate thereof, as raw chemicals in solution they may be administered as part of a pharmaceutically active mixture or preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the -polyhydroxamates or their analogs that can be used pharmaceutically.
  • Suitable formulations for parenteral administration include aqueous solutions of the polyhydroxamates, analogs, or their salts in water- soluble form, for example, water-soluble salts.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • Polyhydroxamates or analogs selected from the library of candidates of the invention are also useful as chelators to form complexes with transition metals and lanthanides for use as imaging agents, radiodiagnostic agents, X-ray contrast agents, and may also be utilized as therapeutic radioactive agents in a complex of an appropriate radionuclide and ligand attached to a suitable targeting moiety.
  • Complexes of the invention with X-ray opaque metals such as lead, tungsten, and bismuth may give suitable X-ray imaging agents.
  • Complexes with gadolinium or other lanthanides, manganese, or iron may give suitable MRI imaging agents.
  • polyhydroxamate ligand molecules used for imaging will have three hydroxamic moieties for complexing with transition metals and four for complexing with lanthanides.
  • Such polyhydroxamates are prepared having varying chain lengths between the hydroxamic complexing units to maximize affinity for the particular target metal. They also may include ionic (amine, acid) groups which do not participate in metal complexation, but affect the overall charge of the complex to enhance excretion, absorption, uptake or other physiological properties as desired. Further, the polyhydroxamate ligand molecules also may include non-ionic groups such as hydroxyl, alkoxide, and ether linkages to enhance solubility.
  • hydrophobic groups alkyl, phenyl , benzyl
  • hydrophobic groups alkyl, phenyl , benzyl
  • polyhydroxamates and analogs of the present invention also have utility for binding metal ions in solution, for example, to achieve quantitative removal of heavy metals from wastewater effluents.
  • candidates are screened and selected for target properties such as enhanced affinity to specified metal (s) (e.g., iron, copper, lead).
  • a mixture of two or more polyhydroxamates may be utilized to achieve highly specific binding of ligands to an array of metal ions found in the source solution.
  • water purification of heavy metals may be achieved (or other separation and concentration of solution-borne metals accomplished) by a variety of methods.
  • the metal-containing solution is brought into contact with a composition which includes the metal-binding hydroxamates by flowing the solution through a porous mesh container housing the polymeric hydroxamate composition.
  • the metal ions are thereby captured by the metal chelators and may be discarded or recycled as desired.
  • the reaction cycle includes introduction of the nosyl-protected metal -binding moiety, nosyl group removal, introduction of a spacer, and repetition of the cycle to elongate the scaffold.
  • washing protocol A typical washing cycle consisted of mechanically stirring the resin in the specified volume of solvent for 3-5 min, followed by decantation of the liquid phase by suction using a gas dispersion tube (Porosity C) and house vacuum. Cleavage protocol for small resin samples.
  • N- [2- ⁇ itrobenzenesulfonyl] -0- benzylhydroxylamine, Bn-O-NH-Ns A 250-mL round bottom flask fitted with an addition funnel was charged with O-benzylhydroxylamine hydrochloride (5 g, 31.32 mmol) and the solid was partially dissolved in 60 mL dry pyridine by stirring with a magnetic bar under a flow of nitrogen. The flask was immersed in an ice-salt water bath and cooled to about -50 °C.
  • N- [2-Nitrobenesulfonyl] -O-tert- butylhydroxyla ine, tBu-0-NH-Ns A 250 mL round bottom flask fitted with an addition funnel was charged with O-tert- butylhydroxyla ine hydrochloride (4.8 g, 38 mmol) and the solid was dissolved in 80 mL of dry chloroform by stirring with a magnetic bar. The flask was immersed in an ice-salt water bath and cooled to about -5 °C and triethylamine (8.08g, 80 mmol) was added dropwise.
  • 2-Nitrobezenesulfonyl chloride (nosyl chloride, 7.8 g, 35 mmol) as a solution in 20 mL of anhydrous pyridine was added dropwise over 30 min. After addition was complete, the dark orange solution was allowed to warm to RT and stirred for 17 h. Pyridine was removed under reduced pressure and the residue taken up in EtOAc (200 mL) and extracted successively with water (2 x 100 mL) , 5% aq. HCl (2 x 100 mL) , and 5% aq. NaHC0 3 (200 mL) . The organic layer was dried over MgS0 4 , filtered, and the solvent removed.
  • O-protected-N- (nosyl) hydroxylamine derivatives may be prepared from commercially available starting materials using synthetic and isolation protocols analogous to those described above for the t- Bu, Bn, 2, 4-dimethoxybenzyl, and THP analogs.
  • reaction (a) Freshly prepared 3.1 (0.996 mmol/g, 4.51 mmol) was suspended in 50 mL DMF and stirred while DIPEA (1.2 mL, 6.77 mmol, 1.5 eq.) was added with a graduated pipette. After five min, 5-aminopentanol (2.5 mL, 22.6 mmol, 5 eq. , 0.45 M in final resin suspension) was added likewise. The suspension was heated to 60 °C with a heating mantle and stirred for 24 hours.
  • reaction (b) Tosylation of terminal hydroxyl group to form compound 3.3, reaction (b) . Freshly prepared 3.2 (0.962 mmol/g, 4.51 mmol) was washed with 60 mL dichloroethane and decanted. Dichloroethane (35 mL) was added and stirred while a light yellow solution of p-toluenesulfonyl chloride (4.3 g, 22.6 mmol, 5 eq. , 0.38 M in final resin suspension) and pyridine (3.57 g,45.2 mmol, 10 eq, 0.76 M) in dichloroethane (25 mL) was added with a pipette.
  • p-toluenesulfonyl chloride 4.3 g, 22.6 mmol, 5 eq. , 0.38 M in final resin suspension
  • pyridine 3.57 g,45.2 mmol, 10 eq, 0.76 M
  • reaction (d) Removal of 2 -nitrobenzene-sulfonyl (nosyl) protective group to form compound 3.5, reaction (d) .
  • Freshly prepared 3.4 (0.742 mmol/g, 4.51 mmol) was suspended in 45 mL DMF, and Cs 2 C0 3 (12 g, 36 mmol, 8 eq.) was added in one portion. The suspension was stirred at room temperature while thiophenol (1.5 mL, 13.6 mmol, 3 eq. , 0.3 M in final suspension) was added with a graduated pipette. The suspension turned orange immediately. After stirring for 4 hours, the brownish-orange supernatant was decanted, and the resin washed with DMF:H 2 0 (7:3, v/v, 3 x 50 mL) , DMF (50 mL) ,
  • HPLC analysis (condition 1) of a crude isolate from cleavage of a small sample ( ca . 2-3 mg resin) indicated 95% purity and complete nosyl group removal.
  • reaction (b) Repetition of a series of reactions (tosylation, displacement of tosyl group by nosyl- protected hydroxylamine, removal of nosyl group, coupling of succinate, and condensation of aminopentanol) to further elongate the polyhydroxamate scaffold and produce compound 3.8.
  • Repetition of reaction (b) Starting from compound 3.7 (0.74 mmol/g 4.51 mmol), tosylation was effected with tosyl chloride (4.3 g, 22.6 mmol, 5 eq. , 0.38 M in final suspension), and pyridine (3.57g, 45.2 mmol) in dichloroethane (60 mL) as described above for the preparation of 3.3.
  • reaction (e) Repetition of reaction (e) .
  • the second succinate unit was added (0.685 mmol/g, 4.51 mmol) by reaction with succinic anhydride (1.36 g, 13.6 mmol, 3 eq., 0.27 M in final suspension) in DMF (50 mL) as described for the preparation of compound 3.6.
  • reaction (f) Repetition of reaction (f) .
  • the terminal succinic acid group (0.641 mmol/g, 4,51 mmol) was activated with CDI (3.65g, 22.6 mmol) or HATU as depicted in Scheme 3 in a mixture of DMF and THF (4:1, 50 mL) and then, after washing, coupled to 5-aminopentanol (2.5 mL, 22.6 mmol, 5 eq., 0.45 M in final suspension) in DMF (50 mL) to yield 3.8.
  • the suspension was decanted and washed with DMF (3 30 mL) , EtOH (30 mL) , CH 2 C1 2 (30 mL) , EtOH (30 mL) , CH 2 C1 2 (30 mL) and DMF (3 x 30 mL) .
  • the derivatized resin was used as such for the next step.
  • reaction (d) Condensation of hydroxylamine intermediate with 6-bromohexanoic acid to form compound 4.4, reaction (d) .
  • Freshly prepared compound 4.3 (4.42 mmol, 5.0 g) was suspended in 50 mL DMF while a solution of 6- bromohexanoic acid (4.32 g, 22.1 mmol, 5 eq.) and DMAP (0.11 g, 0.9 mmol, 0.2 eq. w/respect to resin) in 10 mL DMF was added. After stirring for 2-3 min, DIC (3.5 mL, 22.1 mmol, 5 eq.) was added via pipette.
  • the suspension was heated for 17 hours at 50 °C, cooled, decanted, and washed with DMF (3 x 30 mL) , EtOH (3 x 30 mL) , CH 2 C1 2 (3 x 30 mL) , EtOH (2 x 30 mL) , and DMF (3 x 30 mL) .
  • the resin was washed with DMF (3 x 60 mL) , EtOH (60 mL) , CH 2 C1 2 (60 mL) , EtOH (60 mL) , CH 2 C1 2 (60 mL) , EtOH (2 x 60 mL) , and CH 2 C1 2 (3 x 60 mL) .
  • the library compounds represented by the general structure 10 were characterized by ES-MS and the purity determined by HPLC (condition 2) . Results of the ES-MS and HPLC analyses are set forth in Table 9 below.
  • the CDI -activated Wang resin 13a.1 was prepared from Wang PS resin (1.1 mmol/g) as described in Example 2 and 0.066 g (0.91 mmol/g, 0.06 mmol) of the activated resin was loaded into each well of the 96 well-format reaction block. The resin was swelled in DMF and washed with the solvent in which the reaction was supposed to be performed. A typical washing cycle include mixing the resin with 1.0-1.5 mL of the specified solvent at 600 rpm for 1 min and emptying the block for 4 to 5 min with N 2 pressure of about 9 psi .
  • the reagent solutions prepared in anhydrous solvents whenever necessary
  • solvents were delivered into the reaction wells by robotic arms (except during the cleavage of the compounds from the solid support, which was done manually using a repeater pipette) and all the operations and reactions were carried out in an atmosphere of N 2 .
  • the reaction block was agitated at 600 rpm for specified amount of time. After each reaction, the reaction block was emptied and the resin was washed with one of the following washing protocols given below:
  • Washing protocol 1 THF (x 2) , DMF (x 1) , EtOH (x 1) , and DMF (x 1); Washing protocol 2: DMF (x 2) , EtOH (x 1) , and DMF (x 2); and Washing protocol 3: DMA (x 2) , EtOH (x 1) , and DMA (x 2) . Washing protocol 4: THF (x 3) .
  • CDI -activated Wang resin in each well was reacted with a solution of 0.5 M 5-aminopentanol and 0.3 M N, N- diisopropylethylamine (DIPEA) in DMF for 8 h at 60 °C. Washing protocol 2 and 4.
  • DIPEA N, N- diisopropylethylamine
  • the resin-bound substrate was agitated and heated with a solution of 0.50 M NsNHOBu t (4.0 eq.)in THF (0.4 ml), 1.0 M triphenylphosphine (4.0 eq.) in THF (0.22 ml), and 1.0 M diisopropyl azodicarboxylate (DIAD, 4.0 eq.) in THF (0.22 mL) for 4 h at 37 °C. Washing protocol 1.
  • the substrate 13a.3 was agitated with a solution of 0.20 M 2-mercaptoethanol (0.18 mmol, 3.0 eq.) and 0.40 M DBU (0.36 mmol, 6.0 eq.) in DMF (0.90 mL) for 30 min at room temperature. The yellow colored solution was drained and the resin was washed with 1 mL each of EtOH and DMF. The reaction was repeated with fresh reagents. Washing protocol 2. (d) Coupling with carboxylic acid anhydride or dicarboxylic acid/HATU/DIPEA to from intermediates of the general structure 13a.5
  • the intermediate 13a.4 in each well was reacted with dicarboxylic acid anhydride (succinic anhydride, glutaric anhydride or 3,3- tetramethyleneglutaric anhydride) or with dicarboxylic acid (1 , 4-phenylenedipropionic acid, adipic acid, trans- 1, 4-cyclohexane dicarboxylic acid) depending on its location in the array) .
  • dicarboxylic acid anhydride succinic anhydride, glutaric anhydride or 3,3- tetramethyleneglutaric anhydride
  • dicarboxylic acid 1, 4-phenylenedipropionic acid, adipic acid, trans- 1, 4-cyclohexane dicarboxylic acid
  • Each of the activated intermediate was reacted with 0.5 M solution of amino-alcohol (5-aminopentanol, 4- piperidineethanol, 3-aminopropanol , depending on its location in the array) and 0.3 M DIPEA in DMA for 8 h at room temperature (with agitation) . Washing protocol 2 and 4.
  • amino-alcohol 5-aminopentanol, 4- piperidineethanol, 3-aminopropanol , depending on its location in the array
  • the substrate was agitated with a solution of 0.25 M acetic anhydride (0.30 mmol, 5.0 eq.) and 0.50 M DIPEA (0.60 mmol, 10.0 eq) in DMF (1.2 mL) for 6 h at room temperature.
  • the resin was further washed with DMF (x 2), EtOH ( x 2) , and 1,2- dichloroethane (DCE, x 3) , and dried overnight under vacuum.
  • the compounds were simultaneously cleaved off the resin by agitating the resin-bound intermediate of the general structure 13a.7 with a solution of 90%TFA in CH 2 C1 2 (1.5 mL each; 18:1:1, v/v) for 2 h at room temperature. After filtration, the resin was washed with cleavage cocktail (1.0 mL each), and the combined solution in the collection vial was screw-capped, and left overnight (24 h) at room temeprature to ensure the complete deprotection of the tert-butyl groups . The solutions were then transferred to glass tubes and evaporated to dryness by blowing a stream of N 2 . Acetonitrile (1 mL) was added to each sample and evaporated to dryness with N 2 . Once again acetonitrile (1 mL) was added to each sample and evaporated to dryness on a speedvac concentrator overnight. The samples were further dried under high vacuum overnight .
  • Washing protocol 1 THF (x 2) , DMF (x 1) , EtOH (x 1) , and DMF (x 1); Washing protocol 2: DMF (x 2), EtOH (x 1) , and DMF (x 2); and Washing protocol 3: DMF (x 2) , EtOH (x 1) , and 1,2 -dichloroethane (DCE, x 2).
  • the resin was washed successively with 20 mL portions of DCE (x 2) , DMF (x 2) , EtOH (x 1) , and CH 2 C1 2 (x 3) , which was then dried under high vacuum to give 1.82 g of 14a.2.
  • the reaction can also be carried out in DCE using pyridine as base or in pyridine as solvent without compromising the loading (typically 0.89 to 0.91 mmol/g based on the mass of dried resin) of the resin and the purity of the subsequent reaction products.
  • nosyl resin 14a.2 was converted to intermediates of the general structure 14a.3 either (i) by alkylation using alkyl bromides (leading to the products 14.1 to 14.4) or (ii) by Mitsunobu reaction with alcohols [leading to the products 14.7 to 14.33 (corresponding N-Boc alcohol was used for the analogs 14.27 to 14.33) ] .
  • the substrate 14a.3 was agitated with a solution of 0.20 M 2-mercaptoethanol (0.18 mmol, 3.0 eq.) and 0.40 M DBU (0.36 mmol, 6.0 eq.) in DMF (0.90 mL) for 30 min at room temperature. The yellow colored solution was drained and the resin was washed with 1 mL each of EtOH and DMF. The reaction was repeated with fresh reagents. Washing protocol 3.
  • the substrate 14a.4 was agitated with a solution of the appropriate 0.25 M bromoacid chloride (6- bromohexanoyl chloride or 8-bromooctanoyl chloride; 0.24 mmol, 4.0 eq.) and 0.50 M DIPEA (0.48 mmol, 8.0 eq.) in DCE (0.96 mL) for 4 h at room temperature. Washing protocol 2.
  • the substrate 14a.5 was agitated with a solution of 0.20 M 0- (tert-butyl) -N- (2 -nosyl) hydroxylamine (0.18 mmol, 3.0 eq.) and 0.13 M 1, 1, 3 , 3-tetramethylguanididne (TMG, 0.12 mmol, 2.0 eq.) in DMF (0.90 mL) at 50 °C for 6 h. Washing protocol 2.
  • the nosyl resin 14a.2 was converted to intermediates of the general structure 15a.1 either (i) by Mitsunobu reaction with alcohols [leading to the products 15.1 to 15.6 (Boc-aminopentanol was used) and 15.11 to 15.18] or (ii) by alkylation using alkyl bromides (leading to the products 15.7 to 15.10).
  • End-capping was carried out by agitating the substrate with a solution of 0.25 M acetic anhydride (0.15 mmol, 2.5 eq.) and 0.50 M DIPEA (0.30 mmol, 5.0 eq) in DMF (0.60 mL) for 2 h at room temperature. Washing protocol 2.
  • the substrate 15a.1 was agitated with a solution of 0.20 M 2-mercaptoethanol (0.18 mmol, 3.0 eq.) and 0.40 M DBU (0.36 mmol, 6.0 eq.) in DMF (0.90 mL) for 30 min at room temperature. The yellow colored solution was drained and the resin was washed with 1 mL each of EtOH and DMF. The reaction was repeated with fresh reagents. Washing protocol 2.
  • the substrate 15a.2 was agitated with a solution of appropriate 0.17 M N-Fmoc -amino acid (0.24 mmol, 4.0 eq.), 0.17 M HATU (0.24 mmol, 4.0 eq.), and 0.33 M (DIPEA) in DMA (1.48 mL) for 4 h at room temperature. The solution was drained and the resin was washed with DMF (x 2) . The reaction was repeated with fresh reagents using half the amounts given above. Washing protocol 2.
  • the substrate 15a.4 was agitated with a solution of the appropriate 0.25 M bromoacid chloride (6- bromohexanoyl chloride or 8-bromooctanoyl chloride; 0.24 mmol, 4.0 eq.) and 0.50 M DIPEA (0.48 mmol, 8.0 eq.) in DCE (0.96 mL) for 4 h at room temperature. Washing protocol 2.
  • Bromide displacement was carried out by agitating the substrate 15a.5 with a solution of 0.20 M O- ( ert- butyl) -N- (2 -nosyl) hydroxylamine (0.18 mmol, 3.0 eq.) and 0.13 M TMG (0.12 mmol, 2.0 eq.) in DMF (0.90 mL) at 50 °C for 4 h. Washing protocol 2. The nosyl group was removed as described in the step (b) . Washing protocol 2.
  • the substrate 15a.6 was heated with a solution of 0.50 M succinic anhydride (0.30 mmol, 5.0 eq.), 0.05 M
  • the substrate 15a.7 was agitated with 0.50 M CDI (0.30 mmol, 5.0 eq) in DMA (0.60 mL) for 2 h at room temperature.
  • the solution was drained and the resin was washed with DMA (x 2) , and the intermediate was then reacted with 0.50 M 5-amino-l-pentanol (0.30 mmol, 5.0 eq.) and 0.50 M DIPEA (0.30 mmol, 5.0 eq.) in DMA (0.60 mL) for 8 h at room temperature. Washing protocol 2.
  • the substrate 15a.8 was heated with a solution of the 0.25 M 0- ( ert-butyl) -N- (2-nosyl) hydroxylamine (0.24 mmol, 4.0 eq.), 0.25 M triphenylphosphine (0.24 mmol, 4.0 eq.), and 0.25 M DIAD (0.24 mmol, 4.0 eq.) in THF (0.96 mL) for 4 h at 37 °C. Washing protocol 1.
  • the substrate 15a.10 was agitated with a solution of
  • End-capping was carried out by agitating the substrate with a solution of 0.25 M acetic anhydride (0.15 mmol, 2.5 eq.) and 0.50 M DIPEA (0.30 mmol, 5.0 eq) in DMF (0.60 mL) for 2 h at room temperature. Washing protocol 2. Subsequent transformation of the intermediates of the type 16a.2 to the final products 16 was achieved by repeating the sequence of reactions described earlier in Scheme 15a for the conversion of 15a.1 to 15 [except that TFA-CH 2 C1 2 (9:1, v/v) was used in the final step].
  • the novel examples represented by the general structure 16 were characterized by ES-MS and the purity determined by HPLC (condition 2; gradient: 0% to 100% B in 10 min) and the results are summarized in the following Table 13.
  • a library of DFO analogs were synthesized in IRORI MiniKanTM reactors (polypropylene) using AccuTagTM-100 Combinatorial Chemistry System.
  • the nosyl derivatized resin 14a.2 was prepared (Example 6) , and loaded into each of the sixteen MiniKans containing a radiofrequency tag. Subsequent chemical operations were carried out in round-bottom flasks by sorting MiniKans (whenever necessary) using the AccuTag system. After the addition of solvent or reagent solutions, air bubbles were removed from the MiniKans by applying vacuum (10-20 mm Hg) for 5-10 seconds.
  • the MiniKans were stirred for 15 min with 25 mL or 50 mL of the solvent for 8 and 16 MiniKans respectively. After finishing the wash cycles between the reactions, the MiniKans were dried under vacuum (10-20 mm Hg) for about 30 min.
  • MiniKans containing nosyl-derivatized resin 14a.2 (0.907 mmol/g, 0.061 g each, 8 MiniKans, 0.055 mmol) were suspended in a solution of 0.25 M each of triphenylphoshine (1.64 g, 6.25 mmol), appropriate alcohol MeOH (0.253 mL, 6.25 mmol) or EtOH (0.362 mL,
  • End-capping was carried out by stirring the MiniKans (16) with 0.40 M acetic anhydride (1.89 mL, 20.0 mmol) and 0.80 M DIPEA (6.95 mL, 40.0 mmol) in DMF (50 mL) for 3 h at room temperature. The solution was decanted and the MiniKans were washed with DMF (x 1) and then with EtOH and CH 2 C1 2 alternately (3 cycles) . (b) The nosyl group was removed to form intermediates of the general structure 17a.2
  • the MiniKans (16) containing 17a.1 were stirred with a soluion of 0.20 M 2-mercaptoethanol (0.70 mL, 10.0 mmol) and 0.40 M DBU (2.99 mL, 20.0 mmol) in DMF (50 mL) for 1 h at room temperature in an atmosphere of N 2 .
  • the yellow colored solution was removed and the MiniKans were washed with DMF (50 mL) .
  • the reaction was repeated with fresh reagents and the MiniKans were washed with DMF (x 1) and then with EtOH and CH 2 C1 2 alternately (4 cycles) .
  • the MiniKans (8) were suspended in 20% piperidine in DMF and stirred for 6 min at room temperature. The solution was decanted, fresh deprotection cocktail was added, and stirring continued for 40 min. The solution was decanted and the MiniKans were washed with EtOH and CH 2 C1 2 alternately (4 cycles) .
  • the Minikans (16) containing 17a.5 were stirred with an orange-red colored solution of 0.20 M 0-(2,4- dimethoxybenzyl) -N- (2-nitrobenzenesulfonyl) hydroxylamine (2.94 g, 8.00 mmol) and 0.15 M TMG (0.752 mL, 6.00 mmol) in anhydrous DMF (40 mL) for 12 h at 50 °C in an atmosphere of ⁇ 2 . The solution was removed and the
  • N-Acetylation was carried out by stirring the MiniKans (16) with 0.25 M acetic anhydride (1.18 mL, 12.5 mmol) and 0.50 M DIPEA (4.35 mL, 25.0 mmol) in 1,2- dichloroethane (50 mL) for 12 h at room temperature in an atmosphere of ⁇ 2 .
  • the solution was decanted and the MiniKans were washed with DMF (x 2) and then with EtOH (x 1) and CH 2 C1 2 (x 2) alternately (2 cycles) .
  • the nosyl resin 14a.2 was converted to intermediates of the general structure 18a.1 either (i) by Mitsunobu reaction with alcohols [leading to the final products 18.1, 18.2, and 18.15 (Boc-aminopentanol was used) and 18.7 to 18.14] or (ii) by alkylation using alkyl bromides (leading to the products 18.3 to 18.6).
  • End-capping was carried out by agitating the substrate with a solution of 0.25 M acetic anhydride (0.15 mmol, 2.5 eq.) and 0.50 M DIPEA (0.30 mmol, 5.0 eq) in DMF (0.60 mL) for 2 h at room temperature. Wash protocol 2. (b) The nosyl group was removed to form the intermediates of the general structure 18a.2.
  • the substrate 18a.1 was agitated with a solution of 0.20 M 2-mercaptoethanol (0.18 mmol, 3.0 eq.) and 0.40 M DBU (0.36 mmol, 6.0. eq.) in DMF (0.90 mL) for 30 min at room temperature. The yellow colored solution was drained and the resin was washed with 1 mL each of EtOH and DMF. The reaction was repeated with fresh reagents. Washing protocol 2. (c) Coupling with N-Fmoc -amino acid was carried out to form intermediates of the general structure 18a.3.
  • the substrate 18a.2 was agitated with a solution of appropriate 0.17 M N-Fmoc - amino acid (0.24 mmol, 4.0 eq.), 0.17 M HATU (0.24 mmol, 4.0 eq.), and 0.33 M (DIPEA) in DMA (1.48 mL) for 4 h at room temperature. The solution was drained and the resin was washed with DMF (x 2) . The reaction was repeated with fresh reagents using half the amounts given above. Washing protocol 2.
  • the substrate 18a.3 was agitated with a solution of 25% piperidine in DMF (1.0 mL) for 3 min at room temperature. The solution was drained off and the reaction was repeated with fresh reagents for 15 min. Washing protocol 2.
  • the substrate 18a.4 was agitated with a solution of the appropriate 0.25 M bromoacid chloride (6- bromohexanoyl chloride or 8-bromooctanoyl chloride; 0.24 mmol, 4.0 eq.) and 0.50 M DIPEA (0.48 mmol, 8.0 eq.) in DCE (0.96 mL) for 4 h at room temperature. Washing protocol 2.
  • Bromide displacement was carried out by agitating the substrate 18a.5 with a solution of 0.20 M O- ( tert- butyl) -N- (2 -nosyl) hydroxylamine (0.18 mmol, 3.0 eq.) and 0.13 M TMG (0.12 mmol, 2.0 eq.) in DMF (0.90 mL) at 50 °C for 4 h. Wash protocol 2.
  • the substrate 18a.6 was transformed to compounds of the general structure 18a.7 by repeating the above dsecribed steps (b) to (f) .
  • the substrate was agitated with a solution of 0.25 M acetic anhydride (0.30 mmol, 5.0 eq.) and 0.50 M DIPEA (0.60 mmol, 10.0 eq) in DCE (1.2 mL) for 6 h at room temperature. After completing the washing protocol 2, the resin was further washed with (DCE x 3) , and dried overnight under vacuum.
  • Bromide displacement was carried out by agitating the substrate 19a.1 with a solution of 0.20 M O- ( ert- butyl) -N- (2 -nosyl) hydroxylamine (0.18 mmol, 3.0 eq.) and 0.13 M TMG (0.12 mmol, 2.0 eq.) in DMF (0.90 mL) at 55 °C for 6 h. Washing protocol 2.
  • End-capping was carried out by agitating the substrate with a solution of 0.25 M acetic anhydride (0.15 mmol, 2.5 eq.) and 0.50 M DIPEA (0.30 mmol, 5.0 eq) in DMF (0.60 mL) for 2 h at room temperature. Washing protocol 2.
  • the CAS assay solution was prepared as described by B.Schwyn and J.B. Neilands (Analytical Biochemistry, 160, 47-56, 1987) .
  • a 6-mL volume of 10 mM HDTMA (hexadecyltrimethylammonium bromide) solution was placed in a 100-mL volumetric flask and diluted with water (10 mL) .
  • a mixture of 1.5 mL iron(III) solution (1 mM, FeCl 3 .6H 2 0, 10 mM HC1) and 7.5 mL of a 2 mM aqueous CAS solution was slowly added under stirring.
  • A/A 0 S x [ligand] .
  • the negative value of the descending slope (- S) was used to determine the relative binding affinity of each ligand for iron. A more negative slope corresponds to a higher iron binding affinity.
  • DFO was used as a control ligand.
  • the following relative iron binding affinities (expressed as -S) were determined for each ligand in the library: DFO 3 (0.64), 10.10 (0.46); 10.7 (0.44); 10.4 (0.43); 10.12 (0.42); 10.8 (0.35); 10.11 (0.39); 10.2 (0.37), 10.3 (0.32) 10.5 (0.32), 10.1 (0.27), 10.9 (0.24).
  • Example 12b Determination of relative iron binding affinity for a library of polyhydroxamates (compounds 13, 14, 15, 16, 17, 18 ) using a UV-VIS spectrometric assay- based on sulfoxine.
  • Nanopure water before using The required stock solutions were prepared as described below:
  • Stock solution #2 0.1 M Iron (III) chloride in 0.1 M HCl Dissolved FeCl 3 .6H 2 0 (0.27 g, 0.001 mol) in 5 mL water. Added 1 mL 1 M HCl; mix, and diluted to 10 mL with water.
  • Stock soul tion #3 1 mM FeCl 3 in 1 mM HCl. Prepared fresh. Diluted 50 ⁇ l 0.1 M FeCl 3 in 0.1 M HCl to 5.0 mL with water. Stock solution #4: 0.01 M Sulfoxine sodium salt in water.
  • Stock solution #6 0.5 mM FeCl 3 -6H 2 0 in 0.5 mM HCl Prepared fresh. Diluted 25 ⁇ l of 0.1 M (FeCl 3 - 6H 2 0-HC1) (stock #2) to 5.0 mL with water.
  • Stock solution #7 1.0 mM sulfoxine sodium salt in water. Prepared fresh. Diluted 1 mL of 0.01 M sulfoxine-Na to 10 mL with 0.02 M PIPES (pH 7.0, stock #1)
  • a 0 the absorbance of the control solution
  • a s the absorbance of a sample solution
  • the precentage of iron stripped by the tested ligand of the library 13, 14, 15, 16, 17, and 18, is expressed as a percentage: [A 0 -A s ] / [A 0 ] xlOO
  • a 0 is the absorbance of the initial sulfoxine : Fe complex
  • A is the absorbance of the solution after addition and equilibration of uncharacterized ligand.
  • the calculation is made for both sets of samples, using the appropriate A 0 .
  • the error in the %Fe value has been determined to be ⁇ 2%.
  • the following relative iron binding affinities were determined for each ligand and expressed as % of iron removed from preformed sulfoxine.
  • Fe 3+ complex preformed ligand. Fe 3+ complex
  • the system can be represented as:
  • Fe +3 complex were determined for ligands in the library of the general structure 13. 13.1 (DFO): 58.9; 13.2: 28.2; 13.3: 30.8; 13.6:25.5;
  • ES-MS analysis of 10.5 in the presence of a metal mixture containing iron, copper and nickel 20 ⁇ l of a 5 mM stock solution of 10.5 in methanol was mixed with 20 ⁇ l of a 5 mM stock solution of FeCl 3 in water, 20 ⁇ l of a 5 mM stock solution of Cu(N0 3 ) 2 in waters, and 20 ⁇ l of a 5 mM stock solution of Zn(N0 3 ) 2 in water. The mixture was diluted to 0.5 mL. This solution was allowed to stand for 24 hr to equilibrate before analyzing by ES-MS.

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Abstract

L'invention concerne un procédé de synthèse de polyhydroxamates voulus et d'analogues de polyhydroxamates. Le procédé consiste à lier un premier composant du polyhydroxamate voulu ou d'un analogue de polyhydroxamate à une matrice de support dans des conditions effectives pour former un premier intermédiaire lié à la matrice de ce polyhydroxamate voulu ou de son analogue; à étendre ce premier intermédiaire lié à la matrice au moyen de réactifs dans des conditions de réaction effectives pour former un ou plusieurs intermédiaires supplémentaires liés à la matrice de ce polyhydroxamate voulu ou de son analogue, ce qui permet de former un précurseur lié à la matrice du polyhydroxamate voulu ou de l'analogue de polyhydroxamate. On retire les groupes protecteurs utilisés lors de la synthèse du précurseur et on enlève de la matrice de support le précurseur lié à la matrice, ce qui permet d'effectuer une synthèse du polyhydroxamate voulu ou de l'analogue de polyhydroxamate. L'invention concerne également des procédés de production, de criblage et de sélection de banques de polyhydroxamates candidats, des banques et des polyhydroxamates d'analogues de polyhydroxamates, de leurs intermédiaires, ainsi que des procédés d'utilisation de ces composés et de leurs compositions.
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US9334716B2 (en) 2012-04-12 2016-05-10 Halliburton Energy Services, Inc. Treatment fluids comprising a hydroxypyridinecarboxylic acid and methods for use thereof
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Title
MILLER S C ET AL: "SITE-SELECTIVE N-METHYLATION OF PEPTIDES ON SOLID SUPPORT" JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, AMERICAN CHEMICAL SOCIETY, WASHINGTON, DC, US, vol. 119, 1997, pages 2301-2302, XP001128157 ISSN: 0002-7863 *
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